Background:
Mitochondria take up Ca²⁺ primarily via a Ca²⁺ uniporter (MCU). In mitochondria, Ca²⁺ is required to increase Krebs cycle activity and NADH formation to match energy supply to increased demands, but also to booster the antioxidative capacity through Krebs cycle-dependent NADPH regeneration. Mice lacking a functional MCU are viable, and the heart does not show gross pathology. Therefore, alternative pathways to allow Ca²⁺ entry into mitochondria may exist. The Transmembrane Bax Inhibitor motif -containing protein 5 (TMBIM5) is a pH-dependent Ca²⁺ leak channel that localizes to mitochondria. Our previous studies in HAP1 indicated that its knockout reduces mitochondrial membrane potential, respiration, ATP generation and causes release of pro-apoptotic proteins from the mitochondria. To determine the role of TMIBM5 for mitochondrial Ca²⁺ uptake, function and regulation of redox state in cardiac myocytes, we generated a mouse model with a global, specific mutation in the pore-forming unit of the TMBIM5 protein (D326R) with the Crispr/Cas9-system. The analogous mutation abrogates function in a prokaryotic orthologue of TMBIM5.

Methods and results:
In isolated mitochondria, uptake of Ca²⁺ into mitochondria upon repetitive pulses of extra-mitochondrial Ca²⁺ was unchanged in D326R vs. WT mice. Isolated cardiac myocytes from 20 weeks old D326R mice and wild-type littermates (WT) were exposed to a physiological stress protocol by pacing at 0.5 Hz followed by β-adrenergic stimulation (isoproterenol, 30 nM) and increased stimulation rate at 5 Hz for 3 minutes. Diastolic and systolic sarcomere length and fractional shortening (WT, n=82; D326R, n=73) were unchanged, while diastolic and systolic [Ca²⁺] and Ca²⁺ transient amplitudes (determined by Indo1-AM) were increased with unchanged kinetics in D326R vs. WT myocytes (n=91/80). While the mitochondrial membrane potential (determined by TMRM; WT, n=130; D326R, n=110) was unchanged, the mitochondrial redox states of NAD(P)H/NAD(P)⁺ and FADH₂/FAD were slightly more reduced in D326R vs. WT myocytes (n=45/n=40). While reactive oxygen species (ROS) production (measured with DCF) during the stress protocol was unchanged, the DCF response to external H₂O₂ (1 mM) in D326R (n=43) was only ~60% of that in WT (n=82), indicating a higher antioxidative capacity.

Conclusions:
While TMBIM5 does not contribute to rapid mitochondrial Ca²⁺ uptake and energy supply-and-demand matching in working cardiac myocytes, inactivating the pore-forming unit of TMBIM5 appears to elicit a more reduced redox environment in cardiac mitochondria, augmenting antioxidative capacity. Further studies will have to elucidate the underlying mechanisms of this rather adaptive response and the consequences for excitation-contraction coupling, where force generation requires higher cytosolic Ca²⁺ concentrations.