Significance of Ventricular Myocytes and Nonmyocytes Interaction During Cardiocyte Hypertrophy
Evidence for Endothelin-1 as a Paracrine Hypertrophic Factor From Cardiac Nonmyocytes
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-β1 (TGF-β1) (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-β1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-β1 was associated with the potentiation of ET-1 production in the MC-NMC coculture, and the effect of Ang II or TGF-β1 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-β1–induced cardiocyte hypertrophy.
- growth substances
- atrial natriuretic peptide
- brain natriuretic peptide
The heart functions as a syncytium of cardiac MCs and the surrounding support cells, referred to as NMCs, which consist of fibroblasts, endothelial cells, smooth muscle cells, and macrophages. Although cardiac MCs make up most of the adult myocardial mass, they make up only 30% of the total cell number present in the heart; the rest is composed of NMCs.1 2 During the process of cardiac hypertrophy, proliferation of cardiac fibroblasts and progressive interstitial and perivascular fibrosis were emphasized to account for cardiac hypertrophy or ventricular remodeling.3
We have previously revealed that plasma concentrations of both ANP and BNP are significantly elevated in cardiac overload, including cardiac hypertrophy.4 5 ANP has been well characterized as a cardiac hormone, mainly produced in and released from the atrium in the normal heart.6 On the other hand, BNP, the second member of the natriuretic peptide family, is predominantly synthesized in and secreted from the ventricle.6 7 8 In cardiac overload, however, elevation of both plasma ANP and BNP concentrations is significantly attributed to the induction of their productions in the ventricular myocytes.6 9 10 The augmentation of ANP and BNP productions can be considered a compensation mechanism against cardiac overload, because ANP and BNP serve to reduce both cardiac preload and afterload by their natriuretic, diuretic, and vasodilatory actions.6
Recently, a culture model for cardiac hypertrophy has been established using a primary culture of neonatal rat cardiac ventricular MCs in which phenylephrine, ET-1, or mechanical stress provokes hypertrophic changes of MCs characterized by an increase in cell size, an organization of contractile proteins into sarcomeric units, and an induction of a set of immediate early genes (c-fos, c-jun, c-myc, and Egr-1).11 12 13 14 15 16 17 18 19 20 21 In such a culture model, the induction of ANP mRNA during the process of MC hypertrophy was revealed to be one of the features well indicative of hypertrophic response.20 Furthermore, we have recently reported that gene expression of BNP is also augmented during the process of ET-1–induced MC hypertrophy.21 Therefore, hypertrophy of cultured MCs well mimics the feature of molecular biology of cardiac hypertrophy in vivo.
Although most of these in vitro experiments were investigated in MC-rich culture, cardiac hypertrophy in vivo involves proliferation of NMCs and interstitial fibrosis.22 Recently, some vasoactive substances, including Ang II and TGF-β1, both of which are well known as hypertrophic factors, were revealed to stimulate the proliferation of NMCs. Hypertrophy of MCs and proliferation of NMCs progress simultaneously during the course of cardiac hypertrophy. Cardiac hypertrophy in vivo, therefore, should occur in the interaction of MCs and NMCs. Indeed, there is growing evidence that NMCs may secrete growth factor(s) that stimulate MC hypertrophy.22 23 However, little is known about the interaction of MCs and NMCs during cardiac hypertrophy.
In this study, we examined changes in MC size and secretions of ANP and BNP in cardiocytes culture as markers for MC hypertrophy and revealed that NMCs secrete some hypertrophic factor(s), including ET-1, and furthermore that NMCs are essential for Ang II or TGF-β1 to exhibit a hypertrophic effect on MC.
Preparation of Cardiac MCs and NMCs
Two- to 4-day-old Wistar rats were used for MC and NMC culture. Ventricular cardiac cells were dispersed in the balanced salt solution containing 0.04% collagenase II (Worthington Biochemical Corp) and 0.06% pancreatin (GIBCO Laboratories) as previously reported.21 MCs and NMCs were separately collected by the discontinuous Percoll gradient method.21 The discontinuous gradient of Percoll (Sigma Chemical Co) consisting of 40.5% and 58.5% was prepared in the balanced salt solution described above, and ventricular cells were suspended in the layer of 58.5% Percoll. After centrifugation at 3000 rpm for 30 minutes at 15°C, MCs were migrated to the interface of the discontinuous layers, and NMCs were migrated to the surface of the layer of 40.5% Percoll.
Purified MCs were plated at a density of 3.0×104 cells/cm2 in the gelatin-coated six-well plates (2.9×105 cells per well) in Dulbecco’s modified Eagle’s medium (Flow Laboratories) supplemented with 10% FCS and antibiotics (100 U/mL penicillin G and 100 μg/mL streptomycin) (DME/FCS). After 36 hours of incubation, the cells were maintained in serum-free DME for 12 hours. After this precondition period, the culture was incubated in serum-free DME containing 1 mg/mL BSA (Sigma) with test substances.
NMCs were plated in 10-cm dishes in DME/FCS. After a 30-minute incubation, the dishes were vigorously washed with PBS, and the cells attached to the dishes were incubated in DME/FCS as NMC culture. After a 36-hour incubation, NMCs were removed by trypsinization (0.5 mg/mL in PBS, 0.016% EDTA at 37°C for 1 minute), and added to the MC culture prepared as described above, resulting in an equal number of MCs in the MC-NMC coculture and the MC culture. After a preconditioning incubation in serum-free DME for 12 hours, the culture was incubated in DME/BSA with test substances.
Preparation of NMC-Conditioned Media
NMC culture was prepared as described above and maintained in DME/FCS. To rule out contamination of endothelial cells in our NMC culture, binding of DiI-acetyl-LDL (Biochemical Technologies Inc) was examined. Cultured cells were incubated with DiI-acetyl-LDL for 4 hours and viewed by confocal laser micrography. The subconfluent NMC culture was incubated with serum-free DME for 12 hours. After this preconditioning period, the media was changed to DME/BSA and incubated for 72 hours. Conditioned media of the NMC culture (NMC-conditioned media) was then collected (final cell density of NMC, 5.0×105 cells/cm2).
Immunocytochemistry for Sarcomeric Actin
For immunocytochemistry, cells were prepermeabilized with 0.2% Triton X-100, fixed with 3% formaldehyde for 10 minutes at room temperature in PBS, and reacted for 8 hours with alpha-Sr-1, the anti-rat sarcomeric actin antibody (DAKO A/S), followed by the treatment with peroxidase-conjugated second antibody for visualization with 3,3′-diaminobenzidine tetrahydrochloride. Additionally, the nuclei of cultured cardiac cells were stained with hematoxylin. Phase-contrast microphotographs were scanned by the computed image analyzing system, and the cell size of MCs was estimated by measuring the area of sarcomeric actin–positive cells attached.
Radioimmunoassays for ANP, BNP, and ET-1
Northern Blot Analyses for ANP, BNP, ET-1, and GAPDH
The probes for ANP mRNA, BNP mRNA, and GAPDH mRNA were prepared as previously described6 8 and labeled by the random priming method with [α-32P] dCTP (Amersham International). The probe for ET-1 mRNA was prepared by PCR cloning from rat aortic endothelial cell cDNA. The synthetic primers were corresponding to the rat ET-1 cDNA sequence: 5′-GCGATCAGAGCAACCAGACACCATCCTC-3′ (sense) and 5′-GAATGAGTCAGACACGAACACTAACTAA- 3′ (antisense). The sequence of the amplified product was confirmed to be identical to that of rat ET-1 cDNA by the dideoxy chain termination method.
Total cellular RNA was extracted from the MC culture, MC-NMC coculture, and NMC culture by TRIzol Reagent (GIBCO BRL). Northern blot analyses were performed as previously reported21 at least three times.
RT-PCR Analyses for AT1a and ET-AR
Evaluation of gene expression of AT1a was performed by PCR with cDNA prepared from 1 μg of total RNA extracted from cultured cardiac cells. The synthetic primers were corresponding to the rat AT1a cDNA sequence, 5′-GCACACTGGCAATGTAATGC-3′ (sense) and 5′-GTTGAACAGAACAAGTGACC-3′ (antisense), which generate a 385-bp product.25 The amplification profile involved denaturation at 95°C for 45 seconds, primer annealing at 50°C for 45 seconds, and PCR at 72°C for 1 minute for variable cycles. Then, 10-μL aliquots were subjected to electrophoresis on a 2.5% agarose gel, and the amount of PCR products was evaluated by etidium bromide staining. For the PCR analyses for ET-AR mRNA, the synthetic primers 5′-GACGGCTTTCAAATATATCAACACTGTG-3′ (sense) and 5′-GGAGACAATTTCAATGGCGGT-3′ (antisense), which generate a 397-bp product,26 were used.
Synthetic ET-1, Ang II, and TGF-β1 (Peptide Institute); BQ-123 (provided by Banyu Pharmaceutical Co Ltd); and bosentan (provided by Nippon Roche K.K.) were used.
One-way ANOVA followed by the multiple comparison methods of Scheffé was used for statistical analyses. A value of P<.05 was considered significant. Values were expressed as mean±SD.
Negative Binding of DiI-Acetyl-LDL to NMCs
As shown in Fig 1A⇓ and 1B⇓, in our NMC culture, the binding of DiI-Acetyl-LDL was not observed, which indicates no contamination of endothelial cells in our NMC culture. The images of bovine arterial endothelial cells bounded by DiI-Acetyl-LDL are shown in Fig 1C⇓ and 1D⇓.
MC Hypertrophy in MC-NMC Coculture
Staining of cultured cells with the anti-rat sarcomeric antibody is shown in Fig 2⇓. In our MC culture, >99% cells were stained with the antibody (Fig 2A⇓). On the other hand, when MCs were cocultured with NMCs (MC-NMC coculture), apparent enlargement of MCs was observed around 24 hours of incubation (Fig 2B⇓). Cell size of MCs was significantly greater in the MC-NMC coculture than in the MC culture after a 24-hour incubation (Table 1⇓).
Increase in ANP and BNP Productions in MC-NMC Coculture
In the MC-NMC coculture, the concentrations of ANP and BNP in the culture media were augmented to significantly higher levels than in the MC culture around 12 hours of incubation (Fig 3A⇓). This augmentation of ANP and BNP secretions in MC-NMC coculture was dependent on the ratio of the cell number of NMCs to MCs (5% to 33%) (Fig 3B⇓), and the concentrations of ANP and BNP in the MC-NMC coculture (ratio of cell number, NMC:MC, 33%) were 3.4-fold and 2.7-fold higher than those in the MC culture, respectively, after 48 hours of incubation (Fig 3A⇓ and 3B⇓). Furthermore, as shown in Fig 5A⇓, ANP mRNA and BNP mRNA expression was significantly augmented in the MC-NMC coculture after a 24-hour incubation.
Effect of NMC-Conditioned Media on MC Hypertrophy and Secretions of ANP and BNP
In the MC culture, 10% replacement of culture media with NMC-conditioned media induced a significant increase in cell size of MCs ≈24 hours after stimulation (Fig 1C⇑, Table 1⇑). The ANP and BNP concentrations were significantly higher in the NMC-conditioned media group than in the control MC culture group ≈6 hours after stimulation and reached a 3.9-fold and 3.3-fold increase compared with control, respectively, after a 48-hour incubation (Fig 4A⇓). As Fig 4B⇓ shows, the stimulating effect of NMC-conditioned media on the secretions of ANP and BNP depended on percent replacement of culture media with NMC-conditioned media (2% to 50%). ANP and BNP concentrations in the treatment group were 15-fold and 21-fold, respectively, higher than in control, 48 hours after the replacement of culture media with NMC-conditioned media by 50% (Fig 4B⇓). The expression of ANP mRNA and BNP mRNA was also significantly augmented by the treatment with NMC-conditioned media (Fig 5A⇓).
Detection of ET-1 in NMC-Conditioned Media
ET-1 was not detected in the conditioned media of the MC culture (<1.2 pmol/L). In contrast, a considerable amount of ET-1 was detected in NMC-conditioned media after a 72-hour incubation (196±17.0 pmol/L). In addition, we observed significant expression of ET-1 mRNA in the NMC culture, whereas we did not detect any specific band for ET-1 mRNA in the MC culture (Fig 5B⇑).
Suppressive Effect of ET Receptor Antagonists on MC Hypertrophy Induced by NMC-Conditioned Media or in MC-NMC Coculture
Next, we examined the effect of BQ-123, an ET-AR antagonist, and bosentan, a nonselective ET receptor antagonist, on MC hypertrophy induced by NMC-conditioned media. After the preconditioning period, the MC culture was incubated with DME/BSA containing BQ-123 (10−5 mol/L) or bosentan (10−5 mol/L) for 2 hours and then treated with NMC-conditioned media (50% replacement; the estimated ET concentration was ≈10−10 mol/L). Both BQ-123 and bosentan partially but significantly suppressed the effect of NMC-conditioned media on the secretions of ANP and BNP (Fig 6⇓). Furthermore, BQ-123 also significantly attenuated secretions of ANP and BNP in the MC-NMC coculture (Fig 8⇓).
Responses of MC to Ang II or TGF-β1 in MC Culture and MC-NMC Coculture
Ang II or TGF-β1, which is known to be a cardiac hypertrophic factor, did not increase either cell size or secretions of ANP and BNP in the MC culture (Table 1⇑, Fig 7⇓). In contrast, in the MC-NMC coculture (ratio of cell number, NMC:MC, 33%), further increase in cell size of MC was observed by the treatment with Ang II or TGF-β1 (Table 1⇑), and secretions of ANP and BNP were augmented dose dependently by the treatment with Ang II (10−10 to 10−6 mol/L) or TGF-β1 (10−13 to 10−9 mol/L) (Fig 7⇓). Ang II (10−6 mol/L) significantly increased ANP and BNP concentrations by 2.4-fold and 2.4-fold, respectively, versus control 48 hours after treatment. TGF-β1 (10−9 mol/L) also significantly increased ANP and BNP concentrations by 2.6-fold and 2.9-fold, respectively (Fig 7⇓). Even when MCs were cocultured with a smaller number of NMCs (ratio of cell number, NMC:MC, 5%), Ang II (10−6 mol/L) increased ANP and BNP concentrations by 1.2-fold and 1.2-fold, respectively, versus control, and TGF-β1 (10−9 mol/L) also increased them by 1.5-fold and 1.7-fold, respectively, 48 hours after treatment. Fig 5C⇑ shows Northern blot analyses using total RNA prepared from the MC culture and MC-NMC coculture (NMC:MC, 33%) after the treatment with Ang II (10−6 mol/L) or TGF-β1 (10−9 mol/L). In the MC culture, expression of ANP mRNA and BNP mRNA was not increased by the treatment with Ang II or TGF-β1. In contrast, Ang II and TGF-β1 significantly augmented the expression of ANP mRNA and BNP mRNA in the MC-NMC coculture.
Furthermore, augmentation of the expression of ET-1 mRNA in the MC-NMC co-culture was observed by treatment with Ang II or TGF-β1. As Table 2⇓ shows, ET-1 was detected in culture media of the MC-NMC coculture, and the concentration of ET-1 increased when the MC-NMC coculture was treated with Ang II or TGF-β1. To assess the involvement of ET-1 secreted from NMCs in the mechanism of cardiocyte hypertrophy induced by Ang II or TGF-β1, we examined the effect of BQ-123 in the MC-NMC coculture. After the preconditioning period, the MC-NMC coculture was incubated with DME/BSA containing BQ-123 (10−5 mol/L). The treatment with BQ-123 partially but significantly blocked the Ang II–or TGF-β1–induced increase in ANP and BNP secretions in the MC-NMC coculture (Fig 8⇓).
Differential Expression of AT1a mRNA and ET-AR mRNA in MCs and NMCs
To examine the expression of AT1a or ET-AR, RT-PCR analyses for AT1a mRNA and ET-AR mRNA were performed with cDNAs prepared from MCs or NMCs. As Fig 9⇓ shows, the PCR product specific for AT1a mRNA was amplified in a cycle-dependent manner in cDNA prepared from NMCs, and little or no amplification of the PCR product was detected in cDNA prepared from MCs. Furthermore, the expression of AT1a mRNA was also not observed in MCs after the treatment with ET-1 or NMC-conditioned media (data not shown). The PCR product specific to ET-AR mRNA was amplified dominantly in cDNA prepared from MCs (Fig 9⇓).
In this study, we demonstrated apparent enlargement of MCs in the MC-NMC coculture. During this course of MC hypertrophy in the MC-NMC coculture, production of ANP and BNP was significantly augmented. These results clearly indicate the involvement of NMCs in MC hypertrophy. Furthermore, the dose-dependent effect of NMC-conditioned media on the increase in cell size of MCs and in the secretions of ANP and BNP suggests that NMC secrete some humoral factor(s) regulating MC hypertrophy.
In the present study, we detected significant expression of ET-1 mRNA in NMCs and the secretion of ET-1 from NMC into culture media. In contrast, we could not detect any ET-1 mRNA or ET-1 in MC culture. The ET-1 level detected in NMC-conditioned media was enough to stimulate MC hypertrophy (Table 2⇑),21 and the hypertrophic response induced by NMC-conditioned media was significantly blocked by the treatment with both BQ-123 and bosentan. These results clearly indicate that ET-1 is involved in the hypertrophic effect of NMC-conditioned media on MCs, and this effect of ET-1 is mediated by ET-A receptor, which was demonstrated to be expressed on cardiac myocytes in the present study. The partial blockade of NMC-conditioned media–induced MC hypertrophy by ET receptor antagonists indicates the possibility that other humoral factor(s) are secreted from NMCs and regulating MC hypertrophy. Cardiotrophin-1, a newly isolated cardiocyte hypertrophic factor, may be one of the candidates accounting for this NMC-derived cardiac hypertrophic activity.27
NMCs are reported to consist primarily of fibroblasts and a small amount of other cell types, including vascular endothelial cells, smooth muscle cells, and macrophages.1 2 Actually, it was reported that there exists cell-cell signaling between ventricular MCs and cardiac microvascular endothelial cells.28 In the present study, however, NMCs appeared to be homogeneous, and there was no binding of DiI-acetyl-LDL in our NMCs, which clearly indicates that NMCs in this study consist of fibroblasts and that there is no contamination of vascular endothelial cells. Accordingly, the origin of ET-1 is considered to be cardiac fibroblasts.
Recently, several vasoactive substances, including Ang II, are revealed to stimulate cardiac hypertrophy.17 18 29 30 In culture models, Ang II causes hypertrophy of cultured MCs, representing an increase in cell size and induction of ANP mRNA expression, and causes hyperplasia of NMCs characterized by the increase in cell number, protein synthesis, and DNA synthesis.30 TGF-β1 has been revealed to be expressed in the cardiac tissue31 or cultured microvascular endothelial cells28 and acts a cardioprotective role in myocardial ischemia.32 It has been reported that TGF-β1 induces fetal contractile proteins and ANP gene expression in cultured MCs.33 However, the exact mechanism by which Ang II and TGF-β1 regulate the growth of MCs in the interaction between MCs and NMCs has not been investigated.
In the present study, neither Ang II nor TGF-β1 stimulated MC hypertrophy or increased ANP and BNP production in the MC culture. In contrast, they induced the increase in cell size of MCs and augmentation of ANP and BNP secretions in the MC-NMC coculture. Because ET-1 mRNA expression and ET-1 secretion in the MC-NMC coculture were augmented by treatment with Ang II or TGF-β1 and because the action of Ang II or TGF-β1 in the MC-NMC coculture was inhibited by the ET-A receptor antagonist, the hypertrophic activity of Ang II or TGF-β1 was considered to be at least partly due to increased secretion of ET-1 from NMCs.
Our finding that Ang II or TGF-β1 does not stimulate MCs in the MC culture is apparently not compatible with recent studies that reported that Ang II or TGF-β1 induces cardiocyte hypertrophy in the MC culture.17 18 33 The exact reason for this dissociation is unclear, but it may be attributed to the methods of separation of MCs and NMCs and the purity of the MC culture used. Indeed, we observed that Ang II or TGF-β1 induced the enlargement of MC and increased ANP and BNP secretions when MCs were cocultured with a relatively small number of NMCs (ratio of cell number, NMC:MC, 5%). These data clearly indicate that Ang II–or TGF-β1–induced hypertrophic effect depends on the existence of NMC in a cardiocyte culture. Moreover, little or no expression of AT1a mRNA in MCs and its substantial expression in NMCs confirm the necessity of NMCs for Ang II to exhibit stimulating effect on MCs.
Fig 10⇓ shows a scheme representing the interaction between MCs and NMCs during the process of cardiac hypertrophy suggested by the present study. NMCs modulate hypertrophy of MCs through the secretion of humoral factor(s), including ET-1. Ang II or TGF-β1 regulates the cell size of MCs and the production of ANP and BNP in MCs not directly but indirectly, partly via augmentation of ET-1 secretion from NMCs. ANP and BNP at augmented levels can inhibit the proliferation of NMCs,34 which may be a feedback mechanism against the action of NMCs for MC hypertrophy. We are now conducting an analysis of further molecular mechanisms of this MC-NMC interaction and identification of additional unknown hypertrophic factor(s).
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|ANP||=||atrial natriuretic peptide|
|AT1a||=||Ang II type 1a receptor|
|BNP||=||brain natriuretic peptide|
|DME||=||Dulbecco’s modified Eagle’s medium|
|PCR||=||polymerase chain reaction|
|TGF-β1||=||transforming growth factor-β1|
This work was supported in part by research grants from the Japanese Ministry of Education, Science and Culture; Japanese Ministry of Health and Welfare “Disorders of Adrenal Hormone” Research Committee, Japan; “Molecular Approach for the Pathogenesis of Immunological Disorder” Research Committee; Japanese Society for the Promotion of Science “Research for the Future” program (JSPS-RFTF 96100204); Smoking Research Foundation; Yamanouchi Foundation for Research on Metabolic Disorders; Takeda Science Foundation; Salt Science Research Foundation; Uehara Memorial Foundation; and Japanese Society for Cardiovascular Diseases. We thank H. Kitoh and M. Okumura for their excellent secretarial work.
- Received April 30, 1997.
- Revision received June 20, 1997.
- Accepted July 3, 1997.
- Copyright © 1997 by American Heart Association
Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium: fibrosis and renin-angiotensin-aldosterone system. Circulation. 1991;83:1849-1865.
Sugawara A, Nakao K, Morii N, Yamada T, Itoh H, Shiono S, Saito Y, Mukoyama M, Arai H, Nishimura K, Obata K, Yasue H, Ban T, Imura H. Synthesis of atrial natriuretic polypeptide in human failing hearts: evidence for altered processing of atrial natriuretic polypeptide precursor and augmented synthesis of β-human ANP. J Clin Invest. 1988;81:1962-1970.
Hama N, Itoh H, Shirakami G, Nakagawa O, Suga S, Ogawa Y, Masuda I, Nakanishi K, Yoshimasa T, Hashimoto Y, Yamaguchi M, Hori R, Yasue H, Nakao K. Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction. Circulation. 1995;92:1558-1564.
Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y, Shirakami G, Jougasaki M, Obata K, Yasue H, Kambayashi Y, Inouye K, Imura H. Brain natriuretic peptide (BNP) as a novel cardiac hormone in humans: evidence for an exquisite dual natriuretic peptide system, ANP and BNP. J Clin Invest. 1991;87:1402-1412.
Ogawa Y, Nakao K, Mukoyama M, Hosoda K, Shirakami G, Arai H, Saito Y, Suga S, Jougasaki M, Imura H. Natriuretic peptides as cardiac hormones in normotensive and spontaneously hypertensive rats: the ventricle is a major site of synthesis and secretion of brain natriuretic peptide. Circ Res. 1991;69:491-500.
Yasue H, Obata K, Okumura K, Kurose M, Ogawa H, Matsuyama K, Jougasaki M, Saito Y, Nakao K, Imura H. Increased secretion of atrial natriuretic polypeptide (ANP) from the left ventricle in patients with dilated cardiomyopathy. J Clin Invest. 1989;83:46-51.
Saito Y, Nakao K, Arai H, Nishimura K, Okumura K, Obata K, Takemura G, Fujiwara H, Sugawara A, Yamada T, Itoh H, Mukoyama M, Hosoda K, Kawai C, Ban T, Yasue H, Imura H. Augmented expression of atrial natriuretic polypeptide gene in ventricle of human failing heart. J Clin Invest. 1989;83:298-305.
Long CS, Ordahl CP, Simpson PC. α1-adrenergic receptor stimulation of sarcomeric action isogene transcription in hypertrophy of cultured rat heart muscle cells. J Clin Invest. 1989;83:1078-1082.
Waspe LE, Ordahl CP, Simpson PC. The cardiac β-myosin heavy chain isogene is induced selectively in α1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes. J Clin Invest. 1990;85:1206-1214.
Iwaki K, Sukhatme VP, Shubeita HE Chien KR. α- and β-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells. fos/jun expression is associated with sarcomere assembly; egl-1 induction is primarily an α1-mediated response. J Biol Chem. 1990;265:13809-13817.
Knowlton KU, Baracchini E, Ross RS, Harris AN, Henderson SA, Evans SM, Glembotski CC, Chien KR. Co-regulation of the atrial natriuretic factor and cardiac myosin light chain-2 genes during a-adrenergic stimulation of neonatal rat ventricular cells: identification of cis sequences within an embryonic and a constitutive contractile protein gene which mediate inducible expression. J Biol Chem. 1991;266:7759-7768.
Komuro I, Katoh Y, Kaida T, Shibazaki Y, Kurabayashi M, Hoh E, Takaku F, Yazaki Y. Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes: possible role of protein kinase C activation. J Biol Chem. 1991;266:1265-1268.
Shubeita HE, Martinson EA, Van Bilsen M, Chien KR, Heller Brown J. Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes. Proc Natl Acad Sci U S A. 1992;89:1305-1309.
Sadoshima J, Izumo S. Signal transduction pathways of angiotensin II-induced c-fos gene expression in cardiac myocytes in vitro. Circ Res. 1993;73:424-438.
Ito H, Hirata Y, Adachi S, Tanaka M, Tsujino M, Koike A, Nogami A, Marumo F, Hiroe M. Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes. J Clin Invest. 1993;92:398-403.
Nakagawa O, Ogawa Y, Itoh H, Suga S, Komatsu Y, Kishimoto I, Nishino K, Yoshimasa T, Nakao K. Rapid transcriptional activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy: evidence for brain natriuretic peptide as an ‘emergency’ cardiac hormone against ventricular overload. J Clin Invest. 1995;96:1280-1287.
Saito Y, Nakao K, Mukoyama M, Shirakami G, Itoh H, Yamada T, Arai H, Hosoda K, Suga S, Jougasaki M, Ogawa Y, Nakajima S, Ueda M, Imura H. Application of monoclonal antibodies for endothelin to hypertensive research. Hypertension. 1990;15:734-738.
Matsubara H, Kanasaki M, Murasawa S, Tsukaguchi Y, Nio Y, Inada M. Differential gene expression and regulation of angiotensin II receptor subtypes in rat cardiac fibroblasts and cardiomyocytes in culture. J Clin Invest. 1994;93:1592-1601.
Lin HY, Kaji EH, Winkel GK, Ives HE, Lodish HF. Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor. Proc Natl Acad Sci U S A. 1991;88:3185-3189.
Nishida M, Soringhorn JP, Kelly RA, Smith TW. Cell-cell signaling between adult rat ventricular myocytes and cardiac microvascular endothelial cells in heterotypic primary culture. J Clin Invest. 1993;91:1934-1941.
Rockman HA, Wachhorst SP, Mao L, Ross Jr J. ANG II receptor blockade prevents ventricular hypertrophy and ANP gene expression with pressure overload in mice. Am J Physiol. 1994;266:H2468-H2475.
Schorb W, Booz GW, Dostal DE, Conrad KM, Chang KC, Baker KM. Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts. Circ Res. 1993;72:1245-1254.
Takahashi N, Calderone A, Izzo Jr NJ, Mäki TM, Marsh JD, Colucci WS. Hypertrophic stimuli induce transforming growth factor-β1 expression in rat ventricular myocytes. J Clin Invest. 1994;94:1470-1476.
Lefer AM, Tsao P, Aoki N, Palladino MA Jr. Mediation of cardioprotection by transforming growth factor-β. Science. 1990;249:61-64.
Parker TG, Parker SE, Schneider MD. Peptide growth factors can provoke ‘fetal’ contractile protein gene expression in rat cardiac myocytes. J Clin Invest. 1990;85:507-514.
Cao Li, Gardner DG. Natriuretic peptides inhibit DNA synthesis in cardiac fibroblasts. Hypertension. 1995;25:227-234.