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 are dependent on changes in extracellular matrix fibrillar collagen and/or cardiomyocyte titin. Methods and Results Seventy patients undergoing coronary artery bypass grafting underwent an echocardiogram, plasma biomarker determination, and intra-operative left ventricular (LV) epicardial anterior wall biopsy. Patients were divided into 3 groups: referent control (n=17, no hypertension or diabetes), 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. Compared with controls, patients with HTN(-)HFpEF had no change in LV end diastolic pressure (LVEDP), myocardial passive stiffness, collagen, or titin phosphorylation but had an increase in biomarkers of inflammation (CRP, sST2, TIMP-1). Compared with both control and HTN(-)HFpEF, patients with HTN(+)HFpEF had increased LVEDP, left atrial volume, NT-proBNP, 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 is dependent on changes in both collagen and titin homeostasis.

Multiple mutations in cardiac troponin T (cTnT) can cause familial hypertrophic cardiomyopathy (FHC). Patients with cTnT mutations generally exhibit mild or no ventricular hypertrophy, yet demonstrate a high frequency of early sudden death. To understand the functional basis of these phenotypes, we created transgenic mouse lines expressing 30%, 67%, and 92% of their total cTnT as a missense (R92Q) allele analogous to one found in FHC. Similar to a mouse FHC model expressing a truncated cTnT protein, the left ventricles of all R92Q lines are smaller than those of wild-type. In striking contrast to truncation mice, however, the R92Q hearts demonstrate significant induction of atrial natriuretic factor and β-myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from R92Q mice have increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working heart data are consistent, showing hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC. These mice represent the first disease model to exhibit hypercontractility, as well as a unique model system for exploring the cellular pathogenesis of FHC. The distinct phenotypes of mice with different TnT alleles suggest that the clinical heterogeneity of FHC is at least partially due to allele-specific mechanisms.

Mutations in multiple cardiac sarcomeric proteins including myosin heavy chain (MyHC) and cardiac troponin T (cTnT) cause a dominant genetic heart disease, familial hypertrophic cardiomyopathy (FHC). Patients with mutations in these two genes have quite distinct clinical characteristics. Those with MyHC mutations demonstrate more significant and uniform cardiac hypertrophy and a variable frequency of sudden death. Patients with cTnT mutations generally exhibit mild or no hypertrophy, but a high frequency of sudden death at an early age. To understand the basis for these distinctions and to study the pathogenesis of the disease, we have created transgenic mice expressing a truncated mouse cTnT allele analogous to one found in FHC patients. Mice expressing truncated cTnT at low (< 5%) levels develop cardiomyopathy and their hearts are significantly smaller (18-27%) than wild type. These animals also exhibit significant diastolic dysfunction and milder systolic dysfunction. Animals that express higher levels of transgene protein die within 24 h of birth. Transgenic mouse hearts demonstrate myocellular disarray and have a reduced number of cardiac myocytes that are smaller in size. These studies suggest that multiple cellular mechanisms result in the human disease, which is generally characterized by mild hypertrophy, but, also, frequent sudden death.

We hypothesize that age-related skeletal muscle dysfunction and physical disability may be partially explained by alterations in the function of the myosin molecule. To test this hypothesis, skeletal muscle function at the whole muscle, single fiber, and molecular levels was measured in young (21-35 yr) and older (65-75 yr) male and female volunteers with similar physical activity levels. After adjusting for muscle size, older adults had similar knee extensor isometric torque values compared with young, but had lower isokinetic power, most notably in women. At the single-fiber and molecular levels, aging was associated with increased isometric tension, slowed myosin actin cross-bridge kinetics (longer myosin attachment times and reduced rates of myosin force production), greater myofilament lattice stiffness, and reduced phosphorylation of the fast myosin regulatory light chain; however, the age effect was driven primarily by women (i.e., age-by-sex interaction effects). In myosin heavy chain IIA fibers, single-fiber isometric tension and molecular level mechanical and kinetic indexes were correlated with whole muscle isokinetic power output. Collectively, considering that contractile dysfunction scales up through various anatomical levels, our results suggest a potential sex-specific molecular mechanism, reduced cross-bridge kinetics, contributes to the reduced physical capacity with aging in women. Thus these results support our hypothesis that age-related alterations in the myosin molecule contribute to skeletal muscle dysfunction and physical disability and indicate that this effect is stronger in women.

The force response of activated striated muscle to length perturbations includes the so-called C-process, which has been considered the frequency domain representation of the fast single-exponential force decay after a length step (phases 1 and 2). The underlying molecular mechanisms of this phenomenon, however, are still the subject of various hypotheses. In this study, we derived analytical expressions and created a corresponding computer model to describe the consequences of independent acto-myosin cross-bridges characterized solely by 1), intermittent periods of attachment (t(att)) and detachment (t(det)), whose values are stochastically governed by independent probability density functions; and 2), a finite Hookian stiffness (k(stiff)) effective only during periods of attachment. The computer-simulated force response of 20,000 (N) cross-bridges making up a half-sarcomere (F(hs)(t)) to sinusoidal length perturbations (L(hs)(t)) was predicted by the analytical expression in the frequency domain, (F(hs)(omega)/L(hs)(omega))=(t(att)/t(cycle))Nk(stiff)(iomega/(t(att)(-1)+iomega)), where t(att) = mean value of t(att), t(cycle) = mean value of t(att) + t(det), k(stiff) = mean stiffness, and omega = 2pi x frequency of perturbation. The simulated force response due to a length step (L(hs)) was furthermore predicted by the analytical expression in the time domain, F(hs)(t)=(t(att)/t(cycle))Nk(stiff)L(hs)e(-t/t(att)). The forms of these analytically derived expressions are consistent with expressions historically used to describe these specific characteristics of a force response and suggest that the cycling of acto-myosin cross-bridges and their associated stiffnesses are responsible for the C-process and for phases 1 and 2. The rate constant 2pic, i.e., the frequency parameter of the historically defined C-process, is shown here to be equal to t(att)(-1). Experimental results from activated cardiac muscle examined at different temperatures and containing predominately alpha- or beta-myosin heavy chain isoforms were found to be consistent with the above interpretation.

Abstract-Despite advances in the molecular biology of cardiac myosin binding protein-C (cMyBP-C), little is understood about its precise role in muscle contraction, particularly in the intact heart. We tested the hypothesis that cMyBP-C is central to the time course and magnitude of left ventricular systolic elastance (chamber stiffening), and assessed mechanisms for this influence in intact hearts, trabeculae, and skinned fibers from wild-type (ϩ/ϩ) and homozygous truncated cMyBP-C (t/t) male mice. cMyBP-C protein was not detected by gel electrophoresis or Western blot in t/t myocardium. cMyBP-C t/t ventricles displayed reduced peak elastance, but more strikingly a marked abbreviation of the systolic elastance time course, which peaked earlier (27.6Ϯ2.1 ms) than in ϩ/ϩ controls (47.8Ϯ1.6 ms). Control hearts reached only 42Ϯ4% of maximum elastance at the onset of ejection, with substantial further stiffening during ejection. This contrasted to t/t mutants, which reached 77Ϯ3% of peak elastance before ejection of peak. These unusual findings were not observed in alternative models involving severe cardiomyopathy, but were recapitulated in a cMyBP-C null mouse. The abbreviated elastance time course and lower peak were consistent with earlier time-to-peak trabecular tension, increased unloaded shortening velocity in t/t skinned muscle strips, and dramatically reduced myofilament stiffness at diastolic calcium concentrations. These results provide novel insights into the role of cMyBP-C in myocardial systolic mechanics. Abnormal sarcomere shortening velocity and abbreviated muscle stiffening may underlie development of cardiac dysfunction associated with deficient incorporation of cMyBP-C.

Skeletal muscle function is impaired in heart failure patients due, in part, to loss of myofibrillar protein content, in particular myosin. In the present study, we utilized small-amplitude sinusoidal analysis for the first time in single human skeletal muscle fibres to measure muscle mechanics, including cross-bridge kinetics, to determine if heart failure further impairs contractile performance by altering myofibrillar protein function. Patients with chronic heart failure (n = 9) and controls (n = 6) were recruited of similar age and physical activity to diminish the potentially confounding effects of ageing and muscle disuse. Patients showed decreased cross-bridge kinetics in myosin heavy chain (MHC) I and IIA fibres, partially due to increased myosin attachment time (t on ). The increased t on compensated for myosin protein loss previously found in heart failure patients by increasing the fraction of the total cycle time myosin is bound to actin, resulting in a similar number of strongly bound cross-bridges in patients and controls. Accordingly, isometric tension did not differ between patients and controls in MHC I or IIA fibres. Patients also had decreased calcium sensitivity in MHC IIA fibres and alterations in the viscoelastic properties of the lattice structure of MHC I and IIA fibres. Collectively, these results show that heart failure alters skeletal muscle contraction at the level of the myosin-actin cross-bridge, leading to changes in muscle mechanics which could contribute to impaired muscle function. Additionally, we uncovered a unique kinetic property of MHC I fibres, a potential indication of two distinct populations of cross-bridges, which may have important physiological consequences. Abbreviations A, A-process magnitude; B, B-process magnitude; C, C-process magnitude; CSA, cross-sectional area; E e , elastic modulus; E v , viscous modulus; f , frequency of the length perturbations; F, force; F/CSA, tension; HF, heart failure; k, A-process unitless exponent; L, fibre length; L amp , length oscillation amplitude; MHC, myosin heavy chain; MLC, myosin light chain; n, Hill coefficient; P i , phosphate; t, time needed to perform length perturbations; t on , average myosin attachment time; t 3 , time from the start of stretch activation to the peak tension; Y (ω), complex modulus at peak calcium activation; L, fibre length change; L/L, fibre strain; ω, 2π × the frequency of the length perturbation; 2πb, B-process characteristic rate; 2πc, C-process characteristic rate.

Objectives Tachycardia-induced relaxation abnormalities were evaluated in myocardium from patients with normal ejection fraction (EF). Background Diastolic dysfunction and left ventricular (LV) hypertrophy are closely linked. Tachycardia can induce heart failure symptoms in otherwise asymptomatic patients. To study the effects of tachycardia on myocardial contractility and relaxation we evaluated the effects of increasing pacing rates in myocardial biopsies obtained from patients with normal EF. Methods LV biopsies were obtained during coronary bypass surgery. Myocardial strip preparations were electrically paced at rates from 60/min to180/min. Diastolic resting tone was assessed by crossbridge deactivation. Calcium transporting systems were functionally examined and myofilament calcium sensitivity was studied. Results Seven preparations developed incomplete relaxation with increased diastolic tension development at increasing pacing rates. This was absent in the remaining seven preparations. Incomplete relaxation was found to be associated with increased LV mass and left atrial volume. Crossbridge deactivation showed that these preparations also had a significant resting tone. Additional functional analyses suggest that incomplete relaxation is associated with disproportionately elevated cellular calcium loads due to a reduced sarcolemmal calcium extrusion reserve. Conclusions Tachycardia-induced incomplete relaxation was associated with increased LV mass and left atrial volumes. We also found a disproportionately increased calcium load at high rates and a substantial resting tone due to diastolic crossbridge cycling. These observations may play a role in reduced exercise tolerance and tachycardia induced diastolic dysfunction.