Unexpected maturation of PI3K and MAPK-ERK signaling in fetal ovine cardiomyocytes.

In the first two-thirds of gestation, ovine fetal cardiomyocytes undergo mitosis to increase cardiac mass and accommodate fetal growth. Thereafter, some myocytes continue to proliferate while others mature and terminally differentiate into binucleated cells. At term (145 days gestational age; dGA) about 60% of cardiomyocytes become binucleated and exit the cell cycle under hormonal control. Rising thyroid hormone (T3) levels near term (135 dGA) inhibit proliferation and stimulate maturation. However, the degree to which intracellular signaling patterns change with age in response to T3 is unknown. We hypothesized that in vitro activation of ERK, Akt, and p70(S6K) by two regulators of cardiomyocyte cell cycle activity, T3 and insulin like growth factor-1 (IGF-1), would be similar in cardiomyocytes at gestational ages 100 and 135 dGA. IGF-1 and T3 each independently stimulated phosphorylation of ERK, Akt, and p70(S6K) in cells at both ages. In the younger mononucleated myocytes, the phosphorylation of ERK and Akt was reduced in the presence of IGF-1 and T3. However, the same hormone combination led to a dramatic twofold increase in the phosphorylation of these signaling proteins in the 135 dGA cardiomyocytes-even in cells that were not proliferating. In the older cells, both mono- and binucleated cells were affected. In conclusion, fetal ovine cardiomyocytes undergo profound maturation-related changes in signaling in response to T3 and IGF-1, but not to either factor alone. Differences in age-related response are likely to be related to milestones in fetal cardiac development as the myocardium prepares for ex utero life.

Atrial natriuretic peptide inhibits angiotensin II-stimulated proliferation in fetal cardiomyocytes.

The role of atrial natriuretic peptide (ANP) in regulating fetal cardiac growth is poorly understood. Angiotensin II (Ang II) stimulates proliferation in fetal sheep cardiomyocytes when growth is dependent on the activity of the mitogen-activated protein kinase (MAPK) and phosphoinositol-3-kinase (PI3K) pathways. We hypothesized that ANP would suppress near-term fetal cardiomyocyte proliferation in vitro and inhibit both the MAPK and PI3K pathways. Forty-eight hour 5-bromodeoxyuridine (BrdU) uptake (used as an index of proliferation) was measured in cardiomyocytes isolated from fetal sheep (135 day gestational age) in response to 100 nm Ang II with or without ANP (0.003-100 nm) or 1 microm 8-bromo-cGMP. The effects of these compounds on the MAPK and PI3K pathways were assessed by measuring extracellular signal-regulated kinase (ERK) and AKT phosphorylation following 10 min of treatment with Ang II, ANP or 8-bromo-cGMP. In right ventricular myocytes (RV), the lowest dose of ANP (0.003 nm) inhibited Ang II-stimulated BrdU uptake by 68%. Similarly, 8-bromo-cGMP suppressed Ang II-stimulated proliferation by 62%. The same effects were observed in left ventricular (LV) cardiomyocytes but the RV was more sensitive to the inhibitory effects of ANP than the LV (P < 0.0001). Intracellular cGMP was increased by 4-fold in the presence of 100 nm ANP. Ang II-stimulated ERK and Akt phosphorylation was inhibited by 100 nm ANP. The activity of ANP may in part be cGMP dependent, as 8-bromo-cGMP had similar effects on the cardiomyocytes.

Thyroid hormone inhibits proliferation of fetal cardiac myocytes in vitro.

Thyroid hormone (T(3)) is a key regulator of fetal organ maturation. Premature elevations of thyroid hormone may lead to a 'mature' cardio-phenotype. Thyroid hormone will stimulate maturation of ovine fetal cardiomyocytes in culture by decreasing their proliferative capacity. Group 1 fetal cardiomyocytes (approximately 135 days gestation) were incubated with T3 (1.5, 3, 10, and 100 nM) and bromodeoxyuridine (BrdU; 10 microM) for 24 and 48 h. Group 2 cardiomyocytes were cultured with T3 alone for later protein analysis of cell cycle regulators. At all concentrations, T3 decreased BrdU uptake fourfold in serum media (P<0.001 versus serum, n=5). Following serum-free (SF) T3 treatment, BrdU uptake was inhibited when compared with serum (P<0.001 versus serum, n=5). p21 expression increased threefold (P<0.05 versus serum free, n=4) and cyclin D1 expression decreased twofold (P<0.05 versus serum, n=4) in T3-treated cardiomyocytes. (1) T3 inhibits fetal cardiomyocyte proliferation, while (2) p21 protein levels increase, and (3) cyclin D1 levels decrease. Thus, T3 may be a potent regulator of cardiomyocyte proliferation and maturation in the late gestation fetus.

Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells.

Pulmonary hypertension is characterized by vascular remodeling involving smooth muscle cell proliferation and migration. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators, and the inhibition of aortic smooth muscle cell (ASMC) proliferation by NO has been documented, but less is known about the effects of CGRP. The mechanism by which overexpression of CGRP inhibits proliferation in pulmonary artery smooth muscle cells (PASMC) and ASMC following in vitro transfection by the gene coding for prepro-CGRP was investigated. Increased expression of p53 is known to stimulate p21, which inhibits G(1) cyclin/cdk complexes, thereby inhibiting cell proliferation. We hypothesize that p53 and p21 are involved in the growth inhibitory effect of CGRP. In this study, CGRP was shown to inhibit ASMC and PASMC proliferation. In PASMC transfected with CGRP and exposed to a PKA inhibitor (PKAi), cell proliferation was restored. p53 and p21 expression increased in CGRP-treated cells but decreased in cells treated with CGRP and PKAi. PASMC treated with CGRP and a PKG inhibitor (PKGi) recovered from inhibition of proliferation induced by CGRP. ASMC treated with CGRP and then PKAi or PKGi recovered only when exposed to the PKAi and not PKGi. Although CGRP is thought to act through a cAMP-dependent pathway, cGMP involvement in the response to CGRP has been reported. It is concluded that p53 plays a role in CGRP-induced inhibition of cell proliferation and cAMP/PKA appears to mediate this effect in ASMC and PASMC, whereas cGMP appears to be involved in PASMC proliferation.