Figure 3. MCU is required for acute mitochondrial Ca²⁺ stress signaling.

(A) Time-course for the cardiac ischemia-reperfusion experiment.

(B) Representative images of transverse heart sections stained with 2,3,5-triphenyltetrazolium chloride following ischemia-reperfusion injury from the indicated groups. Ischemic area is outlined in yellow.

(C) Quantification of the ischemic area versus area at risk. *P< 0.05 versus all other groups. All values presented as mean ± SEM.

(D) Cardiomyocyte viability in response to ionomycin treatment (625 nM, 24h). *P<0.05 versus control. All values presented as mean ± SEM.

(E) Mitochondrial swelling in response to Ca²⁺ challenge (200 μM CaCl₂). Controls were 5 μM cyclosporine A (CsA) and 2 μM Ru360. The red arrows show the critical experimental group where swelling is inhibited with Mcu deletion alone.

(F) Quantification of MPTP opening frequency in permeabilized cardiomyocytes. MPTP opening is measured by loss of mitochondrial membrane potential (TMRM signal; inset).

Myocytes were challenged with 100 nM free Ca²⁺ and 1 μM CsA was used as a control. *P< 0.005 versus control. All values presented as mean ± SEM.

(G) Western blots of CypD, VDAC, and ANT protein expression in purified cardiac mitochondria from the indicated genotypes of mice.