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Cardiac muscle cells are equipped with specialized biochemical machineries for the rapid generation of force and movement central to the work generated by the heart. During each heart beat cardiac muscle cells perceive and experience changes in length and load, which reflect one of the fundamental principles of physiology known as the Frank–Starling law of the heart. Cardiac muscle cells are unique mechanical stretch sensors that allow the heart to increase cardiac output, and adjust it to new physiological and pathological situations. In the present review we discuss the mechano-sensory role of the cytoskeletal proteins with respect to their tight interaction with the sarcolemma and extracellular matrix. The role of contractile thick and thin filament proteins, the elastic protein titin, and their anchorage at the Z-disc and M-band, with associated proteins are reviewed in physiologic and pathologic conditions leading to heart failure. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé

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 Ca(2+)-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 Ca(2+)-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca(2+)-sensitivity at physiological Ca(2+)-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM. Electronic supplementary material The online version of this article (doi:10.1007/s00395-014-0451-8) contains supplementary material, which is available to authorized users.

Aims Disease mechanisms regarding hypertrophic cardiomyopathy (HCM) are largely unknown and disease onset varies. Sarcomere mutations might induce energy depletion for which until now there is no direct evidence at sarcomere level in human HCM. This study investigated if mutations in genes encoding myosin-binding protein C (MYBPC3) and myosin heavy chain (MYH7) underlie changes in the energetic cost of contraction in the development of human HCM disease. Methods and results Energetic cost of contraction was studied in vitro by measurements of force development and ATPase activity in cardiac muscle strips from26 manifestHCMpatients (11 MYBPC3mut, 9MYH7mut, and 6 sarcomere mutation-negative,HCMsmn). In addition, in vivo, the ratio between external work (EW) and myocardial oxygen consumption (MVO2) to obtain myocardial external efficiency (MEE) was determined in 28 pre-hypertrophic mutation carriers (14 MYBPC3mut and 14 MYH7mut) and 14 healthy controls using [11C]-acetate positron emission tomography and cardiovascular magnetic resonance imaging.Tension cost (TC), i.e.ATPase activity during force development, was higher in MYBPC3mut andMYH7mut compared with HCMsmn at saturating [Ca 2++]. TC was also significantly higher in MYH7mut at submaximal, more physiological [Ca2++]. EW was significantly lower in both mutation carrier groups, while MVO2 did not differ. MEE was significantly lower in both mutation carrier groups compared with controls, showing the lowest efficiency in MYH7 mutation carriers. Conclusion We provide direct evidence that sarcomere mutations perturb the energetic cost of cardiac contraction. Gene-specific severity of cardiac abnormalities may underlie differences in disease onset and suggests that early initiation of metabolic treatment may be beneficial, in particular, in MYH7 mutation carriers. © The Author 2014.

Introduction: Rbm20 is a RNA-binding protein enriched in heart- and skeletal muscle. Mutations in Rbm20 have been described to cause heart failure. Furthermore, Rbm20 has been shown to regulate alternative splicing of titin (Ttn). This demonstrates that RBM20 has a major role in cardiac biology. However, it is unknown how Rbm20 expression changes in acquired heart disease. Therefore, we investigated expression of Rbm20 on mRNA and protein level and the degree of alternative splicing of Ttn in various forms of heart disease. Methods: We measured mRNA expression and protein levels of Rbm20 by western blotting and qPCR in transverse aorta constrictions (TAC) in mice and pigs, and in human heart disease including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), aortic stenosis (AoS) and pulmonary artery hypertension (PAH). We measured cardiac function in mice with echocardiography, and measured stress markers with qPCR. Furthermore, we analyzed Ttn splicing using qPCR and RT-PCR. Results: Real-time PCR and western blotting revealed that Rbm20 is selectively downregulated in mice after TAC and in patients with aortic stenosis. In contrast, we did not observe a difference in Rbm20 levels in patients with DCM, HCM and PAH. Furthermore, we measured functional parameters such as ejection fraction (EF), systolic left ventricular internal diameter (LVIDsys), and fractional shortening (FS), as well as ANF level in TAC mice, and correlated these to Rbm20 level. We found that the level of downregulation of Rbm20 is correlated with heart failure severity in mice after TAC. Also, we found that Ttn is differentially spliced after loss of Rbm20. Conclusions: Here we show for the first time that Rbm20 decreases in acquired forms of heart failure such as TAC in mice and pigs, and AoS in men. Furthermore, in experimental HF in mice, Rbm20 levels correlate to HF severity. Moreover, loss of Rbm20 leads to skipping of intron retention in Ttn in the region between exon 50-70. However, the biological effect of this event remains unclear. Given its major role in the heart, loss of Rbm20 may play an important role in the development of heart disease.

Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PICA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+](i) transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+](i) signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia.

Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in genes encoding sarcomeric proteins. One of the most frequent affected genes is MYBPC3, which encodes the thick filament protein cardiac myosin binding protein C. Despite the prevalence of HCM, disease pathology and clinical outcome of sarcomeric mutations are largely unknown. We hypothesized that microRNAs (miRNAs) could play a role in the disease process. To determine which miRNAs were changed in expression, miRNA arrays were performed on heart tissue from HCM patients with a MYBPC3 mutation (n = 6) and compared with hearts of non-failing donors (n = 6). 532 out of 664 analyzed miRNAs were expressed in at least one heart sample. 13 miRNAs were differentially expressed in HCM compared with donors (at p < 0.01, fold change >= 2). The genomic context of these differentially expressed miRNAs revealed that miR-204 (fold change 2.4 in HCM vs. donor) was located in an intron of the TRPM3 gene, encoding an aspecific cation channel involved in calcium entry. RT-PCR analysis revealed a trend towards TRPM3 upregulation in HCM compared with donor myocardium (fold change 23, p = 0.078). In silico identification of mRNA targets of differentially expressed miRNAs showed a large proportion of genes involved in cardiac hypertrophy and cardiac beta-adrenergic receptor signaling and we showed reduced phosphorylation of cardiac troponin I in the HCM myocardium when compared with donor. HCM patients with MYBPC3 mutations have a specific miRNA expression profile. Downstream mRNA targets reveal possible involvement in cardiac signaling pathways. (C) 2013 Elsevier Ltd. All rights reserved.

A novel homozygous mutation in the TNNT2 gene encoding cardiac troponin T (cTnT K280N) was identified in one HCM patient undergoing cardiac transplantation. mRNA and Mass Spectrometry analyses revealed expression of the mutant alleles without evidence of haploinsufficiency. Kinetics of contraction and relaxation of myofibrils from a frozen left ventricular sample of the K280N HCM patient were compared to those of “control” myofibrils (from donor hearts, from aortic stenosis patients, and from HCM patients negative for sarcomeric protein mutations). Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (

Background Recently, we observed reduced myofilament length-dependent activation in cardiomyocytes from a patient harboring a homozygous mutation (K280N) in the TNNT2 gene encoding cardiac troponin T (cTnT) compared to non-failing donors. Protein kinase A (PKA)-mediated phosphorylation of cardiac troponin I (cTnI) did not rescue impaired length-dependence. Moreover, cTnI phosphorylation did not differ between TNNT2mut and donor myocardium. In this study we present direct evidence that the K280N mutation in TNNT2 blunts length-dependent activation.Methods Force measurements were performed in single permeabilized cardiomyocytes isolated from the TNNT2mut heart at various [Ca2+] and sarcomere lengths of 1.8 and 2.2 μm, with and without PKA-pretreatment. To investigate if mutant cTnT underlies impaired length-dependent activation, the endogenous mutant troponin complex was partially exchanged with recombinant whole human wild-type troponin (Tnwt) complex. TNNT2mut sample was exchanged with 0.25, 0.5 and 1 mg/mL Tnwt complex, which in accordance with previous studies from our group is predicted to yield ∼40%, ∼50% and ∼70% of troponin exchange, respectively.Results The length-dependent increase in myofilament Ca2+-sensitivity (ΔEC50) was not significantly different between TNNT2mut, TNNT2∼40%, TNNT2∼50%, TNNT2∼70%, (ΔEC50=0.35±0.16, 0.23±0.06, −0.02 ±0.02 and 0.22±0.06 μmol/L, respectively), but was significantly reduced compared to donor (ΔEC50=0.77±0.06 μmol/L). PKA-pretreatment did not restore the reduced length-dependent activation of TNNT2mut and TNNT2∼40% (ΔEC50=0.26±0.19 and 0.32±0.15), but did recover the blunted length-dependent activation of TNNT2∼50% and TNNT2∼70% (ΔEC50=0.71±0.04 and 0.79±0.14) to control values (ΔEC50=0.82±0.09).Conclusions Length-dependent activation of myofilaments is corrected to donor values only when ∼50% of mutant cTnT is replaced by wild-type troponin and subsequent phosphorylation with PKA. Our data show that the TNNT2 mutation K280N underlies impaired length-dependent activation in a dose-dependent manner.

Cardiac troponin (cTn) is an important sarcomeric protein complex situated on the thin filament and is involved in the regulation of cardiac muscle contraction. This regulation is primarily controlled by Ca(2+) binding to troponin C and in addition fine-tuned by the posttranslational modification of cTnI and cTnT. The vast majority of cTnT modifications involve the phosphorylation by protein kinase C (PKC) or other kinases and the N-terminal cleavage by caspase and calpain. In vitro studies employing reconstituted detergent-skinned fiber bundles and cell culture generally show a detrimental effect of cTnT phosphorylation on muscle contraction, which is backed by some in vivo studies finding increased cTnT phosphorylation in heart failure, but contradicted by others. In addition, N-terminal cleavage of cTnT is thought to be another factor influencing cardiac contraction. Time-dependent degradation of cTnT has been observed in human serum upon myocardial infarction. These molecular changes might influence the immunoreactivity of cTnT in the clinical immunoassay and have consequences for the clinical interpretations of these measurements. No consensus has yet been reached on the occurrence and extent of these observations and their underlying processes are subject of intense scientific debate. This review will focus on discussing these modifications, their implications on physiology and disease and summarizes the complex interplays of different enzymes on the molecular forms of cTnT and their associated effects.

Familial hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Energy depletion of the heart is thought to initiate and promote HCM disease development. It has been hypothesized that HCM sarcomere gene mutations increase ATP utilization for myofilament contraction and thereby increase energy demand of the heart. Previous studies in single left ventricular myofibrils from myocardium harbouring the first identified causal HCM mutation R403Q in the gene encoding β-myosin heavy chain revealed increased rates of tension generation and relaxation in R403Q compared to myofibrils from healthy control myocardium. The altered kinetics observed in the mutant sample predicted a 3-fold increase in the energy cost of tension generation in R403Q sarcomeres. In the present study we investigated if tension cost was indeed increased in human myocardium harbouring the R403Q mutation.Maximal force generating capacity (Fmax) and ATP consumption (ATPmax) were simultaneously measured in Triton-permeabilized left ventricular muscle strips (n=8) from a patient carrying the R403Q mutation. Left ventricular muscle strips (n=17) from 5 HCM sarcomere mutation negative patients (HCMsmn) and strips (n=6) from 2 patients with secondary hypertrophy due to aortic stenosis (LVHao) served as a control groups. Economy of myofilament contraction is expressed as tension cost (TC), i.e. amount of ATP used during force development (ATPmax/Fmax). Both Fmax and ATPmax were significantly lower in R403Q compared to HCMsmn and LVHao. TC was significantly increased in R403Q (3.8±0.5 μmolL−1s−1/kNm−2) compared to HCMsmn and LVHao (1.6±0.1 and 1.7±0.1 μmolL−1s-1/kNm−2, respectively).Our data provide direct evidence that TC is increased in myocardium harbouring the MYH7 mutation R403Q, indicating that expression of R403Q in the heart impairs economy of myocardial contraction.Funding: Seventh Framework Program of the European Union “BIG-HEART,” grant agreement 241577

Rationale: High-myofilament Ca 2+ sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca 2+ sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. Objective: To investigate whether high myofilament Ca 2+ sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Methods and Results: Cardiac samples from patients with HCM harboring mutations in genes encoding thick ( MYH7 , MYBPC3 ) and thin ( TNNT2 , TNNI3 , TPM1 ) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca 2+ sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca 2+ sensitivity was similar ( MYBPC3 mut , TPM1 mut , sarcomere mutation-negative HCM), higher ( MYH7 mut , TNNT2 mut ), or even significantly lower ( TNNI3 mut ) compared with 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 sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2 mut and TNNI3 mut 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 with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.

Cardiac remodeling in response to a myocardial infarction or chronic pressure-overload is an independent risk factor for the development of angina pectoris and heart failure. In contrast, cardiac remodeling produced by regular physical exercise is associated with a decreased risk for coronary artery disease and heart failure. There is evidence that exercise training has a beneficial effect on disease progression and survival in patients with cardiac remodeling and dysfunction, but concern has also been expressed that exercise training may aggravate pathological remodeling and dysfunction. Here, we present studies from our laboratory on the effects of exercise training on pathological cardiac remodeling and dysfunction in mice. The results indicate that even in the presence of a large infarct, exercise training exerts beneficial effects on the heart. These effects were mimicked in part by eNOS overexpression and abrogated by eNOS deficiency, demonstrating the importance of nitric oxide signaling in mediating the cardiac effects of exercise. Exercise prior to a myocardial infarction was also cardioprotective. In contrast, exercise tended to aggravate pathological cardiac remodeling and dysfunction in the setting of pressure-overload produced by an aortic stenosis. These observations emphasize the critical importance of the underlying pathological stimulus for cardiac hypertrophy and remodeling, in determining the effects of exercise training. Future studies are needed to define the influence of exercise type, intensity, and duration in different models and severities of pathological cardiac remodeling. Together such studies will aid in optimizing the therapy of exercise training in the setting of cardiovascular disease.

Background— Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (F passive ) and cardiomyocyte hypertrophy. In experimental models, both reacted favorably to raised protein kinase G (PKG) activity. The present study assessed myocardial PKG activity, its downstream effects on cardiomyocyte F passive and cardiomyocyte diameter, and its upstream control by cyclic guanosine monophosphate (cGMP), nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF). Methods and Results— Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese ( P P passive was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Lower PKG activity in HFPEF than in aortic stenosis ( P P passive ( P P P passive in HFPEF was corrected by in vitro PKG administration. Conclusions— Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte F passive and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

International audience; RATIONALE: Patients with idiopathic pulmonary arterial hypertension (iPAH) often have a low cardiac output. To compensate, neurohormonal systems such as the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system are up-regulated, but this may have long-term negative effects on the progression of iPAH. OBJECTIVES: Assess systemic and pulmonary RAAS activity in patients with iPAH and determine the efficacy of chronic RAAS inhibition in experimental PAH. METHODS: We collected 79 blood samples from 58 patients with iPAH in the VU University Medical Center Amsterdam (between 2004 and 2010) to determine systemic RAAS activity. MEASUREMENTS AND MAIN RESULTS: We observed increased levels of renin, angiotensin (Ang)I, and AngII, which were associated with disease progression (P < 0.05) and mortality (P < 0.05). To determine pulmonary RAAS activity, lung specimens were obtained from patients with iPAH (during lung transplantation, n = 13) and control subjects (during lobectomy or pneumonectomy for cancer, n = 14). Local RAAS activity in pulmonary arteries of patients with iPAH was increased, demonstrated by elevated angiotensin-converting enzyme activity in pulmonary endothelial cells and increased AngII type 1 (AT(1)) receptor expression and signaling. In addition, local RAAS up-regulation was associated with increased pulmonary artery smooth muscle cell proliferation via enhanced AT(1) receptor signaling in patients with iPAH compared with control subjects. Finally, to determine the therapeutic potential of RAAS activity, we assessed the chronic effects of an AT(1) receptor antagonist (losartan) in the monocrotaline PAH rat model (60 mg/kg). Losartan delayed disease progression, decreased right ventricular afterload and pulmonary vascular remodeling, and restored right ventricular-arterial coupling in rats with PAH. CONCLUSIONS: Systemic and pulmonary RAAS activities are increased in patients with iPAH and are associated with increased pulmonary vascular remodeling. Chronic inhibition of RAAS by losartan is beneficial in experimental PAH.

Background— In pulmonary arterial hypertension (PH), sympathetic adrenergic activity is highly elevated. Sympathetic overactivity is a compensatory mechanism at first, but might be detrimental for cardiac function in the long run. We therefore investigated whether chronic low-dose treatment with bisoprolol (a cardioselective β-blocker) has beneficial effects on cardiac function in experimental PH. Methods and Results— PH was induced in rats by a single injection of monocrotaline (60 mg/kg). Pressure telemetry in PH rats revealed that 10 mg/kg bisoprolol was the lowest dose that blunted heart rate response during daily activity. Ten days after monocrotaline injection, echocardiography was performed and PH rats were randomized for bisoprolol treatment (oral gavage) or vehicle (n=7/group). At end of study (body mass loss >5%), echocardiography was repeated, with additional pressure-volume measurements and histomolecular analyses. Compared with control, right ventricular (RV) systolic pressure and arterial elastance (measure of vascular resistance) more than tripled in PH. Bisoprolol delayed time to right heart failure ( P P P Conclusions— In experimental PH, treatment with bisoprolol delays progression toward right heart failure, and partially preserves RV systolic and diastolic function. These promising results suggest a therapeutic role for β-blockers in PH that warrants further clinical investigation.

Protein kinase C (PKC) is a family of serine/threonine protein kinases, and alterations have been found in PKC isoform expression and localization in the failing heart. These alterations in PKC activation levels influence the PKC-mediated phosphorylation status of cellular target proteins involved in Ca2+-handling and sarcomeric contraction. The differences observed in the effects due to PKC-mediated phosphorylation may underlie part of the contractile dysfunction observed in the failing heart. It is therefore important to establish the beneficial and detrimental effects of this kinase in the healthy and failing heart. The function of PKC has been studied intensively; however, the complexity of the regulation of this kinase makes the interpretation of the different effects difficult. The main focus of this review is the (patho)physiological impact of phosphorylation of sarcomeric proteins, myosin light chain-2, troponin I and T, desmin, myosin binding protein-C, and titin by PKC.

Chronic pressure-overload and diabetes mellitus are two frequent disorders affecting the heart. We aimed to characterize myocardial structural and functional changes induced by both conditions. Pressure-overload was established in Wistar-han male rats by supra-renal aortic banding. Six-weeks later, diabetes was induced by streptozotocin (65 mg/kg,ip), resulting in four groups: SHAM, banding (BA), diabetic (DM) and diabetic-banding (DB). Six-weeks later, pressure–volume loops were obtained and left ventricular samples were collected to evaluate alterations in insulin signalling pathways, extracellular matrix as well as myofilament function and phosphorylation. Pressure-overload increased cardiomyocyte diameter (BA 22.0 ± 0.4 μm, SHAM 18.2 ± 0.3 μm) and myofilament maximal force (BA 25.7 ± 3.6 kN/m2, SHAM 18.6 ± 1.4 kN/m2), Ca2+ sensitivity (BA 5.56 ± 0.02, SHAM 5.50 ± 0.02) as well as MyBP-C, Akt and Erk phosphorylation, while decreasing rate of force redevelopment (K tr; BA 14.9 ± 1.1 s−1, SHAM 25.2 ± 1.5 s−1). At the extracellular matrix level, fibrosis (BA 10.8 ± 0.9%, SHAM 5.3 ± 0.6%), pro-MMP-2 and MMP-9 activities increased and, in vivo, relaxation was impaired (τ; BA 14.0 ± 0.9 ms, SHAM 12.9 ± 0.4 ms). Diabetes increased cardiomyocyte diameter, fibrosis (DM 21.4 ± 0.4 μm, 13.9 ± 1.8%, DB 20.6 ± 0.4 μm, 13.8 ± 0.8%, respectively), myofilament Ca2+sensitivity (DM 5.57 ± 0.02, DB 5.57 ± 0.01), advanced glycation end-product deposition (DM 4.9 ± 0.6 score/mm2, DB 5.1 ± 0.4 score/mm2, SHAM 2.1 ± 0.3 score/mm2), and apoptosis, while decreasing K tr (DM 13.5 ± 1.9 s−1, DB 15.2 ± 1.4 s−1), Akt phosphorylation and MMP-9/TIMP-1 and MMP-1/TIMP-1 ratios. Diabetic hearts were stiffer (higher end-diastolic-pressure: DM 7.0 ± 1.2 mmHg, DB 6.7 ± 0.7 mmHg, SHAM 5.3 ± 0.4 mmHg, steeper end-diastolic-pressure–volume relation: DM 0.59 ± 0.18, DB 0.83 ± 0.17, SHAM 0.41 ± 0.10), and hypo-contractile (decreased end-systolic-pressure-volume-relation). DB animals presented further pulmonary congestion (Lungs/body-weight: DB 5.23 ± 0.21 g/kg, SHAM 3.80 ± 0.14 g/kg) as this group combined overload-induced relaxation abnormalities and diabetes-induced stiffness. Diabetes mellitus and pressure overload led to distinct diastolic dysfunction phenotypes: while diabetes promoted myocardial stiffening, pressure overload impaired relaxation. The association of these damages accelerates the progression of diastolic heart failure progression in diabetic-banded animals.

Alpha-adrenergic receptor activated protein kinase Cα (PKCα) modifies cardiac contractile function and is implicated in heart failure. Aim of the study: To determine the specific effects of PKCα-mediated phosphorylation of cardiac troponin (cTn) on myofilament function in failing cardiomyocytes. Methods: Endogenous cTn in Triton-permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was partially exchanged (∼69%) with PKCα-treated recombinant human cTn(DD), in which the PKA-sites Ser23/24 on cTnI were mutated into aspartic acids (D). cTn(DD) was used to rule out in-vitro cross-phosphorylation of the PKA-sites by PKCα. A comparison was made between the effects of PKCα-treated and untreated cTn(DD). Isometric force was measured at various [Ca2+] to determine the maximum isometric force per cross-sectional area (Fmax) and the calcium sensitivity of force (pCa50). Results: Fmax after exchange with PKCα-treated cTn(DD) (17.1±1.9 kN/m2) was significantly reduced in comparison with untreated cTn(DD) (26.1±1.9 kN/m2). Subsequent incubation of the cardiomyocytes with activated PKCα in the PKCα-treated cTn(DD) group did not restore Fmax. Cardiomyocytes exchanged with PKCα-treated cTn(DD) were more sensitive to Ca2+ (pCa50=5.59±0.02) than cells exchanged with untreated cTn(DD) (pCa50=5.51±0.02). Surprisingly, subsequent PKCα-incubation of cardiomyocytes exchanged with PKCα-treated cTn(DD) caused a reduction in pCa50 to 5.45±0.02. Western blot analysis of PKCα-treated cTn showed phosphorylation of Ser42 and Thr143 on cTnI. Mass spectrometry revealed two novel phosphorylation sites: Ser198 on cTnI and Ser179 on cTnT. Conclusions: Specific in vitro PKCα-mediated phosphorylation of cTn increases Ca2+-sensitivity and decreases maximal force. Subsequent PKCα-mediated phosphorylation of the myofilaments in situ decreases Ca2+-sensitivity, without affecting Fmax. These results demonstrate the dual, site-specific effects of PKCα-mediated phosphorylation in human myocardium.

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.

Aim: Transmural differences in sarcomeric protein composition and function across the left ventricular (LV) wall have been reported. We studied in pigs sarcomeric function and protein phosphorylation in subepicardial (EPI) and subendocardial (ENDO) layers of remote LV myocardium after myocardial infarction (MI), induced by left circumflex coronary artery ligation. Methods: EPI and ENDO samples were taken 3 weeks after sham surgery (n = 12) or induction of MI (n = 12) at baseline (BL) and during β-adrenergic receptor (βAR) stimulation with dobutamine. Isometric force was measured in single cardiomyocytes at various [Ca2+] and 2.2 μm sarcomere length. Results: In sham hearts, no significant transmural differences were observed in myofilament function or protein phosphorylation. Myofilament Ca2+-sensitivity was significantly higher in both EPI and ENDO of MI compared to sham hearts. Maximal force was significantly reduced in MI compared to sham, but solely in ENDO cells. A higher passive force was observed in MI hearts, but only in EPI cells. The proportion of stiff N2B isoform was higher in EPI than in ENDO in both sham and MI hearts, and a trend toward increased N2B-proportion appeared in MI EPI, but not MI Endo. Analysis of myofilament protein phosphorylation did not reveal significant transmural differences in phosphorylation of myosin binding protein C, desmin, troponin T, troponin I (cTnI), and myosin light chain 2 (MLC-2) both at BL and during βAR stimulation with dobutamine infusion. A significant increase in MLC-2 phosphorylation was observed during dobutamine only in sham. In addition, the increase in cTnI phosphorylation upon dobutamine was twofold lower in MI than in sham. Conclusion: Myofilament dysfunction is present in both EPI and ENDO in post-MI remodeled myocardium, but shows a high degree of qualitative heterogeneity across the LV wall. These heterogeneous transmural changes in sarcomeric properties likely contribute differently to systolic vs. diastolic global LV dysfunction after MI.

Since the discovery of the striation pattern of muscle by Antoni van Leeuwenhoek in the Netherlands, research in muscle physiology has provided an immensely detailed picture of skeletal and cardiac muscle structure and function. Particularly in recent years, molecular techniques, gene expression and imaging techniques have provided a wealth of information about the basic function of muscle and its dysfunction in chronic diseases and congenital myopathies. In this special issue, based on the work presented at the 42nd European Muscle Conference (2013) held in Amsterdam (The Netherlands), an overview of the state-of-the-art in skeletal and cardiac muscle research is presented. Since basic and clinical muscle physiology are two sides of the same coin, combining both perspectives is key to fully profit from advances made in both research fields. The force-generating capacity of muscle is largely determined by the quantity and quality of the contractile apparatus. As striated muscle is generally considered as post-mitotic tissue, hypertrophy relies on a net positive balance between the rate of protein synthesis and degradation. In skeletal muscle including the diaphragm, muscle stem cells, residing between the myofiber basal lamina and sarcolemma (satellite cell, MuSC) have the ability to proliferate, differentiate and fuse with the host myofiber. Whether the pool of myonuclei (i.e. amount of DNA) within a myofiber is limiting protein synthesis, and as such myofibrillar hypertrophy, is still subject of debate. In this issue, Blaauw and Reggiani (2014) discuss the current theories on the role of MuSC in muscle hypertrophy. Is the activation of MuSC and accretion of myonuclei within the myofiber requisite for its hypertrophy? Recent reports suggest that MuSC activation may not be required. Blaauw and Reggiani (2014) argue that local inflammatory responses (elevated cytokines) may attract and activate other muscle resident stem cells, such as mesangioblasts which can differentiate into muscle precursors and possibly contribute to the pool of myonuclei within the myofiber. A key alternative mechanism to increase the rate of protein synthesis is the activation of the mammalian target of rapamycin (mTOR), which is activated by mechanical loading, leucine or growth factors in muscle. Activation of mTOR is generally regarded as a prime stimulator of mRNA translation. In this issue of the journal, Jacobs et al. (2013) reviewed the current literature on how mechanical and biochemical stimuli activate mTOR signaling. Recent evidence from Hornberger’s laboratory suggests that the late endosomal lysosome could serve as a prime regulatory center for controlling the mechanical loading induced activation of mTOR in skeletal muscle. These novel insights suggest that the late endosome/lysosome system plays a critical role in protein synthesis, protein trafficking as well as protein degradation. In addition to muscle fiber size, also muscle quality matters. Both skeletal and cardiac muscle function can be negatively affected by lifestyle and ageing, particularly through an increased oxidative stress. Canton et al. (2014) discuss the role of secondary mechanisms, particularly mediated by oxidative stress, in muscular dystrophies, C. A. C. Ottenheijm R. C. I. Wust J. van der Velden (&) Department of Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands e-mail: j.vandervelden@vumc.nl

BackgroundHypertrophic cardiomyopathy (HCM) is an inherited cardiac disorder with a prevalence of 1:500. In ∼65% of all HCM patients the causative mutation is identified. HCM patients that are sarcomere-mutation negative tend to have a less severe phenotype. Mutations in the MYBPC3 and MYH7 genes encoding cardiac myosin-binding protein C (cMyMP-C) and β-myosin heavy chain (MyHC) represent >80% of all genotyped HCM cases. HCM is characterized by asymmetric hypertrophy of the left ventricle and diastolic dysfunction. In the present study we investigated if passive stiffness of the sarcomeres may underlie diastolic dysfunction.MethodsIn-vitro passive tension measurements were done at sarcomere lengths of 1.8 to 2.2μm in cardiomyocytes from 10 sarcomere mutation-negative patients(SMN: 5 male, 5 female), 17 patients carrying a MYBPC3 mutation(MYBPC3: 10 male, 7 female), and 10 patients carrying a MYH7 mutation (MYH7: 5 male, 5 female). Tissue was obtained during myectomy surgery from the interventricular septum. Cardiomyocytes were mechanically isolated and Triton-permeabilized.ResultsPassive tension over the entire range of sarcomere lengths did not differ between sarcomere-mutation positive and mutation-negative male HCM patients. Passive tension in myocytes from sarcomere mutation-positive women was significantly higher compared to female mutation-negative HCM patients. Female MYH7 cardiomyocytes showed a higher sarcomere stiffness compared to male MYH7.ConclusionOur measurements suggest that high sarcomere passive stiffness may contribute to diastolic dysfunction in female HCM patients harboring a mutation in genes encoding thick filament proteins.

Introduction: Hypertrophic cardiomyopathy(HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Decreased force development was found in human HCM due to mutations in genes encoding myosin binding protein-C(MYBPC3), myosin heavy chain(MYH7) and in sarcomere mutation-negative HCM(HCMsmn). This is partially caused by the mutation (especially MYH7 mutations), increased cell size and reduced myofibrillar density. However, 10% force reduction could not be explained by the mutation or cellular remodeling. In end-stage human heart failure increased actin-carbonylation, induced by oxidative stress, negatively correlated with myocardial function. Therefore, we investigated whether actin is carbonylated in human HCM.Methods and results: Left ventricular tissue of human HCMsmn, MYBPC3mut and MYH7mut patients was obtained. Non-failing donors were used as control. Actin-carbonylation was analyzed using Oxyblot. Carbonyl groups of myofilament proteins were derivatized by 2,4-dinitrophenyl-hydrazine(DNPH). Protein lysates were processed via SDS-page, western blot and chemiluminescence using appropriate antibodies. Actin-carbonylation was higher in HCM patients compared to donors. It was highest in MYH7mut(Figure).Conclusion: Increased actin-carbonylation could partially underlie reduced force development in human HCM. Increased oxidative stress may contribute to impaired contractile function during disease development in sarcomere mutation-positive and mutation-negative HCM patients.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Hypertrophic cardiomyopathy (HCM) is a familial disorder characterized by left ventricular hypertrophy in the absence of other cardiac or systemic disease likely to cause this hypertrophy. HCM is considered a disease of the sarcomere as most causal mutations are identified in genes encoding sarcomeric proteins, although several other disorders have also been linked to the HCM phenotype. The clinical course of HCM is characterized by a large inter- and intrafamilial variability, ranging from severe symptomatic HCM to asymptomatic individuals. The general picture emerges that the underlying pathophysiology of HCM is complex and still scarcely clarified. Recent findings indicated that both functional and morphological (macroscopic and microscopic) changes of the HCM muscle are present before the occurrence of HCM phenotype. This review aims to provide an overview of the myocardial alterations that occur during the gradual process of wall thickening in HCM on a myofilament level, as well as the structural and functional abnormalities that can be observed in genetically affected individuals prior to the development of HCM with state of the art imaging techniques, such as tissue Doppler echocardiography and cardiovascular magnetic resonance imaging. Additionally, present and future therapeutic options will be briefly discussed.

Background: Clinical data on myocardial function in HCM mutation carriers (carriers) is sparse but suggests that subtle functional abnormalities can be measured with tissue Doppler imaging before the development of overt hypertrophy. We aimed to confirm the presence of functional abnormalities using cardiovascular magnetic resonance (CMR), and to investigate if sensitive functional assessment could be employed to identify carriers. Results: 28 carriers and 28 controls were studied. Global left atrial (LA) and left ventricular (LV) dimensions, segmental peak systolic circumferential strain (SCS) and peak diastolic circumferential strain rate (DCSR), as well as the presence of late Gadolinium enhancement (LGE) were determined with CMR. Septal and lateral myocardial velocities were measured with echocardiographic tissue Doppler imaging. lv mass and volumes were comparable between groups. Maximal septal to lateral wall thickness ratio (SL ratio) was larger in carriers than in controls (1.3 ± 0.2 versus 1.1 ± 0.1, p 1.2 and a peak DCSR 105%.s -1 , yielding a negative predictive value of 90%. With multivariate analysis, HCM mutation carriership was an independent determinant of reduced peak SCS and peak DCSR. Conclusions: HCM mutation carriership is an independent determinant of reduced peak SCS and peak DCSR when LV wall thickness is within normal limits, and is associated with increased LA volumes and SL ratio. Using SL ratio and peak DCSR has a high accuracy to identify carriers. However, since carriers also display structural abnormalities and focal LGE, we advocate to also evaluate morphology and presence of LGE when screening for carriers. Background Hypertrophic cardiomyopathy (HCM) is a relatively common cardiomyopathy with an estimated prevalence of 1:500 in the general population [1]. The clinical course has a large inter- and intrafamilial heterogeneity, ranging from mild symptoms of heart failure late in life to the onset of sudden cardiac death at young age. Over 430 mutations in mainly sarcomeric genes have been identified to cause HCM. It has been suggested that the hypertrophy in HCM is a compensatory mechanism for mutant-induced myocardial dysfunction [2-4]. This hypothesis is supported by experimental data demonstrating that sarcomeric dysfunction precedes hypertrophy in HCM animal models [5-7]. Limited clinical data is available on regional myocardial function in human HCM mutation carriers (carriers) when wall thickness is still within normal limits. Several studies using tissue Doppler imaging with

Activation of the beta-adrenergic receptor (beta AR) pathway is the main mechanism of the heart to increase cardiac output via protein kinase A (PKA)-mediated phosphorylation of cellular target proteins, and perturbations therein may contribute to cardiac dysfunction in heart failure. In the present study a comprehensive analysis was made of mediators of the beta AR pathway, myofilament properties and cardiac structure in patients with idiopathic (IDCM; n = 13) and ischemic (ISHD; n = 10) cardiomyopathy in comparison to non-failing hearts (donor; n = 10) for the following parameters: beta AR density, G-coupled receptor kinases 2 and 5, stimulatory and inhibitory G-proteins, phosphorylation of myofilament targets of PKA, protein phosphatase 1, phospholamban, SERCA2a and single myocyte contractility. All parameters exhibited the expected alterations of heart failure, but for most of them the extent of alteration was greater in IDCM than in ISHD. Histological analysis also revealed higher collagen in IDCM compared to ISHD. Alterations in the beta AR pathway are more pronounced in IDCM than in ISHD and may reflect sequential changes in cellular protein composition and function. Our data indicate that cellular dysfunction is more severe in IDCM than in ISHD

We discuss a current controversy regarding the relative role of phosphorylation sites on cardiac troponin I (cTnI) (Fig. 1) in physiological and patho-physiological cardiac function. Studies with mouse models and in vitro studies indicate that multi-site phosphorylations are involved in both control of maximum tension and sarcomeric responsiveness to Ca2+. Thus one hypothesis is that cardiac function reflects a balance of cTnI phosphorylations and a tilt in this balance may be maladaptive in acquired and genetic disorders of the heart. Studies on human heart samples taken mainly at end-stage heart failure, and in depth proteomic analysis of human and rat heart samples demonstrate that Ser23/Ser24 are the major and perhaps the only sites likely to be relevant to control cardiac function. Thus functional significance of Ser23/Ser24 phosphorylation is taken as fact, whereas the function of some other sites is treated as fancy. Maybe the extremes will meet: in any case we both agree that further work needs to be carried out with relatively large mammals and with determination of the time course of changes in phosphorylation to identify transient modifications that may be relevant at a beat-to-beat basis. Moreover, we agree that the changes and effects of cTnI phosphorylation need to be fully integrated into the effects of other phosphorylations in the cardiac myocyte.

Background— The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart. Methods and Results— TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas Conclusion— TSP-2 expression in the heart protects against age-dependent dilated cardiomyopathy.

In coronary artery disease, areas subtended by a severely stenotic artery or by collateral vessels can develop chronic contractile dysfunction in the absence of necrosis. This dysfunction is thought to be adaptive to the reduced flow reserve and can be reversible upon revascularization, hence the term 'hibernating' myocardium. In the present report, the underlying cellular mechanisms were studied in a pig with severe stenosis in the left circumflex coronary artery, resulting in hibernation in the distal myocardium.After six weeks, single cardiomyocytes were isolated enzymatically from the hibernating region (HIB) and their properties compared with those of cardiomyocytes from the same area in matched control pigs (CTRL). The amplitude of cell shortening during field stimulation (1 Hz) was reduced in HIB versus CTRL; the accompanying Ca(2+) transients were only modestly reduced. In whole cell recording, prolongation of action potential was observed in HIB. When this difference was excluded by using depolarizing steps of fixed duration in both HIB and CTRL, the Ca(2+) transients in HIB myocytes were reduced compared with CTRL. There was also a decrease in peak L-type Ca(2+) current in HIB. In intact cells, increasing the available Ca(2+) for contraction did not correct the contractile deficit in HIB, suggesting alterations to the myofilaments.In conclusion, in this pig model for hibernating myocardium, intrinsic remodelling of the myocytes with a unique profile of excitation-contraction coupling is demonstrated. Along with the changes observed in myocytes from the border zone of a myocardial infarction, or in the remote area, this specific phenotype adds to the diversity and complexity of the remodelling processes in ischemic cardiomyopathy.

Background In vitro data suggest that changes in myocardial substrate metabolism may contribute to impaired myocardial function in diabetic cardiomyopathy (DCM). The purpose of the present study was to study in a rat model of early DCM, in vivo changes in myocardial substrate metabolism and their association with myocardial function. Methods Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats underwent echocardiography followed by [11C]palmitate positron emission tomography (PET) under fasting, and [18F]-2-fluoro-2-deoxy-D-glucose PET under hyperinsulinaemic euglycaemic clamp conditions. Isolated cardiomyocytes were used to determine isometric force development. Results PET data showed a 66% decrease in insulin-mediated myocardial glucose utilisation and a 41% increase in fatty acid (FA) oxidation in ZDF vs. ZL rats (both p < 0.05). Echocardiography showed diastolic and systolic dysfunction in ZDF vs. ZL rats, which was paralleled by a significantly decreased maximal force (68%) and maximal rate of force redevelopment (69%) of single cardiomyocytes. Myocardial functional changes were significantly associated with whole-body insulin sensitivity and decreased myocardial glucose utilisation. ZDF hearts showed a 68% decrease in glucose transporter-4 mRNA expression (p < 0.05), a 22% decrease in glucose transporter-4 protein expression (p = 0.10), unchanged levels of pyruvate dehydrogenase kinase-4 protein expression, a 57% decreased phosphorylation of AMP activated protein kinase α1/2 (p < 0.05) and a 2.4-fold increased abundance of the FA transporter CD36 to the sarcolemma (p < 0.01) vs. ZL hearts, which are compatible with changes in substrate metabolism. In ZDF vs. ZL hearts a 2.4-fold reduced insulin-mediated phosphorylation of Akt was found (p < 0.05). Conclusion Using PET and echocardiography, we found increases in myocardial FA oxidation with a concomitant decrease of insulin-mediated myocardial glucose utilisation in early DCM. In addition, the latter was associated with impaired myocardial function. These in vivo data expand previous in vitro findings showing that early alterations in myocardial substrate metabolism contribute to myocardial dysfunction.

High diastolic stiffness of failing myocardium results from interstitial fibrosis and elevated resting tension ( F passive ) of cardiomyocytes. A shift in titin isoform expression from N2BA to N2B isoform, lower overall phosphorylation of titin, and a shift in titin phosphorylation from N2B to N2BA isoform can raise F passive of cardiomyocytes. In left ventricular biopsies of heart failure (HF) patients, aortic stenosis (AS) patients, and controls (CON), we therefore related F passive of isolated cardiomyocytes to expression of titin isoforms and to phosphorylation of titin and titin isoforms. Biopsies were procured by transvascular technique (44 HF, 3 CON), perioperatively (25 AS, 4 CON), or from explanted hearts (4 HF, 8 CON). None had coronary artery disease. Isolated, permeabilized cardiomyocytes were stretched to 2.2-μm sarcomere length to measure F passive . Expression and phosphorylation of titin isoforms were analyzed using gel electrophoresis with ProQ Diamond and SYPRO Ruby stains and reported as ratio of titin (N2BA/N2B) or of phosphorylated titin (P-N2BA/P-N2B) isoforms. F passive was higher in HF (6.1±0.4 kN/m 2 ) than in CON (2.3±0.3 kN/m 2 ; P 2 ; P P P F passive of human HF cardiomyocytes.

Left ventricular (LV) myocardial structure and function differ in heart failure (HF) with normal (N) and reduced (R) LV ejection fraction (EF). This difference could underlie an unequal outcome of trials with beta-blockers in heart failure with normal LVEF (HFNEF) and heart failure with reduced LVEF (HFREF) with mixed results observed in HFNEF and positive results in HFREF. To investigate whether beta-blockers have distinct myocardial effects in HFNEF and HFREF, myocardial structure, cardiomyocyte function, and myocardial protein composition were compared in HFNEF and HFREF patients without or with beta-blockers.Patients, free of coronary artery disease, were divided into beta-(HFNEF) (n = 16), beta+(HFNEF) (n = 16), beta-(HFREF) (n = 17), and beta+(HFREF) (n = 22) groups. Using LV endomyocardial biopsies, we assessed collagen volume fraction (CVF) and cardiomyocyte diameter (MyD) by histomorphometry, phosphorylation of myofilamentary proteins by ProQ-Diamond phosphostained 1D-gels, and expression of beta-adrenergic signalling and calcium handling proteins by western immunoblotting. Cardiomyocytes were also isolated from the biopsies to measure active force (F(active)), resting force (F(passive)), and calcium sensitivity (pCa(50)). Myocardial effects of beta-blocker therapy were either shared by HFNEF and HFREF, unique to HFNEF or unique to HFREF. Higher F(active), higher pCa(50), lower phosphorylation of troponin I and myosin-binding protein C, and lower beta(2) adrenergic receptor expression were shared. Higher F(passive), lower CVF, lower MyD, and lower expression of stimulatory G protein were unique to HFNEF and lower expression of inhibitory G protein was unique to HFREF.Myocardial effects unique to either HFNEF or HFREF could contribute to the dissimilar outcome of beta-blocker therapy in both HF phenotypes.

Cardiomyocyte contraction is regulated by phosphorylation of sarcomeric proteins. Throughout the heart regional and transmural differences may exist in protein phosphorylation. In addition, phosphorylation of sarcomeric proteins is altered in cardiac disease. Heterogeneity in protein phosphorylation may be larger in hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) as it may be caused by multiple mutations in genes encoding different sarcomeric proteins. Moreover, HCM is characterized by asymmetric remodelling of the heart. In the present study we assessed if local differences in sarcomeric protein phosphorylation are more evident in primary HCM or DCM than in non-failing donors. Thereto, phosphorylation of the two main target proteins of the beta-adrenergic receptor pathway, troponin I (cTnI) and myosin binding protein C (cMyBP-C) was analysed in different parts in the free left ventricular wall of end–stage failing HCM and DCM patients and donors obtained during transplant surgery. Intra-patient variability in protein phosphorylation within tissue samples of approximately 2 g wet weight was comparable between donor, HCM and DCM samples and could partly be attributed to the precision of the technique. Thus, our data indicate that within the precision of the measurements small, biopsy-sized cardiac tissue samples are representative for the region of the free left ventricular wall from which they were obtained.

The present study was designed to investigate if antibodies against beta-adrenergic receptors (betaARs) can be used to determine expression of betaAR in human myocardium. Western blotting was performed to investigate the specificity of antibodies directed against beta(1)AR and beta(2)AR in human left ventricular tissue. A comparison was made between cardiac tissue from patients with idiopathic dilated cardiomyopathy and ischemic heart disease and nonfailing donors. The antibodies directed against beta(1)AR and beta(2)AR recognized several protein bands at different molecular weights. Moreover, both antibodies also recognized multiple proteins in Chinese hamster ovary cells expressing beta(1)AR, beta(2)AR, and even beta(3)AR. betaAR antibodies are not specific and are not suited to study expression of betaAR in human myocardium.

Concentric left ventricular (LV) remodeling and cardiomyocyte (CM) hypertrophy characterize heart failure with normal ejection fraction (HFNEF) whereas eccentric LV remodeling and low myofilamentary density characterize heart failure with reduced ejection fraction (HFREF). In rodents, low myocardial protein kinase G (PKG) activity has been implicated in the development of pathological CM hypertrophy induced by LV pressure-overload. The present study therefore compared LV remodeling, CM hypertrophy and myocardial PKG activity in patients (pts) with HFNEF (n=36) and HFREF (n=43). All pts had been admitted to hospital for worsening heart failure (NYHA 3–4) and were free of coronary artery disease. HFNEF pts had a LVEF > 50% and a LV end-diastolic pressure > 16 mmHg. HFREF pts had a LVEF ). As evident from (P)-VASP/VASP ratio, PKG activity was lower in HFNEF than in HFREF (Table ). Conclusion: HFNEF patients have lower myocardial PKG activity than HFREF patients. This low myocardial PKG activity could contribute to CM hypertrophy and concentric LV remodeling observed in HFNEF. Raising myocardial PKG activity by phosphodiesterase 5A inhibition could be useful to limit CM hypertrophy and concentric LV remodeling in HFNEF.

Raised diastolic left ventricular (LV) stiffness importantly contributes to heart failure (HF) in diabetes mellitus (DM) and results from myocardial deposition of advanced glycation endproducts (AGEs), interstitial fibrosis, cardiomyocyte hypertrophy and elevated diastolic resting tension (F passive ) of cardiomyocytes. The relative importance of these mechanisms for diastolic LV stiffness differs between HF with reduced LV ejection fraction (EF) (HFrEF) and HF with normal LV EF (HFnEF) as evident from more fibrosis in HFrEF and more hypertrophy and higher F passive in HFnEF. The present study therefore compared myocardial effects of β-blocker therapy in DM patients suffering of HFrEF (n=11) or of HFnEF (n=15). All patients had DM type 2 and were free of significant coronary artery disease. LV endomyocardial biopsies were used to assess AGEs deposition by carboxymethyllysine immunohistochemistry and collagen volume fraction (CVF) or cardiomyocyte diameter (MyD) by histomorphometry. Cardiomyocytes were also isolated from the biopsies, attached to a force transducer and stretched to 2.2 μm to measure F passive . AGEs deposition was expressed relative to an age and sex matched control group without DM. In both HFrEF and HFnEF groups, β-blocker therapy reduced DM-related myocardial AGEs deposition. In both HFrEF and HFnEF groups, β-blocker therapy also reduced MyD. In HFnEF, β-blocker therapy lowered CVF but raised F passive of cardiomyocytes. Conclusion: Myocardial effects of β-blocker therapy were uniformly beneficial for diastolic LV stiffness in DM patients suffering of HFrEF but not in DM patients suffering of HFnEF. In DM, HF phenotype could therefore affect outcome of β-blocker therapy.

Cardiomyocytes (CM) of diastolic heart failure (DHF) patients (pts) are hypertrophied and have high diastolic resting tension (RT), which elevates left ventricular (LV) diastolic stiffness. In rodent LV pressure overload models, development of pathological CM hypertrophy has been related to low myocardial protein kinase G (PKG) activity. Myocardial PKG activity and in-vitro effect of PKG on RT of isolated CM were therefore determined using LV endomyocardial biopsies procured from DHF pts. All DHF pts (n=36) had been admitted to hospital for worsening heart failure (NYHA 3– 4), were free of coronary artery disease, had a LV ejection fraction > 50%, a LV end-diastolic volume index < 97 ml/m 2 , a LV end-diastolic pressure > 16 mmHg and no histological evidence in the biopsies of myocardial infiltration or inflammation. Myocardial PKG activity was assessed by immunohistochemistry using antibodies against the specific PKG substrate vasodilator stimulated phosphoprotein (VASP) and against phosphorylated VASP ((P)-VASP). Myocardial PKG activity corresponded to the ratio of (P)-VASP/VASP. CM were isolated from the biopsies, treated with Triton X-100 to remove all membranes, attached to a force transducer and stretched to 2.2 μm to measure RT. In DHF pts, (P)-VASP/VASP ratio (0.67±0.15) was lower than in a control group (n=22) (0.98±0.19; p2 ) than CM of a control group (4.8±0.2 kN/m 2 ; p2 ; p2 ). DHF patients had low myocardial PKG activity. This low myocardial PKG activity could have elevated RT of CM as in-vitro administration of PKG corrected the high RT. Absence of an additional in-vitro effect of PKA after PKG suggested that high RT results from hypophosphorylation of myofilamentary proteins, whose phosphorylation sites are targets for both PKA and PKG. In pts with DHF, phosphodiesterase 5A inhibition could raise myocardial PKG activity and thereby correct their high CM RT, which is an important determinant of their high diastolic LV stiffness.

The authors appreciated the thoughtful comment of Connelly et al on their study,1 which indeed did not imply that fibrosis was no determinant of diastolic left ventricular (LV) dysfunction in patients with heart failure and normal LV ejection fraction (HFNEF). A previous study from our group identified both collagen volume fraction (CVF) and cardiomyocyte resting tension to be …

Sarcomeric dysfunction plays a central role in reduced cardiac pump function in heart failure. This review focuses on the alterations in sarcomeric proteins in diseased myocardium that range from altered isoform expression to post-translational protein changes such as proteolysis and phosphorylation. Recent studies in animal models of heart failure and human failing myocardium converge and indicate that sarcomeric dysfunction, including altered maximum force development, Ca(2+) sensitivity, and increased passive stiffness, largely originates from altered protein phosphorylation, caused by neurohumoral-induced alterations in the kinase-phosphatase balance inside the cardiomyocytes. Novel therapies, which specifically target phosphorylation sites within sarcomeric proteins or the kinases and phosphatases involved, might improve cardiac function in heart failure.

Early detection of myocardial dysfunction in Fabry disease (FD) cardiomyopathy suggests the contribution of myofilament structural alterations. Six males with untreated FD cardiomyopathy submitted to cardiac studies, including tissue Doppler imaging and left ventricular endomyocardial biopsy. Active and resting tensions before and after treatment with protein kinase A (PKA) were determined in isolated Triton-permeabilized cardiomyocytes. Cardiomyocyte cross-sectional area, glycosphingolipid vacuole area, myofibrillolysis, and extent of fibrosis were also determined. Biopsies of mitral stenosis in patients with normal left ventricles served as controls. Active tension was four times lower in FD cardiomyocytes and correlated with extent of myofibriflolysis. Resting tension was six times higher in FD cardiomyocytes than in controls. PKA treatment decreased resting tension but did not affect active force. Protein analysis revealed troponin I and desmin degradation products. FD cardiomyocytes were significantly larger and filled with glycosphingolipids. Fibrosis was mildly increased compared with controls. Tissue Doppler imaging lengthening and shortening velocities were reduced in FD cardiomyocytes; compared with controls, correlating with resting and active tensions, respectively, but not with cardiomyocyte area, percentage of glycosphingolipids, or extent of fibrosis. in conclusion, myofilament degradation and dysfunction contribute to FD cardiomyopathy. Partial reversal of high resting tension after pharmacological PKA treatment of cardiomyocytes suggests potential benefits from enzyme replacement therapy and/or energy-releasing agents.

This study sought to characterize the relation between the oxidation of protein sulfhydryl (SH) groups and Ca2+-activated force production in the human myocardium. Triton-permeabilized left ventricular cardiomyocytes from donor hearts were exposed to an oxidative (2,2'-dithiodipyridine, DTDP) agent in vitro, and the changes in isometric force, its Ca2+ sensitivity, the cross-bridge-sensitive rate constant of force redevelopment at saturating [Ca2+] (k(tr,max)), and protein SH oxidation were monitored. DTDP (0.1-10 mM for 2 min) oxidized the myocardial proteins and diminished the Ca2+-activated force with different concentration dependences (EC(50,SH) = 0.17 +/- 0.02 mM and EC(50,force) = 2.46 +/- 0.22 mM; mean +/- SEM). The application of 2.5 mM DTDP decreased the maximal Ca2+-activated force (to 64%), its Ca2+ sensitivity (DeltapCa(50) = 0.22 +/- 0.02), and the steepness of the Ca2+-force relation (n(Hill), from 2.01 +/- 0.08 to 1.76 +/- 0.08). These changes were paralleled by reductions in the free SH content of the proteins (to 15%) and in k(tr,max) (to 75%). SH-specific labeling identified SH oxidation of myosin light chain 1 and actin at DTDP concentrations at which the changes in the contractile parameters occurred. Our data suggest that SH oxidation in selected myofilament proteins diminishes the Ca2+-activated force and its Ca2+ sensitivity through an impaired Ca2+ regulation of the actin-myosin cycle in the human heart.

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.