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This study was designed to examine the effects of interleukin-1 beta (IL-1 beta) on myocyte (MC) hypertrophy and the production of A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in rat ventricular cardiocyte culture, and to investigate the role of nonmyocyte (NMC) in this process. We examined the effects of IL-1 beta on the production of ANP and BNP in comparison with the effects of endothelin-1 (ET-1) by using two types of neonatal rat cardiocyte culture; MC-enriched culture and MC-NMC coculture. In the MC-enriched culture, the increase in secretion of ANP and BNP was small in treatment with IL-1 beta (1000 pg/ml), while ET-1 (10 nM) markedly augmented the secretion of ANP and BNP. In the MC-NMC coculture, IL-1 beta and ET-1 each significantly augmented the secretion of ANP and BNP. The degree of the increase of ANP and BNP was equivalent between IL-1 beta and ET-1. As for the morphological changes of MCs, IL-1 beta induced the star-shaped MC hypertrophy characterized by elongation and pointed edges only in the MC-NMC coculture, while ET-1 induced the MC hypertrophy characterized by shapes of squares, triangles or circles in both cultures. This study shows that IL-1 beta induces unique cardiac hypertrophy and the marked secretion of ANP and BNP, and that NMC is indispensable when treated with IL-1 beta.

Background: The mechanism responsible for cardiac hypertrophy is currently conceptualized as having 2 components, mediated by cardiac myocytes and nonmyocytes, respectively. The interaction between myocytes and nonmyocytes via growth factors and/or cytokines plays an important role in the development of cardiac hypertrophy. We found that cardiac myocytes showed hypertrophic changes when cocultured with cardiac nonmyocytes. Cardiotrophin-1 (CT-1), a new member of the interleukin-6 family of cytokines, was identified by its ability to induce hypertrophic response in cardiac myocytes. In this study, we used the in vitro coculture system to examine how CT-1 is involved in the interaction between cardiac myocytes and nonmyocytes during the hypertrophy process., Methods and Results: RNase protection assay revealed that CT-1 mRNA levels were 3. 5 times higher in cultured cardiac nonmyocytes than in cultured cardiac myocytes. We developed anti-CT-1 antibodies and found that they significantly inhibited the increased atrial and brain natriuretic peptide secretion and protein synthesis characteristic of hypertrophic changes of myocytes in the coculture. In addition, non-myocyte-conditioned medium rapidly elicited tyrosine phosphorylation of STAT3 and induced an increase in natriuretic peptide secretion and protein synthesis in cultured cardiac myocytes; these effects were partially suppressed by anti-CT-1 antibodies. Finally, the hypertrophic effects of CT-1 and endothelin-1, which we had previously implicated in the hypertrophic activity in the coculture, were additive in cardiac myocytes., Conclusions: These results show that CT-1 secreted from cardiac nonmyocytes is significantly involved in the hypertrophic changes of cardiac myocytes in the coculture and suggest that CT-1 is an important local regulator in the process of cardiac hypertrophy.

Background: In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system., Methods and Results: The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC-conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10(-10) to 10(-6) mol/L) and transforming growth factor-beta1 (TGF-beta1) (10(-13) to 10(-9) mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-beta1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-beta1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-beta1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123., Conclusions: These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II- or TGF-beta1-induced cardiocyte hypertrophy.

In cardiac hypertrophy or ventricular remodeling, not only the enlargement of myocytes but also interstitial or perivascular fibrosis are observed simultaneously, which suggests an interaction between cardiac myocytes and fibroblasts. In this study, we examined the mechanism of cyclic mechanical stretch-induced myocyte hypertrophy, highlighting the interaction between myocytes and cardiac nonmyocytes, mostly fibroblasts. Ventricular myocytes (MC) and cardiac nonmyocytes (NMC) were separately extracted from neonatal rat ventricles by the discontinuous Percoll gradient method and primary cultures of cardiac cells were prepared. Cyclic mechanical stretch was applied to the cultures with a Flexercell Stress Unit. In addition to cell size, we examined atrial natriuretic peptide/brain natriuretic peptide (ANP/BNP) production as the most sensitive biological markers for MC hypertrophy. Cyclic stretch did not induce hypertrophic responses in MC when they were cultured without NMC. In contrast, when MC were co-cultured with NMC, cyclic stretch induced further increase in ANP/BNP production (2.2-fold and 2.1-fold increases versus non-stretch group, after 48-h incubation). This increase in ANP/BNP production in the co-culture was significantly suppressed by CV-11974, an angiotensin II type 1 receptor antagonist. Moreover, ANP/BNP production in the co-culture was significantly suppressed by BQ-123, an endothelin A receptor antagonist, whether cyclic stretch was applied or not. This study raised the possibility that NMC mediate the hypertrophic effect of mechanical stress on MC by increasing endothelin production. It was also suggested that, in this process, angiotensin II is involved in the crosstalk between MC and NMC.

To elucidate the regulation of very-low density-lipoprotein (VLDL) receptor, we have studied its gene expression in the heart of spontaneously hypertensive rats-stroke prone (SHR-SP, an animal model for hypertension-induced cardiac hypertrophy) compared with Wistar-Kyoto rats. RNase protection assay showed that ventricular VLDL receptor mRNA falls to 41% of normal levels at 4 weeks when hypertension is not yet fully developed, and drops further to 14% at 13 weeks, when cardiac hypertrophy is established. Lipoprotein lipase mRNA decreases in parallel with VLDL receptor mRNA. In cultured neonatal rat ventricular cardiomyocytes, VLDL receptor mRNA decreases in parallel with the process of cardiocyte hypertrophy during the 24 hours after treatment with 10-8 mol/L endothelin-1, falling to 40% of the initial value. These results demonstrate that there is downregulation of VLDL receptor gene expression in cardiac hypertrophy both in vivo and in vitro and suggest that the regulation of the VLDL receptor is possibly linked with the switch in energy substrate from lipid to glucose known to occur in cardiac hypertrophy.

1. Prostacyclin elicits potent vasodilation and inhibition of platelet aggregation through binding to its membrane receptor. The impairment of prostacyclin receptor activity is implicated in various human cardiovascular diseases. We recently succeeded in molecular cloning of cDNA for the mouse, rat, and human prostacyclin receptors. 2. In the present study, we examined the mRNA expression of the prostacyclin receptor in various rat tissues, and further investigated its gene expression in the hypertrophied cardiac ventricles of stroke-prone spontaneously hypertensive rats (SHRSP). 3. In rat tissues, a single RNA band of approximately 3.7 kb was detected by northern blotting analysis using rat prostacyclin receptor cDNA as a probe. In adult Wistar rats, abundant mRNA expression was observed in the aorta, lung and spleen. Substantial amounts of transcript were expressed in the heart, pancreas, thymus and stomach. In contrast, no mRNA expression was detected in the brain. 4. We further examined the mRNA expression of the prostacyclin receptor in the ventricles of 21 week old SHRSP. The ventricles of SHRSP showed remarkable hypertrophy, compared with those of age-matched Wistar-Kyoto (WKY) rats. The expression of prostacyclin receptor mRNA in the hypertrophied ventricles of SHRSP was almost equivalent to that in the ventricles of WKY. 5. The present study revealed the gene expression of the prostacyclin receptor in various rat tissues, and further demonstrated the receptor mRNA expression in hypertensive cardiac hypertrophy. The present study will give a clue to investigate the clinical implication of prostacyclin and its receptor.

1. We previously demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone mainly produced in the ventricle, while the major production site of atrial natriuretic peptide (ANP) is the atrium. The production and secretion of BNP and ANP in the hypertrophied ventricles were markedly augmented, serving as a compensation mechanism against ventricular overload by their natriuretic, diuretic and vasodilatory actions. 2. In the present study, we prepared an in vitro model of cardiocyte hypertrophy using cultured neonatal rat ventricular cardiocytes and alpha1-adrenergic stimulation, and examined the gene expressions of BNP and ANP during the process of cardiocyte hypertrophy. 3. The treatment of cultured ventricular cardiocytes with phenylephrine evoked cardiocyte hypertrophy around 24 h after the treatment, which was characterized by augmented expression of the myosin light chain-2 gene and increase in cell size. 4. In this model of cardiocyte hypertrophy, the steady-state level of BNP mRNA rapidly increased to the maximal level within 1 h after the treatment. In contrast, ANP mRNA began to increase at 3 h, and accumulated during the course of cardiocyte hypertrophy. The secretion of BNP from ventricular cardiocytes was also stimulated more rapidly than the ANP secretion. 5. These results indicate that the gene expression of BNP is distinctly regulated from that of ANP in cardiocyte hypertrophy, and suggest a discrete pathophysiological role of BNP as an 'emergency' cardiac hormone against ventricular overload.

We previously demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone mainly produced in the ventricle, while the major production site of atrial natriuretic peptide (ANP) is the atrium. To assess the pathophysiological role of BNP in ventricular overload, we have examined the gene expression of BNP, In comparison with that of ANP, in a model of cardiac hypertrophy using cultured neonatal rat ventricular cardiocytes. During cardiocyte hypertrophy evoked by endothelin-1, Phenylephrine, or PMA, the steady state level of BNP mRNA increased as rapidly as the "immediate-early" induction of the c-fos gene expression, and reached a maximal level within 1 h. Actinomycin D, a transcriptional inhibitor, completely diminished the response, while the translational blocked with cycloheximide did not inhibit it. In contrast, ANP mRNA began to increase 3 h after the stimulation, and accumulated during cardiocyte hypertrophy. The BNP secretion from ventricular cardiocytes was also stimulated, more rapidly than the ANP secretion. Furthermore, the turnover of BNP mRNA was significantly faster than that of ANP mRNA, being consistent with the existence of AUUUA motif in the 3'-untranslated region of BNP mRNA. These results demonstrate that the gene expression of BNP is distinctly regulated from that of ANP at transcriptional and posttranscriptional levels, and indicate that the characteristics of the BNP gene expression are suitable for its possible role as an " emergency" cardiac hormone against ventricular overload.