Mechanism Of TRPV1 Activation Mediated Enhancement Of Exercise Endurance

Background and objective:Exercise endurance is the ability to exert oneself to exercise for a long time, which is closely related to energy metabolism in skeletal muscle. Skeletal muscle is the primary tissue of glucose and fat metabolism and plays a major role in energy balance. The muscle fiber types, mitochondrial content and key enzyme activities mainly contribute to the oxidative capacity of skeletal muscle. Enhancement of exercise endurance can promote lipid oxidation, increase energy expenditure, and thus reduce cardio-metabolic risk factors. Therefore, study on the methods to enhance exercise endurance and the molecular mechanism of exercise endurance regulation is of important significance.Several earlier studies showed that administration of a single dose of capsaicin temporally improved exercise endurance in humans and rodents, effects associated with increased catecholamine release. However, it is unclear whether chronic intake of capsaicin enhances exercise endurance on a more permanent basis and whether TRPV1 channels in skeletal muscle are involved in such long-term changes.The TRPV1 ion channel was first cloned as a receptor for capsaicin which causes burning pain by activation of nociceptive pathways. TRPV1 responds to noxious stimuli that include capsaicin, heat, and acidic solutions, which results in an extra-cellular calcium influx. TRPV1 also exists in adipocytes. Our previous study showed that chronic administration of capsaicin up-regulated TRPV1 expression in adipose tissue and prevented diet-induced obesity in mice. Recent research also identified TRPV1 in rat skeletal muscle cells, which was assumed to be related to exercise endurance regulation. As indicated by recent studies, several important molecules were involved in the regulation of exercise endurance, such as AMPK,p-AMPK,PPARδ,PGC-1α,UCP3 and PEPCK-C, most of which could be regulated by calcium signals. Thus, we hypothesize that activation of TRPV1 channels would enhance exercise endurance through upregulating some of the above key molecules.In the present study, we first confirmed the expression and distribution of TRPV1 in mice skeletal muscle, and detected the TRPV1-dependent change of intracellular calcium ([Ca2+]i). Then we examined the exercise endurance capacity, the skeletal muscle fiber types, and serum parameters of wild-type mice and TRPV1 knockout mice fed with or without capsaicin. We also compared the endurance-related phenotypes between transgenic mice with overexpression of TRPV1 and their wild-type littermates. To find out which molecule was regulated by TRPV1 activation, we detected the above endurance-related key molecules in skeletal muscle, and finally studied whether this endurance-related molecule was affected by different calcium signals in vitro.Materials and Methods:The present study includes in vivo and in vitro experiments. In vivo models include wild-type mice and TRPV1 knockout mice fed with or without capsaicin, and the transgenic mice with TRPV1 overexpression (TRPV1-tg) and their wild-type littermates fed with normal diet were also observed. In vitro models were myotubes differentiated from control C2C12 myoblasts and TRPV1-RNAi C2C12 myoblasts.1. Selective silencing of TRPV1 by RNA interference in C2C12 myoblast was performed using a lentiviral system.2. Expressions of TRPV1 protein and mRNA in cells and skeletal muscle were detected by immunoblotting and RT-PCR respectively. Distribution of TRPV1 in C2C12 myotubes and skeletal muscle was shown by immunofluorescence.3. Capsaicin induced [Ca2+]i change was examined by PTI systems.4. TRPV1-tg mice with TRPV1 overexpression were constructed by DNA microinjection.5. Exercise endurance was examined by treadmill test. Skeletal muscle fiber types were distinguished by ATPase metachromia staining. Expression of troponin-ss and FAT/CD36 were detected by immunoblotting. Serum parameters like lactic acid, free fatty acid, triglycerides, total cholesterol, glucose and insulin were examined with special kits.6. Expression of endurance related molecules including AMPK,p-AMPK,PPARδ,PGC-1α,UCP3,PEPCK-C, were detected by immunoblotting and Expression of PEPCK-C mRNA was detected by RT-PCR. 7. Expression of PEPCK-C in C2C12 myotubes after stimulated with capsaicin or thapsigargin, an inhibitor of Ca2+-ATPase, in a medium with or without Ca2+ was detected by immunoblotting.Results:1. TRPV1 existed in both plasma membrane and sarcoplasmic reticulum (SR) of C2C12 myotubes and mice skeletal muscle. Expression of TRPV1 protein was up-regulated by capsaicin treatment and inhibted by TRPV1-RNAi.2. Capsaicin induced a concentration-dependent [Ca2+]i increase in C2C12 myotubes, which was inhibited by TRPV1-RNAi and capsazepine pretreatment.3. Activation of TRPV1 increased [Ca2+]i by Ca2+ entry from the extracellular space, as well as by Ca2+ release from intracellular SR stores.4. Chronic capsaicin administration continuously enhanced exercise endurance of mice with little effect on food intake. Diatery capsaicin also promoted oxygen consumption, increased oxidative muscle fibers, upregulated expression of troponin-ss and FAT/CD36 in skeletal muscle, inhibited visceral fat accumulation, and reduced serum levels of triglycerides, free fatty acid and lactic acid. However, capsaicin had no effect on TRPV1-/- mice.5. TRPV1-tg mice with markedly over-expressed TRPV1 protein were successfully constructed. Compared to wild-type littermates, the exercise endurance and oxygen consumption were impressively higher in TRPV1-tg mice. Expression of troponin-ss protein in skeletal muscle also significantly increased. But the body weight and food intake were similar between two groups.6. Expression of AMPK,p-AMPK,PPARδ,PGC-1αand UCP3 in skeletal muscle of capsaicin treated mice or TRPV1-tg mice were similar with that of control mice.7. Compared to the control mice, Expression of PEPCK-C mRNA and protein were both significantly up-regulated in capsaicin treated mice and TRPV1-tg mice.8. Expression of PEPCK-C protein in C2C12 myotubes was significantly up-regulated by capsaicin, which was inhibited by TRPV1-RNAi and capsazepine treatment.9. Similar to capsaicin, the Ca2+-ATPase inhibitor, thapsigargin, also significantly up-regulated PEPCK-C expression in medium containing 1.5 mmol/L Ca2+; however, neither capsaicin nor thapsigargin up-regulated PEPCK-C expression in Ca2+-free medium. Conclusions:1. Capsaicin induced changes of intracellular free calcium in C2C12 myotubes are composed of calcium release from TRPV1-dependent store in SR and calcium influx from extra-cellular fluid.2. Both capsaicin-induced activation and genetic overexpression of TRPV1 increase exercise endurance, oxygen consumption and type I fibers of mice. Activation of TRPV1 also promotes lipid oxidation in skeletal muscle and improves serum parameters.3. TRPV1 activation up-regulates expression of PEPCK-C through increased calcium influx, but has no effect on expression of AMPK,p-AMPK,PPARδ,PGC-1αand UCP3. It indicates that activation of TRPV1 enhances exercise endurance mainly through up-regulation of PEPCK-C.