Article

Ca2+-calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel α1C-subunit gene (Cacna1c) by DREAM translocation

The Journal of Physiology

June 2011
589(Pt 11):2669-86

DOI:10.1113/jphysiol.2010.201400

Source
PubMed

Authors:

Sandra Hänninen

University of Oulu

Topi Korhonen

Polar Electro

Jussi Koivumäki

Tampere University

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Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

Integrative computational modeling of calcium handling and cardiac arrhythmias

Thesis

Full-text available

Jan 2021

Henry Sutanto

[CLICK DOI ABOVE TO GET THE FREE FULLTEXT] Cardiomyocyte calcium handling is a major determinant of excitation-contraction coupling. Alterations in one or more calcium-handling proteins may induce arrhythmias through the formation of ectopic activity, direct and indirect ion-channel regulation, and structural remodeling. Due to the complex and tight interactions between calcium and other molecules within a cardiomyocyte, it remains experimentally challenging to study the exact contributions of calcium-handling abnormalities to arrhythmogenesis. Multiscale computational studies performed in close collaboration with laboratory experiments create new opportunities to unravel the mechanisms of arrhythmogenesis. This thesis describes the roles of integrative computational modeling in unraveling the arrhythmogenic consequences of calcium- handling abnormalities.

Cardiomyocyte calcium handling in health and disease: Insights from in vitro and in silico studies

Article

Full-text available

Mar 2020
PROG BIOPHYS MOL BIO

Calcium (Ca2+) plays a central role in cardiomyocyte excitation-contraction coupling. To ensure an optimal electrical impulse propagation and cardiac contraction, Ca2+ levels are regulated by a variety of Ca2+-handling proteins. In turn, Ca2+ modulates numerous electrophysiological processes. Accordingly, Ca2+-handling abnormalities can promote cardiac arrhythmias via various mechanisms, including the promotion of afterdepolarizations, ion-channel modulation and structural remodeling. In the last 30 years, significant improvements have been made in the computational modeling of cardiomyocyte Ca2+ handling under physiological and pathological conditions. However, numerous questions involving the Ca2+-dependent regulation of different macromolecular complexes, cross-talk between Ca2+-dependent regulatory pathways operating over a wide range of time scales, and bidirectional interactions between electrophysiology and mechanics remain to be addressed by in vitro and in silico studies. A better understanding of disease-specific Ca2+-dependent proarrhythmic mechanisms may facilitate the development of improved therapeutic strategies. In this review, we describe the fundamental mechanisms of cardiomyocyte Ca2+ handling in health and disease, and provide an overview of currently available computational models for cardiomyocyte Ca2+ handling. Finally, we discuss important uncertainties and open questions about cardiomyocyte Ca2+ handling and highlight how synergy between in vitro and in silico studies may help to answer several of these issues.

Kv channel-interacting proteins as neuronal and non-neuronal calcium sensors

Article

Full-text available

Aug 2018
CHANNELS

Robert Bähring

Kv channel-interacting proteins (KChIPs) belong to the neuronal calcium sensor (NCS) family of Ca²⁺-binding EF-hand proteins. KChIPs constitute a group of specific auxiliary β-subunits for Kv4 channels, the molecular substrate of transient potassium currents in both neuronal and non-neuronal tissues. Moreover, KChIPs can interact with presenilins to control ER calcium signaling and apoptosis, and with DNA to control gene transcription. Ca²⁺ binding via their EF-hands, with the consequence of conformationl changes, is well documented for KChIPs. Moreover, the Ca²⁺ dependence of the presenilin/KChIP complex may be related to Alzheimer’s disease and the Ca²⁺ dependence of the DNA/KChIP complex to pain sensing. However, only in few cases could the Ca²⁺ binding to KChIPs be directly linked to the control of excitability in nerve and muscle cells known to express Kv4/KChIP channel complexes. This review summarizes current knowledge about the Ca²⁺ binding properties of KChIPs and the Ca²⁺ dependencies of macromolecular complexes containing KChIPs, including those with presenilins, DNA and especially Kv4 channels. The respective physiological or pathophysiolgical roles of Ca²⁺ binding to KChIPs are discussed.

Modulation of human Kv4.3/KChIP2 channel inactivation kinetics by cytoplasmic Ca2+

Article

Full-text available

Nov 2017
PFLUG ARCH EUR J PHY

The transient outward current (Ito) in the human heart is mediated by Kv4.3 channels complexed with Kv channel interacting protein (KChIP) 2, a cytoplasmic Ca²⁺-binding EF-hand protein known to modulate Kv4.3 inactivation gating upon heterologous co-expression. We studied Kv4.3 channels co-expressed with wild-type (wt) or EF-hand-mutated (ΔEF) KChIP2 in human embryonic kidney (HEK) 293 cells. Co-expression took place in the absence or presence of BAPTA-AM, and macroscopic currents were recorded in the whole-cell patch-clamp configuration with different free Ca²⁺ concentrations in the patch-pipette. Our data indicate that Ca²⁺ is not necessary for Kv4.3/KChIP2 complex formation. The Kv4.3/KChIP2-mediated current decay was faster and the recovery of Kv4.3/KChIP2 channels from inactivation slower with 50 μM Ca²⁺ than with BAPTA (nominal Ca²⁺-free) in the patch-pipette. The apparent Ca²⁺-mediated slowing of recovery kinetics was still observed when EF-hand 4 of KChIP2 was mutated (ΔEF4) but not when EF-hand 2 (ΔEF2) was mutated, and turned into a Ca²⁺-mediated acceleration of recovery kinetics when EF-hand 3 (ΔEF3) was mutated. In the presence of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 cytoplasmic Ca²⁺ (50 μM) induced an acceleration of Kv4.3/KChIP2 recovery kinetics, which was still observed when EF-hand 2 was mutated (ΔEF2) but not when EF-hand 3 (ΔEF3) or EF-hand 4 (ΔEF4) was mutated. Our results support the notion that binding of Ca²⁺ to KChIP2 EF-hands can acutely modulate Kv4.3/KChIP2 channel inactivation gating, but the Ca²⁺-dependent gating modulation depends on CaMKII action. Our findings speak for an acute modulation of Ito kinetics and frequency-dependent Ito availability in cardiomyocytes under conditions with elevated Ca²⁺ levels and CaMKII activity.

KV Channel-Interacting Proteins in Neurological and Cardiovascular System: An Updated Review

Preprint

Full-text available

Jun 2023

KV channel-interacting proteins (KChIPs) belong to a family of Ca2+-binding EF-hand proteins that are able to bind to the N-terminus of the KV4 channel α-subunits. As the auxiliary subunit, KChIPs are critically involved in regulating the amplitude and gating properties of KV4 channels by modulating their cell surface trafficking, voltage-dependent activation, inactivation kinetics, and recovery rate from inactivation. IKs, ICa,L, and INa can also be regulated by KChIPs. KChIPs are predominantly expressed in the brain and heart, where they contribute to the maintenance of the excitability of neurons and cardiomyocytes by modulating the KV4 currents. Interestingly, all KChIPs can act as transcription factors to control the expression of genes involved in pain, memory, and circadian regulation. Altered expression of KChIPs has been implicated in the pathogenesis of many diseases, such as arrhythmia, heart failure, Alzheimer's disease, etc. In this review, we summarize the research progress of KChIPs in their structural properties, physiological functions, and pathological roles in disease progression, and provide an overview of the therapeutic potential of KChIPs as pharmacological targets for associated disorders.

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling

Article

Full-text available

Jun 2016

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca2+ dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca2+ ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca2+ concentration. Neither increasing nor decreasing intracellular Ca2+ concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2−/− heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca2+. Thus, KChIP2 does not function as a conventional transcription factor in the heart.

Toward a hierarchy of mechanisms in CaMKII-mediated arrhythmia

Article

Full-text available

May 2014

Calcium/calmodulin-dependent protein kinase II (CaMKII) activity has been shown to contribute to arrhythmogenesis in a remarkably broad range of cardiac pathologies. Several of these involve significant structural and electrophysiologic remodeling, whereas others are due to specific channelopathies, and are not typically associated with arrhythmogenic changes to protein expression or cellular and tissue structure. The ability of CaMKII to contribute to arrhythmia across such a broad range of phenotypes suggests one of two interpretations regarding the role of CaMKII in cardiac arrhythmia: (1) some CaMKII-dependent mechanism is a common driver of arrhythmia irrespective of the specific etiology of the disease, or (2) these different etiologies expose different mechanisms by which CaMKII is capable of promoting arrhythmia. In this review, we dissect the available mechanistic evidence to explore these two possibilities and discuss how the various molecular actions of CaMKII promote arrhythmia in different pathophysiologic contexts.

KV Channel-Interacting Proteins in the Neurological and Cardiovascular Systems: An Updated Review

Article

Full-text available

Jul 2023

KV channel-interacting proteins (KChIP1-4) belong to a family of Ca2+-binding EF-hand proteins that are able to bind to the N-terminus of the KV4 channel α-subunits. KChIPs are predominantly expressed in the brain and heart, where they contribute to the maintenance of the excitability of neurons and cardiomyocytes by modulating the fast inactivating-KV4 currents. As the auxiliary subunit, KChIPs are critically involved in regulating the surface protein expression and gating properties of KV4 channels. Mechanistically, KChIP1, KChIP2, and KChIP3 promote the translocation of KV4 channels to the cell membrane, accelerate voltage-dependent activation, and slow the recovery rate of inactivation, which increases KV4 currents. By contrast, KChIP4 suppresses KV4 trafficking and eliminates the fast inactivation of KV4 currents. In the heart, IKs, ICa,L, and INa can also be regulated by KChIPs. ICa,L and INa are positively regulated by KChIP2, whereas IKs is negatively regulated by KChIP2. Interestingly, KChIP3 is also known as downstream regulatory element antagonist modulator (DREAM) because it can bind directly to the downstream regulatory element (DRE) on the promoters of target genes that are implicated in the regulation of pain, memory, endocrine, immune, and inflammatory reactions. In addition, all the KChIPs can act as transcription factors to repress the expression of genes involved in circadian regulation. Altered expression of KChIPs has been implicated in the pathogenesis of several neurological and cardiovascular diseases. For example, KChIP2 is decreased in failing hearts, while loss of KChIP2 leads to increased susceptibility to arrhythmias. KChIP3 is increased in Alzheimer’s disease and amyotrophic lateral sclerosis, but decreased in epilepsy and Huntington’s disease. In the present review, we summarize the progress of recent studies regarding the structural properties, physiological functions, and pathological roles of KChIPs in both health and disease. We also summarize the small-molecule compounds that regulate the function of KChIPs. This review will provide an overview and update of the regulatory mechanism of the KChIP family and the progress of targeted drug research as a reference for researchers in related fields.

Nuclear Calcium in Cardiac (Patho)Physiology: Small Compartment, Big Impact

Article

Full-text available

Mar 2023

The nucleus of a cardiomyocyte has been increasingly recognized as a morphologically distinct and partially independent calcium (Ca2+) signaling microdomain, with its own Ca2+-regulatory mechanisms and important effects on cardiac gene expression. In this review, we (1) provide a comprehensive overview of the current state of research on the dynamics and regulation of nuclear Ca2+ signaling in cardiomyocytes, (2) address the role of nuclear Ca2+ in the development and progression of cardiac pathologies, such as heart failure and atrial fibrillation, and (3) discuss novel aspects of experimental methods to investigate nuclear Ca2+ handling and its downstream effects in the heart. Finally, we highlight current challenges and limitations and recommend future directions for addressing key open questions.

Increased expression of six-large extracellular vesicle-derived miRNAs signature for nonvalvular atrial fibrillation

Article

Full-text available

Jan 2022
J TRANSL MED

Abstract Backgrounds Non-valvular atrial fibrillation (AF) is the most common type of cardiac arrhythmia. AF is caused by electrophysiological abnormalities and alteration of atrial tissues, which leads to the generation of abnormal electrical impulses. Extracellular vesicles (EVs) are membrane-bound vesicles released by all cell types. Large EVs (lEVs) are secreted by the outward budding of the plasma membrane during cell activation or cell stress. lEVs are thought to act as vehicles for miRNAs to modulate cardiovascular function, and to be involved in the pathophysiology of cardiovascular diseases (CVDs), including AF. This study identified lEV-miRNAs that were differentially expressed between AF patients and non-AF controls. Methods lEVs were isolated by differential centrifugation and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), flow cytometry and Western blot analysis. For the discovery phase, 12 AF patients and 12 non-AF controls were enrolled to determine lEV-miRNA profile using quantitative reverse transcription polymerase chain reaction array. The candidate miRNAs were confirmed their expression in a validation cohort using droplet digital PCR (30 AF, 30 controls). Bioinformatics analysis was used to predict their target genes and functional pathways. Results TEM, NTA and flow cytometry demonstrated that lEVs presented as cup shape vesicles with a size ranging from 100 to 1000 nm. AF patients had significantly higher levels of lEVs at the size of 101–200 nm than non-AF controls. Western blot analysis was used to confirm EV markers and showed the high level of cardiomyocyte expression (Caveolin-3) in lEVs from AF patients. Nineteen miRNAs were significantly higher (> twofold, p

Stress-driven cardiac calcium mishandling via a kinase-to-kinase crosstalk

Article

Full-text available

Mar 2021
PFLUG ARCH EUR J PHY

Calcium homeostasis in the cardiomyocyte is critical to the regulation of normal cardiac function. Abnormal calcium dynamics such as altered uptake by the sarcoplasmic reticulum (SR) Ca²⁺-ATPase and increased diastolic SR calcium leak are involved in the development of maladaptive cardiac remodeling under pathological conditions. Ca²⁺/calmodulin-dependent protein kinase II-δ (CaMKIIδ) is a well-recognized key molecule in calcium dysregulation in cardiomyocytes. Elevated cellular stress is known as a common feature during pathological remodeling, and c-jun N-terminal kinase (JNK) is an important stress kinase that is activated in response to intrinsic and extrinsic stress stimuli. Our lab recently identified specific actions of JNK isoform 2 (JNK2) in CaMKIIδ expression, activation, and CaMKIIδ-dependent SR Ca²⁺ mishandling in the stressed heart. This review focuses on the current understanding of cardiac SR calcium handling under physiological and pathological conditions as well as the newly identified contribution of the stress kinase JNK2 in CaMKIIδ-dependent SR Ca²⁺ abnormal mishandling. The new findings identifying dual roles of JNK2 in CaMKIIδ expression and activation are also discussed in this review.

Research progress on the role of CaMKII in heart disease

Article

Dec 2020

In the heart, Ca2+ participates in electrical activity and myocardial contraction, which is closely related to the generation of action potential and excitation contraction coupling (ECC) and plays an important role in various signal cascades and regulates different physiological processes. In the Ca2+ related physiological activities, CaMKII is a key downstream regulator, involving autophosphorylation and post-translational modification, and plays an important role in the excitation contraction coupling and relaxation events of cardiomyocytes. This paper reviews the relationship between CaMKII and various substances in the pathological process of myocardial apoptosis and necrosis, myocardial hypertrophy and arrhythmia, and what roles it plays in the development of disease in complex networks. This paper also introduces the drugs targeting at CaMKII to treat heart disease.

Tiotropium bromide, a long acting muscarinic receptor antagonist triggers intracellular calcium signalling in the heart

Article

Oct 2019

Background and purpose Tiotropium bromide (TB) is a long acting muscarinic receptor antagonist used to manage chronic obstructive pulmonary disease (COPD). Recent meta-analyses suggest an increased risk of cardiovascular events with TB. Ca2+/calmodulin dependent kinase II (CaMKII) and L-type Ca2+ channels regulate Ca2+ concentrations allowing management of Ca2+ across membranes, however pathological increases are initially slow and progressive but once the cytosolic concentration rises >1–3 μM from ~100 nM, calcium overload occurs and can lead to cell death. Ipratropium bromide, a short acting muscarinic receptor antagonist has previously been found to induce Ca2+ mediated eryptosis. The aim of this study was to investigate the role of Ca2+ in Tiotropium bromide mediated cardiotoxicity. Experimental approach Isolated Sprague-Dawley rat hearts were perfused with TB (10–0.1 nM) ± KN-93 (400 nM) or nifedipine (1 nM). Hearts were stained to determine infarct size (%) using triphenyltetrazolium chloride (TTC), or snap frozen to determine p-CaMKII (Thr286) expression. Cardiomyocytes were isolated using a modified Langendorff perfusion and enzymatic dissociation before preparation for Fluo 3-AM staining and flow cytometric analysis. Key results TB increased infarct size compared with controls by 6.91–8.41%, with no effect on haemodynamic function. KN-93/nifedipine with TB showed a 5.90/7.38% decrease in infarct size compared to TB alone, the combined use of KN-93 with TB also showed a significant increase in left ventricular developed pressure whilst nifedipine with TB showed a significant decrease in coronary flow. TB showed a 42.73% increase in p-CaMKII (Thr286) versus control, and increased Ca2+ fluorescence by 30.63% in cardiomyocytes. Conclusions and implications To our knowledge, this is the first pre-clinical study to show that Tiotropium bromide induces Ca2+ signalling via CaMKII and L-type Ca2+ channels to result in cell damage. This has significant clinical impact due to long term use of TB in COPD patients, and warrants assessment of cardiac drug safety.

Ca2+-Dependent Transcriptional Repressors KCNIP and Regulation of Prognosis Genes in Glioblastoma

Article

Full-text available

Dec 2018

Glioblastomas (GBMs) are the most aggressive and lethal primary astrocytic tumors in adults, with very poor prognosis. Recurrence in GBM is attributed to glioblastoma stem-like cells (GSLCs). The behavior of the tumor, including proliferation, progression, invasion, and significant resistance to therapies, is a consequence of the self-renewing properties of the GSLCs, and their high resistance to chemotherapies have been attributed to their capacity to enter quiescence. Thus, targeting GSLCs may constitute one of the possible therapeutic challenges to significantly improve anti-cancer treatment regimens for GBM. Ca2+ signaling is an important regulator of tumorigenesis in GBM, and the transition from proliferation to quiescence involves the modification of the kinetics of Ca2+ influx through store-operated channels due to an increased capacity of the mitochondria of quiescent GSLC to capture Ca2+. Therefore, the identification of new therapeutic targets requires the analysis of the calcium-regulated elements at transcriptional levels. In this review, we focus onto the direct regulation of gene expression by KCNIP proteins (KCNIP1–4). These proteins constitute the class E of Ca2+ sensor family with four EF-hand Ca2+-binding motifs and control gene transcription directly by binding, via a Ca2+-dependent mechanism, to specific DNA sites on target genes, called downstream regulatory element (DRE). The presence of putative DRE sites on genes associated with unfavorable outcome for GBM patients suggests that KCNIP proteins may contribute to the alteration of the expression of these prognosis genes. Indeed, in GBM, KCNIP2 expression appears to be significantly linked to the overall survival of patients. In this review, we summarize the current knowledge regarding the quiescent GSLCs with respect to Ca2+ signaling and discuss how Ca2+ via KCNIP proteins may affect prognosis genes expression in GBM. This original mechanism may constitute the basis of the development of new therapeutic strategies.

Calcium and Ca 2+ /Calmodulin-dependent kinase II as targets for helminth parasite control

Article

Nov 2018

In eukaryotes, effective calcium homeostasis is critical for many key biological processes. There is an added level of complexity in parasites, particularly multicellular helminth worms, which modulate calcium levels while inhabiting the host microenvironment. Parasites ensure efficient calcium homeostasis through gene products, such as the calmodulin-dependent kinases (CaMK), the main focus of this review. The importance of CaMK is becoming increasingly apparent from recent functional studies of helminth and protozoan parasites. Investigations on the molecular regulation of calcium and the role of CaMK are important for both supplementing current drug regimens and finding new antiparasitic compounds. Whereas calcium regulators, including CaMK, are well charac-terised in mammalian systems, knowledge of their functional properties in parasites is increasing but is still in its infancy.

Specific activation of the alternative cardiac promoter of Cacna1c by the mineralocorticoid receptor

Thesis

Dec 2017

Thássio Mesquita

The mineralocorticoid receptor (MR) antagonists belong to the current therapeutic armamentarium for the management of cardiovascular diseases, but the mechanisms conferring their beneficial effects are still poorly understood. Part of these MR effects might be related to the L-type Cav1.2 Ca2+ channel expression regulation, critically involved in heart failure and hypertension. Here, we show that MR acts as a transcription factor triggering aldosterone signal into specific alternative 'cardiac' P1-promoter usage, given rise to long (Cav1.2-LNT) N-terminal transcripts. Aldosterone increases Cav1.2-LNT expression in cardiomyocytes in a time- and dose-dependent manner due to MR-dependent P1-promoter activity, through specific DNA sequence-MR interactions. This cis-regulatory mechanism induced a MR-dependent P1-promoter switch in vascular cells leading to a new Cav1.2-LNT molecular signature with reduced Ca2+ channel blocker sensitivity. These findings uncover Cav1.2-LNT as a specific mineralocorticoid target that might influence the therapeutic outcome of cardiovascular diseases.

KChIP2 is a core transcriptional regulator of cardiac excitability

Article

Full-text available

Mar 2017
eLife

ELife digest The heart pumps blood throughout the body to provide oxygen and nourishment. To do so, proteins in the heart create electrical signals that tell the heart muscles to contract in a coordinated manner. Heart disease can cause cells to lose control of the production or activity of these proteins, creating disorganized electrical signals called arrhythmias that interfere with the heart’s ability to pump. Sometimes these arrhythmias lead to sudden death. Researchers do not know exactly what triggers these changes in the heart’s normal electrical rhythms. This has made it difficult to develop strategies to prevent these disruptions or to fix them when they occur. By studying rat and human heart cells, Nassal et al. now show that a protein called KChIP2 stops working properly during heart disease. Most importantly, because of the decreased level of KChIP2 in heart disease, KChIP2 loses the ability to restrict the production of two microRNA molecules – a role that KChIP2 was not previously known to perform. This loss of activity sets off a cascade of signals that worsens the balance of electrical activity in the heart cells, creating arrhythmias. Treatments that restored proper levels of the fully working KChIP2 protein to the heart cells or that blocked the signals set off by a lack of KChIP2 returned the electrical activity of the cells back to normal. This also stopped the development of arrhythmias. Further studies are now needed to investigate whether these treatments have the same effects in living mammals. If effective, this could ultimately lead to new treatments for heart diseases and arrhythmias. DOI: http://dx.doi.org/10.7554/eLife.17304.002

Calcium Signaling and Transcriptional Regulation in Cardiomyocytes

Article

Sep 2017

Calcium (Ca²⁺) is a universal regulator of various cellular functions. In cardiomyocytes, Ca²⁺ is the central element of excitation–contraction coupling, but also impacts diverse signaling cascades and influences the regulation of gene expression, referred to as excitation–transcription coupling. Disturbances in cellular Ca²⁺-handling and alterations in Ca²⁺-dependent gene expression patterns are pivotal characteristics of failing cardiomyocytes, with several excitation–transcription coupling pathways shown to be critically involved in structural and functional remodeling processes. Thus, targeting Ca²⁺-dependent transcriptional pathways might offer broad therapeutic potential. In this article, we (1) review cytosolic and nuclear Ca²⁺ dynamics in cardiomyocytes with respect to their impact on Ca²⁺-dependent signaling, (2) give an overview on Ca²⁺-dependent transcriptional pathways in cardiomyocytes, and (3) discuss implications of excitation–transcription coupling in the diseased heart.

Calcium Microdomains in Cardiac Cells

Chapter

Sep 2017

Calcium (Ca²⁺) is a universal intracellular second messenger. In the heart, it plays a key role by activating contraction through the excitation-contraction coupling (EC coupling) mechanism. Although this is its key role in the heart, Ca²⁺ has other important functions, not only being involved in cell growth (in the heart named excitation-transcription coupling, ET coupling) but also in mitochondrial function (excitation-metabolism coupling, EM coupling) and cell death. Moreover, as Ca²⁺ is electrically charged, its movement across membranes generates an electrical current, which is important in cardiomyocyte electrophysiology and, if disturbed, may be involved in arrhythmias. The cardiac myocyte may discriminate between Ca²⁺ signals by creating “spaces” where Ca²⁺ diffusion is limited, creating gradients of [Ca²⁺]i at the micrometer scale, which are named microdomains. They are maintained by the cellular architecture and location of Ca²⁺-handling proteins and buffers.

Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization

Article

Dec 2016
BBA-MOL CELL RES

The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve. By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs. In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying vascular disease states, such as aortic aneurysm, as well as novel ideas regarding therapeutic strategies. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

The Stress-Response MAP Kinase Signaling in Cardiac Arrhythmias

Chapter

Full-text available

Nov 2016
REV PHYSIOL BIOCH P

Stress-response kinases, the mitogen-activated protein kinases (MAPKs) are activated in response to the challenge of a myriad of stressors. c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and p38 MAPKs are the predominant members of the MAPK family in the heart. Extensive studies have revealed critical roles of activated MAPKs in the processes of cardiac injury and heart failure and many other cardiovascular diseases. Recently, emerging evidence suggests that MAPKs also promote the development of cardiac arrhythmias. Thus, understanding the functional impact of MAPKs in the heart could shed new light on the development of novel therapeutic approaches to improve cardiac function and prevent arrhythmia development in the patients. This review will summarize the recent findings on the role of MAPKs in cardiac remodeling and arrhythmia development and point to the critical need of future studies to further elucidate the fundamental mechanisms of MAPK activation and arrhythmia development in the heart.

Ca2+ coding and decoding strategies for the specification of neural and renal precursor cells during development

Article

Dec 2015

Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization

Article

Full-text available

Dec 2015

Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hyper-contractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, qPCR and western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers was increased in isolated smooth muscle cells cultured under high compared to low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization and myocardin related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility.

Elevated glucose levels promote contractile gene expression in vascular smooth muscle via Rho/protein kinase C and actin polymerization.

Article

Jan 2015

Spontaneous activity and stretch-induced contractile differentiation are reduced in vascular smooth muscle of miR-143/145 knockout mice

Article

Jun 2015
ACTA PHYSIOL

AimStretch is essential for maintaining the contractile phenotype of vascular smooth muscle cells and small non-coding microRNAs are known to be important in this process. By using a Dicer knockout model we have previously reported that miRNAs are essential for stretch-induced differentiation and regulation of L-type calcium channel expression. The aim of the present study was to investigate the importance of the smooth muscle enriched miR-143/145 microRNA cluster for stretch-induced differentiation of the portal vein.Methods Contractile force and depolarization-induced calcium influx were determined in portal veins from wild type and miR-143/145 knockout mice. Stretch-induced contractile differentiation was investigated by determination of mRNA expression following organ culture for 24 hours under longitudinal load by a hanging weight.ResultsIn the absence of miR-143/145, stretch-induced mRNA expression of contractile markers in the portal vein was reduced. This was associated with decreased amplitude of spontaneous activity and depolarization-induced contractile and intracellular calcium responses, while contractile responses to 5-HT were largely maintained. We found that these effects correlated with a reduced basal expression of the pore-forming subunit of L-type calcium channels and an increased expression of CaMKIIδ and the transcriptional repressor DREAM.Conclusion Our results suggest that the microRNA-143/145 cluster plays a role in maintaining stretch-induced contractile differentiation and calcium signaling in the portal vein. This may have important implications for the use of these microRNAs as therapeutic targets in vascular disease.This article is protected by copyright. All rights reserved.

Sequence characterization of the HSA12/22/12 evolutionary breakpoint region on SSC5q21

Article

May 2012

Natalie Pierce

MiR-103 inhibits osteoblast proliferation mainly through suppressing Cav1.2 expression in simulated microgravity

Article

Full-text available

Apr 2015

SR Calcium Handling Dysfunction, Stress-Response Signaling Pathways, and Atrial Fibrillation

Article

Full-text available

Feb 2015

Xun Ai

Atrial fibrillation (AF) is the most common sustained arrhythmia. It is associated with a markedly increased risk of premature death due to embolic stroke and also complicates co-existing cardiovascular diseases such as heart failure. The prevalence of AF increases dramatically with age, and aging has been shown to be an independent risk of AF. Due to an aging population in the world, a growing body of AF patients are suffering a diminished quality of life and causing an associated economic burden. However, effective pharmacologic treatments and prevention strategies are lacking due to a poor understanding of the molecular and electrophysiologic mechanisms of AF in the failing and/or aged heart. Recent studies suggest that altered atrial calcium handling contributes to the onset and maintenance of AF. Here we review the role of stress-response kinases and calcium handling dysfunction in AF genesis in the aged and failing heart.

Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts

Article

Full-text available

Jan 2015

L-type voltage-sensitive calcium channels (LTCCs), particularly Cav1.2 LTCCs, play fundamental roles in cellular responses to mechanical stimuli in osteoblasts. Numerous studies have shown that mechanical loading promotes bone formation, whereas the removal of this stimulus under microgravity conditions results in a reduction in bone mass. However, whether microgravity exerts an influence on LTCCs in osteoblasts and whether this influence is a possible mechanism underlying the observed bone loss remain unclear. In the present study, we demonstrated that simulated microgravity substantially inhibited LTCC currents and suppressed Cav1.2 at the protein level in MC3T3-E1 osteoblast-like cells. In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells. Moreover, simulated microgravity increased miR-103 expression. Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions. These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression. Furthermore, the down-regulation of Cav1.2 expression and the inhibition of LTCCs caused by mechanical unloading in osteoblasts are partially due to miR-103 up-regulation. Our study provides a novel mechanism for microgravity-induced detrimental effects on osteoblasts, offering a new avenue to further investigate the bone loss induced by microgravity.

CaMKII content affects contractile, but not mitochondrial, characteristics in regenerating skeletal muscle

Article

Full-text available

Dec 2014
BMC Physiol

Background The multi-meric calcium/calmodulin-dependent protein kinase II (CaMKII) is the main CaMK in skeletal muscle and its expression increases with endurance training. CaMK family members are implicated in contraction-induced regulation of calcium handling, fast myosin type IIA expression and mitochondrial biogenesis. The objective of this study was to investigate the role of an increased CaMKII content for the expression of the contractile and mitochondrial phenotype in vivo. Towards this end we attempted to co-express alpha- and beta-CaMKII isoforms in skeletal muscle and characterised the effect on the contractile and mitochondrial phenotype.ResultsFast-twitch muscle m. gastrocnemius (GM) and slow-twitch muscle m. soleus (SOL) of the right leg of 3-month old rats were transfected via electro-transfer of injected expression plasmids for native ¿/ß CaMKII. Effects were identified from the comparison to control-transfected muscles of the contralateral leg and non-transfected muscles. ¿/ß CaMKII content in muscle fibres was 4-5-fold increased 7 days after transfection. The transfection rate was more pronounced in SOL than GM muscle (i.e. 12.6 vs. 3.5%). The overexpressed ¿/ß CaMKII was functional as shown through increased threonine 287 phosphorylation of ß-CaMKII after isometric exercise and down-regulated transcripts COXI, COXIV, SDHB after high-intensity exercise in situ. ¿/ß CaMKII overexpression under normal cage activity accelerated excitation-contraction coupling and relaxation in SOL muscle in association with increased SERCA2, ANXV and fast myosin type IIA/X content but did not affect mitochondrial protein content. These effects were observed on a background of regenerating muscle fibres.Conclusion Elevated CaMKII content promotes a slow-to-fast type fibre shift in regenerating muscle but is not sufficient to stimulate mitochondrial biogenesis in the absence of an endurance stimulus.

CaMKII inhibition with KN93 attenuates endothelin and serotonin receptor-mediated vasoconstriction and prevents subarachnoid hemorrhage-induced deficits in sensorimotor function

Article

Full-text available

Dec 2014
J NEUROINFLAMM

Background It has been suggested that transcriptional upregulation of cerebral artery contractile endothelin (ETB) and 5-hydroxytryptamine (5-HT1B) receptors play an important role in the development of late cerebral ischemia and increased vasoconstriction after subarachnoid hemorrhage (SAH). We tested the hypothesis that inhibition of calcium calmodulin-dependent protein kinase II (CaMKII) may reduce cerebral vasoconstriction mediated by endothelin and serotonin receptors and improve neurological outcome after experimental SAH.MethodsSAH was induced in adult rats by injection of 250 ¿L autologous blood into the basal cisterns. The CaMKII activity in cerebral vessels was studied by Western blot and immunohistochemistry. The vasomotor responses of middle cerebral and basilar arteries were measured in a sensitive myograph system. The functional outcome was examined by the rotating pole test 2 and 3 days after SAH.ResultsSAH induced a rapid early increase in phosphorylated CaMKII protein at 1 h that was attenuated by cisternal administration of the CaMKII inhibitor KN93 (0.501 ¿g/kg) 45 min prior and immediately after SAH as evaluated by Western blot. Application of KN93 at 1 h and every 12 h post-SAH significantly reduced vascular CaMKII immunoreactivity at 72 h. In addition, contractile responses of cerebral arteries to endothelin-1 (ET-1) and 5-hydroxycarboxamide (5-CT) were increased at this time-point. KN93 treatment significantly attenuated the contraction induced by ET-1 and 5-CT. Importantly, treatment with the CaMKII inhibitor prevented SAH-induced deficits in neurological function, as evaluated by the rotating pole test, and similar sensorimotor scores were seen in sham-operated animals.Conclusions The present study has shown that SAH is associated with increased contractile responses to ET-1 and 5-CT in cerebral arteries and enhanced early activation of CaMKII. Treatment with the CaMKII inhibitor KN93 attenuated the contractile responses and prevented impaired sensorimotor function after SAH.

Calcium Channels and Selective Neuronal Vulnerability in Parkinson’s Disease

Chapter

Nov 2022

Ca2+ entry through voltage-dependent ion channels (Cav) in the plasma membrane provides a signal coupling neuronal activity to a wide array of intracellular processes ranging from control of other ion channels to regulation of metabolism and gene expression. In Parkinson’s disease (PD), the degeneration of substantia nigra pars compacta (SNc) dopaminergic neurons, causing the cardinal symptoms, has been tied to the prominent engagement of Cav channels that modulate repetitive spiking, transmitter-release, and mitochondrial function. Here, we summarize the literature underlying this connection. We focus on Cav1 L-type Cav channels, as epidemiological studies indicate that inhibition of these channels reduces the risk of developing PD. In addition, we discuss the translational implications of this literature and the prospect of selective Cav channel modulators as disease-modifying drugs for early-stage PD.KeywordsCa2+ channelParkinson’s diseaseDopamineSubstantia nigraL-typeR-typeT-typeD2 dopamine receptorNeuronal calcium sensors (NCS)MitochondriaBioenergeticsNeurodegenerationHomeostasis

Stress Kinase Signaling in Cardiac Myocytes

Chapter

Sep 2022

Stress-response kinases, the mitogen-activated protein kinases (MAPKs), are activated in response to the challenge of a myriad of stressors. c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and MAPK p38 are the important members of the MAPK family in the heart. Extensive studies have revealed critical roles of activated MAPKs in the processes of cardiac injury, cardiac arrhythmias, heart failure, and other cardiovascular diseases. Advancing our understanding regarding the functional impacts of MAPKs in the development of heart diseases could shed new light on developing novel therapeutic approaches to improve cardiac function and prevent arrhythmia development in patients. This chapter summarizes relevant current knowledge on the pivotal roles of MAPKs in physiopathological and molecular remodeling in cardiac myocytes during the disease development and for the therapeutic potentials of developing MAPK inhibitors and/or activators.KeywordsMitogen-activated protein kinases Stress Arrhythmia Heart failure Calcium homeostasis Cardiovascular diseases

Isoproterenol-induced hypertrophy of neonatal cardiac myocytes and H9c2 cell is dependent on TRPC3-regulated CaV1.2 expression

Article

Dec 2020
CELL CALCIUM

CaV1.2 and transient receptor potential canonical channel 3 (TRPC3) are two proteins known to have important roles in pathological cardiac hypertrophy; however, such roles still remain unclear. A better understanding of these roles is important for furthering the clinical understanding of heart failure. We previously reported that Trpc3-knockout (KO) mice are resistant to pathologic hypertrophy and that their CaV1.2 protein expression is reduced. In this study, we aimed to examine the relationship between these two proteins and characterize their role in neonatal cardiomyocytes. We measured CaV1.2 expression in the hearts of wild-type (WT) and Trpc3−/− mice, and examined the effects of Trpc3 knockdown and overexpression in the rat cell line H9c2. We also compared the hypertrophic responses of neonatal cardiomyocytes cultured from Trpc3−/− mice to a representative hypertrophy-causing drug, isoproterenol (ISO), and measured the activity of nuclear factor of activated T cells 3 (NFAT3) in neonatal cardiomyocytes (NCMCs). We inhibited the L-type current with nifedipine, and measured the intracellular calcium concentration using Fura-2 with 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ba²⁺ influx. When using the Trpc3-mediated Ca²⁺ influx, both intracellular calcium concentration and calcium influx were reduced in Trpc3-KO myocytes. Not only was the expression of CaV1.2 greatly reduced in Trpc3-KO cardiac lysate, but the size of the CaV1.2 currents in NCMCs was also greatly reduced. When NCMCs were treated with Trpc3 siRNA, it was confirmed that the expression of CaV1.2 and the intracellular nuclear transfer activity of NFAT decreased. In H9c2 cells, the ISO activated- and verapamil inhibited- Ca²⁺ influxes were dramatically attenuated by Trpc3 siRNA treatment. In addition, it was confirmed that both the expression of CaV1.2 and the size of H9c2 cells were regulated according to the expression and activation level of TRPC3. We found that after stimulation with ISO, cell hypertrophy occurred in WT myocytes, while the increase in size of Trpc3-KO myocytes was greatly reduced. These results suggest that not only the cell hypertrophy process in neonatal cardiac myocytes and H9c2 cells were regulated according to the expression level of CaV1.2, but also that the expression level of CaV1.2 was regulated by TRPC3 through the activation of NFAT.

Acquired cardiac channelopathies in epilepsy: Evidence, mechanisms, and clinical significance

Article

Jul 2019
EPILEPSIA

There is growing evidence that cardiac dysfunction in patients with chronic epilepsy could play a pathogenic role in sudden unexpected death in epilepsy (SUDEP). Recent animal studies have revealed that epilepsy secondarily alters the expression of cardiac ion channels alongside abnormal cardiac electrophysiology and remodeling. These molecular findings represent novel evidence for an acquired cardiac channelopathy in epilepsy, distinct from inherited ion channels mutations associated with cardiocerebral phenotypes. Specifically, seizure activity has been shown to alter the messenger RNA (mRNA) and protein expression of voltage‐gated sodium channels (Nav1.1, Nav1.5), voltage‐gated potassium channels (Kv4.2, Kv4.3), sodium‐calcium exchangers (NCX1), and nonspecific cation‐conducting channels (HCN2, HCN4). The pathophysiology may involve autonomic dysfunction and structural cardiac disease, as both are independently associated with epilepsy and ion channel dysregulation. Indeed, in vivo and in vitro studies of cardiac pathology reveal a complex network of signaling pathways and transcription factors regulating ion channel expression in the setting of sympathetic overactivity, cardiac failure, and hypertrophy. Other mechanisms such as circulating inflammatory mediators or exogenous effects of antiepileptic medications lack evidence. Moreover, an acquired cardiac channelopathy may underlie the electrophysiologic cardiac abnormalities seen in chronic epilepsy, potentially contributing to the increased risk of malignant arrhythmias and sudden death. Therefore, further investigation is necessary to establish whether cardiac ion channel dysregulation similarly occurs in patients with epilepsy, and to characterize any pathogenic relationship with SUDEP.

Potassium Channels in the Heart

Chapter

Sep 2018

Morten B. Thomsen

DREAM: a multifunctional transcriptional regulator

Article

Oct 2017
Acta Physiol Sin

DREAM (downstream regulatory element antagonist modulator), Calsenilin and KChIP3 (potassium channel interacting protein 3) belong to the neuronal calcium sensor (NCS) superfamily, which transduces the intracellular calcium signaling into a variety of activities. They are encoded by the same gene locus, but have distinct subcellular locations. DREAM was first found to interact with DRE (downstream regulatory element) site in the vicinity of the promoter of prodynorphin gene to suppress gene transcription. Calcium can disassemble this interaction by binding reversibly to DREAM protein on its four EF-hand motifs. Apart from having calcium dependent DRE site binding, DREAM can also interact with other transcription factors, such as cAMP responsive element binding protein (CREB), CREB-binding protein (CBP) and cAMP responsive element modulator (CREM), by this concerted actions, DREAM extends the gene pool under its control. DREAM is predominantly expressed in central nervous system with its highest level in cerebellum, and accumulating evidence demonstrated that DREAM might play important roles in pain sensitivity. Novel findings have shown that DREAM is also involved in learning and memory processes, Alzheimer's disease and stroke. This mini-review provides a brief introduction of its discovery history and protein structure properties, focusing on the mechanism of DREAM nuclear translocation and gene transcription regulation functions.

Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4–NKX2-5 Interaction

Article

Full-text available

Mar 2018

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4–NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4–NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II-mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro- and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4–NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries.

Loss of Vascular Myogenic Tone in miR-143/145 Knockout Mice Is Associated With Hypertension-Induced Vascular Lesions in Small Mesenteric Arteries

Article

Dec 2017

Objective: Pressure-induced myogenic tone is involved in autoregulation of local blood flow and confers protection against excessive pressure levels in small arteries and capillaries. Myogenic tone is dependent on smooth muscle microRNAs (miRNAs), but the identity of these miRNAs is unclear. Furthermore, the consequences of altered myogenic tone for hypertension-induced damage to small arteries are not well understood. Approach and results: The importance of smooth muscle-enriched microRNAs, miR-143/145, for myogenic tone was evaluated in miR-143/145 knockout mice. Furthermore, hypertension-induced vascular injury was evaluated in mesenteric arteries in vivo after angiotensin II infusion. Myogenic tone was abolished in miR-143/145 knockout mesenteric arteries, whereas contraction in response to calyculin A and potassium chloride was reduced by ≈30%. Furthermore, myogenic responsiveness was potentiated by angiotensin II in wild-type but not in knockout mice. Angiotensin II administration in vivo elevated systemic blood pressure in both genotypes. Hypertensive knockout mice developed severe vascular lesions characterized by vascular inflammation, adventitial fibrosis, and neointimal hyperplasia in small mesenteric arteries. This was associated with depolymerization of actin filaments and fragmentation of the elastic laminae at the sites of vascular lesions. Conclusions: This study demonstrates that miR-143/145 expression is essential for myogenic responsiveness. During hypertension, loss of myogenic tone results in potentially damaging levels of mechanical stress and detrimental effects on small arteries. The results presented herein provide novel insights into the pathogenesis of vascular disease and emphasize the importance of controlling mechanical factors to maintain structural integrity of the vascular wall.

DREAM (Downstream Regulatory Element Antagonist Modulator)

Chapter

Apr 2017

Surface-modified particles loaded with CaMKII inhibitor protect cardiac cells against mitochondrial injury

Article

Feb 2017

An excess of calcium (Ca²⁺) influx into mitochondria during mitochondrial re-energization is one of the causes of myocardial cell death during ischemic/reperfusion injury. This overload of Ca²⁺ triggers the mitochondrial permeability transition pore (mPTP) opening which leads to programmed cell death. During the ischemic/reperfusion stage, the activated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) enzyme is responsible for Ca²⁺ influx. To reduce CaMKII-related cell death, sub-micron particles composed of poly(lactic-co-glycolic acid) (PLGA), loaded with a CaMKII inhibitor peptide were fabricated. The CaMKII inhibitor peptide-loaded (CIP) particles were coated with a mitochondria targeting moiety, triphenylphosphonium cation (TPP), which allowed the particles to accumulate and release the peptide inside mitochondria to inhibit CaMKII activity. The fluorescently labeled TPP-CIP were taken up by mitochondria and successfully reduced ROS caused by Isoprenaline (ISO) in a differentiated rat cardiomyocyte-like cell line. When cells were treated with TPP-CIP prior ISO exposure, they maintained mitochondrial membrane potential. The TPP-CIP protected cells from ISO-induced ROS production and decreased mitochondrial membrane potential. Thus, TPP-CIP have the potential to be used in protection against ischemia/reperfusion injury.

CaMKIIδ Meditates Phenylephrine Induced Cardiomyocyte Hypertrophy Through Store-Operated Ca2+ Entry

Article

Dec 2016

Evidence suggests that store-operated Ca2+ entry (SOCE) is involved in the hypertrophy of cardiomyocytes. The signaling mechanisms of SOCE contributing to cardiac hypertrophy following phenylephrine (PE) stimulation are not fully understood. Ca²⁺/calmodulin-dependent protein kinase II δ (CaMKIIδ) plays an important role in regulating intracellular Ca²⁺ hemostasis and function in the cardimyocytes. This study is aimed to determine the role of CaMKIIδ in regulating the PE-induced myocardial hypertrophy and the associated molecular signaling mechanisms. We used primary cultures of neonatal cardimyocytes isolated from the left ventricle of Sprague Dawley rats to investigate the effects of CaMKIIδ on myocardial hypertrophy and intracellular Ca²⁺ mobilization. We found that the expression of CaMKIIδ was enhanced in PE-induced hypertrophic cardiomyocytes. CaMKIIδ siRNA, CaMKII inhibitor KN93, and SOCE blocker BTP2 attenuated the increase in the expression of CaMKIIδ and normalized the hypertrophic markers, atrial natriuretic peptide and brain natriuretic peptide, and size of cardiomyocytes induced by PE stimulation. The protein level of stromal interaction molecule 1 and Orai1, the essential components of the SOCE, is also enhanced in hypertrophic cardiomyocytes, which were normalized by CaMKIIδ siRNA and KN93 treatment. Hypertrophic cardiomyocytes showed an increase in the peak of Ca²⁺ transient following store depletion, which was inhibited by SOCE blocker BTP2, CaMKIIδ siRNA, and KN93. The Ca²⁺ currents through Ca²⁺ release-activated Ca²⁺ channels were increased in PE-treated cardiomyocytes and were attenuated by CaMKIIδ siRNA and KN93. These data indicate that PE-induced myocardial hypertrophy requires a complex signaling pathway that involves activation of both CaMKIIδ and SOCE. In conclusion, these studies reveal that up-regulation of CaMKIIδ may contribute to the PE-induced myocardial hypertrophy through the activation of SOCE expressed in the cardiomyocytes.

PGC-1α1 induces cardiac excitation-contraction coupling phenotype without metabolic remodelling

Article

Sep 2016
J PHYSIOL-LONDON

Key points: Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known. Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy. Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels. These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation-contraction coupling phenotype in the heart. Abstract: The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation-contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart.

Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor

Article

Mar 2016
BIOORG CHEM

Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C2 of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C20 of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC50 of 9μM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC50 values ranging from 9μM to as high as 333μM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pHi). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA.

Specific cytoarchitectureal changes in hippocampal subareas in daDREAM mice

Article

Dec 2016

Background: Transcriptional repressor DREAM (downstream regulatory element antagonist modulator) is a Ca(2+)-binding protein that regulates Ca(2+) homeostasis through gene regulation and protein-protein interactions. It has been shown that a dominant active form (daDREAM) is implicated in learning-related synaptic plasticity such as LTP and LTD in the hippocampus. Neuronal spines are reported to play important roles in plasticity and memory. However, the possible role of DREAM in spine plasticity has not been reported. Results: Here we show that potentiating DREAM activity, by overexpressing daDREAM, reduced dendritic basal arborization and spine density in CA1 pyramidal neurons and increased spine density in dendrites in dentate gyrus granule cells. These microanatomical changes are accompanied by significant modifications in the expression of specific genes encoding the cytoskeletal proteins Arc, Formin 1 and Gelsolin in daDREAM hippocampus. Conclusions: Our results strongly suggest that DREAM plays an important role in structural plasticity in the hippocampus.

Mitochondria, contractile apparatus, and ion channels in the failing myocardium: Special relationships or dangerous liaisons?

Article

Jan 2016

Matteo Vatta

Voltage-Gated Calcium Channel Signaling to the Nucleus

Article

Dec 2012

Excitation-transcription coupling makes use of cellular excitability to produce intracellular signals to the nucleus to control activity-dependent gene expression. Voltage-gated calcium channels are presented here as a signaling platform able to redirect multiple signaling pathways toward the nucleus. Whilst several CaV subunits are implicated in excitation-transcription coupling, each type of CaV nevertheless possesses its own proteome and microenvironment able to promote individualized signaling pathways. L-type calcium channels have structural determinants that favor the initiation of MAPK and CamK pathways for example, but P/Q and N-type channels, in close proximity to the endoplasmic reticulum, promote calcium-induced calcium release-dependent mechanisms. Furthermore, auxiliary CaVβ4 subunits or truncated C-termini of CaV1.2 and CaV2.1 channels can be targeted to the nucleus and become direct messengers involved in the regulation of gene expression. These later discoveries suggest that novel pathways must be inserted in the global description of excitation-transcription coupling and give new clues to the understanding of calcium channelopathies with interesting physiopathological perspectives. © Springer Science+Business Media Dordrecht 2013. All rights are reserved.

Regulation of Smooth Muscle Dystrophin and Synaptopodin 2 Expression by Actin Polymerization and Vascular Injury

Article

Apr 2015
ARTERIOSCL THROM VAS

Actin dynamics in vascular smooth muscle is known to regulate contractile differentiation and may play a role in the pathogenesis of vascular disease. However, the list of genes regulated by actin polymerization in smooth muscle remains incomprehensive. Thus, the objective of this study was to identify actin-regulated genes in smooth muscle and to demonstrate the role of these genes in the regulation of vascular smooth muscle phenotype. Mouse aortic smooth muscle cells were treated with an actin-stabilizing agent, jasplakinolide, and analyzed by microarrays. Several transcripts were upregulated including both known and previously unknown actin-regulated genes. Dystrophin and synaptopodin 2 were selected for further analysis in models of phenotypic modulation and vascular disease. These genes were highly expressed in differentiated versus synthetic smooth muscle and their expression was promoted by the transcription factors myocardin and myocardin-related transcription factor A. Furthermore, the expression of both synaptopodin 2 and dystrophin was significantly reduced in balloon-injured human arteries. Finally, using a dystrophin mutant mdx mouse and synaptopodin 2 knockdown, we demonstrate that these genes are involved in the regulation of smooth muscle differentiation and function. This study demonstrates novel genes that are promoted by actin polymerization, that regulate smooth muscle function, and that are deregulated in models of vascular disease. Thus, targeting actin polymerization or the genes controlled in this manner can lead to novel therapeutic options against vascular pathologies that involve phenotypic modulation of smooth muscle cells. © 2015 American Heart Association, Inc.

Chasing cardiac physiology and pathology down the CaMKII cascade

Article

Mar 2015

Calcium dynamics is central in cardiac physiology, as the key event leading to the excitation-contraction coupling (ECC) and relaxation processes. The primary function of Ca(2+) in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca(2+) signaling explains the subtle and critical control of important events of ECC and relaxation, such Ca(2+) influx and SR Ca(2+) release and uptake. The multifunctional Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule that regulates a diverse array of proteins involved not only in ECC and relaxation, but also in cell death, transcriptional activation of hypertrophy, inflammation and arrhythmias. CaMKII activity is triggered by an increase in intracellular Ca(2+) levels. This activity can be sustained, creating molecular memory after the decline in Ca(2+) concentration, by autophosphorylation of the enzyme, as well as by oxidation, glycosylation and nitrosylation at different sites of the regulatory domain of the kinase. CaMKII activity is enhanced in several cardiac diseases, altering the signaling pathways by which CaMKII regulates the different fundamental proteins involved in functional and transcriptional cardiac processes. Dysregulation of these pathways constitutes a central mechanism of various cardiac disease phenomena, like apoptosis and necrosis during ischemia/reperfusion injury, digitalis exposure, post-acidosis and heart failure arrhythmias, or cardiac hypertrophy. Here we summarize significant aspects of the molecular physiology of CaMKII and provide a conceptual framework for understanding the role of the CaMKII cascade on Ca(2+) regulation and dysregulation in cardiac health and disease. Copyright © 2015, American Journal of Physiology - Heart and Circulatory Physiology.

Regulation of calcium channels in smooth muscle: New insights into the role of myosin light chain kinase

Article

Full-text available

Sep 2014
CHANNELS

Smooth muscle myosin light chain kinase (MLCK) plays a crucial role in artery contraction, which regulates blood pressure and blood flow distribution. In addition to this role, MLCK contributes to Ca(2+) flux regulation in vascular smooth muscle (VSM) and in non-muscle cells, where cytoskeleton has been suggested to help Ca(2+) channels trafficking. This conclusion is based on the use of pharmacological inhibitors of MLCK and molecular and cellular techniques developed to down-regulate the enzyme. Dissimilarities have been observed between cells and whole tissues, as well as between large conductance and small resistance arteries. A differential expression in MLCK and ion channels (either voltage-dependent Ca(2+) channels or non-selective cationic channels) could account for these observations, and is in line with the functional properties of the arteries. A potential involvement of MLCK in the pathways modulating Ca(2+) entry in VSM is described in the present review.

HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte

Article

Full-text available

Mar 1998
P NATL ACAD SCI USA

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase–PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of α-cardiac myosin heavy chain, α-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at ≫+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/− 60 nM and a Bmax of 118 fmol per 105 cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.

Regulation of excitation-contraction coupling in mouse cardiac myocytes: Integrative analysis with mathematical modelling

Article

Full-text available

Sep 2009
BMC Physiol

The cardiomyocyte is a prime example of inherently complex biological system with inter- and cross-connected feedback loops in signalling, forming the basic properties of intracellular homeostasis. Functional properties of cells and tissues have been studied e.g. with powerful tools of genetic engineering, combined with extensive experimentation. While this approach provides accurate information about the physiology at the endpoint, complementary methods, such as mathematical modelling, can provide more detailed information about the processes that have lead to the endpoint phenotype. In order to gain novel mechanistic information of the excitation-contraction coupling in normal myocytes and to analyze sophisticated genetically engineered heart models, we have built a mathematical model of a mouse ventricular myocyte. In addition to the fundamental components of membrane excitation, calcium signalling and contraction, our integrated model includes the calcium-calmodulin-dependent enzyme cascade and the regulation it imposes on the proteins involved in excitation-contraction coupling. With the model, we investigate the effects of three genetic modifications that interfere with calcium signalling: 1) ablation of phospholamban, 2) disruption of the regulation of L-type calcium channels by calcium-calmodulin-dependent kinase II (CaMK) and 3) overexpression of CaMK. We show that the key features of the experimental phenotypes involve physiological compensatory and autoregulatory mechanisms that bring the system to a state closer to the original wild-type phenotype in all transgenic models. A drastic phenotype was found when the genetic modification disrupts the regulatory signalling system itself, i.e. the CaMK overexpression model. The novel features of the presented cardiomyocyte model enable accurate description of excitation-contraction coupling. The model is thus an applicable tool for further studies of both normal and defective cellular physiology. We propose that integrative modelling as in the present work is a valuable complement to experiments in understanding the causality within complex biological systems such as cardiac myocytes.

The delta isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload

Article

Full-text available

Feb 2009
P NATL ACAD SCI USA

Acute and chronic injuries to the heart result in perturbation of intracellular calcium signaling, which leads to pathological cardiac hypertrophy and remodeling. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transduction of calcium signals in the heart, but the specific isoforms of CaMKII that mediate pathological cardiac signaling have not been fully defined. To investigate the potential involvement in heart disease of CaMKIIdelta, the major CaMKII isoform expressed in the heart, we generated CaMKIIdelta-null mice. These mice are viable and display no overt abnormalities in cardiac structure or function in the absence of stress. However, pathological cardiac hypertrophy and remodeling are attenuated in response to pressure overload in these animals. Cardiac extracts from CaMKIIdelta-null mice showed diminished kinase activity toward histone deacetylase 4 (HDAC4), a substrate of stress-responsive protein kinases and suppressor of stress-dependent cardiac remodeling. In contrast, phosphorylation of the closely related HDAC5 was unaffected in hearts of CaMKIIdelta-null mice, underscoring the specificity of the CaMKIIdelta signaling pathway for HDAC4 phosphorylation. We conclude that CaMKIIdelta functions as an important transducer of stress stimuli involved in pathological cardiac remodeling in vivo, which is mediated, at least in part, by the phosphorylation of HDAC4. These findings point to CaMKIIdelta as a potential therapeutic target for the maintenance of cardiac function in the setting of pressure overload.

Expression of dihydropyridine receptor (Ca2+ channel) and calsequestrin genes in the myocardium of patients with end-stage heart failure

Article

Full-text available

Oct 1992

Cytoplasmic free calcium ions (Ca2+) play a central role in excitation-contraction coupling of cardiac muscle. Abnormal Ca2+ handling has been implicated in systolic and diastolic dysfunction in patients with end-stage heart failure. The current study tests the hypothesis that expression of genes encoding proteins regulating myocardial Ca2+ homeostasis is altered in human heart failure. We analyzed RNA isolated from the left ventricular (LV) myocardium of 30 cardiac transplant recipients with end-stage heart failure (HF) and five organ donors (normal control), using cDNA probes specific for the cardiac dihydropyridine (DHP) receptor (the alpha 1 subunit of the DHP-sensitive Ca2+ channel) and cardiac calsequestrin of sarcoplasmic reticulum (SR). In addition, abundance of DHP binding sites was assessed by ligand binding techniques (n = 6 each for the patients and normal controls). There was no difference in the level of cardiac calsequestrin mRNA between the HF patients and normal controls. In contrast, the level of mRNA encoding the DHP receptor was decreased by 47% (P less than 0.001) in the LV myocardium from the patients with HF compared to the normal controls. The number of DHP binding sites was decreased by 35-48%. As reported previously, expression of the SR Ca(2+)-ATPase mRNA was also diminished by 50% (P less than 0.001) in the HF group. These data suggest that expression of the genes encoding the cardiac DHP receptor and SR Ca(2+)-ATPase is reduced in the LV myocardium from patients with HF. Altered expression of these genes may be related to abnormal Ca2+ handling in the failing myocardium, contributing to LV systolic and diastolic dysfunction in patients with end-stage heart failure.

Schreiber E, Mathias P, M??ller MM and Schaffner WRRapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acid Res. 17: 6419

Article

Full-text available

Sep 1989

Mutational analysis of the autoinhibitory domain of calmodulin kinase II

Article

Full-text available

Dec 1994

Calmodulin (CaM)-kinase II is inactive in the absence of Ca2+/CaM due to interaction of its autoinhibitory domain with its catalytic domain. Previous studies using synthetic autoinhibitory domain peptides (residues 281-302) identified several residues as important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. To further examine the autoinhibitory domain, site-specific mutants were expressed using the baculovirus/Sf9 cell system. The purified mutants had many biochemical properties identical to wild-type kinase, but mutants H282Q, H282R, R283E, and T286D had 10-20% constitutive Ca(2+)-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca(2+)-independent activities of the H282Q, H282R, and R283E mutants exhibited 10-fold lower Km values for ATP than the wild-type kinase. Wild-type and mutant kinases, except T286A and T286D, generated Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM, and those mutants having constitutive Ca2+ independence also exhibited enhanced Ca2+/CaM-independent autophosphorylation. This Ca(2+)-independent autophosphorylation resulted in a decrease in total kinase activity, but there was little increase in Ca(2+)-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, we propose a molecular model for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II.

The Nuclear B Isoform of Ca2+/Calmodulin-dependent Protein Kinase II Regulates Atrial Natriuretic Factor Gene Expression in Ventricular Myocytes

Article

Full-text available

Jan 1998

Cultured neonatal ventricular myocytes display features of myocardial hypertrophy including increased cell size, myofilament organization, and reexpression of the embryonic gene for atrial natriuretic factor (ANF). KN-93, an inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II), blocked the induction of these responses by the α1-adrenergic receptor agonist phenylephrine, whereas its inactive analog KN-92 did not. To directly determine whether CaM kinase II could regulate ANF gene expression, we transiently expressed each of three isoforms of CaM kinase II (α, δB, and δC) along with an ANF promoter/luciferase reporter gene. The δB isoform markedly increased luciferase gene expression, whereas comparable levels of the δC and α isoforms were ineffective. Expression of δB-CaM kinase II also potentiated phenylephrine-mediated ANF gene expression, and this effect was blocked by KN-93 but not by KN-92. The ability of δB-CaM kinase II to transactivate a truncated ANF promoter, containing a serum response element (SRE) required for phenylephrine-inducible gene expression, was lost when this SRE was mutated. The δB isoform of CaM kinase II has been shown to exhibit nuclear localization. Coexpression of the non-nuclear δC or α isoforms, which can form multimers with the δB isoform, prevented the nuclear localization of δB-CaM kinase II and also blocked its effects on ANF reporter gene and protein expression. In addition, a chimeric α-CaM kinase II which contains the nuclear localization signal of the δB isoform was able to induce ANF reporter gene expression, albeit to a lesser extent than δB-CaM kinase II. These data are the first to assign a function to the δB isoform of CaM kinase II and to link its nuclear localization to subsequent activation of cardiac gene expression.

DREAM is a Ca2+-regulated transcriptional repressor

Article

Full-text available

Apr 1999

Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.

Calmodulin Is the Ca2+ Sensor for Ca2+-Dependent Inactivation of L-Type Calcium Channels

Article

Full-text available

Apr 1999
NEURON

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.

Modulation of A-type potassium channels by a family of calcium sensors

Article

Full-text available

Mar 2000

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Regulation of DHP receptor expression by elements in the 5'-flanking sequence

Article

Full-text available

May 2000

The alpha(1)-subunit of the cardiac/vascular Ca(2+) channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca(2+) entry. It contains the binding sites for multiple classes of drugs collectively known as Ca(2+) antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac alpha(1C)-subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking sequences and identified the alpha(1C)-subunit gene transcription start site. The rat alpha(1C)-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of alpha(1C)-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5'-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca(2+) channel alpha(1C)-subunit gene. Electrophoretic mobility shift assays identified a region of the alpha(1C)-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.

DREAM-αCREM Interaction via Leucine-Charged Domains Derepresses Downstream Regulatory Element-Dependent Transcription

Article

Full-text available

Jan 2001

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, αCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of αCREM is not required for the interaction, but phospho-αCREM shows higher affinity for DREAM. The interaction with αCREM is independent of the Ca2+-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both αCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca2+.

The Cardiac-specific Nuclear B Isoform of Ca2+/Calmodulin-dependent Protein Kinase II Induces Hypertrophy and Dilated Cardiomyopathy Associated with Increased Protein Phosphatase 2A Activity

Article

Full-text available

Feb 2002

The delta isoform of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) predominates in the heart. To investigate the role of CaMKII in cardiac function, we made transgenic (TG) mice that express the nuclear delta(B) isoform of CaMKII. The expressed CaMKIIdelta(B) transgene was restricted to the myocardium and highly concentrated in the nucleus. Cardiac hypertrophy was evidenced by an increased left ventricle to body weight ratio and up-regulation of embryonic and contractile protein genes including atrial natriuretic factor, beta-myosin heavy chain, and alpha-skeletal actin. Echocardiography revealed ventricular dilation and decreased cardiac function, which was also observed in hemodynamic measurements from CaMKIIdelta(B) TG mice. Surprisingly, phosphorylation of phospholamban at both Thr(17) and Ser(16) was significantly decreased in the basal state as well as upon adrenergic stimulation. This was associated with diminished sarcoplasmic reticulum Ca(2+) uptake in vitro and altered relaxation properties in vivo. The activity and expression of protein phosphatase 2A were both found to be increased in CaMKII TG mice, and immunoprecipitation studies indicated that protein phosphatase 2A directly associates with CaMKII. Our findings are the first to demonstrate that CaMKII can induce hypertrophy and dilation in vivo and indicate that compensatory increases in phosphatase activity contribute to the resultant phenotype.

Olson, E.N. & Schneider, M.D. Sizing up the heart: development redux in disease. Genes Dev. 17, 1937-1956

Article

Full-text available

Sep 2003
GENE DEV

Following gastrulation and establishment of the three embryonic germ layers, the first definitive organ to form in the embryo is the heart, whose morphogenesis, growth, and integrated function are essential to embry- onic survival, even by midgestation. Abnormalities in heart development result in congenital heart disease, the most frequent form of birth defects in humans. At the opposite end of the temporal spectrum, adult cardiac dis- ease is the most common cause of death in the industri- alized world, with congestive heart failure and inad- equate pump function the end result of diverse disorders intrinsic to cardiac muscle cells, cardiac valves, systemic blood pressure, and the coronary blood supply. Despite recent therapeutic advances and mechanical devices to sustain cardiac function, only a minority of heart failure patients lives longer than 5yr. Death from heart disease therefore comprises an epidemic more prevalent than all cancers combined (Ries et al. 2003). Recent studies have begun to reveal the cellular cir- cuitry that controls cardiac growth during development and disease. Intriguingly, many of the molecules and mechanisms that regulate growth of the embryonic heart are redeployed in the adult heart in response to stress signals that provoke cardiac enlargement and heart fail- ure. Thus, understanding the mechanisms involved in heart development promises to provide insights into the molecular basis for pathogenesis of the adult heart, as well as to reveal novel therapeutic targets. In this review, we consider three aspects of cardiac development with significant implications for adult heart disease: (1) nor- mal growth during organogenesis, (2) a "fetal" cardiac gene program reactivated in hypertrophy, and (3) restor- ative growth by undifferentiated progenitor cells that have cardiogenic potential. Each of these aspects of car- diac growth could be, itself, the subject of an in-depth review. Our goal, however, is not to comprehensively review these areas, but to identify common themes in developmental biology that are reiterated in settings of abnormal growth and dysfunction of the adult heart. Al- though we focus on development and disease of the myo- cardium, many of the same principles of allometric growth and homeostasis apply to other organs whose structure and function are influenced by physiological and pathological signaling.

Cooperative activation of muscle gene expression by MEF2 and myogenic BHLH proteins. E

Article

Jan 1996

Members of the Myocyte Enhancer Factor-2 (MEF2) family of MADS-box transcription factors activate muscle gene expression by binding to an A/Trich DNA sequence in the control regions of muscle-specific genes. We generated a series of deletion and site-directed mutants of MEF2C which demonstrated that the MADS and MEF2 domains at the N-terminus mediate DNA binding and dimerization, whereas the C-terminus is required for transcriptional activation. Amino acids were also identified in the MEF2 domain that are essential for MEF2 site-dependent transcription, but which do not affect DNA binding. This type of "positive control" mutant revealed an interdependence between the MEF2 domain and the transcription activation domain. MEF2 factors cannot induce myogenesis in transfected fibroblasts, but when coexpressed with the myogenic basic-helix-loop-helix (bHLH) proteins MyoD or myogenin they dramatically increase the extent of myogenic conversion above that seen with either myogenic bHLH factor alone. This cooperativity required direct interactions between the DNA binding domains (DBDs) of MEF2 and myogenic bHLH factors, but only one of the factors needed a transactivation domain and only one of the factors needed to be bound to DNA. These interactions allow either factor to activate transcription through the other's binding site and reveal a novel mechanism for indirect activation of gene expression via protein-protein interactions between the DNA binding domains of heterologous classes of transcription factors.

Increased open probability of single cardiac L-type calcium channels in patients with chronic atrial fibrillation Role of phosphatase 2A

Article

Jul 2003

Gunnar Klein

Background: The L-type calcium channel (LCC) plays a crucial role in the electrical remodeling of atrial fibrillation (AF). AF is associated with reduction of L-type calcium current density, due to a transcriptional downregulation of the pore forming alpha1c-subunit of LCC. However, it is unclear, whether this current reduction is related to a decrease in channel number or to alterations in channel function. Hence, we performed a single LCC analysis to assess channel gating and function in human AF. Methods and results: We used the cell-attached patch-clamp technique in isolated atrial human cardiomyocytes of 25 patients with sinus rhythm (SR) and 15 patients with chronic AF. Protein expression of the pore-forming α1c-subunit of LCC was reduced by 40% in AF. Single channel peak average current was 1.7-fold higher in AF than in SR, due to a 3.1-fold higher open probability of LCC. Since phosphatase 2A (PP2A) is known to preferentially reduce LCC open probability via channel dephosphorylation, we assessed whether PP2A expression or activity is reduced in AF. Okadaic acid, an inhibitor of phosphatases, increased channel open probability in SR, but not in AF. However, Western blot analysis of atrial homogenates of the same patient population revealed unchanged expression of PP2A. Conclusions: Human AF is characterized by increased single LCC activity, due to an increase of channel open probability. The blunted effect of PP2A on LCC as shown by single channel analysis may be related to a reduction of cytosolic PP2A activity or impaired local interaction between PP2A and LCC in AF.

Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca2+-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban

Article

Apr 1999

OBJECTIVES We investigated the gene expression of calcium-handling genes including L-type calcium channel, sarcoplasmic reticular calcium adenosine triphosphatase (Ca2+-ATPase), ryanodine receptor, calsequestrin and phospholamban in human atrial fibrillation.BACKGROUND Recent studies have demonstrated that atrial electrical remodeling in atrial fibrillation is associated with intracellular calcium overload. However, the changes of calcium-handling proteins remain unclear.METHODSA total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The messenger ribonucleic acid (mRNA) amount of the genes was measured by reverse transcription–polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase.RESULTSThe mRNA of L-type calcium channel and of Ca2+-ATPase was significantly decreased in patients with persistent atrial fibrillation for more than 3 months (0.36 ± 0.26 vs. 0.90 ± 0.88 for L-type calcium channel; 0.69 ± 0.42 vs. 1.21 ± 0.68 for Ca2+-ATPase; both p < 0.05, all data in arbitrary unit). We further demonstrated that there was no spatial dispersion of the gene expression among the four atrial tissue sampling sites. Age, gender and underlying cardiac disease had no significant effects on the gene expression. In contrast, the mRNA levels of ryanodine receptor, calsequestrin and phospholamban showed no significant change in atrial fibrillation.CONCLUSIONSL-type calcium channel and the sarcoplasmic reticular Ca2+-ATPase gene were down-regulated in atrial fibrillation. These changes may be a consequence of, as well as a contributory factor for, atrial fibrillation.

Ca2+-dependent transcriptional repression and derepression: DREAM, a direct effector

Article

Mar 2001

Control of gene expression by Ca2+is a well known phenomenon acting through three major pathways: (i) changes in the transactivating properties of transcription factors after induction of Ca2+-dependent kinases and phosphatases (ii) Ca2+-dependent interaction between calmodulin and S-100 proteins with basic helix–loop–helix (bHLH) transcription factors that prevents binding to DNA and (iii) direct interaction between Ca2+-free DREAM and DNA that represses transcription. Because the first mechanism has been extensively reviewed, (Gallin, W. J., Greenberg, M. E. (1995). Calcium regulation of gene expression in neurons: the mode of entry matters. Curr Opin Neurobiol 5: 367–374; Santella, L., Carafoli, E. (1997). Calcium signaling in the cell nucleus. FASEB J, 11: 1091–1109) this commentary will focus on the other two with special emphasis on DREAM, the first EF-hand protein known to specifically bind DNA and regulate transcription in a Ca2+-dependent manner (Carrion, A. M.; Link, W. A., Ledo, F., Mellstrom, B., Naranjo, J. R. (1999). DREAM is a Ca2+-regulated transcriptional repressor, Nature. 398: 80–84).

Alterations of L-Type Calcium Current and Cardiac Function in CaMKII delta Knockout Mice

Article

Aug 2010
CIRC RES

Recent studies have highlighted important roles of CaMKII in regulating Ca(2+) handling and excitation-contraction coupling. However, the cardiac effect of chronic CaMKII inhibition has not been well understood. We have tested the alterations of L-type calcium current (I(Ca)) and cardiac function in CaMKIIdelta knockout (KO) mouse left ventricle (LV). We used the patch-clamp method to record I(Ca) in ventricular myocytes and found that in KO LV, basal I(Ca) was significantly increased without changing the transmural gradient of I(Ca) distribution. Substitution of Ba(2+) for Ca(2+) showed similar increase in I(Ba). There was no change in the voltage dependence of I(Ca) activation and inactivation. I(Ca) recovery from inactivation, however, was significantly slowed. In KO LV, the Ca(2+)-dependent I(Ca) facilitation (CDF) and I(Ca) response to isoproterenol (ISO) were significantly reduced. However, ISO response was reversed by beta2-adrenergic receptor (AR) inhibition. Western blots showed a decrease in beta1-AR and an increase in Ca(v)1.2, beta2-AR, and Galphai3 protein levels. Ca(2+) transient and sarcomere shortening in KO myocytes were unchanged at 1-Hz but reduced at 3-Hz stimulation. Echocardiography in conscious mice revealed an increased basal contractility in KO mice. However, cardiac reserve to work load and beta-adrenergic stimulation was reduced. Surprisingly, KO mice showed a reduced heart rate in response to work load or beta-adrenergic stimulation. Our results implicate physiological CaMKII activity in maintaining normal I(Ca), Ca(2+) handling, excitation-contraction coupling, and the in vivo heart function in response to cardiac stress.

Gene expression of proteins influencing the calcium homeostasis in patients with persistent and paroxysmal atrial fibrillation

Article

Jun 1999

Objective: Persistent atrial fibrillation (AF) results in an impairment of atrial function. In order to elucidate the mechanism behind this phenomenon, we investigated the gene expression of proteins influencing calcium handling. Methods: Right atrial appendages were obtained from eight patients with paroxysmal AF, ten with persistent AF (> 8 months) and 18 matched controls in sinus rhythm. All controls underwent coronary artery bypass grafting, whereas most AF patients underwent Cox's MAZE surgery (n = 12). All patients had a normal left ventricular function. Total RNA was isolated and reversely transcribed into cDNA. In a semi-quantitative polymerase chain reaction the cDNA of interest and of glyceraldehyde-3-phosphate dehydrogenase were coamplified and separated by ethidium bromide-stained gel electrophoresis. Slot blot analysis was performed to study protein expression. Results: L-type calcium channel alpha 1 and sarcoplasmic reticulum Ca(2+)-ATPase mRNA (-57%, p = 0.01 and -28%, p = 0.04, respectively) and protein contents (-43%, p = 0.02 and -28%, p = 0.04, respectively) were reduced in patients with persistent AF compared to the controls. mRNA contents of phospholamban, ryanodine receptor type 2 and sodium/calcium exchanger were comparable. No changes were observed in patients with paroxysmal AF. Conclusions: Alterations in gene expression of proteins involved in the calcium homeostasis occur only in patients with long-term persistent AF. In the absence of underlying heart disease, the changes are rather secondary than primary to AF.

Role of CaMKII for signaling and regulation in the heart

Article

Feb 2009
FRONT BIOSCI

Lars S Maier

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the CaMK isoform predominantly found in the heart. Cardiac myocytes signaling during excitation-contraction coupling (ECC) is described by the increase in intracellular Ca2+ concentration. In consequence, CaMKII is activated thereby phosphorylating several important Ca2+ handling proteins with multiple functional consequences for cardiac myocytes. Specific CaMKII overexpression in the heart and in isolated myocytes of animals can exert distinct and novel effects on ECC. CaMKII activity and expression are reported to be increased in cardiac hypertrophy, in human heart failure, as well as in animal models thereby contributing to cardiac disease through a regulation process termed excitation-transcription coupling (ETC). In the present review important aspects of the role of CaMKII in ECC and ETC are summarized with an emphasis on recent novel findings.

Reporting ethical matters in The Journal of Physiology

Article

Mar 2009
J PHYSIOL-LONDON

Gordon Blair Drummond

Reporting of ethical matters in The Journal is very important. To advise and assist authors, particularly those who may be less familiar with the legislation in the UK, this article sets out the basic principles and methods that should be used and provides many key web sources of information. It addresses the structure of regulations, and introduces the concept of research governance. The UK law is summarized. Advice is given on the format and description of experiments, and common problems addressed. Aspects of human studies are addressed. Ethical considerations of publication such as authorship and originality, and problems such as plagiarism and fabrication are described. Updates will be published regularly.

Cyclic AMP-dependent modulation of cardiac Ca channels expressed in Xenopus laevis oocytes

Article

Mar 1992

Cyclic AMP-dependent modulation of cardiac L-type voltage-dependent Ca channel (VDCC) has been probed in Xenopus laevis oocytes injected with poly(A+) RNA from rat heart. A 2 to 3 fold increase of the Ba current amplitude was routinely obtained upon microinjection of cAMP (50-500 microM). Inhibition of protein kinase A (PKA) dramatically reduced the Ba current amplitude, indicating that cAMP-dependent modulation plays an important role in maintaining the basal activity of expressed Ca channels. Moreover, the effects of the DHP agonist Bay K 8644 on kinetic properties of expressed Ba current (IBa,C) were dependent on PKA activation. The results suggest that most expressed cardiac L-type VDCCs are phosphorylated and demonstrate that reconstitution in Xenopus oocytes is a suitable approach to address how phosphorylation regulates VDCC activity.

Luciferase gene vectors for analysis of promoters and enhancers

Article

Jun 1988

Steven Nordeen

Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins

Article

Jan 1996
CELL

Members of the myocyte enhancer factor-2 (MEF2) family of MADS domain transcription factors cannot induce myogenesis in transfected fibroblasts, but when coexpressed with the myogenic basic-helix-loop-helix (bHLH) proteins MyoD or myogenin they dramatically increase the extent of myogenic conversion above that seen with either myogenic bHLH factor alone. This cooperativity required direct interactions between the DNA-binding domains of MEF2 and the myogenic bHLH factors, but only one of the factors needed a transactivation domain, and only one of the factors needed to be bound to DNA. These interactions allow either factor to activate transcription through the other's binding site and reveal a novel mechanism for indirect activation of gene expression via protein-protein interactions between the DNA-binding domains of heterologous classes of transcription factors.

Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction

Article

Dec 1998

Ionic currents and unloaded cell shortening were recorded from guinea‐pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 °C. Patch pipettes (1–3 MΩ) were used to provide intracellular dialysis with pipette solutions. Na ⁺ currents were blocked with 200 μ m lidocaine. Contractions initiated by the voltage‐sensitive release mechanism (VSRM) and Ca ²⁺ ‐induced Ca ²⁺ release (CICR) in response to L‐type Ca ²⁺ current ( I Ca,L ) were separated with voltage clamp protocols. Without 8‐bromo cyclic adenosine 3′,5′‐monophosphate (8‐Br‐cAMP) in the pipette, small VSRM‐induced contractions occurred transiently in only 13 % of myocytes. In contrast, large I Ca,L ‐induced contractions were demonstrable in 100 % of cells. Addition of 10 or 50 μ m 8‐Br‐cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93 %, respectively. With 50 μ m 8‐Br‐cAMP, contractions initiated by the VSRM and I Ca,L were not significantly different in amplitude. 8‐Br‐cAMP‐supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd ²⁺ (100 μ m ) blocked I Ca,L and I Ca,L contractions but not VSRM contractions. 8‐Br‐cAMP‐supported contractions exhibited steady‐state inactivation with parameters characteristic of the VSRM, as well as sigmoidal contraction‐voltage relations. Without 8‐Br‐cAMP in the pipette, contraction‐voltage relations determined with steps from a post‐conditioning potential ( V pc ) of either −40 or −65 mV were bell shaped, with a threshold near −35 mV. With 50 μ m 8‐Br‐cAMP in the pipette, contraction‐voltage relations from a V pc of −65 mV were sigmoidal and the threshold shifted to near −55 mV. Contraction‐voltage relations remained bell shaped in the presence of 8‐Br‐cAMP when the V pc was −40 mV. H‐89, which inhibits cAMP‐dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84 % with 50 μ m 8‐Br‐cAMP in the pipette. H‐89 also significantly reduced the amplitudes of peak I Ca,L and I Ca,L contractions, although to a lesser extent. We conclude that intracellular dialysis with patch pipettes disrupts the adenylyl cyclase‐PKA phosphorylation cascade, and that the VSRM requires intracellular phosphorylation to be available for activation. Intracellular dialysis with solutions that do not maintain phosphorylation levels inhibits a major mechanism in cardiac excitation‐ contraction coupling.

Emergent Properties of Networks of Biological Signaling Pathways

Article

Feb 1999

Many distinct signaling pathways allow the cell to receive, process, and respond to information. Often, components of different pathways interact, resulting in signaling networks. Biochemical signaling networks were constructed with experimentally obtained constants and analyzed by computational methods to understand their role in complex biological processes. These networks exhibit emergent properties such as integration of signals across multiple time scales, generation of distinct outputs depending on input strength and duration, and self-sustaining feedback loops. Feedback can result in bistable behavior with discrete steady-state activities, well-defined input thresholds for transition between states and prolonged signal output, and signal modulation in response to transient stimuli. These properties of signaling networks raise the possibility that information for “learned behavior” of biological systems may be stored within intracellular biochemical reactions that comprise signaling pathways.

Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca2+ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban An insight into the mechanism of atrial electrical remodeling

Article

Apr 1999
J AM COLL CARDIOL

We investigated the gene expression of calcium-handling genes including L-type calcium channel, sarcoplasmic reticular calcium adenosine triphosphatase (Ca(2+)-ATPase), ryanodine receptor, calsequestrin and phospholamban in human atrial fibrillation. Recent studies have demonstrated that atrial electrical remodeling in atrial fibrillation is associated with intracellular calcium overload. However, the changes of calcium-handling proteins remain unclear. A total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The messenger ribonucleic acid (mRNA) amount of the genes was measured by reverse transcription-polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase. The mRNA of L-type calcium channel and of Ca(2+)-ATPase was significantly decreased in patients with persistent atrial fibrillation for more than 3 months (0.36+/-0.26 vs. 0.90+/-0.88 for L-type calcium channel; 0.69+/-0.42 vs. 1.21+/-0.68 for Ca(2+)-ATPase; both p < 0.05, all data in arbitrary unit). We further demonstrated that there was no spatial dispersion of the gene expression among the four atrial tissue sampling sites. Age, gender and underlying cardiac disease had no significant effects on the gene expression. In contrast, the mRNA levels of ryanodine receptor, calsequestrin and phospholamban showed no significant change in atrial fibrillation. L-type calcium channel and the sarcoplasmic reticular Ca(2+)-ATPase gene were down-regulated in atrial fibrillation. These changes may be a consequence of, as well as a contributory factor for, atrial fibrillation.

Retraction: Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels

Article

Apr 2000

A dynamic positive feedback mechanism, known as 'facilitation', augments L-type calcium-ion currents (ICa) in response to increased intracellular Ca2+ concentrations. The Ca2+-binding protein calmodulin (CaM) has been implicated in facilitation, but the single-channel signature and the signalling events underlying Ca2+/CaM-dependent facilitation are unknown. Here we show that the Ca2+/CaM-dependent protein kinase II (CaMK) is necessary and possibly sufficient for ICa facilitation. CaMK induces a channel-gating mode that is characterized by frequent, long openings of L-type Ca2+ channels. We conclude that CaMK-mediated phosphorylation is an essential signalling event in triggering Ca2+/CaM-dependent ICa facilitation.

Decoding calcium signals involved in cardiac growth and function

Article

Dec 2000

Calcium is central in the regulation of cardiac contractility, growth and gene expression. Variations in the amplitude, frequency and compartmentalization of calcium signals are decoded by calcium/calmodulin-dependent enzymes, ion channels and transcription factors. Understanding the circuitry for calcium signaling creates opportunities for pharmacological modification of cardiac function.

Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation

Article

Dec 2000

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors associate with myogenic basic helix-loop-helix transcription factors such as MyoD to activate skeletal myogenesis. MEF2 proteins also interact with the class II histone deacetylases HDAC4 and HDAC5, resulting in repression of MEF2-dependent genes. Execution of the muscle differentiation program requires release of MEF2 from repression by HDACs, which are expressed constitutively in myoblasts and myotubes. Here we show that HDAC5 shuttles from the nucleus to the cytoplasm when myoblasts are triggered to differentiate. Calcium/calmodulin-dependent protein kinase (CaMK) signalling, which stimulates myogenesis and prevents formation of MEF2-HDAC complexes, also induces nuclear export of HDAC4 and HDAC5 by phosphorylation of these transcriptional repressors. An HDAC5 mutant lacking two CaMK phosphorylation sites is resistant to CaMK-mediated nuclear export and acts as a dominant inhibitor of skeletal myogenesis, whereas a cytoplasmic HDAC5 mutant is unable to block efficiently the muscle differentiation program. Our results highlight a mechanism for transcriptional regulation through signal- and differentiation-dependent nuclear export of a chromatin-remodelling enzyme, and suggest that nucleo-cytoplasmic trafficking of HDACs is involved in the control of cellular differentiation.

Interleukin 3-dependent activation of DREAM is involved in transcriptional silencing of the apoptotic Hrk gene in hematopoietic progenitor cells

Article

Jun 2001

The apoptotic protein Hrk is expressed in hematopoietic progenitors after growth factor deprivation. Here we identify a silencer sequence in the 3' untranslated region of the hrk gene that binds to the transcriptional repressor DREAM in interleukin-3 (IL-3)-dependent hematopoietic progenitor cells, and abrogates the expression of reporter genes when located downstream of the open reading frame. In addition, the binding of DREAM to the hrk gene is reduced or eliminated when cells are cultured in the absence of IL-3 or treated with a calcium ionophore or a phosphatidylinositol 3-kinase-specific inhibitor, suggesting that both calcium mobilization and phosphorylation can regulate the transcriptional activity of DREAM. Furthermore, we have shown that DREAM is phosphorylated by a phosphatidylinositol 3-kinase-dependent, but Akt-independent pathway. In all cases, loss of the DREAM-DNA binding complex was correlated with increased levels of Hrk and apoptosis. These data suggest that IL-3 may trigger the activation of DREAM through different signaling pathways, which in turn binds to a silencer sequence in the hrk gene and blocks transcription, avoiding inappropriate cell death in hematopoietic progenitors.

Calmodulin kinase and a calmodulin-binding ‘IQ’ domain facilitate L-type Ca 2+ current in rabbit ventricular myocytes by a common mechanism

Article

Oct 2001

1. Ca2+-calmodulin-dependent protein kinase II (CaMK) and a calmodulin (CaM)-binding 'IQ' domain (IQ) are both implicated in Ca2+-dependent regulation of L-type Ca2+ current (I(Ca)). We used an IQ-mimetic peptide (IQmp), under conditions in which CaMK activity was controlled, to test the relationship between these CaM-activated signalling elements in the regulation of L-type Ca2+ channels (LTCCs) and I(Ca) in rabbit ventricular myocytes. 2. A specific CaMK inhibitory peptide nearly abolished I(Ca) facilitation, but the facilitation was 'rescued' by cell dialysis with IQmp. 3. IQmp significantly enhanced I(Ca) facilitation and slowed the fast component of I(Ca) inactivation, compared with an inactive control peptide. Neither effect could be elicited by a more avid CaM-binding peptide, suggesting that generalized CaM buffering did not account for the effects of IQmp. 4. I(Ca) facilitation was abolished and the fast component of inactivation eliminated by ryanodine, caffeine or thapsigargin, suggesting that the sarcoplasmic reticulum (SR) is an important source of Ca2+ for I(Ca) facilitation and inactivation. IQmp did not restore I(Ca) facilitation under these conditions. 5. Engineered Ca2+-independent CaMK and IQmp each markedly increased LTCC open probability (P(o)) in excised cell membrane patches. The LTCC P(o) increases with CaMK and IQmp were non-additive, suggesting that CaMK and IQmp are components of a shared signalling pathway. 6. Both CaMK and IQmp induced a modal gating shift in LTCCs that favoured prolonged openings, indicating that CaMK and IQmp affect LTCCs through a common biophysical mechanism. 7. These findings support the hypothesis that CaMK is required for physiological I(Ca) facilitation in cardiac myocytes. Both CaMK and IQmp were able to induce a modal gating shift in LTCCs, suggesting that each of these signalling elements is important for Ca2+-CaM-dependent LTCC facilitation in cardiac myocytes.

DREAM Is a Critical Transcriptional Repressor for Pain Modulation

Article

Feb 2002
CELL

Control and treatment of chronic pain remain major clinical challenges. Progress may be facilitated by a greater understanding of the mechanisms underlying pain processing. Here we show that the calcium-sensing protein DREAM is a transcriptional repressor involved in modulating pain. dream(-/-) mice displayed markedly reduced responses in models of acute thermal, mechanical, and visceral pain. dream(-/-) mice also exhibited reduced pain behaviors in models of chronic neuropathic and inflammatory pain. However, dream(-/-) mice showed no major defects in motor function or learning and memory. Mice lacking DREAM had elevated levels of prodynorphin mRNA and dynorphin A peptides in the spinal cord, and the reduction of pain behaviors in dream(-/-) mice was mediated through dynorphin-selective kappa (kappa)-opiate receptors. Thus, DREAM appears to be a critical transcriptional repressor in pain processing.

Bers, DM. Cardiac excitation-contraction coupling. Nature 415: 198-205

Article

Feb 2002

Donald Bers

Of the ions involved in the intricate workings of the heart, calcium is considered perhaps the most important. It is crucial to the very process that enables the chambers of the heart to contract and relax, a process called excitation-contraction coupling. It is important to understand in quantitative detail exactly how calcium is moved around the various organelles of the myocyte in order to bring about excitation-contraction coupling if we are to understand the basic physiology of heart function. Furthermore, spatial microdomains within the cell are important in localizing the molecular players that orchestrate cardiac function.

The role of calcium-binding proteins in the control of transcription: Structure to function

Article

Jul 2002

Transcriptional regulation is coupled with numerous intracellular signaling processes often mediated by second messengers. Now, growing evidence points to the importance of Ca(2+), one of the most versatile second messengers, in activating or inhibiting gene transcription through actions frequently mediated by members of the EF-hand superfamily of Ca(2+)-binding proteins. Calmodulin and calcineurin, representative members of this EF-hand superfamily, indirectly regulate transcription through phosphorylation/dephosphorylation of transcription factors in response to a Ca(2+) increase in the cell. Recently, a novel EF-hand Ca(2+)-binding protein called DREAM has been found to interact with regulatory sequences of DNA, thereby acting as a direct regulator of transcription. Finally, S100B, a dimeric EF-hand Ca(2+)-binding protein, interacts with the tumor suppressor p53 and controls its transcriptional activity. In light of the structural studies reported to date, this review provides an overview of the structural basis of EF-hand Ca(2+)-binding proteins linked with transcriptional regulation.

L-Type Ca2+ Channel Density and Regulation Are Altered in Failing Human Ventricular Myocytes and Recover After Support With Mechanical Assist Devices

Article

Oct 2002
CIRC RES

Ca2+ influx through the L-type calcium channel (LTCC) induces Ca2+ release from the sarcoplasmic reticulum (SR) and maintains SR Ca2+ loading. Alterations in LTCC properties, their contribution to the blunted adrenergic responsiveness in failing hearts and their recovery after support with LV assist devices (LVAD) were studied. L-type Ca2+ current (I(Ca,L)) was measured under basal conditions and in the presence of isoproterenol (ISO), dibutyryl-cAMP (db-cAMP), Bay K 8644 (BayK), Okadaic acid (OA, a phosphatase inhibitor), and phosphatase 2A (PP2A) in nonfailing (NF), failing (F), and LVAD-supported human left ventricular myocytes (HVMs). Basal I(Ca,L) density was not different in the 3 groups but I(Ca,L) was activated at more negative voltages in F- and LVAD- versus NF-HVMs (V(0.5): -7.18+/-1.4 and -7.0+/-0.9 versus 0.46+/-1.1 mV). Both ISO and db-cAMP increased I(Ca,L) in NF- and LVAD- significantly more than in F-HVMs (NF >LVAD> F: ISO: 90+/-15% versus 77+/-19% versus 24+/-12%; db-cAMP: 235%>172%>90%). ISO caused a significant leftward shift of the I(Ca,L) activation curve in NF- and LVAD- but not in F-HVMs. After ISO and db-cAMP, the I(Ca,L) activation was not significantly different between groups. BayK also increased I(Ca,L) more in NF- (81+/-30%) and LVAD- (70+/-15%) than in F- (51+/-8%) HVMs. OA increased I(Ca, L) by 85.6% in NF-HVMs but had no effect in F-HVMs, while PP2A decreased I(Ca, L) in F-HVMs by 35% but had no effect in NF-HVMs. These results suggest that the density of LTCC is reduced in F-HVMs but basal I(Ca,L) density is maintained by increasing in LTCC phosphorylation.

A 27 bp cis-acting sequence is essential for L-type calcium channel α1C subunit expression in vascular smooth muscle cells

Article

Oct 2002
Biochim Biophys Acta

Expression of L-type calcium channels in cardiac myocytes and vascular smooth muscle cells (VSMC) critically regulates the contractile state of these cells. In order to discover the elements in the promoter region of the Ca(v)1.2 gene encoding the vascular/cardiac calcium channel alpha(1C) subunit that are important for the basal gene expression, approximately 2 kb of the 5'-flanking sequence of the Ca(v)1.2 gene has been cloned in our lab. In this study, using various lengths of the 5'-flanking DNA fused with a luciferase gene as a reporter, we have defined a 493-bp fragment of the cis-regulatory DNA which carries the majority of promoter activity in pulmonary artery smooth muscle (PAC1) cells. DNase I footprinting analysis of this 493-bp DNA using nuclear extracts from PAC1 cells revealed a 27-bp DNA sequence that contains a c-Ets like motif (CAGGATGC). Mutation of the Ets-like site and the respective flanking sequence within the DNase I footprinting protection region induced a marked change in the promoter activity in PAC1 cells. Electrophoretic mobility shift assays (EMSA) confirmed the presence of specific binding factor(s) in PAC1 cells' nuclear extracts for this 27-bp DNA. Competition studies with the wild-type and mutated DNA fragments established the importance of the 27 bp DNA sequence for high-affinity binding of the nuclear proteins to the promoter. We conclude that there is a 27 bp region in the promoter of the Ca(v)1.2 gene to which nuclear proteins from VSMC bind and strongly regulate the basal promoter activity.

Abnormal Ca2+ Release, but Normal Ryanodine Receptors, in Canine and Human Heart Failure

Article

Dec 2002
CIRC RES

Sarcoplasmic reticulum (SR) Ca2+ transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca2+ transport. Intracellular Ca2+ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca2+]i transient were reduced in HF. Ca2+ uptake assays showed 44+/-8% reduction of Vmax in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30+/-5% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The P(o) of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor beta-adrenergic stimulation of Ca2+ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca2+ uptake, more than Ca2+ release, contributing to the depressed and slow Ca2+ transient characteristic of HF.

Cellular Basis of Abnormal Calcium Transients of Failing Human Ventricular Myocytes

Article

May 2003
CIRC RES

Depressed contractility is a central feature of the failing human heart and has been attributed to altered [Ca2+]i. This study examined the respective roles of the L-type Ca2+ current (ICa), SR Ca2+ uptake, storage and release, Ca2+ transport via the Na+-Ca2+ exchanger (NCX), and Ca2+ buffering in the altered Ca2+ transients of failing human ventricular myocytes. Electrophysiological techniques were used to measure and control V(m) and measure I(m), respectively, and Fluo-3 was used to measure [Ca2+]i in myocytes from nonfailing (NF) and failing (F) human hearts. Ca2+ transients from F myocytes were significantly smaller and decayed more slowly than those from NF hearts. Ca2+ uptake rates by the SR and the amount of Ca2+ stored in the SR were significantly reduced in F myocytes. There were no significant changes in the rate of Ca2+ removal from F myocytes by the NCX, in the density of NCX current as a function of [Ca2+]i, ICa density, or cellular Ca2+ buffering. However, Ca2+ influx during the late portions of the action potential seems able to elevate [Ca2+]i in F but not in NF myocytes. A reduction in the rate of net Ca2+ uptake by the SR slows the decay of the Ca2+ transient and reduces SR Ca2+ stores. This leads to reduced SR Ca2+ release, which induces additional Ca2+ influx during the plateau phase of the action potential, further slowing the decay of the Ca2+ transient. These changes can explain the defective Ca2+ transients of the failing human ventricular myocyte.

The ??C Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure

Article

Jun 2003
CIRC RES

Recent studies have demonstrated that transgenic (TG) expression of either Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIdeltaB, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIdeltaB but also a cytoplasmic isoform, CaMKIIdeltaC. In the present study, we demonstrate that expression of the deltaC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic deltaC isoform of CaMKII can lead to phosphorylation of Ca2+ regulatory proteins and induce hypertrophy, we generated TG mice that expressed the deltaC isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2+ handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIdeltaC can mediate phosphorylation of Ca2+ regulatory proteins in vivo and provide evidence for the involvement of CaMKIIdeltaC activation in the pathogenesis of dilated cardiomyopathy and heart failure.

Transgenic CaMKIIδC Overexpression Uniquely Alters Cardiac Myocyte Ca2+ Handling Reduced SR Ca2+ Load and Activated SR Ca2+ Release

Article

May 2003
CIRC RES

Ca2+/calmodulin-dependent protein kinase II (CaMKII) delta is the predominant cardiac isoform, and the deltaC splice variant is cytoplasmic. We overexpressed CaMKIIdeltaC in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKIIdeltaC transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by approximately 50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKIIdeltaC overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Article

Sep 2003

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca2+ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca2+ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro, but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca2+] ([Ca2+]i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca2+]i by ~25 %, but had no significant effect on the rate of SR Ca2+ uptake or the force-[Ca2+]i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca2+]i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca2+ release in the basal state and during repeated contractions, providing a positive feedback between [Ca2+]i and SR Ca2+ release.

Increased open probability of single cardiac L-type calcium channels in patients with chronic atrial fibrillation: Role of phosphatase 2A

Article

Aug 2003

The L-type calcium channel (LCC) plays a crucial role in the electrical remodeling of atrial fibrillation (AF). AF is associated with reduction of L-type calcium current density, due to a transcriptional downregulation of the pore forming alpha(1c)-subunit of LCC. However, it is unclear, whether this current reduction is related to a decrease in channel number or to alterations in channel function. Hence, we performed a single LCC analysis to assess channel gating and function in human AF. We used the cell-attached patch-clamp technique in isolated atrial human cardiomyocytes of 25 patients with sinus rhythm (SR) and 15 patients with chronic AF. Protein expression of the pore-forming alpha(1c)-subunit of LCC was reduced by 40% in AF. Single channel peak average current was 1.7-fold higher in AF than in SR, due to a 3.1-fold higher open probability of LCC. Since phosphatase 2A (PP2A) is known to preferentially reduce LCC open probability via channel dephosphorylation, we assessed whether PP2A expression or activity is reduced in AF. Okadaic acid, an inhibitor of phosphatases, increased channel open probability in SR, but not in AF. However, Western blot analysis of atrial homogenates of the same patient population revealed unchanged expression of PP2A. Human AF is characterized by increased single LCC activity, due to an increase of channel open probability. The blunted effect of PP2A on LCC as shown by single channel analysis may be related to a reduction of cytosolic PP2A activity or impaired local interaction between PP2A and LCC in AF.

Roles of calcineurin and calcium/calmodulin-dependent protein kinase II in pressure overload-induced cardiac hypertrophy

Article

Oct 2003

Calcineurin and calcium/calmodulin-dependent protein kinase (CaMK) II have been suggested to be the signaling molecules in cardiac hypertrophy. It was not known, however, whether these mechanisms are involved in cardiac hypertrophy induced by pressure overload without the influences of blood-derived humoral factors, such as angiotensin II. To elucidate the roles of calcineurin and CaMK II in this situation, we examined the effects of calcineurin and CaMK II inhibitors on pressure overload-induced expression of c-fos, an immediate-early gene, and protein synthesis using heart perfusion model. The hearts isolated from Sprague-Dawley rats were perfused according to the Langendorff technique, and then subjected to the acute pressure overload by raising the perfusion pressure. The activation of calcineurin was evaluated by its complex formation with calmodulin and by its R-II phosphopeptide dephosphorylation. CaMK II activation was evaluated by its autophosphorylation. Expression of c-fos mRNA and rates of protein synthesis were measured by northern blot analysis and by 14C-phenylalanine incorporation, respectively. Acute pressure overload significantly increased calcineurin activity, CaMK II activity, c-fos expression and protein synthesis. Cyclosporin A and FK506, the calcineurin inhibitors, significantly inhibited the increases in both c-fos expression and protein synthesis. KN62, a CaMK II inhibitor, also significantly prevented the increase in protein synthesis, whereas it failed to affect the expression of c-fos. These results suggest that both calcineurin and CaMK II pathways are critical in the pressure overload-induced acceleration of protein synthesis, and that transcription of c-fos gene is regulated by calcineurin pathway but not by CaMK II pathway.

Mice overexpressing the cardiac sodium-calcium exchanger: Defects in excitation-contraction coupling

Article

Feb 2004

Homozygous overexpression of the cardiac Na(+)-Ca(2+) exchanger causes cardiac hypertrophy and increases susceptibility to heart failure in response to stress. We studied the functional effects of homozygous overexpression of the exchanger at the cellular level in isolated mouse ventricular myocytes. Compared with patch-clamped myocytes from wild-type animals, non-failing myocytes from homozygous transgenic mice exhibited increased cell capacitance (from 208 +/- 16 pF to 260 +/- 15 pF, P < 0.05). Intracellular Ca(2+) oscillations were readily elicited in homozygous transgenic animals during depolarizations to +80 mV, consistent with rapid Ca(2+) overload caused by reverse Na(+)-Ca(2+) exchange. After normalization to cell capacitance, transgenic myocytes had significant increases in Na(+)-Ca(2+) exchange activity (318%) and peak L-type Ca(2+) current (8.2 +/- 0.7 pA pF(-1) at 0 mV test potential) compared to wild-type (5.8 +/- 0.9 pA pF(-1) at 0 mV, P < 0.02). The peak Ca(2+) current amplitude and its rate of inactivation could be modulated by rapid reversible block of the exchanger. Thus, we describe an unexpected direct influence of Na(+)-Ca(2+) exchange activity on the L-type Ca(2+) channel. Despite intact sarcoplasmic reticular Ca(2+) content and larger peak L-type Ca(2+) currents, homozygous transgenic animals exhibited smaller Ca(2+) transients (Delta[Ca(2+)](i)= 466 +/- 48 nm in transgenics versus 892 +/- 104 nm in wild-type, P < 0.0005) and substantially reduced gain of excitation-contraction coupling. These alterations in excitation-contraction coupling may underlie the tendency for these animals to develop heart failure following haemodynamic stress.

CaM kinase II-dependent phosphorylation of myogenin contributes to activity-dependent suppression of nAChR gene expression in developing rat myotubes

Article

Jun 2004
CELL SIGNAL

During development of the neuromuscular junction (NMJ), extrajunctional expression of genes, whose products are destined for the synapse, is suppressed by muscle activity. One of the best-studied examples of activity-dependent gene regulation in muscle are those encoding nicotinic acetylcholine receptor (nAChR) subunits. We recently showed that nAChR gene expression is inhibited by calcium/calmodulin-dependent protein kinase II (CaMKII) and CaMKII inhibitors block activity-dependent suppression of these genes. Here we report results investigating the mechanism by which CaMKII suppresses nAChR gene expression. We show that the muscle helix-loop-helix transcription factor, myogenin, is necessary for activity-dependent control of nAChR gene expression in cultured rat myotubes and is a substrate for CaMKII both in vitro and in vivo. CaMKII phosphorylation of myogenin is induced by muscle activity and this phosphorylation influences DNA binding and transactivation. Thus we have identified a signaling mechanism by which muscle activity controls nAChR gene expression in developing muscle.

Altered Calcium Handling Is Critically Involved in the Cardiotoxic Effects of Chronic -Adrenergic Stimulation

Article

Apr 2004
CIRCULATION

Chronic adrenergic stimulation leads to cardiac hypertrophy and heart failure in experimental models and contributes to the progression of heart failure in humans. The pathways mediating the detrimental effects of chronic beta-adrenergic stimulation are only partly understood. We investigated whether genetic modification of calcium handling through deletion of phospholamban in mice would affect the development of heart failure in mice with transgenic overexpression of the beta1-adrenergic receptor. We crossed beta1-adrenergic receptor transgenic (beta1TG) mice with mice homozygous for a targeted deletion of the phospholamban gene (PLB-/-). Phospholamban ablation dramatically enhanced survival of beta1TG mice. The decrease of left ventricular contractility typically observed in beta1TG mice was reverted back to normal by phospholamban ablation. Cardiac hypertrophy and fibrosis were significantly inhibited in beta1TG/PLB-/- mice compared with beta1TG mice, and the heart failure-specific gene expression pattern was normalized. Analysis of intracellular calcium transients revealed increased diastolic calcium levels and decreased rate constants of diastolic calcium decline in beta1TG mice. In beta1TG/PLB-/- mice, diastolic calcium concentration was normal and rate constants of diastolic calcium decline were greater than in wild-type mice. We conclude that modification of abnormal calcium handling in beta1TG mice through ablation of phospholamban resulted in a rescue of functional, morphological, and molecular characteristics of heart failure in beta1-adrenergic receptor-transgenic mice. These results imply altered calcium handling as critical for the detrimental effects of beta1-adrenergic signaling.

Intracellular calcium modulates the nuclear translocation of calsenilin

Article

Jun 2004

Calsenilin, which was originally identified as a presenilin interacting protein, has since been shown to be involved in the processing of presenilin(s), the modulation of amyloid beta-peptide (Abeta) levels and apoptosis. Subsequent to its original identification, calsenilin was shown to act as a downstream regulatory element antagonist modulator (and termed DREAM), as well as to interact with and modulate A-type potassium channels (and termed KChIP3). Calsenilin is primarily a cytoplasmic protein that must translocate to the nucleus to perform its function as a transcriptional repressor. This study was designed to determine the cellular events that modulate the translocation of calsenilin from the cytoplasm to the nucleus. The nuclear translocation of calsenilin was found to be enhanced following serum deprivation. A similar effect was observed when cells were treated with pharmacological agents that directly manipulate the levels of intracellular calcium (caffeine and the calcium ionophore A23187), suggesting that the increased levels of calsenilin in the nucleus are mediated by changes in intracellular calcium. A calsenilin mutant that was incapable of binding calcium retained the ability to translocate to the nucleus. Taken together, these findings indicate that the level of intracellular calcium can modulate the nuclear translocation of calsenilin and that this process does not involve the direct binding of calcium to calsenilin.

Calmodulin Kinase and L-Type Calcium ChannelsA Recipe for Arrhythmias?

Article

Jun 2004
TRENDS CARDIOVAS MED

Mark E Anderson

L-type Ca2+ channels (LTCCs) are the main portal for Ca2+ entry into cardiac myocytes. These ion channel proteins open in response to cell membrane depolarizations elicited by action potentials, and LTCC current (I(Ca)) flows during the action potential plateau, to increase cellular Ca2+ (Ca2+(i)) and trigger myocardial contraction. I(Ca) is also implicated in the genesis of cardiac arrhythmias under conditions such as heart failure and cardiac hypertrophy, in which the action potential plateau and QT interval are prolonged. This article reviews recent findings about the molecular regulation of LTCCs by the Ca2+-dependent signaling molecule, calmodulin kinase II (CaMKII), and compares this form of regulation with regulation by calmodulin-binding domains and beta-adrenergic receptor agonists. LTCC dysregulation is discussed in the context of new results showing that CaMKII can be a proarrhythmic signal in disease conditions in which Ca2+(i) is disordered and cardiac repolarization is excessively prolonged.

Ca2+-calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel α1C-subunit gene (Cacna1c) by DREAM translocation

Abstract

No full-text available

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