Decreased Force Generation Research Articles

Chloroquine impairs maximal transdiaphragmatic pressure generation in old mice.

Aging results in increased neuromuscular transmission failure and denervation of the diaphragm muscle, as well as decreased force generation across a range of motor behaviors. Increased risk for respiratory complications in old age is a major health problem. Aging impairs autophagy, a tightly regulated multistep process responsible for clearing misfolded or aggregated proteins and damaged organelles. In motor neurons, aging-related autophagy impairment may contribute to deficits in neurotransmission, subsequent muscle atrophy, and loss of muscle force. Chloroquine is commonly used to inhibit autophagy. We hypothesized that chloroquine decreases transdiaphragmatic pressure (Pdi) in mice. Old mice (16-28 mo old; n = 26) were randomly allocated to receive intraperitoneal chloroquine (50 mg/kg) or vehicle 4 h before measuring Pdi during eupnea, hypoxia (10% O2)-hypercapnia (5% CO2) exposure, spontaneous deep breaths ("sighs"), and maximal activation elicited by bilateral phrenic nerve stimulation (Pdimax). Pdi amplitude and ventilatory parameters across experimental groups and behaviors were evaluated using a mixed linear model. There were no differences in Pdi amplitude across treatments during eupnea (∼8 cm H2O), hypoxia-hypercapnia (∼10 cm H2O), or sigh (∼36 cm H2O), consistent with prior studies documenting a lack of aging effects on ventilatory behaviors. In vehicle and chloroquine-treated mice, average Pdimax was 61 and 46 cm H2O, respectively. Chloroquine decreased Pdimax by 24% compared to vehicle (P < 0.05). There were no sex or age effects on Pdi in older mice. The observed decrease in Pdimax suggests aging-related susceptibility to impairments in autophagy, consistent with the effects of chloroquine on this important homeostatic process.NEW & NOTEWORTHY Recent findings suggest that autophagy plays a role in the development of aging-related neuromuscular dysfunction; however, the contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. This study shows that in old mice, chloroquine administration decreases maximal transdiaphragmatic pressure generation. These chloroquine effects suggest a susceptibility to impairments in autophagy in old age.

Evaluation of Senescence and Its Prevention in Doxorubicin-Induced Cardiotoxicity Using Dynamic Engineered Heart Tissues

BackgroundDoxorubicin is an essential cancer treatment, but its usefulness is hampered by the occurrence of cardiotoxicity. Nevertheless, the pathophysiology underlying doxorubicin-induced cardiotoxicity and the respective molecular mechanisms are poorly understood. Recent studies have suggested involvement of cellular senescence. ObjectivesThe aims of this study were to establish whether senescence is present in patients with doxorubicin-induced cardiotoxicity and to investigate if this could be used as a potential treatment target. MethodsBiopsies from the left ventricles of patients with severe doxorubicin-induced cardiotoxicity were compared with control samples. Additionally, senescence-associated mechanisms were characterized in 3-dimensional dynamic engineered heart tissues (dyn-EHTs) and human pluripotent stem cell–derived cardiomyocytes. These were exposed to multiple, clinically relevant doses of doxorubicin to recapitulate patient treatment regimens. To prevent senescence, dyn-EHTs were cotreated with the senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol. ResultsSenescence-related markers were significantly up-regulated in the left ventricles of patients with doxorubicin-induced cardiotoxicity. Treatment of dyn-EHTs resulted in up-regulation of similar senescence markers as seen in the patients, accompanied by tissue dilatation, decreased force generation, and increased troponin release. Treatment with senomorphic drugs led to decreased expression of senescence-associated markers, but this was not accompanied by improved function. ConclusionsSenescence was observed in the hearts of patients with severe doxorubicin-induced cardiotoxicity, and this phenotype can be modeled in vitro by exposing dyn-EHTs to repeated clinically relevant doses of doxorubicin. The senomorphic drugs 5-aminoimidazole-4-carboxamide ribonucleotide and resveratrol prevent senescence but do not result in functional improvements. These findings suggest that preventing senescence by using a senomorphic during doxorubicin administration might not prevent cardiotoxicity.

Use of a novel technique to assess impact of age-related denervation on mouse soleus muscle function

Denervation contributes to loss of force-generating capacity in aged skeletal muscles, but problems with quantification of denervated fibers mean the precise impact of denervation on muscle function remains unclear. This study therefore looked to develop a reliable assay for identifying denervated muscle fibers, and used this to explore the impact of denervation on age-related force-generation in mouse skeletal muscle. Thirteen young (6-month-old) and 10 old (24-months-old) C57Bl/6 J female mice were utilized. Anaesthetized mice were infused with the fluorescent deoxyglucose analog 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) and the tibial nerve was repeatedly stimulated to label active skeletal muscle fibers by activity-dependent uptake of 2-NBDG. Data on muscle force generation were acquired as part of the stimulation routine. Labeled muscles were removed, snap frozen, sectioned, and slide mounted. Sections were imaged to show accumulation of 2-NBDG in activated fibers and lack of 2-NBDG accumulation in quiescent (denervated) fibers, then processed using immunohistochemistry to allow collection of data on fiber number and morphology. Soleus muscles from older mice had nine times as many denervated fibers as those from young mice (average n = 36 vs 4, old vs young). Older muscles developed significantly more passive force and less specific force, but denervation only partly accounted for age-related deficits in specific force. Further investigations are required to definitively identify contributors to the decrease in force generation that remain unaccounted for.

Abnormal kinetics of contraction caused by an α-actinin 2 missense variant in the EF-3-4 hand domain in human myocardium.

ACTN2 gene encodes α-actinin 2 protein, which binds actin. In cardiac muscle, α-actinin 2 is located in the Z-disc of the sarcomere, where it anchors myofibrillar actin filaments. A 48-years old female presented with out-of-hospital ventricular fibrillation arrest. Echocardiogram showed stage II diastolic dysfunction, elevated RVSP, and abnormal global longitudinal strain consistent with early LV systolic dysfunction. Cardiopulmonary testing indicated AHA functional class IV HF. Genetic testing identified a missense heterozygous variant of unknown significance in ACTN2 (gene previously associated with HCM and DCM), and SCNB2 (β-2 subunit of type II voltage-gated sodium channel). Histopathology demonstrated mild interstitial fibrosis of the LV free wall. We investigated the effects of the heterozygous A868T variant in α-actinin 2 on cardiac muscle mechanics. LV free wall samples were obtained from the patient's explanted heart at the time of cardiac transplantation. Permeabilized cardiac muscle preparations (CMPs) containing A868T and WT proteins displayed increased myofilament Ca2+-sensitivity of isometric force and lower maximal isometric specific force when compared to human donor samples. Sinusoidal stiffness was decreased at all levels of Ca2+-activation in A8686T CMPs. The rate of tension redevelopment (kTR) was faster at all levels of Ca2+-activation in A8686T CMPs. Computational modeling suggests that faster kTR in A868T CMPs is mainly due to faster cross-bridge detachment (g). The kinetic changes are consistent with decreased force generation by A868T CMPs being due to abnormalities in the cross-bridge interaction. Molecular dynamics simulation did not find significant differences in the interaction of A868T variant with titin and α-actinin. This project establishes the importance of α-actinin 2 variant in the Z-disc mechanosensitive interaction of thin- and thick-filament distal from its structural role in the sarcomere.

When fat meets the engine: implications of dietary rumenic acid on myosin-targeting therapies in heart failure.

When fat meets the engine: implications of dietary rumenic acid on myosin-targeting therapies in heart failure.

Traction Force Microscopy of Human Neutrophils During Critical Illness

Mapping of tractional forces produced by human neutrophils enables a precise understanding of mechanisms responsible for well-regulated cell motility. In collaboration with the Henann Lab at Brown University's Department of Solid Mechanics and the Franck Lab at University of Wisconsin's College of Engineering, we have developed a novel method of quantifying cellular force generation. Previously, Traction Force Microscopy (TFM) methods have required a 3D imaging modality to capture 3D forces. Here, we demonstrate a new method that can capture 3D forces using only a 2D imaging modality. Therefore, both forces in-plane with the substrate as well as forces out-of-plane to the substrate can now be seen with a standard epifluorescent microscope. Our technique involves embedding a single layer of fiducial markers under the surface of a soft fibronectin-coated polyacrylamide gel, followed by our previously published single particle tracking algorithm (T-PT) and a novel finite element method to quantify forces from substrate displacement. To validate this system, PMNs were obtained from healthy donors with ex vivo activation and from septic donors. Sepsis is a potentially fatal systemic inflammatory response to infection that can progress to multiorgan system failure. We hypothesize that ex vivo activation of PMNs from healthy donors and different substrate stiffnesses will lead to differential force production. On soft substrates (1kPa), when comparing PMN force generation from healthy and septic donors, we found that neutrophils from septic donors produce greater forces. However, ex vivo activation of PMNs from healthy donors with Lipopolysaccharide (LPS) had no effect, while a stimulant cocktail of LPS, Interferon Gamma (IFN-g), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) decreased force generation. Further, ex vivo activation with Phorbol Myristate Acetate (PMA) and N-Formylmethionine-leucyl-phenylalanine (fMLP) increased force generation. However, on stiff substrates (10kPa) LPS, PMA, and fMLP all had no effect on force generation, while the stimulant cocktail decreased total forces. Taken together with our novel TFM technique, these findings indicate that PMNs from septic patients are abnormal in their spatiotemporal generation of 3D forces. Further, secondary stimulation leads to differential dysregulation of force generation that is also mechanosensitive to substrate stiffness. Because there is still the need to further elucidate the deleterious molecular mechanisms during PMN-mediated sepsis, our methods will help bridge the divide between understanding dysregulation both in its biochemical and its mechanical interactions with the microenvironment.

Microstructural analysis of skeletal muscle force generation during aging.

Human aging results in a progressive decline in the active force generation capability of skeletal muscle. While many factors related to the changes of morphological and structural properties in muscle fibers and the extracellular matrix (ECM) have been considered as possible reasons for causing age-related force reduction, it is still not fully understood why the decrease in force generation under eccentric contraction (lengthening) is much less than that under concentric contraction (shortening). Biomechanically, it was observed that connective tissues (endomysium) stiffen as ages, and the volume ratio of connective tissues exhibits an age-related increase. However, limited skeletal muscle models take into account the microstructural characteristics as well as the volume fraction of tissue material. This study aims to provide a numerical investigation in which the muscle fibers and the ECM are explicitly represented to allow quantitative assessment of the age-related force reduction mechanism. To this end, a fiber-level honeycomb-like microstructure is constructed and modeled by a pixel-based Reproducing Kernel Particle Method (RKPM), which allows modeling of smooth transition in biomaterial properties across material interfaces. The numerical investigation reveals that the increased stiffness of the passive materials of muscle tissue reduces the force generation capability under concentric contraction while maintains the force generation capability under eccentric contraction. The proposed RKPM microscopic model provides effective means for the cellular-scale numerical investigation of skeletal muscle physiology. NOVELTY STATEMENT: A cellular-scale honeycomb-like microstructural muscle model constructed from a histological cross-sectional image of muscle is employed to study the causal relations between age-associated microstructural changes and age-related force loss using Reproducing Kernel Particle Method (RKPM). The employed RKPM offers an effective means for modeling biological materials based on pixel points in the medical images and allow modeling of smooth transition in the material properties across interfaces. The proposed microstructure-informed muscle model enables quantitative evaluation on how cellular-scale compositions contribute to muscle functionality and explain differences in age-related force changes during concentric, isometric and eccentric contractions.

Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers.

3299 Dynamic Afterload Cardiac Microtissue Model To Examine Molecular Pathways of Heart Failure

OBJECTIVES/SPECIFIC AIMS: This project aims to determine the key molecular pathways that link increased myocardial wall stress to cardiomyocyte hypertrophy and subsequent heart failure. We will use a cardiac microtissue (CMT) model with dynamically tunable cantilever stiffness to examine changes in CMT hypertrophy and electro-mechanical properties in response to increased afterload (cantilever stiffness). Subsequently, we will determine if inhibition of pro-hypertrophic or anti-hypertrophic pathways alter the hypertrophic response to increased afterload. Primary outcomes for this study are static/dynamic force, minimum electric field strength (VT), maximum capture rate (MCR), average cell area, and tissue cross-sectional thickness, and secondary outcomes are degree of myoblast activation and apoptosis. METHODS/STUDY POPULATION: CMT platforms will be fabricated using iron-doped polydimethylsiloxane (PDMS) to create magnetically tunable cantilevers. Cantilever stiffness will be increased with the application of an external magnetic field. Cantilever stiffness will be measured using a capacitance probe, where the force required to deflect both the cantilever and calibration probe is in accordance with Hooke’s Law. Human induced pluripotent stem cell cardiomyocyte (hiPSC-CMs) will be cultured and matured as 3D CMTs. In-vitro static/dynamic force generation will also be calculated by measuring the deflection of the cantilevers and applying Hooke’s law. CMTs will be paced using carbon electrodes to obtain VT and MCR. Structural data will be obtained using immunostaining and confocal microscopy. Finally, we will use pharmacologic inhibitors to inhibit molecular pathways that we identified in prior genetic screens such as ABCC8 (anti-hypertrophic mediator) and C1QTNF9 (pro-hypertrophic mediator). We will examine each of these pathways in low- and high-stiffness conditions. RESULTS/ANTICIPATED RESULTS: We believe increased afterload will cause significant hypertrophy, measured by increases in CMT cross-sectional thickness, cardiac myocyte area, myofibroblast activation, and myocyte apoptosis. In addition, we expect to see increases in static/dynamic force, increased voltage threshold, and decreased maximum capture rate. Preliminary results show a 64.3% increase in force generation when stiffness is increased by approximately 30%, and a 44.4% decrease in force generation when stiffness is decreased by approximately 30%. Finally, we expect that inhibiting a pro- or anti-hypertrophic molecular pathway will weaken or strengthen the hypertrophic response to increased afterload, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: To our knowledge, our lab is the first to create a dynamically tunable afterload system in the cantilever CMT model. This advance provides us with a robust platform to determine the molecular pathways that cause increased myocardial wall stress to result in cardiomyocyte hypertrophy and heart failure, which remain a critical knowledge gap in our understanding of cardiovascular disease. With more precise understanding of these pathways, we will equip ourselves with the knowledge to develop novel therapeutic agents to prevent the development or progression of heart failure.

Changing muscle function with sustained glial derived neurotrophic factor treatment of rabbit extraocular muscle

Recent microarray and RNAseq experiments provided evidence that glial derived neurotrophic factor (GDNF) levels were decreased in extraocular muscles from human strabismic subjects compared to age-matched controls. We assessed the effect of sustained GDNF treatment of the superior rectus muscles of rabbits on their physiological and morphological characteristics, and these were compared to naïve control muscles. Superior rectus muscles of rabbits were implanted with a sustained release pellet of GDNF to deliver 2μg/day, with the contralateral side receiving a placebo pellet. After one month, the muscles were assessed using in vitro physiological methods. The muscles were examined histologically for alteration in fiber size, myosin expression patterns, neuromuscular junction size, and stem cell numbers and compared to age-matched naïve control muscles. GDNF resulted in decreased force generation, which was also seen on the untreated contralateral superior rectus muscles. Muscle relaxation times were increased in the GDNF treated muscles. Myofiber mean cross-sectional areas were increased after the GDNF treatment, but there was a compensatory increase in expression of developmental, neonatal, and slow tonic myosin heavy chain isoforms. In addition, in the GDNF treated muscles there was a large increase in Pitx2-positive myogenic precursor cells. One month of GDNF resulted in significant extraocular muscle adaptation. These changes are interesting relative to the decreased levels of GDNF in the muscles from subjects with strabismus and preliminary data in infant non-human primates where sustained GDNF treatment produced a strabismus. These data support the view that GDNF has the potential for improving eye alignment in subjects with strabismus.

Role of Oxidative Stress as Key Regulator of Muscle Wasting during Cachexia.

Skeletal muscle atrophy is a pathological condition mainly characterized by a loss of muscular mass and the contractile capacity of the skeletal muscle as a consequence of muscular weakness and decreased force generation. Cachexia is defined as a pathological condition secondary to illness characterized by the progressive loss of muscle mass with or without loss of fat mass and with concomitant diminution of muscle strength. The molecular mechanisms involved in cachexia include oxidative stress, protein synthesis/degradation imbalance, autophagy deregulation, increased myonuclear apoptosis, and mitochondrial dysfunction. Oxidative stress is one of the most common mechanisms of cachexia caused by different factors. It results in increased ROS levels, increased oxidation-dependent protein modification, and decreased antioxidant system functions. In this review, we will describe the importance of oxidative stress in skeletal muscles, its sources, and how it can regulate protein synthesis/degradation imbalance, autophagy deregulation, increased myonuclear apoptosis, and mitochondrial dysfunction involved in cachexia.

Deletion of the Ste20-like kinase SLK in skeletal muscle results in a progressive myopathy and muscle weakness

BackgroundThe Ste20-like kinase, SLK, plays an important role in cell proliferation and cytoskeletal remodeling. In fibroblasts, SLK has been shown to respond to FAK/Src signaling and regulate focal adhesion turnover through Paxillin phosphorylation. Full-length SLK has also been shown to be essential for embryonic development. In myoblasts, the overexpression of a dominant negative SLK is sufficient to block myoblast fusion.MethodsIn this study, we crossed the Myf5-Cre mouse model with our conditional SLK knockout model to delete SLK in skeletal muscle. A thorough analysis of skeletal muscle tissue was undertaken in order to identify defects in muscle development caused by the lack of SLK. Isometric force analysis was performed on adult knockout mice and compared to age-matched wild-type mice. Furthermore, cardiotoxin injections were performed followed by immunohistochemistry for myogenic markers to assess the efficiency muscle regeneration following SLK deletion.ResultsWe show here that early deletion of SLK from the myogenic lineage does not markedly impair skeletal muscle development but delays the regenerative process. Interestingly, adult mice (~6 months) display an increase in the proportion of central nuclei and increased p38 activation. Furthermore, mice as young as 3 months old present with decreased force generation, suggesting that the loss of SLK impairs myofiber stability and function. Assessment of structural components revealed aberrant localization of focal adhesion proteins, such as FAK and paxillin. Our data show that the loss of SLK results in unstable myofibers resulting in a progressive myopathy. Additionally, the loss of SLK resulted in a delay in muscle regeneration following cardiotoxin injections.ConclusionsOur results show that SLK is dispensable for muscle development and regeneration but is required for myofiber stability and optimal force generation.

Calcium homeostasis alterations in a mouse model of the Dynamin 2-related centronuclear myopathy.

ABSTRACTAutosomal dominant centronuclear myopathy (CNM) is a rare congenital myopathy characterized by centrally located nuclei in muscle fibers. CNM results from mutations in the gene encoding dynamin 2 (DNM2), a large GTPase involved in endocytosis, intracellular membrane trafficking, and cytoskeleton regulation. We developed a knock-in mouse model expressing the most frequent DNM2-CNM mutation; i.e. the KI-Dnm2R465W model. Heterozygous (HTZ) KI-Dnm2 mice progressively develop muscle atrophy, impairment of contractile properties, histopathological abnormalities, and elevated cytosolic calcium concentration. Here, we aim at better characterizing the calcium homeostasis impairment in extensor digitorum longus (EDL) and soleus muscles from adult HTZ KI-Dnm2 mice. We demonstrate abnormal contractile properties and cytosolic Ca2+ concentration in EDL but not soleus muscles showing that calcium impairment is correlated with muscle weakness and might be a determinant factor of the spatial muscle involvement. In addition, the elevated cytosolic Ca2+ concentration in EDL muscles is associated with an increased sarcolemmal permeability to Ca2+ and releasable Ca2+ content from the sarcoplasmic reticulum. However, amplitude and kinetics characteristics of the calcium transient appear unchanged. This suggests that calcium defect is probably not a primary cause of decreased force generation by compromised sarcomere shortening but may be involved in long-term deleterious consequences on muscle physiology. Our results highlight the first pathomechanism which may explain the spatial muscle involvement occurring in DNM2-related CNM and open the way toward development of a therapeutic approach to normalize calcium content.

Activation Of Lower Limb Deep Muscles During Deep Squats On Different Unstable Surfaces

Although there are many studies about the effect of unstable conditions on surface muscles during deep squats, a key human movement, the conclusions were not consistent, e.g., while some studies showed that unstable conditions decreased force generation and the muscular activities increased with the level of instability, others concluded that there were not significant changes in muscle patterns among unstable surfaces. PURPOSE: To evaluate the effect of unstable conditions on both deep and surface muscles activation during deep squat posture. METHODS: Eighteen healthy males (22.3±1.8 years, height: 173±4 cm, mass: 67.4±5.8 kg) volunteered for the experiment. One repetition maximum (1RM) tests (69.5±7.6 kg) were performed for squats on stable surface. The sEMG of the tibialis anterior (TA) and rectus femoris (RF) were collected by a Trigno EMG system when holding the deep squat posture (thigh and calf at an 80 degree angle ) for 5s on Balance Disc (BD, unstable) and on ground (stable) with 30%RM. The thickness of TA, RF, hallucis longus (HL) and shares muscle (SM) were measured by APLIO Doppler 400 at the same time. A polar heart rate monitor was used to ensure participants’ heart rates were under 100 beats/minute.Paired t-test and effect size (ES, Cohen’s d) were calculated to evaluate the difference between stable and unstable conditions. RESULTS: 1. sEMG and thickness of muscle responded similarly during deep squating. 2. Significant difference was observed for TA, HL, and SM between stable and unstable surfaces (Table 1).Table 1: The difference of muscle activation between unstable and stable conditionsCONCLUSIONS: Compared with the stable condition, deep muscle activation increased to adapt to the unstable condition. Study was supported by Independent scientific research plan of Ministry of Education (20121088023)

Functional in situ assessment of muscle contraction in wild-type and mdx mice.

Reports of muscle testing are frequently limited to maximal force alone. The experiments reported here show that force generation and relaxation rates can be obtained from the same experiments and provide a more complete functional characterization. Partial in situ testing was performed on the tibialis anterior of young wild-type (WT) mice, young mdx mice, and old mdx mice. Force, force generation rate, and relaxation rates were measured during a fatigue test, 2 frequency-force tests, and a passive tension test. We measured increased force but decreased force generation rate in WT compared with mdx muscles, and increased force but decreased relaxation rate of old compared with young mdx muscles. Young mdx muscles were the most sensitive to increases in passive tension. These measurements offer an improved understanding of muscle capability and are readily acquired by further analysis of the same tests used to obtain force measurements.

Structure and Dynamics of the Acidosis-Resistant A162H Mutant of the Switch Region of Troponin I Bound to the Regulatory Domain of Troponin C.

Intracellular acidosis lowers the Ca²⁺ sensitivity of cardiac muscle, which results in decreased force generation, decreased cardiac output, and, eventually, heart failure. The A162H mutant of cardiac troponin I in the thin filament turns the heart acidosis-resistant. Physiological and structural studies have provided insights into the mechanism of protection by the A162H substitution; however, the effect of other native residues of cardiac troponin I is not fully understood. In this study, we determined the structure of the A162H mutant of the switch region of cardiac troponin I bound to the regulatory domain of troponin C at pH 6.1, and the dynamics as a function of pH, by NMR spectroscopy to evaluate the changes induced by protonation of A162H. The results indicate that A162H induces a transitory curved conformation on troponin I that promotes contraction, but it is countered by residue E164 to ensure proper relaxation. Our model explains the absence of diastolic impairment in the gain-of-function phenotype induced by the A162H substitution as well as the effects of a variety of mutants studied previously. The description of this mechanism underlines the fine quality of regulation on cardiac muscle contraction and anticipates pharmacological agents that induce modest changes in the contraction-relaxation equilibrium to produce marked effects in cardiac performance.

Glucose concentration and streptomycin alter in vitro muscle function and metabolism.

Cell culture conditions can vary between laboratories and have been optimised for 2D cell culture. In this study, engineered muscle was cultured in 5.5 mM low glucose (LG) or 25 mM high glucose (HG) and in the absence or presence (+S) of streptomycin and the effect on C2C12 tissue-engineered muscle function and metabolism was determined. Following 2 weeks differentiation, streptomycin (3-fold) and LG (0.5-fold) significantly decreased force generation. LG and/or streptomycin resulted in upward and leftward shifts in the force-frequency curve and slowed time-to-peak tension and half-relaxation time. Despite changes in contractile dynamics, no change in myosin isoform was detected. Instead, changes in troponin isoform, calcium sequestering proteins (CSQ and parvalbumin) and the calcium uptake protein SERCA predicted the changes in contractile dynamics. Culturing in LG and/or streptomycin resulted in increased fatigue resistance despite no change in the mitochondrial enzymes SDH, ATPsynthase and cytochrome C. However, LG resulted in increases in the β-oxidation enzymes LCAD and VLCAD and the fatty acid transporter CPT-1, indicative of a greater capacity for fat oxidation. In contrast, HG resulted in increased GLUT4 content and the glycolytic enzyme PFK, indicative of a more glycolytic phenotype. These data suggest that streptomycin has negative effects on force generation and that glucose can be used to shift engineered muscle phenotype via changes in calcium-handling and metabolic proteins.

The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function.

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.

The R249Q and R146N Hypertrophic Cardiomyopathy Myosin Mutations Decrease Muscle Force and Power Generation

Hypertrophic cardiomyopathy (HCM) is an inheritable disease in humans that causes the heart's ventricular walls to thicken. There are over 200 identified mutations that cause HCM, and the generally proposed hypothesis is that HCM mutations increase heart muscle contractility. To test this hypothesis, we expressed two HCM myosin mutations, R249Q and R146N, in Drosophila and performed skinned indirect flight muscle (IFM) fiber mechanics to measure power, force and muscle kinetics. The R146N and R249Q mutant fibers significantly (p<0.05) decreased power generation by 45% and 67%, respectively, from control fibers. The decrease in power by R146N resulted from a decrease in force generation because isometric tension and work production decreased, 47% and 42%, respectively, proportional to power reduction. R249Q fibers produced less power because both force and muscle speed decreased. Isometric tension decreased by 37% and the frequency at which maximum power generation occurred was 20% lower. Muscle mechanics were performed on mutant and control fibers from two-hour-old Drosophila, prior to degradation of myofilament structure, which started at two-days-old and increased with age. Electron microscopy showed that degradation was more pronounced in the R146N line. Decreased power output from the fibers caused a 60% decrease in two-day-old Drosophila flight ability for both lines. Molecular modeling suggests R249Q could be interfering with communication between the nucleotide and thin filament binding sites, while R146N may alter the N-terminal domain's interactions with the lever arm. Overall, our results contradict the increased contractility HCM hypothesis and instead suggest ventricular hypertrophy is a compensatory response to a decrease in heart muscle power generating ability. Supported by NIH grants R01 AR055611 to D.M.S. and R01 GM322433 to S.I.B.

Cellular Mechanisms of Cardiac Dysfunction in Volume Overload Induced Heart Failure

Left ventricular (LV) volume overload (VO) in valve regurgitation patients can be only treated by surgical intervention. The cellular mechanisms involved in cardiac dysfunction are not well known. The aim of this study was to understand the mechanical properties of myocytes isolated from VO‐induced heart failure and the possible mechanisms of dysfunction. Male Sprague‐Dawley rats underwent aorta‐caval fistula (ACF) surgery to induce VO heart failure or sham procedure. The rats with ACF went into failure at ~21wks post‐surgery. LV myocytes were isolated. Myocyte cell‐shortening fraction (CS) and Ca[i]2+ transients were measured at different frequency field stimulations to study cellular mechanical properties and molecular components of excitation‐contraction (EC) coupling. SERCA, pRYR and pPLB were reduced in ACF hearts while NCX was unchanged. Regardless of the stimulation frequency, CS of ACF myocytes was significantly lower than those of sham myocytes (~45% reduction). Interestingly, rapid stimulation at higher frequencies did not alter the Ca2+ area of relengthening in ACF myocytes, suggesting the activity of RYR and SERCA are intact despite reduction in protein levels. These data suggest that EC coupling is not a causative factor for cardiac dysfunction seen in VO and may possibly be due to reduced Ca2+ sensitivity or decreased force generation in elongated myocytes of ACF rats.