Figure 1. Cardiomyocyte-specific deletion of Mcu impairs mitochondrial Ca²⁺uptake.

(A) Targeting strategy for the Mcu locus to generate the Mcu^fl/fl mice where exons 5 and 6 were flanked with LoxP sites (triangles). Mcu^fl/fl mice were crossed to α-MHC MerCreMer (MCM) mice to generate the Mcu^fl/fl-MCM animals.

(B) Tamoxifen dosing to induce MerCreMer activity was given to 8 week-old animals for 4 weeks, followed by examination at 18 and 52 weeks of age.

(C) Western blots of MCU and mNCX expression in cardiac mitochondria. The COXI subunit of mitochondrial Complex IV was used as a protein loading control.

(D) Quantification of Ca²⁺ content from isolated cardiac mitochondria from the indicated genotypes of mice.

(E) Quantification of baseline mitochondrial Ca²⁺ content in permeabilized myocytes from the indicated genotypes of mice.

(F) The effect of Ru360 (1 μM) on mitochondrial Ca²⁺ uptake as measured by calcium-green 5N fluorescence in the solution. Mitochondria were challenged with 100 μM CaCl₂ additions (arrows).

(G) Mitochondrial Ca²⁺ uptake in mitochondria from hearts of Mcu^fl/fl vs. Mcu^fll/fl-MCM mice. Mitochondria were challenged with 200 μM CaCl₂ additions (arrows).

(H) Measurement of mitochondrial Ca²⁺ uptake in permeabilized myocytes as assessed by Rhod-2 fluorescence in the indicated groups of mice, with or without Ru360.

(I) Quantification of Rhod-2 signal 14 min after Ca²⁺ addition as shown in H. *P<0.05 vs Mcu^fl/fl. All values reported as mean ± SEM.

(J) Measurement of mitochondrial Ca²⁺ efflux as mediated by mNCX and leak, assessed by Rhod-2 fluorescence in adult cardiomyocytes.

(K) Quantification of rates of mNCX Ca²⁺ efflux as shown in J. All values reported as mean ± SEM. *P<0.05 versus Mcu^fl/fl

See also Figure S1