LIMITATIONS OF USING CYTOCHROME C AS AN INDICATOR OF MITOCHONDRIA OUTER MEMBRANE INTEGRITY IN HIGH-RESOLUTION RESPIROMETRY EXPERIMENTS

Freshly isolated mitochondria from tissues are believed to undergo partial structural disruption. In high-resolution respirometry, the addition of cytochrome c is commonly used as an indicator of outer mitochondrial membrane integrity, based on the principle that disrupted mitochondria lose their endogenous cytochrome c pool. A resulting increase in mitochondrial oxygen consumption rates (OCR) upon cytochrome c addition is typically interpreted as evidence of outer membrane permeabilization. However, we observed that mitochondrial responsiveness to cytochrome c varies depending on the supplied substrate. Here, we aimed to evaluate these differences using freshly isolated liver mitochondria. High-resolution respirometry was performed in the presence of pyruvate (5 mM)/malate (5 mM) (to generate the complex I substrate NADH) or succinate (10 mM, a direct complex II substrate), followed by ADP and cytochrome c addition. When pyruvate/malate was the sole substrate, cytochrome c addition did not significantly alter OCR. In contrast, succinate supplementation led to an average OCR increase of ~20%. We next plan to assess whether disrupted mitochondria—subjected to freeze–thaw cycles—exhibit a similar response to NADH, a direct complex I substrate, upon cytochrome c addition. This will help determine whether the succinate-responsive fraction corresponds to mitochondria with both inner and outer membrane disruption or if a dedicated cytochrome c pool preferentially transfers electrons through the CII–CIII–CIV pathway. Our findings highlight that cytochrome c responsiveness is substrate-dependent, suggesting that its addition should be carefully interpreted when assessing mitochondrial integrity. Moreover, these observations may provide insights into unique aspects of mitochondrial structure and function.