Skeletal muscle calcium homeostasis during fatigue -modulation by kinases and mitochondria

The use of skeletal muscles in daily activities and even during strenuous exercise resulting in fatigue requires precise regulation of force and the timing of contraction. To achieve such performance characteristics, vertebrate skeletal muscles have developed a unique control mechanism for regulating the free Ca2+ concentration in their myoplasm ([Ca2+]i). A basic knowledge of cellular and molecular mechanisms regulating these mechanisms is essential in understanding pathological alterations in Ca2+ handling. This thesis deals with how these mechanisms are modulated by (i) mitochondria, (ii) Ca2+/calmodulin dependent protein kinase II (CaMKII) and (iii) protein kinase A (PKA). (i) Mitochondrial free [Ca2+] ([Ca2+]mit) increased during fatiguing stimulation in most, but not all, slow‐twitch soleus fibres and fast‐twitch extensor digitorum longus (EDL) fibres and was back to pre‐fatiguing levels within 20 min in both fibre types. [Ca2+]mit did not affect tetanic [Ca2+]i and thus, mitochondria do not acutely modulate tetanic [Ca2+]i. [Ca2+]mit was also investigated in mice with skeletal muscle specific disruption of mitochondrial transcription factor A (Tfam KO). Fibres from fast‐twitch flexor digitorum brevis (FDB) muscle normally do not accumulate Ca2+ in their mitochondria during fatigue, but in Tfam KO FDB fibres there was a marked increase in [Ca2+]mit. Tetanic [Ca2+]i was significantly lower in Tfam KO compared to controls, due to downregulation of the sarcoplasmic reticulum (SR) Ca2+ buffering protein calsequestrin‐1. The increased [Ca2+]mit in Tfam KO could be a means by which ATP‐production is boosted. These data suggest that mitochondria do not acutely affect tetanic [Ca2+]i, but that they play a more long‐term role in regulation of Ca2+‐handling by modulating sarcoplasmic reticulum proteins responsible for Ca2+ buffering. (ii) The modulatory role of CaMKII on Ca2+ handling was investigated by inhibiting CaMKII in FDB fibres using either KN‐93 or an inhibitory peptide. CaMKII inhibition resulted in a significant decrease in tetanic [Ca2+]i when contractions occurred at intervals of 2 s or 300 ms, but not 5 s. Mathematical modelling shows that there is some activation of CaMKII using all protocols but suggests that there is an activity threshold that has to be surpassed to permit sustained SR Ca2+ release when contractions occur close together in time. (iii) During cold exposure there is an increase in systemic sympathetic activity so the modulatory role of PKA on Ca2+‐ handling was investigated in cold‐acclimatized mice. FDB muscles fibres from coldacclimatized mice display increased resting [Ca2+]i, which was shown to be due to an increased SR Ca2+ leak. This increased SR Ca2+ leak was shown to be associated with PKA‐mediated phosphorylation of the SR Ca2+ channel, the ryanodine receptor (RyR), on ser2844 and moderate dissociation of the RyR regulatory protein calstabin‐1. An increased leak results in increased SR Ca2+ cycling and could be a local means of generating heat in the distally and superficially located FDB muscles. Our results show that there are several factors involved in the shaping of skeletal muscle [Ca2+]i handling, some which do so acutely and some are of more importance in the long term