Abstract
Metabolic stress diminishes smooth muscle contractile strength by a poorly defined mechanism. To test the hypothesis that metabolic stress activates a compensatory cell signaling program to reversibly downregulate contraction, arterial rings and bladder muscle strips in vitro were deprived of O2 and glucose for 30 and 60 min (“starvation”) to induce metabolic stress, and the phosphorylation status of proteins involved in regulation of contraction and metabolic stress were assessed in tissues under basal and stimulated conditions. A 15–30 min recovery period (O2 and glucose repletion) tested whether changes induced by starvation were reversible. Starvation decreased basal phosphorylation of myosin regulatory light chain (MLC-pS19) and of the rho kinase (ROCK) downstream substrates cofilin (cofilin-pS3) and myosin phosphatase targeting subunit MYPT1 (MYPT1-pT696 and MYPT1-pT853), and abolished the ability of contractile stimuli to cause a strong, sustained contraction. Starvation increased basal phosphorylation of AMPK (AMPK-pT172) and 3 downstream AMPK substrates, acetyl-CoA carboxylase (ACC-pS79), rhoA (rhoA-pS188), and phospholamban (PLB-pS16). Increases in rhoA-pS188 and PLB-pS16 would be expected to inhibit contraction. Recovery restored basal AMPK-pT172 and MLC-pS19 to control levels, and restored contraction. In AMPKα2 deficient mice (AMPK), the basal level of AMPK-pT172 was reduced by 50%, and MLC-pS19 was elevated by 50%, but AMPK did not prevent starvation-induced contraction inhibition nor enhance recovery from starvation. These results indicate that constitutive AMPK activity participates in constitutive regulation of contractile proteins, and suggest that AMPK activation is necessary, but may not be sufficient, to cause smooth muscle contraction inhibition during metabolic stress.
Highlights
Skeletal muscle fatigue is a term used to describe the phenomenon of progressive decline in skeletal muscle force during intensive contractile activity, followed by recovery after a period of rest (Kent-Braun et al, 2012)
The present study showed that, in smooth muscle, metabolic stress caused a strong increase in constitutive AMP-dependent protein kinase (AMPK) activity concomitantly with dramatic reductions in basal levels of phosphoproteins involved in ensuring that contractile proteins are able to become activated, and reductions in the ability of stimuli to cause contraction
These data extend those showing that starvation of rabbit bladder smooth muscle causes metabolic stress by reducing the concentration of ATP to ∼60% of control within 15 min (Levin et al, 1996), that metabolic stress of smooth muscle activates AMPK and inhibits smooth muscle contraction (Rubin et al, 2005; Goirand et al, 2007; Horman et al, 2008; Gayard et al, 2011; Wang et al, 2011; Davis et al, 2012; Lee and Choi, 2013; Pyla et al, 2015; Schneider et al, 2015; Schubert et al, 2017), and that inhibition of contraction by hypoxia-induced metabolic stress is due to reductions in rho kinase (ROCK) activity and myosin regulatory light chain (MLC) phosphorylation (Gu et al, 2005; Wardle et al, 2006, 2007)
Summary
Skeletal muscle fatigue is a term used to describe the phenomenon of progressive decline in skeletal muscle force during intensive contractile activity, followed by recovery after a period of rest (Kent-Braun et al, 2012). Smooth muscle fatigue is understudied and poorly understood This is likely because smooth muscle contraction consumes far less ATP than even slow skeletal muscle (Walker et al, 1994), and smooth muscle is generally considered fatigue-resistant. Smooth muscle is dependent on an immediate supply of ATP because, unlike striated muscles that maintain high levels of phosphocreatine as an energy reserve to resupply ATP, the levels of phosphocreatine and ATP in smooth muscle are approximately equal (Ishida et al, 1994) Metabolic stress, such as occurs during ischemia, hypoxia, and hypoglycemia, has the potential to compromise the ATP supply for smooth muscle contraction and, especially, for maintaining Na+, K+, and Ca2+ ion gradients far from their equilibrium values to prevent cell death. AMPK is activated rapidly by metabolic stress in smooth muscle (Rubin et al, 2005; Pyla et al, 2015), and activation is associated with a decline in contractile activity (Rubin et al, 2005; Goirand et al, 2007; Pyla et al, 2015)
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