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Background: The pathogenesis of MYBPC3 -associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3 :c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3 -HCM with a comprehensive translational approach. Methods: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3 :c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. Results: Haplotype analysis revealed MYBPC3 :c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca 2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. Conclusions: HCM-related MYBPC3 :c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

Full muscle relaxation happens when [Ca2+] falls below the threshold for force activation. Several experimental models, from whole muscle organs and intact muscle down to skinned fibers, have been used to explore the cascade of kinetic events leading to mechanical relaxation. The use of single myofibrils together with fast solution switching techniques, has provided new information about the role of cross-bridge (CB) dissociation in the time course of isometric force decay. Myofibril’s relaxation is biphasic starting with a slow seemingly linear phase, with a rate constant, slow kREL, followed by a fast mono-exponential phase. Sarcomeres remain isometric during the slow force decay that reflects CB detachment under isometric conditions while the final fast relaxation phase begins with a sudden give of few sarcomeres and is then dominated by intersarcomere dynamics. Based on a simple two-state model of the CB cycle, myofibril slow kREL represents the apparent forward rate with which CBs leave force generating states (gapp) under isometric conditions and correlates with the energy cost of tension generation (ATPase/tension ratio); in short slow kREL ~ gapp ~ tension cost. The validation of this relationship is obtained by simultaneously measuring maximal isometric force and ATP consumption in skinned myocardial strips that provide an unambiguous determination of the relation between contractile and energetic properties of the sarcomere. Thus, combining kinetic experiments in isolated myofibrils and mechanical and energetic measurements in multicellular cardiac strips, we are able to provide direct evidence for a positive linear correlation between myofibril isometric relaxation kinetics (slow kREL) and the energy cost of force production both measured in preparations from the same cardiac sample. This correlation remains true among different types of muscles with different ATPase activities and also when CB kinetics are altered by cardiomyopathy-related mutations. Sarcomeric mutations associated to hypertrophic cardiomyopathy (HCM), a primary cardiac disorder caused by mutations in genes encoding sarcomeric proteins, have been often found to accelerate CB turnover rate and increase the energy cost of myocardial contraction. Here we review data showing that faster CB detachment results in a proportional increase in the energetic cost of tension generation in heart samples from both HCM patients and mouse models of the disease.

AimsIncreased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have been associated to total anomalous pulmonary venous return and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease is not understood. To get insight into this problem, we have generated a gain of function ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium.Methods and resultsAnkrd1 is expressed non-homogeneously in the embryonic myocardium, with a dynamic nucleo-sarcomeric localization in developing cardiomyocytes. ANKRD1 transgenic mice present sinus venosus defect, which originates during development by impaired remodelling of early embryonic heart. Adult transgenic hearts develop diastolic dysfunction with preserved ejection fraction, which progressively evolves into heart failure, as shown histologically and haemodynamically. Transgenic cardiomyocyte structure, sarcomeric assembly, and stability are progressively impaired from embryonic to adult life. Postnatal transgenic myofibrils also present characteristic functional alterations: impaired compliance at neonatal stage and impaired lusitropism in adult hearts. Altogether, our combined analyses suggest that impaired embryonic remodelling and adult heart dysfunction in ANKRD1 transgenic mice present a common ground of initial cardiomyocyte defects, which are exacerbated postnatally. Molecular analysis showed transient activation of GATA4-Nkx2.5 transcription in early transgenic embryos and subsequent dynamic transcriptional modulation within titin gene.ConclusionsANKRD1 is a fine mediator of cardiomyocyte response to haemodynamic load in the developing and adult heart. Increased ANKRD1 levels are sufficient to initiate an altered cellular phenotype, which is progressively exacerbated into a pathological organ response by the high ventricular workload during postnatal life. Our study defines for the first time a unifying picture for ANKRD1 role in heart development and disease and provides the first mechanistic link between ANKRD1 overexpression and cardiac disease onset.

We investigated the functional impact of α-tropomyosin (Tm) substituted with one (D137L) or two (D137L/G126R) stabilizing amino acid substitutions on the mechanical behavior of rabbit psoas skeletal myofibrils by replacing endogenous Tm and troponin (Tn) with recombinant Tm mutants and purified skeletal Tn. Force recordings from myofibrils (15°C) at saturating [Ca2+] showed that Tm-stabilizing substitutions did not significantly affect the maximal isometric tension and the rates of force activation (kACT) and redevelopment (kTR). However, a clear effect was observed on force relaxation: myofibrils with D137L/G126R or D137L Tm showed prolonged durations of the slow phase of relaxation and decreased rates of the fast phase. Both Tm-stabilizing substitutions strongly decreased the slack sarcomere length (SL) at submaximal activating [Ca2+] and increased the steepness of the SL-passive tension relation. These effects were reversed by addition of 10 mM 2,3-butanedione 2-monoxime. Myofibrils also showed an apparent increase in Ca2+ sensitivity. Measurements of myofibrillar ATPase activity in the absence of Ca2+ showed a significant increase in the presence of these Tms, indicating that single and double stabilizing substitutions compromise the full inhibition of contraction in the relaxed state. These data can be understood with the three-state (blocked-closed-open) theory of muscle regulation, according to which the mutations increase the contribution of the active open state in the absence of Ca2+ (M−). Force measurements on myofibrils substituted with C-terminal truncated TnI showed similar compromised relaxation effects, indicating the importance of TnI-Tm interactions in maintaining the blocked state. It appears that reducing the flexibility of native Tm coiled-coil structure decreases the optimum interactions of the central part of Tm with the C-terminal region of TnI. This results in a shift away from the blocked state, allowing myosin binding and activity in the absence of Ca2+. This work provides a basis for understanding the effects of disease-producing mutations in muscle proteins.

Myofibril based mechanical studies allow evaluation of sarcomeric protein function. We describe a novel method of obtaining myofibrils from primary cardiomyocyte culture. Adult rat ventricular myocytes (ARVMs) were obtained by enzymatic digestion and maintained in serum free condition. ARVMs were homogenized in relaxing solution (pCa 9.0) with 20% sucrose, and myofibril suspension was made. Myofibrils were Ca2+-activated and relaxed at 15°C. Results from ARVM myofibrils were compared to myofibrils obtained from ventricular tissue skinned with Triton X-100. At maximal Ca2+-activation (pCa 4.5) myofibril mechanical parameters from ARVMs were 6.8 ± 0.9 mN/mm2 (resting tension), 146.8 ± 13.8 mN/mm2 (maximal active tension, P0), 5.4 ± 0.22 s−1 (rate of force activation), 53.4 ± 4.4 ms (linear relaxation duration), 0.69 ± 0.36 s−1 (linear relaxation rate), and 10.8 ± 1.3 s−1 (exponential relaxation rate). Force-pCa curves were constructed from Triton skinned tissue, ARVM culture day 1, and ARVM culture day 3 myofibrils, and pCa50 were 5.79 ± 0.01, 5.69 ± 0.01, and 5.71 ± 0.01, respectively. Mechanical parameters from myofibrils isolated from ARVMs treated with phenylephrine were compared to myofibrils isolated from time-matched non-treated ARVMs. Phenylephrine treatment did not change the kinetics of activation or relaxation but decreased the pCa50 to 5.56 ± 0.03 (vehicle treated control: 5.67 ± 0.03). For determination of protein expression and post-translational modifications, myofibril slurry was re-suspended and resolved for immunoblotting and protein staining. Troponin I phosphorylation was significantly increased at serine 23/24 in phenylephrine treated group. Myofibrils obtained from ARVMs are a viable method to study myofibril mechanics. Phenylephrine treatment led to significant decrease in Ca2+-sensitivity that is due to increased phosphorylation of TnI at serine 23/24. This culture based approach to obtaining myofibrils will allow pharmacological and genetic manipulation of the cardiomyocytes to correlate biochemical and biophysical properties.

Abnormalities of cardiomyocyte Ca2 + homeostasis and excitation–contraction (E–C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E–C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca2 + transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca2 + release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (> 20%) fails to propagate action potentials, with consequent delay of local Ca2 + release. At variance with wild-type, we also observe significantly increased variability of local Ca2 + transient rise as well as higher Ca2 +-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca2 + release and delayed myofilament activation that significantly contribute to mechanical dysfunction., Highlights • Contraction and Ca2 + transient kinetics are impaired in myocardial preparations from mice carrying the cardiac troponin T ∆ 160E mutation. • T-tubules architecture is mildly altered in ∆160E cardiomyocytes. • 20% of T-tubules fail to propagate action potential and produce delay of local Ca2 + rise. • Higher spatio-temporal variability of local Ca2 + rise and increased Ca2 + sparks frequency are found in ∆160E cardiomyocytes.

A ryanodine receptor 2 mutation associated with catecholaminergic polymorphic ventricular tachycardia renders cardiomyocytes incapable of mediating a positive inotropic response., Ryanodine receptor (RyR2) is the major Ca2+ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca2+ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca2+]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca2+]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca2+ transient was normal, despite the reduced SR Ca2+ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca2+ transients because they are incapable of properly increasing the amount of Ca2+ stored in the SR because of a larger SR Ca2+ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca2+ channel current (ICa-L) and SR Ca2+ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po and fractional Ca2+ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca2+ content, but cannot compensate for the inability to increase SR Ca2+ content during inotropic challenge.

Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in three main isoforms in skeletal and cardiac muscles. In the present work we studied the functional role of a- and bTm on force development by modifying the isoform composi- tion of rabbit psoas skeletal muscle myofibrils and of regulated thin filaments for in vitro motility measurements. Skeletal myofibril regulatory proteins were extracted (78 %) and replaced (98 %) with Tm isoforms as homog- enous aaTm or bbTm dimers and the functional effects were measured. Maximal Ca 2? activated force was the same in aaTm versus bbTm myofibrils, but bbTm myo- fibrils showed a marked slowing of relaxation and an impairment of regulation under resting conditions com- pared to aaTm and controls. bbTm myofibrils also showed a significantly shorter slack sarcomere length and a marked increase in resting tension. Both these mechanical features were almost completely abolished by 10 mM 2,3-butane- dione 2-monoxime, suggesting the presence of a significant degree of Ca 2? -independent cross-bridge formation in bbTm myofibrils. Finally, in motility assay experiments in the absence of Ca 2? (pCa 9.0), complete regulation of thin filaments required greater bbTm versus aaTm concentra- tions, while at full activation (pCa 5.0) no effect was observed on maximal thin filament motility speed. We infer from these observations that high contents of bbTm in skeletal muscle result in partial Ca 2? -independent activation of thin filaments at rest, and longer-lasting and less complete tension relaxation following Ca 2? removal.

Background In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation‐driven changes in myofilament function combined with excitation‐contraction (E‐C) coupling abnormalities related to adverse remodeling. Whether myofilament or E‐C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E‐C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. Methods and Results Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca 2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca 2+ activation, the energy cost of tension generation, and myofilament Ca 2+ sensitivity were increased compared with that in wild‐type mice. No sarcomeric changes were observed in R92Q versus wild‐type mice, except for a large increase in myofilament Ca 2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca 2+ transients, reduced SERCA function, and increased Ca 2+ /calmodulin kinase II activity. Contrarily, secondary alterations of E‐C coupling and signaling were minimal in E163R myocardium. Conclusions In E163R models, mutation‐driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocyte signaling and E‐C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s-1, respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l-1 s-1 kN-1 m-2, respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.

Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomeric proteins, but the pathogenic mechanism is unclear. Piroddi et al. find impairment of cross-bridge kinetics and energetics in human sarcomeres with a TNNT2 mutation, suggesting that HCM involves inefficient ATP utilization., Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (kACT) and the rate constant of isometric relaxation (slow kREL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces kACT, slow kREL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases kACT, slow kREL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.

Aims Familial hypertrophic cardiomyopathy (HCM), frequently caused by sarcomeric gene mutations, is characterized by cellular dysfunction and asymmetric left-ventricular (LV) hypertrophy. We studied whether cellular dysfunction is due to an intrinsic sarcomere defect or cardiomyocyte remodelling. Methods and results Cardiac samples from 43 sarcomere mutation-positive patients (HCMmut: mutations in thick ( MYBPC3 , MYH7 ) and thin ( TPM1 , TNNI3 , TNNT2 ) myofilament genes) were compared with 14 sarcomere mutation-negative patients (HCMsmn), eight patients with secondary LV hypertrophy due to aortic stenosis (LVHao) and 13 donors. Force measurements in single membrane-permeabilized cardiomyocytes revealed significantly lower maximal force generating capacity (Fmax) in HCMmut (21 ± 1 kN/m2) and HCMsmn (26 ± 3 kN/m2) compared with donor (36 ± 2 kN/m2). Cardiomyocyte remodelling was more severe in HCMmut compared with HCMsmn based on significantly lower myofibril density (49 ± 2 vs. 63 ± 5%) and significantly higher cardiomyocyte area (915 ± 15 vs. 612 ± 11 μm2). Low Fmax in MYBPC3 mut, TNNI3 mut, HCMsmn, and LVHao was normalized to donor values after correction for myofibril density. However, Fmax was significantly lower in MYH7 mut, TPM1 mut, and TNNT2 mut even after correction for myofibril density. In accordance, measurements in single myofibrils showed very low Fmax in MYH7 mut, TPM1 mut, and TNNT2 mut compared with donor (respectively, 73 ± 3, 70 ± 7, 83 ± 6, and 113 ± 5 kN/m2). In addition, force was lower in MYH7 mut cardiomyocytes compared with MYBPC3 mut, HCMsmn, and donor at submaximal [Ca2+]. Conclusion Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca2+]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations.

α-Tropomyosin (Tm) carrying hypertrophic cardiomyopathy mutation D175N or E180G was expressed in Escherichia coli. We have assembled dimers of two polypeptide chains in vitro that carry one (αα*) or two (α*α*) copies of the mutation. We found that the presence of the mutation has little effect on dimer assembly, thereby predicting that individuals heterozygous for the Tm mutations are likely to express both αα* and α*α* Tm. Depending on the expression level, the heterodimer may be the predominant form in individuals carrying the mutation. Thus, it is important to define differences in the properties of Tm molecules carrying one or two copies of the mutation. We examined the Tm homo- and heterodimer properties: actin affinity, thermal stability, calcium regulation of myosin subfragment 1 binding, and calcium regulation of myofibril force. We report that the properties of the heterodimer may be similar to those of the wild-type homodimer (actin affinity, thermal stability, D175N αα*), similar to those of the mutant homodimer (calcium sensitivity, D175N αα*), intermediate between the two (actin affinity, E180G αα*), or different from both (thermal stability, E180G αα*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.

The influence of Ca2+ binding properties of individual troponin versus cooperative regulatory unit interactions along thin filaments on the rate tension develops and declines was examined in demembranated rabbit psoas fibres and isolated myofibrils. Native skeletal troponin C (sTnC) was replaced with sTnC mutants having altered Ca2+ dissociation rates (koff) or with mixtures of sTnC and D28A, D64A sTnC, that does not bind Ca2+ at sites I and II (xxsTnC), to reduce near-neighbour regulatory unit (RU) interactions. At saturating Ca2+, the rate of tension redevelopment (kTR) was not altered for fibres containing sTnC mutants with decreased koff or mixtures of sTnC:xxsTnC. We examined the influence of koff on maximal activation and relaxation in myofibrils because they allow rapid and large changes in [Ca2+]. In myofibrils with M80Q sTnCF27W (decreased koff), maximal tension, activation rate (kACT), kTR and rates of relaxation were not altered. With I60Q sTnCF27W (increased koff), maximal tension, kACT and kTR decreased, with no change in relaxation rates. Surprisingly, the duration of the slow phase of relaxation increased or decreased with decreased or increased koff, respectively. For all sTnC reconstitution conditions, Ca2+ dependence of kTR in fibres showed Ca2+ sensitivity of kTR (pCa50) shifted parallel to tension and low-Ca2+kTR was elevated. Together the data suggest the Ca2+-dependent rate of tension development and the duration (but not rate) of relaxation can be greatly influenced by koff of sTnC. This influence of sTnC binding kinetics occurs primarily within individual RUs, with only minor contributions of RU interactions at low Ca2+.

The R403Q mutation in β-myosin heavy chain was the first mutation to be identified as responsible for familial hypertrophic cardiomyopathy (FHC). In spite of extensive work on the functional sequelae of this mutation, the mechanism by which the mutant protein causes the disease has not been definitely identified. Here we directly compare contraction and relaxation mechanics of single myofibrils from left ventricular samples of one patient carrying the R403Q mutation to those from a healthy control heart. Tension generation and relaxation following sudden increase and decrease in [Ca2+] were much faster in the R403Q myofibrils with relaxation rates being the most affected parameters. The results show that the R403Q mutation leads to an apparent gain of protein function but a greater energetic cost of tension generation. Increased energy cost of tension generation may be central to the FHC disease process, help explain some unresolved clinical observations, and carry significant therapeutic implications.

We employed single myofibril techniques to test whether the presence of slow skeletal troponin-I (ssTnI) is sufficient to induce increased myofilament calcium sensitivity (EC50) and whether modulation of EC50 affects the dynamics of force development. Studies were performed using rabbit psoas myofibrils activated by rapid solution switch and in which Tn was partially replaced for either recombinant cardiac Tn(cTn) or Tn composed of recombinant cTn-T (cTnT) and cTn-C (cTnC), and recombinant ssTnI (ssTnI-chimera Tn). Tn exchange was performed in rigor solution (0.5 mg/ml Tn; 20°C; 2 h) and confirmed by SDS-PAGE. cTnI exchange induced a decrease in EC50; ssTnI-chimera Tn exchange induced a further decrease in EC50 (in μM: endogenous Tn, 1.35 ± 0.08; cTnI, 1.04 ± 0.13; ssTnI-chimera Tn, 0.47 ± 0.03). EC50 was also decreased by application of 100 μM bepridil (control: 2.04 ± 0.03 μM; bepridil 1.35 ± 0.03 μM). Maximum tension was not different between any groups. Despite marked alterations in EC50, none of the dynamic activation-relaxation parameters were affected under any condition. Our results show that 1) incorporation of ssTnI into the fast skeletal sarcomere is sufficient to induce increased myofilament Ca2+ sensitivity, and 2) the dynamics of actin-myosin interaction do not correlate with EC50. This result suggests that intrinsic cross-bridge cycling rate is not altered by the dynamics of thin-filament activation.

In cardiac muscle from HCM patients primary changes in myofilament function, related to the presence of disease-causing mutations in sarcomeric proteins, are always associated with secondary abnormalities due to adverse remodeling of cardiomyocyte EC-coupling(Coppini et al,Circulation 2013). The latter are likely major contributors of the mechanical dysfunction and arrhythmogeneity of HCM human hearts. Here we characterize the changes in sarcomere function and EC-coupling that occur in two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Echocardiography showed LV hypertrophy, enhanced contractility, diastolic dysfunction and enlarged left atria in both HCM models; the phenotype was more pronounced in the R92Q mice. In E163R ventricular myofibrils, in spite of a significant increase in the rate of the initial isometric slow phase of relaxation, overall relaxation from maximal activation was impaired and prolonged vs WT and R92Q myofibrils that exhibited similar relaxation kinetics. Resting tension was higher in the E163Q compared to WT and R92Q myofibrils. Isometric ATPase both at rest and at maximal Ca2+-activation and the energy cost of tension generation were increased in E163R vs WT and R92Q skinned trabeculae. Myofilament Ca2+-sensitivity was increased in both mutant lines compared to WT; the change was larger in the R92Q preparations. R92Q intact cardiomyocytes and trabeculae compared to WT and E163R preparations showed blunted response to inotropic interventions, reduced amplitude and slower decay of Ca2+-transients with reduced SERCA function. Twitch kinetics were prolonged in both HCM mouse models, despite Ca2+-transient kinetics was faster and SERCA function unchanged in the E163R mice. Intact preparations of both HCM mouse models showed increased probability of arrhythmogenic behavior that increased in response to isoproterenol. The results suggest that similar HCM phenotypes can be generated through different pathogenic pathways. Grant Telethon-GGP13162.

Fast solution switching techniques in single myofibrils offer the opportunity to dissect and directly examine the sarcomeric mechanisms responsible for force generation and relaxation. The feasibility of this approach is tested here in human cardiac myofibrils isolated from small samples of atrial and ventricular tissue. At sarcomere lengths between 2.0 and 2.3 mum, resting tensions were significantly higher in ventricular than in atrial myofibrils. The rate constant of active tension generation after maximal Ca(2+) activation (k (ACT)) was markedly faster in atrial than in ventricular myofibrils. In both myofibril types k (ACT) was the same as the rate of tension redevelopment after mechanical perturbations and decreased significantly by decreasing [Ca(2+)] in the activating solution. Upon sudden Ca(2+) removal, active tension fully relaxed. Relaxation kinetics were (1) much faster in atrial than in ventricular myofibrils, (2) unaffected by bepridil, a drug that increases the affinity of troponin for Ca(2+), and (3) strongly accelerated by small increases in inorganic phosphate concentration. The results indicate that myofibril tension activation and relaxation rates reflect apparent cross-bridge kinetics and their Ca(2+) regulation rather than the rates at which thin filaments are switched on or off by Ca(2+) binding or removal. Myofibrils from human hearts retain intact mechanisms for contraction regulation and tension generation and represent a viable experimental model to investigate function and dysfunction of human cardiac sarcomeres.

In hypertrophic cardiomyopathy (HCM) atrial dilatation (AD) and fibrillation (AF) are very common and associated with worse outcome. The cellular and molecular basis of atrial remodeling in HCM remain undefined. We previously characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and E-C coupling that occur in ventricular myocardium of two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Both models exhibited diastolic dysfunction that was, however, related to different mechanisms i.e. E-C coupling abnormalities in R92Q and sarcomere changes in E163R. Here we employ these mouse models to study whether atrial remodeling is a consequence of diastolic dysfunction or is also influenced by the specific underlying mutation.Echocardiographic measurements of left atrial (LA) dimensions showed that LA area was severely increased in R92Q hearts while it was only mildly increased in E163R (in mm2 : 6.73±0.5 in R92Q, 4.82±0.16 in E163R vs 3.97±0.26 in WT). Left atrial trabeculae were dissected and mounted isometrically to record twitch tension. We studied the steady-state force-frequency relationship and the response to positive inotropic stimuli such as Isoproterenol 10-7 mM (ISO) and 8 mM extracellular [Ca2+]. Compared to WT, R92Q atrial trabeculae showed: (i) slower kinetics of both force development and relaxation (e.g. at 1 Hz, 50% relaxation was prolonged by 35%), (ii) impaired twitch amplitude at high pacing rates (50% reduction), (iii) depressed rested-state contractions and (iv) blunted increase of twitch tension in ISO and high [Ca2+]. None of these changes were observed in intact E163R atrial trabeculae. These findings suggest that atrial remodeling in R92Q is more pronounced compared to E163R, and related to E-C coupling alterations. Supported by the Italian Ministry of Health (WFR GR-2011-02350583).

Changes in myofilament function related to HCM-associated mutations contribute to the diastolic dysfunction observed in the in vivo patient heart and in intact ventricular preparations from patient samples. HCM mutations that are ubiquitously expressed in the heart (e.g. cMyBP-C or cTnT) could also affect atrial function. Here we investigate whether HCM-associated atrial myopathy is a consequence of mutation-driven sarcomere dysfunction or results from atrial remodeling due to the diastolic dysfunction and increased LV filling pressures. In one HCM patient carrying the Lys814del cMyB-C mutation, changes in sarcomere function (increased myofilament Ca2+ sensitivity and increased cross bridges detachment rate under isometric conditions) were similar in atrial and ventricular myofibrils compared to donor preparations. However, isometric twitch mechanics and kinetics of intact trabeculae from the right atrium of 4 cMyB-C-mutant patients were unaffected as compared to trabeculae from non-HCM patients (N=8), or mutation negative HCM patients (N=3), or HCM patients carrying mutations in beta-myosin (N=2). We extended the study to HCM mouse models carrying mutations in cTnT. In the E163R mouse, atrial and ventricular sarcomere kinetics and energetics were similarly altered compared to WT mice. Isometric ATPase, both at rest and at maximal Ca2+-activation and the energy cost of tension generation were increased in both atrial and ventricular preparations of E163R vs WT. However, isometric twitch kinetics were prolonged in intact ventricular trabeculae of E163R mice vs WT while they were unaffected in atrial trabeculae. In the R92Q mouse model, that is associated with a much more severe degree of LV diastolic dysfunction and left atrial dilatation compared to the E163R, left atrial trabeculae showed prolonged twitch contractions, increased spontaneous activity and a number of E-C coupling alterations that resemble those observed in ventricular preparations. HCM-mutations in cMyBP-C and cTnT induce similar alterations in both atrial and ventricular sarcomeres. However, likely due to the different working conditions of the two chambers, sarcomere dysfunction can significantly alter the mechanics and kinetics of the intact myocardium only in the ventricles. Atrial muscle dysfunction in HCM is induced by remodeling processes that depend on the increased filling pressures.

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s(-1), respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l(-1) s(-1) kN(-1) m(-2), respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.

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 (

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

Tropomyosin (Tm) is an essential component in the regulation of striated muscle contraction. Questions about Tm functional role have been difficult to study because sarcomere Tm content is not as easily manipulated as Troponin (Tn). Here we describe the method we recently developed to replace Tm-Tn of skeletal and cardiac myofibrils from animals and humans to generate an experimental model of homogeneous Tm composition and giving the possibility to measure a wide range of mechanical parameters of contraction (e.g. maximal force and kinetics of force generation). The success of the exchange was determined by SDS-PAGE and by mechanical measurements of calcium dependent force activation on the reconstituted myofibrils. In skeletal and cardiac myofibrils, the percentage of Tm replacement was higher than 90%. Maximal isometric tension was 30-35% lower in the reconstituted myofibrils than in control myofibrils but the rate of force activation (k(ACT)) and that of force redevelopment (k(TR)) were not significantly changed. Preliminary results show the effectiveness of Tm replacement in human cardiac myofibrils. This approach can be used to test the functional impact of Tm mutations responsible for human cardiomyopathies.

We have engineered the Ca2+ binding properties of troponin C (TnC) to study the role of increased (I60Q sTnC) and decreased (M80Q sTnCF27W) Ca2+ dissociation rate (koff) on activation and relaxation of skeletal muscle. Previously we reported that myofibril force development kinetics (kACT) are not influenced by decreasing koff from Tn, but are slowed by an increase in koff (Kreutziger et al. 2008 JPhsiol. 586;3683-3700) at low [Pi] (5 μm). The time to initiation of force (kAlag) following a rapid (∼10ms) switch from pCa 9.0 to pCa 3.5 provides information about thin filament activation rate and our preliminary data suggest this rate may also be sensitive to koff. In rabbit psoas myofibrils (15°C) kAlag (∼20 ms for native or WT sTnC) is almost eliminated for M80Q sTnCF27W and increased by I60Q sTnC (∼40-50 ms). Additionally, though kACT is similar for force increases from either full or partial activation to full activation, kAlag disappears when starting from partial activation. We have also reported that fast and slow phase rates of relaxation are not affected by koff, but that duration of the slow phase is affected in skeletal myofibrils. Here we report that lag prior to initiation of the slow phase (kRlag) may be also influenced by koff. Opposite to kAlag, kRlag (∼20 ms for WT or native sTnC) was increased (∼40-50 ms) by decreased koff (M80Q sTnCF27W) and almost eliminated by increased koff (I60QsTnC). These experiments demonstrate a potential approach to study thin filament activation/deactivation kinetics without the need for fluorescent probes attached to thin filament proteins that can affect their function. Supported by Telethon GGP07133, MIUR (CP, CT), NIH-HL65497 (MR).

Tropomyosin (Tm) is an essential component in the regulation of striated muscle contraction. Questions about Tm functional role have been difficult to study because sarcomere Tm content is not as easily manipulated as Troponin (Tn). Here we describe the method we recently developed to replace Tm-Tn of skeletal and cardiac myofibrils from animals and humans to generate an experimental model of homogeneous Tm composition and giving the possibility to measure a wide range of mechanical parameters of contraction (e.g. maximal force and kinetics of force generation). The success of the exchange was determined by SDS-PAGE and by mechanical measurements of calcium dependent force activation on the reconstituted myofibrils. In skeletal and cardiac myofibrils, the percentage of Tm replacement was higher than 90%. Maximal isometric tension was 30-35% lower in the reconstituted myofibrils than in control myofibrils but the rate of force activation (k(ACT)) and that of force redevelopment (k(TR)) were not significantly changed. Preliminary results show the effectiveness of Tm replacement in human cardiac myofibrils. This approach can be used to test the functional impact of Tm mutations responsible for human cardiomyopathies.

Hypertrophic cardiomyopathy (HCM) was the first inherited heart disease to be characterized at the molecular genetic level with the demonstration that it is caused by mutations in genes that encode different components of the cardiac sarcomere. Early functional in vitro studies have concluded that HCM mutations cause a loss of sarcomere mechanical function. Hypertrophy would then follow as a compensatory mechanism to raise the work and power output of the affected heart. More recent in vitro and mouse model studies have suggested that HCM mutations enhance contractile function and myofilament Ca(2+) sensitivity and impair cardiac myocyte energetics. It has been hypothesized that these changes may result in cardiac myocyte energy depletion due to inefficient ATP utilization and also in altered myoplasmic Ca(2+) handling. The problems encountered in reaching a definitive answer on the effects of HCM mutations are discussed. Though direct analysis of the altered functional characteristics of HCM human cardiac sarcomeres has so far lagged behind the in vitro and mouse studies, recent work with mechanically isolated skinned myocytes and myofibrils from affected human hearts seem to support the energy depletion hypothesis. If further validated in the human heart, this hypothesis would identify tractable therapeutic targets that suggest that HCM, perhaps more than any other cardiomyopathy, will be amenable to disease-modifying therapy.

In this study we investigated the molecular mechanism of glutathionylation on isolated human cardiac myofibrils using several pro-glutathionylating agents. Total glutathionylated proteins appeared significantly enhanced with all the pro-oxidants used. The increase was completely reversed by the addition of a reducing agent, demonstrating that glutathione binding occurs by a disulfide and that the process is reversible. A sensitive target of glutathionylation was alpha-actin, showing a different reactivity to the several pro-glutathionylating agents by ELISA. Noteworthy, myosin although highly sensitive to the in vitro glutathionylation does not represent the primary glutathionylation target in isolated myofibrils. Light scattering measurements of the glutathionylated alpha-actin showed a slower polymerisation compared to the non-glutathionylated protein and force development was depressed after glutathionylation, when the myofibrils were mounted in a force recording apparatus. Interestingly, confocal laser scanning microscopy of cardiac cryosections indicated, for the first time, the constitutive glutathionylation of alpha-cardiac actin in human heart. Due to the critical location of alpha-actin in the contractile machinery and to its susceptibility to the oxidative modifications, glutathionylation may represent a mechanism for modulating sarcomere assembly and muscle functionality under patho-physiological conditions in vivo.

The influence of Ca(2+) binding properties of individual troponin versus cooperative regulatory unit interactions along thin filaments on the rate tension develops and declines was examined in demembranated rabbit psoas fibres and isolated myofibrils. Native skeletal troponin C (sTnC) was replaced with sTnC mutants having altered Ca(2+) dissociation rates (k(off)) or with mixtures of sTnC and D28A, D64A sTnC, that does not bind Ca(2+) at sites I and II (xxsTnC), to reduce near-neighbour regulatory unit (RU) interactions. At saturating Ca(2+), the rate of tension redevelopment (k(TR)) was not altered for fibres containing sTnC mutants with decreased k(off) or mixtures of sTnC:xxsTnC. We examined the influence of k(off) on maximal activation and relaxation in myofibrils because they allow rapid and large changes in [Ca(2+)]. In myofibrils with M80Q sTnC(F27W) (decreased k(off)), maximal tension, activation rate (k(ACT)), k(TR) and rates of relaxation were not altered. With I60Q sTnC(F27W) (increased k(off)), maximal tension, k(ACT) and k(TR) decreased, with no change in relaxation rates. Surprisingly, the duration of the slow phase of relaxation increased or decreased with decreased or increased k(off), respectively. For all sTnC reconstitution conditions, Ca(2+) dependence of k(TR) in fibres showed Ca(2+) sensitivity of k(TR) (pCa(50)) shifted parallel to tension and low-Ca(2+) k(TR) was elevated. Together the data suggest the Ca(2+)-dependent rate of tension development and the duration (but not rate) of relaxation can be greatly influenced by k(off) of sTnC. This influence of sTnC binding kinetics occurs primarily within individual RUs, with only minor contributions of RU interactions at low Ca(2+).

No drugs are capable to prevent phenotype development and adverse cardiac remodeling in hypertrophic cardiomyopathy (HCM). Ranolazine, a late Na+ current blocker, reduced arrhythmogenicity and improved relaxation in cardiomyocytes and trabeculae from HCM patients (Coppini et al., Circulation 2013). Employing a transgenic mouse model carrying the HCM-associated R92Q mutation in the TNNT2 gene, we previously showed that acute in vitro treatment with ranolazine is capable to reverse some electromechanical alterations, including the prolonged kinetics of Ca2+ transients, the higher diastolic [Ca2+] and the increased frequency of arrhythmogenic spontaneous activity (Pioner et al., Biophys J 2014, 106, 644a). Here we employed the same mouse model to assess whether long-term oral treatment with ranolazine since birth is capable to prevent the HCM phenotype and the associated myocardial remodeling. We compared the behavior of WT, R92Q-untreated and R92Q-treated 1 year old mice. Echocardiographic measurements showed that the R92Q-treated in vivo hearts lacked the left ventricular hypertrophy, hypercontractility and diastolic dysfunction found in the R92Q-untreated mice. Gadolinium-contrast magnetic resonance showed that the intramyocardial fibrosis of the R92Q-untreated hearts was largely reduced in the treated mice.Both amelioration of cardiomyocyte function and reduction of extracellular fibrosis may contribute to the positive effect of the long-term treatment with ranolazine that could represent a candidate for preventive treatment of phenotype-negative mutation carriers. Mechanical experiments in intact left and right ventricular trabeculae confirmed the alterations we had previously reported in R92Q-untreated mice compared to WT and showed that those alterations were mostly reversed in the R92Q-treated mice. In the R92Q-treated preparations the inotropic response to isoproterenol was preserved and the occurrence of spontaneous activity was markedly reduced compared to the untreated trabeculae and was comparable to that of WT trabeculae.

The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI 1–192 ) and full-length cTnI were expressed in Escherichia coli , purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 μm, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic β-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI 1–192 in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca 2+ sensitivity of the contractile apparatus was increased in preparations containing cTnI 1–192 (pCa 50 : 5.73±0.03 versus 5.52±0.03 for cTnI 1–192 and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI 1–192 -exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.