Correction: Winter et al. Z-Disk-Associated Plectin (Isoform 1d): Spatial Arrangement, Interaction Partners, and Role in Filamin C Homeostasis. Cells 2023, 12, 1259.

The authors wish to make the following changes to their paper [...].

Z-Disk-Associated Plectin (Isoform 1d): Spatial Arrangement, Interaction Partners, and Role in Filamin C Homeostasis.

Plectin, a highly versatile cytolinker protein, is crucial for myofiber integrity and function. Accordingly, mutations in the human gene (PLEC) cause several rare diseases, denoted as plectinopathies, with most of them associated with progressive muscle weakness. Of several plectin isoforms expressed in skeletal muscle and the heart, P1d is the only isoform expressed exclusively in these tissues. Using high-resolution stimulated emission depletion (STED) microscopy, here we show that plectin is located within the gaps between individual α-actinin-positive Z-disks, recruiting and bridging them to desmin intermediate filaments (Ifs). Loss of plectin in myofibril bundles led to a complete loss of desmin Ifs. Loss of Z-disk-associated plectin isoform P1d led to disorganization of muscle fibers and slower relaxation of myofibrils upon mechanical strain, in line with an observed inhomogeneity of muscle ultrastructure. In addition to binding to α-actinin and thereby providing structural support, P1d forms a scaffolding platform for the chaperone-assisted selective autophagy machinery (CASA) by directly interacting with HSC70 and synpo2. In isoform-specific knockout (P1d-KO) mouse muscle and mechanically stretched plectin-deficient myoblasts, we found high levels of undigested filamin C, a bona fide substrate of CASA. Similarly, subjecting P1d-KO mice to forced swim tests led to accumulation of filamin C aggregates in myofibers, highlighting a specific role of P1d in tension-induced proteolysis activated upon high loads of physical exercise and muscle contraction.

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils.

In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.

Hypertrophic and Dilated Cardiomyopathy-Associated Troponin T Mutations R130C and ΔK210 Oppositely Affect Length-Dependent Calcium Sensitivity of Force Generation.

Length-dependent activation of calcium-dependent myocardial force generation provides the basis for the Frank-Starling mechanism. To directly compare the effects of mutations associated with hypertrophic cardiomyopathy and dilated cardiomyopathy, the native troponin complex in skinned trabecular fibers of guinea pigs was exchanged with recombinant heterotrimeric, human, cardiac troponin complexes containing different human cardiac troponin T subunits (hcTnT): hypertrophic cardiomyopathy-associated hcTnTR130C, dilated cardiomyopathy-associated hcTnTΔK210 or the wild type hcTnT (hcTnTWT) serving as control. Force-calcium relations of exchanged fibers were explored at short fiber length defined as 110% of slack length (L 0) and long fiber length defined as 125% of L 0 (1.25 L 0). At short fiber length (1.1 L 0), calcium sensitivity of force generation expressed by -log [Ca2+] required for half-maximum force generation (pCa50) was highest for the hypertrophic cardiomyopathy-associated mutation R130C (5.657 ± 0.019), intermediate for the wild type control (5.580 ± 0.028) and lowest for the dilated cardiomyopathy-associated mutation ΔK210 (5.325 ± 0.038). Lengthening fibers from 1.1 L 0 to 1.25 L 0 increased calcium sensitivity in fibers containing hcTnTR130C (delta-pCa50 = +0.030 ± 0.010), did not alter calcium sensitivity in the wild type control (delta-pCa50 = -0.001 ± 0.010), and decreased calcium sensitivity in fibers containing hcTnTΔK210 (delta-pCa50 = -0.034 ± 0.013). Length-dependent activation indicated by the delta-pCa50 was highly significantly (P < 0.001) different between the two mutations. We hypothesize that primary effects of mutations on length-dependent activation contribute to the development of the diverging phenotypes in hypertrophic and dilated cardiomyopathy.

Cycling Cross-Bridges Contribute to Thin Filament Activation in Human Slow-Twitch Fibers.

It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart. To explore myosin-induced activation of the thin filament in isometrically contracting human slow-twitch fibers, the endogenous troponin complex was exchanged for a well-characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (fsTn-IANBD). The exchange was ≈70% complete (n = 8). The relative contributions of calcium and strong binding cross-bridges to thin filament activation were dissected by increasing the concentration of calcium from relaxing (pCa 7.5) to saturating levels (pCa 4.5) before and after incubating the exchanged fibers in the myosin inhibitor para-aminoblebbistatin (AmBleb). At pCa 4.5, the relative contributions of calcium and strong binding cross-bridges to thin filament activation were ≈69 and ≈31%, respectively. Additionally, switching from isometric to isotonic contraction at pCa 4.5 revealed that strong binding cross-bridges contributed ≈29% to thin filament activation (i.e., virtually the same magnitude obtained with AmBleb). Thus, we showed through two different approaches that lowering the number of strong binding cross-bridges, at saturating calcium, significantly reduced the activation of the thin filament in human slow-twitch fibers. The contribution of myosin to activation resembled that which was previously reported in rat cardiac and rabbit fast-twitch muscle preparations. This method could be applied to slow-twitch human fibers obtained from the soleus muscle of cardiomyopathy patients. Such studies could lead to a better understanding of the effect of point mutations of the cardiac myosin head on the regulation of muscle contraction and could lead to better management by pharmacological approaches.

Infantile restrictive cardiomyopathy: cTnI-R170G/W impair the interplay of sarcomeric proteins and the integrity of thin filaments.

TNNI3 encoding cTnI, the inhibitory subunit of the troponin complex, is the main target for mutations leading to restrictive cardiomyopathy (RCM). Here we investigate two cTnI-R170G/W amino acid replacements, identified in infantile RCM patients, which are located in the regulatory C-terminus of cTnI. The C-terminus is thought to modulate the function of the inhibitory region of cTnI. Both cTnI-R170G/W strongly enhanced the Ca2+-sensitivity of skinned fibres, as is typical for RCM-mutations. Both mutants strongly enhanced the affinity of troponin (cTn) to tropomyosin compared to wildtype cTn, whereas binding to actin was either strengthened (R170G) or weakened (R170W). Furthermore, the stability of reconstituted thin filaments was reduced as revealed by electron microscopy. Filaments containing R170G/W appeared wavy and showed breaks. Decoration of filaments with myosin subfragment S1 was normal in the presence of R170W, but was irregular with R170G. Surprisingly, both mutants did not affect the Ca2+-dependent activation of reconstituted cardiac thin filaments. In the presence of the N-terminal fragment of cardiac myosin binding protein C (cMyBPC-C0C2) cooperativity of thin filament activation was increased only when the filaments contained wildtype cTn. No effect was observed in the presence of cTn containing R170G/W. cMyBPC-C0C2 significantly reduced binding of wildtype troponin to actin/tropomyosin, but not of both mutant cTn. Moreover, we found a direct troponin/cMyBPC-C0C2 interaction using microscale thermophoresis and identified cTnI and cTnT, but not cTnC as binding partners for cMyBPC-C0C2. Only cTn containing cTnI-R170G showed a reduced affinity towards cMyBPC-C0C2. Our results suggest that the RCM cTnI variants R170G/W impair the communication between thin and thick filament proteins and destabilize thin filaments.

Kinetic coupling of phosphate release, force generation and rate-limiting steps in the cross-bridge cycle.

A basic goal in muscle research is to understand how the cyclic ATPase activity of cross-bridges is converted into mechanical force. A direct approach to study the chemo-mechanical coupling between Pi release and the force-generating step is provided by the kinetics of force response induced by a rapid change in [Pi]. Classical studies on fibres using caged-Pi discovered that rapid increases in [Pi] induce fast force decays dependent on final [Pi] whose kinetics were interpreted to probe a fast force-generating step prior to Pi release. However, this hypothesis was called into question by studies on skeletal and cardiac myofibrils subjected to Pi jumps in both directions (increases and decreases in [Pi]) which revealed that rapid decreases in [Pi] trigger force rises with slow kinetics, similar to those of calcium-induced force development and mechanically-induced force redevelopment at the same [Pi]. A possible explanation for this discrepancy came from imaging of individual sarcomeres in cardiac myofibrils, showing that the fast force decay upon increase in [Pi] results from so-called sarcomere 'give'. The slow force rise upon decrease in [Pi] was found to better reflect overall sarcomeres cross-bridge kinetics and its [Pi] dependence, suggesting that the force generation coupled to Pi release cannot be separated from the rate-limiting transition. The reasons for the different conclusions achieved in fibre and myofibril studies are re-examined as the recent findings on cardiac myofibrils have fundamental consequences for the coupling between Pi release, rate-limiting steps and force generation. The implications from Pi-induced force kinetics of myofibrils are discussed in combination with historical and recent models of the cross-bridge cycle.

Early signs of architectural and biomechanical failure in isolated myofibers and immortalized myoblasts from desmin-mutant knock-in mice.

In striated muscle, desmin intermediate filaments interlink the contractile myofibrillar apparatus with mitochondria, nuclei, and the sarcolemma. The desmin network's pivotal role in myocytes is evident since mutations in the human desmin gene cause severe myopathies and cardiomyopathies. Here, we investigated skeletal muscle pathology in myofibers and myofibrils isolated from young hetero- and homozygous R349P desmin knock-in mice, which carry the orthologue of the most frequent human desmin missense mutation R350P. We demonstrate that mutant desmin alters myofibrillar cytoarchitecture, markedly disrupts the lateral sarcomere lattice and distorts myofibrillar angular axial orientation. Biomechanical assessment revealed a high predisposition to stretch-induced damage in fiber bundles of R349P mice. Notably, Ca2+-sensitivity and passive myofibrillar tension were decreased in heterozygous fiber bundles, but increased in homozygous fiber bundles compared to wildtype mice. In a parallel approach, we generated and subsequently subjected immortalized heterozygous R349P desmin knock-in myoblasts to magnetic tweezer experiments that revealed a significantly increased sarcolemmal lateral stiffness. Our data suggest that mutated desmin already markedly impedes myocyte structure and function at pre-symptomatic stages of myofibrillar myopathies.

Force Responses and Sarcomere Dynamics of Cardiac Myofibrils Induced by Rapid Changes in [Pi].

The second phase of the biphasic force decay upon release of phosphate from caged phosphate was previously interpreted as a signature of kinetics of the force-generating step in the cross-bridge cycle. To test this hypothesis without using caged compounds, force responses and individual sarcomere dynamics upon rapid increases or decreases in concentration of inorganic phosphate [Pi] were investigated in calcium-activated cardiac myofibrils. Rapid increases in [Pi] induced a biphasic force decay with an initial slow decline (phase 1) and a subsequent 3-5-fold faster major decay (phase 2). Phase 2 started with the distinct elongation of a single sarcomere, the so-called sarcomere "give". "Give" then propagated from sarcomere to sarcomere along the myofibril. Propagation speed and rate constant of phase 2 (k+Pi(2)) had a similar [Pi]-dependence, indicating that the kinetics of the major force decay (phase 2) upon rapid increase in [Pi] is determined by sarcomere dynamics. In contrast, no "give" was observed during phase 1 after rapid [Pi]-increase (rate constant k+Pi(1)) and during the single-exponential force rise (rate constant k-Pi) after rapid [Pi]-decrease. The values of k+Pi(1) and k-Pi were similar to the rate constant of mechanically induced force redevelopment (kTR) and Ca2+-induced force development (kACT) measured at same [Pi]. These results indicate that the major phase 2 of force decay upon a Pi-jump does not reflect kinetics of the force-generating step but results from sarcomere "give". The other phases of Pi-induced force kinetics that occur in the absence of "give" yield the same information as mechanically and Ca2+-induced force kinetics (k+Pi(1) ∼ k-Pi ∼ kTR ∼ kACT). Model simulations indicate that Pi-induced force kinetics neither enable the separation of Pi-release from the rate-limiting transition f into force states nor differentiate whether the "force-generating step" occurs before, along, or after the Pi-release.

Postnatal Development of Right Ventricular Myofibrillar Biomechanics in Relation to the Sarcomeric Protein Phenotype in Pediatric Patients with Conotruncal Heart Defects.

BACKGROUND: The postnatal development of myofibrillar mechanics, a major determinant of heart function, is unknown in pediatric patients with tetralogy of Fallot and related structural heart defects. We therefore determined the mechanical properties of myofibrils isolated from right ventricular tissue samples from such patients in relation to the developmental changes of the isoforms expression pattern of key sarcomere proteins involved in the contractile process. METHODS AND RESULTS: Tissue samples from the infundibulum obtained during surgery from 25 patients (age range 15 days to 11 years, median 7 months) were split into half for mechanical investigations and expression analysis of titin, myosin heavy and light chain 1, troponin-T, and troponin-I. Of these proteins, fetal isoforms of only myosin light chain 1 (ALC-1) and troponin-I (ssTnI) were highly expressed in neonates, amounting to, respectively, 40% and 80%, while the other proteins had switched to the adult isoforms before or around birth. ALC-1 and ssTnI expression subsequently declined monoexponentially with a halftime of 4.3 and 5.8 months, respectively. Coincident with the expression of ssTnI, Ca(2+) sensitivity of contraction was high in neonates and subsequently declined in parallel with the decline in ssTnI expression. Passive tension positively correlated with Ca(2+) sensitivity but not with titin expression. Contraction kinetics, maximal Ca(2+)-activated force, and the fast phase of the biphasic relaxation positively correlated with the expression of ALC-1. CONCLUSIONS: The developmental changes in myofibrillar biomechanics can be ascribed to fetal-to-adult isoform transition of key sarcomeric proteins, which evolves regardless of the specific congenital cardiac malformations in our pediatric patients.

Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics.

Cardiac titin is the main determinant of sarcomere stiffness during diastolic relaxation. To explore whether titin stiffness affects the kinetics of cardiac myofibrillar contraction and relaxation, we used subcellular myofibrils from the left ventricles of homozygous and heterozygous N2B-knockout mice which express truncated cardiac titins lacking the unique elastic N2B region. Compared with myofibrils from wild-type mice, myofibrils from knockout and heterozygous mice exhibit increased passive myofibrillar stiffness. To determine the kinetics of Ca(2+)-induced force development (rate constant kACT), myofibrils from knockout, heterozygous and wild-type mice were stretched to the same sarcomere length (2.3 µm) and rapidly activated with Ca(2+). Additionally, mechanically induced force-redevelopment kinetics (rate constant kTR) were determined by slackening and re-stretching myofibrils during Ca(2+)-mediated activation. Myofibrils from knockout mice exhibited significantly higher kACT, kTR and maximum Ca(2+)-activated tension than myofibrils from wild-type mice. By contrast, the kinetic parameters of biphasic force relaxation induced by rapidly reducing [Ca(2+)] were not significantly different among the three genotypes. These results indicate that increased titin stiffness promotes myocardial contraction by accelerating the formation of force-generating cross-bridges without decelerating relaxation.

Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy.

Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.

Kinetic mechanism of Ca²âº-controlled changes of skeletal troponin I in psoas myofibrils.

Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca(2+)] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca(2+)-activation kinetics. Whereas the fast phase (k(obs)∼1000 s(-1)) is only observed in this study, where kinetics were induced by Ca(2+), the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692-2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca(2+) ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca(2+) binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca(2+) sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation.

ATP binding and cross-bridge detachment steps during full Ca²âº activation: comparison of myofibril and muscle fibre mechanics by sinusoidal analysis.

Single myofibrils 50­60 µm length and 2­3 µm diameter were isolated from rabbit psoas muscle fibres, and cross-bridge kinetics were studied by small perturbations of the length (∼0.2%) over a range of 15 frequencies (1­250 Hz). The experiments were performed at 15◦C in the presence of 0.05­10 mM MgATP, 8mM phosphate (Pi), 200 mM ionic strength with KAc (acetate), pCa 4.35­4.65, and pH 7.0. Two exponential processes, B and C, were resolved in tension transients. Their apparent rate constants (2πb and 2πc) increased as the [MgATP] was raised from 0.05 mM to 1mM, and then reached saturation at [MgATP] ≥ 1. Given that these rate constants were similar (c/b ∼1.7) at [Pi] ≥ 4 mM, they were combined to achieve an accurate estimate of the kinetic constants: their sum and product were analysed as functions of [MgATP]. These analyses yielded K1 =2.91 ± 0.31 mM −1, k2 =288 ± 36 s−1, and k−2 =10 ± 21 s−1 (±95% confidence limit, n =13 preparations), based on the cross-bridge model: AM+ATP ↔ (step 1) AM.ATP ↔ (step 2) A+M.ATP, where K1 is the ATP association constant (step 1), k2 is the rate constant of the cross-bridge detachment (step 2), and k−2 is the rate constant of its reversal step. These kinetic constants are respectively comparable to those observed in single fibres from rabbit psoas (K1 =2.35 ± 0.31 mM −1, k2 =243 ± 22 s−1, and k−2 =6 ± 14 s−1; n =8 preparations) when analysed by the same methods and under the same experimental conditions. These values are respectively not significantly different from those obtained in myofibrils, indicating that the same kinetic constants can be deduced from myofibril and muscle fibre studies, in terms of ATP binding and cross-bridge detachments steps. The fact that K1 in myofibrils is 1.2 times that in fibres (P≈0.05) may be explained by a small concentration gradient of ATP, ADP and/or Pi in single fibres.

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish.

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.

Kinetics of cardiac sarcomeric processes and rate-limiting steps in contraction and relaxation.

The sarcomere is the core structure responsible for active mechanical heart function. It is formed primarily by myosin, actin, and titin filaments. Cyclic interactions occur between the cross-bridges of the myosin filaments and the actin filaments. The forces generated by these cyclic interactions provide the molecular basis for cardiac pressure, while the motion produced by these interactions provides the basis for ejection. The cross-bridge cycle is controlled by upstream mechanisms located in the membrane and by downstream mechanisms inside the sarcomere itself. These downstream mechanisms involve the Ca(2+)-controlled conformational change of the regulatory proteins troponin and tropomyosin and strong cooperative interactions between neighboring troponin-tropomyosin units along the actin filament. The kinetics of upstream and downstream processes have been measured in intact and demembranated myocardial preparations. This review outlines a conceptual model of the timing of these processes during the individual mechanical heart phases. Particular focus is given to kinetic data from studies on contraction-relaxation cycles under mechanical loads. Evidence is discussed that the dynamics of cardiac contraction and relaxation are determined mainly by sarcomeric downstream mechanisms, in particular by the kinetics of the cross-bridge cycle. The rate and extent of ventricular pressure development is essentially subjected to the mechanistic principles of cross-bridge action and its upstream and downstream regulation. Sarcomere relengthening during myocardial relaxation plays a key role in the rapid decay of ventricular pressure and in early diastolic filling.

The cardiac troponin C mutation Leu29Gln found in a patient with hypertrophic cardiomyopathy does not alter contractile parameters in skinned murine myocardium.

The present study investigates the effects of the first mutation of troponin C (hcTnC(L29Q)) found in a patient with hypertrophic cardiomyopathy (HCM) on force-pCa relations and the interplay with phosphorylation of sarcomeric PKA substrates. In triton-skinned murine cardiac fibers, the endogenous mcTnC was extracted and the fibers were subsequently reconstituted with recombinant wild-type and mutant hcTnC. Force-pCa relations of preparations containing hcTnC(L29Q) or hcTnC(WT) were similar. Incubation of fibers reconstituted with the recombinant proteins with phosphatase to dephosphorylate sarcomeric PKA substrates induced an increase in Ca2+ sensitivity, slightly more pronounced (0.04 pCa units) in hcTnC(L29Q)-containing fibers. Incubation of the dephosphorylated fibers with PKA induced significant rightward shifts of force-pCa relations of similar magnitude with both, hcTnC(L29Q) and hcTnC(WT). No significant effects of hcTnC(L29Q) on the velocity of unloaded shortening were observed. In conclusion, no major differences in contractile parameters of preparations containing hcTnC(L29Q) compared to hcTnC(WT) were observed. Therefore, it appears unlikely that hcTnC(L29Q) induces the development of HCM by affecting the regulation of Ca2+-activated force and interference with PKA-mediated modulation of the Ca2+ sensitivity of contraction.