Standardization and validation of novel ex-vivo method for mitochondrial bioenergetics using mitochondrial modulators

Abstract

Mitochondria is an essential organelle; it produces 95% of the adenine triphosphate (ATP) of cells, their dysfunction is related to the pathogenesis of multiple diseases, such as diabetes mellitus, cardiovascular and neurological disorders. Various pharmacologic agents are known to target mitochondrial function. Moreover, the toxic side effects of multiple drugs used to treat diseases are related to the impairment of mitochondrial function. Thus, there is a need to develop a method to evaluate the effect of pharmacologic agents for their potential and side effects to identify effective mitochondrial-modulating agents. Therefore, the objective of this study was to develop and validate an ex-vivo method for studying the effect of pharmacologic agents on mitochondrial function and rescue of dysfunction. Dimethyl sulfoxide (DMSO) concentrations that drugs were soluble in and maintained mitochondrial function were determined. Metformin (MET) is a known mitochondrial complex-1 inhibitor tested for its ability to compromise mitochondrion function. Coenzyme Q10 (Q10) and Resveratrol (RSV), which are known to enhance mitochondrial function, were added alone and dose-dependent, tested for the ability to rescue metformin-induced mitochondrial dysfunction. Ex-vivo liver and brain mitochondrial function was assessed using an oxytherm Clark-type oxygen electrode. DMSO was found to be toxic above 10% and drugs insoluble below 5%. The addition of 0.5 mg/ml MET decreased liver and brain mitochondrial respiratory control rate (RCR). At the same time, Q10 improved RCR in normal mitochondria and a concentration-dependent manner in MET-induced dysfunctional mitochondria. RSV was added in the last step of the experiment to confirm that compromised function is due to MET. Hence this method can be used to screen pharmacological agents for their potential therapeutics or toxic effect on mitochondria.