Andrés Hernández

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

•  Dietary supplementation with inorganic nitrate has beneficial effects on skeletal muscle responses to exercise. •  Both mitochondrial and extra‐mitochondrial explanations have been proposed. •  Contractile force of fast‐twitch muscles was enhanced in mice supplemented with 1 mm NaNO3 in drinking water for 7 days. •  Myoplasmic free [Ca2+] during tetanic stimulation was increased in fast‐twitch muscles of nitrate‐supplemented mice and this was accompanied by increased expression of calsequestrin 1 and the dihydropyridine receptor. •  These results provide a new mechanism by which nitrate exerts beneficial effects on muscle function with applications to sports performance and a potential therapeutic role in conditions with muscle weakness.

A prior bout of contractions speeds V̇o2 and blood flow on-kinetics and reduces the V̇o2 slow-component amplitude in canine skeletal muscle contracting in situ

It was the purpose of this study to examine the effect of a priming contractile bout on oxygen uptake (VO2) on-kinetics in highly oxidative skeletal muscle. Canine gastrocnemii (n=12) were stimulated via their sciatic nerves (8 V, 0.2-ms duration, 50 Hz, 200-ms train) at a rate of 2 contractions/3 s (approximately 70% peak VO2) for two 2-min bouts, separated by 2 min of recovery. Blood flow was recorded with an ultrasonic flowmeter, and muscle oxygenation monitored via near-infrared spectroscopy. Compared with the first bout (bout 2 vs. bout 1), the VO2 primary time constant (mean+/-SD, 9.4+/-2.3 vs. 12.0+/-3.9 s) and slow-component amplitude (5.9+/-6.3 vs. 12.1+/-9.0 ml O2.kg wet wt(-1).min(-1)) were significantly reduced (P<0.05) during the second bout. Blood flow on-kinetics were significantly speeded during the second bout (time constant=7.7+/-2.6 vs. 14.8+/-5.8 s), and O2 extraction was greater at the onset of contractions (0.050+/-0.030 vs. 0.020+/-0.010 ml O2/ml blood). Kinetics of muscle deoxygenation were significantly slower at the onset of the second bout (7.2+/-2.2 vs. 4.4+/-1.2 s), while relative oxyhemoglobin concentration was elevated throughout the second bout. These results suggest that better matching of O2 delivery to VO2 speeds Vo(2) on-kinetics at this metabolic rate, but do not eliminate a potential role for enhanced metabolic activation. Additionally, altered motor unit recruitment at the onset of a second bout is not a prerequisite for reductions in the VO2 slow-component amplitude after a priming contractile bout in canine muscle in situ.

Antioxidants and Skeletal Muscle Performance: “Common Knowledge” vs. Experimental Evidence

Antioxidants are assumed to provide numerous benefits, including better health, a reduced rate of aging, and improved exercise performance. Specifically, antioxidants are commonly “prescribed” by the media, supplement industry, and “fitness experts” for individuals prior to training and performance, with assumed benefits of improved fatigue resistance and recovery. This has provoked expansion of the supplement industry which responded by creation of a plethora of products aimed at facilitating the needs of the active individual. However, what does the experimental evidence say about the efficacy of antioxidants on skeletal muscle function? Are antioxidants actually as beneficial as the general populous believes? Or, could they in fact lead to deleterious effects on skeletal muscle function and performance? This Mini Review addresses these questions with an unbiased look at what we know about antioxidant effects on skeletal muscle, and what we still need to know before conclusions can be made.

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

•  Defective mitochondrial function has been shown to cause muscle weakness and exercise intolerance. •  We used a mouse model of premature ageing with defective proofreading of mitochondrial DNA (mtDNA): the mtDNA mutator mouse. •  Muscles of young (3–5 months) mtDNA mutator mice showed reduced endurance, which was caused by decreased mitochondrial ATP production accompanied by decreased levels of factors stimulating mitochondrial biogenesis. •  The dominant defect in muscles of old (11 months) mtDNA mutator mice was severe weakness, which was caused by decreased intracellular Ca2+ stores. •  These results underline two important aspects of mitochondria‐to‐nucleus signalling in skeletal muscle: (1) it fails to respond adequately to decreased mitochondrial ATP production in sedentary animals; and (2) it can induce decreased intracellular Ca2+ stores and hence muscle weakness. •  These results have implication for normal ageing and suggest that the decreased mitochondrial function induced by a sedentary lifestyle may predispose to muscle weakness later in life.

Contraction-by-contraction VO2 and computer-controlled pump perfusion as novel techniques to study skeletal muscle metabolism in situ.

The purpose of this research was to develop new techniques to 1) rapidly sample venous O(2) saturation to determine contraction-by-contraction oxygen uptake (Vo(2)), and 2) precisely control the rate and pattern of blood flow adjustment from one chosen steady state to another. An indwelling inline oximeter probe connected to an Oximetrix 3 meter was used to sample venous oxygen concentration ([O(2)]) (via fractional saturation of Hb with O(2)). Data from the Oximetrix 3 were filtered, deconvolved, and processed by a moving average second by second. Computer software and a program written in-house were used to control blood flow with a peristaltic pump. The isolated canine gastrocnemius muscle complex (GS) in situ was utilized to test these techniques. A step change in metabolic rate was elicited by stimulating GS muscles via their sciatic nerves (supramaximal voltage, 8 V; 50 Hz, 0.2-ms pulse width; train duration 200 ms) at a rate of either 1 contraction/2 s, or 2 contractions/3 s. With arterial [O(2)] maintained constant, blood flow and calculated venous [O(2)] were averaged over each contraction cycle and used in the Fick equation to calculate contraction-by-contraction Vo(2). About 5-8 times more data points were obtained with this method compared with traditional manual sampling. Software-controlled pump perfusion enabled the ability to mimic spontaneous blood flow on-kinetics (tau: 14.3 s) as well as dramatically speed (tau: 2.0 s) and slow (tau: 63.3 s) on-kinetics. These new techniques significantly improve on existing methods for mechanistically altering blood flow kinetics as well as accurately measuring muscle oxygen consumption kinetics during transitions between metabolic rates.

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.

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.

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. FASEB J. 31, 4809–4820 (2017). www.fasebj.org