Department of Cardiology, Huai'An First People's Hospital, Nanjing Medical University, Huai'an, Jiangsu 223300, P.R. China.
Department of Breast and Thyroid, Huai'An First People's Hospital, Nanjing Medical University, Huai'an, Jiangsu 223300, P.R. China.
Mol Med Rep. 2017 Aug;16(2):1376-1382. doi: 10.3892/mmr.2017.6728. Epub 2017 Jun 8.
Acute influenza-associated myocarditis varies in clinical severity ranging between asymptomatic and fulminant varieties. The most severe cases can result in impaired cardiac function‑associated mortality; however, the mechanism underlying the development of viral myocarditis has yet to be fully elucidated. The present study investigated the apoptosis induced in H9C2 cardiomyocytes by infection with the H1N1pdm2009 virus. The H9C2 cells were transfected with nucleoprotein (NP)‑specific short hairpin (sh) RNA, and viral replication was re‑evaluated in H9C2 cells infected with the H1N1pdm2009 virus, as was the apoptosis induced by the virus. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were performed to measure the expression of NP and apoptosis‑associated molecules. A plaque forming assay was used to quantify viral replication in H9C2 cells. An MTT assay and flow cytometric analysis were performed to determine the virus‑associated alterations in cellular viability and apoptosis, respectively. Results demonstrated that the H1N1pdm2009 virus replicated effectively in H9C2 cells and promoted apoptosis in association with the viral infection. The expression levels of apoptosis‑associated markers, including released cytochrome c and activated caspase‑3 were markedly promoted in the H1N1pdm2009‑infected H9C2 cells. However, the NP‑specific shRNA‑mediated NP knockdown significantly inhibited viral infection in the cells. The virus‑induced apoptosis of the H9C2 cells was also significantly reduced by the shRNA, which occurred via a decrease in the number of apoptotic cells through downregulating the levels of apoptosis‑associated markers. Taken together, the present study demonstrated the key pathogenic role of NP in H1N1pdm2009‑induced apoptosis of cardiomyocytes, and this marker of the influenza virus may be important in influenza virus‑associated acute myocarditis. In addition, NP‑specific shRNA may be an effective agent for inhibiting influenza virus‑induced apoptosis in cardiomyocytes or in influenza virus‑associated acute myocarditis.
甲型 H1N1 流感相关性心肌炎的临床严重程度不同，可表现为无症状和暴发性。最严重的情况可导致心功能障碍相关死亡率升高；然而，病毒心肌炎发展的机制尚未完全阐明。本研究探讨了甲型 H1N1 流感病毒感染对 H9C2 心肌细胞凋亡的影响。将 H9C2 细胞转染核蛋白（NP）特异性短发夹（sh）RNA，评估 H9C2 细胞感染甲型 H1N1 流感病毒后病毒复制情况以及病毒诱导的细胞凋亡情况。采用反转录-定量聚合酶链反应和 Western blot 分析检测 NP 及凋亡相关分子的表达。采用空斑形成试验检测 H9C2 细胞中病毒复制情况。MTT 试验和流式细胞术分别用于检测细胞活力和凋亡相关的病毒诱导变化。结果表明，甲型 H1N1 流感病毒可在 H9C2 细胞中有效复制，并与病毒感染相关促进细胞凋亡。甲型 H1N1 流感病毒感染的 H9C2 细胞中，凋亡相关标志物，包括释放的细胞色素 c 和活化的 caspase-3 的表达水平明显上调。然而，NP 特异性 shRNA 介导的 NP 敲低可显著抑制细胞中的病毒感染。shRNA 还可显著降低病毒诱导的 H9C2 细胞凋亡，这是通过下调凋亡相关标志物的水平减少凋亡细胞数量而实现的。综上所述，本研究表明 NP 在甲型 H1N1 流感病毒诱导的心肌细胞凋亡中起关键致病作用，该流感病毒标志物可能在流感病毒相关性急性心肌炎中起重要作用。此外，NP 特异性 shRNA 可能是抑制心肌细胞中流感病毒诱导的凋亡或流感病毒相关性急性心肌炎的有效药物。
