No abstract

The mammalian EGLN family contains three paralagous genes (EGLN1, EGLN2, and EGLN3) encoding prolyl hydroxylase isoforms that mediate the oxygen-dependent targeting of the transcription factor hypoxia inducible factor alpha to the proteosome. The rat orthologue of EGLN3 (SM-20) exhibits tissue-restricted expression, is induced by growth factors in cultured vascular smooth muscle, and is up-regulated during myogenesis. To determine if all three EGLN genes are coordinately regulated, we examined their mRNA expression in murine tissues and in cultured cells. We now report that the three murine EGLN mRNAs have unique but overlapping patterns of tissue expression. The most striking differences were in the heart, where EGLN3 had its highest levels of expression, and the testis, where EGLN2 was the only isoform expressed. In cultured vascular smooth muscle cells, serum treatment led to up-regulation of EGLN1 and EGLN3, but not EGLN2, and only EGLN3 was superinduced by cyclohexamide. In cultured C2C12 myocytes, EGLN3 was up-regulated during differentiation, whereas EGLN1 and EGLN2 were constitutively expressed. The abundance of EGLN3 mRNA in the heart, its induction by growth factors in vascular smooth muscle, and its regulation during C2C12 differentiation suggest a unique role for EGLN3 and might justify the development of isoformspecific inhibitors.

The molecular basis of human heart failure is unknown. Alterations in calcium homeostasis have been observed in failing human heart muscles. Intracellular calcium-release channels regulate the calcium flux required for muscle contraction. Two forms of intracellular calcium-release channels are expressed in the heart: the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (LP3R).In the present study we showed that these two cardiac intracellular calcium release channels were regulated in opposite directions in failing human hearts. In the left ventricle, RyR mRNA levels were decreased by 31% (P < 0.025) whereas IP3R mRNA levels were increased by 123% (P < 0.005). In situ hybridization localized both RyR and IP3R mRNAs to human cardiac myocytes. The relative amounts of IP3 binding sites increased -40% compared with ryanodine binding sites in the failing heart. RyR down-regulation could contribute to impaired contractility; IP3R up regulation may be a compensatory'response providing an alternative'pathway for mobilizing intracellular calcium release, possibly contributing to the increased. diastolic tone associated with heart failure and the hypertrophic response of failing myocardium. (J. Clin. Invest. 1995. 95:888-894.)

Abstract. Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca 2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca2+-induced Ca 2+ release via the RYR, a role for IP3-mediated Ca 2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non-muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were ,,o50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation-contraction coupling in the heart.