RESUMO
Recent remarkable studies have reported that clonogenic putative cardiac stem cells (CSCs) with cardiomyogenic potential migrate from heart tissue biopsies during ex vivo culture, and that these CSCs self-organize into spontaneously beating cardiospheres (CSs). Such data have provided clear promise that injured heart tissue may be repaired by stem cell therapy using autologous CS-derived cells. By further examining CSs from the original CS protocol using immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and microscopic analysis, we here report a more mundane result: that spontaneously beating CSs from neonatal rats likely consist of contaminating myocardial tissue fragments. Thus, filtering away these tissue fragments resulted in CSs without cardiomyogenic potential. Similar data were obtained with CSs derived from neonatal mice as wells as adult rats/mice. Additionally, using in vitro culture, fluorescence-activated cell sorting, and immunofluorescence, we demonstrate that these CSs are generated by cellular aggregation of GATA-4(+)/collagen I(+)/alpha-smooth muscle actin (SMA)(+)/CD45(-) cells rather than by clonal cell growth. In contrast, we found that the previously proposed CS-forming cells, dubbed phase bright cells, were GATA-4(-)/collagen I(-)/alpha-SMA(-)/CD45(+) and unable to form CSs by themselves. Phenotypically, the CS cells largely resembled fibroblasts, and they lacked cardiomyogenic as well as endothelial differentiation potential. Our data imply that the murine CS model is unsuitable as a source of CSCs with cardiomyogenic potential, a result that is in contrast to previously published data. We therefore suggest, that human CSs should be further characterized with respect to phenotype and differentiation potential before initiating human trials.
Assuntos
Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND PURPOSE: The angiotensin II type 1 receptor (AT(1)R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we determined whether miRNAs might be regulated by AT(1)R signals in a Gαq/11-dependent or -independent manner. EXPERIMENTAL APPROACH: We performed a global miRNA array analysis of angiotensin II (Ang II)-mediated miRNA regulation in HEK293N cells overexpressing the AT(1)R and focused on separating the role of Gαq/11-dependent and -independent pathways. MiRNA regulation was verified with quantitative PCR in both HEK293N cells and primary cardiac myocytes and fibroblasts. KEY RESULTS: Our studies revealed five miRNAs (miR-29b, -129-3p, -132, -132* and -212) that were up-regulated by Ang II in HEK293N cells. In contrast, the biased Ang II analogue, [Sar1, Ile4, Ile8] Ang II (SII Ang II), which selectively activates Gαq/11-independent signalling, failed to regulate miRNAs in HEK293N cells. Furthermore, Ang II-induced miRNA regulation was blocked following Gαq/11 and Mek1 inhibition. The observed Ang II regulation of miRNA was confirmed in primary cultures of adult cardiac fibroblasts. Interestingly, Ang II did not regulate miRNA expression in cardiac myocytes, but SII Ang II significantly down-regulated miR-129-3p. CONCLUSIONS AND IMPLICATIONS: Five miRNAs were regulated by Ang II through mechanisms depending on Gαq/11 and Erk1/2 activation. These miRNAs may be involved in Ang II-mediated cardiac biology and disease, as several of these miRNAs have previously been associated with cardiovascular disease and were found to be regulated in cardiac cells.