Figure 4.

Mitochondrial pools of HSP90 regulate pulmonary arterial hypertension–pulmonary artery smooth muscle cell (PAH-PASMC) bioenergetics. (A) The cellular aerobic glycolysis was assessed by measures of the extracellular acidification rate from control and PAH-PASMCs treated or not with Gamitrinib or its vehicle (DMSO) using the Seahorse XF96 analyzer. (B) Effects of Gamitrinib on real-time mitochondrial oxygen consumption rate (OCR). Control PASMCs and PAH-PASMCs exposed or not to Gamitrinib or its vehicle (DMSO) for 48 hours were evaluated after sequential injection of the ATPase synthase inhibitor oligomycin (1 μM), uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (5 μM), complex I inhibitor rotenone (1 μM), and complex III inhibitor antimycin A (1 μM). (C–F) Effects of Gamitrinib on (C) basal OCR, (D) spare respiratory capacity, (E) maximal OCR, and (F) ATP production. Experiments were performed in four control and four PAH-PASMC cell lines. (G) Representative Western blots and corresponding densitometric analyses of succinate dehydrogenase complex, subunit B, Survivin, phospho–pyruvate dehydrogenase, pyruvate dehydrogenase (PDH), and PDH kinases-1 and -2 expression in PAH-PASMCs (n = 6) treated or not with Gamitrinib (2 μM) or its vehicle (DMSO) for 48 hours. Protein expression was normalized to Amido black. Data are presented as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. AB = Amido black; CTRL = control; ECAR = extracellular acidification rate; FCCP = carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; Gami = Gamitrinib; NT = nontreated; PDK = pyruvate dehydrogenase kinase; P-PDH = phospho–pyruvate dehydrogenase; SDHB = succinate dehydrogenase complex, subunit B; Veh = vehicle.