Ju-Chi Liu¹ ， Jin-Jer Chen²  and Tzu-Hurng Cheng^3
　　1 Department of Medicine， Taipei Medical University-Shuang Ho Hospital， Taipei， Taiwan;
　　² Department of Internal Medicine， National Taiwan University Hospital and National Taiwan University College of Medicine， Taipei， Taiwan;
　　³ Department of Biological Science and Technology， College of Life Sciences， China Medical University， Taichung， Taiwan

　　Cardiac remodeling is the changes of size， shape， and function of the ventricle response to injures to the heart (ischemia/reperfusion injury， pressure overload， volume overload， etc). Cardiac remodeling includes hypertrophy， fibrosis， and atrophy. These changes of heart will cause impaired ventricular systolic and diastolic function.
　　Angiotensin converting enzyme inhibitor， aldosterone receptor antagonist and beta-blocker has been proved their beneficial effects on cardiac remodeling. Many studies showed that oxidative stress plays an important role in cardiac remodeling. However， the underlying molecular mechanism is still unclear. In our laboratory， we performed several studies for search the possible pathway of effect of reactive oxygen species (ROS) on cardiac remodeling. And we found the beneficial effect of antioxidants on cardiac remodeling.

　　Cardiac Hypertrophy
　　Mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced beta-myosin heavy chain gene expression and cardiomyocyte hypertrophy:
　　Endothelin-1 (ET-1) has been found to increase cardiac beta-myosin heavy chain (beta-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced beta-MyHC gene expression and hypertrophy in cardiomyocytes， however， has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced beta-MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1， cultured neonatal rat cardiomyocytes were examined for 3H-leucine incorporation and beta-MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and beta-MyHC gene expression. ET-1 increased 3H-leucine incorporation and beta-MyHC promoter activities， which were blocked by the specific ET(A) receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced 3H-leucine incorporation， beta-MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase， p38， and c-Jun NH2 -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased 3H-leucine incorporation and beta-MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras， Raf， and MEK1 decreased the ET-1-induced beta-MyHC promoter activities， suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the beta-MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced beta-MyHC gene expression. Moreover， ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and beta-MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways， which culminates in hypertrophic responses and beta-MyHC expression.
　　Inhibitory effect of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy:
　　The myocardial protective effects of trilinolein， isolated from the Chinese herb Sanchi (Panax notoginseng)， may be related to its antioxidant effects. We investigated the effects of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with angiotensin II， [3H]leucine incorporation and the beta-myosin heavy chain promoter activity were examined. We also examined the effects of trilinolein on angiotensin II-induced intracellular reactive oxygen species generation. Trilinolein significantly inhibited angiotensin II-increased protein synthesis， beta-myosin heavy chain promoter activity， and intracellular reactive oxygen species generation. Antioxidant N-acetylcysteine also decreased angiotensin II-increased protein synthesis and beta-myosin heavy chain promoter activity. Furthermore， trilinolein and N-acetylcysteine decreased angiotensin II- or hydrogen peroxide (H2O2)-activated mitogen-activated protein kinases (MAPKs) phosphorylation， and activator protein-1 (AP-1)- [or nuclear factor-kappaB (NF-kappaB)]-reporter activities. These data indicate that trilinolein inhibits angiotensin II-induced cardiomyocyte hypertrophy and beta-myosin heavy chain promoter activity via attenuation of reactive oxygen species generation. We also demonstrated trilinolein could inhibit cardiac hypertrophy induced by epinephrine and endothelium.
　　Urotensin II induces rat cardiomyocyte hypertrophy via the transient oxidization of Src homology 2-containing tyrosine phosphatase and transactivation of epidermal growth factor receptorUrotensin II induces rat cardiomyocyte hypertrophy via the transient oxidization of Src homology 2-containing tyrosine phosphatase and transactivation of epidermal growth factor receptor:
　　Urotensin II (U-II) is implicated in cardiomyocyte hypertrophy， which results in cardiac remodeling. We recently demonstrated that both reactive oxygen species (ROS) generation and epidermal growth factor receptor (EGFR) transactivation play critical roles in U-II signal transduction. However， the detailed intracellular mechanism(s) underlying cardiac hypertrophy and remodeling remain unclear. In this study， we used rat cardiomyocytes treated with U-II to investigate the association between ROS generation and EGFR transactivation. U-II treatment was found to stimulate cardiomyocyte hypertrophy through phosphorylation of EGFR and ROS generation. Apocynin， an NAD(P)H oxidase inhibitor， and N-acetyl cysteine (NAC)， an ROS scavenger， both inhibited EGFR transactivation induced by U-II. In contrast， 4-(3’-chloroanilino)-6，7-dimethoxy-quinazoline (AG1478， an EGFR inhibitor) failed to inhibit intracellular ROS generation induced by U-II. Src homology 2-containing tyrosine phosphatase (SHP-2)， but not protein tyrosine phosphatase 1B (PTP 1B)， was shown to be associated with EGFR during U-II treatment by EGFR coimmunoprecipitation. ROS have been reported to transiently oxidize the catalytic cysteine of phosphotyrosine phosphatases， subsequently inhibiting their activity. We examined the effect of U-II on SHP-2 and PTP 1B in cardiomyocytes using a modified malachite green phosphatase assay. SHP-2， but not PTP 1B， was transiently oxidized during U-II treatment， which could be repressed by NAC treatment. In SHP-2 knockdown cells， U-II-induced phosphorylation of EGFR and myocyte hypertrophy were dramatically elevated， and these effects were not influenced by NAC. Our data suggest that U-II-mediated ROS generation can transiently inhibit SHP-2 activity， thereby facilitating EGFR transactivation and hypertrophic signal transduction in rat cardiomyocytes.

　　Cardiac Fibrosis
　　Uric acid activates extracellular signal-regulated kinases and thereafter endothelin-1 expression in rat cardiac fibroblasts:
　　The association between hyperuricemia and cardiovascular diseases has long been recognized. Elevated levels of uric acid may have a causal role in hypertension and cardiovascular diseases. However， the direct effect of uric acid on cardiac cells remains unclear. Therefore， this study was aimed to examine the effect of uric acid in rat cardiac fibroblasts and to identify the putative underlying signaling pathways. Cultured rat cardiac fibroblasts were stimulated with uric acid; cell proliferation and endothelin-1 (ET-1) gene expression were examined. The effect of uric acid on NADPH oxidase activity， reactive oxygen species (ROS) formation， and extracellular signal-regulated kinases (ERK) phosphorylation were tested to elucidate the intracellular mechanism of uric acid in ET-1 gene expression. Uric acid-increased cell proliferation and ET-1 gene expression. Uric acid also increased NADPH oxidase activity， ROS formation， ERK phosphorylation， and activator protein-1 (AP-1)-mediated reporter activity. Antioxidants suppressed uric acid-induced ET-1 gene expression， and ERK phosphorylation， and AP-1 reporter activities. Mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in uric acid-induced ET-1 gene expression. These results suggest that uric acid-induced ET-1 gene expression， partially by the activation of ERK pathway via ROS generation in cardiac fibroblasts.
　　Involvement of reactive oxygen species in urotensin II-induced proliferation of cardiac fibroblasts:
　　Urotensin II， a cyclic dodecapeptide， has recently been demonstrated to play an important role in cardiac remodeling and fibrosis. Cardiac fibroblast is the cell type known to proliferate during cardiac fibrosis and to produce the excess matrix proteins characteristic of cardiac remodeling. However， the effect of urotensin II on cardiac fibroblast proliferation and the intracellular mechanisms remain to be clarified. Cultured neonatal rat cardiac fibroblasts were stimulated with urotensin II， cell proliferation and the reactive oxygen species generation were examined. We also examined the effects of antioxidant pretreatment on urotensin II-induced cell proliferation， extracellular signal-regulated kinase phosphorylation， and the tyrosine phosphorylation of epidermal growth factor receptor， to elucidate the redox-sensitive pathway in urotensin II-induced cell proliferation. Urotensin II-increased cell proliferation and intracellular reactive oxygen species levels which were inhibited by antioxidants N-acetylcysteine， and the flavin inhibitor diphenyleneiodonium. Urotensin II potently activated the tyrosine phosphorylation of epidermal growth factor receptors and extracellular signal-regulated kinase. Pretreatment of cells with U0126， an inhibitor of the upstream activator of mitogen-activated protein kinase kinase， or with AG1478， a selective epidermal growth factor receptor kinase inhibitor， reduced the urotensin II-increased extracellular signal-regulated kinase phosphorylation. Antioxidants， U0126， and AG1478， all significantly inhibited urotensin II-increased cell proliferation in cardiac fibroblasts. Our data suggest that the redox-sensitive intracellular signaling pathway plays a role in urotensin II-induced proliferation in rat cardiac fibroblasts.
　　Magnolol depresses urotensin-II-induced cell proliferation in rat cardiac fibroblasts:
　　Accumulating evidence suggests that oxidative stress plays a key role in the development of cardiac fibrosis. Urotensin-II (U-II) has been reported to play an important role in cardiac remodelling and fibrosis. Recently， we demonstrated the involvement of reactive oxygen species (ROS) production in U-II-induced cardiac fibroblast proliferation. Magnolol is an anti-oxidant compound extracted from the cortices of Magnolia officinalis. Thus， it is feasible that magnolol may attenuate cardiac fibroblast proliferation by inhibiting ROS production. Therefore， the aims of the present study were to determine whether magnolol alters U-II-induced cell proliferation and to identify the putative underlying signalling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pretreated with magnolol (1， 3 and 10 micromol/L) for 30 min， followed by exposure to U-II (30 nmol/L) for 24 h， after which cell proliferation and endothelin-1 (ET-1) protein secretion was examined. The effects of magnolol on U-II-induced ROS formation and extracellular signal-regulated kinase (ERK) phosphorylation were examined to elucidate the intracellular mechanisms by which magnolol affects cell proliferation and ET-1 expression.  Urotensin-II (30 nmol/L) stimulated cell proliferation， ET-1 protein secretion and ERK phosphorylation， all of which were inhibited by magnolol (10 micromol/L). Pretreatment of cardiac fibroblasts with N-acetylcysteine (5 mmol/L) for 30 min prior to exposure to U-II resulted in inhibition of U-II increased ROS formation. Similar effects were observed with 10 micromol/L magnolol. The results suggest that magnolol inhibits cardiac fibroblast proliferation by interfering with ROS generation. Thus， the present study provides important new insights into the molecular pathways involved， which may contribute to our understanding of the effects of magnolol on the cardiovascular system.
　　Tanshinone IIA inhibits angiotensin II-induced cell proliferation in rat cardiac fibroblasts:
　　Tanshinone IIA extracted from danshen， a popular medicinal herb used in traditional Chinese medicine， exhibits cardio-protective effects. However， the mechanism of its cardioprotective effect is not well established. The aims of this study were to examine whether tanshinone IIA may alter angiotensin II (Ang II)-induced cell proliferation and to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pre-treated with tanshinone IIA and stimulated with Ang II， cell proliferation and endothelin-1 (ET-1) expression were examined. The effect of tanshinone IIA on Ang II-induced reactive oxygen species (ROS) formation， and extracellular signal-regulated kinase (ERK) phosphorylation were also examined. In addition， the effect of tanshinone IIA on nitric oxide (NO) production， and endothelial nitric oxide synthase (eNOS) phosphorylation were tested to elucidate the intracellular mechanism. The increased cell proliferation and ET-1 expression by Ang II (100 nM) were partially inhibited by tanshinone IIA. Tanshinone IIA also inhibited Ang II-increased ROS formation， and ERK phosphorylation. In addition， tanshinone IIA was found to increase the NO generation， and eNOS phosphorylation. N(G)-nitro-L-arginine methyl ester (L-NAME)， an inhibitor of NOS， and the short interfering RNA transfection for eNOS markedly attenuated the inhibitory effect of tanshinone IIA on Ang II-induced cell proliferation. The results suggest that tanshinone IIA prevents cardiac fibroblast proliferation by interfering with the generation of ROS and involves the activation of the eNOS-NO pathway.

　　Cardiomyocyte Apoptosis
　　L-carnitine reduces doxorubicin-induced apoptosis through a prostacyclin-mediated pathway in neonatal rat cardiomyocytes:
　　Clinical use of doxorubicin is greatly limited by its severe cardiotoxic side effects. L-carnitine is a vitamin-like substance which has been successfully used in many cardiomyopathies， however， the intracellular mechanism(s) remain unclear. The objective of this study was set to evaluate the protective effect of L-carnitine on doxorubicin-induced cardiomyocyte apoptosis， and to explore its intracellular mechanism(s). Primary cultured neonatal rat cardiomyocytes were treated with doxorubicin (1 ?M) with or without pretreatment with L-carnitine (1-30 mM). Lactate dehydrogenase assay， terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining， and flow cytometry measurement were used to assess cytotoxicity and apoptosis. Fluorescent probes 2’，7’-dichlorofluorescein diacetate and chemiluminescence assay of superoxide production were used to detect the production of reactive oxygen species. Western blotting was used to evaluate the quantity of cleaved caspase-3， cytosol cytochrome c， and Bcl-x(L) expression. L-carnitine inhibited doxorubicin-induced reactive oxygen species generation and NADPH oxidase activation， reduced the quantity of cleaved caspase-3 and cytosol cytochrome c， and increased Bcl-x(L) expression， resulting in protecting cardiomyocytes from doxorubicin-induced apoptosis. In addition， L-carnitine was found to increase the prostacyclin (PGI(2)) generation in cardiomyocytes. The siRNA transfection for PGI(2) synthase significantly reduced L-carnitine-induced PGI(2) and L-carnitine’s protective effect. Furthermore， blockade the potential PGI(2) receptors， including PGI(2) receptors (IP receptors)， and peroxisome proliferator-activated receptors alpha and delta (PPARα and PPARδ)， revealed that the siRNA-mediated blockage of PPARα considerably reduced the anti-apoptotic effect of L-carnitine. These findings suggest that L-carnitine protects cardiomyocytes from doxorubicin-induced apoptosis in part through PGI(2) and PPARα-signaling pathways， which may potentially protect the heart from the severe toxicity of doxorubicin.
　　Tanshinone IIA prevents doxorubicin-induced cardiomyocyte apoptosis through Akt-dependent pathway:
　　Doxorubicin， one of the original anthracyclines， remains among the most effective anticancer drugs ever developed. Clinical use of doxorubicin is， however， greatly limited by its serious adverse cardiac effects that may ultimately lead to cardiomyopathy and heart failure. Tanshinone IIA is the main effective component of Salvia miltiorrhiza known as ’Danshen’ in traditional Chinese medicine for treating cardiovascular disorders. The objective of this study was set to evaluate the protective effect of tanshinone IIA on doxorubicin-induced cardiomyocyte apoptosis， and to explore its intracellular mechanism(s). Primary cultured neonatal rat cardiomyocytes were treated with the vehicle， doxorubicin (1μM)， tanshinone IIA (0.1， 0.3， 1 and 3μM)， or tanshinone IIA plus doxorubicin. We found that tanshinone IIA (1 and 3μM) inhibited doxorubicin-induced reactive oxygen species generation， reduced the quantity of cleaved caspase-3 and cytosol cytochrome c， and increased BcL-x(L) expression， resulting in protecting cardiomyocytes from doxorubicin-induced apoptosis. In addition， Akt phosphorylation was enhanced by tanshinone IIA treatment in cardiomyocytes. The wortmannin (100nM)， LY294002 (10nM)， and siRNA transfection for Akt significantly reduced tanshinone IIA-induced protective effect. These findings suggest that tanshinone IIA protects cardiomyocytes from doxorubicin-induced apoptosis in part through Akt-signaling pathways， which may potentially protect the heart from the severe toxicity of doxorubicin. We also demonstrate tanshinone IIA can attenuate angiotensin II-induced cardiomycyte apoptosis via its antioxidant effect.
　　Tanshinone IIA attenuates angiotensin II-induced apoptosis via Akt pathway in neonatal rat cardiomyocytes:
　　We also examine the effects of tanshinone IIA， the main effective component of Salvia miltiorrhiza (known as ’Danshen’ in traditional Chinese medicine) on angiotensin II (Ang II)-mediated cardiomyocyte apoptosis. rat neonatal cardiomyocytes were primarily cultured with Ang II or Ang II plus tanshinone IIA. Myocyte apoptosis was evaluated by caspase-3 activity and DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining. Western blot analysis was employed to determine the related protein expression and flow cytometry assay was used to determine the TUNEL positive cells and the intracellular reactive oxygen species (ROS) production. SiRNA targeted to Akt was used. Ang II (0.1 micromol/L) remarkably increased caspase-3 activity， TUNEL positive cells， and cleaved caspase-3 and cytochrome c expression， but reduced Bcl-X(L) expression. These effects were effectively antagonized by pretreatment with tanshione IIA (1-3 micromol/L). Tanshinone IIA had no effect on basal ROS level， while attenuated the ROS production by Ang II. Interestingly， tanshione IIA significantly increased the phosphorylated Akt level， which was countered by the PI3K antagonist wortmannin or LY294002. Knockdown of Akt with Akt siRNA significantly reduced Akt protein levels and tanshinone IIA protective effect. Tanshinone IIA prevents Ang II-induced apoptosis， thereby suggesting that tanshinone IIA may be used for the prevention of the cardiac remodeling process.

　　Conclusion
　　In conclusion， our studies demonstrated oxidative stress plays an important role in the process of cardiac remodeling. Reactive oxygen species causes hypertrophy， fibrosis and apoptosis of heart. Antioxidants may be useful in attenuation of injury of ROS. Underlying intracellular molecular mechanism may explore further studies in potential drugs development and clinical application.

                                                                                                                                                                              （参考文献备索）