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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. 7. CaMKIIδB phosphorylated CREB to modulate MCU gene expression.

Figure. 7

A-B, Luciferase activity assay showing the activity of truncated MCU promoter (A, *: P=0.0003, 0.0149 or 0.0414 +ISO vs. −ISO for −1780, −1221 or-1020 groups, respectively) or the MCU promoter fragments that contain mutated sequences of CRE I-IV (B, *: P=0.0131, 0.0053 or 0.022 +ISO vs. −ISO for I, II or III groups, respectively) in H9C2 myoblasts. n=4. C, Chromatin immunoprecipitation assay showing the binding of CRE IV with CREB and CRE IV with CaMKII in adult cardiomyocytes. The binding between RNA polymerase II and a sequence in GAPDH promoter was used as positive control. n=3. D, Electrophoretic mobility shift assay (EMSA) showing the binding between CREB and double-stranded and biotin-labeled oligonucleotides corresponding to CRE IV sequence (Biotin-CRE IV) in vitro, which was blocked by 100 fold unlabeled CRE IV sequence (100xCRE IV) but not by mutated biotin-CRE IV (Mut-CRE IV). E, EMSA showing that addition of CREB antibody caused a shift of the band toward higher molecular weight (arrow head). n=3. F, Effects of KN93 on ISO-induced CREB phosphorylation at S133 in adult mouse cardiomyocytes. n=4. G, Co-IP showing the binding between endogenous CaMKII and CREB in adult mouse cardiomyocytes. Representative of 4 repeats. H, Pull down analysis showing the binding between HA-CaMKIIδB and CREB in vitro. Representative of 3 repeats. I, In vitro phosphorylation assay showing the effects of wild type (δB), S332A, S332E or K43A mutations of CaMKIIδB on CREB phosphorylation at S133. Representative of 3 repeats. J, Effects of dominant negative CREB (DNCREB) on ISO-induced MCU mRNA upregulation in adult mouse cardiomyocytes. *: P=0.0087 LacZ+ISO vs. LacZ−ISO. n=6. K, Effects of DNCREB on HA-δB-induced MCU upregulation. n=4. L, Effects of DNCREB on ISO-induced changes in mitochondrial Ca2+ concentrations ([Ca2+]m, measured by mt-PeriCam. *: P=5.440E-06 LacZ+ISO vs. LacZ−ISO. #: P=0.0003 DNCREB+ISO vs. LacZ+ISO, n=76–93 cells from 4 mice.