Impaired Ang II mediated activation of ERK1/ERK2 and SAPK/JNK pathways with normal AT1 receptor binding and mRNA expression in myocytes from failing human hearts

Background: Recent studies demonstrated that cardiac angiotensin II (Ang II) type 1 (AT1) receptors are downregulated in human failing ventricles but characteristics of Ang II receptors on human myocytes are to be defined. Methods: Ang II receptors and Ang II mediated activation of intracellular pathways were investigated on enzymatically dissociated ventricular myocytes from 6 potential transplant donors (NF) and 17 heart failure (HF) patients who underwent cardiac transplantation. Ang II receptors were characterized using 125I-Ang II (2,000 Ci/mmol) as radioligand and Ang II or valsartan and PD123319 as cold displacers. mRNA for AT1 receptor was quantified with RT-PCR using specific primers and GAPDH as internal control. AT1 mediated ERK1/ERK2 and SAPK/JNK activation following Ang II stimulation (10-8M) was investigated in isolated myocytes using selective receptor antagonists. Results: 125I-Ang-II binding to membranes from homogenated failing hearts revealed a significant downregulation of both total Ang II receptors (2.4±0.5 vs 5.1±0.7 fmol/mg protein, in HF and NF hearts respectively, p<0.01) and AT1 subtype (p<0.01) with unchanged affinity. Conversely, isolated cardiomyocytes showed no differences in density of both total Ang II receptor (0.58±0.10 vs 0.57±0.21 fmol/mg protein, ns) and AT1 subtype. Also at RT-PCR studies, AT1 mRNA levels were not significantly different between HF and NF isolated myocytes. However, AT1 mediated activation of both ERK1/ERK2 and SAPK/JNK in HF myocytes was reduced by 88% and 48% respectively, when compared to control cells (p<0.01 for both). Conclusion: In conclusion AT1 receptors are not downregulated in human ventricular myocytes from failing hearts but show a blunted functional response to Ang II.