Electrical Pulse Stimulation of Cultured Human Skeletal Muscle Cells as an In Vitro Model of Exercise

Abstract

Background and AimsPhysical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes.MethodsElectrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting.ResultsHigh-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTracker®Red FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells.ConclusionsOur results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise.

Are cultured human myotubes far from home?

“…Satellite cells may be heterogenous, and it is not known if there are distinct populations of fast twitch (type 2) and slow twitch (type 1) cells. However, when grown in culture, human satellite cells mature to myotubes that express MYH1, MYH2, and MYH7 (Nikolic, et al, 2012a). In accordance with our observations in human myotubes, murine satellite cells isolated from different muscles were uniform regardless of muscle origin, and the dominant muscle fiber type both in primary murine myoblast and in the murine muscle cell lines was the intermediate MyHC-2A (LaFramboise, et al, 2003).…”

“…In our hands, myotube cultures stained at day 8 after differentiation contained a noteworthy amount of slow muscle fibers as well (Fig. 1), and the cell content of MyHC-β proteins increased after chronic electrical pulse stimulation of the cells, showing plasticity potential (Nikolic, et al, 2012a). When satellite cells were isolated from pools of single human skeletal muscle fibers exclusively expressing either fast or slow MyHC isoform the isolated cells differentiated into myotubes that co-expressed both fast and slow MyHC isoforms (Bonavaud, et al, 2001).…”

“…We have recently shown that chronic low-frequency electrical pulse stimulation of human myotubes increases glucose and fatty acid oxidation and mitochondrial content (Nikolic, et al, 2012a). Protein content of MyHC-β increased, indicating a fiber type switch.…”

“…Ca 2+ /calmodulin dependent kinase also seems to be involved in contraction-stimulated glucose uptake, and a link between muscle glycogen content and contraction-stimulated glucose uptake has been suggested (Aslesen, et al, 2001). Recently, it has been shown that human myotubes respond to "exercise", induced by electrical pulse stimulation, in a similar way as to exercising muscle in vivo (Lambernd, et al, 2012, Nikolic, et al, 2012a, and this model may be very important to clarify the mechanisms for contraction-stimulated glucose uptake. Chronic low-frequency stimulation (CLF) increases glucose uptake and oxidation, oleic acid oxidation, mitochondrial content, AMPK activation, and interleukin-6 (IL-6) production (Lambernd, et al, 2012, Nikolic, et al, 2012a.…”

“…Recently, it has been shown that human myotubes respond to "exercise", induced by electrical pulse stimulation, in a similar way as to exercising muscle in vivo (Lambernd, et al, 2012, Nikolic, et al, 2012a, and this model may be very important to clarify the mechanisms for contraction-stimulated glucose uptake. Chronic low-frequency stimulation (CLF) increases glucose uptake and oxidation, oleic acid oxidation, mitochondrial content, AMPK activation, and interleukin-6 (IL-6) production (Lambernd, et al, 2012, Nikolic, et al, 2012a. In fact, insulin resistance induced by adipocyte-conditioned media, chemerin and monocyte chemotactic protein-1 (MCP-1) was abrogated by electrical pulse stimulation (Lambernd, et al, 2012).…”