SERCA2 and ANF Promoter-Activity Studies in Hypertrophic Cardiomyocytes Using Liposome-, Gene Gun-, and Adenovirus-Mediated Gene Transfer

Abstract

Myocardial hypertrophy is known as the process of enlargement of ventricular cells, which is also accompanied by changes in the phenotype. The latter changes include, e.g., downregulation of the expression of sarcoplasmatic reticulum Ca2+ ATPase (SERCA2), phospholamban (PL), and β-adrenergic receptor and upregulation of the expression of atrial natriuretic factor (ANF) and β-myosin heavy chain (s-MHC). Analysis of the transcriptional regulation of a promoter fragment of the SERCA2 gene using liposome-mediated transfection revealed that the SERCA2 gene may not respond to the general increase in transcription upon stimulation of neonatal rat cardiomyocytes by 10-8M endothelin-1. Liposome-mediated transfection used in these promoter activity studies yields no more than 1% transfection efficiency (the percentage of cells expressing the transgene). To obtain higher efficiency, we set out to develop the gene-gun biolistics method for transfection of cardiomyocytes. An efficiency up to approximately 10% can be achieved by the gene gun as tested using a RSV-β-Gal construct. Here, we demonstrate the efficacy of the method by use of an endothelin-1 inducible ANF promoter fragment coupled to a CAT reporter. Therefore, gene-gun biolistics is ideally suited as a quick and reliable method to test DNA constructs on their activity. The ANF promoter is normally only active to a very low extent in ventricular adult cells; it is upregulated by hypertrophic stimuli. We used the latter property for generating DNA constructs encompassing the antisense PL gene under the control of the endothelin-1 inducible ANF promoter fragment. Adenovirus infection with almost 100% efficiency is required to measure the functional consequences of the overexpressed antisense PL. Here, we present the first results with the ANF-promoter-PL-antisense adenovirus infection of rat neonatal cardiomyocytes.