Effects of Tanshinone VI on the Hypertrophy of Cardiac Myocytes and Fibrosis of Cardiac Fibroblasts of Neonatal Rats

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Animals
• Extracts
• Cell culture
• Measurement of protein synthesis by cardiac myocytes or collagen synthesis by cardiac fibroblasts
• Statistics
• Results
• Effects of tsh on the incorporation of [³ H]-leucine into
  cardiac myocytes
• Effects of tsh on PE-, ET-1-, or IGF-1-induced increase in
  [³ H]-leucine incorporation into cardiac myocytes
• Effect of tsh on collagen synthesis in cardiac fibroblasts
• Effect of tsh on the FBS- or IGF-1-induced increase in collagen synthesis in cardiac fibroblasts
• Effect of FB-CM on protein synthesis in cardiac myocytes
• Discussion
• References

Abstract

The possible effects of tanshinone VI (tsh), a diterpene from the root of Tan-Shen (Salvia miltiorrhiza, Bunge (Labiatae)) on hypertrophy and fibrosis in cultured neonatal rat cardiac myocytes and fibroblasts were examined. Tsh had no significant effect on protein synthesis, which was evaluated by [³ H]-leucine incorporation into the acid insoluble fraction in the cells, in the absence of stimulatory factors in cardiac myocytes. The amount of protein produced in cardiac myocytes was increased by 10 ^{- 8} M endothelin-1 (ET-1), 10 ^{- 6} M phenylephrine (PE), or 10 ^{- 8} M insulin-like growth factor-1 (IGF-1), suggesting that hypertrophy of cardiac myocytes in vitro was induced by these factors. The ET-1-, PE-, or IGF-1-induced increase in protein synthesis was attenuated by treatment with 10 ^{- 5} M tsh. Treatment with 10 ^{- 5} M tsh significantly decreased the synthesis of collagen by cardiac fibroblasts, which was evaluated by [³ H]-proline incorpolation into acid-insoluble fraction of the fiblobrasts, in the absence of stimulatory factors for the production. Fetal bovine serum (FBS) or IGF-1 increased collagen synthesis in a concentration-dependent manner. The increase at 5 % FBS or 10 ^{- 8} M IGF-1 was inhibited by 10 ^{- 5} M tsh. Fibroblast-conditioned medium (FB-CM) increased protein synthesis in cardiac myocytes in a concentration-dependent manner (10˜ - 100 %). Tsh attenuated the FB-CM-induced increase in protein synthesis by cardiac myocytes. These results show that tsh may attenuate the humoral factor-induced hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts. The findings suggest that tsh may improve the development of cardiac remodeling under pathophysiological conditions.

Abbreviations

ANP:atrial natriuretic peptide

DMEM:Dulbecco-modified Eagle's medium

ET-1:endothelin-1

FB-CM:fibroblast-conditioned medium

FBS:fetal bovine serum

IGF-1:insulin-like growth factor-1

PE:phenylephrine

tsh:tanshinone VI

Introduction

The cardiac hypertrophy or remodeling that occurs in response to chronic pressure overload or myocardial infarction is often accompanied by a disproportional increase in the deposition of collagen. Cardiac hypertrophy ultimately leads to heart failure, although initially it is an adaptive process that occurs in response to mechanical load or tissue injury. Thus, inhibition of the development of cardiac fibrosis and hypertrophy may exert a favorable effect on cardiac integrity. Recently, a model for cardiac hypertrophy and fibrosis has been established using a primary culture of neonatal rat ventricular cells. In this model, exogenous phenylephrine (PE)- or endothelin-1 (ET-1)-induced hypertrophy of myocytes [1] and angiotensin II-induced fibrosis of cardiac fibroblasts [2] were observed. Furthermore, the induction of atrial natriuretic peptide (ANP) mRNA during the process of myocyte hypertrophy is considered to be well indicative of a hypertrophic response [3]. Therefore, the cardiac hypertrophy and fibrosis of cultured cells well mimic the features of molecular biology of cardiac remodeling in vivo.

Tanshinone VI (tsh, Fig. [1]) is a diterpene compound extracted from the root of Salvia miltiorrhiza, Bunge (Labiatae), a Chinese traditional crude drug, ”Tan-Shen”. In China, extract from Tan-Shen is used to treat patients with cardiac disease [4], [5]. In a previous study, we showed that treatment of perfused rat heart with tsh enhanced the post-hypoxic contractile recovery [6]. The finding implies that tsh is a possible agent for protecting the myocardium against ischemic injury. Very little information, however, is available concerning other effects of tsh on cardiac metabolism. In the present study, we examined the effects of tsh on the protein synthesis of cardiac myocytes and on the collagen synthesis of cardiac fibroblasts.

During the development of cardiac hypertrophy, an increase in the size of cardiac myocytes and a progressive interstitial and perivascular fibrosis occur simultaneously. Several investigators have postulated a possible interaction between cardiac myocytes and fibroblasts in the hypertrophied myocardium. Some studies have suggested that ET-1 and cardiotrophin-1 secreted from cardiac fibroblasts contribute to the hypertrophic response observed in cultured cardiac myocytes [7], [8]. Recently, Maki et al. [9] have shown that insulin-like growth factor-1 (IGF-1) secreted from cardiac fibroblasts, induced hypertrophy of cardiac myocytes and simultaneously increased collagen synthesis by cardiac fibroblasts. Therefore, cardiac hypertrophy may affect the interaction between myocytes and surrounding fibroblasts. In the present study, we examined whether tsh affects the paracrine action of cardiac fibroblasts on myocytes through humoral factors.

Materials and Methods

Animals

Male and female Wistar rats at 1- or 2-days of age (SLC, Hamamatsu, Japan) were used in the present study. The experimental protocol was designed according to The Guide for the Care and Use of Laboratory Animals as promulgated by the National Research Council and approved by The Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Science.

Extracts

Extraction of tsh was conducted by the method described previously [10]. The crushed root of Tan-Shen (300 g) was successively extracted with hexane (700 ml), EtOAc (700 ml) and MeOH (700 ml). The EtOAc extract (5.3 g) was chromatographed over silica gel (50 g) column using C₆H₆ and CHCl₃ as solvents and CHCl₃ eluate (0.6 g) was rechromatographed over slica gel (10 g) column using CHCl₃ as a solvent to give tsh (0.02 g). The purity of tsh in the present study was more than 99 % (w/w) on the basis of the analysis by HPLC [11]. The optical rotation value of tsh was [α]_D ³⁰: + 47° (c 0.1, CHCl₃-MeOH, 1 : 1) [10].

Cell culture

Primary cultures for isolated cardiac myocytes and cardiac fibroblasts from neonatal rats were prepared using discontinous centrifugation method as described previously [12]. Cardiac myocytes were plated at a density of 1 × 10⁵ cells/well onto gelatin-coated 24-well culture dishes with Dulbecco-modified Eagle’s medium (DMEM) including 10 % fetal bovine serum (FBS). Cardiac fibroblasts at the 2nd or 3rd passage were plated at a density of 2.5 × 10⁴ cells/well. This purification procedure has well been established in previous studies [1], [7]. In fact, >95 % cardiac myocytes and almost 100 % cardiac fibroblasts were obtained by this method.

Measurement of protein synthesis by cardiac myocytes or collagen synthesis by cardiac fibroblasts

To evaluate the protein synthesis of cardiac myocytes and collagen synthesis of cardiac fibroblasts, the incorporation of [³ H]-leucine into cardiac myocytes and of [³ H]-proline into cardiac fibroblasts were measured [12]. The incubation medium was changed and cardiac myocytes were incubated in DMEM without FBS (serum-free DMEM). After a 24-h incubation, the serum-free DMEM was renewed. Phenylephrine (PE), ET-1, FBS, or IGF-1 was added to the medium. We examined the effect of tsh on cell viability in cardiac myocytes and fibroblasts. The number of these cells was not changed by treatment with 10^-7 - 10^-5 M tsh. These concentrations of tsh were used in subsequent experiments. Cardiac myocytes or fibroblasts were incubated for 24-h with [³ H]-leucine (0.5μ Ci/ml) or [³ H]-proline (0.5 μCi/ml), respectively. After 24-h incubation, the cells were washed twice with ice-cold PBS and then fixed with ice-cold 10 % trichloroacetic acid. The resultant acid-insoluble residue was solubilized with 1 M NaOH and then the radioactivity of the solution was determined by liquid scintillation counter (ALOCA, Tokyo, Japan), from estimates of the amount of [³ H]-leucine incorporated into cardiac myocytes and of [³ H]-proline incorporated into cardiac fibroblasts.

Statistics

Each value represents the mean ± SEM of 6 experiments. The statistical significance of differences between groups was tested by one-way ANOVA followed by Fisher’s PLSD or Dunnett’s methods. The statistical significance of the FB-CM-induced protein synthesis between the presence and absence of tsh was determined by the two-way ANOVA for repeated measures. P values for the post-hoc test in the present study were calculated using StatView^R (SAS Institute, Tokyo). P values of less than 0.05 were considered statistically significant.

Results

Effects of tsh on the incorporation of [³ H]-leucine into
cardiac myocytes

To determine whether tsh directly modulates protein synthesis in cardiac myocytes, we examined its effects on the incorporation of [³ H]-leucine into cardiac myocytes in the absence of stimulatory factors for protein synthesis (Table [1]). The uptake of [³ H]-leucine into the cells was not affected by treatment with any concentration of tsh (10^-7 - 10^-5 M).

Effects of tsh on PE-, ET-1-, or IGF-1-induced increase in
[³ H]-leucine incorporation into cardiac myocytes

Effects of tsh on exogenous PE-, ET-1-, or IGF-1-induced increases in protein synthesis in cardiac myocytes were examined. As shown in Fig. [2] A, exogenous PE (10^-6 and 10^-5 M) increased the amount of [³ H]-leucine incorporated into cardiac myocytes. The 10^-6 M PE-induced increase in the uptake of [³ H]-leucine was completely inhibited by 10^-5 M tsh (P = 0.003, Fig. [2] B).

As shown in Fig. [2] C, the incorporation of [³ H]-leucine into cardiac myocytes was increased by treatment with exogenous ET-1 from 10^-9 to 10^-7 M. Tsh at the concentration of 10^-5 M significantly inhibited the 10^-8 M ET-1-induced increase in protein synthesis by 35.8 % (P = 0.0005, Fig. [2] D).

Exogenous IGF-1 increased [³ H]-leucine incorporation in a concentration-dependent manner (Fig. [2] E). The 10^-8 M IGF-1-induced increase in the uptake of [³ H]-leucine incorporation was inhibited by 10^-5 M tsh by 24.6 % (P = 0.0034, Fig. [2] F).

Effect of tsh on collagen synthesis in cardiac fibroblasts

To determine whether tsh directly modulates collagen synthesis, we examined its effects on the incorporation of [³ H]-proline into cardiac fibroblasts under basal conditions. Treatment with 10^-5 M tsh significantly decreased the incorporation, whereas tsh at 10^-7 to 10^-6 M did not affect the synthesis of collagen (Table [1]).

Effect of tsh on the FBS- or IGF-1-induced increase in collagen synthesis in cardiac fibroblasts

The incorporation of [³ H]-proline into cardiac fibroblasts was increased by FBS from 0.2 to 5 % in a concentration-dependent manner (Fig. [3] A). The FBS-induced increase in [³ H]-proline incorporation was inhibited by treatment with 10^-5 M tsh (P = 0.0018, Fig. [3] B).

As shown in Fig. [3] C, exogenous IGF-1 increased the incorporation in a concentration-dependent manner. Tsh at 10^-5 M attenuated the IGF-1-induced increase in the uptake of [³ H]-proline by 48.6 % (P < 0.0001, Fig. [3] D).

Effect of FB-CM on protein synthesis in cardiac myocytes

To determine whether the action of cardiac fibroblasts on myocytes through humoral factors is paracrine or not, the effects of FB-CM on protein synthesis in cardiac myocytes were examined (Fig. [4]). The [³ H]-leucine incorporation was increased dose-dependently by treatment with 10 to 100 % FB-CM. Tsh attenuated the FB-CM-induced increase in protein synthesis in cardiac myocytes (Fig. [4]).

Discussion

In the present study, we examined whether tsh modulates protein synthesis induced by stimulatory factors such as ET-1, PE, and IGF-1. Although tsh did not affect protein synthesis in the absence of stimulatory factors, it attenuated the exogenous ET-1-, PE-, or IGF-1-induced increase in protein synthesis in cardiac myocytes. It is known that activation of the myocardial ET, sympathetic nerve, and IGF-1 systems leads to cardiac hypertrophy [13], [14], [15]. It has been reported that the blockade of these systems improves cardiac function and metabolism in experimental models of heart failure [16]. Therefore, our findings suggest that tsh suppresses the cardiac hypertrophy when the cardiac ET, sympathetic nerve, and IGF-1 systems are activated.

We showed in a previous study that endogenous ANP released from cardiac myocytes inhibited collagen synthesis in a paracrine manner in cultured cardiac fibroblasts [12]. ET-1 and cardiotrophin-1 secreted from cardiac fibroblasts are implicated as factors for the FB-CM-induced hypertrophy in cardiac myocytes [7], [8]. In addition, we have shown in a previous study that IGF-1 was produced in and released from cardiac fibroblasts and that endogenous IGF-1 enhanced not only protein synthesis in cardiac myocytes but also collagen synthesis in cardiac fibroblasts [9]. We also found that tsh attenuated the collagen synthesis in cardiac fibroblasts in the absence of stimulatory factors such as FCS and IGF-1. Tsh also attenuated the exogenous IGF-1-induced increase in collagen synthesis. Furthermore, we prepared FB-CM from cultured cardiac fibroblasts in the presence and absence of tsh. FB-CM without tsh increased the protein synthesis in a concentration-dependent manner. In contrast, when cardiac myocytes were incubated with FB-CM treated with tsh, the increase in protein synthesis was attenuated, as shown in Fig. [4]. Since endogenous IGF-1 modulates both protein synthesis in cardiac myocytes and collagen synthesis in cardiac fibroblasts, it is likely that the inhibitory effects of tsh on cardiac hypertrophy and its fibrosis are, at least in part, due to attenuation of the endogenous IGF-1 system. We cannot exclude the possibility that other humoral factors modulate the tsh-induced changes in protein and collagen synthesis. Further investigation concerning the effects of tsh on the synthesis, secretion, and degradation of these humoral factors is required.

Although 10^-5 M tsh improved cardiac remodeling, other concentrations of this drug (10^-7 and 10^-6 M) did not affect the humoral factor-induced increase in protein and collagen synthesis. We examined in a previous study the effect of tsh on the post-hypoxic recovery of cardiac contractile force of hypoxic/reoxygenated rat hearts. The maximal beneficial effect of tsh was observed at 42 nM, whereas appreciable effects were not seen in hearts perfused with other concentrations of this drug. Although we cannot explain exactly why tsh did not suppress the humoral factor-induced hypertrophy and fibrosis in a concentration-dependent manner, the findings suggest that the effective dose of tsh would be considerably limited.

In human failing or hypertrophied hearts, activation of the ET-1, sympathetic nerve, and IGF-1 systems in cardiac tissue was markedly increased and considerable levels of these humoral factors are detected in the ventricles [15], [17], [18]. Under such conditions, tsh is a possible agent to attenuate the activities of these systems and thereby partially suppress the development of protein and collagen synthesis in the heart. Therefore, the inhibition of cardiac hypertrophy and interstitial fibrosis in the heart by tsh may exert a favorable effect on cardiac integrity. The present study is the first report that tsh may function as a local modulator during the processes of cardiac remodeling and heart failure.

References

• 1 Horio T, Nishikimi T, Yoshihara F, Matsuo H, Takishita S, Kangawa K. Inhibitory regulation of hypertrophy by endogenous atrial natriuretic peptide in cultured cardiac myocytes.  Hypertension. 2000;  35 19-24
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 18 Maki T, Nasa Y, Yamaguchi F, Yoshida H, Mori M, Takada T, Horikawa E, Okano K, Takeo S. Long-term treatment with neutral endopeptidase inhibitor improves cardiac function and reduces natriuretic peptides in rats with chronic heart failure.  Cardiovasc Res. 2001;  51 608-17
  CrossrefPubMedSearch in Google Scholar

Satoshi Takeo, Ph. D.

Department of Pharmacology,

Tokyo University of Pharmacy and Life Science

1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Phone: +81-426-76-4583

Fax: +81-426-76-5560

Email: takeos@ps.toyaku.ac.jp

References

• 1 Horio T, Nishikimi T, Yoshihara F, Matsuo H, Takishita S, Kangawa K. Inhibitory regulation of hypertrophy by endogenous atrial natriuretic peptide in cultured cardiac myocytes.  Hypertension. 2000;  35 19-24
  PubMedSearch in Google Scholar
• 2 Yoshida H, Takahashi M, Tanonaka K, Maki T, Nasa Y, Takeo S. Effects of ACE inhibition and angiotensin II type 1 receptor blockade on cardiac function and G proteins in rats with chronic heart failure.  Br J Pharmacol. 2001;  134 150-60
  CrossrefPubMedSearch in Google Scholar
• 3 Chien K R, Zhu H, Knowlton K U, Miller-Hance W, van-Bilsen M, O’Brien T X, Evans S M. Transcriptional regulation during cardiac growth and development.  Ann Rev Physiol. 1993;  55 77-95
  PubMedSearch in Google Scholar
• 4 Huang X, Yang B, Huang H, Xu Y, Hu Z. Studies on the active principles of Dan-Shen III. Search for plant resources containing tanshinone II-A.  Chih Wu Hsueh. 1980;  22 98-100
  PubMedSearch in Google Scholar
• 5 Chen Z. Chinese traditional medicines. Some recent studies of Chinese medical plants.  Kagaku no Ryoiki. 1981;  35 494-501
  PubMedSearch in Google Scholar
• 6 Takeo S, Tanonaka K, Hirai K, Kawaguchi K, Ogawa M, Yagi A, Fujimoto K. Beneficial effect of tan-shen, an extract from the root of Salvia, on post-hypoxic recovery of cardiac contractile force.  Biochem Pharmacol. 1990;  40 1137-43
  CrossrefPubMedSearch in Google Scholar
• 7 Harada M, Itoh H, Nakagawa O, Ogawa Y, Miyamoto Y, Kuwahara K, Ogawa E, Igaki T, Yamashita J, Masuda I, Yoshimasa T, Tanaka I, Saito Y, Nakao K. Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy: evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes.  Circulation. 1997;  96 3737-44
  CrossrefPubMedSearch in Google Scholar
• 8 Kuwahara K, Saito Y, Harada M, Ishikawa M, Ogawa E, Miyamoto Y, Hamanaka I, Kamitani S, Kajiyama N, Takahashi N, Nakagawa O, Masuda I, Nakao K. Involvement of cardiotrophin-1 in cardiac myocyte-nonmyocyte interactions during hypertrophy of rat cardiac myocytes in vitro .  Circulation. 1999;  100 1116-24
  PubMedSearch in Google Scholar
• 9 Maki T, Horio T, Yoshihara F, Tokudome T, Suga S, Kawano Y, Takeo S, Kangawa K. Endogenous insulin-like growth factor-1 promotes collagen production and myocyte hypertrophy in an autocrine and paracrine manner in cultured neonatal rat cardiac fibroblasts and myocytes.  Circulation. 2001;  104 II-186
  PubMedSearch in Google Scholar
• 10 Yagi A, Fujimoto K, Tanonaka K, Hirai K, Takeo S. Possible active components of Tan-Shen (Salvia miltiorrhiza) for protection of the myocardium against ischemia-induced derangements.  Planta Med. 1989;  55 51-4
  PubMedSearch in Google Scholar
• 11 Okamura N, Kobayashi K, Yagi A, Kitazawa T, Shimomura K. High-performance liquid chromatography of abietane-type compounds.  J Chromatogr. 1991;  542 317-26
  CrossrefPubMedSearch in Google Scholar
• 12 Maki T, Horio T, Yoshihara F, Suga S, Takeo S, Matsuo H, Kangawa K. Effect of neutral endopeptidase inhibitor on endogenous atrial natriuretic peptide as a paracrine factor in cultured cardiac fibroblasts.  Br J Pharmacol. 2000;  131 1204-10
  CrossrefPubMedSearch in Google Scholar
• 13 Ebensperger R, Acevedo E, Melendez J, Corbalan R, Acevedo M, Sapag-Hagar M, Jalil J E, Lavandero S. Selective increase in cardiac IGF-1 in a rat model of ventricular hypertrophy.  Biochem Biophys Res Commun. 1998;  243 20-4
  CrossrefPubMedSearch in Google Scholar
• 14 Mulder P, Richard V, Derumeaux G, Hogie M, Henry J P, Lallemand F, Compagnon P, Mace B, Comoy E, Letac B, Thuillez C. Role of endogenous endothelin in chronic heart failure: effect of long- term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling.  Circulation. 1997;  96 1976-82
  PubMedSearch in Google Scholar
• 15 Esler M, Kaye D. Sympathetic nervous system activation in essential hypertension, cardiac failure and psychosomatic heart disease.  J Cardiovasc Pharmacol. 2000;  35 S1-7
  CrossrefPubMedSearch in Google Scholar
• 16 Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, Sugishita Y. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure.  Nature. 1996;  384 353-5
  CrossrefPubMedSearch in Google Scholar
• 17 Donohue T J, Dworkin L D, Lango M N, Fliegner K, Lango R P, Benstein J A, Slater W R, Catanese V M. Induction of myocardial insulin-like growth factor-I gene expression in left ventricular hypertrophy.  Circulation. 1994;  89 799-809
  CrossrefPubMedSearch in Google Scholar
• 18 Maki T, Nasa Y, Yamaguchi F, Yoshida H, Mori M, Takada T, Horikawa E, Okano K, Takeo S. Long-term treatment with neutral endopeptidase inhibitor improves cardiac function and reduces natriuretic peptides in rats with chronic heart failure.  Cardiovasc Res. 2001;  51 608-17
  CrossrefPubMedSearch in Google Scholar

Satoshi Takeo, Ph. D.

Department of Pharmacology,

Tokyo University of Pharmacy and Life Science

1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Phone: +81-426-76-4583

Fax: +81-426-76-5560

Email: takeos@ps.toyaku.ac.jp