Abstract
ObjectiveCardiac 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.MethodsMitochondrial 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.ResultsCompared 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).ConclusionThese 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.
Highlights
Two distinct mitochondrial subpopulations – subsarcolemmal (SSM), situated underneath the sarcolemmal membrane, and interfibrillar (IFM), distributed between myofibrils – have been previously identified in myocardium [1,2,3,4,5]. These mitochondrial subpopulations differ in respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and in their sensitivity to metabolic challenge [4,5,6,7,8,9,10,11,12,13]
The content of citrate synthase (CS) and ANT1, specific mitochondrial matrix and membrane proteins were all determined by Western blot (Fig. 1B, top panels)
Ca2+ handling and oxidative phosphorylation capacity of SSM and IFM The sensitivity of mitochondria toward Ca2+-induced mitochondrial permeability transition (MPT) pore opening was determined from the number of Ca2+ pulses required to reach the threshold for rapid and spontaneous Ca2+ release [31]
Summary
Two distinct mitochondrial subpopulations – subsarcolemmal (SSM), situated underneath the sarcolemmal membrane, and interfibrillar (IFM), distributed between myofibrils – have been previously identified in myocardium [1,2,3,4,5]. These mitochondrial subpopulations differ in respect to their protein and lipid compositions, capacities for respiration and protein synthesis, and in their sensitivity to metabolic challenge [4,5,6,7,8,9,10,11,12,13]. These results provide evidence of distinct sensitivity of cardiac mitochondrial subpopulations toward the protective effect of diazoxide, indicating that SSM could be the preferred target for drug treatment
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