Figure 4.

NO and cGMP stimulate myogenesis through inhibition of mitochondrial fission. (a, b) Myogenic precursor cells were transfected with the vector coding for either mitoGFP (green) or mitoDsRed (red), mixed, differentiated for 6 h and exposed for 1 h to l-NAME, ODQ, DETA-NO, 8Br-cGMP or vehicle (C) as indicated. Plasma membrane fusion was induced by addition of PEG 1500 and mitochondrial fusion events were quantified after 2, 4 and 6 h in the heteropolykaryons by measuring the fraction of mitochondria simultaneously positive for both mtGFP and mitoDsRed (colocalization index %; n = 3). Bar: 10 μM. (c) Myogenic precursor cells were transfected with vectors coding for the cytosolic marker pEYFP-N1 (green) or the dominant-negative Drp1, pEYFP-N1-DRPK38A, and differentiated in the presence of l-NAME and ODQ. Mitochondrial morphology was assessed after 6 h. Bar: 10 μM. (d) Expression of the myogenic differentiation markers Mef-2A, MyoD, myogenin and sarcomeric myosin (MyHC) was determined by western blotting in myogenic precursors transfected with either the empty pCDNA3 vector or the dominant-negative Drp1 (pcDNA3-Drp1 K38A) at the indicated time points. The result of one out of three reproducible experiments is shown