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

Background and Aims Physical 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. Methods Electrical 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. Results High-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. Conclusions Our 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.This work was funded by the University of Oslo, Oslo University College, the Norwegian Diabetes Foundation, the Freia Chocolade Fabriks Medical Foundation and the Anders Jahre’s Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Effekter av elektrisk stimulering av humane skjelettmuskelceller : Utprøving av en in vitro-treningsmodell

Bakgrunn/formål: I de senere årene har det vært en kraftig økning i forekomsten av kroniske sykdommer som inkluderer fedme og type 2-diabetes. Hovedårsaken til dette er en stillesittende livsstil kombinert med for høyt næringsinntak. Fysisk aktivitet har en beskyttende funksjon mot disse sykdommene og har vist seg å være en effektiv behandlingsform. De positive effektene av trening på glukose- og fettsyremetabolisme i skjelettmuskel spiller trolig en rolle, men de cellulære mekanismene bak disse effektene er vanskelig å studere in vivo. For å kunne studere direkte effekter av trening på muskel uten påvirkning av systemiske faktorer, har det tidligere blitt utviklet en in vitro modell for trening ved bruk av elektrisk pulsstimulering (EPS) av myotuber i kultur. Formålet med denne masteroppgaven var å teste ut denne modellen, ved å undersøke om EPS av humane skjelettmuskelceller i kultur ville vise de samme effektene som er sett etter trening in vivo. Metode: Satelittceller ble isolert fra biopsier fra M. obliquus internus abdominis. Etter differensiering til myotuber ble de kontinuerlig stimulert med lav-frekvent EPS (1 Hz, 30 V, 2 ms) de siste 24-48 timene av differensieringen. I forbehandlingsforsøkene ble myotubene kontinuerlig behandlet med PPARδ agonisten GW 501516 eller oljesyre i henholdsvis de siste 48 eller 24 timene av differensieringen. Fettsyremetabolismen i celler behandlet med EPS ble undersøkt ved bruk av radioaktivt merket [14C]oljesyre. Forandringer i genekspresjon ble undersøkt ved RT-real-time-PCR. Immunoblotting ble brukt for å detektere og kvantifisere proteiner av interesse. Resultater: Ved å gi en signifikant økning i oljesyreoksidasjonen, økte kronisk lav-frekvent elektrisk pulsstimulering den oksidative kapasiteten til humane skjelettmuskelceller. Videre ga EPS en signifikant økning i ekspresjonen av CPT1b og en tendens til økt ekspresjon av PDK4 og PGC-1α som er gener involvert i glukose- og lipidmetabolisme. EPS ga en tendens til økt ekspresjon av IL-6, dette kan indikere at humane skjelettmuskelceller in vitro kontraherer under elektrisk stimulering. Forbehandling med GW 501516 eller oljesyre ga ingen økning utover den EPS-stimulerte økningen i oksidasjonen av oljesyre. Konklusjon: Vår modell for elektrisk stimulering av humane skjelettmuskelceller i kultur viser mange av de samme effektene som er sett etter trening in vivo. Vi kan dermed konkludere med at vår modell kan brukes for å studere effekter av trening in vitro

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

Background and Aims Physical 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. Methods Electrical 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. Results High-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. Conclusions Our 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

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

[This corrects the article DOI: 10.1371/journal.pone.0033203.]

Effects of chronic, low-frequency EPS on gene expression.

<p>Low-frequency EPS was applied to cultured myotubes for the last 24 h or 48 h of the eight days differentiation period. mRNA was isolated and expression assessed by real time RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>, and values are presented as means±SEM of 3–6 experiments, normalized to levels of housekeeping genes 36B4. The ranges of the fold changes of the mRNA expression levels in the control groups normalized to the level of housekeeping gene 36B4 were as follows: 0.3–1.9 for CPT1b, 0.4–1.3 for cytochrome C, 0.001–0.6 for PGC-1α, 0.6–1.2 for GLUT1, 0.2–2.6 for GLUT4, 0.7–1.0 for PDK4 and 0.1–1.1 for IL-6. *Statistically significant vs. unstimulated control cells (<i>P = </i>0.04, non-parametric Kruskall-Wallis test).</p

Effects of chronic, low-frequency EPS on markers of slow-oxidative

<p>(<b>MHCI</b>) <b>and fast-glycolytic</b> (<b>MHCIIa</b>) <b>muscle fiber types.</b> Low-frequency EPS was applied to cultured myotubes for the last 24 h or 48 h of the eight days differentiation period as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a> before the cells were harvested. (<b>A</b>) <b>MHCI/MHCIIa mRNA ratio:</b> mRNA was isolated from cultured myotubes after the EPS treatment. Expression was assessed by RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>, and values are presented as means±SEM of 4 experiments, normalized to levels of housekeeping genes 36B4. (<b>B</b>) (<b>C) Immunoblot analysis of MHCI after 24</b>–<b>48 </b><b>h of EPS:</b> Aliquots of 40 µg cell protein from total cell lysates prepared in Laemmli buffer were electrophoretically separated on NuPAGE® 4–12% (w/v) Bis-Tris Gel, followed by immunoblotting with specific antibody for slow-oxidative MHCI. (B) One representative immunoblot. (C) Densitometric analysis of immunoblots, values are presented as means±SEM of 6 experiments. *Statistically significant vs. unstimulated control cells (<i>P = </i>0.03, non-parametric Wilcoxon matched pair test).</p

Effects of chronic, low-frequency EPS on glucose

<p>(<b>A</b>) <b>and oleic acid metabolism</b> (<b>B</b>)<b>.</b> Cultured myotubes were electrically stimulated (1 Hz, 2 ms pulses, 30 V), for the last 24 h or 48 h of the differentiation period. (A) <b>Deoxyglucose uptake</b>: After termination of electrical stimulation (day 8 of differentiation), uptake of [³H]deoxyglucose (1 µCi/mL) was measured for 1 h as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>. Values are presented as means±SEM of 6 experiments. *Significantly different from unstimulated control cells (absolute values 28.0–170.5 nmol/mg protein) (<i>P = </i>0.004, non-parametric Kruskall-Wallis test). <b>Glucose oxidation</b>: After termination of electrical stimulation (day 8 of differentiation), the rate of D-[¹⁴C(U)]glucose (2 µCi/mL) oxidation was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>. Values are presented as means±SEM of 4 experiments. ^*Statistically significant compared to unstimulated control cells (absolute values 2.7–28.4 nmol/mg protein) (<i>P = </i>0.008, non-parametric Kruskall-Wallis test). (B) <b>Oleic acid metabolism</b>: Eight or nine days after the onset of differentiation, myotubes were exposed to [1-¹⁴C]OA (1 µCi/mL) for 2 h, and CO₂, ASMs and cell-associated (CA) radioactivity were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>. Values are presented as means±SEM of 8 experiments. *Statistically significant vs. unstimulated control myotubes (absolute values 15.0–166.8 nmol/mg protein for CA, 0.6–4.0 nmol/mg protein for ASMs and 0.2–2.9 nmol/mg protein for CO₂) (<i>P = </i>0.04, non-parametric Kruskall-Wallis test).</p

Effects of 48 h of chronic, low-frequency EPS on mitochondrial content and citrate synthase activity.

<p>Low-frequency EPS was applied to cultured myotubes for the last 48 h of the eight days differentiation period as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>. (<b>A</b>) <b>Live imaging of mitochondria:</b> The cells were stained for nuclei (blue) and mitochondria (red) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>. Scale bar is 50 µm. Left: Unstimulated control myotubes. Right: Myotubes after 48 h of chronic, low-frequency EPS. (<b>B</b>) <b>Mitochondrial content in electrically stimulated myotubes</b>: Mitochondrial content was measured by live imaging after 48 h of chronic, low-frequency EPS. Values are presented as means±SEM of 6 experiments. *Statistically significant vs. unstimulated control (<i>P = </i>0.03, non-parametric Wilcoxon matched pair test). (<b>C</b>) <b>Citrate synthase activity in electrically stimulated myotubes:</b> Enzyme activity was determined spectrophotometrically from cell homogenates prepared from the myotubes after 48 h of chronic, low-frequency EPS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033203#s2" target="_blank">Materials and Methods</a>, and compared to activity in unstimulated control cells. Values are presented as means±SEM of 5 experiments.</p