The Mitochondrial Calcium Uniporter Selectively Matches Metabolic Output to Acute Contractile Stress in the Heart

Abstract

In the heart, augmented Ca(2+) fluxing drives contractility and ATP generation through mitochondrial Ca(2+) loading. Pathologic mitochondrial Ca(2+) overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca(2+) uptake is primarily mediated by the mitochondrial Ca(2+) uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca(2+) uptake, with impaired ATP production, and inhibited MPTP opening upon acute Ca(2+) challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca(2+) levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca(2+) after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca(2+) loading underlying a "fight-or-flight" response that acutely matches cardiac workload with ATP production.

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

Figure 2. Loss of MCU from the adult heart does not lead to pathology at baseline or with pathologic stress stimulation

(A) Time-course for the analyses of cardiac function in response to aging following Mcu deletion. (B) Transverse H&E heart sections at 200X magnification. (C) Representative electron micrographs from heart sections. Scale bar is 500 nm. (D and F) Heart-weight normalized to body-weight ratios (HW/BW) at 18 and 52 weeks of age. (E and G) Echocardiographic measurement of fractional shortening (FS%) at 18 and 52 weeks of age. (H) Time course for generation of mice and analyses of cardiac function following TAC surgery. (I) H&E-stained transverse heart sections 8 weeks after TAC surgery, at 200X magnification. (J-L) HW/BW, (K) cardiomyocyte cross-sectional area and (L) FS% in the indicated groups of mice 8 weeks following TAC. All values reported as mean ± SEM. *P<0.05 versus Mcu^fl/fl sham

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.

Figure 4. Acute versus chronic regulation of mitochondrial Ca²⁺ and metabolism due to MCU activity

(A) State 3 mitochondrial oxygen consumption in purified cardiac mitochondria from the indicated mice stimulated with 100 μM Ca²⁺ with or without 2 μM Ru360. *P<0.05 versus ADP baseline; #P<0.05 versus Mcu^fl/fl Ca²⁺. All values presented as mean ± SEM. (B) Mitochondrial ATP synthesis in isolated mitochondria at baseline and stimulated with 400 μM CaCl₂. 5 μM Ru360 was used as a control. *P<0.05 versus ADP baseline; #P<0.05 versus Mcu^fl/fl Ca²⁺. All values presented as mean ± SEM. (C) Relative oxygen consumption rates (OCR) of Mcu^fl/fl control adult cardiomyocytes with or without Ru360 (5 μM) in response to 3.125 nM isoproterenol. (D) OCR in Mcu^fl/fl-MCM vs. Mcu^fl/fl adult cardiomyocytes in response to 3.125 nM isoproterenol. *P<0.05 versus control. All values presented as mean ± SEM. (E) Maximal rates of cardiac contraction as measured in a closed chest mouse in response to increasing doses of dobutamine. Dobutamine was increased from 0 to 32 ng/g/min and then maintained at 32 ng/g/min for an additional hour. *P<0.05 versus control. All values presented as mean ± SEM. (F) Total mitochondrial Ca²⁺ content measured from hearts taken at the indicated time-points from indicated groups of mice following dobutamine administration of the experiment shown in E. *P<0.05 versus control. All values presented as mean ± SEM. (G) Treadmill performance as quantified by maximum sprint time in the Mcu^fl/fl-MCM vs. Mcu^fl/fl controls. Animals were subjected to two different protocols where they were allowed either a 2 minute warm-up or a 30 minute warm-up before reaching maximum sprint speed. *P<0.05 versus control. Number of mice used is shown in the bars. All values presented as mean ± SEM. See also Figures S2 and S3.