Rate Constant Of Force Redevelopment Research Articles

Cooperative mechanisms underlie differences in myocardial contractile dynamics between large and small mammals.

Ca2+ binding to troponin C (TnC) and myosin cross-bridge binding to actin act in a synergistic cooperative manner to modulate myocardial contraction and relaxation. The responsiveness of the myocardial thin filament to the activating effects of Ca2+ and myosin cross-bridge binding has been well-characterized in small mammals (e.g., mice). Given the nearly 10-fold difference in resting heart rates and twitch kinetics between small and large mammals, it is unlikely that the cooperative mechanisms underlying thin filament activation are identical in these two species. To test this idea, we measured the Ca2+ dependencies of steady-state force and the rate constant of force redevelopment (ktr) in murine and porcine permeabilized ventricular myocardium. While murine myocardium exhibited a steep activation-dependence of ktr, the activation-dependent profile of ktr was significantly reduced in porcine ventricular myocardium. Further insight was attained by examining force-pCa and ktr-pCa relationships. In the murine myocardium, the pCa50 for ktr was right-shifted compared with the pCa50 for force, meaning that increases in steady-state force occurred well before increases in the rate of force redevelopment were observed. In the porcine myocardium, we observed a tighter coupling of the force-pCa and ktr-pCa relationships, as evidenced by near-maximal rates of force redevelopment at low levels of Ca2+ activation. These results demonstrate that the molecular mechanisms underlying the cooperative activation of force are a dynamic property of the mammalian heart, involving, at least in part, the species- and tissue-specific expression of cardiac myosin heavy chain isoforms.

Rapamycin treatment increases survival, autophagy biomarkers and expression of the anti-aging klotho protein in elderly mice.

Previous investigations have demonstrated that treatment of animals with rapamycin increases levels of autophagy, which is a process by which cells degrade intracellular detritus, thus suppressing the emergence of senescent cells, whose pro-inflammatory properties, are primary drivers of age-associated physical decline. A hypothesis is tested here that rapamycin treatment of mice approaching the end of their normal lifespan exhibits increased survival, enhanced expression of autophagic proteins; and klotho protein-a biomarker of aging that affects whole organism senescence, and systemic suppression of inflammatory mediator production. Test groups of 24-month-old C57BL mice were injected intraperitoneally with either 1.5 mg/kg/week rapamycin or vehicle. All mice administered rapamycin survived the 12-week course, whereas 43% of the controls died. Relative to controls, rapamycin-treated mice experienced minor but significant weight loss; moreover, nonsignificant trends toward decreased levels of leptin, IL-6, IL-1β, TNF-α, IL-1α, and IGF-1, along with slight elevations in VEGF, MCP-1 were observed in the blood serum of rapamycin-treated mice. Rapamycin-treated mice exhibited significantly enhanced autophagy and elevated expression of klotho protein, particularly in the kidney. Rapamycin treatment also increased cardiomyocyte Ca2+ -sensitivity and enhanced the rate constant of force re-development, which may also contribute to the enhanced survival rate in elderly mice.

Developmental increase in β-MHC enhances sarcomere length-dependent activation in the myocardium.

Shifts in myosin heavy chain (MHC) isoforms in cardiac myocytes have been shown to alter cardiac muscle function not only in healthy developing hearts but also in diseased hearts. In guinea pig hearts, there is a large age-dependent shift in MHC isoforms from 80% α-MHC/20% β-MHC at 3 wk to 14% α-MHC/86% β-MHC at 11 wk. Because kinetic differences in α- and β-MHC cross-bridges (XBs) are known to impart different cooperative effects on thin filaments, we hypothesize here that differences in α- and β-MHC expression in guinea pig cardiac muscle impact sarcomere length (SL)-dependent contractile function. We therefore measure steady state and dynamic contractile parameters in detergent-skinned cardiac muscle preparations isolated from the left ventricles of young (3 wk old) or adult (11 wk old) guinea pigs at two different SLs: short (1.9 µm) and long (2.3 µm). Our data show that SL-dependent effects on contractile parameters are augmented in adult guinea pig cardiac muscle preparations. Notably, the SL-mediated increase in myofilament Ca2+ sensitivity (ΔpCa50) is twofold greater in adult guinea pig muscle preparations (ΔpCa50 being 0.11 units in adult preparations but only 0.05 units in young preparations). Furthermore, adult guinea pig cardiac muscle preparations display greater SL-dependent changes than young muscle preparations in (1) the magnitude of length-mediated increase in the recruitment of new force-bearing XBs, (2) XB detachment rate, (3) XB strain-mediated effects on other force-bearing XBs, and (4) the rate constant of force redevelopment. Our findings suggest that increased β-MHC expression enhances length-dependent activation in the adult guinea pig cardiac myocardium.

Myosin heavy chain and cardiac troponin T damage is associated with impaired myofibrillar ATPase activity contributing to sarcomeric dysfunction in Ca2+-paradox rat hearts

This study aimed to explore the potential contribution of myofibrils to contractile dysfunction in Ca2+-paradox hearts. Isolated rat hearts were perfused with Krebs–Henseleit solution (Control), followed by Ca2+-depletion, and then Ca2+-repletion after Ca2+-depletion (Ca2+-paradox) by Langendorff method. During heart perfusion left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), rate of pressure development (+ dP/dt), and pressure decay (-dP/dt) were registered. Control LVDP (127.4 ± 6.1 mmHg) was reduced during Ca2+-depletion (9.8 ± 1.3 mmHg) and Ca2+-paradox (12.9 ± 1.3 mmHg) with similar decline in +dP/dt and –dP/dt. LVEDP was increased in both Ca2+-depletion and Ca2+-paradox. Compared to Control, myofibrillar Ca2+-stimulated ATPase activity was decreased in the Ca2+-depletion group (12.08 ± 0.57 vs. 8.13 ± 0.19 µmol Pi/mg protein/h), besides unvarying Mg2+ ATPase activity, while upon Ca2+-paradox myofibrillar Ca2+-stimulated ATPase activity was decreased (12.08 ± 0.57 vs. 8.40 ± 0.22 µmol Pi/mg protein/h), but Mg2+ ATPase activity was increased (3.20 ± 0.25 vs. 7.21 ± 0.36 µmol Pi/mg protein/h). In force measurements of isolated cardiomyocytes at saturating [Ca2+], Ca2+-depleted cells had lower rate constant of force redevelopment (ktr,max, 3.85 ± 0.21) and unchanged active tension, while those in Ca2+-paradox produced lower active tension (12.12 ± 3.19 kN/m2) and ktr,max (3.21 ± 23) than cells of Control group (25.07 ± 3.51 and 4.61 ± 22 kN/m2, respectively). In biochemical assays, α-myosin heavy chain and cardiac troponin T presented progressive degradation during Ca2+-depletion and Ca2+-paradox. Our results suggest that contractile impairment in Ca2+-paradox partially resides in deranged sarcomeric function and compromised myofibrillar ATPase activity as a result of myofilament protein degradation, such as α-myosin heavy chain and cardiac troponin T. Impaired relaxation seen in Ca2+-paradoxical hearts is apparently not related to titin, rather explained by the altered myofibrillar ATPase activity.

Cross-bridge Kinetics Impairment In Elite Octogenarian Masters Athletes

The association between adult normal aging and impairments of the neuromuscular system as well as the application of exercise training to attenuate these problems have already been reported by several studies. However, it is still not clear whether intrinsic cross-bridge kinetics, especially of the ‘slow’ resilient fibre type, are affected in aging muscle, and if this kinetics would be better preserved in high functioning elderly athletes. PURPOSE: to compare cross-bridge kinetics in healthy non-athlete octogenarians to age-matched world class masters athletes, and young controls. METHODS: Skinned muscle fibres obtained from vastus lateralis biopsies of young (∼23y), old non-athlete (NA) adults (∼80y), and age-matched world class masters athletes (MA; ∼80y) were first isometrically activated to assess force per cross-sectional (Po), then submitted to slack test, and shortening-re-stretch protocol, by which unloaded shortening velocity (Vo), and the rate constant of force redevelopment (ktr) were respectively obtained. A post-hoc analysis was performed, and only the mechanical properties of ‘slow type’ fibres based on unloaded shortening velocity measurements were used (the level of significance was set at P<0.05). RESULTS: MA and NA produced ∼55% and 45% lower Po than young. Vo for both MA and NA old groups were ∼62% and 42% slower, respectively, compared with young. Both MA and NA adults had ∼2 times slower values for ktr compared with young. CONCLUSION: The lower ktr in both old groups relative to young suggests that impaired cross-bridge kinetics was, at least in part, responsible for impaired single fibre contractile properties, i.e Vo, Po, with aging. Apparently, masters athletes’ higher performance cannot be attributed to their intrinsic cross-bridge kinetics.

Functional Effects of the β-Myosin Mutation Arg453Cys in Familial Hypertrophic Cardiomyopathy

In Familial Hypertrophic Cardiomyopathy (FHC) 1/3 of the patients are affected by mutations in the ß-myosin heavy chain (ß-MyHC), the myosin isoform of the ventricle and of slow skeletal muscle fibers in humans. Yet, not much is known about (i) direct effects of specific ß-MyHC-mutations on acto-myosin function, and (ii) how these mutations may trigger development of the FHC-phenotype. To address these questions, we analyzed the effects of the ß-MyHC-mutation R453C on contractile properties of slow (type I) fibers from the M. soleus of a severely affected FHC patient. We found an about 12% higher isometric force, 15% faster rate constant of force redevelopment (ktr), 10% higher isometric ATPase activity and essentially unchanged tension cost. Together with only slightly higher fiber stiffness in rigor, a main part of the increase in isometric force appears to be due to altered cross-bridge cycling kinetics, specifically an increase in fapp, the rate constant for the cross-bridge transition into force generating states. Currently we investigate whether increased force generated per myosin head e.g., by a larger y0-value also contributes. Relative quantification of mutated vs. wildtype ß-MyHC-mRNA of the heterozygous patient revealed a fraction of about 35% for the R453C-mRNA. Assuming similar abundance of mutated MyHC at the protein level, as we had found in other FHC-mutations, about a third of the ß-myosin heads in the sarcomeres carry the mutation. Thus, force contribution of the mutated myosin head population is about 40% increased. A larger variability in pCa50 among individual fibers with mutation R453C vs. control fibers, as we had seen for other FHC-mutations, suggests unequal expression of mutated myosin. We hypothesize that unequal expression of mutated myosin in individual cardiomyocytes causes imbalanced force generation and initiates functional impairment of the myocardium in FHC.

Fast Skeletal Muscle Troponin Activation Increases Force of Mouse Fast Skeletal Muscle and Ameliorates Weakness Due to Nebulin-Deficiency

The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension–pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring ktr (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased ktr at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.

Impaired Cross Bridge Formation Contributes to Reduced Cardiac Contraction in the Infarct Border Zone

After myocardial infarction, adjacent to the infarct there is a poorly contracting non-ischemic border zone. The mechanism of border zone dysfunction is unclear. We found that border zone dysfunction does not involve myocardial fibrosis, or a decreased content of either myofibrils or myosin, suggesting there is a defect in the contractile mechanism. Goal: Determine the mechanism for border zone dysfunction. Methods: We used sheep hearts 2 weeks after apical infarction. Cardiac muscle strips were dissected from the border zone adjacent to the infarct and from a zone remote from the infarct. Myofilament contraction was assessed using in-vitro isometric and isotonic contractions of demembranated cardiac muscle strips bathed in activating solutions. To assess strongly bound cross-bridge formation, we measured muscle stiffness using high frequency, low amplitude oscillations of muscle length in the absence of ATP (in this rigor state, cross bridge formation should be maximal). Results: In border zone myocardium, maximal force development (Fmax) was reduced by 31±2% (n=6, P<0.01) compared to Fmax of remote zone myocardium (85±1 mN/mm2). The stiffness in the rigor state was reduced by 34±6% (n=5, P<0.05) in border zone myocardium versus remote zone myocardium, suggesting impaired cross bridge formation in the border zone. There was no difference between border zone and remote zone myocardium in the maximum velocity of muscle shortening (∼0.35 muscle lengths/s), or in the rate constant of force redevelopment (Ktr, ∼2.5 s−1) after briefly mechanically disrupting cross bridges with a rapid perturbation of muscle length. Conclusions: Impaired contraction of border zone myocardium involves a reduction in cross bridge formation, without effects on cross bridge kinetics. Preliminary studies suggest proteolytic cleavage of contractile proteins may play a role.

Cardiomyopathy - Associated W4R Variant of Muscle LIM Protein affects Skeletal Muscle Passive Mechanics

Muscle LIM protein (MLP) is located at the Z-disc and the M-line of the sarcomere of striated muscle. It has multiple protein interaction partners. The MLP-W4R variant was shown to be associated with cardiomyopathy in humans. MLP-W4R knock-in mice showed myopathic changes in skeletal muscle and other mutations of MLP were associated with a skeletal myopathy in affected human individuals. Therefore, we investigated the influence of MLP-W4R on skeletal muscle function and ultrastructure.Skinned single muscle fibers of M. vastus lat. of wildtype and MLP-W4R knock-in mice were used to characterize passive and active parameters like relaxed fiber stiffness, passive force-sarcomere length curve, isometric force generation, rate constant of force redevelopment and maximal shortening velocity. Thin sections of the muscle fibers were analyzed by transmission electron microscopy.In comparison to wildtype, MLP-W4R muscle fibers showed a significant decrease in relaxed fiber stiffness and passive force-sarcomere length curve, whereas no significant changes occurred in isometric force generation, force redevelopment, and shortening velocity. Ultrastructurally, the alignment of myofilaments within the sarcomere was normal with MLP-W4R, however, Z-discs and M-lines were broadened.MLP-W4R knock-in mice exhibit discrete morphological changes of skeletal myofibers consistent with a skeletal myopathy. The disturbed passive properties of the muscle fibers support the hypothesis that the MLP-W4R variant contributes to the disorder.

Effects of low-level α-myosin heavy chain expression on contractile kinetics in porcine myocardium

Myosin heavy chain (MHC) isoforms are principal determinants of work capacity in mammalian ventricular myocardium. The ventricles of large mammals including humans normally express ∼10% α-MHC on a predominantly β-MHC background, while in failing human ventricles α-MHC is virtually eliminated, suggesting that low-level α-MHC expression in normal myocardium can accelerate the kinetics of contraction and augment systolic function. To test this hypothesis in a model similar to human myocardium we determined composite rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) in porcine myocardium expressing either 100% α-MHC or 100% β-MHC in order to predict the MHC isoform-specific effect on twitch kinetics. Right atrial (∼100% α-MHC) and left ventricular (∼100% β-MHC) tissue was used to measure myosin ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentration. The rate of ATP utilization and k(tr) were approximately ninefold higher in atrial compared with ventricular myocardium, while tension cost was approximately eightfold greater in atrial myocardium. From these values, we calculated f(app) to be ∼10-fold higher in α- compared with β-MHC, while g(app) was 8-fold higher in α-MHC. Mathematical modeling of an isometric twitch using these rate constants predicts that the expression of 10% α-MHC increases the maximal rate of rise of force (dF/dt(max)) by 92% compared with 0% α-MHC. These results suggest that low-level expression of α-MHC significantly accelerates myocardial twitch kinetics, thereby enhancing systolic function in large mammalian myocardium.

Altered myofilament function depresses force generation in patients with nebulin-based nemaline myopathy (NEM2)

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is clinically characterized by muscle weakness. However, the mechanisms underlying this weakness are poorly understood. Here, we studied the contractile phenotype of skeletal muscle from NM patients with nebulin mutations (NEM2). SDS-PAGE and Western blotting studies revealed markedly reduced nebulin protein levels in muscle from NM patients, whereas levels of other thin filament-based proteins were not significantly altered. Muscle mechanics studies indicated significantly reduced calcium sensitivity of force generation in NM muscle fibers compared to control fibers. In addition, we found slower rate constant of force redevelopment, as well as increased tension cost, in NM compared to control fibers, indicating that in NM muscle the rate of cross-bridge attachment is reduced, whereas the rate of cross-bridge detachment is increased. The resulting reduced fraction of force generating cross-bridges is expected to greatly impair the force generating capacity of muscle from NM patients. Thus, the present study provides important novel insights into the pathogenesis of muscle weakness in nebulin-based NM.

Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics

The ventricles of small mammals express mostly alpha-myosin heavy chain (alpha-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express approximately 10% alpha-MHC on a predominately beta-MHC (slow isoform) background. In failing human ventricles, the amount of alpha-MHC is dramatically reduced, leading to the hypothesis that even small amounts of alpha-MHC on a predominately beta-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) for myosins composed of 100% alpha-MHC or beta-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% alpha-MHC or 100% beta-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentrations. The rate of ATP utilization was approximately 2.5-fold higher in preparations expressing 100% alpha-MHC compared with those expressing only beta-MHC, whereas k(tr) was 2-fold faster in the alpha-MHC myocardium. From these variables, we calculated f(app) to be approximately threefold higher for alpha-MHC than beta-MHC and g(app) to be twofold higher in alpha-MHC. Mathematical modeling of isometric twitches predicted that small increases in alpha-MHC significantly increased the rate of force development. These results suggest that low-level expression of alpha-MHC has significant effects on contraction kinetics.

Different Molecular Mechanisms of Force Enhancement by Myosin Head Domain Mutations R723G and R453C

The myosin head transforms chemical energy from ATP hydrolysis into mechanical work to generate isometric force or to drive muscle shortening. During the power stroke, small changes in the catalytic ATPase site of the actin-bound myosin head are coupled to tilting of the light-chain binding domain relative to the catalytic domain. In this mechanism distortion of an elastic element within the actomyosin complex is essential for strain to develop prior to movement.We have studied missense mutations naturally occurring in Familial Hypertrophic Cardiomyopathy (FHC) to test molecular mechanisms and functional roles of specific domains of the myosin head. Slow soleus muscle fibers (expressing the ventricular myosin isoform) of FHC-patients were used for these studies. Previously we found that mutation R723G which is located in the converter domain of the myosin head increases active force and resistance to elastic distortion (fiber stiffness) during contraction, relaxation, and rigor, while cross-bridge cycling kinetics were unchanged. This indicated that the converter is the part of the actomyosin complex where most of the elastic distortion occurs.We now included mutation R453C located near the nucleotide binding pocket of myosin in our functional studies. This mutation was found to also increase active force, however, without affecting fiber stiffness. Instead, mutation R453C increased fiber ATPase activity and the rate constant of force redevelopment (kredev) significantly, which were unchanged by mutation R723G. Thus, both FHC mutations cause force enhancement in muscle fibers from FHC patients, however, by distinctly different mechanisms. R723G affects resistance to elastic distortion of the myosin head while R453C alters cross-bridge cycling kinetics. The data underline the different functional roles of the domains within the myosin head and point to an involvement of residue R453 in human ventricular myosin ATPase activity.

Oxidation of Myofilament Protein Sulfhydryl Groups Reduces the Contractile Force and Its Ca2+Sensitivity in Human Cardiomyocytes

This study sought to characterize the relation between the oxidation of protein sulfhydryl (SH) groups and Ca2+-activated force production in the human myocardium. Triton-permeabilized left ventricular cardiomyocytes from donor hearts were exposed to an oxidative (2,2'-dithiodipyridine, DTDP) agent in vitro, and the changes in isometric force, its Ca2+ sensitivity, the cross-bridge-sensitive rate constant of force redevelopment at saturating [Ca2+] (k(tr,max)), and protein SH oxidation were monitored. DTDP (0.1-10 mM for 2 min) oxidized the myocardial proteins and diminished the Ca2+-activated force with different concentration dependences (EC(50,SH) = 0.17 +/- 0.02 mM and EC(50,force) = 2.46 +/- 0.22 mM; mean +/- SEM). The application of 2.5 mM DTDP decreased the maximal Ca2+-activated force (to 64%), its Ca2+ sensitivity (DeltapCa(50) = 0.22 +/- 0.02), and the steepness of the Ca2+-force relation (n(Hill), from 2.01 +/- 0.08 to 1.76 +/- 0.08). These changes were paralleled by reductions in the free SH content of the proteins (to 15%) and in k(tr,max) (to 75%). SH-specific labeling identified SH oxidation of myosin light chain 1 and actin at DTDP concentrations at which the changes in the contractile parameters occurred. Our data suggest that SH oxidation in selected myofilament proteins diminishes the Ca2+-activated force and its Ca2+ sensitivity through an impaired Ca2+ regulation of the actin-myosin cycle in the human heart.

Defective regulation of contractile function in muscle fibres carrying an E41K β‐tropomyosin mutation

A novel E41K beta-tropomyosin (beta-Tm) mutation, associated with congenital myopathy and muscle weakness, was recently identified in a woman and her daughter. In both patients, muscle weakness was coupled with muscle fibre atrophy. It remains unknown, however, whether the E41K beta-Tm mutation directly affects regulation of muscle contraction, contributing to the muscle weakness. To address this question, we studied a broad range of contractile characteristics in skinned muscle fibres from the two patients and eight healthy controls. Results showed decreases (i) in speed of contraction at saturated Ca(2+) concentration (apparent rate constant of force redevelopment (k(tr)) and unloaded shortening speed (V(0))); and (ii) in contraction sensitivity to Ca(2+) concentration, in fibres from patients compared with controls, suggesting that the mutation has a negative effect on contractile function, contributing to the muscle weakness. To investigate whether these negative impacts are reversible, we exposed skinned muscle fibres to the Ca(2+) sensitizer EMD 57033. In fibres from patients, 30 mum of EMD 57033 (i) had no effect on speed of contraction (k(tr) and V(0)) at saturated Ca(2+) concentration but (ii) increased Ca(2+) sensitivity of contraction, suggesting a potential therapeutic approach in patients carrying the E41K beta-Tm mutation.

Effects of a preferential myosin loss on Ca2+ activation of force generation in single human skeletal muscle fibres

Preferential loss of the motor protein myosin, as observed in patients with acute quadriplegic myopathy (AQM) or cancer cachexia, causes generalized muscle wasting, muscle weakness and a decrease in muscle fibre force normalized to cross-sectional area. It remains unclear, however, whether this myosin loss influences other important features of muscle fibre function, such as Ca(2+) activation of the contractile proteins. To address this question, we have studied Ca(2+) sensitivity of force generation using skinned muscle fibres from four patients with AQM or cancer cachexia and a preferential loss of myosin; and from seven healthy control individuals. Force and apparent rate constant of force redevelopment (k(tr)) were assessed in solutions with varying Ca(2+) concentrations (pCa), allowing construction of relative force-pCa and k(tr)-pCa relationships. Results showed a rightward shift of the relative force-pCa relationship and a leftward shift of the relative k(tr)-pCa curve in muscle fibres with a preferential myosin loss. To improve the understanding of the mechanisms underlying these alterations, the relative stiffness-pCa relationship was evaluated. A rightward shift of this curve was observed, suggesting that the changes in the Ca(2+) activation of force and k(tr) were predominantly due to a decrease in the relative number of attached cross-bridges at different pCa values. Thus, a change in Ca(2+) activation of the contractile apparatus in patients with preferential myosin loss is proposed as an additional factor contributing to the muscle function impairment in these patients.

Effects of a R133W β‐tropomyosin mutation on regulation of muscle contraction in single human muscle fibres

A novel R133W beta-tropomyosin (beta-Tm) mutation, associated with muscle weakness and distal limb deformities, has recently been identified in a woman and her daughter. The muscle weakness was not accompanied by progressive muscle wasting or histopathological abnormalities in tibialis anterior muscle biopsy specimens. The aim of the present study was to explore the mechanisms underlying the impaired muscle function in patients with the beta-Tm mutation. Maximum force normalized to fibre cross-sectional area (specific force, SF), maximum velocity of unloaded shortening (V0), apparent rate constant of force redevelopment (ktr) and force-pCa relationship were evaluated in single chemically skinned muscle fibres from the two patients carrying the beta-Tm mutation and from healthy control subjects. Significant differences in regulation of muscle contraction were observed in the type I fibres: a lower SF (P<0.05) and ktr (P<0.01), and a faster V0 (P<0.05). The force-pCa relationship did not differ between patient and control fibres, indicating an unaltered Ca2+ activation of contractile proteins. Collectively, these results indicate a slower cross-bridge attachment rate and a faster detachment rate caused by the R133W beta-Tm mutation. It is suggested that the R133W beta-Tm mutation induces alteration in myosin-actin kinetics causing a reduced number of myosin molecules in the strong actin-binding state, resulting in overall muscle weakness in the absence of muscle wasting.

Rate of tension redevelopment is not modulated by sarcomere length in permeabilized human, murine, and porcine cardiomyocytes

The increase in Ca(2+) sensitivity of isometric force development along with sarcomere length (SL) is considered as the basis of the Frank-Starling law of the heart, possibly involving the regulation of cross-bridge turnover kinetics. Therefore, the Ca(2+) dependencies of isometric force production and of the cross-bridge-sensitive rate constant of force redevelopment (k(tr)) were determined at different SLs (1.9 and 2.3 mum) in isolated human, murine, and porcine permeabilized cardiomyocytes. k(tr) was also determined in the presence of 10 mM inorganic phosphate (P(i)), which interfered with the force-generating cross-bridge transitions. The increases in Ca(2+) sensitivities of force with SL were very similar in human, murine, and porcine cardiomyocytes (DeltapCa(50): approximately 0.11). k(tr) was higher (P < 0.05) in mice than in humans or pigs at all Ca(2+) concentrations ([Ca(2+)]) [maximum k(tr) (k(tr,max)) at a SL of 1.9 mum and pCa 4.75: 1.33 +/- 0.11, 7.44 +/- 0.15, and 1.02 +/- 0.05 s(-1), in humans, mice, and pigs, respectively] but k(tr) did not depend on SL in any species. Moreover, when the k(tr) values for each species were expressed relative to their respective maxima, similar Ca(2+) dependencies were obtained. Ten millimolar P(i) decreased force to approximately 60-65% and left DeltapCa(50) unaltered in all three species. P(i) increased k(tr,max) by a factor of approximately 1.6 in humans and pigs and by a factor of approximately 3 in mice, independent of SL. In conclusion, species differences exert a major influence on k(tr), but SL does not appear to modulate the cross-bridge turnover rates in human, murine, and porcine hearts.

Hypoxia-induced skeletal muscle fiber dysfunction: role for reactive nitrogen species

Hypoxia impairs skeletal muscle function, but the precise mechanisms are incompletely understood. In hypoxic rat diaphragm muscle, generation of peroxynitrite is elevated. Peroxynitrite and other reactive nitrogen species have been shown to impair contractility of skinned muscle fibers, reflecting contractile protein dysfunction. We hypothesized that hypoxia induces contractile protein dysfunction and that reactive nitrogen species are involved. In addition, we hypothesized that muscle reoxygenation reverses contractile protein dysfunction. In vitro contractility of rat soleus muscle bundles was studied after 30 min of hyperoxia (Po2 approximately 90 kPa), hypoxia (Po2 approximately 5 kPa), hypoxia + 30 microM N(G)-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor), hyperoxia + 30 microM L-NMMA, and hypoxia (30 min) + reoxygenation (15 min). One part of the muscle bundle was used for single fiber contractile measurements and the other part for nitrotyrosine detection. In skinned single fibers, maximal Ca2+-activated specific force (Fmax), fraction of strongly attached cross bridges (alphafs), rate constant of force redevelopment (ktr), and myofibrillar Ca2+ sensitivity were determined. Thirty minutes of hypoxia reduced muscle bundle contractility. In the hypoxic group, single fiber Fmax, alphafs, and ktr were significantly reduced compared with hyperoxic, L-NMMA, and reoxygenation groups. Myofibrillar Ca2+ sensitivity was not different between groups. Nitrotyrosine levels were increased in hypoxia compared with all other groups. We concluded that acute hypoxia induces dysfunction of skinned muscle fibers, reflecting contractile protein dysfunction. In addition, our data indicate that reactive nitrogen species play a role in hypoxia-induced contractile protein dysfunction. Reoxygenation of the muscle bundle partially restores bundle contractility but completely reverses contractile protein dysfunction.

Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium.

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT(trunc)) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT(trunc) (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca(2+) sensitivity of force development [pCa for half-maximal tension generation (pCa(50))] and the rate constant of force redevelopment (k(tr)) in cTnT(trunc) and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT(trunc) mice exhibited greater pCa(50), reduced steepness of the force-pCa relationship [Hill coefficient (n(H))], and faster k(tr) at submaximal Ca(2+) concentration ([Ca(2+)]), i.e., reduced activation dependence of k(tr). Treatment with NEM-S1 elicited similar increases in pCa(50) and similar reductions in n(H) in WT and cTnT(trunc) myocardium but elicited greater increases in k(tr) at submaximal activation in cTnT(trunc) myocardium. Contrary to our initial hypothesis, cTnT(trunc) appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca(2+)-activated force and the rate of cross-bridge attachment at submaximal [Ca(2+)]. Although these mechanisms would not be expected to depress systolic function per se in cTnT(trunc) hearts, they would account for slowed rates of myocardial relaxation during early diastole.