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

Abstract

N‑terminal truncated peroxisome proliferator‑activated receptor‑γ coactivator‑1α (NT‑PGC‑1α) is an alternative splice variant of PGC‑1α. NT‑PGC‑1α exhibits stronger anti‑obesity effects in adipose tissue than PGC‑1α; however, NT‑PGC‑1α has not yet been investigated in neonatal rat cardiomyocytes (NRCMs). The present study aimed to investigate the role of NT‑PGC‑1α in mitochondrial fatty acid metabolism and its possible regulatory mechanism in NRCMs. NRCMs were exposed to phenylephrine (PE) or angiotensin II (Ang II) to induce cardiac hypertrophy. Following this, NRCMs were infected with adenovirus expressing NT‑PGC‑1α, and adenosine 5'‑triphsophate (ATP) levels, reactive oxygen species (ROS) generation and mitochondrial membrane potential were subsequently detected. In addition, western blotting, lipid droplet staining and oxygen consumption assays were performed to examine the function of NT‑PGC‑1α in fatty acid metabolism. NT‑PGC‑1α was demonstrated to be primarily expressed in the cytoplasm, which differed from full‑length PGC‑1α, which was predominantly expressed in the nucleus. NT‑PGC‑1α overexpression alleviated mitochondrial function impairment, including ATP generation, ROS production and mitochondrial membrane potential integrity. Furthermore, NT‑PGC‑1α overexpression alleviated the PE‑induced suppression of fatty acid metabolism‑associated protein expression, increased extracellular oxygen consumption and decreased lipid droplet accumulation in NRCMs. Taken together, the present study demonstrated that NT‑PGC‑1α alleviated PE‑induced mitochondrial impairment and decreased lipid droplet accumulation in NRCMs, indicating that NT‑PGC‑1α may have ameliorated mitochondrial energy defects in NRCMs, and may be considered as a potential target for the treatment of heart failure.

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.

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.

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.

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.

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.