Microtubule destabilization with colchicine increases the work output of myocardial slices.

Cardiac microtubules have recently been implicated in mechanical dysfunction during heart failure. However, systemic intolerance and non-cardiac effects of microtubule-depolymerizing compounds have made it challenging to determine the effect of microtubules on myocardial performance. Herein, we leverage recent advancements in living myocardial slices to develop a stable working preparation that recapitulates the complexity of diastole by including early and late phases of diastolic filling. To determine the effect of cardiac microtubule depolymerization on diastolic performance, myocardial slices were perfused with oxygenated media to maintain constant isometric twitch forces for more than 90 min. Force-length work loops were collected before and after 90 min of treatment with either DMSO (vehicle) or colchicine (microtubule depolymerizer). A trapezoidal stretch was added prior to the beginning of ventricular systole to mimic late-stage-diastolic filling driven by atrial systole. Force-length work loops were obtained at fixed preload and afterload, and tissue velocity was obtained during diastole as an analog to trans-mitral Doppler. In isometric twitches, microtubule destabilization accelerated force development, relaxation kinetics, and decreased end diastolic stiffness. In work loops, microtubule destabilization increased stroke length, myocardial output, accelerated isometric contraction and relaxation, and increased the amplitude of early filling. Taken together, these results indicate that the microtubule destabilizer colchicine can improve diastolic performance by accelerating isovolumic relaxation and early filling leading to increase in myocardial work output.

Strain rate of stretch affects crossbridge detachment during relaxation of intact cardiac trabeculae.

Mechanical Control of Relaxation refers to the dependence of myocardial relaxation on the strain rate just prior to relaxation, but the mechanisms of enhanced relaxation are not well characterized. This study aimed to characterize how crossbridge kinetics varied with strain rate and time-to-stretch as the myocardium relaxed in early diastole. Ramp-stretches of varying rates (amplitude = 1% muscle length) were applied to intact rat cardiac trabeculae following a load-clamp at 50% of the maximal developed twitch force, which provides a first-order estimate of ejection and coupling to an afterload. The resultant stress-response was calculated as the difference between the time-dependent stress profile between load-clamped twitches with and without a ramp-stretch. The stress-response exhibited features of the step-stretch response of activated, permeabilized myocardium, such as distortion-dependent peak stress, rapid force decay related to crossbridge detachment, and stress recovery related to crossbridge recruitment. The peak stress was strain rate dependent, but the minimum stress and the time-to-minimum stress values were not. The initial rapid change in the stress-response indicates enhanced crossbridge detachment at higher strain rates during relaxation in intact cardiac trabeculae. Physiologic considerations, such as time-varying calcium, are discussed as potential limitations to fitting these data with traditional distortion-recruitment models of crossbridge activity.

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by ~24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.

The distinctive mechanical and structural signatures of residual force enhancement in myofibers.

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titin's function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE.

Heart rate changes and myocardial sodium.

Historic studies with sodium ion (Na+ ) micropipettes and first-generation fluorescent probes suggested that an increase in heart rate results in higher intracellular Na+ -levels. Using a dual fluorescence indicator approach, we simultaneously assessed the dynamic changes in intracellular Na+ and calcium (Ca2+ ) with measures of force development in isolated excitable myocardial strip preparations from rat and human left ventricular myocardium at different stimulation rates and modeled the Na+ -effects on the sodium-calcium exchanger (NCX). To gain further insight into the effects of heart rate on intracellular Na+ -regulation and sodium/potassium ATPase (NKA) function, Na+ , and potassium ion (K+ ) levels were assessed in the coronary effluent (CE) of paced human subjects. Increasing the stimulation rate from 60/min to 180/min led to a transient Na+ -peak followed by a lower Na+ -level, whereas the return to 60/min had the opposite effect leading to a transient Na+ -trough followed by a higher Na+ -level. The presence of the Na+ -peak and trough suggests a delayed regulation of NKA activity in response to changes in heart rate. This was clinically confirmed in the pacing study where CE-K+ levels were raised above steady-state levels with rapid pacing and reduced after pacing cessation. Despite an initial Na+ peak that is due to a delayed increase in NKA activity, an increase in heart rate was associated with lower, and not higher, Na+ -levels in the myocardium. The dynamic changes in Na+ unveil the adaptive role of NKA to maintain Na+ and K+ -gradients that preserve membrane potential and cellular Ca2+ -hemostasis.

Inorganic phosphate accelerates cardiac myofilament relaxation in response to lengthening.

Myocardial relaxation in late systole is enhanced by increasing velocities of lengthening. Given that inorganic phosphate (Pi) can rebind to the force-producing myosin enzyme prior to MgADP release and hasten crossbridge detachment, we hypothesized that myocardial relaxation in late systole would be further enhanced by lengthening in the presence of Pi. Wistar rat left ventricular papillary muscles were attached to platinum clips, placed between a force transducer and a length motor at room temperature, and bathed in Krebs solution with 1.8 mM Ca2+ and varying Pi of 0, 1, 2, and 5 mM. Tension transients were elicited by electrical stimulation at 1 Hz. Peak tension was significantly enhanced by Pi: 0.593 ± 0.088 mN mm-2 at 0 mM Pi and 0.817 ± 0.159 mN mm-2 at 5 mM Pi (mean ± SEM, p < 0.01 by ANCOVA). All temporal characteristics of the force transient were significantly shortened with increasing Pi, e.g., time-to-50% recovery was shortened from 305 ± 14 ms at 0 mM Pi to 256 ± 10 ms at 5 mM Pi (p < 0.01). A 1% lengthening stretch with varying duration of 10-200 ms was applied at time-to-50% recovery during the descending phase of the force transient. Matching lengthening stretches were also applied when the muscle was not stimulated, thus providing a control for the passive viscoelastic response. After subtracting the passive from the active force response, the resulting myofilament response demonstrated features of faster myofilament relaxation in response to the stretch. For example, time-to-70% relaxation with 100 ms lengthening duration was shortened by 8.8 ± 6.8 ms at 0 Pi, 19.6 ± 4.8* ms at 1 mM Pi, 31.0 ± 5.6* ms at 2 Pi, and 25.6 ± 5.3* ms at 5 mM Pi (*p < 0.01 compared to no change). Using skinned myocardium, half maximally calcium-activated myofilaments underwent a 1% quick stretch, and the tension response was subjected to analysis for sensitivity of myosin detachment rate to stretch, g 1, at various Pi concentrations. The parameter g 1 was enhanced from 15.39 ± 0.35 at 0 Pi to 22.74 ± 1.31 s-1/nm at 8 Pi (p < 0.01). Our findings suggest that increasing Pi at the myofilaments enhances lengthening-induced relaxation by elevating the sensitivity of myosin crossbridge detachment due to lengthening and thus speed the transition from late-systole to early-diastole.

Defects in the Proteome and Metabolome in Human Hypertrophic Cardiomyopathy.

BACKGROUND: Defects in energetics are thought to be central to the pathophysiology of hypertrophic cardiomyopathy (HCM); yet, the determinants of ATP availability are not known. The purpose of this study is to ascertain the nature and extent of metabolic reprogramming in human HCM, and its potential impact on contractile function. METHODS: We conducted proteomic and targeted, quantitative metabolomic analyses on heart tissue from patients with HCM and from nonfailing control human hearts. RESULTS: In the proteomic analysis, the greatest differences observed in HCM samples compared with controls were increased abundances of extracellular matrix and intermediate filament proteins and decreased abundances of muscle creatine kinase and mitochondrial proteins involved in fatty acid oxidation. These differences in protein abundance were coupled with marked reductions in acyl carnitines, byproducts of fatty acid oxidation, in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, branched chain amino acids, and their breakdown products, were all significantly increased in HCM hearts. ATP content, phosphocreatine, nicotinamide adenine dinucleotide and its phosphate derivatives, NADP and NADPH, and acetyl CoA were also severely reduced in HCM compared with control hearts. Functional assays performed on human skinned myocardial fibers demonstrated that the magnitude of observed reduction in ATP content in the HCM samples would be expected to decrease the rate of cross-bridge detachment. Moreover, left atrial size, an indicator of diastolic compliance, was inversely correlated with ATP content in hearts from patients with HCM. CONCLUSIONS: HCM hearts display profound deficits in nucleotide availability with markedly reduced capacity for fatty acid oxidation and increases in ketone bodies and branched chain amino acids. These results have important therapeutic implications for the future design of metabolic modulators to treat HCM.

Preparing Excitable Cardiac Papillary Muscle and Cardiac Slices for Functional Analyses.

While the reductionist approach has been fruitful in understanding the molecular basis of muscle function, intact excitable muscle preparations are still important as experimental model systems. We present here methods that are useful for preparing cardiac papillary muscle and cardiac slices, which represent macroscopic experimental model systems with fully intact intercellular and intracellular structures. The maintenance of these in vivo structures for experimentation in vitro have made these model systems especially useful for testing the functional effects of protein mutations and pharmaceutical candidates. We provide solutions recipes for dissection and recording, instructions for removing and preparing the cardiac papillary muscles, as well as instruction for preparing cardiac slices. These instructions are suitable for beginning experimentalists but may be useful for veteran muscle physiologists hoping to reacquaint themselves with macroscopic functional analyses.

Inertial artifact in viscoelastic measurements of striated muscle: Modeling and experimental results.

Viscoelastic properties of striated muscle are often measured using length perturbation analysis and quantified as a complex modulus, whose elastic and viscous components reflect the energy-storage and energy-absorbing properties of the tissue, respectively. The energy stored as inertia is commonly ignored due to the small size of samples examined, typically <1 mm. Considering recent advances in tissue engineering to generate muscle tissues of larger sizes, we questioned whether ignoring the inertial artifact was still reasonable in these samples. To answer this question, we derived and solved the one-dimensional wave equation that describes the propagation of strain along the length of a sample. The inertial artifact was predicted to contaminate the elastic modulus with (2πf)2L02ρ/6, where f is perturbation frequency, L0 is muscle length, and ρ is muscle density. We then measured viscoelastic properties up to 500 Hz in mouse skeletal muscle fibers at long (4.8 mm) and short (<1 mm) lengths and up to 100 Hz in rat cardiac slices at long (10-12 mm) and short (<2 mm) lengths. We found the elastic modulus of long preparations was elevated as frequency increased and was about half the magnitude of that predicted by the model. While the prediction tended to overestimate the measured inertial artifact, these results provided some validity to the model. We used the predicted artifact as an overly conservative estimate of error that might arise in a mechanics assay of mammalian striated muscle, whose nominal resting stiffness is on the order 100 kN m-2. We found that muscle lengths of <1 mm resulted in negligible inertial artifact (<0.5% error) for perturbation frequencies under 250 Hz. Muscle samples longer than 5 mm, on the other hand, would result in >5% error at frequencies of 200 Hz and higher.

Cardiac myosin binding protein-C phosphorylation accelerates ß-cardiac myosin detachment rate in mouse myocardium.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the ß-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0-12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca2+ activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates ß-myosin detachment in the intact myofilament lattice.NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that ß-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C.

Mechanical Characteristics of Ultrafast Zebrafish Larval Swimming Muscles.

Zebrafish (Danio rerio) swim within days of fertilization, powered by muscles of the axial myotomes. Forces generated by these muscles can be measured rapidly in whole, intact larval tails by adapting protocols developed for ex vivo muscle mechanics. But it is not known how well these measurements reflect the function of the underlying muscle fibers and sarcomeres. Here, we consider the anatomy of the 5-day-old, wild-type larval tail, and implement technical modifications to measuring muscle physiology in intact tails. Specifically, we quantify fundamental relationships between force, length, and shortening velocity, and capture the extreme contractile speeds required to swim with tail-beat frequencies of 80-100 Hz. Therefore, we analyze 1000 frames/s videos to track the movement of structures, visible in the transparent tail, which correlate with sarcomere length. We also characterize the passive viscoelastic properties of the preparation to isolate forces contributed by nonmuscle structures within the tail. Myotomal muscles generate more than 95% of their maximal isometric stress (76 ± 3 mN/mm2) over the range of muscle lengths used in vivo. They have rapid twitch kinetics (full width at half-maximal stress: 11 ± 1 ms) and a high twitch/tetanus ratio (0.91 ± 0.05), indicating adaptations for fast excitation-contraction coupling. Although contractile stress is relatively low, myotomal muscles develop high net power (134 ± 20 W/kg at 80 Hz) in cyclical work loop experiments designed to simulate the in vivo dynamics of muscle fibers during swimming. When shortening at a constant speed of 7 ± 1 muscle lengths/s, muscles develop 86 ± 2% of isometric stress, whereas peak instantaneous power during 100 Hz work loops occurs at 18 ± 2 muscle lengths/s. These approaches can improve the usefulness of zebrafish as a model system for muscle research by providing a rapid and sensitive functional readout for experimental interventions.

Enhancing diastolic function by strain-dependent detachment of cardiac myosin crossbridges.

The force response of cardiac muscle undergoing a quick stretch is conventionally interpreted to represent stretching of attached myosin crossbridges (phase 1) and detachment of these stretched crossbridges at an exponential rate (phase 2), followed by crossbridges reattaching in increased numbers due to an enhanced activation of the thin filament (phases 3 and 4). We propose that, at least in mammalian cardiac muscle, phase 2 instead represents an enhanced detachment rate of myosin crossbridges due to stretch, phase 3 represents the reattachment of those same crossbridges, and phase 4 is a passive-like viscoelastic response with power-law relaxation. To test this idea, we developed a two-state model of crossbridge attachment and detachment. Unitary force was assigned when a crossbridge was attached, and an elastic force was generated when an attached crossbridge was displaced. Attachment rate, f(x), was spatially distributed with a total magnitude f0. Detachment rate was modeled as g(x) = g0+ g1x, where g0 is a constant and g1 indicates sensitivity to displacement. The analytical solution suggested that the exponential decay rate of phase 2 represents (f0 + g0) and the exponential rise rate of phase 3 represents g0. The depth of the nadir between phases 2 and 3 is proportional to g1. We prepared skinned mouse myocardium and applied a 1% stretch under varying concentrations of inorganic phosphate (Pi). The resulting force responses fitted the analytical solution well. The interpretations of phases 2 and 3 were consistent with lower f0 and higher g0 with increasing Pi. This novel scheme of interpreting the force response to a quick stretch does not require enhanced thin-filament activation and suggests that the myosin detachment rate is sensitive to stretch. Furthermore, the enhanced detachment rate is likely not due to the typical detachment mechanism following MgATP binding, but rather before MgADP release, and may involve reversal of the myosin power stroke.

Electrical stimulation prevents doxorubicin-induced atrophy and mitochondrial loss in cultured myotubes.

Muscle contraction may protect against the effects of chemotherapy to cause skeletal muscle atrophy, but the mechanisms underlying these benefits are unclear. To address this question, we utilized in vitro modeling of contraction and mechanotransduction in C2C12 myotubes treated with doxorubicin (DOX; 0.2 µM for 3 days). Myotubes expressed contractile proteins and organized these into functional myofilaments, as electrical field stimulation (STIM) induced intracellular calcium (Ca2+) transients and contractions, both of which were prevented by inhibition of membrane depolarization. DOX treatment reduced myotube myosin content, protein synthesis, and Akt (S308) and forkhead box O3a (FoxO3a; S253) phosphorylation and increased muscle RING finger 1 (MuRF1) expression. STIM (1 h/day) prevented DOX-induced reductions in myotube myosin content and Akt and FoxO3a phosphorylation, as well as increases in MuRF1 expression, but did not prevent DOX-induced reductions in protein synthesis. Inhibition of myosin-actin interaction during STIM prevented contraction and the antiatrophic effects of STIM without affecting Ca2+ cycling, suggesting that the beneficial effect of STIM derives from mechanotransductive pathways. Further supporting this conclusion, mechanical stretch of myotubes recapitulated the effects of STIM to prevent DOX suppression of FoxO3a phosphorylation and upregulation of MuRF1. DOX also increased reactive oxygen species (ROS) production, which led to a decrease in mitochondrial content. Although STIM did not alter DOX-induced ROS production, peroxisome proliferator-activated receptor-γ coactivator-1α and antioxidant enzyme expression were upregulated, and mitochondrial loss was prevented. Our results suggest that the activation of mechanotransductive pathways that downregulate proteolysis and preserve mitochondrial content protects against the atrophic effects of chemotherapeutics.

Relaxation and the Role of Calcium in Isolated Contracting Myocardium From Patients With Hypertensive Heart Disease and Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Relaxation characteristics and Ca2+ homeostasis have not been studied in isolated myocardium from patients with hypertensive heart disease (HHD) and heart failure with preserved ejection fraction (HFpEF). Prolonged myocardial relaxation is believed to play an important role in the pathophysiology of these conditions. In this study, we evaluated relaxation parameters, myocardial calcium (Ca2+), and sodium (Na+) handling, as well as ion transporter expression and tested the effect of Na+-influx inhibitors on relaxation in isolated myocardium from patients with HHD and HFpEF. METHODS AND RESULTS: Relaxation characteristics were studied in myocardial strip preparations under physiological conditions at stimulation rates of 60 and 180 per minute. Intracellular Ca2+ and Na+ were simultaneously assessed using Fura-2 and AsanteNATRIUMGreen-2, whereas elemental analysis was used to measure total myocardial concentrations of Ca, Na, and other elements. Quantitative polymerase chain reaction was used to measure expression levels of key ion transport proteins. The lusitropic effect of Na+-influx inhibitors ranolazine, furosemide, and amiloride was evaluated. Myocardial left ventricular biopsies were obtained from 36 control patients, 29 HHD and 19 HHD+HFpEF. When compared with control patients, half maximal relaxation time (RT50) at 60 per minute was prolonged by 13% in HHD and by 18% in HHD+HFpEF (both P<0.05). Elevated resting Ca2+ levels and a tachycardia-induced increase in diastolic Ca2+ were associated with incomplete relaxation and an increase in diastolic tension in HHD and HHD+HFpEF. Na+ levels were not increased, and expression levels of Ca2+- or Na+-handling proteins were not altered. Na+-influx inhibitors did not improve relaxation or prevent incomplete relaxation at high stimulation rates. CONCLUSIONS: Contraction and relaxation are prolonged in isolated myocardium from patients with HHD and HHD+HFpEF. This leads to incomplete relaxation at higher rates. Elevated calcium levels in HFpEF are neither a result of an impaired Na+ gradient nor expression changes in key ion transporters and regulatory proteins.

Mimicking the cardiac cycle in intact cardiomyocytes using diastolic and systolic force clamps; measuring power output.

AIMS: A single isolated cardiomyocyte is the smallest functional unit of the heart. Yet, all single isolated cardiomyocyte experiments have been limited by the lack of proper methods that could reproduce a physiological cardiac cycle. We aimed to investigate the contractile properties of a single cardiomyocyte that correctly mimic the cardiac cycle. METHODS AND RESULTS: By adjusting the parameters of the feedback loop, using a suitably engineered feedback system and recording the developed force and the length of a single rat cardiomyocyte during contraction and relaxation, we were able to construct force-length (FL) relations analogous to the pressure-volume (PV) relations at the whole heart level. From the cardiac loop graphs, we obtained, for the first time, the power generated by one single cardiomyocyte. CONCLUSION: Here, we introduce a new approach that by combining mechanics, electronics, and a new type optical force transducer can measure the FL relationship of a single isolated cardiomyocyte undergoing a mechanical loop that mimics the PV cycle of a beating heart.

Dynamics of cross-bridge cycling, ATP hydrolysis, force generation, and deformation in cardiac muscle.

Despite extensive study over the past six decades the coupling of chemical reaction and mechanical processes in muscle dynamics is not well understood. We lack a theoretical description of how chemical processes (metabolite binding, ATP hydrolysis) influence and are influenced by mechanical processes (deformation and force generation). To address this need, a mathematical model of the muscle cross-bridge (XB) cycle based on Huxley's sliding filament theory is developed that explicitly accounts for the chemical transformation events and the influence of strain on state transitions. The model is identified based on elastic and viscous moduli data from mouse and rat myocardial strips over a range of perturbation frequencies, and MgATP and inorganic phosphate (Pi) concentrations. Simulations of the identified model reproduce the observed effects of MgATP and MgADP on the rate of force development. Furthermore, simulations reveal that the rate of force re-development measured in slack-restretch experiments is not directly proportional to the rate of XB cycling. For these experiments, the model predicts that the observed increase in the rate of force generation with increased Pi concentration is due to inhibition of cycle turnover by Pi. Finally, the model captures the observed phenomena of force yielding suggesting that it is a result of rapid detachment of stretched attached myosin heads.

Myocardial stiffness in patients with heart failure and a preserved ejection fraction: contributions of collagen and titin.

BACKGROUND: The purpose of this study was to determine whether patients with heart failure and a preserved ejection fraction (HFpEF) have an increase in passive myocardial stiffness and the extent to which discovered changes depend on changes in extracellular matrix fibrillar collagen and cardiomyocyte titin. METHODS AND RESULTS: Seventy patients undergoing coronary artery bypass grafting underwent an echocardiogram, plasma biomarker determination, and intraoperative left ventricular epicardial anterior wall biopsy. Patients were divided into 3 groups: referent control (n=17, no hypertension or diabetes mellitus), hypertension (HTN) without (-) HFpEF (n=31), and HTN with (+) HFpEF (n=22). One or more of the following studies were performed on the biopsies: passive stiffness measurements to determine total, collagen-dependent and titin-dependent stiffness (differential extraction assay), collagen assays (biochemistry or histology), or titin isoform and phosphorylation assays. In comparison with controls, patients with HTN(-)HFpEF had no change in left ventricular end-diastolic pressure, myocardial passive stiffness, collagen, or titin phosphorylation but had an increase in biomarkers of inflammation (C-reactive protein, soluble ST2, tissue inhibitor of metalloproteinase 1). In comparison with both control and HTN(-)HFpEF, patients with HTN(+)HFpEF had increased left ventricular end-diastolic pressure, left atrial volume, N-terminal propeptide of brain natriuretic peptide, total, collagen-dependent, and titin-dependent stiffness, insoluble collagen, increased titin phosphorylation on PEVK S11878(S26), reduced phosphorylation on N2B S4185(S469), and increased biomarkers of inflammation. CONCLUSIONS: Hypertension in the absence of HFpEF did not alter passive myocardial stiffness. Patients with HTN(+)HFpEF had a significant increase in passive myocardial stiffness; collagen-dependent and titin-dependent stiffness were increased. These data suggest that the development of HFpEF depends on changes in both collagen and titin homeostasis.

Molecular determinants of force production in human skeletal muscle fibers: effects of myosin isoform expression and cross-sectional area.

Skeletal muscle contractile performance is governed by the properties of its constituent fibers, which are, in turn, determined by the molecular interactions of the myofilament proteins. To define the molecular determinants of contractile function in humans, we measured myofilament mechanics during maximal Ca(2+)-activated and passive isometric conditions in single muscle fibers with homogenous (I and IIA) and mixed (I/IIA and IIA/X) myosin heavy chain (MHC) isoforms from healthy, young adult male (n = 5) and female (n = 7) volunteers. Fibers containing only MHC II isoforms (IIA and IIA/X) produced higher maximal Ca(2+)-activated forces over the range of cross-sectional areas (CSAs) examined than MHC I fibers, resulting in higher (24-42%) specific forces. The number and/or stiffness of the strongly bound myosin-actin cross bridges increased in the higher force-producing MHC II isoforms and, in all isoforms, better predicted force than CSA. In men and women, cross-bridge kinetics, in terms of myosin attachment time and rate of myosin force production, were independent of CSA, although women had faster (7-15%) kinetics. The relative proportion of cross bridges and/or their stiffness was reduced as fiber size increased, causing a decline in specific force. Results from our examination of molecular mechanisms across the range of physiological CSAs explain the variation in specific force among the different fiber types in human skeletal muscle, which may have relevance to understanding how various physiological and pathophysiological conditions modulate single-fiber and whole muscle contractility.