Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control.

Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.

Rational antibody design for undruggable targets using kinetically controlled biomolecular probes.

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

Loss of α-actinin-3 during human evolution provides superior cold resilience and muscle heat generation.

The protein α-actinin-3 expressed in fast-twitch skeletal muscle fiber is absent in 1.5 billion people worldwide due to homozygosity for a nonsense polymorphism in ACTN3 (R577X). The prevalence of the 577X allele increased as modern humans moved to colder climates, suggesting a link between α-actinin-3 deficiency and improved cold tolerance. Here, we show that humans lacking α-actinin-3 (XX) are superior in maintaining core body temperature during cold-water immersion due to changes in skeletal muscle thermogenesis. Muscles of XX individuals displayed a shift toward more slow-twitch isoforms of myosin heavy chain (MyHC) and sarcoplasmic reticulum (SR) proteins, accompanied by altered neuronal muscle activation resulting in increased tone rather than overt shivering. Experiments on Actn3 knockout mice showed no alterations in brown adipose tissue (BAT) properties that could explain the improved cold tolerance in XX individuals. Thus, this study provides a mechanism for the positive selection of the ACTN3 X-allele in cold climates and supports a key thermogenic role of skeletal muscle during cold exposure in humans.

A Mechanism for Statin-Induced Susceptibility to Myopathy.

This study aimed to identify a mechanism for statin-induced myopathy that explains its prevalence and selectivity for skeletal muscle, and to understand its interaction with moderate exercise. Statin-associated adverse muscle symptoms reduce adherence to statin therapy; this limits the effectiveness of statins in reducing cardiovascular risk. The issue is further compounded by perceived interactions between statin treatment and exercise. This study examined muscles from individuals taking statins and rats treated with statins for 4 weeks. In skeletal muscle, statin treatment caused dissociation of the stabilizing protein FK506 binding protein (FKBP12) from the sarcoplasmic reticulum (SR) calcium (Ca2+) release channel, the ryanodine receptor 1, which was associated with pro-apoptotic signaling and reactive nitrogen species/reactive oxygen species (RNS/ROS)-dependent spontaneous SR Ca2+ release events (Ca2+ sparks). Statin treatment had no effect on Ca2+ spark frequency in cardiac myocytes. Despite potentially deleterious effects of statins on skeletal muscle, there was no impact on force production or SR Ca2+ release in electrically stimulated muscle fibers. Statin-treated rats with access to a running wheel ran further than control rats; this exercise normalized FKBP12 binding to ryanodine receptor 1, preventing the increase in Ca2+ sparks and pro-apoptotic signaling. Statin-mediated RNS/ROS-dependent destabilization of SR Ca2+ handling has the potential to initiate skeletal (but not cardiac) myopathy in susceptible individuals. Importantly, although exercise increases RNS/ROS, it did not trigger deleterious statin effects on skeletal muscle. Indeed, our results indicate that moderate exercise might benefit individuals who take statins.

Intact single muscle fibres from SOD1G93A amyotrophic lateral sclerosis mice display preserved specific force, fatigue resistance and training-like adaptations.

KEY POINTS: How defects in muscle contractile function contribute to weakness in amyotrophic lateral sclerosis (ALS) were systematically investigated. Weakness in whole muscles from late stage SOD1G93A mice was explained by muscle atrophy as seen by reduced mass and maximal force. On the other hand, surviving single muscle fibres in late stage SOD1G93A have preserved intracellular Ca2+ handling, normal force-generating capacity and increased fatigue resistance. These intriguing findings provide a substrate for therapeutic interventions to potentiate muscular capacity and delay the progression of the ALS phenotype. ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by degeneration and loss of motor neurons, leading to severe muscle weakness and paralysis. The SOD1G93A mouse model of ALS displays motor neuron degeneration and a phenotype consistent with human ALS. The purpose of this study was to determine whether muscle weakness in ALS can be attributed to impaired intrinsic force generation in skeletal muscles. In the current study, motor neuron loss and decreased force were evident in whole flexor digitorum brevis (FDB) muscles of mice in the late stage of disease (125-150 days of age). However, in intact single muscle fibres, specific force, tetanic myoplasmic free [Ca2+ ] ([Ca2+ ]i ), and resting [Ca2+ ]i remained unchanged with disease. Fibre-type distribution was maintained in late-stage SOD1G93A FDB muscles, but remaining muscle fibres displayed greater fatigue resistance compared to control and showed increased expression of myoglobin and mitochondrial respiratory chain proteins that are important determinants of fatigue resistance. Expression of genes central to both mitochondrial biogenesis and muscle atrophy where increased, suggesting that atrophic and compensatory adaptive signalling occurs simultaneously within the muscle tissue. These results support the hypothesis that muscle weakness in SOD1G93A is primarily attributed to neuromuscular degeneration and not intrinsic muscle fibre defects. In fact, surviving muscle fibres displayed maintained adaptive capacity with an exercise training-like phenotype, which suggests that compensatory mechanisms are activated that can function to delay disease progression.

SR Ca2+ leak in skeletal muscle fibers acts as an intracellular signal to increase fatigue resistance.

Effective practices to improve skeletal muscle fatigue resistance are crucial for athletes as well as patients with dysfunctional muscles. To this end, it is important to identify the cellular signaling pathway that triggers mitochondrial biogenesis and thereby increases oxidative capacity and fatigue resistance in skeletal muscle fibers. Here, we test the hypothesis that the stress induced in skeletal muscle fibers by endurance exercise causes a reduction in the association of FK506-binding protein 12 (FKBP12) with ryanodine receptor 1 (RYR1). This will result in a mild Ca2+ leak from the sarcoplasmic reticulum (SR), which could trigger mitochondrial biogenesis and improved fatigue resistance. After giving mice access to an in-cage running wheel for three weeks, we observed decreased FKBP12 association to RYR1, increased baseline [Ca2+]i, and signaling associated with greater mitochondrial biogenesis in muscle, including PGC1α1. After six weeks of voluntary running, FKBP12 association is normalized, baseline [Ca2+]i returned to values below that of nonrunning controls, and signaling for increased mitochondrial biogenesis was no longer present. The adaptations toward improved endurance exercise performance that were observed with training could be mimicked by pharmacological agents that destabilize RYR1 and thereby induce a modest Ca2+ leak. We conclude that a mild RYR1 SR Ca2+ leak is a key trigger for the signaling pathway that increases muscle fatigue resistance.

Exercise training during chemotherapy preserves skeletal muscle fiber area, capillarization, and mitochondrial content in patients with breast cancer.

Exercise has been suggested to ameliorate the detrimental effects of chemotherapy on skeletal muscle. The aim of this study was to compare the effects of different exercise regimens with usual care on skeletal muscle morphology and mitochondrial markers in patients being treated with chemotherapy for breast cancer. Specifically, we compared moderate-intensity aerobic training combined with high-intensity interval training (AT-HIIT) and resistance training combined with high-intensity interval training (RT-HIIT) with usual care (UC). Resting skeletal muscle biopsies were obtained pre- and postintervention from 23 randomly selected women from the OptiTrain breast cancer trial who underwent RT-HIIT, AT-HIIT, or UC for 16 wk. Over the intervention, citrate synthase activity, muscle fiber cross-sectional area, capillaries per fiber, and myosin heavy chain isoform type I were reduced in UC, whereas RT-HIIT and AT-HIIT were able to counteract these declines. AT-HIIT promoted up-regulation of the electron transport chain protein levels vs. UC. RT-HIIT favored satellite cell count vs. UC and AT-HIIT. There was a significant association between change in citrate synthase activity and self-reported fatigue. AT-HIIT and RT-HIIT maintained or improved markers of skeletal muscle function compared with the declines found in the UC group, indicating a sustained trainability in addition to the preservation of skeletal muscle structural and metabolic characteristics during chemotherapy. These findings highlight the importance of supervised exercise programs for patients with breast cancer during chemotherapy.-Mijwel, S., Cardinale, D. A., Norrbom, J., Chapman, M., Ivarsson, N., Wengström, Y., Sundberg, C. J., Rundqvist, H. Exercise training during chemotherapy preserves skeletal muscle fiber area, capillarization, and mitochondrial content in patients with breast cancer.

Post-exercise recovery of contractile function and endurance in humans and mice is accelerated by heating and slowed by cooling skeletal muscle.

KEY POINTS: We investigated whether intramuscular temperature affects the acute recovery of exercise performance following fatigue-induced by endurance exercise. Mean power output was better preserved during an all-out arm-cycling exercise following a 2 h recovery period in which the upper arms were warmed to an intramuscular temperature of Ì´ 38°C than when they were cooled to as low as 15°C, which suggested that recovery of exercise performance in humans is dependent on muscle temperature. Mechanisms underlying the temperature-dependent effect on recovery were studied in intact single mouse muscle fibres where we found that recovery of submaximal force and restoration of fatigue resistance was worsened by cooling (16-26°C) and improved by heating (36°C). Isolated whole mouse muscle experiments confirmed that cooling impaired muscle glycogen resynthesis. We conclude that skeletal muscle recovery from fatigue-induced by endurance exercise is impaired by cooling and improved by heating, due to changes in glycogen resynthesis rate. ABSTRACT: Manipulation of muscle temperature is believed to improve post-exercise recovery, with cooling being especially popular among athletes. However, it is unclear whether such temperature manipulations actually have positive effects. Accordingly, we studied the effect of muscle temperature on the acute recovery of force and fatigue resistance after endurance exercise. One hour of moderate-intensity arm cycling exercise in humans was followed by 2 h recovery in which the upper arms were either heated to 38°C, not treated (33°C), or cooled to ∼15°C. Fatigue resistance after the recovery period was assessed by performing 3 × 5 min sessions of all-out arm cycling at physiological temperature for all conditions (i.e. not heated or cooled). Power output during the all-out exercise was better maintained when muscles were heated during recovery, whereas cooling had the opposite effect. Mechanisms underlying the temperature-dependent effect on recovery were tested in mouse intact single muscle fibres, which were exposed to ∼12 min of glycogen-depleting fatiguing stimulation (350 ms tetani given at 10 s interval until force decreased to 30% of the starting force). Fibres were subsequently exposed to the same fatiguing stimulation protocol after 1-2 h of recovery at 16-36°C. Recovery of submaximal force (30 Hz), the tetanic myoplasmic free [Ca2+ ] (measured with the fluorescent indicator indo-1), and fatigue resistance were all impaired by cooling (16-26°C) and improved by heating (36°C). In addition, glycogen resynthesis was faster at 36°C than 26°C in whole flexor digitorum brevis muscles. We conclude that recovery from exhaustive endurance exercise is accelerated by raising and slowed by lowering muscle temperature.

Prolonged force depression after mechanically demanding contractions is largely independent of Ca2+ and reactive oxygen species.

Increased production of reactive oxygen/nitrogen species (ROS) and impaired cellular Ca2+ handling are implicated in the prolonged low-frequency force depression (PLFFD) observed in skeletal muscle after both metabolically and mechanically demanding exercise. Metabolically demanding high-intensity exercise can induce PLFFD accompanied by ROS-dependent fragmentation of the sarcoplasmic reticulum Ca2+ release channels, the ryanodine receptor 1s (RyR1s). We tested whether similar changes occur after mechanically demanding eccentric contractions. Human subjects performed 100 repeated drop jumps, which require eccentric knee extensor contractions upon landing. This exercise caused a major PLFFD, such that maximum voluntary and electrically evoked forces did not recover within 24 h. Drop jumps induced only minor signs of increased ROS, and RyR1 fragmentation was observed in only 3 of 7 elderly subjects. Also, isolated mouse muscle preparations exposed to drop-jump-mimicking eccentric contractions showed neither signs of increased ROS nor RyR1 fragmentation. Still, the free cytosolic [Ca2+] during tetanic contractions was decreased by ∼15% 1 h after contractions, which can explain the exaggerated force decrease at low-stimulation frequencies but not the major frequency-independent force depression. In conclusion, PLFFD caused by mechanically demanding eccentric contractions does not involve any major increase in ROS or RyR1 fragmentation.-Kamandulis, S., de Souza Leite, F., Hernandez, A., Katz, A., Brazaitis, M., Bruton, J. D., Venckunas, T., Masiulis, N., Mickeviciene, D., Eimantas, N., Subocius, A., Rassier, D. E., Skurvydas, A., Ivarsson, N., Westerblad, H. Prolonged force depression after mechanically demanding contractions is largely independent of Ca2+ and reactive oxygen species.

Exercise differentially affects metabolic functions and white adipose tissue in female letrozole- and dihydrotestosterone-induced mouse models of polycystic ovary syndrome.

Here we hypothesized that exercise in dihydrotestosterone (DHT) or letrozole (LET)-induced polycystic ovary syndrome mouse models improves impaired insulin and glucose metabolism, adipose tissue morphology, and expression of genes related to adipogenesis, lipid metabolism, Notch pathway and browning in inguinal and mesenteric fat. DHT-exposed mice had increased body weight, increased number of large mesenteric adipocytes. LET-exposed mice displayed increased body weight and fat mass, decreased insulin sensitivity, increased frequency of small adipocytes and increased expression of genes related to lipolysis in mesenteric fat. In both models, exercise decreased fat mass and inguinal and mesenteric adipose tissue expression of Notch pathway genes, and restored altered mesenteric adipocytes morphology. In conclusion, exercise restored mesenteric adipocytes morphology in DHT- and LET-exposed mice, and insulin sensitivity and mesenteric expression of lipolysis-related genes in LET-exposed mice. Benefits could be explained by downregulation of Notch, and modulation of browning and lipolysis pathways in the adipose tissue.

Dietary nitrate markedly improves voluntary running in mice.

Nitrate supplementation is shown to increase submaximal force in human and mouse skeletal muscles. In this study, we test the hypothesis that the increased submaximal force induced by nitrate supplementation reduces the effort of submaximal voluntary running, resulting in increased running speed and distance. C57Bl/6N male mice were fed nitrate in the drinking water and housed with or without access to an in-cage running wheel. Nitrate supplementation in sedentary mice had no effect on endurance in a treadmill test, nor did it enhance mitochondrial function. However, after three weeks with in-cage running wheel, mice fed nitrate ran on average 20% faster and 30% further than controls (p<0.01). Compared to running controls, this resulted in ~13% improved endurance on a subsequent treadmill test (p<0.05) and increased mitochondrial oxidative capacity, as judged from a mean increase in citrate synthase activity of 14% (p<0.05). After six weeks with nitrate, the mice were running 58% longer distances per night. When nitrate supplementation was removed from the diet, the running distance and speed decreased to the control level, despite the improved endurance achieved during nitrate supplementation. In conclusion, low-frequency force improvement due to nitrate supplementation facilitates submaximal exercise such as voluntary running.

Impaired Ca(2+) release contributes to muscle weakness in a rat model of critical illness myopathy.

BACKGROUND: Critical illness myopathy is an acquired skeletal muscle disorder with severe myosin loss and muscle weakness frequently seen in intensive care unit (ICU) patients. It is unknown if impaired excitation-contraction coupling contributes to the muscle weakness. METHODS: We used a unique ICU model where rats were deeply sedated, post-synaptically pharmacologically paralyzed, mechanically ventilated and closely monitored for up to ten days. Single intact fibers from the flexor digitorum brevis muscle were isolated and used to measure force and free myoplasmic [Ca(2+)] ([Ca(2+)]i) during tetanic contractions. RESULTS: Fibers from ICU rats had 80 % lower tetanic [Ca(2+)]i and produced only 15 % of the force seen in fibers from sham-operated (SHAM) rats. In the presence of 5 mM caffeine, tetanic [Ca(2+)]i was similar in fibers from ICU and SHAM rats but force was 50 % lower in fibers from ICU rats than SHAM rats. Confocal imaging showed disrupted tetanic [Ca(2+)]i transients in fibers from ICU rats compared to SHAM rats. Western blots showed similar levels of Na(+) channel and dihydropyridine receptor (DHPR) protein expression, whereas ryanodine receptor (RyR) and sarco-endoplasmic reticulum Ca(2+) ATPase 1 (SERCA1) expression was markedly lower in muscle of ICU rats than in SHAM rats. Immunohistochemical analysis showed that distribution of Na(+) channel and DHPR protein on the sarcolemma was disrupted in fibers from ICU rats compared with SHAM rats. CONCLUSIONS: These results suggest that impaired SR Ca(2+) release contributes to the muscle weakness seen in patients in ICU.

Dietary nitrate improves cardiac contractility via enhanced cellular Ca²âº signaling.

The inorganic anion nitrate (NO3 (-)), which is naturally enriched in certain vegetables (e.g., spinach and beetroot), has emerged as a dietary component that can regulate diverse bodily functions, including blood pressure, mitochondrial efficiency, and skeletal muscle force. It is not known if dietary nitrate improves cardiac contractility. To test this, mice were supplemented for 1-2 weeks with sodium nitrate in the drinking water at a dose similar to a green diet. The hearts from nitrate-treated mice showed increased left ventricular pressure and peak rate of pressure development as measured with the Langendorff heart technique. Cardiomyocytes from hearts of nitrate-treated and control animals were incubated with the fluorescent indicator Fluo-3 to measure cytoplasmic free [Ca(2+)] and fractional shortening. Cardiomyocytes from nitrate-treated mice displayed increased fractional shortening, which was linked to larger Ca(2+) transients. Moreover, nitrate hearts displayed increased protein expression of the L-type Ca(2+) channel/dihydropyridine receptor and peak L-type Ca(2+) channel currents. The nitrate-treated hearts displayed increased concentration of cAMP but unchanged levels of cGMP compared with controls. These findings provide the first evidence that dietary nitrate can affect the expression of important Ca(2+) handling proteins in the heart, resulting in increased cardiomyocyte Ca(2+) signaling and improved left ventricular contractile function. Our observation shows that dietary nitrate impacts cardiac function and adds understanding to inorganic nitrate as a physiological modulator.

Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy.

Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca(2+) uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca(2+) handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy.

Impaired mitochondrial respiration and decreased fatigue resistance followed by severe muscle weakness in skeletal muscle of mitochondrial DNA mutator mice.

Mitochondrial dysfunction can drastically impair muscle function, with weakness and exercise intolerance as key symptoms. Here we examine the time course of development of muscle dysfunction in a mouse model of premature ageing induced by defective proofreading function of mitochondrial DNA (mtDNA) polymerase (mtDNA mutator mouse). Isolated fast-twitch muscles and single muscle fibres from young (3-5 months) and end-stage (11 months) mtDNA mutator mice were compared to age-matched control mice. Force and free myoplasmic [Ca(2+)] ([Ca(2+)](i)) were measured under resting conditions and during fatigue induced by repeated tetani. Muscles of young mtDNA mutator mice displayed no weakness in the rested state, but had lower force and [Ca(2+)](i) than control mice during induction of fatigue. Muscles of young mtDNA mutator mice showed decreased activities of citrate synthase and ß-hydroxyacyl-coenzyme A dehydrogenase, reduced expression of cytochrome c oxidase, and decreased expression of triggers of mitochondrial biogenesis (PGC-1α, PPARα, AMPK). Muscles from end-stage mtDNA mutator mice showed weakness under resting conditions with markedly decreased tetanic [Ca(2+)](i), force per cross-sectional area and protein expression of the sarcoplasmic reticulum Ca(2+) pump (SERCA1). In conclusion, fast-twitch muscles of prematurely ageing mtDNA mutator mice display a sequence of deleterious mitochondrial-to-nucleus signalling with an initial decrease in oxidative capacity, which was not counteracted by activation of signalling to increase mitochondrial biogenesis. This was followed by severe muscle weakness in the end stage. These results have implication for normal ageing and suggest that decreased mitochondrial oxidative capacity due to a sedentary lifestyle may predispose towards muscle weakness developing later in life.

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca(2+) handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca(2+)] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤ 50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca(2+) handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca(2+) handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

AICAR reverses ketone body mediated insulin resistance in isolated oxidative muscle.

Recently it was demonstrated that the ketone body ß-hydroxybutyrate (BOH) inhibits insulin-mediated glucose transport in isolated oxidative muscle, which was associated with decreased phosphorylation of Akt/protein kinase B. The purpose of the present study was to determine if activation of AMP-dependent protein kinase by the pharmacological activator AICAR could reverse the insulin resistance induced by BOH. Isolated mouse soleus muscle was incubated in vitro in the absence or presence of 5mM BOH for ∼20 h. Following prolonged incubation, insulin increased 2-deoxyglucose glucose (2-DG) uptake 3-fold, but in the presence of BOH most of the insulin response was lost (only ∼30% remained). Addition of 2mM AICAR during the last 2h of prolonged incubation increased the insulin response in the presence of BOH to ∼80% of the normal insulin effect on 2-DG uptake. The AICAR-mediated reversal of the insulin resistance was not associated with a restoration of the insulin effect on Akt/protein kinase B phosphorylation. However, AICAR enhanced the insulin-induced phosphorylation of the Akt substrate, AS160. In conclusion, these data demonstrate that AICAR reverses the negative effect of BOH on insulin-mediated glucose uptake and this is attributed to activation of a late step in insulin signaling.

Enhanced cardiomyocyte Ca(2+) cycling precedes terminal AV-block in mitochondrial cardiomyopathy Mterf3 KO mice.

AIMS: Heart disease is commonly associated with altered mitochondrial function and signs of oxidative stress. This study elucidates whether primary cardiac mitochondrial dysfunction causes changes in cardiomyocyte handling of reactive oxygen species (ROS) and Ca(2+). We used a mouse model with a tissue-specific ablation of the recently discovered mtDNA transcription regulator Mterf3 (Mterf3 KO). These mice display a cardiomyopathy with severe respiratory chain dysfunction, cardiac hypertrophy, and shortened lifespan. ROS and Ca(2+) handling were measured using fluorescent indicators and confocal microscopy. RESULTS: Mterf3 KO hearts displayed no signs of increased ROS production or oxidative stress. Surprisingly, Mterf3 KO cardiomyocytes showed enlarged Ca(2+) transient amplitudes, faster sarcoplasmic reticulum (SR) Ca(2+) reuptake, and increased SR Ca(2+) load, resembling increased adrenergic stimulation. Furthermore, spontaneous releases of Ca(2+) were frequent in Mterf3 KO cardiomyocytes. Electrocardiography (measured with telemetry in freely moving mice) showed a terminal state in Mterf3 KO mice with gradually developing bradycardia and atrioventricular block. CONCLUSION: In conclusion, mitochondrial dysfunction induced by Mterf3 KO leads to a cardiomyopathy without signs of oxidative stress but with increased cardiomyocyte Ca(2+) cycling and an arrhythmogenic phenotype. These findings highlight the complex interaction between mitochondrial function, cardiomyocyte contractility, and compensatory mechanisms, such as activation of adrenergic signaling.