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. Author manuscript; available in PMC: 2023 Apr 5.
Published in final edited form as: Circulation. 2022 Feb 15;145(14):1067–1083. doi: 10.1161/CIRCULATIONAHA.121.055841

Figure. 3. MCU modulated Ca2+ and energy homeostasis in rat cardiomyocytes during chronic ISO incubation.

Figure. 3

A-D, Mitochondrial Ca2+ concentrations (A, [Ca2+]m, measured by mt-PeriCam, *: P=0.0013 LacZ+ISO vs. LacZ−ISO, #: P=2.979E-05 DNMCU−ISO vs. LacZ−ISO, #: P=2E-15 DNMCU+ISO vs. LacZ+ISO, n=48–71 cells from 5–7 rats), cytosolic Ca2+ concentrations (B, [Ca2+]c, measured by Twitch2B, *: P=8.32E-12 DNMCU+ISO vs. DNMCU−ISO, #: P=2.71E-23 DNMCU+ISO vs. LacZ+ISO, n=36–69 cells from 4–7 rats), SR Ca2+ content (C, [Ca2+]SR, measured by D1ER, #: P=0.0121 DNMCU−ISO vs. LacZ−ISO, *: P=0.0155 DNMCU+ISO vs. DNMCU−ISO, n=33–41 cells from 4 rats), and the decay rate of pacing-induced cytosolic Ca2+ transients (D, measured by GCaMP6f, *: P=0.0202 LacZ+ISO vs. LacZ−ISO, #: P=0.0113 DNMCU−ISO vs. LacZ−ISO, *: P=0.0002 DNMCU+ISO vs. DNMCU−ISO, #: P=0.0003 DNMCU+ISO vs. LacZ+ISO, n=33–60 cells from 5 rats) in adult rat cardiomyocytes overexpressing dominant negative MCU (DNMCU) and incubated with ISO (0.1 μmol/L, 24 hr). E, Western blotting images showing the protein levels of overexpressed flag-tagged MCU in adult rat cardiomyocytes. F-H, [Ca2+]m, [Ca2+]c, and [Ca2+]SR in adult rat cardiomyocytes overexpressing MCU and with ISO (0.1 μmol/L, 24 hr) incubation. In F, *: P=3.86E-06 LacZ+ISO vs. LacZ−ISO, #: P=0.0964 MCU+ISO vs. LacZ+ISO, n=48–71 cells from 5–7 rats. In G, #: P=1.5E-05 MCU−ISO vs. LacZ−ISO, *: P=8.9E-08 MCU+ISO vs. MCU−ISO, #: P=3.27E-16 MCU+ISO vs. LacZ+ISO, n=36–69 cells from 4–7 rats. In H, #: P=0.0238 MCU−ISO vs. LacZ−ISO, #: P=0.0006 MCU+ISO vs. LacZ+ISO, n=33–41 cells from 4 rats. I-J, Calpain 1/2 activity in adult rat cardiomyocytes. In I, *: P=8.4E-10 DNMCU+ISO vs. DNMCU−ISO, #: P=2.70E-10 DNMCU+ISO vs. mGFP+ISO. n=4. For J, n=6. K-N, Cleaved caspase 3 (K, M) and caspase 3/7 activity (L, N) in adult rat cardiomyocytes. n=3 for K and M. In L, *: P=1.13E-07 mGFP+ISO vs. mGFP−ISO, *: P=2.44E-09 DNMCU+ISO vs. DNMCU−ISO, #: P=6.89E-09 DNMCU+ISO vs. mGFP+ISO, n=4. In N, *: P=0.0077 mGFP+ISO vs. mGFP−ISO, #: P=0.0232 MCU+ISO vs. mGFP+ISO, n=6. O, Cytosolic ATP levels measured by ATeam in adult rat cardiomyocytes. *: P=0.0048 LacZ+ISO vs. LacZ−ISO. n=51–53 cells from 5 rats. For A-O, LacZ or mGFP was expressed in Control cells. P, Representative confocal images and summarized data showing mitochondrial ATP measurements in adult rat cardiomyocytes overexpressing MCU-ATeam or DNMCU-ATeam and with ISO incubation. *: P=0.003 MCU-ATeam +ISO vs. MCU-ATeam −ISO. n=23–30 cells from 4–6 rats.