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The giant myofilament protein titin has an extendable region that functions as a molecular spring. Cardiomyocytes have exquisite control over the length of titin, through splicing, enabling it to regulate passive stiffness. We hypothesized that titin as it sets the preload on the cardiomyocyte when stretched, together with diastolic Ca2+ pre-activates the cardiomyocyte during diastole and that this pre-activation is a major determinant for force production in the subsequent systolic phase. Via this route titin is thought to play an important role in active force development. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. In the present study we aimed to investigate the effect of long, highly compliant titin on the contractile properties of single cardiomyocytes. We measured single cardiomyocyte work-loops that mimic the cardiac cycle, in wildtype (WT) and heterozygous (HET) RBM20 deficient rats. In addition we studied detergent-permeabilized human patient samples that had known variations in titin based passive stiffness. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies (>6 Hz). HET myocytes operated at higher SL to achieve the same level of work (2.1µm vs. 1.94µm at 6 Hz). In detergent-permeabilized cardiomyocytes isolated from human and rat heart we simulated cardiac twitches by transiently (0.5 s) exposing the cell to a physiological calcium concentration of pCa 5.7. Increasing pre-activation by bathing the cells in pCa 6.7 or pre-stretching the myocyte increased the kinetics of force development and thus the total force development within a transient activation. This is consistent with our hypothesis that pre-activation can increase force development in a time limited contraction such as a cardiac twitch. Pre-activation was pre-load dependent as the sarcomere length to which the myocytes had to be stretched for equivalent levels of pre-activation varied with the compliance of titin.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disorder. Mutations in the gene (MYBPC3) encoding cardiac myosin binding protein C (cMyBP-C) are a frequent cause of HCM. Clinical as well as animal-model studies have reported sex-related differences in HCM disease onset and severity. In addition, it has been established that physiological stimuli such as exercise may elicit a sexually dimorphic cardiac response. However, less attention has been paid to the sex-specific differences in the cellular pathophysiologic mechanisms underlying HCM. Therefore, we studied functional properties of the heart and sarcomeres in male and female sedentary and exercise (exposed to 8 weeks voluntary wheel running) mice.Echocardiography and isometric force measurements in mechanically isolated left ventricular (LV) membrane-permeabilized cardiomyocytes were performed in Wild-type (WT) and heterozygous (HET) knock-in mice carrying a Mybpc3 point mutation (G>A transition) associated with HCM.The LV mass was significantly lower in female WT and HET mice (23% in WT and 25% in HET), compared to corresponding male mice. Isometric force measurements revealed a significant lower maximal generated tension (Fmax) in HET male (13.0 ±1.1 kN/m2), than in females (20.0 ±2.2 kN/m2). Exercise induced a higher fractional shortening in HET male mice, which is correlated with an increased Fmax in exercised HET males. In contrast, LV weight was significantly increased in exercised HET females compared to sedentary females (7% in WT and 15% in HET). Ca2+-sensitivity was increased in exercised male and females WT mice. Similarly, Ca2+-sensitivity was enhanced in HET females, however not in exercised HET mice.In conclusion, exercise training improved cardiac and myofilament performance particularly in HET male mice, indicating that physiological stimuli may elicit a sexually dimorphic cardiac response in heterozygous Mybpc3-targeted knock-in mice.

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.

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.

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

BackgroundThe Frank-Starling mechanism describes the intrinsic relationship between ventricular filling (end-diastolic volume) and contraction (stroke volume) and is based on length-dependent activation of cardiac sarcomeres. An increase in sarcomere length with increased end-diastolic volume increases myofilament Ca2+-sensitivity and thereby contractility. In the present study we investigated if length-dependent activation is altered in human hypertrophic cardiomyopathy (HCM) with known mutations in thin filament proteins.MethodsComparisons were made between cardiac samples from a patient harboring a homozygous TNNT2 (troponin T) mutation, a patient carrying a heterozygous TPM1 (tropomyosin) mutation, two patients harboring a heterozygous TNNI3 (troponin I) mutation and non-failing donors. Force measurements were performed in single Triton-permeabilized cardiomyocytes at various [Ca2+] and sarcomere lengths of 1.8 and 2.2 μm, with and without protein kinase A (PKA)-pretreatment.ResultsMyofilament Ca2+-sensitivity at 2.2 μm did not significantly differ between TNNT2, TPM1 and TNNI3 (EC50 = 2.51±0.10, 2.16±0.19 and 2.69±0.16 μmol/L, respectively), while it was significantly higher compared to donors (EC50 = 3.09±0.09 μmol/L).The length-dependent increase in myofilament Ca2+-sensitivity was not significantly different between TNNT2, TPM1 and TNNI3 (ΔEC50 = 0.41±0.17, 0.29±0.13 and 0.29±0.15 μmol/L, respectively), but significantly reduced compared to donors (ΔEC50 = 0.76±0.06 μmol/L).PKA-treatment did not alter myofilament Ca2+-sensitivity in the TNNT2 and TPM1 samples, whereas Ca2+-sensitivity was significantly reduced in the TNNI3 to values observed in donor.PKA-pretreatment did not restore the reduced length-dependent activation of HCM-groups.ConclusionsPerturbed length-dependent activation may represent a feature of HCM and contribute to impaired cardiac function in HCM-patients. Our data show that responsiveness to β-adrenergic receptor stimulation mimicked by PKA-treatment is absent in a HCM-homozygous TNNT2 mutation. Although hearts with a mutation in TPM1 and TNNI3 responded to PKA, evident from a reduction in myofilament Ca2+-sensitivity, the blunted length-dependent activation was not corrected by PKA.

During recent years it has become increasingly evident that cardiac myosin binding protein C (cMyBP-C) exerts an important role in regulation of sarcomere function with consequences for in vivo cardiac performance. The functional role of cMyBP-C is tightly regulated by kinase-mediated phosphorylation, of which protein kinase A (PKA)-mediated phosphorylation is important during cardiac stress and exercise. To date, three serines have been identified on which cMyBP-C can be phosphorylated in vivo, all of which are located in the cardiac isoform specific M region, i.e. Ser273, Ser282 and Ser 302. However, previous experiments have indicated that at least one more phosphorylation site should exist in humans. The aim of the present study was to identify the unknown phosphorylation site on human cMyBP-C. cMyBP-C was semi-purified from human non-failing donor and end-stage failing cardiac tissue. Tandem mass spectrometry was used to identify phosphorylation sites. Besides the already known Ser282 site, a phosphorylation site was detected on Ser133 in donor tissue. For characterization of this site, a phospho-specific antibody was developed. Levels of Ser133 phosphorylation are lower in end-stage heart failure samples as well as in samples from hypertrophic and dilated cardiomyopathy patients compared to donor. Initial experiments suggest that unlike the three previously known phosphorylation sites, Ser133 is not phosphorylated by PKA.In conclusion, we identified a fourth phosphorylation site (Ser133) in human cMyBP-C, which is present in the Pro-Ala rich region that links the C0 and C1 domains. This site is less phosphorylated in various cardiac pathologies and does not seem to be a target of PKA.Supported by the 7th Framework Program of the European Union (“BIG-HEART,” grant agreement 241577).

The first mutation found to be associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain. R403Q is located in the globular head of myosin (S1), responsible for actin interaction, and hence motor function of myosin. According to the 2-state model of actin-myosin interaction total cross-bridge turnover rate can be described by the sum of the cross-bridge association (fapp) and detachment (gapp) rates. Gapp is equivalent to the slow rate of cross-bridge relaxation (slow krel), which equals the energetic cost of tension generation, i.e. tension cost (expressed by ATP utilization/tension). In the present study we investigated to what extent cross-bridge kinetics in HCM with the R403Q mutation correlated with cross-bridge energetics.Left ventricular multicellular muscle strips and myofibrillar preparations were isolated from 3 HCM patients with the R403Q mutation. 8 HCM sarcomere mutation negative (HCMsmn) patients served as control. In the muscle strips force generating capacity and ATP utilization were measured to assess tension cost and in the myofibrillar preparations cross-bridge kinetics was analyzed. The fraction of mutated R403Q mRNA was analyzed using specific restriction digests and realtime-PCR. Compared to HCMsmn tension cost was higher in the muscle strips of the 3 R403Q patients which showed a positive linear correlation with relaxation kinetics in the corresponding myofibrillar preparations. Variation in cross-bridge function among the 3 R403Q patients could not be explained by differences in R403Q mRNA levels.Cross-bridge cycling efficiency was decreased in HCM due to the R403Q mutation, while cross-bridge kinetics was increased. This suggests that the apparent gain in function evident from increased kinetics leads to a loss of function with respect to cross-bridge cycling efficiency in HCM caused by the R403Q mutation.

Mismatch between energy supply and demand may limit cardiac performance of the remodeled heart after myocardial infarction. To assess whether economy of myofilament contraction is altered in the post-infarct remodeled myocardium, force and ATP utilization were measured simultaneously in permeabilized muscle strips from pigs with a myocardial infarction (MI). Remote left ventricular subendocardial muscle strips were taken 3 weeks after sham surgery (n=5) or induction of MI (n=5), by ligation of the left circumflex coronary artery. Isometric force and ATP consumption were measured at various [Ca2+] in 17 permeabilized muscle strips in each group.Three weeks after infarction, significant LV remodeling had occurred, reflected by LV dilation and hypertrophy of the surviving myocardium. LV systolic dysfunction was evident from the significantly reduced ejection fraction. Maximal force was significantly lower in MI (14±1 kN/m2) compared to sham (29±2 kN/m2), while maximal ATPase activity was slightly, though not significantly lower in MI (33±5 μM/s) compared to sham (45±5 μM/s). Tension cost, the rate of ATP splitting divided by isometric force is a measure of muscle economy. Mean tension cost at maximal activation was almost two-times higher in MI compared to sham (P

Perturbations in Protein kinase C (PKC) isoform expression and localization have been reported in failing cardiomyocytes. Previous studies suggested that PKC-mediated phosphorylation of cardiac troponin I (cTnI) underlies myofilament dysfunction in cardiac disease. PKC phosphorylates cTnI at various sites; however, the functional effects of PKC-mediated cTnI phosphorylation at these different sites in human myocardium are unknown. In the present study, we investigated the effects of cTnI phosphorylation at well-known PKC-sites Ser42/44 and Thr143, and the recently identified PKC-site Ser199 on myofilament function in human failing cardiomyocytes.Myofilament force development was measured at various [Ca2+] and sarcomere lengths of 1.8 and 2.2 µm in permeabilized end-stage failing cardiomyocytes in which endogenous troponin complex was partially exchanged (66.2±1.6%) with recombinant whole human troponin complexes. Site-directed mutagenesis was used to replace the Ser/Thr of the PKC-sites by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In addition, wild-type troponin (Wt) was used as a control.Myofilament Ca2+-sensitivity (pCa50) was significantly reduced by pseudophosphorylation of Ser42/44 (pCa50=5.36±0.01), while an increase was found with pseudophosphorylated Thr143 (pCa50=5.68±0.02) and Ser199 (pCa50=5.72±0.01) in comparison to Wt (pCa50=5.59±0.02). Only at the Ser42/44 sites, alanine mutations affected Ca2+-sensitivity (pCa50 = 5.50±0.02) compared to Wt. The length-dependent increase in myofilament Ca2+-sensitivity and maximal force was not significantly affected by pseudophosphorylation of the different PKC sites.These opposite site-specific effects suggest that the collective effect of various PKC isoforms on myofilament Ca2+-sensitivity depends on the degree at which the different sites are phosphorylated.

Protein kinase A (PKA)-mediated phosphorylation of contractile proteins upon activation of β-adrenergic receptors decreases myofilament Ca2+-sensitivity and accelerates relaxation of the heart. Phosphorylation of the PKA sites (Ser23/24) of cardiac troponin I (cTnI) is decreased in end-stage failing compared to non-failing human hearts. However, the site-specific functional consequences of phosphorylation at these sites in human myocardium are unknown. Therefore, we studied the effect of phosphorylation of cTnI-Ser23/24 on myofilament function in explanted human heart tissue.Myofilament force development was measured at various [Ca2+] in permeabilized cardiomyocytes in which endogenous troponin complex was partially exchanged (69±2%) with recombinant (Myc-tag labeled) whole human troponin complexes (1 mg/ml). Site-directed mutations were used to mimic phosphorylation of both Ser23 and Ser24 (cTnI-DD), Ser24 only (cTnI-AD), Ser23 only (cTnI-DA) or dephosphorylation of both sites (cTnI-AA).In donor cardiomyocytes,myofilament Ca2+-sensitivity (pCa50) was significantly reduced in cTnI-DD (pCa50=5.39±0.01) compared to cTnI-AA (pCa50=5.50±0.01), cTnI-AD (pCa50=5.48±0.01) and cTnI-DA (pCa50=5.51±0.01). The maximal rate of tension redevelopment (ktr) was decreased by 15.1-19.8% in cTnI-AD compared to cTnI-AA, cTnI-DA and cTnI-DD. Pseudo-phosphorylation of Ser23/24 had no effect on maximal or passive force nor on the steepness of the force-pCa relation. In end-stage failing cardiomyocytes, using cTnI-DD concentrations between 0 and 1 mg/ml in order to vary the degree of exchange, a linear decline in the pCa50 vs cTnI-DD content by 0.13±0.03 units was observed, saturating at 52±7% of cTnI-DD.Our data indicate that (1) pseudo-phosphorylation of both PKA-sites on cTnI is required to reduce myofilament Ca2+-sensitivity, (2) pseudo-phosphorylation of Ser24 on cTnI results in a slowing of crossbridge kinetics and (3) the maximal reduction in pCa50 is reached at ∼50% bis-phosphorylated cTnI.

Background: One of the mutated sarcomeric proteins in familial hypertrophic cardiomyopathy (FHC) is Troponin I (cTnI), a down-stream target of the β-adrenergic receptor pathway. Activation of this pathway leads to protein kinase A (PKA)-mediated phosphorylation of cTnI and sarcomere desensitization to Ca2+ to improve muscle relaxation. Moreover, cTnI phosphorylation was shown to enhance sarcomere length-dependent activation (i.e. Frank-Starling mechanism). We investigated if the responsiveness to PKA and to an increase in sarcomere length is impaired by mutant cTnI in human FHC.Methods: A comparison was made between cardiac samples from two FHC patients harboring the R145W mutation in the TNNI3 gene (TNNI3mut) and non-failing donor hearts. Isometric force was measured in Triton-permeabilized cardiomyocytes at different [Ca2+] and sarcomere lengths of 1.8 and 2.2 μm before and after treatment with exogenous PKA.Results: At a sarcomere length of 2.2 μm, TNNI3mut cells (n=30) showed lower maximal force (26.3±4.9 kN/m2) and similar passive force (2.1±0.2 kN/m2) compared to donor cells (n=48; 36.4±2.3 and 2.4±0.3 kN/m2, respectively). In addition, a significantly higher Ca2+-sensitivity (EC50)(2.69±0.16 μM) and a decreased steepness (2.17±0.10) of the force-Ca2+ relation were found in TNNI3mut compared to donor (3.09±0.09 μM and 3.75±0.12, respectively). The increase in Ca2+-sensitivity (i.e. length-dependent activation; ΔEC50) upon an increase in sarcomere length was significantly smaller in TNNI3mut (0.29±0.15) compared to donor (0.76±0.06) cells. Treatment with PKA decreased myofilament Ca2+-sensitivity in TNNI3mut to values observed in donor, but did not correct the reduced length-dependent activation.Conclusions: Our data show preserved responsiveness to PKA in FHC harboring the R145W mutation, but a blunted length-dependent activation, which was not corrected by PKA. Reduced maximal force and perturbed Frank-Starling mechanism, both at baseline and after PKA, may impair contractile performance in human FHC with mutant cTnI.

Background Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. The main target of the thin filament is cardiac Troponin T (cTnT) accounting for ∼15% of the known HCM-causing mutations. Studies with recombinant proteins and transgenic animals showed that cTnT mutations increase Ca2+-sensitivity of the myofilaments. However, data in human myocardium are lacking.Methods We investigated sarcomeric function and protein phosphorylation in a HCM patient with a homozygous cTnT charge mutation (K273N). Myofilament force was measured at various calcium concentrations in demembranated single cardiomyocytes from the septum and left ventricular wall. To investigate myofilament responsiveness to β-adrenergic stimulation force measurements were repeated after incubation with exogenous protein kinase A (PKA). Protein composition was analysed by 1- and 2-dimensional gel electrophoresis.Results Maximal and passive force were significantly lower in K273N cells (21±2 and 1.9±0.1 kN/m2, respectively) compared to non-failing cardiomyocytes (33±4 and 2.9±0.4 kN/m2, respectively), while Ca2+-sensitivity was significantly higher in K273N (pCa50=5.58±0.02) compared to control (pCa50=5.52±0.02). K273N cardiomyocytes did not respond to PKA, while a significant reduction in Ca2+-sensititivity was observed in non-failing cells.Phosphorylation of cTnT was lower in K273N compared to non-failing myocardium and previously studied end-stage failing hearts. Phosphorylation of the PKA target proteins, cardiac myosin-binding protein-C was similar as found in non-failing myocardium, while troponin I phosphorylation was slightly lower in K273N.Conclusion Our data reveal reduced maximal force generating capacity and increased Ca2+-sensitivity of human myofilaments harbouring the HCM-associated cTnT mutation K273N. The lower force generating capacity and absence of myofilament Ca2+-desensitization upon PKA may impair cardiac function in human HCM with mutant cTnT at baseline and during exercise.Funding: Seventh Framework Program of the European Union “BIG-HEART,” grant agreement 241577.

Protein kinase C (PKC) isoforms have been shown to play an important role in the development of heart failure. Most research performed on PKCα has been done in rodents and direct evidence in human heart failure is limited. Our previous study showed a decrease in Ca2+-sensitivity in failing tissue upon PKCα treatment of cardiomyocytes via phosphorylation of cardiac troponin I (cTnI), cardiac troponin T (cTnT) and myosin binding protein C (cMyBP-C). This study aims to determine the targets of PKCα on cTnI and cTnT. Western immunoblotting revealed that PKCα is less abundant but more active in failing compared to donor tissue. PKCα treatment of donor and failing tissue was able to phosphorylate Thr-143, which is a known PKCα site on cTnI, but endogenous phosphorylation levels were very low. LC-MS analysis of purified human recombinant cTn complex incubated with PKCα identified two novel phosphorylation sites, Ser-199 located on cTnI and Ser-189 on cTnT. Both sites are located in conserved regions on cTnI and cTnT. The PKA sites Ser23/24 on cTnI are phosphorylated by PKCα in purified human recombinant cTn complex, but there is no cross phosphorylation in donor and failing tissue. In conclusion, endogenous Thr-143 phosphorylation is low, which makes its involvement in heart failure unlikely. Exogenous PKCα phosphorylation of Thr-143 and Ser-199 on cTnI and Ser-189 on cTnT could possibly explain the decrease in Ca2+-sensitivity observed and further research on the site-specific effects is warranted.

Previously we have shown that in vivo cardiac responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a myocardial infarction (MI), consistent with defects in β-adrenergic signaling. Here we tested the hypothesis that the blunted increase in pump function with exercise after MI is due to reduced myofilament responsiveness, and is prevented by β-blocker therapy. In pigs with a MI induced by ligation of the left circumflex coronary artery, β-blocker therapy (bisoprolol, MI+β) was initiated on the first day after MI. Myofilament force measurements and protein analysis were performed in left ventricular subendocardial biopsies taken at baseline, and upon dobutamine stimulation 3 weeks after MI or sham (n=6). Isometric force was measured in single permeabilized cardiomyocytes. At baseline, maximal force (Fmax) was lower in MI compared to sham, while Ca2+-sensitivity (pCa50) was higher (both P

Phosphorylation status appears to be a key determinant of excitation-contraction coupling ion channel and pump function. Dysfunction of the ryanodine receptor (RyR) secondary to catecholaminergic drive and phosphorylation has been proposed as a factor in contractile dysfunction and arrhythmia patho-physiology in the failing heart. The phosphorylation states of RyR, along with those of phospholamban and troponin I have been investigated by immunoblotting, and quantitated by comparing levels in failing hearts with basal levels, minimum levels after beta-blocker treatment and maximal levels achieved by ex vivo treatment with isoprenaline. We found that RyR residue Ser2809 was phosphorylated to 124 ± 11 % (n = 5, P > 0.05) of control (sham-operated, basal) in heart failure under basal conditions and 143 ± 12 % (n = 6, P 0.05) for heart failure and 199 ± 9 % (n=6, P < 0.05) for isoprenaline treatment. Phosphorylation levels at Ser16 of phospholamban were higher: 159 ± 17 % (heart failure, n = 7, P < 0.05) and 366 ± 95 % (isoprenaline treatment, n = 5, P < 0.05). At Ser23/24 of troponin I there is no significant change in heart failure (n = 5, P < 0.05) but a 230 ± 92 % increase with isoprenaline treatment (n = 6, P < 0.05). Basal levels of phosphorylation are thus relatively low at RyR Ser2030, phospholamban Ser16, and troponin I Ser23/24, and are not significantly increased in heart failure, but are substantially increased by isoprenaline treatment. In contrast, the phosphorylation level at Ser 2809 is already high and can be increased only moderately by isoprenaline.

Upon β-adrenergic stimulation, protein kinase A (PKA) enhances cardiac Troponin I (cTnI) phosphorylation at ser23/24. PKA treatment leads to a decrease in myofilament Ca2+-sensitivity. However, the specific effect of PKA-mediated phosphorylation of cTnI in human myocardium is unclear since PKA phosphorylates a broader set of contractile proteins, in particular myosin binding protein C (cMyBP-C).To address this issue, a selective exchange procedure was used in which 50% and 70% of the endogenous cTn complex in permeabilized human cardiomyocytes was exchanged with recombinant unphosphorylated human cTn. Cardiomyocytes isolated from healthy donor hearts showed almost saturated phosphorylation levels at the ser23/24 of cTnI. Endogenous phosphorylated cTn of donor cardiomyocytes (pCa50= 5.45±0.03) was exchanged with 0.5 and 1.0 mg/ml unphosphorylated recombinant human cTn (cTn-U), which resulted in an increase in Ca2+-sensitivity (ΔpCa50=0.08). Subsequent incubation of the cells with PKA reversed Ca2+-sensitivity to baseline levels (pCa50= 5.46±0.03).To study if the effect of PKA-mediated phosphorylation on cTnI ser23/24 depends on phosphorylation of other contractile proteins, failing human cardiac tissue was used in which phosphorylation of cTnI and cMyBP-C is depressed. Cells from failing tissue showed increased Ca2+-sensitivity (pCa50 5.56±0.03) compared to donor cells. Endogenous cTn of failing cardiomyocytes was exchanged with 0.5 and 1.0 mg/ml cTn pre-treated with PKA to fully saturate ser23/24 (cTn-bisP). However, upon exchange with the cTn-bisP complex, Ca2+-sensitivity did not decrease. Subsequent PKA incubation reduced pCa50 back to the level observed in donor myocardium. This indicates that the effect of cTnI ser23/24 bis-phosphorylation on Ca2+-sensitivity is dependent on PKA-mediated phosphorylation of other contractile protein(s). Preliminary protein phosphorylation data point towards the involvement of cMyBP-C.

During recent years it has become increasingly evident that cardiac myosin binding protein C (cMyBP-C) exerts an important role in regulation of sarcomere function with consequences for in vivo cardiac performance. The functional role of cMyBP-C is tightly regulated by kinase-mediated phosphorylation. The most important kinase which phosphorylates cMyBP-C in vivo is protein kinase A (PKA), which is activated upon stimulation of the β-adrenergic receptors during exercise. In end-stage failing human myocardium, reduced phosphorylation of cMyBP-C has been reported using 1-dimensional and 2-dimensional gel electrophoresis. This reduced phosphorylation has been attributed to down-regulation and desensitization of the β-adrenergic receptor pathway in end-stage human heart failure. Low levels of cMyBP-C phosphorylation were also found in patients with familial hypertrophic cardiomyopathy (FHC), which is frequently caused by mutations in genes encoding sarcomeric proteins, with one exception: in FHC patients with mutations in the gene which encodes cMyBP-C (MYBPC3), phosphorylation of cMyBP-C was unaltered compared to non-failing donor hearts. cMyBP-C can be phosphorylated in vivo on at least three serine sites (Ser273, Ser282 and Ser 302), all of which are located in the cardiac isoform specific M region. At least one other site should exist in humans. With tandem mass spectrometry we recently identified a fourth phosphorylation site on Ser133, which is present in the Pro-Ala rich region that links the C0 and C1 domains. Preliminary data indicate that Ser133 is not phosphorylated by PKA. Analysis of phosphorylation on Ser133 in failing and donor samples revealed lower levels of Ser133 phosphorylation in several forms of cardiac disease compared to non-failing myocardium. Overall, our studies indicate that diverse cMyBP-C phosphorylation patterns exist in human cardiomyopathies, which may in part underlie sarcomere dysfunction observed in human heart failure.

Hypertrophic and familial dilated cardiomyopathies are arguably the most common forms of inherited myocardial dysfunction. Both disease states result in deterioration of cardiac function and quality of life as a consequence of extensive remodelling of the chamber walls.Here we use a novel technique SPontaneous Oscillatory Contractions - or SPOCs to assess changes in contraction and relaxation phases using a range of explanted human heart samples. We examine left ventricle (LV) samples from: (1) patients with hypertrophic cardiomyopathy (HCM); (2) patients with familial dilated cardiomyopathy (FDCM); and (3) and aged-matched non-failing donors. The SPOC parameters of interest are: (i) SPOC amplitudes; (ii) the rates of lengthening (relaxation); (iii) the rates of shortening (contraction); and (iv) their respective SPOC periods.On average, samples from HCM patients exhibited significantly slower rates of lengthening and shorter SPOC periods, while FDCM patients displayed significantly longer lengthening and shortening SPOC periods, compared to donors. Impaired shortening is indicative of diastolic dysfunction while impaired shortening indicates a systolic dysfunction. We observed extensive changes in SPOC parameters that were mutation-specific. The MYBPC3 mutation exhibited shorter SPOC period and faster shortening rates while the samples with the TNNI3 mutation had a higher amplitude and slower shortening rates. Their SPOC data are consistent with the relatively mild phenotype associated with their respective mutations. Furthermore, SPOC is also sensitive to the progressive deterioration in LV ejection fraction.SPOC analysis is a promising tool that provides a quantitative insights into cardiac contractility. It may allow us to unravel other significant differences between familial cardiomyopathies and donor hearts. The SPOC data agree well with patient clinical phenotypes.

Background: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Previously, we observed a reduced maximal force generating capacity (Fmax) of cardiomyocytes from HCM patients with mutations in myosin-binding protein-C (MYBPC3). In this study we investigated the contribution of myofibril density (MyoD) and cross-sectional area (CSA) to the reduction in Fmax.Methods: We measured Fmax and CSA of single permeabilized ventricular cardiomyocytes of 27 HCM patients with mutations in MYBPC3, myosin heavy chain: MYH7, troponin T: TNNT2 and troponin I: TNNI3 and 10 non-failing donor hearts. MyoD was calculated as the sum of myofibril area related to cell area assessed by electron microscopy.Results: Average Fmax was significantly lower in HCM groups compared to donor (Figure A). Figure B shows that MyoD of HCM hearts was reduced compared to donors. CSA of HCM hearts (529±48μm2) was significantly higher compared to donors (342±12μm2). Linear regression analysis revealed a significant negative linear relationship between CSA and Fmax (R2= 0.50, β=-0.023, p=0.014).Conclusion: Cardiomyocyte hypertrophy and reduced myofibrillar density underlie depressed Fmax in HCM with mutations in thick and thin sarcomeric proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

We isolated troponin from left ventricular muscle of a patient with a homozygous K273N mutation in troponin T and compared it with troponin from donor heart muscle using the quantitative in vitro motility assay. Motility of thin filaments reconstituted with skeletal muscle actin, human tropomyosin and mutant native troponin was indistinguishable from thin filaments containing donor heart troponin (Sliding speed at 4µM Ca2+, K273N/donor = 0.98±0.09, n=9; Ca2+- sensitivity, EC50 fraction filaments motile K273N/donor = 0.94±0.28, n=9).This pattern of results was also observed with troponin from human HCM muscle caused by mutations in myosin and MyBP-C and in this case it was ascribed to a secondary modification of troponin not related to the mutation. To test for this possibility with our K273N mutant sample, we exchanged recombinant K273N TnT into troponin from a donor heart. In the non-HCM context the K273N mutation caused a substantial increase in Ca2+-sensitivity (EC50 fraction filaments motile K273N/donor = 0.54±0.17, n=5, p=0.004). We also exchanged recombinant K273N TnT into troponin from a patient with HCM due to a mutation in MyBP-C.We conclude that the K273N TnT mutation increases thin filament Ca2+-sensitivity, in common with other HCM-causing mutations, but in the heart muscle of patients with HCM this effect is largely masked by secondary changes in troponin function due to post-translational modifications. We are investigating whether troponin I phosphorylation is uncoupled from decreased Ca2+-sensitivity in this sample, since this dysfunction was observed in troponin from HCM patients with MYH7 and MYBPC3 mutations.Supported by Seventh Framework Program of the European Union “BIG-HEART,” grant agreement 241577.

SPontaneous Oscillatory Contraction (SPOC) describes repetitive cycles of rapid lengthening (relaxation) and slow shortening (contraction) observed in small bundles of striated muscle cells (myocytes). SPOCs can be induced in both skeletal and cardiac muscles. Here we describe SPOCs in human cardiac muscle, and in particular focus on SPOCS from left ventricles. All samples were frozen in liquid N either within minutes of the loss of the blood supply to the heart (i.e. soon after the aorta was clamped), or within four hours of surgical removal of non-failing hearts that were perfused with cardioplegic solution, thus reducing post-mortem damage to inhibit spontaneous contractions. SPOC is best considered as a third state that is intermediate to contraction and relaxation. SPOC frequency (SPOC period) and shortening velocities correlate well body size in various animals thus, SPOC is likely to reflect the physiology of the heart as it functioned in life. Tiny samples (

A method is presented to measure the power output of isolated intact cardiac myocytes. The mycoytes were attached to a very sensitive and fast force transducer on one side (OptiForce form Ionoptix) and a direct drive piezo-translator on the other side to stretch or shorten the myocyte. This allowed feedback based force control. Force clamps were used to impose a minimum ‘pre-load’ level and maximum ‘after-load’ level between which the myocyte was allowed to operate, mimicking the aortic and atrial pressure respectively. As long as the myocyte reaches the after load level in systole, the loaded shortening results in a force versus length curve that forms a work loop, analogous to the pressure volume curve in the whole heart. Within the measured range (0-1.5 µNewton pre-load, corresponding to sarcomere lengths of 1.86±0.07µm through 1.96±0.08µm) the end-systolic and end-diastolic force-length relations were linear. Measuring the area within each work loop provided the external work performed by the mycoyte for each contraction. A power curve was created by varying the after load for a given pre-load and multiplying the work per contraction with the pacing frequency. For rat myocytes at 37°C the peak power output increased linearly with pre-load within the range measured, and at a pre-load of 1.5 µNewton varied between 25 and 90 pJ.s-1. The large variation was mostly due to differences in cell size. Maximum power output was achieved at pacing frequencies between 6-8 Hz.