N‑terminal truncated peroxisome proliferator‑activated receptor‑γ coactivator‑1α alleviates phenylephrine‑induced mitochondrial dysfunction and decreases lipid droplet accumulation in neonatal rat cardiomyocytes

Figures

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  Figure 1.. Subcellular localization of NT-PGC-1α. (A) NRCMs were infected with mCherry-NT-PGC-1α adenovirus and observed by confocal microscopy. NT-PGC-1α was primarily located in the cytoplasm. (B) Immunofluorescence staining revealed the different distribution patterns for endogenous total PGC-1α and NT-PGC-1α. (C) Western blot analysis of nuclear and cytoplasmic fractions demonstrated the subcellular location of endogenous PGC-1α and NT-PGC-1α expression. Experiments were repeated three times. NRCMs, neonatal rat cardiomyocytes; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; NT, N-terminal truncated.

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  Figure 2.. Effect of NT-PGC-1α overexpression on MMP, ATP and ROS generation in cells infected with NT-PGC-1a or control adenovirus and exposed to PE or Ang II. (A) Intracellular ATP levels (n=7–8 per group). (B) MMP was detected using the JC-1 assay and (C) the ratio of red/green fluorescence was calculated (n=12 per group). (D) ROS generation was measured by DCFH-DA fluorescence and (E) relative fluorescence levels were calculated (n=16 per group). *P<0.05 and **P<0.01, as indicated. AdV, adenovirus; NT-PGC-1α, N-terminal truncated peroxisome proliferator-activated receptor-γ coactivator-1α; MMP, mitochondrial membrane potential; ATP, adenosine 5′-triphosphate; ROS, reactive oxygen species; DCFH-DA, dichlorofluorescin diacetate.

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  Figure 3.. NT-PGC-1α overexpression increases fatty acid metabolism-associated gene expression. (A) NT-PGC-1α overexpression in NRCMs induced by adenoviral infection (n=6–8 per group). (B) Effect of NT-PGC-1α overexpression on the expression of downstream target genes, including enzymes involved in fatty acid metabolism and anti-oxidant enzymes (n=6–8 per group). (C) Alterations in the expression of PGC-1a, NT-PGC-1α, CPT-2, PPAR-α, Acadm and SOD2 in response to treatment with MK886, a PPAR-α inhibitor. (D) Quantification of western blot results by densitometry (n=3–5 per group). (E-G) Effect of NT-PGC-1α overexpression, PE and MK886 (500 nM) on fatty acid metabolism-associated protein expression (n=5 per group). *P<0.05 and **P<0.01, as indicated or compared with the control. AdV, adenovirus; NT, N-terminal truncated; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; CPT-2, carnitine palmitoyltransferase 2; PPAR-α, peroxisome proliferator-activated receptor α; Acadm, medium-chain specific acyl-coenzyme A dehydrogenase, mitochondrial; SOD2, superoxide dismutase 2; PE, phenylephrine; ns, not significant.

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  Figure 4.. NT-PGC-1α overexpression reduces LD accumulation in NRCMs. Representative images of LD staining of NRCMs cultured in high lipid medium from (A) the BSA+vehicle group, (B) OA-BSA+vehicle group, (C) OA-BSA+AdV-NT-PGC-1α group and (D) OA-BSA+AdV-NT-PGC-1α+MK886 group. (E) NT-PGC-1α overexpression reduced LD accumulation in NRCMs. The number of LDs in each group was counted. **P<0.01, as indicated. BSA+vehicle, n=27; OA-BSA+vehicle, n=30; OA-BSA+AdV-NT-PGC-1α, n=50 and OA-BSA+AdV-NT-PGC-1α+MK886, n=31. AdV, adenovirus; NT-PGC-1α, N-terminal truncated peroxisome proliferator-activated receptor-γ coactivator-1α; LDs, lipid droplets; NRCMs, neonatal rat cardiomyocytes; OA, oleic acid; BSA, bovine serum albumin.

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  Figure 5.. NT-PGC-1α overexpression in NRCMs increases extracellular oxygen consumption. (A) Variations in oxygen consumption in response to different stimuli. (B) Oxygen consumption following 1 h compared with the baseline (n=7–8 per group). *P<0.05 and **P<0.01, as indicated. NT-PGC-1α, N-terminal truncated peroxisome proliferator-activated receptor-γ coactivator-1α; NRCMs, neonatal rat cardiomyocytes; PE, phenylephrine; AdV, adenovirus; F, fluorescence.