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The signals that regulate cardiac myocyte maturation in the neonatal heart are not completely understood. In our study we examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on primary cultures of ventricular myocytes isolated from neonatal rat hearts. Our data show that 1,25-(OH)2D3 inhibited an increase in both the muscle-specific form of creatine kinase and V1 myosin isoenzyme levels in myocytes induced to mature by serum withdrawal. Thus, in contrast to other cell types studied to date, in the heart, 1,25-(OH)2D3 blocks cell maturation. Treating cultures with phorbol 12-myristate 13-acetate induced a decrease in the muscle-specific isoenzyme of creatine kinase similar to the effects of 1,25-(OH)2D3. Interestingly, we found that staurosporine, a protein kinase-C inhibitor, blunts the effects of 1,25-(OH)2D3 and has the opposite effect of phorbol 12-myristate 13-acetate on cultured myocytes induced to mature by serum withdrawal. Thus, our data identify a novel role for 1,25-(OH)2D3 in the regulation of myocardial development and suggest that 1,25-(OH)2D3 may be acting through a protein kinase-dependent mechanism to maintain cardiac myocytes in an immature state.

Thyroid hormones (THs) have many effects on the cardiovascular system including cardiac hypertrophy. Although THs induce cardiac hypertrophy, the mechanism through which they exert this effect is unknown. We previously found that THs activate signaling related to increased protein synthesis [mammalian target of rapamycin (mTOR) and p70 S6 kinase] in the heart. It is unknown whether this activation contributes to TH-induced hypertrophy or whether it is merely incidental. In this study, we used rapamycin to inhibit mTOR function in mice and neonatal cardiomyocyte cultures treated with THs to test whether mTOR/S6 kinase signaling is involved in TH-mediated cardiac hypertrophy. C57 mice were treated with T4 for 3 d, 1 wk, 2 wk, or 1 month with either placebo, T4 (50 μg/100 g body weight·d), rapamycin (200 μg/100 g body weight·d) or T4/rapamycin by sc slow-release pellets. At the end of the treatment period, hemodynamics and physical data were collected and hearts were frozen for Western blot analysis or myocytes were isolated. The effects of T3 and rapamycin were also investigated using neonatal cardiomyocytes. THs activated specific components of the AKT signaling pathway in vivo and in vitro. THs induced cardiac hypertrophy, which was completely inhibited by rapamycin. Our results suggest that TH-induced hypertrophy is mediated by AKT/mTOR/S6 kinase signaling, which is important in the regulation of protein synthesis, a hallmark of cardiac hypertrophy.

The steroid hormone 1,25-dihydroxyvitamin D3[1,25-(OH)2D3] induces maturation of many cells, including HL-60 promyelocytic leukemia cells that are differentiated to monocytes/macrophages. This process involves changes in transcription of genes such as c-myc. We investigated the effects of 1,25-(OH)2D3 on nuclear protein binding to the c-myc intron element (MIE), a region of DNA within the c-myc gene. A mutation in this MIE sequence has been shown to be associated with uncontrolled expression of the c-myc gene in various cell lines. In this report, we demonstrate for the first time that 1,25-(OH)2D3 induces increased binding of nuclear proteins to this MIE in HL-60 cells. The major MIE-binding proteins were approximately 32 kDa in size. Interestingly, phorbol 12-myristate 13-acetate induced a similar increase in binding of the 32-kDa doublet protein to the MIE. In addition, we showed that the level of 138-kDa MIE-binding protein was increased by 1,25-(OH)2D3 and phorbol 12-myristate 13-acetate. However, the extent of MIE binding by the 138-kDa protein is significantly less than that of the 32-kDa doublet binding species. MIE binding by the 32-kDa doublet protein was significantly increased within 12 h of 1,25-(OH)2D3 treatment. The time course of this increase was similar to that of 1,25-(OH)2D3-induced inhibition of c-myc gene transcription. We also demonstrated that dephosphorylation of the 32-kDa doublet protein inhibited its binding to the MIE. Thus, this study shows that the mechanism employed by 1,25-(OH)2D3 for regulation of c-myc expression may involve an increase in protein binding to the MIE, which has been shown to be the site for control of c-myc gene expression.

In this report, we demonstrate a significant inverse correlation between contractility and serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels and no correlation between contractility and serum levels of calcium, phosphate, or PTH. We also examined myosin isozyme distribution in vitamin D3-deficient rats, because myosin isozyme distribution can alter contractility. There was a significant increase in the levels of the V1 myosin isozyme in animals raised on a vitamin D3-deficient diet that maintained normal serum calcium and phosphate levels. There was no difference in the relative myosin isozyme distribution in animals raised on a hypocalcemia-yielding vitamin D3-deficient diet vs. animals raised on a control diet. As increased contractility has been observed in both groups of vitamin D3-deficient animals, a shift in myosin isozyme distribution cannot solely explain the increase in contractility previously observed in the vitamin D3-deficient rat heart. To determine whether 1,25-(OH)2D3 directly regulates myosin isozyme levels, we analyzed myosin isozyme distribution in primary cultures of ventricular myocytes. We found that 1,25-(OH)2D3 reduces total myosin levels, but does not alter myosin isozyme distribution. Thus, we show that the influence of vitamin D3 status on myosin isozyme expression in the intact rat involves a complex regulatory system of direct and indirect effects.