Salusins promote cardiomyocyte growth but does not affect cardiac function in rats

Regulatory Peptides 122 (2004) 191 – 197 www.elsevier.com/locate/regpep

Salusins promote cardiomyocyte growth but does not affect cardiac function in rats Fang Yu a, Jing Zhao a, Jinghui Yang a, Bin Gen a, Shuheng Wang a, Xinheng Feng b, Chaoshu Tang a,c,d, Lin Chang c,d,* a

Department of Physiology, Health Science Center, Peking University, Beijing 100083, PR China b Echo Lab of Cardiology, Third Hospital, Peking University, Beijing 100083, PR China c Institute of Cardiovascular Disease Research, First Hospital, Peking University, Beijing 100034, PR China d Key Lab for Cardiovascular Molecular Biology of Health Ministry of China, Beijing 100083, PR China Received 23 April 2004; accepted 9 June 2004 Available online 18 August 2004

Abstract Salusin-a and -h are newly found polypeptides that stimulate proliferation, hypotension and bradycardia in vascular smooth muscle cells (VSMCs) and fibroblasts. Propresalusin mRNA is widespread, and positive stains for salusins have been observed in many human tissues such as endothelium and ventricular tissue. To investigate the bio-effect of salusins on cardiovascular function, 20 nmol/kg salusin-a or 2 nmol/kg salusin-h was intravenously (i.v.) injected into rats, and isolated rat hearts were perfused with 10 12 to 10 7 mol/l salusin-a or -h. 45 Ca2 + uptake and 3H-Leucine incorporation were determined in cultured neonatal rat cardiomyocytes. Neither salusin-a nor -h affected cardiac function in vivo or in vitro but salusin-h decreased mean arterial blood pressure (MAP). The polypeptides’ stimulation of 45Ca2 + uptake and 3H-Leucine incorporation was concentration-dependent, and the incorporation was inhibited by nicardipine (Nic) and FK-506 [FK; an inhibitor of calcineurin (CaN)]. PD98059 [PD; inhibitor of mitogen-activated protein kinase (MAPK)] and chelerythrine [inhibitor of protein kinase C (PKC)] inhibited salusin-stimulated 3H-Leucine incorporation. Endothelin-1 (ET) synergistically increased salusin-induced 45 Ca2 + uptake. Our results suggest that salusin-a and -h did not directly affect cardiac function in the rat heart but that they improved calcium uptake and protein synthesis in neonatal rat cardiomyocytes through the calcium, calcineurin, MAPK and PKC signal pathways. Salusins may be regulatory factors for myocardial growth and hypertrophy. D 2004 Elsevier B.V. All rights reserved. Keywords: Salusin; Rat; Cardiac function; Cardiomyocyte

1. Introduction Shichiri et al. [1] recently identified and characterized two related peptides, of 28 and 20 amino acids, designated salusin-a and -h, respectively. Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. TOR2A is predicted to have five exons and four introns and to produce a 321-amino acid protein with 39% amino acid identity to

* Corresponding author. Institute of Cardiovascular Disease Research, First Hospital, Peking University, Xishiku Street 38, Xichen District, Beijing 100034 PR China. Tel.: +86-10-82802851; fax: +86-10-66176255. E-mail address: [email protected] (L. Chang). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.06.013

the 332-amino acid TOR1A protein. Preprosalusin mRNA may be generated by alternatively splicing TOR2A with a different reading frame in exon 5 which encodes the salusin peptides. The N-terminal 26 amino acids of preprosalusin are predicted to be a signal peptide, the removal of which generates a 216-amino acid prosalusin. Salusins are expressed and synthesized within human tissues, including vessels and kidney, and are present in human body fluids. Salusins stimulate the proliferation of quiescent vascular smooth muscle cells (VSMCs) and fibroblasts, increase the level of [Ca2 +]i via the influx of extracellular Ca2 + into cells and induce the expression of growth-associated genes such as c-myc and c-fos. Salusins also cause rapid and profound decreases in blood pressure and heart rate (HR) [1]. These data suggest that salusins may

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be new regulatory peptides relevant to the cardiovascular system. However, the cardiovascular effects of salusins are not well known. In the present study, we observed the effects of salusins on rat hemodynamics in vivo and perfused isolated rat hearts and on cellular growth in cultured neonatal rat cardiomyocytes in vitro.

2. Materials and methods 2.1. Materials Salusin-a and -h and endothelin-1 (ET) were purchased from Phoenix Pharmaceuticals (CA, USA), nicardipine (Nic) and chelerythrine (Ch) chlorine from Sigma (MO, USA), Dulbecco’s modified eagle medium (DMEM) from Gibco (Grand Island, NY, USA), 45CaCl2 (185 MBq/mL) and 3H-Leucine (3H-Leu; 37 MBq/ml) from NEN Life Sci. (Boston, MA, USA) and FK-506 (FK) and PD98059 (PD) from Calbiochem – Novabiochem (San Diego, CA, USA). All other reagents were analytical.

Echocardiography System (Acuson, Mountain View, CA) equipped with a 7.0-MHz mechanical probe (focus depth set at 3.0 cm, sectorial angle of 60j). For the procedure, animals were anesthetized with sodium pentobarbital (30 mg/kg, i.p.). The rat’s chest wall was shaved, and the rat was placed in a semileft– lateral decubitus position. The transducer was placed on the left thorax, and M-mode recordings were made by directing the ultrasound beam at the midpapillary muscle level. For all animals, three to four beats were recorded by using the same transducer position. After acquired the normal echocardiograms, salusin-a (2 or 20 nmol/kg) or -h (2 nmol/kg) were injected into the left femoral vein to record the echocardiograms. Mean values were used for analysis. The left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) were obtained with the leading-edge method [3]. All parameters were measured with electronic calipers, and mean calculations were obtained from three or more consecutive cardiac cycles. 2.4. Perfusion of isolated hearts

2.2. Measurement of hemodynamic parameters in rats in vivo Hemodynamic parameters were determined with a polygraph via the femoral artery and intraventricular cannula [2]. Male Sprague – Dawley rats (200 to 250 g) were purchased from the Experimental Animal Center, Peking University (Beijing, China). Animal use was in accordance with the Chinese Council on animal care guidelines. Hemodynamic parameters were determined with use of a polygraph via an arterial and intraventricular cannula. The rats were anesthetized by intraperitoneal (i.p.) injection of urethane (1 g/kg). Two PE-50 tubes were inserted into the left femoral and right carotid arteries, respectively. The latter tube was inserted further into the left ventricle. All catheters were filled with 0.9% NaCl containing 10 kU/l of heparin. The ventricular and arterial catheters were separately connected to pressure transducers. Heart rate (HR), mean arterial blood pressure (MAP), left ventricular end diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP) and the maximal rates of left ventricular pressure development (LVdp/dtmax) were recorded on a microcomputer-controlled physiological polygraph (Biolab, Australia). After 10-min equilibration, salusin-a (2 or 20 nmol/kg) and -h (2 nmol/kg) were injected into the left femoral vein of the salusin-a and -h rats, respectively. After the MAP returned to basal level, 0.3 Ag/kg isoprotorenol as a positive control was intravenously (i.v.) injected to observe hemodynamic parameters. 2.3. Echocardiographic studies All echocardiograms were obtained by the same experienced sonographer by using the Sequoia C256

Male Sprague –Dawley rats (200 to 250 g) were anesthetized with urethane and promptly decapitated. The hearts were quickly removed and placed in ice-cold Krebs-Henseleit (KH) buffer (118 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4, 25 mmol/l NaHCO3, 11 mmol/l glucose, pH 7.4) and mounted on a Langendorff setup as described [4]. The hearts were randomly divided into control, salusina and salusin-h groups (n = 8 in each group). All hearts were preperfused with KH buffer oxygenated with 95% O2 –5% CO2 for 20 min at 37 jC with constant pressure of 80 cm H2O. The hearts in the control group were continuously perfused with KH buffer, and the KH buffers contained 10 12 to 10 7 mol/l salusin-a or -h for the salusin-a and -h groups, respectively. The changes in the left ventricular pressure were recorded on a physiological polygraph through a catheter with a latex balloon inserted into the left ventricle. 2.5. Culture of neonatal rat cardiomyocytes Cardiomyocytes were isolated from 1-day-old SD rats by the methods previously described, with minor modification [5]. In brief, the ventricles were minced and digested with 1 mmol/l pancreatase at 37 jC for 30 min. The digested fractions were filtered through a sterile wire mesh. The pooled cells were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and were centrifuged (10 min at 200  g), washed in DMEM containing 10% FBS and the preplated in 100-mm culture disses at 37 jC for 30 min to remove the majority of fibroblasts. The nonattached cells were then removed, counted and plated in 35-mm2 plates at an initial plating

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than 95% after each of the following treatments, as observed on trapan blue dying (data not shown). 2.6. Measurement of

45

Ca2+ uptake in cardiomyocytes

45

Ca2 + uptake in cardiomyocytes was determined as previously reported [6]. Cultured cardiomyocytes (5  104/ well) were incubated with salusin-a and -h in different concentrations (10 12 to 10 7 mol/l) for 4 h. A total of 37 KBq 45CaCl2/well was then added, and cells were further cultured for 8 h. To observe the effects of extracellular Ca2 + and calcineurin (CaN) signaling on 45Ca2 + uptake, the cells were pretreated for 20 min with 10 5 mol/ l nicardipine (L-type calcium channel blocker) or 10 6 mol/l FK-506 (a inhibitor of CaN) before treatment with 10 8 mol/l of salusin-a or -h. At the end of the culture, the cells were washed with cold phosphate buffer solution (PBS) three times and were then collected by use of a Millipore filter (Type HA; pore size, 0.45 Am) under vacuum negative pressure. After the addition of 5-ml scintillation fluid, radioactivity was determined by a hscintillation counter (Beckman LS 6500). The cells incubated with 10 8 mol/l endothelin-1 served as positive control. 2.7. Measurement for 3H-Leu incorporation in cardiomyocytes 3

H-Leu incorporation in cardiomyocytes was determined as previously reported [7]. After being starved for 24 h in free FBS medium, cardiomyocytes were incubated for 12 h with salusin-a or -h (10 12, 10 10, 10 8 and 10 7 mol/l) and salusin-a or -h (10 8 mol/l) plus various inhibitors: 10 6 mol/l FK-506, 10 5 mol/ l nicardipine, 10 6 mol/l PD98059 and 10 5 mol/l chelerythrine. The various inhibitors were preadded for 20 min. The cells treated with free or 15% FBS served as controls and positive controls, respectively. A total of 37 KBq 3H-Leu/well was then added, and cells were further

Fig. 1. Effects of salusin on hemodynamic parameters in rats in vivo. Hemodynamic parameters were determined as described in Section 2. Intravenous administration of 2 nmol/kg salusin-h to rats in vivo caused a profound decrease in mean arterial blood pressure (MAP) 30 s after administration, which returned to baseline 10 min later. However, intravenous administration of salusin-a (2 or 20 nmol/kg, only shown 20 nmol/kg) had no effect on MAP (A). Neither salusin-a nor -h changed left ventricular end systolic pressure (LVESP), left ventricular end diastolic pressure (LVEDP; B) and the maximal rates of left ventricular pressure development (LVdp/dtmax; C).

density of 10 000 cells/mm2. Myocytes were allowed to attach for 18 h in DMEM containing 10% FBS, 0.1 mmol/l bromodeoxyuridine (BdU) and 100 units/ml penicillin and streptomycin. The viability of cells was higher

Table 1 Echocardiographic data Salusin-a

HR (bpm) LVEDD (cm) LVESD (cm) LVEDV (ml) LVSV (ml/stroke) LV EF (%)

Salusin-h

Baseline

Salusin-a injection

Baseline

Salusin-h injection

340 F 22 0.68 F 0.03 0.26 F 0.01 0.48 F 0.1 0.45 F 0.06 63.9 F 1.03

343 F 26 0.65 F 0.01 0.25 F 0.03 0.43 F 0.15 0.43 F 0.15 60.8 F 2.5

345 F 18 0.67 F 0.06 0.28 F 0.03 0.68 F 0.15 0.44 F 0.18 61.7 F 1.58

341 F 25 0.64 F 0.01 0.25 F 0.03 0.63 F 0.05 0.46 F 0.08 63.5 F 2.05

HR: heart rate; LVEDD: Left ventricular end-diastolic dimension; LVESD: Left ventricular end-systolic dimension; LVEDV: Left ventricular end-diastolic volume; LVSV: left ventricular stroke volume; LV EF: Left ventricular ejection fraction. Data are means F S.E.M. n = 6 in each group.

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Fig. 2. Echocardiograms. An M-mode echocardiogram from a rat injected with salusins [before (A) and after (B) 20 nmol/kg salusin-a injection, before (C) and after (D) 2 nmol/kg salusin-h injection].

cultured for 8 h. The cells were washed with cold PBS three times and were then collected by use of a Millipore filter (Type HA; pore size, 0.45 Am) under vacuum negative pressure. After the addition of 5-ml scintillation fluid, radioactivity was determined by use of a hscintillation counter.

2.8. Statistical analysis All data were expressed as mean F SEM. Data were first analyzed by use of one-way ANOVA and then the Student – Newman – Keuls test. A difference of P < 0.05 was considered significant.

Table 2 Effects of salusin on isolated cardiac function in rats LVESP (mmHg) Control Salusin-a 10 12 mol/l 10 10 mol/l 10 8 mol/l 10 7 mol/l Salusin-h 10 12 mol/l 10 10 mol/l 10 8 mol/l 10 7 mol/l Iosproterenol

LVEDP (mmHg)

+ LVdp/dtmax (mmHg)

LVdp/dtmax (mmHg)

CF(ml/min)

HR (bpm)

78 F 8

8F2

2481 F 289

1253 F 112

12 F 3

286 F 14

76 F 8 81 F 8 76 F 9 74 F 7

7F3 8F3 6F2 7F3

2466 F 252 2499 F 237 2438 F 253 2450 F 260

1208 F 122 1276 F 118 1258 F 130 1263 F 128

11 F 3 11 F 4 13 F 3 12 F 4

277 F 18 281 F 21 276 F 18 291 F 21

8F2 8F3 7F3 8F3 2 F 1**

2443 F 241 2469 F 254 2456 F 248 2438 F 254 3389 F 264**

1233 F 131 1241 F 148 1233 F 140 1277 F 133 2045 F 276**

11 F 5 13 F 3 12 F 2 12 F 3 16 F 2*

287 F 19 285 F 24 282 F 23 280 F 23 355 F 22*

78 F 8 77 F 6 83 F 8 84 F 7 121 F 6**

LVESP: left ventricular end systolic pressure, LVEDP: left ventricular end diastolic pressure, LVdp/dtmax: the maximal rates of left ventricular pressure development, CF: coronary flow; HR: heart rate. Data are means F S.E.M. n = 6 in each group. * P < 0.05 versus control. ** P < 0.01 versus control.

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3. Results 3.1. Effect of salusins on rat cardiac function Intravenous administration of 2 nmol/kg salusin-h to rats in vivo caused a profound decrease in MAP ( P < 0.01) 30 s after administration, which returned to baseline 10 min later. However, intravenous administration of salusin-a (2 or 20 nmol/kg) had no effect on MAP. Neither salusin-a nor -h changed intraventricular pressure (LVESP, LVEDP and F LVdp/dtmax) or heart rate (Fig. 1). However, intravenous injection of 0.3 Ag/kg isoproterenol significantly increased LVESP, LVEDP, F LVdp/dtmax and heart rate, by 21%, 43%, 48% and 22%, respectively. Cardiac function (as measured by echocardiogram) of the rats was not differed from the baseline after intravenously injection the 20 nmol/ kg salusin-a or 2 nmol/kg salusin-h (Table 1, Fig. 2). Perfusion with salusin-a and -h (from 10 12 to 10 7 mol/l) to isolated rat hearts did not change the cardiac contractility. The F LVdp/dtmax, LVEDP, LVESP and coronary flow were not changed after infusion with salusin-a and -h than baseline (Table 2). 3.2. Effect of salusins on

45

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cardiomyocytes with FK-506 plus 10 8 mol/l salusin-a was lower, by 19% ( P < 0.01), than that with salusin-a alone, and that with FK-506 plus 10 8 mol/l salusin-h was lower by 32% ( P < 0.01), than that with salusin-h alone. Nicardipine also inhibited the hypertrophic effect of salusins. Nicardipine plus 10 8 mol/l salusin-a gave 39% ( P < 0.01) lower results than that with salulsin-a alone and nicardipine plus 10 8 mol/l salusin-h gave 48% ( P < 0.01) lower results (Fig. 4B). Thus, the Ca2 +/CaN signal pathway mediates the effect of salusin-stimulated hypertrophy and growth of cardiomyocytes.

Ca2+ uptake in cardiomyocytes

Salusin-a and -h (from 10 12 to 10 7mol/l) significantly increased 45Ca2 + uptake in cultured cardiomyocytes in a concentration-dependent manner. Salusin-h increased 45 Ca2 + uptake more potently than did salusin-a (Fig. 3A). Nicardipine and FK-506 significantly inhibited salusinstimulated 45Ca2 + uptake, which suggests that the L-type calcium channel and CaN pathway mediates salusin-induced calcium transport in cardiomyocytes. It is well known that endothelin-1 is a potent stimulator of calcium influx into cells [8]. The 10 8-mol/l endothelin-1-stimulated 45 Ca2 + uptake was markedly higher (81%) than that of controls. The effects of endothelin-1 and salusin were additive (Fig. 3B). 3.3. Effect of salusins on cardiomyocytes

3

H-Leu incorporation in

Treatment in 15% FBS significantly stimulated 3H-Leu incorporation in cardiomyocytes by 4.6 times compared with FBS-free culture. Salusin-a and -h (from 10 12 to 10 7 mol/l) stimulated 3H-Leu incorporation similarly in a concentration-dependent manner, and the effects peaked at 10 8 mol/l. Treatment with 10 6 mol/l salusin-a increased 3 H-Leu incorporation, same as with 10 8 mol/l ( P>0.05). At the same concentrations, the effects of salusin-a and -h did not differ ( P>0.05; Fig. 4A). Thus, salusins promote growth and hypertrophy of cardiomyocytes. The Ca2 +/CaN signal pathway is important for stimulating cardiomyocyte hypertrophy [9]. Treatment with FK506, a CaN inhibitor, significantly decreased the salusinstimulated 3H-Leu incorporation. 3H-Leu incorporation in

Fig. 3. Salusin-stimulated 45Ca2 + uptake in cardiomyocytes. Cardiomyocytes were incubated with salusin-a and -h in different concentrations (10 12 to 10 7 mol/l) for 4 h. A total of 37 KBq 45CaCl2/well was then added, and cells were further cultured for 8 h (A). The cells were pretreated for 20 min with 10 5 mol/l nicardipine (Nic) or 10 6 mol/L FK-506 (FK) before treatment with 10 8 mol/l of salusin-a or -h (B). Endothelin-1 (ET) served as a positive control. Salusin-a and -h significantly increased 45Ca2 + uptake in a concentration-dependent manner. Salusin-h increased 45Ca2 + uptake more potently than did salusin-a (A). Nicardipine and FK-506 significantly inhibited salusin-stimulated 45Ca2 + uptake. The effects of endothelin-1 and salusin were additive (B). The data were shown as mean F S.E.M for six independent experiments. **P < 0.01 versus salusina control and ##P < 0.01 versus salusin-h control. Salusin 0 mol/l as control in panel A; **P < 0.01 versus control, ##P < 0.01 versus salusin-a alone and DP < 0.01 versus salusin-h alone in panel B.

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4. Discussion

Fig. 4. Salusin-stimulated 3H-leu incorporation in cardiomyocytes. After being starved for 24 h in free FBS medium, cardiomyocytes were incubated for 12 h with salusin-a or -h (10 12, 10 10, 10 8 and 10 7 mol/l) and salusin-a or -h (10 8 mol/l) plus various inhibitors: 10 6 mol/l FK-506 (FK), 10 5 mol/l nicardipine(Nic), 10 6 mol/l PD98059(PD) and 10 5 mol/ l chelerythrine (Ch). The various inhibitors were preadded for 20 min. A total of 37 KBq 3H-Leu/well was then added, and cells were further cultured for 8 h. Salusin-a and -h stimulated 3H-Leu incorporation similarly in a concentration-dependent manner. At the same concentrations, the effects of salusin-a and -h did not differ (A). FK-506, Nicardipine, PD98059 and chelerythrine attenuated the salusin-stimulated 3H-Leu incorporation (B). The data were shown as mean F S.E.M for six independent experiments. **P < 0.01 versus salusin-a control and ## P < 0.01 versus salusin-h control. Salusin 0 mol/l as control in panel A and salusin alone as control in panel B.

It is well known that mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) participate in myocardial growth and hypertrophy [10,11]. Treatment with PD98059, a MAPK inhibitor, significantly decreased 10 8 mol/lsalusin-a-induced 3H-Leu incorporation by 59%, compared with salusin-a alone ( P < 0.01). It also decreased 10 8 mol/l salusin-h-stimulated 3H-Leu incorporation by 60%, compared with salusin-h alone ( P < 0.01). Chelerythrine, a PKC inhibitor, attenuated the salusin-stimulated 3HLeu incorporation (Fig. 4B). Thus, the MAPK and PKC pathways could participate in salusin-stimulated hypertrophy and growth of cardiomyocytes.

The physiological function of salusins is poorly known as yet. Shichiri et al. [1] reported that intravenous administration of salusin-a (10 nmol/kg) or salusin-h (1 nmol/kg) to rats caused a profound decrease in MAP and heart rate. Salusins increased [Ca2 +]i in rat VSMCs and fibroblasts, and salusin-h increased [Ca2 +]i more potently than did salusin-a, endothelin-1 or angiotensin II in VSMCs. In addition, salusin-a and -h induced c-myc and c-fos expressions in VSMCs and fibroblasts but not in human endothelial cells. Salusin-h, not -a, stimulated the generation of intracellular cAMP in rat and human VSMCs but not in rat cardiomyocytes. Pretreatment with salusin-a did not block salusin-h binding to VSMCs, which suggests that salusin-a and -h do not share common cell surface receptors [1]. We concentrated on the cardiac action of salusins. The present study showed that perfusing isolated rat hearts with salusin-a or -h did not change cardiac contractility (LVESP, LVEDP and F LVdp/dtmax) and coronary flow and heart rate. However, rats intravenously injected in vivo with salusin-h, not -a, caused a rapid decrease of MAP, not cardiac function, which suggests not a direct cardiac action but decreased MAP in dilatory peripheral vessels. Although cardiomyocytes do not synthesize and secrete salusins [1], it is still unclear whether cardiomyocytes have salusin receptors. In this study, treating cultured cardiomyocytes with salusins significantly stimulated 3H-Leu incorporation which suggests that cellular protein synthesis increases and salusin receptors could exist on cardiomyocytes. Salusins could promote cardiomyocyte growth. Ca2 + is an important signal molecule in regulating cell growth, differentiation and proliferation. Our results showed that nicardipine and FK-506 significantly revised the salusininduced Ca2 + influx into cells. Activated CaN, a Ca2 +dependent kinase, promotes dephosphorylation and translocation of intracellular transcription factor NF-AT3 to the nucleus and regulates myocardial hypertrophy gene expression with CATA4 [12]. Our results showed that the Ca2 +/ CaN signal pathway participates in salusin-induced cardiomyocyte growth. Endothelin-1 is a strong stimulator of the calcium channel [8]. We found that the 45Ca2 + uptake effects of salusins were similar to that of endothelin-1 and had some additive effects, which suggests that salusins and endothelin-1 could coregulate cardiomyocyte growth. MAPK and PKC are other important kinases in regulating cardiomyocyte growth and hypertrophy [13]. MAPK, which is widespread in eukaryocytes, could lead to serine and tyrosine phosphorylation. Activated MAPK induces c-fos and c-jun expressions and promotes DNA synthesis and cardiomyocyte hypertrophy in VSMCs [11]. Chelerythrine (as inhibitor of PKC) and PD98059 (an inhibitor of MAPK) significantly inhibited salusin-induced 3H-Leu incorporation, which suggests that the MAPK and PKC signal pathways are involved in cardiomyocyte protein synthesis and growth.

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Our results showed that the signal pathways by which salusins promoted protein synthesis and growth were related to Ca2 +/CaN, MAPK and PKC. These pathways can crosstalk and may have an intracellular network-regulation role [10,11]. Although cardiomyocytes do not produce salusins, endothelium-generated salusins in the coronary artery may act on cardiomyocytes in a paracrine manner. We speculate that salusins could be important regulatory peptides in myocardial growth and hypertrophy. It is valuable to further investigate the physiological and pathophysiological significance of salusins on heart development and hypertrophy.

Acknowledgements This work was supported by the Major State Basic Research Development Program of the People’s Republic of China (G2000056905). We also thank Profs. Chaokang Chang and Jun Yang of Phoenix Pharmaceuticals (CA, USA) for the free use of salusin-a and -h.

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