Mic60/Mitofilin Inhibitor, Miclxin, Induces Rat H9C2 Cardiomyoblast Death by Inhibiting the Removal of Damaged Mitochondria

Mitochondrial dysfunction is a hallmark in a variety of disease conditions, including ischemia/reperfusion (I/R) injury, stroke, and myocardial infarction (MI), metabolic syndrome, and aging. As with all biological organelles, the function of mitochondria is tightly linked with their structure. The inner mitochondrial membrane is a highly regulated, large surface area membrane that hosts the electron transport chain (ETC) machinery, generates membrane potential necessary for ATP generation, and forms the signature cristae folds of mitochondria. Mitochondrial inner membrane protein (Mitofilin/Mic60) is part of a large complex that constitutes the mitochondrial inner membrane organizing system (MICOS or MINOS), which plays a critical role in maintaining mitochondrial architecture and function. Recent evidence has highlighted the importance of cristae morphology in mitochondrial function and cell survival. Mic60/Mitofilin elimination during reperfusion was reported to determine the extent of myocardial infarct size after I/R. Here, we investigated the effects and mechanism of a novel Mic60/Mitofilin inhibitor, Miclxin, in cell viability and death using H9c2 cardiomyoblasts. Cultured rat H9c2 cardiomyoblasts were incubated in the presence of different concentrations (0, 5, 10, and 20 mM) of Miclxin. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and cell death determined by flow cytometry using Propidium iodide dye. Mitochondrial membrane potential measurement was assessed using MitoTracker Red CMXROS assay kit, and Mitophagy in mitochondria was detected by using a Mitophagy Detection Kit. Mitochondrial morphology was assessed using electron microscopy, and proteins quantification was measured by Western blot analysis and immunofluorescence staining. After 24 hours of treatment, Miclxin concentration-dependently decreased cell viability and reduced the number of viable cells measured using MTT assay. This effect was associated with pronounced reduction of Mic60 protein levels measured by Western blot, and immunocytochemistry. We found that the mechanism of Miclxin-reduction of cell viability was related to inhibition of the elimination of mitochondria by mitophagy. Our finding suggests that treatment of cells with Miclxin decreases the levels of Mic60, which reduces cell viability that is associated with cell death by increasing mitochondria structural damage and dysfunction via impairment of mitophagy.