title: Cardiac subsarcolemmal and interfibrillar mitochondria display distinct responsiveness to protection by diazoxide.
abstract: OBJECTIVE
  Cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial subpopulations possess distinct biochemical properties and differ with respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and sensitivity to metabolic challenge, yet their responsiveness to mitochondrially active cardioprotective therapeutics has not been characterized. This study assessed the differential responsiveness of the two mitochondrial subpopulations to diazoxide, a cardioprotective agent targeting mitochondria.
  
  
  METHODS
  Mitochondrial subpopulations were freshly isolated from rat ventricles and their morphologies assessed by electron microscopy and enzymatic activities determined using standard biochemical protocols with a plate reader. Oxidative phosphorylation was assessed from State 3 respiration using succinate as a substrate. Calcium dynamics and the status of Ca²⁺-dependent mitochondrial permeability transition (MPT) pore and mitochondrial membrane potential were assessed using standard Ca²⁺ and TPP⁺ ion-selective electrodes.
  
  
  RESULTS
  Compared to IFM, isolated SSM exhibited a higher sensitivity to Ca²⁺ overload-mediated inhibition of adenosine triphosphate (ATP) synthesis with decreased ATP production (from 375±25 to 83±15 nmol ATP/min/mg protein in SSM, and from 875±39 to 583±45 nmol ATP/min/mg protein in IFM). In addition, SSM exhibited reduced Ca²⁺-accumulating capacity as compared to IFM (230±13 vs. 450±46 nmol Ca²⁺/mg protein in SSM and IFM, respectively), suggestive of increased Ca²⁺ sensitivity of MPT pore opening. Despite enhanced susceptibility to stress, SSM were more responsive to the protective effect of diazoxide (100 μM) against Ca²⁺ overload-mediated inhibition of ATP synthesis (67% vs. 2% in SSM and IFM, respectively).
  
  
  CONCLUSIONS
  These results provide evidence for the distinct sensitivity of cardiac SSM and IFM toward Ca²⁺-dependent metabolic stress and the protective effect of diazoxide on mitochondrial energetics.
authors.affiliation: Center for Integrative Research on Cardiovascular Aging, Aurora University of Wisconsin Medical Group, Aurora Health Care, Milwaukee, Wisconsin, United States of American.
authors.forename: Ekhson L
authors.initials: EL
authors.lastname: Holmuhamedov
authors.forename: Andrew
authors.initials: A
authors.lastname: Oberlin
authors.forename: Kevin
authors.initials: K
authors.lastname: Short
authors.forename: Andre
authors.initials: A
authors.lastname: Terzic
authors.forename: Arshad
authors.initials: A
authors.lastname: Jahangir
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002316:Cardiotonic Agents / Q000494:pharmacology*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D003981:Diazoxide / Q000494:pharmacology*; D008297:Male; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009206:Myocardium / Q000166:cytology*; D009210:Myofibrils / Q000378:metabolism*; D010085:Oxidative Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012508:Sarcolemma / Q000378:metabolism*
chemical_list: D002316:Cardiotonic Agents; D000255:Adenosine Triphosphate; D002950:Citrate (si)-Synthase; D003981:Diazoxide; D002118:Calcium