Peroxynitrite (ONOO-) is a reactive nitrogen species implicated in oxidative injury and impaired calcium handling. We examined whether ONOO- inactivates the cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and whether heat shock protein 70 (Hsp70) preserves SERCA2a function during ONOO- exposure. Human embryonic kidney (HEK-293) cells were co-transfected with SERCA2a and either empty pMT2 vector (S2a-pMT2) or Hsp70 (S2a-Hsp70). Cells were untreated or exposed to ten nominal 250 μM additions of ONOO-, delivered once per minute for 10 min. Hsp70 abundance was greater in S2a-Hsp70 cells, although the magnitude of this difference varied by treatment (interaction, p = 0.0015). SERCA2a abundance was unaffected by transfection condition or ONOO- exposure. Co-immunoprecipitation showed that Hsp70 and SERCA2a were present in the same protein complex under the conditions examined. ONOO- reduced maximal SERCA2a activity by approximately 20% in S2a-pMT2 cells (p = 0.03), whereas activity was preserved in S2a-Hsp70 cells. ONOO- also increased SERCA2a-associated reactive carbonyl content by approximately 80% in S2a-pMT2 cells (p = 0.01), with no increase in S2a-Hsp70 cells. SERCA2a 3-nitrotyrosine content and FITC-binding capacity did not differ among groups. These findings show that Hsp70 overexpression preserves SERCA2a activity during acute ONOO- exposure and that this protection is associated with reduced SERCA2a carbonyl accumulation.

OBJECTIVE:To investigate the recovery of prolonged low-frequency force depression (PLFFD) within the first hour post-volitional fatigue following a sustained isometric triceps contraction performed at high (70 % of maximum voluntary force (MVF)) and low (20 % of MVF) intensities. BACKGROUND:High threshold and low threshold motor units contribute differently to force generation capability and fatigue at different rates. However, we know little about how high and low threshold motor units recover following fatiguing contractile activity. METHODS:Fourteen males and fourteen females sustained an isometric elbow extensor contraction until volitional fatigue at high (70 % MVF) and low (20 % MVF) intensities. The ratio of force generated by transcutaneous muscle stimulation of the triceps at 20 Hz and 100 Hz frequencies was measured at baseline and intermittently for 1 h post fatigue to characterize PLFFD. RESULTS:The 20/100 Hz force ratio was initially (0-4 min) significantly greater (meaning less force depression) following the high intensity protocol. However, the 20/100 Hz ratio for both intensities was similar for the duration of recovery thereafter. CONCLUSION:Initial differences in the 20/100 Hz ratio within the first 0-4 min are likely explained by post-activation potentiation following the higher intensity submaximal contraction. However, the similarity in the recovery of 20/100 Hz ratio thereafter supports the hypothesis that once PLFFD is present, the recovery rate may be independent from how fatigue and corresponding PLFFD developed. APPLICATION:The practical impact is that recovery of prolonged frequency fatigue may not depend on the intensity of the contraction that caused the fatigue.

Skeletal muscle atrophy involves significant remodeling of fibers and is characterized by deficits in mitochondrial content and function. These changes are intimately connected to shifts in mitochondrial turnover, encompassing processes such as mitophagy and mitochondrial biogenesis. However, the role of these mitochondrial turnover processes in muscle atrophy remains poorly understood. We used a novel mitophagy reporter model, mt-Keima mice, to perform hindlimb immobilization and accurately measure mitophagy. A comprehensive set of analyses were conducted to investigate biochemical and molecular changes at the muscle and mitochondrial levels. We also performed image analyses to determine mitophagic flux. To further explore the role of mitophagy in immobilization-induced atrophy, we treated animals with N-acetylcysteine (NAC; 150 mg/kg/day) to modify reactive oxygen species (ROS) signaling and colchicine (0.4 mg/kg/day) to inhibit autophagy. Our study revealed that hindlimb immobilization leads to muscle weakness and atrophy of fast-twitch muscle fibers (types IIA, IIX, and IIB), with recovery observed in IIA fibers following remobilization. This atrophy was accompanied by a significant increase in mitophagic flux. Additionally, immobilization induced notable mitochondrial dysfunction, as shown by diminished respiration, increased mitochondrial ROS, and greater whole muscle lipid peroxidation. Treatment of immobilized mice with NAC enhanced mitochondrial respiration and reduced ROS generation but suppressed mitophagic flux and intensified atrophy of type IIX and IIB fibers. Additionally, administration of colchicine to immobilized mice suppressed mitophagic flux, which also exacerbated atrophy of IIX and IIB fibers. Colchicine treatment led to significant reductions in mitochondrial function, accompanied by CASP9 and CASP3 activation. These findings emphasize the role of mitophagy in limiting excessive muscle atrophy during immobilization. Targeting mitophagy may offer new strategies to preserve muscle function during prolonged periods of immobilization.

Acute mountain sickness (AMS) occurs due to rapid altitude ascents and/or insufficient acclimatization. Acetazolamide (AZ) is commonly prescribed for AMS prophylaxis but inhibits exercise performance. Methazolamide (MZ), an analogous drug, has similar prophylactic benefits but does not impair isolated muscle mass exercise performance in normoxia. We sought to compare whole body exercise performance in acute hypoxia (fraction of inspired oxygen, [Formula: see text] = 0.15) between AZ, MZ, and placebo (PLA). Fifteen healthy participants completed five testing visits: day 1 for maximal exercise test, day 2 for familiarization, and days 3-5 were the experimental visits. Each experimental visit involved a 5-km hypoxic cycling time trial (TT) performed after a 2-day dosing protocol of either AZ (250 mg three times a day), MZ (100 mg twice a day), or PLA (three times a day); the order was randomized and double-blinded. Before exercise, capillary blood samples were taken, and maximal voluntary contractions of quadriceps were performed. AZ and MZ resulted in a partially compensated metabolic acidosis at rest compared with PLA [capillary hydrogen ions (H+) 47 ± 3, 43 ± 2, and 39 ± 2 nmol for AZ, MZ, and PLA respectively, P < 0.01]. Time to complete 5 km with PLA (562 ± 32 s, P < 0.01) was significantly faster than AZ and MZ (577 ± 38 vs. 581 ± 37 s, respectively), with no differences between AZ and MZ (P = 0.96). There were no differences in average ventilation (124 ± 27, 127 ± 24, 127 ± 19 L/min) and oxyhemoglobin saturation (87 ± 2, 88 ± 2, 88 ± 3%) between AZ, MZ, and PLA, respectively (P > 0.05). Overall, both AZ and MZ impair whole body exercise performance in acute normobaric hypoxia.NEW & NOTEWORTHY Administration of acetazolamide (AZ) and methazolamide (MZ) both resulted in a significantly slower 5-km time trial in acute normobaric hypoxia compared with a placebo. Both drugs lead to a partially compensated metabolic acidosis, but ventilation and oxyhemoglobin saturation were not different across the conditions. Overall, acetazolamide and methazolamide both impaired whole body exercise performance in acute normobaric hypoxia but potentially have different mechanisms of action.

The sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) are responsible for inducing muscle relaxation and are integral to the maintenance of intracellular calcium (Ca2+) homeostasis. As such, their activity is modulated by multiple regulatory proteins. Myoregulin (MLN) is a newly discovered protein inhibitor of SERCAs that physically interacts with the pump to regulate Ca2+-handling in muscle. While MLN has emerged as a key regulator of Ca2+ homeostasis and muscle contractility, it is unknown whether MLN can uncouple Ca2+ transport from ATP hydrolysis by SERCAs and in doing so, alter SERCAs’ apparent coupling ratio. To that end, HEK-293 cells were co-transfected with cDNA encoding SERCA1a alone or with SERCA1a and MLN and SERCA2a alone or with SERCA2a and MLN. Both Ca2+ uptake and Ca2+-ATPase activity were measured on crude cell homogenate prepared from the transfected cells. Ca2+-dependent SERCA activity was assessed over Ca2+ concentrations ranging from pCa 6.85 to 4.80 in presence and absence of the Ca2+ ionophore A23187 using a spectrophotometric plate reader assay. SERCA-mediated Ca2+ uptake was measured in the presence and absence of the precipitating anion, oxalate, using the fluorescent dye Indo-1 and a fluorometer. SERCA coupling ratio was calculated by dividing Ca2+ uptake by Ca2+-ATPase rates across different pCa values. In both the presence (p < 0.05) and absence of ionophore (p < 0.05), MLN significantly depressed the maximal rate of ATP consumption (VMAX) and SERCA’s Ca2+ affinity, with this effect being more pronounced in the presence of ionophore. Similarly, MLN significantly reduced SERCA Ca2+ uptake in both conditions, with a greater effect in the presence of oxalate. The ability of MLN to reduce VMAX and impede Ca2+ uptake was greater for SERCA1a compared to SERCA2a. These results indicate that MLN does not affect the Ca2+/ATP coupling ratio of SERCA1a and SERCA2a pumps at maximal (presence of ionophore/oxalate) and physiological (absence of ionophore/oxalate) conditions. NSERC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sarcolipin (SLN), a small protein inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), is dynamically upregulated in atrophic unloading and disease states where it promotes muscle health by activating calcineurin (Cn) and the dephosphorylation and subsequent nuclear translocation of nuclear factor of activated T-cells (NFAT). This study sought to examine how the ablation of SLN impacted Cn signalling, fibre type profile and muscle mass in both male and female mice with aging. Male and female, wild type (WT) and SLN knockout (SLNKO) mice were assessed at two age groups (young adult (4-6M) and older adult (18+M)) for muscle mass (soleus:body weight ratio, fibre type specific cross-sectional area), fibre type profile and protein expression (SLN, Cn, and NFAT). SLN content was significantly greater in WT females (p <00.1) and older animals (5<0.05) and absent from SLNKO. As SLN has been linked to Cn signalling previously, we hypothesized that the increased SLN content in female and aged animals may promote Cn signalling. While there was a trend for lower Cn expression with aging (p=0.10), neither sex nor genotype significantly impacted Cn expression. To examine activation of Cn, the ratio of inactive phosphorylated NFAT to total NFAT was assessed via Western Blotting. We found that aging significantly increased the activation of Cn (p<0.01) and there was a trend (p=0.10) towards greater NFATp/NFAT with SLN ablation signifying a reduction in Cn signalling. As Cn is a known regulator of muscle fibre type, we examined fibre type profile using immunofluorescence and found aging increased the percentage of type I fibres (p<0.001), which was witnessed more predominately in female mice regardless of genotype. Furthermore, when examining type II fibres, we found a significant sex by age interaction (p<0.05) in which both WT and SLNKO females displayed reduced type II fibres with aging while males did not display this apparent shift. With respect to muscle mass, aging, regardless of sex or genotype, was shown to significantly reduce relative muscle mass (p<0.001), however no significant reductions were found in total fibre count, or cross-sectional area of Type I or Type II fibres. In summary, this study found that WT female and aged animals display increased SLN content. Aligning with our hypothesis, there was a trend towards lower Cn signalling with SLN ablation, regardless of age or sex. However, this does not seem to be linked to changes in muscle fibre type or muscle mass, as SLNKO animals did not display significant differences from their WT counterparts. Further studies should seek to examine the role of SLN in both sex and aging further, with special focus on metabolic energy expenditure and SERCA protection. NSERC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiomyopathy has become one of the leading causes of death in patients with Duchenne muscular dystrophy (DMD). We recently reported that the inhibition of the interaction between the receptor activator of nuclear factor κB ligand (RANKL) and receptor activator of nuclear factor κB (RANK) significantly improves muscle and bone functions in dystrophin-deficient mdx mice. RANKL and RANK are also expressed in cardiac muscle. Here, we investigate whether anti-RANKL treatment prevents cardiac hypertrophy and dysfunction in dystrophic mdx mice. Anti-RANKL treatment significantly reduced LV hypertrophy and heart mass, and maintained cardiac function in mdx mice. Anti-RANKL treatment also inhibited NFκB and PI3K, two mediators implicated in cardiac hypertrophy. Furthermore, anti-RANKL treatment increased SERCA activity and the expression of RyR, FKBP12, and SERCA2a, leading possibly to an improved Ca2+ homeostasis in dystrophic hearts. Interestingly, preliminary post hoc analyses suggest that denosumab, a human anti-RANKL, reduced left ventricular hypertrophy in two patients with DMD. Taken together, our results indicate that anti-RANKL treatment prevents the worsening of cardiac hypertrophy in mdx mice and could potentially maintain cardiac function in teenage or adult patients with DMD.

Caloric restriction that promotes weight loss is an effective strategy for treating non-alcoholic fatty liver disease and improving insulin sensitivity in people with type 2 diabetes 1 . Despite its effectiveness, in most individuals, weight loss is usually not maintained partly due to physiological adaptations that suppress energy expenditure, a process known as adaptive thermogenesis, the mechanistic underpinnings of which are unclear 2 , 3 . Treatment of rodents fed a high-fat diet with recombinant growth differentiating factor 15 (GDF15) reduces obesity and improves glycaemic control through glial-cell-derived neurotrophic factor family receptor α-like (GFRAL)-dependent suppression of food intake 4 – 7 . Here we find that, in addition to suppressing appetite, GDF15 counteracts compensatory reductions in energy expenditure, eliciting greater weight loss and reductions in non-alcoholic fatty liver disease (NAFLD) compared to caloric restriction alone. This effect of GDF15 to maintain energy expenditure during calorie restriction requires a GFRAL–β-adrenergic-dependent signalling axis that increases fatty acid oxidation and calcium futile cycling in the skeletal muscle of mice. These data indicate that therapeutic targeting of the GDF15–GFRAL pathway may be useful for maintaining energy expenditure in skeletal muscle during caloric restriction.

In healthy muscle, the rapid release of calcium ions (Ca2+) with excitation-contraction (E-C) coupling, results in elevations in Ca2+ concentrations which can exceed 10-fold that of rest-ing values. The sizable transient changes in Ca2+ concentrations are necessary for the ac-tivation of signaling pathways, which rely on Ca2+ as a second messenger, including those involved with force generation, fiber type distribution and hypertrophy. However, prolonged elevations in intracellular Ca2+ can result in the unwanted activation of Ca2+ signaling path-ways that cause muscle damage, dysfunction, and disease. Muscle employs several cal-cium handling and calcium transport proteins that function to rapidly return Ca2+ concen-trations back to resting levels following contraction. This review will detail our current under-standing of calcium handling during the decay phase of intracellular calcium transients in healthy skeletal and cardiac muscle. We will also discuss how impairments in Ca2+ transport can occur and how mishandling of Ca2+ can lead to the pathogenesis and/or progression of skeletal muscle myopathies and cardiomyopathies.

Pancreatic β-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell-specific knockout (KO) mice for all 3 isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in β-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Sarcolipin (SLN) is a small regulatory protein that inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. When bound to SERCA, SLN reduces the apparent Ca2+ affinity of SERCA and uncouples SERCA Ca2+ transport from its ATP consumption. As such, SLN plays a direct role in altering skeletal muscle relaxation and energy expenditure. Interestingly, the expression of SLN is dynamic during times of muscle adaptation, in that large increases in SLN content are found in response to development, atrophy, overload, and disease. Several groups have suggested that increases in SLN, especially in dystrophic muscle, are deleterious as it may reduce muscle function and exacerbate already abhorrent intracellular Ca2+ levels. However, there is also significant evidence to show that increased SLN content is a beneficial adaptive mechanism that protects the SERCA pump and activates Ca2+ signaling and adaptive remodeling during times of cell stress. In this review, we first discuss the role for SLN in healthy muscle during both development and overload, where SLN has been shown to activate Ca2+ signaling to promote mitochondrial biogenesis, fiber-type shifts, and muscle hypertrophy. Then, with respect to muscle disease, we summarize the discrepancies in the literature as to whether SLN upregulation is adaptive or maladaptive in nature. This review is the first to offer the concept of SLN hormesis in muscle disease, wherein both too much and too little SLN are detrimental to muscle health. Finally, the underlying mechanisms which activate SLN upregulation are discussed, specifically acknowledging a potential positive feedback loop between SLN and Ca2+ signaling molecules.

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25–35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Dietary nitrate has been shown to increase cytosolic calcium concentrations within the heart, which would necessitate greater calcium sequestration for relaxation. In the present study we demonstrate that while nitrate supplementation reduced blood pressure, calcium-handling protein content, sarco(endo)plasmic reticulum Ca-ATPase 2a (SERCA) enzymatic properties, and left ventricular function were not altered. In addition, nitrite did not alter in vitro SERCA activity. Combined, these data suggest that in healthy rats, dietary nitrate does not increase left ventricle SERCA-related calcium-handling properties. Novelty Dietary nitrate decreases blood pressure but does not alter left ventricular calcium-handling protein content or SERCA activity in healthy rats.

Phospholamban (PLN) is an important Ca2+ modulator at the sarcoplasmic reticulum (SR) of striated muscles. It physically interacts and inhibits sarcoplasmic reticulum Ca2+ ATPase (SERCA2) function, whereas a protein kinase A (PKA)-dependent phosphorylation at its serine 16 reverses the inhibition. The underlying mechanism of this post-translational modification, however, remains not fully understood. Using publicly available databases, we identified A-kinase anchoring protein 6 (AKAP6) as a candidate that might play some roles in PLN phosphorylation. Immunofluorescence showed colocalization between GFP-AKAP6 and PLN in transfected HEK-293T cells and cultured mouse neonatal cardiomyocytes (CMNCs). Co-immunoprecipitation confirmed the functional interaction between AKAP6 and PLN in HEK-293T and isolated adult rat cardiomyocytes in response to isoproterenol stimulation. Functionally, AKAP6 promoted Ca2+ uptake activity of SERCA1 in cotransfected HEK-293T cells despite the presence of PLN. These results were further confirmed in adult rat cardiomyocytes. Immunofluorescence showed colocalization of both proteins around the perinuclear region, while protein-protein interaction was corroborated by immunoprecipitation of the nucleus-enriched fraction of rat hearts. Our findings suggest AKAP6 as a novel interacting partner to PLN in HEK-293T and murine cardiomyocytes.

The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1β (ARNT/HIF1β) plays a key role in maintaining β-cell function and has been shown to be one of the most down regulated transcription factors in islets from type 2 diabetic patients. We have shown a role for ARNT/HIF1β in glucose sensing and insulin secretion in vitro and no defects in in vivo glucose homeostasis. In order to gain a better understanding of the role of ARNT/HIF1β in the development of diabetes, we placed control (+/+/Cre) and β-cell specific ARNT/HIF1β knockout (fl/fl/Cre) mice on a high fat (HF) diet. Unlike the control (+/+/Cre) mice, fl/fl/Cre mice on a HF diet had no impairment in in vivo glucose tolerance. The lack of impairment in HF fed fl/fl/Cre mice was partly due to an improved islet glucose-stimulated NADPH/NADP+ ratio and glucose-stimulated insulin secretion (GSIS). The effects of the HF diet rescued insulin secretion in fl/fl/Cre islets could be reproduced by treating low fat diet fed fl/fl/Cre islets with the lipid signalling molecule 1-monoacylglcyerol. This suggests that the defects seen in low fat diet fed fl/fl/Cre islet insulin secretion involves lipid signalling molecules. Overall, mice lacking ARNT/HIF1β in β-cells have altered lipid signalling in vivo and are resistant to a HF diets ability to induce diabetes.

The amount of calcium released from the sarcoplasmic reticulum in skeletal muscle rapidly declines during repeated twitch contractions. In this study, we test the hypothesis that caffeine can mitigate these contraction-induced declines in calcium release. Lumbrical muscles were isolated from male C57BL/6 mice and loaded with the calcium-sensitive indicator, AM-furaptra. Muscles were then stimulated at 8 Hz for 2.0 s in the presence or absence of 0.5 mM caffeine, at either 30 °C or 37 °C. The amplitude and area of the furaptra-based intracellular calcium transients and force produced during twitch contractions were calculated. For each of these measures, the values for twitch 16 relative to twitch 1 were higher in the presence of caffeine than in the absence of caffeine at both temperatures. We conclude that caffeine can attenuate contraction-induced diminutions of calcium release during repeated twitch contractions, thereby contributing to the inotropic effects of caffeine.

Calcineurin is a Ca 2+ /calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

A single session of aerobic exercisemay offer one means to "prime" motor regions to bemore receptive to the acquisition of a motor skill; however, the mechanisms whereby this priming may occur are not clear. One possible explanation may be related to the post-translational modification of plasticity-related receptors and their associated intracellular signaling molecules, given that these proteins are integral to the development of synaptic plasticity. In particular, phosphorylation governs the biophysical properties (e.g., Ca2+ conductance) and the migratory patterns (i.e., trafficking) of plasticity-related receptors by altering the relative density of specific receptor subunits at synapses. We hypothesized that a single session of exercise would alter the subunit phosphorylation of plasticity-related receptors (AMPA receptors, NMDA receptors) and signaling molecules (PKA, CaMKII) in amanner that would serve to prime motor cortex. Young, male Sprague-Dawley rats (n = 24) were assigned to either exercise (Moderate, Exhaustion), or non-exercising (Sedentary) groups. Immediately following a single session of treadmill exercise, whole tissue homogenates were prepared from both the motor cortex and hippocampus. We observed a robust (1.2-2.0x greater than sedentary) increase in tyrosine phosphorylation of AMPA (GluA1,2) and NMDA (GluN2A, B) receptor subunits, and a clear indication that exercise preferentially affects pPKA over pCaMKII. The changes were found, specifically, following moderate, but not maximal, acute aerobic exercise in bothmotor cortex and hippocampus. Given the requirement for these proteins during the early phases of plasticity induction, the possibility exists that exercise-induced priming may occur by altering the phosphorylation of plasticity-related proteins. (C) 2019 IBRO. Published by Elsevier Ltd. All rights reserved.

We are currently facing an "obesity epidemic" worldwide. Promoting inefficient metabolism in muscle represents a potential treatment for obesity and its complications. Sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA) pumps in muscle are responsible for maintaining low cytosolic Ca2+ concentration through the ATP-dependent pumping of Ca2+ from the cytosol into the SR lumen. SERCA activity has the potential to be a critical regulator of body mass and adiposity given that it is estimated to contribute upwards of 20% of daily energy expenditure. More interestingly, this fraction can be modified physiologically in the face of stressors, such as ambient temperature and diet, through its physical interaction with several regulators known to inhibit Ca2+ uptake and muscle function. In this review, we discuss advances in our understanding of Ca2+-cycling thermogenesis within skeletal muscle, focusing on SERCA and its protein regulators, which were thought previously to only modulate muscular contractility. Novelty ATP consumption by SERCA pumps comprises a large proportion of resting energy expenditure in muscle and is dynamically regulated through interactions with small SERCA regulatory proteins. SERCA efficiency correlates significantly with resting metabolism, such that individuals with a higher resting metabolic rate have less energetically efficient SERCA Ca2+ pumping in muscle (i.e., lower coupling ratio). Futile Ca2+ cycling is a versatile heat generating mechanism utilized by both skeletal muscle and beige fat.