Mitochondrial magnesium regulates MCU activity and PTP opening during ischemic reperfusion injury

PROJECT SUMMARY Magnesium (Mg2+) is a ubiquitous intracellular divalent cation and a crucial co-factor in the machinery that replicates, transcribes, and translates genomic information. Like calcium, Mg2+ is compartmentalized to mitochondria, and Mrs2 is the only known molecular machinery associated with mitochondrial Mg2+ influx. Apart from its conventional function of activating enzymes within the TCA cycle and electron transport chain, our previous research showed mitochondrial Mg2+ (mMg2+) to bind the N-terminal domain of the mitochondrial Ca2+ Uniporter (MCU) regulating acidic patch (MRAP) and regulates MCU-mediated mitochondrial Ca2+ (mCa2+) uptake. The overload of mCa2+ is associated with heart failure (HF) and because mMg2+ regulates mCa2+ uptake, we anticipate a loss of mMg2+ homeostasis in ischemic reperfusion (IR) injury, which can overload mCa2+ and predispose the cardiomyocytes to mitochondrial permeability transition pore (mPTP)-mediated cell death. Our preliminary results indicated a significant reduction in mMg2+ uptake in NRVMs exposed to hypoxia/reoxygenation relative to the control. Intriguingly, the decrease in mMg2+ was not due to a decrease in mRNA or protein levels, but rather post-translational modification of Mrs2 by oxidation, resulting in a loss of function. Our preliminary results also showed that the loss of mMg2+ uptake was related to increased mCa2+ overload, bioenergetics crisis, and mPTP opening. To determine whether impaired mMg2+ influx causes mCa2+ overload and mPTP opening, we expressed a cysteine null (Mrs2C269A) mutant in NRVMs. We observed that cysteine nullification rendered Mrs2 insensitive to oxidation and oxidative inhibition, protecting NRVMs from mCa2+ overload-mediated cell death during HR injury. Based on these observations, we hypothesize that oxidative stress inactivates Mrs2, resulting in the loss of mMg2+ uptake. This, in turn, relieves MCU from Mg2+- dependent negative feedback inhibition, ultimately leading to an increase in MCU-mediated mCa2+ overload and making cardiomyocytes more susceptible to mPTP-mediated cell death. To evaluate whether impaired mMg2+ influx is causally linked to mCa2+ overload-mediated cardiomyocyte death and heart failure, we propose three aims: Aim 1: Define the mechanism by which post-translational oxidative modification regulates Mrs2 activity. Aim 2: Investigate the regulatory role of Mrs2 oxidation in cardiac mitochondrial bioenergetics. Aim 3: Define the ionic link between Mrs2 and MCU in regulating cardiac function during IR injury. The completion of the proposed experiments will provide a missing link between the "ionic dysregulation" and "mitochondrial dysfunction" hypotheses and advocate targeting mitochondrial ion homeostasis as a powerful therapeutic strategy to inhibit or reverse HF progression, even though the role of mCa2+ overload and mPTP opening in cardiac diseases are already known.