Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

Substrate- and Calcium-Dependent Differential Regulation of Mitochondrial Oxidative Phosphorylation and Energy Production in the Heart and Kidney

“…The bioenergetic aspects of mitochondrial functions have been challenging, and reports of studies even of the same organ, such as the heart, have been conflicting since many key factors that influence ATP production have not been consistently controlled or quantitatively accounted for in most studies [ 5 , 10 , 15 , 35 , 61 , 63 ]. In the present study, we demonstrated that different respiratory substrates resulted in dramatically different respiratory rates in the heart and kidney OM mitochondria, consistent with our recent study [ 65 ]. In addition, the effects of Ca 2+ upon mitochondrial respiration and ATP production in the heart and kidney OM were distinctly dependent on the respiratory substrates [ 63 ].…”

“…Mitochondria were freshly isolated from the ventricles of the heart and the OM of the kidneys of adult SD rats (8–9 weeks, 300–350 gm), as detailed in our recent study [ 65 ]. Following an intraperitoneal injection of pentobarbital to achieve a deep plane of anesthesia, the hearts and kidneys were excised, and the mitochondria were isolated, as briefly described below.…”

“…Cardiac mitochondria were isolated via the differential centrifugation method, as described previously [ 10 , 65 , 66 , 67 ]. Briefly, the ventricles of the heart were isolated and immediately placed in ice-cold isolation buffer (IB) containing 200 mM mannitol, 50 mM sucrose, 5 mM KH 2 PO 4 , 5 mM MOPS, 1 mM EGTA, and 0.1% BSA, with the pH adjusted to 7.15 with KOH.…”

“…Renal OM mitochondria were isolated through the differential centrifugation method with a slight modification from that for the cardiac mitochondria [ 65 ]. Briefly, OM were dissected from both kidneys, weighed, and minced in the 4 °C IB.…”

“…In the second set of experiments, the time-courses of states 2, 3, and 4 respiration were monitored by adding a variety of respiratory substrates ( Table 1 ) to isolated mitochondria (0.1 mg protein/mL for heart and 0.2 mg protein/mL for kidney OM) [ 65 ], including pyruvate + malate (PM; 5:2.5 mM), glutamate + malate (GM; 5:2.5 mM), alpha-ketoglutarate + malate (AM; 5:2.5 mM), palmitoyl- l -carnitine + malate (PCM; 0.025:2.5 mM), and succinate + rotenone (SUC + ROT; 10 mM + 1 μM) at time t = 2 min, and ADP (200 and 100 μM for heart and kidney OM mitochondria; respectively) at time t = 4 min, which initiates state 3 respiration proceeding to state 4 respiration after the conversion of the added ADP to ATP. To assess the effect of Ca 2+ on mitochondrial O 2 consumption, various concentrations of Ca 2+ (250, 400, 700, 750, or 800 μM of CaCl 2 in the presence of the Ca 2+ chelator EGTA at 1 mM; negligible changes in total Cl − concentration of 130 mM) were added a minute after state 4 was reached.…”