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Leptin Increases Cardiomyocyte Hyperplasia via Extracellular Signal-Regulated Kinase- and Phosphatidylinositol 3-Kinase-Dependent Signaling Pathways

Department of Biology (P.T., R.B.C., I.R.C., G.S.), York University, and Toronto General Research Institute (R.-K.L.), Toronto General Hospital, Toronto, Ontario, Canada M3J 1P3

Address all correspondence and requests for reprints to: Gary Sweeney, Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3. E-mail: gsweeney{at}yorku.ca.

Abstract

Obesity is a major risk factor for the development of heart failure. Importantly, it is now appreciated that a change in the number of myocytes is one of multiple structural and functional alterations (remodeling) leading to heart failure. Here we investigate the effect of leptin, the product of the obese (ob) gene, on proliferation of human and murine cardiomyocytes. Leptin caused a time- and dose-dependent significant increase in proliferation of HL-1 cells that was inhibited by preincubation with PD98059 and LY294002, suggesting that leptin mediated proliferation via extracellular signal-regulated kinase-1/2- and phosphatidylinositol-3-kinase-dependent signaling pathways. We confirmed that leptin activates both extracellular signal-regulated kinase-1/2 phosphorylation and association of phosphatidylinositol-3-kinase (regulatory p85 subunit) with phosphotyrosine immunoprecipitates. We also examined bromodeoxyuridine incorporation as a measure of new DNA synthesis and demonstrated a stimulatory effect of leptin in both HL-1 cells and human cardiomyocytes. Bromodeoxyuridine incorporation in HL-1 cells was inhibited by PD98059 and LY294002. Our results establish a mitogenic effect of leptin in cardiomyocytes and provide additional evidence for a potential direct link between leptin and cardiac remodeling in obesity.

THE EMERGING OBESITY epidemic (1) is recognized as an independent risk factor for congestive heart failure, coronary artery disease, hypertension, diabetes, and other diseases (2, 3, 4). Indeed, obesity is now classified by the American Heart Association as a major modifiable risk factor for cardiovascular complications (3). The Framingham heart study found that the risk of heart failure was increased 34% in overweight [body mass index (BMI) of 25.0–29.9) and by 104% in obese (BMI > 30.0) individuals (5). However, the pathogenesis of heart failure in obese individuals is still unclear.

Heart failure is believed to manifest due to multiple structural and functional alterations (remodeling) that interact over a sustained period of time (6). Broadly speaking, the changes that occur include those affecting size or number of myocytes and those altering the composition and structure of the extracellular matrix (7). Importantly, the longstanding concept that ventricular myocytes are terminally differentiated cells has recently been revised to incorporate myocyte replication as a significant component of remodeling (8, 9). Whether cardiac remodeling leading to left ventricular hypertrophy occurs as a result of an increased hemodynamic load or via direct neurohumoral signaling has generated much debate.

Adipose tissue, once considered simply a lipid storage depot, is now known to be a dynamic endocrine organ secreting factors such as leptin, the product of the obese (ob) gene (4, 10). Fasting plasma leptin concentrations are associated with increased myocardial wall thickness, independent of body weight or blood pressure (11), and the role of leptin as a link between obesity and cardiovascular disease is rapidly emerging as an important research focus (12). In recent years it has become clear that leptin mediates many direct effects on peripheral tissues (13). Leptin has been shown to directly attenuate contraction (14) and induce (15) or prevent (16) hypertrophy in isolated myocytes. The effect of leptin on proliferation of cardiomyocytes has not yet been examined. Here we provide additional evidence for a potential direct link between leptin and cardiac remodeling in obesity by demonstrating a mitogenic effect of leptin on primary human pediatric cardiomyocytes and a stable murine cardiomyocyte cell line.

Materials and Methods

Culture of HL-1 cells
HL-1 cells (17) were maintained as previously described (18) using Claycomb medium (JRH Biosciences, Lenexa, KS), supplemented with 10% (vol/vol) fetal bovine serum (Wisent, Quebec, Canada), 4 mM L-glutamine (Wisent), 10 µM noradrenaline (norepinephrine) (Sigma Chemical Co., St. Louis, MO), and 1% (vol/vol) penicillin-streptomycin (Wisent). During leptin treatment periods, the cells were cultured in media containing 1% fetal bovine serum. Cells were grown at 37 C in an atmosphere of 5% CO₂. During continued growth, the medium was changed routinely every 48 h.

Isolation of human ventricular cardiomyocytes
The method used here for isolation of human ventricular cardiomyocytes has been described many times (19, 20, 21). Biopsy samples were obtained from children undergoing elective surgical repair of tetralogy of Fallot. All patients gave written informed consent to obtain biopsies through a protocol approved by the University of Toronto Human Experimentation Committee. Briefly, myocardial ventricular resection biopsies (20 mg) from children (6 months or older) were immediately washed in PBS and then digested by 0.1% collagenase/0.2% trypsin in PBS. Cardiomyocytes were subsequently purified by dilution cloning and maintained at 37 C (95% air/5% CO₂) in Iscove’s modified Dulbecco’s medium (Canada Life Technologies Inc., Burlington, Ontario, Canada) containing 10% fetal calf serum, 0.1 mM ß-mercaptoethanol, 0.4 mM L-arginine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Purification of a cardiomyocyte population can be confirmed by fluorescent staining with a monoclonal antibody for human ventricular myosin heavy chain (20, 22). During the leptin treatment period, cells were cultured in media containing 1% fetal bovine serum. Based on previous experience, cells were used for experiments only at passage 3 or 4 (23).

Cell counting
To measure cell number, 0.5 x 10⁶ cells were added per well of a six-well culture dish and allowed to grow for up to 96 h in the presence or absence of leptin (mouse recombinant, endotoxin: 1.0 EU/µg) (Calbiochem, La Jolla, CA) at the concentrations indicated in figure legends. Where indicated, cells were also pretreated for 1 h with PD98059 (20 µM) or LY294002 (20 µM). PD98059 and LY294002 were from Bioshop Canada Inc. (Burlington, Ontario, Canada). At each time point, cells from each well were harvested by trypsinization and counted using a hemocytometer. Eight fields of view were analyzed in a blinded manner for each of three experiments. Trypan blue dye exclusion was used to confirm viability of cells. To reinforce the accuracy of this approach, cell number was also determined using a Coulter counter.

Bromodeoxyuridine (BrdU) uptake assay
The effect of leptin on DNA synthesis was assessed by measuring BrdU incorporation into newly synthesized DNA using a colorimetric ELISA-based assay kit according to the manufacturer’s instructions (Oncogene Research Products, San Diego, CA). Briefly, HL-1 or human pediatric cardiomyocytes cultured in 96-well plates were stimulated with leptin (6–60 nM) for 12 h and then BrdU was added with subsequent incubation for an additional 12 h. Where indicated in HL-1 cell experiments, cells were also pretreated for 1 h with PD98059 (20 µM) or LY294002 (20 µM) before the addition of leptin. Thereafter, binding of anti-BrdU antibody to BrdU incorporated in DNA was detected by measuring absorbance at dual wavelengths of 450–540 nm using a spectrophotometer.

Analysis of extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI) 3-kinase activation
Cells grown on six-well plates were treated with leptin (6 nM, up to 15 min) and then washed quickly with ice-cold PBS. Lysates to assess ERK phosphorylation were prepared as previously described (24). Equal amounts of protein (40 µg) were then immunoblotted using a phospho-ERK antibody that specifically recognizes the kinase phosphorylated on Thr202/Tyr204 (Cell Signaling Technology, Beverly, MA). To determine association of p85 with phosphotyrosine, cell lysates were prepared and immunoprecipitation using antiphosphotyrosine antibody performed essentially as described previously (25). Immunoprecipitated proteins were immunoblotted with an antibody specifically recognizing the p85 regulatory subunit of PI 3-kinase (Santa Cruz Biotechnology, Santa Cruz, CA). Higher association of this subunit with phosphotyrosine immunoprecipitates is indicative of PI 3-kinase activation.

Statistical analysis
Statistical analysis was performed using either one- or two-way ANOVA with Tukey-Kramer multiple comparison test or Bonferroni post hoc tests where appropriate. The level of significance was set at P < 0.05.

Results

To investigate the effect of leptin on cardiomyocyte hyperplasia we have used both isolated pediatric human cardiomyocytes and an established murine cardiomyocyte cell line (17, 19). The cultured human cardiomyocytes isolated by clonal dilution measured approximately 40 x 80 µm, proliferate in vitro, and have been used extensively for analysis of myocyte function (19, 20, 21, 22). Characteristics of the murine cardiomyocytes used in this study include a mature cardiomyocyte phenotype, an ultrastructure similar to primary cultures of cardiomyocytes, cytoplasmic reorganization and myofibrillogenesis similar to that observed in mitotic cardiomyocytes in the developing heart, presence of highly ordered myofibrils, the ability to undergo spontaneous contraction, expression of cardiac-specific genes, and the presence of several cardiac-specific voltage-dependent characteristics (17). Furthermore, transplantation studies suggest HL-1 cells act as a functional equivalent of endogenous adult cardiomyocytes (26). A previous study using mouse heart suggests that both the short and long leptin receptor isoform transcripts are expressed and in a ratio of approximately 11:1, respectively (27). Before using HL-1 cells for this study we demonstrated expression of long and short isoforms in a similar ratio (data not shown). Hence, these cells represent an excellent model (28) to begin to elucidate the effects of leptin on the heart.

Effect of leptin on cardiomyocyte hyperplasia
To determine whether leptin altered cardiomyocyte growth, we treated cells with leptin (6 nM, 0–96 h) and then analyzed cell number using a hemocytometer. Leptin caused a time-dependent increase in proliferation of these cells (Fig. 1A). A small increase was evident after 4 h of leptin treatment and a statistically significant increase observed after 24 h. The magnitude of response observed is significant and typical of growth responses by other cells in response to leptin (29, 30). We also investigated the sensitivity of HL-1 cell growth to leptin, and Fig. 1B shows that when cells were incubated for 24 h in varying concentrations of leptin, the hormone caused a statistically significant increase in cell growth at all concentrations tested (0.3–60 nM). The magnitude of response elicited reached approximately 2.3-fold above control at 60 nM leptin. To further support our observations, results presented in Fig. 2 were confirmed using a Coulter counter (data not shown). Subsequent experiments were performed with 6 nM leptin.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Effect of leptin on proliferation of HL-1 cells. Cells were grown in the presence or absence of leptin and then cell number was determined. A, Time course showing untreated cells (white bars) and cells treated with 6 nM leptin for up to 4 d (black bars); B, dose response, varying leptin concentrations for 24 h. Results are expressed relative to initial number at time zero. Three independent experiments were conducted; *, P < 0.05 with respect to control at same time point.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Role of ERK and PI 3-kinase in stimulation of HL-1 cell growth by leptin. The mitogenic effect of leptin was examined in cells pretreated for 1 h with 20 µM PD98059 (A) or 20 µM LY294002 (B) before stimulation with 6 nM leptin for 24 h in the continued presence of inhibitor. Results are expressed relative to cell growth seen in control (C) samples. Results shown are mean ± SEM of three independent experiments; *, P < 0.05 with respect to control; #, P < 0.05 with respect to leptin alone. Western blots to determine the effect of leptin (6 nM) on ERK phosphorylation (C) and association of p85 with phosphotyrosine (D) are shown.

Effect of leptin on BrdU incorporation in HL-1 and human cardiomyocytes
To further investigate the effect of leptin on cardiomyocyte growth we have also measured incorporation of BrdU into DNA as an index of cell proliferation. Results shown in Fig. 3 confirm that leptin caused an approximately 2.3-fold increase in BrdU uptake in HL-1 cells. This effect was sensitive to inhibition by both PD98059 and LY294002 (Fig. 3). Importantly, we also used primary human cardiomyocytes and demonstrate that treatment with leptin for up to 48 h significantly increased BrdU uptake approximately 1.4-fold above control (Fig. 4). These results further support the conclusion that leptin directly stimulates cardiomyocyte cell division and thus increases cell number.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Effect of leptin on BrdU incorporation in HL-1 cells. HL-1 cells were treated with or without leptin (6 nM) for 24 h before analysis of BrdU incorporation into newly synthesized DNA. The inhibitors PD98059 (20 µM) and LY294002 (20 µM) were added 1 h before leptin and their presence maintained during subsequent incubation. Values are expressed relative to control, and the results represent mean ± SEM of three independent experiments. *, P < 0.05 with respect to control; #, P < 0.05 with respect to leptin alone.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Effect of leptin on BrdU incorporation in human cardiomyocytes. Primary human cardiomyocytes were incubated in the absence (white bars) or presence (black bars) of leptin (60 nM) for up to 48 h before analysis of BrdU incorporation into newly synthesized DNA. Values are expressed relative to BrdU uptake in untreated cells after 24 h. Data presented represent mean ± SEM of four independent experiments; *, P < 0.05 with respect to control at the corresponding time.

Although still controversial, it is likely that endocrine effects of factors secreted by fat tissue play an important role in the pathogenesis of heart failure associated with obesity (2, 12). Indeed, current treatment of heart failure is largely based on inhibition of neuroendocrine activation to limit progression of heart failure (36). Many studies have suggested that leptin plays a role in the pathogenesis of cardiovascular disease. Elevated plasma leptin levels are found in patients with congestive heart failure (37). Fasting plasma leptin concentrations in hypertensive men were significantly associated with myocardial wall thickness, independent of hypertension (11, 38). Plasma leptin levels correlate with heart rate, independently of plasma insulin, BMI, waist:hip ratio, and percentage body fat in men (39). The ability of leptin to attenuate cardiac muscle contraction has been proposed to explain the decrease in diastolic compliance and prolonged relaxation observed in obese individuals (14). Activation of fatty acid oxidation, and thus decreased triglyceride content, or altered adenylate cyclase function by leptin have been suggested to contribute to reduced cardiac function in obese individuals (40, 41). Intriguingly, a direct impact of leptin on remodeling was recently demonstrated by correction of ventricular hypertrophy in ob/obmice upon injection of leptin (16). Whereas this implies an antihypertrophic effect of leptin, another study in cultured myocytes suggested that leptin induces hypertrophy via a p38MAPK-dependent mechanism (15).

Only recently was the longstanding paradigm of the adult heart as a postmitotic organ challenged and the importance of myocyte death and regeneration realized (42). However, many studies have now noted an ability of fetal human cardiomyocytes (19, 21, 22), cells isolated from 30- to 65-yr-old adults with hypertrophic cardiomyopathy (43), and rodent cardiomyocytes (15, 17) to proliferate in culture. It is interesting to note that transplantation of fetal rat cardiomyocytes, which demonstrate hyperplasia in vitro, to sc tissue of an adult rat lose their ability to proliferate (44). This suggests that non-cardiomyocyte-derived factors act in vivo to regulate cardiomyocyte hyperplasia. Thus, investigations of the effects of adipose-derived factors on cardiomyocyte proliferation are timely and may enhance our understanding of the mechanisms responsible for the association between obesity and heart failure. The ability of leptin to regulate cell growth was demonstrated recently in a surprisingly wide variety of cell types including vascular smooth muscle cells (29), glomerular endothelial cells (45), tracheal epithelial cells (33), monocytes (46), and ß-cells (30). Here we make the novel observation that leptin increases proliferation of both human and murine cardiomyocytes. Importantly, our observations are made over a range of leptin concentrations that are similar to those found in human plasma of obese individuals: 0.06 nM (1 ng/ml) in normal to 6.1 nM (100 ng/ml) in obese (47, 48).

The signaling pathways activated by leptin (49) to mediate effects in cardiomyocytes are only beginning to be characterized. We show here that leptin activated both ERK1/2 and PI 3-kinase in cardiomocytes. It is known that sustained activation of ERK is sufficient to induce the proliferation of several cell types (31) and in fact three studies have demonstrated that the proliferative effect of leptin was reduced by the MAPK kinase1 inhibitor, PD98059 (30, 32, 33). Many growth factors also promote proliferation via phosphatidylinositol (PI) 3-kinase-mediated signaling pathways (34). Here we show that inhibitors of ERK1/2 activation and PI 3-kinase action reduced leptin-stimulated cardiomyocyte proliferation, implicating a role for these kinases in mediating this effect of leptin.

BrdU labeling has recently been used to demonstrate the existence of cycling myocytes in the adult myocardium (50). Thus, whereas many myocytes in the adult heart may be terminally differentiated there is a continuously renewing pool of myocytes that do enter the cell cycle in response to appropriate physiological stimuli (8, 9). It is likely that cell proliferation declines over time or with risk factors such as obesity, contributing to heart failure (51). Here we measured incorporation of BrdU into DNA as an index of cell proliferation and found that, in keeping with cell number analysis, leptin caused an increase in BrdU uptake in HL-1 cells, an effect that was sensitive to inhibition by both PD98059 and LY294002. Importantly, we also demonstrated the ability of leptin to increase BrdU uptake in human cardiomyocytes. This suggests that our studies are likely to be of direct physiological relevance in humans.

In summary, we demonstrate that leptin increased DNA synthesis and proliferation of human and murine cardiomyocytes. This likely occurs via ERK- and PI 3-kinase-dependent signaling pathways. The implications of our results are that leptin may promote myocardial growth and thus help repair the failing myocardium. However, as recently discussed (12), obese individuals are predominantly hyperleptinemic and leptin resistant. Under such circumstances we may speculate from our results that the contribution made by leptin to myocyte regeneration would be lost in obese individuals, thus causing or exacerbating heart failure. Although it is tempting to extrapolate observations made in isolated myocytes to in vivo physiology, care must be extended and appropriate in vivo experiments performed before definite conclusions are drawn. Hopefully our results will provide impetus for such studies and help establish a direct role of leptin on cardiac remodeling and thus in the pathogenesis of heart failure.

Acknowledgments

We are extremely grateful to Shuhong Li, Toronto General Hospital for assistance in providing human pediatric cardiomyocytes.

Footnotes

This work was supported by the Canadian Institutes of Health Research, Institute of Nutrition, Metabolism, and Diabetes, and the Canadian Diabetes Association (G.S. is recipient of a scholarship in honor of the late Mary A. Bodington and R.B.C. a postdoctoral fellowship in honor of the late Norman J. Newell).

Abbreviations: BMI, Body mass index; BrdU, bromodeoxyuridine; ERK, extracellular signal-regulated kinase; PI, phosphatidylinositol.

Received August 28, 2003.

Accepted for publication December 29, 2003.

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