Evaluation of mitochondrial respiration

Mitochondrial respiration was tested at 25 °C by using a Clark-type electrode connected to a respiration chamber (YSI Incorporated, Yellow Springs, OH, USA) and a linear chart recorder, in buffer (pH 7.4) containing 20 mM HEPES, 10 mM KCl, 5 mM KH2PO4, 2.5 mM MgCl2, 0.25 M sucrose, 0.2 mM EDTA and 1 mg/ml fatty acid-free BSA. Intact cardiac mitochondria were added to 0.5 mg protein per ml. For glutamate/malate respiration evaluation, substrates were added each at 5 mM. With the addition of 0.5 mM ADP, state 3 respiration was tested. In the absence of ADP, state 4 respiration was determined. The respiratory control ratio was calculated as the ratio of the state 3 to state 4 respiration.⁴¹