[Show abstract][Hide abstract] ABSTRACT: -A hallmark of heart failure is impaired cytoplasmic Ca(2+) handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca(2+) handling -- via altered excitation-transcription coupling -- contribute to the development and progression of heart failure.
[Show abstract][Hide abstract] ABSTRACT: Both β-adrenergic (β-AR) and Gq-coupled agonist (GqR) driven signaling play key roles in the events leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear.
To assess the spatiotemporal dynamics of PKD activation in response to β-AR signaling alone and upon co-activation with GqR agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of protein kinase A (PKA) facilitation and inhibition of PKD activation.
We report on the spatial and temporal profiles of PKD activation using GFP-tagged PKD (wildtype or mutant S427E) and targeted FRET based biosensors (DKARs) in adult cardiomyocytes. We find that β-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of β-AR/cAMP/PKA signaling on GqR-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes GqR-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits GqR-evoked cell growth and expression of hypertrophic markers. Lastly, we show altered S427 phosphorylation in TAC-induced hypertrophy.
β-AR signaling triggers local nuclear signaling and inhibits GqR-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD S427 fine-tunes the PKD responsiveness to GqR-agonists, serving as a key integration point for β-adrenergic and Gq-coupled stimuli.
Circulation Research 03/2014; · 11.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium (Ca(2+)) has long been recognized as a crucial intracellular messenger attaining stimuli-specific cellular outcomes via localized signaling. Ca(2+)-binding proteins, such as calmodulin (CaM), and its target proteins are key to the segregation and refinement of these Ca(2+)-dependent signaling events. This review not only summarizes the recent technological advances enabling the study of subcellular Ca(2+)-CaM and Ca(2+)-CaM-dependent protein kinase (CaMKII) signaling events but also highlights the outstanding challenges in the field.
Journal of Molecular Medicine 06/2013; · 4.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5's newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription and . Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.
PLoS ONE 01/2013; 8(3):e57324. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: Mitochondrial [Ca(2+)] ([Ca(2+)](mito)) regulates mitochondrial energy production, provides transient Ca(2+) buffering under stress and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes and evidence for crosstalk exists. However, quantitative measurements of [Ca(2+)](mito) are limited and spatial [Ca(2+)](mito) gradients have not been directly measured. Objective: To directly measure local [Ca(2+)](mito) during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca(2+)](mito) gradients. Methods and Results: We used in-situ calibration of the mitochondrially targeted inverse pericam indicator Mitycam and directly measured [Ca(2+)](mito) during SR Ca(2+) release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing &[Delta]Ca(2+)](mito) amplitude was 29 &[plusmn] 3 nM, rising rapidly (similar to cytosolic [Ca(2+)](i)) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca(2+)](mito) near SR Ca(2+) release sites (Z-lines) vs. mid sarcomere (M-line) reached a higher peak ampli¬tude (37 &[plusmn] 4 vs. 26 &[plusmn] 4 nM, respectively P < 0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca(2+) release sites, because the mitochondrial Ca(2+) uniporter was homogeneously distributed and elevated [Ca(2+)] applied laterally did not produce longitudinal [Ca(2+)](mito) gradients. Conclusions: We developed methods to measure spatiotemporal [Ca(2+)](mito) gradients quanti¬tatively during excitation-contraction coupling. The amplitude and kinetics of [Ca(2+)](mito) transients differ significantly from those in the cytosol and are higher and faster near the Z- vs. M-line. This approach will help clarify SR-mitochondrial Ca(2+) signaling.
Circulation Research 12/2012; · 11.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Na(+)-K(+)-ATPase (NKA) establishes the transmembrane [Na(+)] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na(+) affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM-NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP-NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance energy transfer (FRET) with WT-PLM-YFP in HEK293 cells. NKA-PLM FRET was unaltered with F956A or F967A, reduced with L964A, and nearly abolished with E960A. Mutating the PLM site (F28A) identified by structural analysis to interact with E960-NKA also nearly abolished NKA-PLM FRET. In contrast, NKA-PLM coimmunoprecipitation was only slightly reduced by E960A-NKA or F28A-PLM mutants, consistent with an additional interaction site. FRET titrations indicate that the additional site has higher affinity than that between E960-NKA and F28-PLM. To test whether the FRET-preventing mutations also prevent PLM functional effects, we measured NKA-mediated Na(+)-transport in intact cells. For WT-NKA, PLM reduced apparent Na(+)-affinity of NKA and PLM phosphorylation reversed the effect. In contrast, for E960A-NKA the apparent Na(+)-affinity was unaltered by either PLM or forskolin-induced PLM phosphorylation. We conclude that E960 on NKA and F28 on PLM are critical for PLM effects on both NKA function and NKA-PLM FRET, but also there is at least one additional site that is critical for tethering PLM to NKA.
Proceedings of the National Academy of Sciences 11/2012; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During hemodynamic stress, catecholamines and neurohumoral stimuli may induce co-activation of Gq-coupled receptors and beta-adrenergic receptors (β-AR) leading to cardiac remodeling. Dynamic regulation of histone deacetylase 5 (HDAC5), a transcriptional repressor, is crucial during stress signaling due to its role in epigenetic control of fetal gene markers. Little is known about its regulation during acute and chronic β-AR stimulation and its cross interaction with Gq-signaling in adult cardiac myocytes. We aim to evaluate the potential crosstalk between Gq-driven and β-AR mediated signaling at the level of nucleocytoplasmic shuttling of HDAC5. We study the translocation of GFP-tagged wildtype HDAC5 or mutants (S279A and S279D) in response to β-AR or Gq agonists. Isopreterenol (ISO) or PKA activation results in strong nuclear accumulation of HDAC5 in contrast to the nuclear exports driven by Ca2+-calmodulin protein kinase II (CaMKII) and protein kinase D (PKD). Moreover, nuclear accumulation of HDAC5 under acute ISO/PKA signaling is dependent on phosphorylation of its serine 279 (S279) and can block the following Gq-mediated nuclear HDAC5 export. Intriguingly, the attenuation of Gq-induced export is abolished after chronic PKA activation, yet nuclear HDAC5 remains elevated. Lastly, the effect of chronic β-AR signaling on HDAC5 translocation was examined using heart failure rabbit myocytes, where ISO-induced nuclear import is ablated, but Gq-agonist mediated export is preserved. Acute β-AR/PKA activation protects against hypertrophic signaling by delaying Gq-mediated transcriptional activation. This serves as a key physiological control switch before allowing genetic re-programming via HDAC5 nuclear export during more severe stress conditions, such as heart failure.
Journal of Biological Chemistry 11/2012; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: Intracellular Ca(2+) concentration ([Ca(2+)]i) is regulated and signals differently in various subcellular microdomains, which greatly enhances its second messenger versatility. In the heart, sarcoplasmic reticulum (SR) Ca(2+) release and signaling is controlled by local [Ca(2+)]i in the junctional cleft ([Ca(2+)]Cleft), the small space between sarcolemma and junctional SR. However, methods to directly measure [Ca(2+)]Cleft are needed. Objective: To construct novel sensors that allow direct measurement of [Ca(2+)]Cleft. Methods and Results: We constructed cleft-targeted [Ca(2+)] sensors by fusing Ca(2+)-sensor GCaMP2.2 and a new lower Ca(2+)-affinity variant GCaMP2.2Low to FKBP12.6, which binds with high affinity and selectivity to ryanodine receptors (RyRs). The fluorescence pattern, affinity for RyRs and competition by un-tagged FKBP12.6 demon¬strated that FKBP12.6-tagged sensors are positioned to measure local [Ca(2+)]Cleft in adult rat myocytes. Using GCaMP2.2Low-FKBP12.6, we showed that [Ca(2+)]Cleft reaches higher levels with faster kinetics than global [Ca(2+)]i during excitation-contraction coupling. Diastolic SR Ca(2+) leak or sarcolemmal Ca(2+) entry may raise local [Ca(2+)]Cleft above bulk cytosolic [Ca(2+)]i ([Ca(2+)]Bulk), an effect that may contribute to triggered arrhythmias and even transcriptional regulation. We measured this diastolic standing [Ca(2+)]Cleft-[Ca(2+)]Bulk gradient using GCaMP2.2-FKBP12.6 vs. GCaMP2.2, using [Ca(2+)] measured without gradients as a reference point. This diastolic difference ([Ca(2+)]Cleft=194 nmol/L vs. [Ca(2+)]Bulk=100 nmol/L) is dictated mainly by the SR Ca(2+) leak, rather than sarcolemmal Ca(2+) flux. Conclusions: We have developed junctional cleft targeted sensors to measure [Ca(2+)]Cleft vs. [Ca(2+)]Bulk, and demonstrated dynamic differences during electrical excitation and a standing diastolic [Ca(2+)]i gradient which could influence local Ca(2+)-dependent signaling within the junctional cleft.
[Show abstract][Hide abstract] ABSTRACT: Depressed Ca-handling in cardiomyocytes is frequently attributed to impaired sarcoplasmic reticulum (SR) function in human and experimental heart failure. Phospholamban (PLN) is a key regulator of SR and cardiac function, and PLN mutations in humans have been associated with dilated cardiomyopathy (DCM). We previously reported the deletion of the highly conserved amino acid residue arginine 14 (nucleic acids 39, 40 and 41) in DCM patients. This basic amino acid is important in maintaining the upstream consensus sequence for PKA phosphorylation of Ser 16 in PLN. To assess the function of this mutant PLN, we introduced the PLN-R14Del in cardiac myocytes of the PLN null mouse. Transgenic lines expressing mutant PLN-R14Del at similar protein levels to wild types exhibited no inhibition of the initial rates of oxalate-facilitated SR Ca uptake compared to PLN-knockouts (PLN-KO). The contractile parameters and Ca-kinetics also remained highly stimulated in PLN-R14Del cardiomyocytes, similar to PLN-KO, and isoproterenol did not further stimulate these hyper-contractile basal parameters. Consistent with the lack of inhibition on SR Ca-transport and contractility, confocal microscopy indicated that the PLN-R14Del failed to co-localize with SERCA2a. Moreover, PLN-R14Del did not co-immunoprecipitate with SERCA2a (as did WT-PLN), but rather co-immunoprecipitated with the sarcolemmal Na/K-ATPase (NKA) and stimulated NKA activity. In addition, studies in HEK cells indicated significant fluorescence resonance energy transfer between PLN-R14Del-YFP and NKAα1-CFP, but not with the NKA regulator phospholemman. Despite the enhanced cardiac function in PLN-R14Del hearts (as in PLN-knockouts), there was cardiac hypertrophy (unlike PLN-KO) coupled with activation of Akt and the MAPK pathways. Thus, human PLN-R14Del is misrouted to the sarcolemma, in the absence of endogenous PLN, and alters NKA activity, leading to cardiac remodeling.
Journal of Molecular and Cellular Cardiology 12/2011; 52(3):773-82. · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein kinase D (PKD) is a nodal point in cardiac hypertrophic signaling. It triggers nuclear export of class II histone
deacetylase (HDAC) and regulates transcription. Although this pathway is thought to be critical in cardiac hypertrophy and
heart failure, little is known about spatiotemporal aspects of PKD activation at the myocyte level. Here, we demonstrate that
in adult cardiomyocytes two important neurohumoral stimuli that induce hypertrophy, endothelin-1 (ET1) and phenylephrine (PE),
trigger comparable global PKD activation and HDAC5 nuclear export, but via divergent spatiotemporal PKD signals. PE-induced
HDAC5 export is entirely PKD-dependent, involving fleeting sarcolemmal PKD translocation (for activation) and very rapid subsequent
nuclear import. In contrast, ET1 recruits and activates PKD that remains predominantly sarcolemmal. This explains why PE-induced
nuclear HDAC5 export in myocytes is totally PKD-dependent, whereas ET1-induced HDAC5 export depends more prominently on InsP3 and CaMKII signaling. Thus α-adrenergic and ET-1 receptor signaling via PKD in adult myocytes feature dramatic differences
in cellular localization and translocation in mediating hypertrophic signaling. This raises new opportunities for targeted
therapeutic intervention into distinct limbs of this hypertrophic signaling pathway.
Journal of Biological Chemistry 09/2011; 286(38):33390-33400. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited.
We use Camui, a novel FRET-based biosensor in which full-length CaMKII is flanked by CFP and YFP, to measure CaMKII activation state in living rabbit myocytes. MethoDS AND RESULTS: We show that Camui and mutant variants that lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKIIδ activation state. Camui (wild-type or mutant) was expressed in isolated adult cardiac myocytes, and localization and CaMKII activation state were determined using confocal microscopy. Camui, like CaMKIIδ, is concentrated at the z-lines, with low baseline activation state. Camui activation increased directly with pacing frequency, but the maximal effect was blunted with the T286A, consistent with frequency-dependent phosphorylation of CaMKII at T286 mainly at high-frequency and high-amplitude Ca transients. Camui was also activated by 4 neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely through an oxidation-dependent mechanism, whereas isoproterenol- and phenylephrine-mediated mechanisms had a significant autophosphorylation-dependent component.
Camui is a novel, nondestructive tool that allows spatiotemporally resolved measurement of CaMKII activation state in physiologically functioning myocytes. This represents a first step in using Camui to elucidate key mechanistic details of CaMKII signaling in live hearts and myocytes.
Circulation Research 08/2011; 109(7):729-38. · 11.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The small GTPase RhoA serves as a nodal point for signaling through hormones and mechanical stretch. However, the role of RhoA signaling in cardiac pathophysiology is poorly understood. To address this issue, we generated mice with cardiomyocyte-specific conditional expression of low levels of activated RhoA (CA-RhoA mice) and demonstrated that they exhibited no overt cardiomyopathy. When challenged by in vivo or ex vivo ischemia/reperfusion (I/R), however, the CA-RhoA mice exhibited strikingly increased tolerance to injury, which was manifest as reduced myocardial lactate dehydrogenase (LDH) release and infarct size and improved contractile function. PKD was robustly activated in CA-RhoA hearts. The cardioprotection afforded by RhoA was reversed by PKD inhibition. The hypothesis that activated RhoA and PKD serve protective physiological functions during I/R was supported by several lines of evidence. In WT mice, both RhoA and PKD were rapidly activated during I/R, and blocking PKD augmented I/R injury. In addition, cardiac-specific RhoA-knockout mice showed reduced PKD activation after I/R and strikingly decreased tolerance to I/R injury, as shown by increased infarct size and LDH release. Collectively, our findings provide strong support for the concept that RhoA signaling in adult cardiomyocytes promotes survival. They also reveal unexpected roles for PKD as a downstream mediator of RhoA and in cardioprotection against I/R.
The Journal of clinical investigation 08/2011; 121(8):3269-76. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein kinase D (PKD) is a nodal point in cardiac hypertrophic signaling. It triggers nuclear export of class II histone deacetylase (HDAC) and regulates transcription. Although this pathway is thought to be critical in cardiac hypertrophy and heart failure, little is known about spatiotemporal aspects of PKD activation at the myocyte level. Here, we demonstrate that in adult cardiomyocytes two important neurohumoral stimuli that induce hypertrophy, endothelin-1 (ET1) and phenylephrine (PE), trigger comparable global PKD activation and HDAC5 nuclear export, but via divergent spatiotemporal PKD signals. PE-induced HDAC5 export is entirely PKD-dependent, involving fleeting sarcolemmal PKD translocation (for activation) and very rapid subsequent nuclear import. In contrast, ET1 recruits and activates PKD that remains predominantly sarcolemmal. This explains why PE-induced nuclear HDAC5 export in myocytes is totally PKD-dependent, whereas ET1-induced HDAC5 export depends more prominently on InsP(3) and CaMKII signaling. Thus α-adrenergic and ET-1 receptor signaling via PKD in adult myocytes feature dramatic differences in cellular localization and translocation in mediating hypertrophic signaling. This raises new opportunities for targeted therapeutic intervention into distinct limbs of this hypertrophic signaling pathway.
Journal of Biological Chemistry 07/2011; 286(38):33390-400. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.
Journal of Biological Chemistry 01/2011; 286(11):9120-6. · 4.65 Impact Factor