Validation of mitochondrial function measurements in permeabilized myocardial tissue from porcine left ventricular specimens with ischemia/reperfusion

Abstract

Abstract Background Mitochondrial function is critical to myocardial ischemia/reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, permeabilized myocardial tissue can be used for quantification of mitochondrial respiration in small myocardial specimens of about only 2-5 mg, which can be retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. Whether or not mitochondrial dysfunction during ischemia/reperfusion is reflected to the same extent in saponin-permeabilized myocardial tissue specimens (PMT) and in isolated mitochondria is still unclear. Purpose We attempted to validate measurements of mitochondrial respiration from PMT by comparison to those from isolated mitochondria in left ventricular myocardium from anesthetized pigs undergoing coronary occlusion/reperfusion. Methods Göttingen open-chest, anesthetized minipigs (n=10) were subjected to 60 min distal left anterior descending artery occlusion and 180 min reperfusion. At the end of reperfusion, PMT and isolated mitochondria were prepared from remote and ischemic/reperfused myocardium. Baseline, adenosine diphosphate-stimulated complex I (without/with cytochrome c, to account for absence or presence of the outer mitochondrial membrane integrity) and complex IV respiration was measured at 37°C during magnetic stirring using a high-resolution respirometer. Mitochondrial respiration in PMT and isolated mitochondria was normalized to protein content and the content of the mitochondrial marker protein cytochrome c oxidase 4. Results Mitochondrial respiration measurements in PMT and isolated mitochondria correlated well (Figure 1A) and agreed well in Bland-Altman plot (Figure 1B). While baseline respiration in PMT was somewhat higher than that in isolated mitochondria (Figure 2A), mitochondrial dysfunction during ischemia/reperfusion was equally reflected in PMT and isolated mitochondria (Figure 2B & 2C). Complex IV respiration was not different between remote myocardium and ischemic/reperfused myocardium, independently of the preparation, indicating a comparable amount of viable mitochondria (Figure 2D). Conclusion Mitochondrial function measurements in PMT can substitute for that in isolated mitochondria to reflect their dysfunction in ischemia/reperfusion. Thus, mitoprotective strategies targeting ischemia/reperfusion injury in small tissue samples and also in human myocardial biopsies can be followed in PMT.Figure 1Figure 2