[Show abstract][Hide abstract] ABSTRACT: Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.
[Show abstract][Hide abstract] ABSTRACT: Cardiac troponin I (cTnI) is well known as a biomarker for the diagnosis of myocardial damage. However, because of its central role in the regulation of contraction and relaxation in heart muscle, cTnI may also be a potential target for the treatment of heart failure. Studies in rodent models of cardiac disease and human heart samples showed altered phosphorylation at various sites on cTnI (i.e. site-specific phosphorylation). This is caused by altered expression and/or activity of kinases and phosphatases during heart failure development. It is not known whether these (transient) alterations in cTnI phosphorylation are beneficial or detrimental. Knowledge of the effects of site-specific cTnI phosphorylation on cardiomyocyte contractility is therefore of utmost importance for the development of new therapeutic strategies in patients with heart failure. In this review we focus on the role of cTnI phosphorylation in the healthy heart upon activation of the beta-adrenergic receptor pathway (as occurs during increased stress and exercise) and as a modulator of the Frank-Starling mechanism. Moreover, we provide an overview of recent studies which aimed to reveal the functional consequences of changes in cTnI phosphorylation in cardiac disease.
[Show abstract][Hide abstract] ABSTRACT: Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca2+-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.
Archives of Biochemistry and Biophysics 07/2014; · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca(2+)-sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length-dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca(2+)-concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (Wt) troponin or troponin mutated at the PKC site Thr143, or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca(2+)-sensitivity compared to Wt without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca(2+)-sensitivity after Wt and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca(2+)-sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca(2+)-sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca(2+)-sensitivity.
AJP Heart and Circulatory Physiology 02/2014; · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: High myofilament Ca(2+)-sensitivity has been proposed as trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) based on in vitro and transgenic mice studies. However, myofilament Ca(2+)-sensitivity depends on protein phosphorylation and muscle length and at present data in human are scarce. Objective:To investigate if high myofilament Ca(2+)-sensitivity and perturbed length-dependent activation is characteristic for human HCM with mutations in thick and thin filament proteins. Methods and Results:: Cardiac samples from HCM patients harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM (HCMsmn) and non-failing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+)-sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA)-targets compared to donors. After exogenous PKA treatment, myofilamentCa(2+)-sensitivity was either similar (MYBPC3mut, TPM1mut, HCMsmn), higher (MYH7mut, TNNT2mut) or even significantly lower (TNNI3mut) compared to donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in HCMsmn and HCM with truncating MYBPC3 mutations, but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mu and TNNI3mut corrected length-dependent activation to donor values. Conclusions: High myofilament Ca(2+)-sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA-targets compared to donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via post-translational modifications other than PKA-hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM.
Circulation Research 03/2013; · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.
Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca(2+)] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m(2)) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m(2)). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca(2+)-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.
PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca(2+)-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca(2+)-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.
PLoS ONE 01/2013; 8(10):e74847. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: Cardiac myosin binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and thereby fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However recent work has shown that up to four phosphate groups are present in human cMyBP-C. Objective: To identify and characterize additional phosphorylation sites in human cMyBP-C. Methods and Results: Cardiac MyBP-C was semi-purified from human heart tissue. Tandem mass-spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine (Pro-Ala) rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared to PKA-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. Conclusions: GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the Pro-Ala rich region and increases kinetics of force development, suggesting a non-canonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.
Circulation Research 12/2012; · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein kinase A (PKA)-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin I (cTnI) results in a decrease in myofilament Ca(2+)-sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser23 and Ser24 of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated; cTnI-AD: Ser23 unphosphorylated and Ser24 phosphorylated; cTnI-DA: Ser23 phosphorylated and Ser24 unphosphorylated and AA: both sites unphosphorylated. Force development was measured at various [Ca(2+)] in permeabilized cardiomyocytes in which endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca(2+)-sensitivity (pCa(50)) was significantly lower in cTnI-DD (pCa(50)=5.39±0.01) compared to cTnI-AA (pCa(50)=5.50±0.01), cTnI-AD (pCa(50)=5.48±0.01) and cTnI-DA (pCa(50)=5.51±0.01) at ~70% cTn exchange. No effects were observed on rate of tension redevelopment (k(tr)). In cardiomyocytes from idiopathic dilated cardiomyopathy tissue, a linear decline in pCa(50) with cTnI-DD content was observed, saturating at ~55% bisphosphorylation. Our data suggest that in human myocardium phosphorylation of both PKA-sites on cTnI is required to reduce myofilament Ca(2+)-sensitivity, which is maximal at approximately 55% bisphosphorylated cTnI. Implications for in vivo cardiac function in health and disease will be discussed.
AJP Heart and Circulatory Physiology 11/2012; · 4.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS.
FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry.
CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1β and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55.
Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.
PLoS ONE 05/2012; 7(5):e35606. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation-contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50=0.05±0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50=0.10±0.03) and dilated (DCM, ΔpCa50=0.06±0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.
Journal of Muscle Research and Cell Motility 09/2011; 32(3):221-33. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background and objectivesCD55 (decay-accelerating factor) is a complement-regulatory protein, which is highly expressed by fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leucocytes. We recently showed that lack of either CD55 or CD97 ameliorates disease in murine collagen-induced and K/BxN serum transfer models of arthritis.1 Little is known regarding the regulation of CD55 expression in FLS. We therefore investigated the effect of toll-like receptors ligation and pro-inflammatory cytokines on CD55 expression.Materials and methodsSynovial fibroblasts, obtained from biopsy samples of arthritis patients, were cultured and stimulated with cytokines (TNF, IFNγ, IL-1β, IL-6, IFNα) or TLR ligands (LTA, poly (I:C), LPS, imiquimod, CpG). Expression of CD55 was measured by flow cytometry using domain-specific monoclonal antibodies and recombinant CD97-loaded fluorescent beads. Chloroquine was used to inhibit TLR3 activity. Upregulation and functionality of dsRNA sensors in response to poly (I:C) or 5'-triphosphate RNA was analysed by PCR. Apoptosis was measured by PI/annexinV staining and was blocked with the pan-caspase inhibitor Q-VD-OPH.ResultsCultured synovial fibroblasts of patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis, and spondylarthritis express equal amount of CD55. Stimulation of RA-FLS with IL-1β (p=0.02) and poly (I:C) (p=0.001) induced a significant upregulation of CD55. Engagement of TLR3 by the dsRNA analog poly (I:C) was confirmed using chloroquine, an inhibitor of endosomal acidification that impairs TLR3 signaling. Synovial fibroblasts also expressed the cytoplasmic dsRNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Stimulation of these receptors with either poly (I:C) or 5'-triphosphate RNA induced CD55 expression, but, in case of MDA5, also induced significant cell death (p
Journal of Translational Medicine 02/2011; 70(2). · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes.
The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22(phox) and p47(phox), was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy.
NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22(phox), p47(phox) and nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine.
We for the first time show that NOX2, p22(phox) and p47(phox) are targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes.
Cellular Physiology and Biochemistry 01/2011; 27(5):471-8. · 3.55 Impact Factor