To elucidate the cellular mechanism by which angiotensin II (ANG II) induces cardiac hypertrophy, we investigated the possible autocrine/paracrine role of endogenous endothelin-1 (ET-1) in ANG II-induced hypertrophy of neonatal rat cardiomyocytes by use of synthetic ET-1 receptor antagonist and antisense oligonucleotides to preproET-1 (ppET-1) mRNA. Northern blot analysis and in situ hybridization revealed that ppET-1 mRNA was expressed in cardiomyocytes, but, to a lesser extent, in nonmyocytes as well. ANG II upregulated ppET-l mRNA level by threefold over control level as early as 30 min, and it stimulated release of immunoreactive ET-1 from cardiomyocytes in a dose-and time-dependent manner. ET-1 stimulated ppET-1 mRNA levels after 30 min in a similar fashion as ANG II. Tetradecanoylphorbol-acetate (lo-7 M) mimicked the effects of ANG II and ET-1 on induction of ppET-1 mRNA. ANG II-induced ppET-1 gene expression was completely blocked by protein kinase C inhibitor H-7 or by downregulation ofendogenous protein kinase C by pretreatment with phorbol ester. ET-1 and ANG II stimulated twofold increase I13Hlleucine incorporation into cardiomyocytes, whose effects were similarly and dose dependently inhibited by endothelin A receptor antagonist (BQ123). Introduction of antisense sequence against coding region of ppET-l mRNA into cardiomyocytes resulted in complete blockade with ppET-l mRNA levels and 13Hlleucine incorporation stimulated by ANG II. These results suggest that endogenous ET-l locally generated and secreted by cardiomyocytes may contribute to ANG II-induced cardiac hypertrophy via an autocrine / paracrine fashion. (J. Clin. Invest. 1993.92:398403.) Key words: preproendothelin-l mRNA * neonatal rat cardiomyocytes * endothelin A receptor antagonist * antisense nucleotides

We examined whether apoptosis occurs in cardiomyocytes by hypoxia in vitro. Neonatal rat cardiomyocytes and nonmyocytes were cultured in 95% N2-5% CO2 atmosphere to produce hypoxic conditions. DNA fragmentation into ultraviolet exposure,2,6 anticancer drugs,8-10 viral infection, 11-3 and hyperthermia,14 in a variety of tissues. Ischemia of the heart, which is usually caused by reduction of coronary flow, induces the death of cardiomyocytes and replacement by fibrosis. Although this cardiomyocyte death is classically believed to be induced via necrosis, which is the mechanism of accidental cell death, its precise mechanism remains unclear. It has been recently reported that ischemia induces apoptotic death in the liver15 and in the brain16 in vivo. Thus, in addition to necrotic death, the mechanism of cardiomyocyte death may be involved in the apoptotic process. © 1994 American Heart Association, Inc.myocytes and nonmyocytes as revealed by Northern blotting and in situ hybridization. In hypoxic conditions, Fas messenger RNA levels in cardiomyocytes were upregulated by twofold over controls, whereas those of nonmyocytes were downregulated. These results indicate that cardiomyocyte death by hypoxia can occur via apoptosis and that Fas antigen may be associated with the mechanism of this apoptotic process. (Circ Res. 1994;75:426-433.)

BACKGROUND Cardiac hypertrophy is commonly observed in acromegalic patients, in whom serum insulin-like growth factor-I (IGF-I) levels are elevated. In the present study, we examined whether IGF-I induces hypertrophy in cultured neonatal rat cardiomyocytes through its specific receptor and whether IGF binding protein-3 (IGFBP-3), which is a major circulating carrier protein for IGF-I, inhibits IGF-I-induced cardiac hypertrophy in vitro. METHODS AND RESULTS Because the response of cardiac hypertrophy is characterized by the induction of expression for muscle-specific genes, the effect of IGF-I on steady-state levels of mRNA for myosin light chain-2 (MLC-2) and troponin I and for skeletal and cardiac alpha-actin isoforms was evaluated by Northern blot analysis. IGF-I (10(-7) M) increased mRNA levels for MLC-2 and troponin I as early as 60 minutes with maximum levels by 6 hours, which were maintained for as long as 24 hours. IGF-I (10(-7) M) also increased transcripts for skeletal alpha-actin but not for cardiac alpha-actin. The cell size as evaluated morphometrically was almost doubled after 48-hour treatment with IGF-I. IGF-I induction of protein synthesis was dose dependent (10(-10) to 10(-7) M) with a maximal 2.2-fold increase seen at 10(-8) M. In contrast to the hypertrophic effect of IGF-I, growth hormone affected neither protein synthesis nor expression for muscle-specific genes. Binding study using 125I-IGF-I revealed the presence of specific binding sites for IGF-I in rat cardiomyocytes. IGFBP-3 induced a dose-dependent inhibition of protein synthesis stimulated by IGF-I; IGFBP-3 (10(-7) M) completely inhibited the [3H]leucine uptake stimulated by IGF-I (10(-8) M). IGFBP-3 similarly inhibited the IGF-I-stimulated gene expressions for MLC-2 and troponin I. CONCLUSIONS These results suggest that IGF-I directly causes cardiac hypertrophy and that its effect can be blocked by IGFBP-3.

To determine whether endothelin-1 (ET-1) induces hypertrophy of cardiomyocytes, the effects of ET-1 on the expression of muscle-specific genes and a proto-oncogene, c-fos, in cultured neonatal rat cardiomyocytes were examined by Northern blot analysis. ET-1 (10(-7) M) induced about twofold to fourfold increases in the gene expression of myosin light chain 2, alpha-actin, and troponin I after 6 hours, which continued up to 24 hours. The ET-1-induced increases in mRNA levels for these muscle-specific genes were dose dependent (10(-9) to 10(-7) M). Run-on transcriptional assay showed that the changes in mRNA level for three muscle-specific genes were regulated, at least in part, at the transcriptional level. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a potent protein kinase C activator, and the Ca2+ ionophore ionomycin also increased mRNA levels of three muscle-specific genes. ET-1, TPA, and ionomycin similarly induced the expression of c-fos after 30 minutes, which returned to an undetectable level after 6 hours. ET-1 remarkably and dose-dependently stimulated accumulation of total inositol phosphates in cardiomyocytes. Morphometrical evaluation showed that ET-1 significantly increased surface area of cardiomyocytes without cell proliferation. ET-1 also dose-dependently stimulated the synthesis of protein and DNA, which was unaffected by the L-type calcium channel blocker nicardipine. These data suggest that ET-1 induces hypertrophy of cardiomyocytes associated with the induction of muscle-specific gene transcripts through the possible involvement of protein kinase C activation or intracellular Ca2+ mobilization.

SummaryCellular senescence is an important phenomenon in decreased cellular function. Recently, it was shown that cellular senescence is induced in proliferating cells within a short period of time by oxidative stresses. This phenomenon is known as premature senescence. However, it is still unknown whether premature senescence can be also induced in cardiomyocytes. The aim of the present study was to investigate whether a senescence-like phenotype can be induced in cardiomyocytes by oxidative stress. In cardiomyocytes obtained from aged rats (24 months of age), the staining for senescence-associated β β β β -galactosidase increased significantly and the protein or RNA levels of cyclin-dependent kinase inhibitors increased compared to those of young rats. Decreased cardiac troponin I phosphorylation and telomerase activity were also observed in aged cardiomyocytes. Treatment of cultured neonatal rat cardiomyocytes with a low concentration of doxorubicin (DOX) (10 -7 mol L -1 ) did not induce apoptosis but did induce oxidative stress, which was confirmed by 2′ ′ ′ ′ ,7′ ′ ′ ′ -dichlorofluorescin diacetate staining. In DOX-treated neonatal cardiomyocytes, increased positive staining for senescence-associated β β β β -galactosidase, cdk-I expression, decreased cardiac troponin I phosphorylation, and decreased telomerase activity were observed, as aged cardiomyocytes. Alterations in mRNA expression typically seen in aged cells were observed in DOX-treated neonatal cardiomyocytes. We also found that promyelocytic leukemia protein and acetylated p53, key proteins involved in stressinduced premature senescence in proliferating cells, were associated with cellular alterations of senescence in DOX-treated cardiomyocytes. In conclusion, cardiomyocytes treated with DOX showed characteristic changes similar to cardiomyocytes of aged rats. promyelocytic leukemiarelated p53 acetylation may be an underlying mechanism of senescence-like alterations in cardiomyocytes. These findings indicate a novel mechanism of myocardial dysfunction induced by oxidative stress.