Skeletal muscle adaptation: The roles of AMP-activated protein kinase and calcium signals

Sungkwon Park, Purdue University

Abstract

The purpose of this research was to determine the roles of AMP-activated protein kinase (AMPK) and Ca2+ signals in skeletal muscle adaptations, including energy metabolism and myosin heavy chain (MyHC) profiles. The effect of muscle contraction on adaptation has been studied extensively, yet the specific mechanisms by which AMPK and Ca2+ signals modulate energy metabolism and MyHC isoform expression have not been elucidated. The first study determined the long-term effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), a known activator of AMPK, on MyHC isoform composition and energy metabolism in growing pigs. Administration of AICAR for 10 d increased phosphorylation of AMPK and was followed by an increase in muscle specific glucose transporter 4 (GLUT4). AMPK activation was confirmed by measuring the phosphorylation level of acetyl CoA carboxylase (ACC), a known target of AMPK. AICAR treatment induced a shift to faster phenotype as evidenced by MyHC transition from IIx to IIb, and increased lactate dehydrogenase (LDH) activity, which is a glycolytic enzyme that is greater in fast twitching fibers than in slow twitching fibers. To study the effects of both AMPK and Ca2+ signals on muscle adaptation, we utilized pigs including Rendement Napole (RN) bearing a point mutation in AMPK γ3 subunit, Halothane (Hal) bearing a leaky Ca2+ channel due to the mutation in ryanodine receptor, and double mutant (Hal-RN). Combined use of real-time PCR, gel electrophoresis, and western blot analysis, indicated that myosin heavy chain (MyHC) isoform shifted from IIb to IIx/a in RN bearing mutant pigs. In addition, protein level of MyHC assessed by Western blot correlated with mRNA level by real-time PCR, supporting the hypothesis that MyHC genes are transcriptionally controlled. However, transcript abundance of genes involved in energy metabolism including LDH, citrate synthase, glycogen synthase, and PPARα, was not different between genotypes, suggesting that regulation of metabolism and structural phenotype are not coordinately regulated. Muscles from RN pigs showed higher AMPK phosphorylation and GLUT4 expression compared to normal. High cytosolic [Ca2+] in Hal pigs did not affect AMPK or GLUT4 expression. Interestingly, RN-induced AMPK phosphorylation and GLUT4 expression were blunted by Hal gene in Hal-RN double mutant pigs. These data suggested calcium has an inhibitory effect on AMPK-induced GLUT4 expression. To further evaluate this phenomenon, we utilized AICAR and caffeine to enhance AMPK activity and Ca2+ level in cultured C2C12 myotubes. Increase in GLUT4 expression induced by AICAR was positively regulated by short term (< 4h) caffeine (3 mM) treatment, but this increase in GLUT4 was blocked by long term (8 to 72 h) exposure to 3 mM caffeine. This result confirmed our hypothesis that chronic high Ca2+ level blocks AMPK-induced GLUT4 expression. To further specify the mechanisms by which Ca2+ signals modulate AMPK activity, we utilized various inhibitors and siRNA technique to knock-down Ca2+-induced signals. Also, because activities of Ca2+/calmodulin dependent kinases (CaMKs) are affected by the pattern of Ca2+ oscillation, we compared AMPK activities from C2C12 myotubes exposed to different Ca2+ frequencies. This was accomplished by switching cells between caffeine-containing and dantrolene-containing media. Inhibitory effect of long-term caffeine exposure was diminished by treatment with CaMKII inhibitors. Continuous incubation of C2C12 myotubes with caffeine had a negative effect on AICAR-induced AMPK activity. Ca 2+ oscillation produced by switching caffeine- and dantrolene-containing media reduced the inhibitory effect of long term Ca2+ treatment on AICAR-induced AMPK activity. Taken together, Ca2+ and AMPK signals modulate skeletal muscle adaptation, including MyHC expression and activities of enzymes involved in energy metabolism. In addition, Ca2+ appears to regulate AMPK activity in a frequency dependent manner through the CaMKK-CaMKII signaling cascade.

Degree

Ph.D.

Advisors

Gerrard, Purdue University.

Subject Area

Molecular biology|Cellular biology|Physiology

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