Abstract 19126: Discovery of TMEM65 as a Positive Regulator of Mitochondrial Calcium Efflux

Abstract

Introduction: The balance between mitochondrial calcium ( m Ca 2+ ) uptake and efflux regulates ATP production, but if perturbed causes m Ca 2+ depletion and energy starvation or m Ca 2+ overload and cell death. Disrupted m Ca 2+ homeostasis is implicated in ischemia-reperfusion injury, heart failure, and neurodegeneration. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of m Ca 2+ efflux in excitable tissues, supporting NCLX as a promising therapeutic target to limit pathogenic m Ca 2+ overload in organs like the heart and brain. However, a critical barrier to translation is that little is known about the mechanisms controlling NCLX activity. Goals: We used proximity biotinylation screening to identify the NCLX interactome and proteins that may regulate NCLX function. We identified TMEM65, a mitochondrial inner membrane protein of unknown function, as an NCLX-proximal protein. Hypothesis: Null mutation of TMEM65 causes mitochondrial encephalomyopathy and phenocopies genetic NCLX disruption. Therefore, we hypothesized that TMEM65 modulates m Ca 2+ efflux through NCLX. Approach: Here, we used measurements of m Ca 2+ exchange in AC16 cardiomyocytes with TMEM65 overexpression (OE) or CRISPR/Cas9 genetic disruption of TMEM65 to test TMEM65’s effect on m Ca 2+ flux and its functional dependence on NCLX. We used AAV-mediated Tmem65 knockdown (KD) and TMEM65 OE in mice to assess the effects of gain- or loss of TMEM65 expression on cardiac function. Results: TMEM65 OE potently enhanced m Ca 2+ efflux in vitro , and NCLX inhibition with CGP-37157 mitigated this effect. Knockout of TMEM65 attenuated m Ca 2+ efflux. In vivo Tmem65 KD reduced left ventricular fractional shortening and reduced S293 phosphorylation of pyruvate dehydrogenase in the heart, indicative of increased m Ca 2+ loading. In vivo TMEM65 OE did not alter cardiac function. Conclusions: We conclude that TMEM65 has an essential function to promote m Ca 2+ efflux, likely via positive regulation of NCLX, and is critical to limiting deleterious m Ca 2+ overload in the intact heart. Ongoing work is defining the functional interactions between TMEM65 and NCLX, and establishing the utility of targeting TMEM65 to restore m Ca 2+ homeostasis in heart failure and other excitable tissue diseases.