L, Immunofluorescence images showing cTnT+ cardiomyocytes incubated with Con and CsA (2 g/mL). cardiac regeneration. The mechanisms of mitochondrial metabolic and redox regulation for efficient cardiomyocyte differentiation are, however, still poorly understood. Here, we show that inhibition of the mitochondrial permeability transition pore (mPTP) by Cyclosporin A (CsA) promotes cardiomyocyte differentiation from PSCs. Methods and Results We induced cardiomyocyte differentiation from mouse and human PSCs and examined the effect of CsA on the AM-2099 differentiation process. The cardiomyogenic effect of CsA mainly resulted from mPTP inhibition rather than from calcineurin inhibition. The mPTP inhibitor NIM811, which does not have an inhibitory effect on calcineurin, promoted cardiomyocyte differentiation as much as CsA did, but calcineurin inhibitor FK506 only slightly increased cardiomyocyte differentiation. CsA\treated cells showed an increase in mitochondrial calcium, mitochondrial membrane potential, oxygen consumption rate, ATP level, and expression Rabbit Polyclonal to ELOVL1 of genes related to mitochondrial function. Furthermore, inhibition of mitochondrial oxidative metabolism reduced the cardiomyogenic AM-2099 effect of CsA while antioxidant treatment augmented the cardiomyogenic effect of CsA. Conclusions Our data show that mPTP inhibition by CsA alters mitochondrial oxidative metabolism and redox signaling, which leads to differentiation of functional cardiomyocytes from PSCs. test or analysis of variance with 1\way ANOVA followed by the Student\Newman\Keuls test. The Mann\Whitney test and Kruskal\ Wallis ANOVA were performed when data were not normally distributed. Statistical significance was set at and cardiomyocyte differentiation (nkx2.5and in the differentiating Flk1+ MPCs. The expression levels of all these genes were variably up\regulated by CsA compared with control vehicle (Figure 4I), suggesting that CsA regulates the cardiomyogenic effect by alterations of the gene transcriptions related to mitochondrial function. Observation of individual cardiomyocytes after FACS sorting using MHC\GFP revealed that the shape, beating rate, mitochondria content, and cTnT+ sarcomere structure of each cardiomyocyte was quite varied (Video S5, Figure 5A). Interestingly, cardiomyocytes with loosely organized sarcomere structure also showed less developed, fragmented mitochondria that are located in the perinuclear region (Figure 5A\1 and ?and5B\1),5B\1), while cardiomyocytes with densely organized sarcomere structure contained well\developed, elongated mitochondria which are distributed along the sarcomere (Figures ?(Figures5A\25A\2 and ?and5B\2).5B\2). These data indicate that mitochondrial development and function are closely related to cardiomyogenesis. Open in a separate window Figure 5. Mitochondrial development and function are closely related to cardiomyogensis. A, Immunofluorescence images showing Mitotracker+ and cTnT+ cardiomyocytes and DAPI+ nuclei in sorted MHC\GFP+ cardiomyocytes at day 11.5. 1 and 2 are magnified views of square\dotted area in the left AM-2099 side. Scale bar represent 100 m in the left side and 50 m in the right side. B, Co\localized signals of Mitotracker+ and cTnT+ in reconstructed image of (A). cTnT indicates cardiac Troponin T; DAPI, 4,6\diamidino\2\phenylindole; GFP, green fluorescent protein; MHC, myosin heavy chain. To confirm whether the effect of CsA to mitochondria in differentiating Flk1+ MPCs is a direct effect rather than a secondary effect due to cardiomyogenesis, we treated CsA to H9C2 cardiac cell line (Figure 6A). Consistent with the data from differentiating Flk1+ MPCs, CsA increased the mean fluorescence intensity of Calcein AM (4.0\fold), mitochondrial Ca2+ (1.5\fold) and m (1.8\fold) compared with control vehicle in FACS analysis (Figures ?(Figures6B6B through ?through6D).6D). CsA also increased the fluorescence of Calcein AM, TMRM and Mitotracker in live cell and immunofluorescence images (Figures ?(Figures6E6E and ?and6F).6F). Additionally, electron microscope images revealed that CsA increased the mitochondrial size (1.7\fold) and yielded more matured cristae structure (Figures ?(Figures7A7A and ?and7B).7B). These data suggest that an increase of mitochondrial function is a direct effect of CsA rather than a secondary effect due to cardiomyogenesis in differentiating Flk1+ MPCs. Open in a separate window Figure 6. Inhibition of mPTP by CsA directly increases mitochondrial function in H9C2 cardiac cell line. A, Protocol for proliferation and differentiation of H9C2 cells. H9C2 cells were incubated for 2 days in growth medium and then incubated with control vehicle (Con) and CsA (1 g/mL) in differentiation medium for 4 days. B, Representative FACS analysis of Calcein AM fluorescence intensity and the percentage of AM-2099 relative mean fluorescence intensity of Calcein AM. C, Representative FACS analysis.