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 resting values. The sizable transient changes in Ca2+ concentrations are necessary for the activation 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 pathways that cause muscle damage, dysfunction, and disease. Muscle employs several calcium handling and calcium transport proteins that function to rapidly return Ca2+ concentrations back to resting levels following contraction. This review will detail our current understanding 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.
The sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) pump is a major regulator of cytosolic Ca2+ in striated muscle. Sarcolipin (SLN) reduces SERCA-mediated Ca2+ pumping efficiency and is a key regulator of muscle metabolism and contractile function. With SERCA activity comprising approximately 12% of whole-body metabolic rate, the inefficient SERCA Ca2+ cycling promoted by SLN would be predicted to increase metabolic rate. Although SLN is involved in adaptive diet-induced thermogenesis and overexpression of SLN increases metabolic rate and protects against diet-induced obesity, we found previously that SLN ablation had no effect on whole body metabolic rate or body composition in male mice that were fed a standard chow diet. However, unpublished work from our lab indicates that SLN expression in mouse soleus is significantly higher in females compared with males. Therefore, the purpose of this study was to investigate the effects of SLN ablation on body composition and metabolic rate of female mice. Adult (4-6mo) female wild type (WT) and SLN knockout (KO) mice (n=4 per group) were used to collect anthropometric measures (adiposity and body weight) and were housed in a comprehensive lab animal monitoring system (CLAMS) over a 48-hour period to assess daily whole body VO2 and total cage activity. With SLN ablation, total whole body VO2 (ml/kg/hr) was lower (p<0.05) in the KO females (3534 ± 95.1) compared to WT females (5080 ± 98.8), with no differences (p>0.05) found for total cage activity (counts) (WT, 14935.42 ± 1799.6 vs. KO, 11860.83 ± 679.48), body weight (grams) (WT, 20.97 ± 0.76 vs. KO, 22.18 ± 0.80), or adiposity (arbitrary units) (WT, 5.18 ± 1.71 vs. KO, 2.12 ± 0.36). These results showing that high levels of endogenous muscle SLN expression in female mice influence whole-body metabolism even under chow-fed conditions, demonstrate a potential important sex difference in whole body physiology and metabolism. Natural Sciences and Engineering Research Council of Canada (NSERC) [grant number 311922-05 (to A.R.T.)] 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.
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.
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