shRNA-mediated NP knockdown inhibits the apoptosis of cardiomyocytes induced by H1N1pdm2009 influenza virus

摘要

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.