Low serum magnesium (Mg) is a predictor for cardiovascular and all-cause mortality, while Mg supplementation has shown significant therapeutic effects in heart failure. Cardiac Mg homeostasis is maintained by a series of sarcolemmal and mitochondrial transporters such as the transient receptor potential melastatin 7 channel (TRPM7). Mitochondria act as stores of Mg. In hypomagnesemia (HypoMg), mitochondrial Ca (mitoCa) is increased and mitochondrial Mg (mitoMg) is reduced. Increased mitoCa is associated with mitochondrial reactive oxygen species (mitoROS) overproduction. Recently, we have shown that diabetes mellitus (DM)-mediated diastolic dysfunction (DD) is associated with HypoMg.31, 47, 48 Mg supplementation relieves DD by reducing mitoROS production and mitoCa overload, increasing ATP production, and improving mitochondrial morphology.18 In preliminary data, we show that HypoMg alone, without DM, is sufficient to cause DD49 by inducing electron transport chain (ETC) dysfunction and increasing mitoROS. The increase in mitoROS49 is associated with increased TRPM7 and the mitochondrial Ca uniporter (MCU), while another Mg transporter solute carrier family 41A1 is unchanged. We show that TRPM7 kinase regulates MCU phosphorylation. Again, Mg repletion, MCU knockdown, TRPM7 knockdown, and TRPM7 kinase inactivation reverse effects of HypoMg. The novel findings include that HypoMg alone can cause DD, that HypoMg can cause dysregulation of TRPM7, MCU, and mitoROS, and that Mg supplementation can act as a mitochondrial antioxidant. Hypothesis: Therefore, we hypothesize that changes in TRPM7 (both channel and kinase functions) and MCU result in mitoMg depletion, ETC dysfunction, mitoROS overproduction, and DD and that mitigating these changes will prevent mitoMg depletion and the subsequent cardiomyopathy. In this study, we will test this hypothesis in three aims. Aim 1. To determine the role of TRPM7 in HypoMg-induced mitoMg depletion, ETC dysfunction, mitoROS overproduction and DD. Aim 2. To determine the role of TRPM7 kinase activity in HypoMg-induced mitoMg depletion, ETC dysfunction, mitoROS overproduction and DD. Aim 3. To study the role of MCU in HypoMg-induced mitoMg depletion, ETC dysfunction, mitoROS overproduction and DD. The outcome of this project is expected to lead to novel understandings and treatment strategies for cardiac diastolic dysfunction and heart failure with preserved ejection fraction (HFpEF). Moreover, elucidation of the role of TRPM7 and MCU will provide us with novel insights into the molecular basis of Mg and Ca handling under physiological and pathological conditions.