Aberrant Zn2+ homeostasis is associated with dysregulated intracellular Ca2+ release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+. Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.

Mitochondrial Ca 2þ regulates a wide variety of physiological processes, including ATP production and cell death. Rapid Ca 2þ uptake into mitochondria is mediated by the mitochondrial calcium uniporter (MCU) complex, which is composed of the pore forming MCU protein, and the regulatory proteins EMRE, and MICU1/MICU2. Currently, the submitochondrial localization and transmembrane orientation of these proteins are either unknown or under debate. We attack these issues using a classical thiol-modification approach. We removed native MCU complex proteins in HEK293 cells using CRISPR/Cas9, and reintroduced mutants containing a single cysteine at defined positions. Treatments of the mitoplast with a bulky, thiol-reactive compound polyethylene glycol (PEG) maleimide would alter the molecular weight of the protein if the engineered Cys is exposed to the intermembrane space. Using this strategy along with other biochemical methods, we demonstrated that (1) MCU adopts an orientation with the signature DIME motif facing intermembrane space (IMS), ( 2) the conserved C-terminal polyaspartic tail of EMRE is in the IMS, (3) both MICU1 and MICU2 are associated with the outer leaflet of the inner membrane, and (4) although MICU1 is a peripheral membrane protein, MICU2 is an integral component of the inner membrane. Then, applying domain interaction analysis and mutagenesis screening, we identified molecular contacts that govern the Ca 2þ transport behavior of the MCU complex. In particular, we demonstrate that EMRE interacts with MCU through the transmembrane helices to activate the Ca 2þ pore, while using the polyaspartic tail to recruit MICU1/2 to gate the pore. This dual functionality of EMRE ensures that all functional MCU complexes respond appropriately to Ca 2þ stimuli from the cytosol, safeguarding against dangerous Ca 2þ leakage, which could diminish mitochondrial energy output and potentially trigger apoptotic cell death.