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During ischemic heart failure (IHF), cardiac muscle contraction is typically impaired, though the molecular changes within the myocardium are not fully understood. Thus, we aimed to characterize the biophysical properties of cardiac myosin in IHF. Cardiac tissue was harvested from 10 age-matched males, either with a history of IHF or nonfailing (NF) controls that had no history of structural or functional cardiac abnormalities. Clinical measures before cardiac biopsy demonstrated significant differences in measures of ejection fraction and left ventricular dimensions. Myofibrils and myosin were extracted from left ventricular free wall cardiac samples. There were no changes in myofibrillar ATPase activity or calcium sensitivity between groups. Using isolated myosin, we found a 15% reduction in the IHF group in actin sliding velocity in the in vitro motility assay, which was observed in the absence of a myosin isoform shift. Oxidative damage (carbonylation) of isolated myosin was compared, in which there were no significant differences between groups. Synthetic thick filaments were formed from purified myosin and the ATPase activity was similar in both basal and actin-activated conditions (20 mM actin). Correlation analysis and Deming linear regression were performed between all studied parameters, in which we found statistically significant correlations between clinical measures of contractility with molecular measures of sliding velocity and ELC carbonylation. Our data indicate that subtle deficits in myosin mechanochemical properties are associated with reduced contractile function and pathological remodeling of the heart, suggesting that the myosin motor may be an effective pharmacological intervention in ischemia. [ABSTRACT FROM AUTHOR]

The interaction between actin and myosin is the basis of contraction and force production in muscle fibers. Studies have shown that actin and myosin oxidation cause myofibrillar weakness in healthy and diseased muscles. The degree to which oxidation of each of these proteins contributes to an attenuated force in myofibrils is unclear. In this study, we show that exposure of actin and myosin to the chemical 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1), an NO and O2*- donor, affected actinmyosin interactions, as shown by a decreased myosin-propelled actin velocity in the in vitro motility assay. We also observed that oxidation of actin and myosin resulted in a decrease in force generated by myosin and actin filaments, as determined by a system of microfabricated cantilevers. Although myosin is more sensitive to oxidative modifications than actin, as indicated by a steeper decrease in velocity and force by the filaments, modifications on actin are sufficient to affect force and velocity and also contribute to a decrease in contractile activity in muscles. [ABSTRACT FROM AUTHOR]

Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single-thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFPMyh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging. [ABSTRACT FROM AUTHOR]

When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca2+ sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca2+ sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca2+ sensitivity as shown by a change in the force-pCa2+ curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations. [ABSTRACT FROM AUTHOR]

Factors underpinning the time-course of resistance-type exercise training (RET) adaptations are not fully understood. This study hypothesized that consuming a twice-daily protein-polyphenol beverage (PPB; n = 15; age, 24 ± 1 yr; BMI, 22.3 ± 0.7 kg·m-2) previously shown to accelerate recovery from muscle damage and increase daily myofibrillar protein synthesis (MyoPS) rates would accelerate early (10 sessions) improvements in muscle function and potentiate quadriceps volume and muscle fiber cross-sectional area (fCSA) following 30 unilateral RET sessions in healthy, recreationally active, adults. Versus isocaloric placebo (PLA; n = 14; age, 25 ± 2 yr; BMI, 23.9 ± 1.0 kg·m-2), PPB increased 48 h MyoPS rates after the first RET session measured using deuterated water (2.01 ± 0.15 vs. 1.51 ± 0.16%·day-1, respectively; P < 0.05). In addition, PPB increased isokinetic muscle function over 10 sessions of training relative to the untrained control leg (%U) from 99.9 ± 1.8 pretraining to 107.2 ± 2.4%U at session 10 (vs. 102.6 ± 3.9 to 100.8 ± 2.4%U at session 10 in PLA; interaction P < 0.05). Pre to posttraining, PPB increased type II fCSA (PLA: 120.8 ± 8.2 to 109.5 ± 8.6%U; PPB: 92.8 ± 6.2 to 108.4 ± 9.7%U; interaction P < 0.05), but the gain in quadriceps muscle volume was similar between groups. Similarly, PPB did not further increase peak isometric torque, muscle function, or MyoPS measured posttraining. This suggests that although PPB increases MyoPS and early adaptation, it may not influence longer term adaptations to unilateral RET. [ABSTRACT FROM AUTHOR]

Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, how these transformed mesothelial cells contribute to lung fibrosis remains unclear. Here, we investigated the mechanism of contractile myofibroblast differentiation of PMCs. Transforming growth factor-β (TGF-β) induced marked upregulation of calponin 1 expression, which was correlated with notable cytoskeletal rearrangement in human PMCs (HPMCs) to produce stress fibers. Downregulation of calponin 1 expression reduced stress fiber formation. Interestingly, induced stress fibers predominantly contain α-smooth muscle actin (αSMA) associated with calponin 1 but not β-actin. Calponin 1-associated stress fibers also contained myosin II and α-actinin. Furthermore, focal adhesions were aligned with the produced stress fibers. These results suggest that calponin 1 facilitates formation of stress fibers that resemble contractile myofibrils. Supporting this notion, TGF-β significantly increased the contractile activity of HPMCs, an effect that was abolished by downregulation of calponin 1 expression. We infer that differentiation of HPMCs to contractile myofibroblasts facilitates stiffness of scar tissue in pleura to promote pleural fibrosis (PF) and that upregulation of calponin 1 plays a central role in this process. [ABSTRACT FROM AUTHOR]

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca 2+ flux and Ca 2+ levels in diastole, the troponin-tropomyosin complex role as a Ca 2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

Mitochondria from skeletal muscle of humans with obesity often display alterations with respect to their morphology, proteome, biogenesis, and function. These changes in muscle mitochondria are considered to contribute to metabolic abnormalities observed in humans with obesity. Most of the evidence describing alterations in muscle mitochondria in humans with obesity, however, lacks reference to a specific subcellular location. This is despite data over the years showing differences in the morphology and function of subsarcolemmal (found near the plasma membrane) and intermyofibrillar (nested between the myofibrils) mitochondria in skeletal muscle. Recent studies reveal that impairments in mitochondrial function in obesity with respect to the subcellular location of the mitochondria in muscle are more readily evident following exposure of the skeletal muscle to physiological stimuli. In this review, we highlight the need to understand skeletal muscle mitochondria metabolism in obesity in a subpopulation-specific manner and in the presence of physiological stimuli that modify mitochondrial function in vivo. Experimental approaches employed under these conditions will allow for more precise characterization of impairments in skeletal muscle mitochondria and their implications in inducing metabolic dysfunction in human obesity. [ABSTRACT FROM AUTHOR]

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO−) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO− donor (SIN-1) or directly with ONOO−, which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO− treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate (gapp) in combination with a conservation of the force redevelopment rate (kTr) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology. [ABSTRACT FROM AUTHOR]

Understanding the gross organization of skeletal muscle is critical to understanding the mechanism of action of muscle physiology. Due to coronavirus disease-19 (COVID-19), many colleges have had to discontinue or curtail teaching and laboratory activities. Whether students are in the classroom or learning online, it is important for them to understand the basics of skeletal muscle organization that allows for movement. Manipulatives have been shown to enhance student learning and understanding in many fields, including physiology. This gives instructors an easy-to-follow tool for making a manipulative that allows students to see the organization of the skeletal muscle. Students can make this manipulative themselves from supplies commonly found in the home or office. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the role of titin in preventing the development of sarcomere length nonuniformities following activation and after active and passive stretch by determining the effect of partial titin degradation on sarcomere length nonuniformities and force in passive and active myofibrils. Selective partial titin degradation was performed using a low dose of trypsin. Myofibrils were set at a sarcomere length of 2.4 µm and then passively stretched to sarcomere lengths of 3.4 and 4.4 µm. In the active condition, myofibrils were set at a sarcomere length of 2.8 µm, activated, and actively stretched by 1 µm/sarcomere. The extent of sarcomere length nonuniformities was calculated for each sarcomere as the absolute difference between sarcomere length and the mean sarcomere length of the myofibril. Our main finding is that partial titin degradation does not increase sarcomere length nonuniformities after passive stretch and activation compared with when titin is intact but increases the extent of sarcomere length nonuniformities after active stretch. Furthermore, when titin was partially degraded, active and passive stresses were substantially reduced. These results suggest that titin plays a crucial role in actively stretched myofibrils and is likely involved in active and passive force production. [ABSTRACT FROM AUTHOR]

Myofibrils made up of actin, myosin, and associated proteins generate the contractile force in muscle, and, consequently, mutations in these proteins may lead to heart failure. Septins are a conserved family of small GTPases that associate with actin filaments, microtubules, and cellular membranes. Despite the importance of septins in cytoskeleton organization, their role in cardiomyocyte organization and function is poorly characterized. Here, we show that septin 7 is expressed in both embryonic and adult zebrafish hearts and elucidate the physiological significance of sept7b, the zebrafish ortholog of human septin 7, in the heart in embryonic and larval zebrafish. Knockdown of sept7b reduced F-actin and α-cardiac actin expression in the heart and caused disorganization of actin filaments. Electron microscopy of sept7bdepleted larvae showed disorganization of heart myofibrils and partial detachment from Z-disks. Functional studies revealed that knockdown of sept7b leads to reduced ventricular dimensions, contractility, and cardiac output. Furthermore, we found that depletion of sept7b diminished the expression of retinaldehyde dehydrogenase 2, which catalyzes the synthesis of retinoic acid necessary for heart morphogenesis. We further observed that the sept7b and retinoic acid signaling pathways converge to regulate cardiac function. Together, these results specify an essential role for sept7b in the contractile function of the heart. [ABSTRACT FROM AUTHOR]

We recently reported that skeletal muscle fibers of obscurin knockout (KO) mice present altered distribution of ankyrin B (ankB), disorganization of the subsarcolemmal microtubules, and reduced localization of dystrophin at costameres. In addition, these mice have impaired running endurance and increased exercise-induced sarcolemmal damage compared with wild-type animals. Here, we report results from a combined approach of physiological, morphological, and structural studies in which we further characterize the skeletal muscles of obscurin KO mice. A detailed examination of exercise performance, using different running protocols, revealed that the reduced endurance of obscurin KO animals on the treadmill depends on exercise intensity and age. Indeed, a mild running protocol did not evidence significant differences between control and obscurin KO mice, whereas comparison of running abilities of 2-, 6-, and 11-mo-old mice exercised at exhaustion revealed a progressive age-dependent reduction of the exercise tolerance in KO mice. Histological analysis indicated that heavy exercise induced leukocyte infiltration, fibrotic connective tissue deposition, and hypercontractures in the diaphragm of KO mice. On the same line, electron microscopy revealed that, in the diaphragm of exercised obscurin KO mice, but not in the hindlimb muscles, both M-line and H-zone of sarcomeres appeared wavy and less defined. Altogether, these results suggest that obscurin is required for the maintenance of morphological and ultrastructural integrity of skeletal muscle fibers against damage induced by intense mechanical stress and point to the diaphragm as the skeletal muscle most severely affected in obscurindeficient mice. [ABSTRACT FROM AUTHOR]

This study presents a structure-function analysis of the mammalian left ventricle and examines the performance of the cardiac capillary network, mitochondria, and myofibrils at rest and during simulated heavy exercise. Left ventricular external mechanical work rate was calculated from cardiac output and systemic mean arterial blood pressure in resting sheep (Ovis aries; n = 4) and goats (Capra hircus; n = 4) under mild sedation, followed by perfusion-fixation of the left ventricle and quantification of the cardiac capillary-tissue geometry and cardiomyocyte ultrastructure. The investigation was then extended to heavy exercise by increasing cardiac work according to published hemodynamics of sheep and goats performing sustained treadmill exercise. Left ventricular work rate averaged 0.017 W/cm3 of tissue at rest and was estimated to increase to ~0.060 W/cm3 during heavy exercise. According to an oxygen transport model we applied to the left ventricular tissue, we predicted that oxygen consumption increases from 195 nmol O2.s-1.cm-3 of tissue at rest to ~600 nmol O2.s-1.cm-3 during heavy exercise, which is within 90% of the oxygen demand rate and consistent with work remaining predominantly aerobic. Mitochondria represent 21-22% of cardiomyocyte volume and consume oxygen at a rate of 1,150 nmol O2.s-1.cm-3 of mitochondria at rest and ~3,600 nmol O2.s-1.cm-3 during heavy exercise, which is within 80% of maximum in vitro rates and consistent with mitochondria operating near their functional limits. Myofibrils represent 65-66% of cardiomyocyte volume, and according to a Laplacian model of the left ventricular chamber, generate peak fiber tensions in the range of 50 to 70 kPa at rest and during heavy exercise, which is less than maximum tension of isolated cardiac tissue (120-140 kPa) and is explained by an apparent reserve capacity for tension development built into the left ventricle. [ABSTRACT FROM AUTHOR]

This study examined the contribution of muscle fiber orientation at different knee angles to regional activation identified with high-density surface electromyography (HDsEMG). Monopolar HDsEMG signals were collected using a grid of 13 x 5 electrodes placed over the vastus medialis (VM). Intramuscular electrical stimulation was used to selectively activate two regions within VM. The distribution of EMG responses to stimulation was obtained by calculating the amplitude of the compound action potential for each channel; the position of the peak amplitude was tracked across knee angles to describe shifts of the active muscle regions under the electrodes. In a separate experiment, regional activation was investigated in 10 knee flexion-extension movements against a fixed resistance. Intramuscular stimulation of different VM regions resulted in clear differences in amplitude distribution along the columns of the electrode grid (P < 0.001); changes in knee angle resulted in consistent shifts along the rows (P < 0.01) and negligible shifts along the columns of the electrode grid. Regional VM activation was identified in dynamic movement, with distal shifts of the EMG distribution in the eccentric phase of the movement (P < 0.05) and at more flexed knee angles (P < 0.05). HDsEMG was used to describe regional activation across the VM that was not attributable to anatomic factors. Changes in muscle fiber orientation associated with knee joint angle mainly influence the amplitude distribution along the fiber direction. Future studies are needed to understand possible functional roles for regional activation within the VM in dynamic tasks. [ABSTRACT FROM AUTHOR]

Skeletal muscle contains one of the largest and the most dynamic pools of Na,K-ATPase (NKA) in the body. Under resting conditions, NKA in skeletal muscle operates at only a fraction of maximal pumping capacity, but it can be markedly activated when demands for ion transport increase, such as during exercise or following food intake. Given the size, capacity, and dynamic range of the NKA pool in skeletal muscle, its tight regulation is essential to maintain whole body homeostasis as well as muscle function. To reconcile functional needs of systemic homeostasis with those of skeletal muscle, NKA is regulated in a coordinated manner by extrinsic stimuli, such as hormones and nerve-derived factors, as well as by local stimuli arising in skeletal muscle fibers, such as contractions and muscle energy status. These stimuli regulate NKA acutely by controlling its enzymatic activity and/or its distribution between the plasma membrane and the intracellular storage compartment. They also regulate NKA chronically by controlling NKA gene expression, thus determining total NKA content in skeletal muscle and its maximal pumping capacity. This review focuses on molecular mechanisms that underlie regulation of NKA in skeletal muscle by major extrinsic and local stimuli. Special emphasis is given to stimuli and mechanisms linking regulation of NKA and energy metabolism in skeletal muscle, such as insulin and the energy-sensing AMP-activated protein kinase. Finally, the recently uncovered roles for glutathionylation, nitric oxide, and extracellular K+ in the regulation of NKA in skeletal muscle are highlighted. [ABSTRACT FROM AUTHOR]

The article presents a study conducted by the authors related to effect of muscle disuse on myofibrillar protein synthesis in human beings. Topics discussed include decline in postabsorptive myofibrillar protein synthesis and skeletal muscle atrophy due to muscle disuse; loss of muscle mass from quadriceps and muscle disuse leading to sarcopenia. The study was published in the November 17, 2015 issue of the "American Journal of Physiology: Endocrinology and Metabolism."

Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles. [ABSTRACT FROM AUTHOR]

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca2+ activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca2+-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 µm for the psoas, 2.68 and 4.21 µm for the soleus, and 1.51 and 2.86 µm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca2+-dependent change in titin properties and not associated with changes in titin-actin interactions. [ABSTRACT FROM AUTHOR]

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca2 sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of crossbridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM. [ABSTRACT FROM AUTHOR]

Strategies to enhance weight loss with a high fat-to-lean ratio in overweight/obese older adults are important since lean loss could exacerbate sarcopenia. We examined how dietary protein distribution affected muscle protein synthesis during: energy balance (EB); energy restriction (ER); and ER plus resistance training (ER+RT). A 4-wk ER diet was provided to overweight/obese older men (66 ± 4 y, 31 ± 5 kg/m²) who were randomized to either a balanced (BAL: 25% daily protein/meal x 4) or skewed pattern (SKEW: 7:17:72:4% daily protein/meal; n = 10/group). Myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR) were measured during a 13-h primed continuous infusion of L-[ring-13C6] phenylalanine with BAL and SKEW pattern of protein intake in EB, after 2-wk ER, and after 2-wk ER+RT. Fed-state myofibrillar FSR was lower in ER than EB in both groups (p < 0.001), but was greater in BAL than SKEW (p = 0.014). In ER+RT, fed-state myofibrillar FSR increased above ER in both groups and in BAL was not different from EB (p = 0.903). In SKEW myofibrillar FSR remained lower than EB (p = 0.002) and lower than BAL (p = 0.006). Fed-state sarcoplasmic protein FSR was reduced similarly in ER and ER+RT compared to EB (p < 0.01) in both groups. During ER in overweight/obese older men a BAL consumption of protein stimulated the synthesis of muscle contractile proteins more effectively than traditional, SKEW distribution. Combining RT with a BAL protein distribution 'rescued' the lower rates of myofibrillar protein synthesis during moderate ER. [ABSTRACT FROM AUTHOR]

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγ charge and L-type Ca2+ current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture. [ABSTRACT FROM AUTHOR]

Age-related loss of skeletal muscle mass and function is implicated in the development of disease and physical disability. However, little is known about how age affects skeletal muscle structure at the cellular and ultrastructural levels or how such alterations impact function. Thus we examined skeletal muscle structure at the tissue, cellular, and myofibrillar levels in young (21-35 yr) and older (65-75 yr) male and female volunteers, matched for habitual physical activity level. Older adults had smaller whole muscle tissue cross-sectional areas (CSAs) and mass. At the cellular level, older adults had reduced CSAs in myosin heavy chain II (MHC II) fibers, with no differences in MHC I fibers. In MHC II fibers, older men tended to have fewer fibers with large CSAs, while older women showed reduced fiber size across the CSA range. Older adults showed a decrease in intermyofibrillar mitochondrial size; however, the age effect was driven primarily by women (i.e., age by sex interaction effect). Mitochondrial size was inversely and directly related to isometric tension and myosin-actin cross-bridge kinetics, respectively. Notably, there were no intermyo- fibrillar or subsarcolemmal mitochondrial fractional content or myo- filament ultrastructural differences in the activity-matched young and older adults. Collectively, our results indicate age-related reductions in whole muscle size do not vary by sex. However, age-related structural alterations at the cellular and subcellular levels are different between the sexes and may contribute to different functional phenotypes in ways that modulate sex-specific reductions in physical capacity with age. [ABSTRACT FROM AUTHOR]

Through its upregulation and/or translocation, heat shock protein 72 (HSP72) is involved in protection and repair of key proteins after physiological stress. In human skeletal muscle we investigated HSP72 protein after eccentric (ECC1) and concentric (CONC) exercise and repeated eccentric exercise (ECC2; 8 wk later) and whether it translocated from its normal cytosolic location to membranes/myofibrils. HSP72 protein increased ~ 2-fold 24 h after ECC1, with no apparent change after CONC or ECC2. In resting (nonstressed) human skeletal muscle the total pool of HSP72 protein was present almost exclusively in the cytosolic fraction, and after each exercise protocol the distribution of HSP72 protein remained unaltered. Overall, the amount of HSP72 protein in the cytosol increased 24 h after ECC1, matching the fold increase that was measured in total HSP72 protein. To better ascertain the capabilities and limitations of HSP72, using quantitative Western blotting we determined the HSP72 protein content to be 11.4 p,mol/kg wet weight in resting human vastus lateralis muscle, which is comprised of Type I (slow-twitch) and Type II (fast-twitch) fibers. HSP72 protein content was similar in individual Type I or II fiber segments. After physiological stress, HSP72 content can increase and, although the functional consequences of increased amounts of HSP72 protein are poorly understood, it has been shown to bind to and protect protein pumps like SERCA and Na+-K+-ATPase. Given no translocation of cytosolic HSP72, these findings suggest eccentric contractions, unlike other forms of stress such as heat, do not trigger tight binding of HSP72 to its primary membrane-bound target proteins, in particular SERCA. [ABSTRACT FROM AUTHOR]

Contraction regulates heart development via a complex mechanotransduction process controlled by various mechanical forces. Here, we exploit zebrafish embryos as an in vivo animal model to discern the contribution from different mechanical forces and identify the underlying mechanotransductive signaling pathways of cardiogenesis. We treated 2 days postfertilization zebrafish embryos with Blebbistatin, a myosin II inhibitor, to stop cardiac contraction, which induces a response termed cessation of contraction-induced cardiomyocyte (CM) enlargement (CCE). Accompanying the CCE, lateral fusion of myofibrils was attenuated within CMs. The CCE can be blunted by loss of blood in tail-docked zebrafish but not in cloche mutant fish, suggesting that transmural pressure rather than shear stress is accountable for the chamber enlargement. By screening a panel of small molecule inhibitors, our data suggested essential functions of phosphoinositide 3-kinase signaling and protein synthesis in CCE, which are independent of the sarcomere integrity. In summary, we defined a unique CCE response in genetically tractable zebrafish embryos. A panel of assays was established to verify the contribution extrinsic forces and interrogate underlying signaling pathways. [ABSTRACT FROM AUTHOR]

Electrical dyssynchrony leads to prestretch in late-activated regions and alters the sequence of mechanical contraction, although prestretch and its mechanisms are not well defined in the failing heart. We hypothesized that in heart failure, fiber prestretch magnitude increases with the amount of early-activated tissue and results in increased end-systolic strains, possibly due to length-dependent muscle properties. In five failing dog hearts with scars, three-dimensional strains were measured at the anterolateral left ventricle (LV). Prestretch magnitude was varied via ventricular pacing at increasing distances from the measurement site and was found to increase with activation time at various wall depths. At the subepicardium, prestretch magnitude positively correlated with the amount of early-activated tissue. At the subendocardium, local end-systolic strains (fiber shortening, radial wall thickening) increased proportionally to prestretch magnitude, resulting in greater mean strain values in late-activated compared with early-activated tissue. Increased fiber strains at end systole were accompanied by increases in preejection fiber strain, shortening duration, and the onset of fiber relengthening, which were all positively correlated with local activation time. In a dog-specific computational failing heart model, removal of length and velocity dependence on active fiber stress generation, both separately and together, alter the correlations between local electrical activation time and timing of fiber strains but do not primarily account for these relationships. [ABSTRACT FROM AUTHOR]

We compared the contractile performance of papillary muscle from a mouse model of hypertrophic cardiomyopathy [α-cardiac actin (ACTC) E99K mutation] with nontransgenic (non-TG) littermates. In isometric twitches, ACTC E99K papillary muscle produced three to four times greater force than non-TG muscle under the same conditions independent of stimulation frequency and temperature, whereas maximum isometric force in myofibrils from these muscles was not significantly different. ACTC E99K muscle relaxed slower than non-TG muscle in both papillary muscle (1.4×) and myofibrils (1.7×), whereas the rate of force development after stimulation was the same as non-TG muscle for both electrical stimulation in intact muscle and after a Ca2+ jump in myofibrils. The EC50 for Ca2+ activation of force in myofibrils was 0.39 ± 0.33 μmol/l in ACTC E99K myofibrils and 0.80 ± 0.11 μmol/l in non-TG myofibrils. There were no significant differences in the amplitude and time course of the Ca2+ transient in myocytes from ACTC E99K and non-TG mice. We conclude that hypercontractility is caused by higher myofibrillar Ca2+ sensitivity in ACTC E99K muscles. Measurement of the energy (work + heat) released in actively cycling heart muscle showed that for both genotypes, the amount of energy turnover increased with work done but with decreasing efficiency as energy turnover increased. Thus, ACTC E99K mouse heart muscle produced on average 3.3-fold more work than non-TG muscle, and the cost in terms of energy turnover was disproportionately higher than in non-TG muscles. Efficiency for ACTC E99K muscle was in the range of 11-16% and for non-TG muscle was 15-18%. [ABSTRACT FROM AUTHOR]

Among its many biological roles, fibroblast growth factor-2 (FGF2) acutely protects the heart from dysfunction associated with ischemia/reperfusion (I/R) injury. Our laboratory has demonstrated that this is due to the activity of the low molecular weight (LMW) isoform of FGF2 and that FGF2-mediated cardioprotection relies on the activity of protein kinase C (PKC); however, which PKC isoforms are responsible for LMW FGF2-mediated cardioprotection, and their downstream targets, remain to be elucidated. To identify the PKC pathway(s) that contributes to postischemic cardiac recovery by LMW FGF2, mouse hearts expressing only LMW FGF2 (HMWKO) were bred to mouse hearts not expressing PKCα (PKCαKO) or subjected to a selective PKCε inhibitor (εV1-2) before and during I/R. Hearts only expressing LMW FGF2 showed significantly improved postischemic recovery of cardiac function following I/R (P < 0.05), which was significantly abrogated in the absence of PKCα (P < 0.05) or presence of PKCε inhibition (P < 0.05). Hearts only expressing LMW FGF2 demonstrated differences in actomyosin ATPase activity as well as increases in the phosphorylation of troponin I and T during I/R compared with wild-type hearts; several of these effects were dependent on PKCα activity. This evidence indicates that both PKCα and PKCε play a role in LMW FGF2-mediated protection from cardiac dysfunction and that PKCα signaling to the contractile apparatus is a key step in the mechanism of LMW FGF2-mediated protection against myocardial dysfunction. [ABSTRACT FROM AUTHOR]

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of a-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure. [ABSTRACT FROM AUTHOR]

When a stretch is imposed to activated muscles, there is a residual force enhancement that persists after the stretch; the force is higher than that produced during an isometric contraction in the corresponding length. The mechanisms behind the force enhancement remain elusive, and there is disagreement if it represents a sarcomeric property, or if it is associated with length nonuniformities among sarcomeres and half-sarcomeres. The purpose of this study was to investigate the effects of stretch on single sarcomeres and myofibrils with predetermined numbers of sarcomeres (n = 2, 3. . . , 8) isolated from the rabbit psoas muscle. Sarcomeres were attached between two precalibrated microneedles for force measurements, and images of the preparations were projected onto a linear photodiode array for measurements of half-sarcomere length (SL). Fully activated sarcomeres were subjected to a stretch (5-10% of initial SL, at a speed of 0.3 μm-s-1·SL-1) after which they were maintained isometric for at least 5 s before deactivation. Single sarcomeres showed two patterns: 31 sarcomeres showed a small level of force enhancement after stretch (10.46 ± 0.78%), and 28 sarcomeres did not show force enhancement (-0.54 ± 0.17%). In these preparations, there was not a strong correlation between the force enhancement and half-sarcomere length nonuniformities. When three or more sarcomeres arranged in series were stretched, force enhancement was always observed, and it increased linearly with the degree of half-sarcomere length nonuniformities. The results show that the residual force enhancement has two mechanisms: 1) stretch-induced changes in sarcomeric structure(s); we suggest that titin is responsible for this component, and 2) stretch-induced nonuniformities of half-sarcomere lengths, which significantly increases the level of force enhancement. [ABSTRACT FROM AUTHOR]

For the past half century, the sliding filament-based cross-bridge theory has been the cornerstone of our understanding of how muscles contract. According to this theory, active force can only occur if there is overlap between the contractile filaments, actin and myosin. Otherwise, forces are thought to be caused by passive structural elements and are assumed to vary solely because of the length of the muscle. We observed increases in muscle force by a factor of 3 to 4 above the purely passive forces for activated and stretched myofibrils in the absence of actin-myosin overlap. We show that this dramatic increase in force is crucially dependent on the presence of the structural protein titin, cannot be explained with calcium activation, and is regulated by actin-myosin-based cross-bridge forces before stretching. We conclude from these observations that titin is a strong regulator of muscle force and propose that this regulation is based on cross-bridge force-dependent titin-actin interactions. These results suggest a mechanism for stability of sarcomeres on the "inherently unstable" descending limb of the force-length relationship, and they further provide an explanation for the protection of muscles against stretch-induced muscle injuries. [ABSTRACT FROM AUTHOR]

The aim of the present study was to test whether titin is a calcium-dependent spring and whether it is the source of the passive force enhancement observed in muscle and single fiber preparations. We measured passive force enhancement in troponin C (TnC)-depleted myofibrils in which active force production was completely eliminated. The TnC-depleted construct allowed for the investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils (n = 6) were 35.0 ± 2.9 nN/ μm2 when stretched to an average sarcomere length of 3.4 μm in a solution with low calcium concentration (pCa 8.0). Passive forces in the same myofibrils increased by 25% to 30% when stretches were performed in a solution with high calcium concentration (pCa 3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only ∼25% of the passive force enhancement observed in intact myofibrils. Therefore, ∼75% of the normally occurring passive force enhancement remains unexplained. The findings of the present study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness but also requires cross-bridge formation and/or active force production for full manifestation. [ABSTRACT FROM AUTHOR]