NLRC5 enhances autophagy via inactivation of AKT/mTOR pathway and ameliorates cardiac hypertrophy

Abstract

The aim of this study was to investigate the effect of nucleotide-binding oligomerization domain (NOD)-like receptor family CARD domain containing 5 (NLRC5) in cardiac hypertrophy, and to explore the mechanism implicated in this effect Cardiac hypertrophy was induced in neonatal rat cardiac myocytes using 1 μM of angiotensin II (Ang II) for 12, 24 and 48 h. Overexpression of NLRC5 was induced in H9C2 cells, and the NLRC5 + Ang II-treated cells were exposed to SC9 and 3-methyladenine (3MA). An immunofluorescence assay was used for α-actinin staining, and quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for NLRC5, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) determination. Western blot analysis was applied to measure the levels of NLRC5, microtubule-associated protein 1A/1B-light chain 3 type I (LC3I), LC3II, sequestosome 1 (p62), protein kinase B (AKT), phosphorylated Akt (pAKT), mammalian target of rapamycin (mTOR) and phosphorylated mTOR (pmTOR). The level of NLRC5 was significantly decreased after Ang II treatment in cardiomyocytes, but the levels of ANP and BNP were increased. Overexpression of NLRC5 reduced the cell size, downregulated the levels of ANP and BNP, increased LC3II / LC3I, but decreased p62 in Ang II-induced cardiomyocyte hypertrophy. In addition, the results from Western blot showed that overexpression of NLRC5 distinctly decreased the ratios of pAKT/AKT and pmTOR/mTOR in cardiomyocyte hypertrophy. SC79 and 3MA significantly downregulated the ratio of LC3I/LC3II but increased the level of p62 in NLRC5 + Ang II-treated cells. These results provide a possible novel therapeutic strategy for cardiac hypertrophy that might be useful in a clinical setting.

Keywords: autophagy; cardiac hypertrophy; nod-like receptor family CARD domain containing 5; protein kinase B/mammalian target of rapamycin pathway.

FIGURE 1

Ang II application represses NLRC5 expression in rat cardiomyocytes. A, The relative expression levels of NLRC5 after 12‐, 24‐ and 48 h Ang II treatment were measured using qRT‐PCR. B, The protein levels of NLRC5 after 12‐, 24‐ and 48 h Ang II treatment were determined by Western blot analysis. C‐D, The relative expression levels of ANP and BNP after 12‐, 24‐ and 48 h Ang II treatment were measured using qRT‐PCR. NLRC5, NOD‐like receptor family CARD domain containing 5; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; Con, control group; Ang II, angiotensin II–treated group. ***p < 0.001, compared with the control group; and ^# p < 0.05 and ^## p < 0.01, compared with the 12‐h Ang II–treated group

FIGURE 2

Overexpression of NLRC5 inhibits Ang II–induced cardiomyocyte hypertrophy. A, The relative expression level of NLRC5 after NLRC5 treatment was determined by qRT‐PCR. B, The protein expression level of NLRC5 after NLRC5 treatment was determined by Western blot analysis. C, The α‐actinin immunostaining was performed by the immunofluorescence assay after Ang II and NLRC5 treatment. D‐E, The transcription levels of ANP and BNP were determined by qRT‐PCR assay after Ang II and NLRC5 treatment. NLRC5, NOD‐like receptor family CARD domain containing 5; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; Ang II, angiotensin II–treated group. ^## p < 0.01, compared with the vector group; and **p < 0.01, compared with the Ang II–treated group

FIGURE 3

Overexpression of NLRC5 promotes autophagy in hypertrophic cardiomyocytes. Autophagy‐related proteins including LC3I, LC3II and p62 were assessed by Western blot analysis after Ang II and NLRC5 treatment. NLRC5, NOD‐like receptor family CARD domain containing 5; LC3, microtubule‐associated protein 1A/1B‐light chain 3; p62, sequestosome 1; Ang II, angiotensin II–treated group. ^## p < 0.01, compared with the vector group; and **p < 0.01, compared with the Ang II–treated group

FIGURE 4

Overexpression of NLRC5 attenuated Ang II–induced cardiomyocyte hypertrophy through inhibiting Akt/mTOR signalling pathway. The protein expression levels of pAKT, AKT, pmTOR and mTOR were determined by Western blot analysis. NLRC5, NOD‐like receptor family CARD domain containing 5; AKT, protein kinase B; pAKT, phosphorylated Akt; mTOR, mammalian target of rapamycin; pmTOR, phosphorylated mTOR; Ang II, angiotensin II–treated group. ^## p <0.01, compared with the vector group; and **p < 0.01, compared with the Ang II–treated group

FIGURE 5

Akt agonist reverses the effect of NLRC5 on cardiac hypertrophy. A, The α‐actinin immunostaining was performed by the immunofluorescence assay after SC79 and 3MA in NLRC5 + Ang II–treated cells. B‐C, The transcription levels of ANP and BNP were determined by qRT‐PCR assay after SC79 and 3MA in NLRC5 + Ang II–treated cells. D, the ratio of LC3II/LC3I and level of p62 were determined by Western blot analysis after SC79 and 3MA in NLRC5 + Ang II–treated cells. NLRC5, NOD‐like receptor family CARD domain containing 5; LC3, microtubule‐associated protein 1A/1B‐light chain 3; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; p62, sequestosome 1; Ang II, angiotensin II–treated group. ^## p < 0.01, compared with the NLRC5 + Ang II–treated group