title: Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle.
abstract: Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.
authors.affiliation: East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, NC 27858, USA. perrych@ecu.edu
authors.forename: Christopher G R
authors.initials: CG
authors.lastname: Perry
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Kane
authors.forename: Chien-Te
authors.initials: CT
authors.lastname: Lin
authors.forename: Rachel
authors.initials: R
authors.lastname: Kozy
authors.forename: Brook L
authors.initials: BL
authors.lastname: Cathey
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Lark
authors.forename: Constance L
authors.initials: CL
authors.lastname: Kane
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Brophy
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Gavin
authors.forename: Ethan J
authors.initials: EJ
authors.lastname: Anderson
authors.forename: P Darrell
authors.initials: PD
authors.lastname: Neufer
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000328:Adult; D000818:Animals; D003401:Creatine / Q000378:metabolism; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D008297:Male; D008931:Mitochondria, Muscle / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects; D018485:Muscle Fibers, Skeletal; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology*; D009218:Myosins / Q000037:antagonists & inhibitors*; D010725:Phosphocreatine / Q000378:metabolism; D051381:Rats; D012119:Respiration; D013696:Temperature
chemical_list: D006576:Heterocyclic Compounds, 4 or More Rings; D010725:Phosphocreatine; C472645:blebbistatin; D000244:Adenosine Diphosphate; D009218:Myosins; D003401:Creatine