Mic60/Mitofilin inhibitor, Miclxin, induces rat H9C2 cardiomyoblast death by inhibiting the removal of damaged mitochondria.

Mitochondrial dysfunction is a hallmark of various pathologic conditions, including ischemia/reperfusion injury, stroke, myocardial infarction, neurodegeneration and metabolic syndrome. As with all biological organelles, the function of mitochondria is tightly linked to their structure. The inner mitochondrial membrane is a highly regulated membrane with a large surface area that hosts the electron transport chain machinery, generates the membrane potential necessary for ATP generation, and forms the signature cristae folds of mitochondria. The mitochondrial inner membrane protein (Mitofilin/Mic60) is part of a large complex that constitutes the mitochondrial inner membrane organizing system, which is critical in maintaining mitochondrial architecture and function. Recent evidence has shown that Mic60/Mitofilin elimination during reperfusion determines the extent of myocardial infarct size after ischemia/reperfusion. Here, we investigated the effects and mechanisms of action of Miclxin, a novel Mic60/Mitofilin inhibitor using H9c2 cardiomyoblasts. Cultured rat H9c2 cardiomyoblasts were incubated with 0, 5, 10, or 20 μM of Miclxin. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays, and cell death was determined by flow cytometry using propidium iodide dye. Mitochondrial membrane potential was measured using MitoTracker Red CMXROS assay kits, and mitophagy in mitochondria was detected using Mitophagy Detection Kits. Mitochondrial morphology was assessed using electron microscopy, and proteins were measured by Western blot analyses and immunofluorescence staining. After 24 hours of treatment, Miclxin decreased cell viability in a dose-dependent manner and reduced the number of viable cells measured with MTT assays. This effect was associated with pronounced reduction of Mic60 protein levels measured by Western blots and immunocytochemistry. Miclxin's reduction of cell viability was related to its inhibition of mitochondrial elimination by mitophagy. Our findings suggest that Miclxin decreases levels of Mic60, and thereby reduces cell viability by increasing structural damage and dysfunction in mitochondria via impairment of mitophagy.