Recombinant Sindbis virus allows expression and precise targeting of proteins of the contractile apparatus in cultured cardiomyocytes

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• 作者：Daniel A. D?twyler ; Josef P. Magyar ; Vincenzo Busceti ; Alain Hirschy ; Jean-Claude Perriard ; James E. Bailey and Hans M. Eppenberger
• 关键词：Key words Cultured cardiomyocytes – Sindbis virus – gene transfer – contractile proteins – ectopic expression
• 刊名：Basic Research in Cardiology
• 出版年：2001
• 出版时间：December 2001
• 年：2001
• 卷：96
• 期：6
• 页码：630-635
• 全文大小：240 KB
• 刊物类别：Medicine
• 刊物主题：Medicine & Public Health
  Cardiology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1435-1803

文摘

Expression of epitope-tagged sarcomeric proteins in cardiomyocytes is a powerful approach for the characterization of interacting domains. Here, we report a new strategy for the study of the targeting of contractile proteins in cardiomyocytes by Sindbis virus (SIN)-mediated gene transfer. Two recombinant SIN were generated, one encoding the myosin-light chain MLC3f-eGFP fusion protein (SINrep5/MLC3f-eGFP), and the other encoding the α-actinin-DsRed fusion protein (SINrep5/α-actinin-DsRed). After infection of long-term cultured neonatal and adult rat cardiomyocytes with SINrep5/MLC3f-eGFP, the exogenous MLC3f-eGFP fusion protein localized to the sarcomeres. Freshly isolated rod-shaped ventricular cardiomyocytes infected with SINrep5/α-actinin-DsRed exhibited a correct incorporation of the newly synthesized α-actinin-DsRed fusion protein at the Z-band of the sarcomere. This allows the assumption that the exogenous protein is assembled into myofibrils in living cardiomyocytes using the same molecular interactions equally to the endogenous counterpart. It has been thus demonstrated that the SIN expression system makes possible the straightforward analysis of the localization of sarcomeric proteins in cultured cardiomyocytes and may offer new possibilities for the characterization of mutant proteins involved in hypertrophic cardiomyopathies.