ß-Adrenergic signaling drives structural and functional maturation of mouse cardiomyocytes.

Cardiac maturation represents the last phase of heart development and is characterized by morphofunctional alterations that optimize the heart for efficient pumping. Its understanding provides important insights into cardiac regeneration therapies. Recent evidence implies that adrenergic signals are involved in the regulation of cardiac maturation, but the mechanistic underpinnings involved in this process are poorly understood. Herein, we explored the role of ß-adrenergic receptor (ß-AR) activation in determining structural and functional components of cardiomyocyte maturation. Temporal characterization of tyrosine hydroxylase and norepinephrine levels in the mouse heart revealed that sympathetic innervation develops during the first 3 wk of life, concurrent with the rise in ß-AR expression. To assess the impact of adrenergic inhibition on maturation, we treated mice with propranolol, isolated cardiomyocytes, and evaluated morphofunctional parameters. Propranolol treatment reduced heart weight, cardiomyocyte size, and cellular shortening, while it increased the pool of mononucleated myocytes, resulting in impaired maturation. No changes in t-tubules were observed in cells from propranolol mice. To establish a causal link between ß-AR signaling and cardiomyocyte maturation, mice were subjected to sympathectomy, followed or not by restoration with isoproterenol treatment. Cardiomyocytes from sympathectomyzed mice recapitulated the salient immaturity features of propranolol-treated mice, with the additional loss of t-tubules. Isoproterenol rescued the maturation deficits induced by sympathectomy, except for the t-tubule alterations. Our study identifies the ß-AR stimuli as a maturation promoting signal and implies that this pathway can be modulated to improve cardiac regeneration therapies.NEW & NOTEWORTHY Maturation involves a series of morphofunctional alterations vital to heart development. Its regulatory mechanisms are only now being unveiled. Evidence implies that adrenergic signaling regulates cardiac maturation, but the mechanisms are poorly understood. To address this point, we blocked ß-ARs or performed sympathectomy followed by rescue experiments with isoproterenol in neonatal mice. Our study identifies the ß-AR stimuli as a maturation signal for cardiomyocytes and highlights the importance of this pathway in cardiac regeneration therapies.

Time-of-day affects MrgD-dependent modulation of cardiomyocyte contractility.

The renin-angiotensin system (RAS) is comprised of a series of peptides, receptors, and enzymes that play a pivotal role in maintaining cardiovascular homeostasis. Among the most important players in this system are the Angiotensin-II and Angiotensin-(1-7) peptides. Our group has recently demonstrated that alamandine (ALA), a peptide with structural and functional similarities to Angiotensin-(1-7), interacts with cardiomyocytes, enhancing contractility via the Mas-related G protein-coupled receptor member D (MrgD). It is currently unknown whether this modulation varies along the distinct phases of the day. To address this issue, we assessed the ALA-induced contractility response of cardiomyocytes from mice at four Zeitgeber times (ZTs). At ZT2 (light phase), ALA enhanced cardiomyocyte shortening in an MrgD receptor-dependent manner, which was associated with NO production. At ZT14 (dark phase), ALA induced a negative modulation on the cardiomyocyte contraction. ß-Alanine, an MrgD agonist, reproduced the time-of-day effects of ALA on myocyte shortening. L-NG-Nitroarginine Methyl Ester (L-NAME), an NO synthase inhibitor, blocked the increase in fractional shortening induced by ALA at ZT2. No effect of ALA on myocyte shortening was observed at ZTs 8 and 20. Our results show that ALA/MrgD signaling in cardiomyocytes is subject to temporal modulation. This finding has significant implications for pharmacological approaches that combine chronotherapy for cardiac conditions triggered by disruption of circadian rhythms and hormonal signaling.

Transcription factor SOX3 upregulated pro-apoptotic genes expression in human breast cancer.

BACKGROUND: Sex-determining region Y-box 3 (SOX3) protein, a SOX transcriptions factors group, has been identified as a key regulator in several diseases, including cancer. Downregulation of transcriptions factors in invasive ductal carcinoma (IDC) can interfere in neoplasia development, increasing its aggressiveness. We investigated SOX3 protein expression and its correlation with apoptosis in the MDA-MB-231 cell line, as SOX3 and Pro-Caspase-3 immunoexpression in paraffin-embedded invasive ductal carcinoma tissue samples from patients (n = 27). Breast cancer cell line MDA-MD-231 transfected with pEF1-SOX3 + and pEF1-Empty vector followed by cytotoxicity assay (MTT), Annexin-V FITC PI for apoptosis percentage assessment by flow cytometry, qPCR for apoptotic-related gene expression, immunofluorescence, and immunohistochemistry to SOX3 immunolocalization in culture cells, and paraffin-embedded invasive ductal carcinoma tissue samples. RESULTS: Apoptotic rate was higher in cells transfected with pEF1-SOX3 + (56%) than controls (10%). MDA-MB-231 transfected with pEF1-SOX3 + presented upregulation of pro-apoptotic mRNA from CASP3, CASP8, CASP9, and BAX genes, contrasting with downregulation antiapoptotic mRNA from BCL2, compared to non-transfected cells and cells transfected with pEF1-empty vector (p < 0.005). SOX3 protein nuclear expression was detected in 14% (4/27 cases) of ductal carcinoma cases, and pro-Caspase-3 expression was positive in 50% of the cases. CONCLUSION: Data suggest that SOX3 transcription factor upregulates apoptosis in breast cancer cell line MDA-MB-231, and has a down nuclear expression in ductal carcinoma cases, and need to be investigated as a tumor suppressor protein, and its loss of expression and non-nuclear action turn the cells resistant to apoptosis. Further studies are necessary to understand how SOX3 protein regulates the promoter regions of genes involved in apoptosis.