Heat therapy preserves myofibre size and SERCA-mediated Ca2+ uptake in the mouse soleus after tenotomy surgery

Abstract

Heat therapy (HT) has been shown to induce physiological adaptations in muscle, including a reduction in the severity of muscle atrophy resulting from unloading. The muscle atrophy caused by unloading can be partially attributed to the dysregulation of Ca²⁺ in the muscle cell, which can activate calpain-mediated proteolysis. The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) is a primary regulator of Ca²⁺ in muscle, and SERCA dysfunction has been repeatedly demonstrated in various models of muscle unloading. Heat shock protein 70 (HSP70) is a heat-inducible chaperone protein that binds to SERCA and protects against its dysfunction. While previous research has shown HT to upregulate HSP70 in rodent muscle, even in the unloaded state, the effects of HT on SERCA function in rodent skeletal muscle under these conditions remain unknown. Here, we characterized the effects of 4 weeks of HT on soleus muscle size, HSP70 expression, SERCA function, and maximal calpain activity in male C57BL/6J mice subjected to muscle unloading through tenotomy. Four weeks of HT preserved the cross-sectional area of soleus myofibres following tenotomy, while also upregulating HSP70, maintaining SERCA-mediated Ca²⁺ uptake, and reducing maximal calpain activity. Therefore, our research offers new insights into the advantages of HT for muscle health and physiology.

Keywords: HSP70; SERCA; calpain; muscle unloading.

FIGURE 1

Heat therapy (HT) preserves soleus myofibre cross‐sectional area (CSA) after tenotomy. (a) Absolute soleus muscle mass (mg). (b) Soleus muscle mass (mg) relative to body mass (g). (c) Body mass (g). (d) Representative H&E images. Scale bars are set to 200 μm. (e) Frequency distribution graphs plotted by CSA (μm²). (f) Average myofibre CSA. For each n, 3 images were randomly taken with a total of 30 myofibres measured. (g) % reduction in myofibre CSA (Tny vs. sham) in the CON and HT groups. For a, b, and f, data was analyzed with a two‐way plot ANOVA (n = 3–5 males per group). For c and g, data was analyzed with a Student's t‐test. CON, control; Tny, tenotomy. All values are presented as mean ± SD, and exact p‐values are reported.

FIGURE 2

Heat therapy (HT) upregulates HSP70 and maintains SERCA function in the tenotomized soleus. (a) Representative western blot images and semi‐quantitative analysis of HSP70 (b, n = 6–7 per group) and protein tyrosine nitration (c, n = 4–5 per group). (d) Representative Ca²⁺ uptake curves across all experimental groups. Data are presented as the change in Indo‐1_405/485nm, which is indicative of the amount of free intracellular Ca²⁺. (e) AUC analyses for the Ca²⁺ uptake curve. (f) Normalized rates of Ca²⁺ uptake with the slopes obtained via linear regression analysis within the first 88 s of Ca²⁺ uptake, which was then divided by protein content. For d–f, n = 4–5 per group. (g) Representative western blot images and semi‐quantitative analysis of SERCA1 (h, n = 4–5 per group) and SERCA2 (i, n = 5 per group). (j) Maximal calpain activity in the soleus homogenates, n = 4–5 per group. For b, c; e, f; and h‐j; data were analyzed with a two‐way ANOVA and a Tukey's post‐hoc test. All values are presented as mean ± SD, and exact p‐values are reported.