Expression and regulation of angiotensinogen mRNA by quantitative reverse transcription-polymerase chain reaction analysis in cultured cardiomyocytes

Background. The local renin-angiotensin system (RAS) is functionally significant in cardiac hypertrophy. However, it is difficult to detect angiotensinogen mRNA in the rat ventricle by conventional methods. The aim of the study was to measure angiotensinogen mRNA in cultured cardiomyocytes, following cyclical mechanical stretch by reverse transcription-polymerase chain reaction (RT-PCR). Methods and Results. Cultured rat cardiocytes, grown on a flexible membrane base, were deformed by vacuum to 20% of their maximal elongation, at 60 cycles/min in a serum-free medium. To examine the expression of the angiotensinogen gene, total RNA was isolated from cultured cardiocytes and 2 μg of total RNA was reverse-transcribed; the resulting cDNA was coamplified by PCR, using specific primers for angiotensinogen and for GAPDH as an internal control. The specificity of PCR products obtained was confirmed by Southern hybridization. Angiotensinogen gene levels in stretched cells increased 1.9-, 2.9-, 3.9- and 4.8-fold (p <0.01, versus control) after 3, 6, 12, and 24 hours of stretch, respectively. Conclusion. This study demonstrated that cyclical mechanical stretch upregulates the expression of angiotensinogen mRNA, and quantitative RT-PCR is a powerful approach for detection of the expression and regulation of the local RAS gene in cultured cardiomyocytes.