Regulating Striated Muscle Contraction: Through Thick and Thin.

Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular calcium concentration. However, recent studies have elucidated a new class of regulatory mechanisms, based on the myosin-containing thick filament, that control the strength and speed of contraction by modulating the availability of myosin motors for the interaction with actin. This review summarizes the mechanisms of thin and thick filament activation that regulate the contractility of skeletal and cardiac muscle. A novel dual-filament paradigm of muscle regulation is emerging, in which the dynamics of force generation depends on the coordinated activation of thin and thick filaments. We highlight the interfilament signaling pathways based on titin and myosin-binding protein-C that couple thin and thick filament regulatory mechanisms. This dual-filament regulation mediates the length-dependent activation of cardiac muscle that underlies the control of the cardiac output in each heartbeat.

Titin force in muscle cells alters lattice order, thick and thin filament protein formation.

Skeletal muscle force production is increased at longer compared to shorter muscle lengths because of length-dependent priming of thick filament proteins in the contractile unit before contraction. Using small-angle X-ray diffraction in combination with a mouse model that specifically cleaves the stretch-sensitive titin protein, we found that titin cleavage diminished the length-dependent priming of the thick filament. Strikingly, a titin-sensitive, length-dependent priming was also present in thin filaments, which seems only possible via bridge proteins between thick and thin filaments in resting muscle, potentially myosin-binding protein C. We further show that these bridges can be forcibly ruptured via high-speed stretches. Our results advance a paradigm shift to the fundamental regulation of length-dependent priming, with titin as the key driver.

Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A.

PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to ß-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca2+ sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive. Here, we specifically altered the phosphorylation level of cTnI in heart muscle cells and characterized the structural and functional effects at different levels of background phosphorylation of cMyBP-C and with two different SLs. We found Ser22/23 bisphosphorylation of cTnI was indispensable for the enhancement of length-dependent activation by PKA, as was cMyBP-C phosphorylation. This high level of coordination between cTnI and cMyBP-C may suggest coupling between their regulatory mechanisms. Further evidence for this was provided by our finding that cardiac troponin (cTn) can directly interact with cMyBP-C in vitro, in a phosphorylation- and Ca2+-dependent manner. In addition, bisphosphorylation at Ser22/Ser23 increased Ca2+ sensitivity at long SL in the presence of endogenously phosphorylated cMyBP-C. When cMyBP-C was dephosphorylated, bisphosphorylation of cTnI increased Ca2+ sensitivity and decreased cooperativity at both SLs, which may translate to deleterious effects in physiological settings. Our results could have clinical relevance for disease pathways, where PKA phosphorylation of cTnI may be functionally uncoupled from cMyBP-C phosphorylation due to mutations or haploinsufficiency.

Stretch-induced reactive oxygen species contribute to the Frank-Starling mechanism.

Myocardial stretch physiologically activates NADPH oxidase 2 (NOX2) to increase reactive oxygen species (ROS) production. Although physiological low-level ROS are known to be important as signalling molecules, the role of stretch-induced ROS in the intact myocardium remains unclear. To address this, we investigated the effects of stretch-induced ROS on myocardial cellular contractility and calcium transients in C57BL/6J and NOX2-/- mice. Axial stretch was applied to the isolated cardiomyocytes using a pair of carbon fibres attached to both cell ends to evaluate stretch-induced modulation in the time course of the contraction curve and calcium transient, as well as to evaluate maximum cellular elastance, an index of cellular contractility, which is obtained from the end-systolic force-length relationship. In NOX2-/- mice, the peak calcium transient was not altered by stretch, as that in wild-type mice, but the lack of stretch-induced ROS delayed the rise of calcium transients and reduced contractility. Our mathematical modelling studies suggest that the augmented activation of ryanodine receptors by stretch-induced ROS causes a rapid and large increase in the calcium release flux, resulting in a faster rise in the calcium transient. The slight increase in the magnitude of calcium transients is offset by a decrease in sarcoplasmic reticulum calcium content as a result of ROS-induced calcium leakage, but the faster rise in calcium transients still maintains higher contractility. In conclusion, a physiological role of stretch-induced ROS is to increase contractility to counteract a given preload, that is, it contributes to the Frank-Starling law of the heart. KEY POINTS: Myocardial stretch increases the production of reactive oxygen species by NADPH oxidase 2. We used NADPH oxidase 2 knockout mice to elucidate the physiological role of stretch-induced reactive oxygen species in the heart. We showed that stretch-induced reactive oxygen species modulate the rising phase of calcium transients and increase myocardial contractility. A mathematical model simulation study demonstrated that rapid activation of ryanodine receptors by reactive oxygen species is important for increased contractility. This response is advantageous for the myocardium, which must contract against a given preload.

Effects of vigorous isometric muscle contraction on titin stiffness-related contractile properties in rat fast-twitch muscles.

This study was conducted to examine the effects of an acute bout of vigorous isometric contractions on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ until the force was reduced to ∼50% of the initial force. Immediately after cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. The stimulation resulted in a decrease in the titin-based passive force. The amounts of fragmented titin were unchanged by the stimulation. Protein kinase Cα-treatment increased the passive force in stimulated fibers to resting levels. The stimulation had no effect on the maximum Ca2+-activated force (max Ca2+ force) at a sarcomere length (SL) of 2.4 µm and decreased myofibrillar (my)-Ca2+ sensitivity at 2.6-µm SL. Stretching the SL to 3.0 µm led to the augmentation of the max Ca2+ force and my-Ca2+ sensitivity in both rested and stimulated fibers. For the max Ca2+ force, the extent of the increase was smaller in stimulated than in rested fibers, whereas for my-Ca2+ sensitivity, it was higher in stimulated than in rested fibers. These results suggest that vigorous isometric contractions decrease the titin-based passive force, possibly because of a reduction in phosphorylation by protein kinase Cα, and that the decreased titin stiffness may contribute, at least in part, to muscle fatigue.

Mechanisms of Frank-Starling law of the heart and stretch activation in striated muscles may have a common molecular origin.

Vertebrate cardiac muscle generates progressively larger systolic force when the end diastolic chamber volume is increased, a property called the "Frank-Starling Law", or "length dependent activation (LDA)". In this mechanism a larger force develops when the sarcomere length (SL) increased, and the overlap between thick and thin filament decreases, indicating increased production of force per unit length of the overlap. To account for this phenomenon at the molecular level, we examined several hypotheses: as the muscle length is increased, (1) lattice spacing decreases, (2) Ca2+ sensitivity increases, (3) titin mediated rearrangement of myosin heads to facilitate actomyosin interaction, (4) increased SL activates cross-bridges (CBs) in the super relaxed state, (5) increased series stiffness at longer SL promotes larger elementary force/CB to account for LDA, and (6) stretch activation (SA) observed in insect muscles and LDA in vertebrate muscles may have similar mechanisms. SA is also known as delayed tension or oscillatory work, and universally observed among insect flight muscles, as well as in vertebrate skeletal and cardiac muscles. The sarcomere stiffness observed in relaxed muscles may significantly contributes to the mechanisms of LDA. In vertebrate striated muscles, the sarcomere stiffness is mainly caused by titin, a single filamentary protein spanning from Z-line to M-line and tightly associated with the myosin thick filament. In insect flight muscles, kettin connects Z-line and the thick filament to stabilize the sarcomere structure. In vertebrate cardiac muscles, titin plays a similar role, and may account for LDA and may constitute a molecular mechanism of Frank-Starling response.

Dilated cardiomyopathy mutation (R174W) in troponin T attenuates the length-mediated increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Alterations in length-dependent activation (LDA) may constitute a mechanism by which cardiomyopathy mutations lead to deleterious phenotypes and compromised heart function, because LDA underlies the molecular basis by which the heart tunes myocardial force production on a beat-to-beat basis (Frank-Starling mechanism). In this study, we investigated the effect of DCM-linked mutation (R173W) in human cardiac troponin T (TnT) on myofilament LDA. R173W mutation is associated with left ventricular dilatation and systolic dysfunction and is found in multiple families. R173W mutation is in the central region (residues 80-180) of TnT, which is known to be important for myofilament cooperativity and cross-bridge (XB) recruitment. Steady-state and dynamic contractile parameters were measured in detergent-skinned guinea pig left ventricular muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or mutant TnT (TnTR174W; guinea pig analog of human R173W mutation) at two different sarcomere lengths (SL): short (1.9 µm) and long (2.3 µm). TnTR174W decreased pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at long than at short SL; for example, pCa50 decreased by 0.12 pCa units at long SL and by 0.06 pCa units at short SL. Differential changes in pCa50 at short and long SL attenuated the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTR174W fibers; ΔpCa50 was 0.10 units in TnTWT fibers but only 0.04 units in TnTR174W fibers. Furthermore, TnTR174W blunted the SL-dependent increase in the magnitude of XB recruitment. Our observations suggest that the R173W mutation in human cardiac TnT may impair Frank-Starling mechanism.NEW & NOTEWORTHY This work characterizes the effect of dilated cardiomyopathy mutation in cardiac troponin T (TnTR174W) on myofilament length-dependent activation. TnTR174W attenuates the length-dependent increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins.

The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 µm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

Altered myofilament structure and function in dogs with Duchenne muscular dystrophy cardiomyopathy.

AIM: Duchenne Muscular Dystrophy (DMD) is associated with progressive depressed left ventricular (LV) function. However, DMD effects on myofilament structure and function are poorly understood. Golden Retriever Muscular Dystrophy (GRMD) is a dog model of DMD recapitulating the human form of DMD. OBJECTIVE: The objective of this study is to evaluate myofilament structure and function alterations in GRMD model with spontaneous cardiac failure. METHODS AND RESULTS: We have employed synchrotron X-rays diffraction to evaluate myofilament lattice spacing at various sarcomere lengths (SL) on permeabilized LV myocardium. We found a negative correlation between SL and lattice spacing in both sub-epicardium (EPI) and sub-endocardium (ENDO) LV layers in control dog hearts. In the ENDO of GRMD hearts this correlation is steeper due to higher lattice spacing at short SL (1.9µm). Furthermore, cross-bridge cycling indexed by the kinetics of tension redevelopment (ktr) was faster in ENDO GRMD myofilaments at short SL. We measured post-translational modifications of key regulatory contractile proteins. S-glutathionylation of cardiac Myosin Binding Protein-C (cMyBP-C) was unchanged and PKA dependent phosphorylation of the cMyBP-C was significantly reduced in GRMD ENDO tissue and more modestly in EPI tissue. CONCLUSIONS: We found a gradient of contractility in control dogs' myocardium that spreads across the LV wall, negatively correlated with myofilament lattice spacing. Chronic stress induced by dystrophin deficiency leads to heart failure that is tightly associated with regional structural changes indexed by increased myofilament lattice spacing, reduced phosphorylation of regulatory proteins and altered myofilament contractile properties in GRMD dogs.

Role of the C-terminus mobile domain of cardiac troponin I in the regulation of thin filament activation in skinned papillary muscle strips.

The C-terminus mobile domain of cTnI (cTnI-MD) is a highly conserved region which stabilizes the actin-cTnI interaction during the diastole. Upon Ca2+-binding to cTnC, cTnI-MD participates in a regulatory switching that involves cTnI to switch from interacting with actin toward interacting with the Ca2+-regulatory domain of cTnC. Despite many studies targeting the cTnI-MD, the role of this region in the length-dependent activation of cardiac contractility is yet to be determined. The present study investigated the functional consequences of losing the entire cTnI-MD in cTnI(1-167) truncation mutant, as it was exchanged for endogenous cTnI in skinned rat papillary muscle fibers. The influence of cTnI-MD truncation on the extent of the N-domain of cTnC hydrophobic cleft opening and the steady-state force as a function of sarcomere length (SL), cross-bridge state, and [Ca2+] was assessed using the simultaneous in situ time-resolved FRET and force measurements at short (1.8 µm) and long (2.2 µm) SLs. Our results show the significant role of cTnI-MD in the length dependent thin filament activation and the coupling between thin and thick filament regulations affected by SL. Our results also suggest that cTnI-MD transmits the effects of SL change to the core of troponin complex.

Cardiomyopathies and Related Changes in Contractility of Human Heart Muscle.

About half of hypertrophic and dilated cardiomyopathies cases have been recognized as genetic diseases with mutations in sarcomeric proteins. The sarcomeric proteins are involved in cardiomyocyte contractility and its regulation, and play a structural role. Mutations in non-sarcomeric proteins may induce changes in cell signaling pathways that modify contractile response of heart muscle. These facts strongly suggest that contractile dysfunction plays a central role in initiation and progression of cardiomyopathies. In fact, abnormalities in contractile mechanics of myofibrils have been discovered. However, it has not been revealed how these mutations increase risk for cardiomyopathy and cause the disease. Much research has been done and still much is being done to understand how the mechanism works. Here, we review the facts of cardiac myofilament contractility in patients with cardiomyopathy and heart failure.

Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres.

The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation.

Hypertrophic cardiomyopathy mutation in cardiac troponin T (R95H) attenuates length-dependent activation in guinea pig cardiac muscle fibers.

The central region of cardiac troponin T (TnT) is important for modulating the dynamics of muscle length-mediated cross-bridge recruitment. Therefore, hypertrophic cardiomyopathy mutations in the central region may affect cross-bridge recruitment dynamics to alter myofilament Ca2+ sensitivity and length-dependent activation of cardiac myofilaments. Given the importance of the central region of TnT for cardiac contractile dynamics, we studied if hypertrophic cardiomyopathy-linked mutation (TnTR94H)-induced effects on contractile function would be differently modulated by sarcomere length (SL). Recombinant wild-type TnT (TnTWT) and the guinea pig analog of the human R94H mutation (TnTR95H) were reconstituted into detergent-skinned cardiac muscle fibers from guinea pigs. Steady-state and dynamic contractile measurements were made at short and long SLs (1.9 and 2.3 µm, respectively). Our results demonstrated that TnTR95H increased pCa50 (-log of free Ca2+ concentration) to a greater extent at short SL; TnTR95H increased pCa50 by 0.11 pCa units at short SL and 0.07 pCa units at long SL. The increase in pCa50 associated with an increase in SL from 1.9 to 2.3 µm (ΔpCa50) was attenuated nearly twofold in TnTR95H fibers; ΔpCa50 was 0.09 pCa units for TnTWT fibers but only 0.05 pCa units for TnTR95H fibers. The SL dependency of rate constants of cross-bridge distortion dynamics and tension redevelopment was also blunted by TnTR95H Collectively, our observations on the SL dependency of pCa50 and rate constants of cross-bridge distortion dynamics and tension redevelopment suggest that mechanisms underlying the length-dependent activation cardiac myofilaments are attenuated by TnTR95HNEW & NOTEWORTHY Mutant cardiac troponin T (TnTR95H) differently affects myofilament Ca2+ sensitivity at short and long sarcomere length, indicating that mechanisms underlying length-dependent activation are altered by TnTR95H TnTR95H enhances myofilament Ca2+ sensitivity to a greater extent at short sarcomere length, thus attenuating the length-dependent increase in myofilament Ca2+ sensitivity.

Functional significance of C-terminal mobile domain of cardiac troponin I.

Ca2+-regulation of cardiac contractility is mediated through the troponin complex, which comprises three subunits: cTnC, cTnI, and cTnT. As intracellular [Ca2+] increases, cTnI reduces its binding interactions with actin to primarily interact with cTnC, thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD), the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD, we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1-167) and cTnI(1-193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers, and their influence on Ca2+-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9 µm) and long (2.2 µm) sarcomere lengths (SLs). Our results show that the cTnI(1-167) truncation diminished the SL-induced increase in Ca2+-sensitivity of contraction, but not the SL-dependent increase in maximal tension, suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT), both truncations displayed greater Ca2+-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore, cTnI(1-167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.

Recent developments in understanding the length dependence of contractile response of skeletal muscle.

Maximal active force of skeletal muscle contraction occurs at a sarcomere length where overlap of thick and thin filaments is optimal. However, the interaction of muscle length and active force is complicated. Active force, is the force generated by energy-requiring processes. To calculate active force, passive force provided by in-parallel structures must be subtracted from total force. Sarcomere length will change during a contraction with constant muscle-tendon length, due to tendon stretch. Passive force therefore changes during the contraction. Taking this into account, it has been demonstrated that there is less length dependence of fatigue than previously thought. The remaining difference may be associated with length dependence of activation, a property that is evident with submaximal activation. The sarcomere length at which peak contraction occurs is longer than the length that gives optimal overlap of the filaments and this shift of optimal length appears to be due to increased Ca2+ sensitivity. The increased Ca2+ sensitivity occurs because at longer lengths, the myofilaments are closer together allowing greater force than expected. However, the potential for length-dependent activation has been challenged. Submaximal contractions obtained by recruitment of fewer motor units but with maximal stimulation across different muscle lengths still demonstrate length-dependent activation. In contrast, contractions with similar absolute electromyographic signal magnitude at different lengths do not demonstrate length-dependent activation. Recent work has improved our understanding of how sarcomere length impacts the force of contraction but also reveals inadequacies in our knowledge that need to be addressed by additional research.

Sarcomere length dependent effects on the interaction between cTnC and cTnI in skinned papillary muscle strips.

Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how the sarcomeres detect the length changes and convert them into altered activation of thin filament. In this study we investigated how the C-domain of cardiac troponin I (cTnI) functionally and structurally responds to the comprehensive effects of the Ca(2+), crossbridge, and sarcomere length of chemically skinned myocardial preparations. Using our in situ technique which allows for simultaneous measurements of time-resolved FRET and mechanical force of the skinned myocardial preparations, we measured changes in the FRET distance between cTnI(167C) and cTnC(89C), labeled with FRET donor and acceptor, respectively, as a function of [Ca(2+)], crossbridge state and sarcomere length of the skinned muscle preparations. Our results show that [Ca(2+)], cross-bridge feedback and sarcomere length have different effects on the structural transition of the C-domain cTnI. In particular, the interplay between crossbridges and sarcomere length has significant impacts on the functional structural change of the C-domain of cTnI in the relaxed state. These novel observations suggest the importance of the C-domain of cTnI and the dynamic and complex interplay between various components of myofilament in the LDA mechanism.

Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 µm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 µm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 µm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 µm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart.

Dystrophic cardiomyopathy: role of the cardiac myofilaments.

Dystrophic cardiomyopathy arises from mutations in the dystrophin gene. Dystrophin forms part of the dystrophin glycoprotein complex and is postulated to act as a membrane stabilizer, protecting the sarcolemma from contraction-induced damage. Duchenne muscular dystrophy (DMD) is the most severe dystrophinopathy, caused by a total absence of dystrophin. Patients with DMD present with progressive skeletal muscle weakness and, because of treatment advances, a cardiac component of the disease (i.e., dystrophic cardiomyopathy) has been unmasked later in disease progression. The role that myofilaments play in dystrophic cardiomyopathy is largely unknown and, as such, this study aimed to address cardiac myofilament function in a mouse model of muscular dystrophy. To assess the effects of DMD on myofilament function, isolated permeabilized cardiomyocytes of wild-type (WT) littermates and Dmdmdx-4cv mice were attached between a force transducer and motor and subjected to contractile assays. Maximal tension and rates of force development (indexed by the rate constant, k tr) were similar between WT and Dmdmdx-4cv cardiac myocyte preparations. Interestingly, Dmdmdx-4cv cardiac myocytes exhibited greater sarcomere length dependence of peak power output compared to WT myocyte preparations. These results suggest dystrophin mitigates length dependence of activation and, in the absence of dystrophin, augmented sarcomere length dependence of myocyte contractility may accelerate ventricular myocyte contraction-induced damage and contribute to dystrophic cardiomyopathy. Next, we assessed if mavacamten, a small molecule modulator of thick filament activation, would mitigate contractile properties observed in Dmdmdx-4cv permeabilized cardiac myocyte preparations. Mavacamten decreased maximal tension and k tr in both WT and Dmdmdx-4cv cardiac myocytes, while also normalizing the length dependence of peak power between WT and Dmdmdx-4cv cardiac myocyte preparations. These results highlight potential benefits of mavacamten (i.e., reduced contractility while maintaining exquisite sarcomere length dependence of power output) as a treatment for dystrophic cardiomyopathy associated with DMD.

Eccentric muscle contraction potentiates titin stiffness-related contractile properties in rat fast-twitch muscles.

This study was conducted to examine the effects of an acute bout of eccentric muscle contraction (ECC) on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ to undergo 200 repeated ECCs. Immediately after the cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. Small heat shock protein αB-crystallin in the muscle fraction enriched for myofibrillar proteins was increased by ECC. ECC resulted in an increase in the titin-based passive force. Protein kinase A-treatment decreased the passive force only in ECC-subjected but not in rested fibers. ECC decreased the maximum Ca2+-activated force at a sarcomere length (SL) of 2.4 µm and had no effect on myofibrillar-Ca2+ sensitivity at 2.6-µm SL. In both rested and ECC-subjected fibers, these two variables were higher at 3.0-µm SL than at 2.4- or 2.6-µm SL. The differences in the two variables between the short and long SLs were greater in ECC-subjected than in rested fibers. These results indicate that an acute bout of ECC potentiates titin-based passive force, maximum active force at long SLs, and length-dependent activation and suggest that this potentiation may resist muscle fatigue in the muscles of the exercising body.NEW & NOTEWORTHY It remains unclear whether eccentric contraction of skeletal muscle affects titin stiffness-related contractile properties. Here, we provide evidence that an acute bout of eccentric contraction can potentiate titin-based passive force, maximum active force at long sarcomere lengths, and length-dependent activation. This potentiation may resist muscle fatigue in the muscles of the exercising body.