| Summary: | Objective To investigate the effect of phosphofurin acidic cluster sorting protein 2 (PACS-2) on angiotensin Ⅱ (Ang Ⅱ)-induced cardiac hypertrophy and its mechanism. Methods The heart tissue of neonatal Sprague-Dawley rats aged 1-2 d was clipped and digested, and the differential adhesion method was used to obtain neonatal rat cardiomyocytes (NRCMs), which were primary cultured for 24 h. After NRCMs were treated with 1.5 μmol/L Ang Ⅱ for 0, 3, 6, 12, and 24 h, Western blot was used to measure the protein expression levels of FUN14 domain-containing protein 1 (FUNDC1), PACS-2, and inositol 1,4,5-trisphosphate receptor (IP3R) in cells, and RT-qPCR was used to measure the mRNA expression levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) in cells. NRCMs were divided into groups A-C, group A was cultured with serum-free medium, and groups B and C were transfected with si-NC and si-PACS-2, respectively, Western blot was used to measure the protein expression level of PACS-2 in each group. NRCMs were divided into groups D-G, group D was cultured with serum-free medium, group E was cultured with 0.15 μmol/L Ang Ⅱ, and groups F and G were transfected with si-NC and si-PACS-2, respectively, and were then cultured with 0.15 μmol/L Ang Ⅱ, TRITC-phalloidin staining was used to measure the surface area of cardiomyocytes in each group, RT-qPCR was used to measure the mRNA expression levels of ANP, BNP, and β-MHC in each group, and Fluo-4,AM probe was used to measure the level of cytosolic Ca2+ in each group. NRCMs were divided into groups H-K, group H was cultured with serum-free medium, groups I and J were transfected with si-NC and si-PACS-2, respectively, and group K was transfected with si-PACS-2 and cultured by 1 μmol/L CaM inhibitor; RT-qPCR was used to measure the mRNA expression levels of ANP, BNP, and β-MHC in each group. Results After treatment of NRCMs with Ang Ⅱ at a concentration of 1.5 μmol/L for 0, 3, 6, 12, and 24 h, the mRNA expression levels of ANP, BNP, and β-MHC in NRCMs gradually increased over time (F=25.73-58.30,P<0.05), and the levels at 24 hours of treatment were significantly upregulated compared with those at 0 hour (t=5.35-37.50,P<0.05). After treatment of NRCMs with Ang Ⅱ at a concentration of 1.5 μmol/L for 0, 3, 6, 12, and 24 h, the protein expression levels of IP3R, PACS-2, and FUNDC1 in NRCMs gradually decreased over time (F=5.37-9.07,P<0.05), and the levels at 24 h of treatment were significantly downregulated compared with those at 0 h (t=6.55-7.42,P<0.05). Compared with group B, group C had a significant reduction in the protein expression level of PACS-2 in NRCMs (t=5.92,P<0.05). Compared with group F, group G had significant increases in the surface area of cardiomyocytes, the mRNA expression levels of ANP, BNP, and β-MHC, and the level of cytosolic Ca2+ in NRCMs (t=3.50-26.60,P<0.05). Compared with group J, group K had significant reductions in the mRNA expression levels of ANP, BNP, and β-MHC in NRCMs (t=3.27-5.13,P<0.05). Conclusion Knockdown of PACS-2 can increase the level of cytosolic Ca2+ in NRCMs and aggravate Ang Ⅱ-induced cardiac hypertrophy in a Ca2+-CaM-dependent manner.
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