Essential role of Rho/ROCK-dependent processes and actin dynamics in mediating leptin-induced hypertrophy in rat neonatal ventricular myocytes

Abstract

Background: Obesity is associated with increased leptin production, which may contribute to cardiac hypertrophy. Although leptin has been shown to produce cardiomyocyte hypertrophy, its mechanism of action is far from clear. Rho proteins have been suggested as major contributors to cardiac hypertrophy, although their potential role in mediating the effect of leptin has not been studied.

Methods: We determined the role of Rho and Rho-associated kinase (ROCK) as mediators of leptin-induced cell hypertrophy in cultured neonatal rat ventricular myocytes.

Results: Leptin (3.1 nmol/L) significantly increased cell surface area by 32±5% and leucine incorporation by 43±7%. These effects were associated with significant activation of RhoA to 450±40% of pre-leptin levels that was attenuated by pretreatment with an anti-leptin receptor (anti-OBR) antibody (166 ng/mL) to 120±20% of control values. Both the RhoA inhibitor C3 exoenzyme and ROCK inhibitor Y-27632 potently attenuated leptin-induced increased cell surface area and leucine incorporation. The hypertrophic effect of leptin was associated with an increase in phosphorylation of the actin binding protein cofilin to 290±20% of control values. In addition, the increase in polymerization of actin, as reflected by a decrease in the G/F-actin ratio, was significantly inhibited by both the anti-OBR antibody and Y-27632. Leptin-induced hypertrophy was also attenuated by disruption of actin filaments with 50 nmol/L latrunculin B. RhoA pathway inhibitors and latrunculin B also both attenuated leptin-induced ERK1/2 and p38 activation.

Conclusion: Our results indicate that the activation of Rho and actin dynamics play a pivotal role in leptin signaling leading to the development of cardiomyocyte hypertrophy.

1. Introduction

Obesity is associated with increased cardiovascular risk as well as markedly elevated circulating plasma leptin levels. Leptin is a 16-kDa protein predominantly produced by adipose tissue [1], but also by heart [2], vascular smooth muscle [3], placental tissue [4], digestive epithelia, and gastric mucosa [5]. The physiological effects of leptin are mediated via membrane-bound receptors (OBR) located not only in the central nervous system but also in many peripheral tissues [6,7] including in cardiomyocytes [2]. Activation of central OBR plays a key role regulating food intake and body weight [8], however recent studies have shown various peripheral physiological effects of leptin involving several signaling cascades including JAK/STAT [9], MAPK [10], and protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) [11]. Work from our laboratory as well as from others has demonstrated the ability of leptin to directly produce cell hypertrophy in rodent and human cardiomyocytes [12–14] as well as vascular smooth muscle [3,15]. The precise mechanisms underlying leptin's hypertrophic effects either in cardiomyocytes or vascular tissue are unclear at present although this appears to be related to MAPK activation [3,12,14,16], indeed inhibition of leptin-induced MAPK activation significantly attenuated leptin-induced cardiomyocyte hypertrophy [12] as well as leptin-induced proliferation in cardiac-derived HL-1 cells [16]. Others have suggested that leptin-induced cardiomyocyte hypertrophy may also be mediated by endothelin-1 (ET₁) dependent stimulation in the production of reactive oxygen species [13]. In this regard, either pharmacological blockade of the endothelin ET_A receptor or administration of hydrogen peroxide scavenging agent catalase attenuated both leptin-induced hypertrophy as well as the increased generation of reactive oxygen species [13]. Thus, there appears to be substantial complexity underlying the hypertrophic effects of leptin in cardiomyocytes.

Hypertrophic growth of neonatal cardiac muscle cells is associated with distinct morphological changes in cell shape and remodeling of the actin cytoskeleton. Rho/ROCK pathway, a downstream target protein of small GTP-binding protein Rho, is one of the major pathways that affect cell morphology [reviewed in Ref. 11], produces modifications in actin cytoskeletal apparatus [17] and regulates transcription factors leading to cellular hypertrophy [18]. RhoA appears to be an important mediator in hypertrophic responses [reviewed in 19]. Although its potential contribution to the hypertrophic effect leptin has hitherto not been investigated. Cardiomyocyte hypertrophy and myofibrillar assembly are blocked by inhibitory mutants of Rho-kinase (ROCK) [20], suggesting that this small G protein is an important mediator in the signal transduction events regulating hypertrophy. However, the mechanism leading to activation of Rho GTPases and subsequently to cardiac hypertrophy has not been well characterized. RhoA activates several protein kinases, including Rho kinases (ROCK) and the Rho effector protein mDia [21,22].

Activation of LIM kinase-2 (LIMK2) by Rho/ROCK results in phosphorylation (inactivation) of the actin binding protein cofilin, an important factor in the regulation of actin dynamics which in turn leads to depletion of globular actin (G-actin) pools and enhanced actin polymerization (F-actin). A strong relationship between actin dynamics and the activity of serum response factor (SRF) has been demonstrated in studies using either actin-binding drugs (actin-specific C2 toxin) or actin overexpression [23]. SRF is a transcription factor and has the ability to regulate the expression of different growth factors [24]. Recent investigations have shown that the activity of SRF in cardiac and smooth muscle is regulated by a coactivator called myocardin [25], a member of the SAP domain family of nuclear proteins which activates cardiac muscle promoters by associating with SRF.

In view of the critical role of the actin cytoskeleton and Rho/ROCK signaling pathways in cardiomyocytes hypertrophy [26] and because of the direct effect of leptin in cardiac and vascular hypertrophy [3,12,13] we hypothesized that leptin might be implicated in the modulation of actin cytoskeleton through RhoA/ROCK pathways. Our results indicate that the Rho/ROCK pathway and G/F-actin ratio are important components of the leptin-induced hypertrophic signaling in cardiac myocytes. Moreover, inhibiting the Rho/ROCK pathway and decreasing the G-actin pool might represent a critical target to reduce hypertrophy and remodeling associated with hyperleptinemia which may have important implications for reducing cardiac risk in obesity.

2. Materials and methods

2.1. Cardiomyocyte cultures

Neonatal cardiomyocytes were prepared from the ventricles of 1- to 3-day-old Sprague–Dawley rats (Charles River Canada, Montreal, Quebec, Canada), as previously described [12]. Briefly, ventricular myocytes were dissociated enzymatically and the cell suspension was centrifuged at 1000 rpm for 5 min. The washed cells were preplated for 60 min and unattached myocytes were collected with the medium. The cardiomyocytes were plated at 37 °C in humidified atmosphere with 5% CO₂. The myocytes were cultured under serum-free conditions for 24 h before initiating the study at which time the cells were cultured with or without 3.1 nmol/L leptin (Sigma–Aldrich, Oakville, Canada) for 24 h. For some experiments, myocytes were treated with various concentrations (1.66, 16.6 or 166 ng/mL) of leptin receptor antibody (OBR-Ab, Alpha Diagnostic International, Inc. San Antonio, TX), C3 exoenzyme (30 ng/mL; Alexis Biochemicals, Carlsbad, CA) a clostridial toxin that selectively inhibits Rho, Y-27632 (10 μmol/L, Sigma–Aldrich, Oakville, Canada) a selective ROCK inhibitor, and latrunculin B, (10 nmol/L; Calbiochem, San Diego, CA) an actin depolymerization agent, all agents being added 1 h before leptin administration and throughout the leptin treatment period. The protocols for the use of animals were approved by the University of Western Ontario Animal Care and Use Committee and conformed to guidelines in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.2. Measurement of cell surface area

To determine cell surface area, cells were visualized with a Leica inverted microscope and surface area was quantified by imaging the complete boundary of 45–50 individual cells/experiment using Mocha software (SPSS Inc., Chicago, IL).

2.3. Leucine incorporation

Serum starved cardiomyocytes treated for 24 h were subjected to [³H]-leucine (1 μCi/mL,) with or without leptin and the cells were incubated for an additional 24 h. Cell precipitates were solubilized in 0.2 N NaOH. The total radioactivity of incorporated [³H]-leucine into proteins was measured by liquid scintillation counting as described previously [3].

2.4. Western immunoblotting

Cardiomyocytes were lysed in lysis buffer as previously described [12]. Equal amounts of extracted proteins were separated on 12% SDS-PAGE, transferred to nitrocellulose membrane, and the Western blots probed with antibodies specific for the total or phosphorylated forms of ERK1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p38 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or cofilin-2 (Upstate, Lake Placid, NY). The proteins transferred to the membrane were detected using the ECL immunoblotting detection system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

2.5. RhoA activity assay

Following treatment, the culture media was removed and the cells were washed twice in ice-cold PBS and lysed. Thereafter, Rho activity was measured using the Rho activation assay kit (Upstate, Lake Placid, NY) according to the manufacturer's instructions. GTP-Rho in cell lysates was adsorbed to GST-Rhotekin Rho binding domain, which binds selectively to GTP-Rho, not GDP-Rho. After precipitation, samples were processed for Western blotting with a specific anti-Rho antibody (Upstate, Lake Placid, NY).

2.6. Measurement of G-actin/F-actin ratio

Determination of the amount F-actin content compared with free G-actin content was performed using the G-actin/F-actin in vivo assay kit (Cytoskeleton Inc., Denver, CO) according to the manufacturer's instructions. Briefly, upon exposure to various stimuli and/or inhibitors, the cardiomyocytes were homogenized in cell lysis and F-actin stabilization buffer (50 mmol/L PIPES, 50 mmol/L NaCl, 5 mmol/L MgCl₂, 5 mmol/L EGTA, 5% (v/v) lyceral, 0.1% (v/v) Nonidet P-40, 0.1% (v/v) Triton X-100, 0.1% (v/v) Tween 20, 0.1% (v/v) 2-mercaptoethanol and 0.001% (v/v) antifoam and a protease inhibitor cocktail followed by centrifugation for 1 h at 100000 g to separate the F-actin from G-actin pool. Supernatants of the protein extracts were collected after centrifugation at 100000 g for 60 min at 30 °C. The pellets were resuspended in ice-cold dH₂O plus 1 μmol/L cytochalasin D and then incubated on ice for 1 h to dissociate F-actin. The resuspended pellets were gently mixed every 15 min. Equal amounts of both the supernatant (G-actin) and the resuspended pellet (F-actin) were subjected to analysis of immunoblot with the use of an actin antibody (Cytoskeleton Inc., Denver, CO).

2.7. Immunofluorescent cell staining

Myocytes grown on glass coverslips were fixed for 10 min with freshly prepared 3.7% (w/v) paraformaldehyde, then permeabilized for 15 min with 0.2% (v/v) Triton X-100 in PBS and washed twice again with PBS. Thereafter the cells were blocked with blocking solution (1% BSA, 0.1% Triton x-100 in PBS) for 10 min and washed with PBS. The cells were incubated with 1 μg/mL Phalloidin–Fluorescin isothiocyanate (phalloidin-(FITC)) in order to stain F-actin and with Deoxyribonuclease I, Texas Red conjugate (10 μg/mL; Texas Red-labeled DNase I) to stain G-actin. Confocal images of F-actin and G-actin were captured simultaneously with a fluorescence microscope Zeiss LSM 510 (Carl Zeiss, Oberkochen, Germany) at 490 nm (excitation) and 525 nm (emission) for FITC-phalloidin and at 596 nm (excitation) and 615 nm (emission) for Texas Red-labeled DNase I, respectively.

2.8. Statistics

Values are presented as mean±S.E. Data were analyzed using one-way ANOVA followed by a post-hoc Student's t-test. P<0.05 was considered to represent significant differences between groups.

3. Results

3.1. Leptin-induced cardiomyocytes hypertrophy is associated with Rho activation

We first determined the effects of leptin on cardiac cell surface area and leucine incorporation either on its own or in the presence of different concentrations of OBR-Ab. As shown in Fig. 1A and B, 24 h treatment with leptin increased cell surface area by 32±5% compared to the untreated cells and stimulated incorporation of leucine into newly formed protein synthesis by 43±7% (Fig. 1B). The OBR-Ab inhibited the leptin-induced hypertrophic response in a concentration-dependent manner with total inhibition seen at 166 ng/mL, thus demonstrating an OBR-dependent hypertrophic response to the peptide (Fig. 1B and C).

Fig. 2 shows the effect of leptin on Rho activation in the absence or presence of the OBR-Ab. Since RhoA cycles between an inactive form (GDP-bound) and an active form (GTP-bound) we investigated if leptin can induce RhoA activation by using a GTP pull-down assay for the active RhoA. Fig. 2A shows the time course of RhoA activation induced by leptin which was characterized by a greater than four-fold increase which peaked after 5 min of leptin addition and declining thereafter. Subsequently, increasing concentrations of OBR-Ab were used to examine whether leptin directly mediates RhoA activation. As shown in Fig. 2B, the ability of leptin to activate RhoA was attenuated by the OBR-Ab in a concentration-dependent manner with RhoA activation completely prevented with OBR-Ab concentrations that were effective in inhibiting the hypertrophic response to leptin.

3.2. Rho/ROCK pathway activation is necessary for leptin-induced hypertrophic response

To determine whether RhoA activation by leptin is necessary for the hypertrophic responses to leptin, we examined the capacity of the peptide to induce hypertrophy following either RhoA inhibition with C3 exoenzyme or ROCK inhibition with Y-27632 as depicted in the top right panel of Fig. 3. C3 exoenzyme (at 30 ng/mL) significantly inhibited the ability of leptin to increase cell surface area (Fig. 3B) and protein synthesis (Fig. 3C). Similarly, the ROCK inhibitor Y-27632 (at 10 μmol/L) also inhibited leptin-induced hypertrophy as assessed by cell surface area and leucine incorporation (Fig. 3). Taken together, the ability of leptin to induce RhoA activation coupled with the elimination of the hypertrophic response by both a RhoA and ROCK inhibitor strongly implicate the Rho/ROCK pathway in leptin-induced cardiomyocyte hypertrophy.

3.3. Cofilin-2 phosphorylation is associated with leptin-induced hypertrophy

We next examined whether leptin modulates the activity of cofilin-2, a ubiquitous regulator of actin dynamics and a downstream effector following Rho/ROCK activation. To address the capacity of leptin to phosphorylate of cofilin-2, cardiomyocytes were stimulated with leptin for various time points (0, 15, 30, 60, 120, 240 and 480 min). Leptin had a direct effect on cofilin-2 phosphorylation in a time-dependent manner which reached maximum effects after 2 h of leptin treatment (Fig. 4A) with gradual dephosphorylation to near basal level after 8 h of stimulation, although values at this time point were still significantly greater from pre-leptin values. The OBR-Ab significantly, but incompletely, attenuated leptin-induced cofilin phosphorylation whereas both C3 and Y-27632 completely prevented leptin-induced cofilin phosphorylation when measured 2 h after leptin administration (Fig. 4B).

3.4. Leptin decreases G-actin/F-actin ratio

Actin cytoskeleton dynamics is the major target of the Rho/ROCK pathway and several transcription factors important for hypertrophy are likely controlled by free G-actin. Accordingly, we next investigated the effect of leptin on free G-actin to F-actin ratio. Cells were treated with leptin for 24 h and fractionated cell extracts containing nonpolymerized G-actin and F-actin were prepared and analyzed for G and F-actin contents. As shown in Fig. 5A and B, there was a significant decrease in the G to F-actin ratio in cells treated with 3.1 nmol/L leptin compared with control untreated cells indicating that a larger pool of actin exists as filamentous actin in leptin treated cells. These effects were completely prevented by the ObR-Ab as well as by the ROCK inhibitor Y-27632.

The effect of leptin on the actin cytoskeleton of cardiomyocytes was also analyzed with direct fluorescence microscopy using phalloidin and Texas Red-labeled DNase I to stain F-actin and G-actin, respectively. In agreement with the G to F-actin ratio studies using Western blotting, Fig. 5C shows that the G-actin pool was decreased after the addition of leptin compared to the controls cells but not in myocytes pretreated with the OBR-Ab or Y-27632 (Fig. 5C).

3.5. Disruption of the actin cytoskeleton attenuates leptin-induced hypertrophic response

To investigate whether disruption of the actin cytoskeleton affected leptin-induced hypertrophy, cardiomyocytes were pretreated with latrunculin B (50 nmol/L) before stimulation with leptin. Latrunculin B significantly attenuated leptin-induced increase in cell surface area (Fig. 6A and B) and protein synthesis (Fig. 6C). In order to confirm the effect latrunculin B at 50 nmol/L on actin cytoskeleton dynamics, measurement of the G to F-actin ratio was carried out with or without latrunculin B. Fig. 6E shows that latrunculin B significantly elevated the G to F-actin ratio in leptin treated cells.

3.6. Mediation of leptin-induced phosphorylation of ERK1/2 and p38 by Rho/ROCK pathway

To further elucidate the downstream signaling pathways associated with the activated Rho GTPases we examined the possible contribution of ERK1/2 and p38 activation in mediating Rho/ROCK-dependent leptin-induced hypertrophy. ERK1/2 and p38 phosphorylation were determined in cardiomyocytes pretreated with leptin for 5 min which represented peak kinase phosphorylation. As shown in Fig. 7, leptin produced a two-fold increase in ERK phosphorylation (Fig. 7A) whereas approximately a 70% increase in p38 phosphorylation (Fig. 7B) was seen. These effects were completely prevented by the RhoA inhibitor C3, the ROCK inhibitor Y-27632 (10 μmol/L) as well as latrunculin B.

4. Discussion

Recent evidence has suggested a potential critical role of the Rho system plays in mediating the hypertrophic responses in cardiac myocytes [27–29]. The ability of hypertrophic factors including angiotensin II and ET₁ to activate RhoA has indeed been previously reported suggesting the emergence of this pathway as a critical component of the hypertrophy program [reviewed in Ref. 19]. The present study shows for the first time that activation of the Rho system mediates the hypertrophic effect of the satiety factor leptin through a mechanism involving MAP kinase and regulation of actin dynamics. Thus, it appears that RhoA could represent a common pathway mediating the hypertrophic effect not only of leptin but also well-established hypertrophic agents. The nature of the hypertrophic response seen with leptin as manifested by increased cell size and protein synthesis as well as MAPK activation are quite similar to the nature of hypertrophy observed with various hypertrophic stimuli including angiotensin II, endothelin-1 or hypertrophy produced by stretch [reviewed in 19].

Our study was carried out in cells subjected to 24 h serum-starvation prior to leptin administration since serum components potently stimulates RhoA activity. Indeed, in our study we found a six fold elevation in RhoA activation in cells cultured in 10% serum-containing medium with no further activation following leptin administration. When cells were exposed to medium containing 1% serum a 2.5 increase in RhoA activity was observed with a further elevation, albeit not significant, following leptin addition (data not shown). Thus, to circumvent the potential effect of serum in terms of its ability to activate RhoA directly and mitigate the effect of exogenous leptin experiments were done in serum-starved cells. Taken together, our results indicate that activation of the RhoA is necessary for leptin-induced cardiomyocyte hypertrophy as assessed by cell surface area and protein synthesis. We utilized a multifaceted approach to determine the role of the RhoA/ROCK system in leptin-induced hypertrophy. First, our study shows that the hypertrophic effect of leptin is associated with RhoA activation with both responses prevented by an antibody directed against the leptin receptor. It should be noted that this antibody recognizes the extracellular domain of OBR and hence the nature of the specific OBR subtype mediating either the hypertrophic response to leptin or the activation of RhoA is presently not known with certainty.

To further examine the role of RhoA in leptin-induced cardiomyocyte hypertrophy, we utilized Clostridium botulinum C3 exoenzyme, an ADP-ribosyltransferase, which has been extensively used as a direct and specific inhibitor of RhoA in cardiac myocytes [30]. Our study indeed shows that C3 completely abolished the hypertrophic effect of leptin. Moreover, treatment of cardiomyocytes with the pyridine derivative Y-27632, one of the most extensively used inhibitors of ROCK, a downstream effector of RhoA, markedly attenuated leptin-induced hypertrophy. Taken together, the ability of leptin to phosphorylate RhoA coupled with the anti-hypertrophic effects of inhibitors of the Rho/ROCK pathway to prevent leptin-induced hypertrophy suggest an important role of this system in mediating the cardiomyocyte hypertrophic effect of the peptide.

The interaction between RhoA and ROCK is further supported by a previous study demonstrating that ROCK can phosphorylate cofilin via a RhoA-dependent process [31]. Cofilin, is an actin-depolymerizing factor which acts by severing actin filaments and increase treadmilling of actin [32,33]. As a result of the ability of cofilin to promote actin depolymerization, the net effect of RhoA through this pathway is to promote formation of actin microfilaments and reduce the G-actin pool. Our results demonstrate that leptin inactivates cofilin via Rho/ROCK-dependent phosphorylation, an effect which was abolished by C3 exoenzyme and the anti-OBR antibody.

A dynamic equilibrium exists between G-actin and F-actin [34]. Our results obtained using both Western blotting as well as confocal microscopy show that intracellular actin distribution is markedly affected by leptin which was manifested by an increase in the G/F-actin ratio. The polymerization effect of leptin was prevented by the OBR-Ab as well as the C3 exoenzyme indicating the involvement of the Rho/ROCK pathway in leptin-induced change in actin dynamics. Recent studies have highlighted the essential function of Rho family of small GTPases in actin dynamics in fibroblasts through effector proteins including cofilin [35]. Our results support the concept of cofilin phosphorylation in mediating leptin signaling in cardiac myocyte which was dependent on Rho/ROCK activation. Further evidence for an association between leptin and actin cytoskeleton was also demonstrated in studies using latrunculin B to disrupt actin cytoskeleton. Treatment of cardiomyocytes with latrunculin B increased the pool of soluble (monomeric) actin within the cells indicating disruption of actin filaments. This was accompanied by a substantial inhibition of leptin-induced hypertrophy as assessed by both cell surface area and protein synthesis suggesting that the influence of Rho/ROCK/cofilin signaling on cardiomyocytes hypertrophy is dependent on actin filament integrity.

Actin can affect serum inducible genes through transcription factors [23,36], or modifying chromosome structure [37]. There is abundant evidence demonstrating that a variety of growth factor-inducible genes are positively regulated through increases in actin polymerization [23,35] and transcription serum response factor (SRF). Sotiropoulos et al. [23] have found that SRF activity is reduced by overexpression of actin in cells and it is controlled by the cofactor myocardin. In their investigation, G-actin showed an inhibitory effect on the interaction between SRF and its cofactor, which led to inhibition of SRF activity.

A number of studies have shown the potential importance for Rho/ROCK pathway for MAPK activation [38,39]. There is increasing evidence that MAP kinase activation mediates the direct hypertrophic effect of leptin as demonstrated in both rat and human cardiomyocytes [12,14]. Accordingly, we determined whether phosphorylation of the p38 and ERK1/2 pathway by leptin could be affected by RhoA or ROCK inhibition or by actin cytoskeleton depolymerization. Interestingly, both Rho and ROCK inhibition completely abrogated the early activation of ERK1/2 and p38 in response to leptin suggesting Rho/ROCK as upstream mediators of leptin-induced p38 and ERK1/2 phosphorylation. Furthermore, disruption of actin cytoskeleton by latrunculin B also completely prevented leptin-induced ERK1/2 and p38 phosphorylation, suggestive of the involvement of intact actin cytoskeleton in leptin-induced ERK1/2 and p38 phosphorylation.

4.1. Conclusion and study limitations

In conclusion, our study demonstrates for the first time the involvement of the Rho/ROCK system in mediating the hypertrophic effect of leptin. As summarized in Fig. 8, the key components of this pathway involve a ROCK-mediated inactivation of cofilin, leading to an increase in the cytoplasmic G-actin pool which then results in MAP kinase activation resulting in hypertrophy. Future investigations are required to identify more precisely the downstream transcriptional changes which occur subsequent to leptin-induced Rho/ROCK activation which mediate the hypertrophic response. We propose that our results will advance our current understanding of the cell signalling mechanisms underlying leptin-induced cardiomyocyte hypertrophy. It is also important to elucidate the role of leptin under in vivo conditions. For example, in an obese mouse model of leptin deficiency, exogenous leptin was found to produce an anithypertrophic effect [40]. We have not been able to demonstrate an antihypertrophic effect of leptin in the postinfarcted rat and indeed, antibodies directed against the leptin receptor prevented the development of hypertrophy 1 month following coronary artery ligation [43]. Thus, it is possible that leptin exerts diverse effects on hypertrophy which are dependent on the nature of the insult. Although our study also provides further evidence for the emerging role of the Rho/ROCK system in cardiovascular disease and as a potential target for cardiovascular therapeutics (reviewed in Refs. [41,42]), it should be emphasized that the implication of the RhoA/ROCK system reflects studies done on neonatal cardiomyocytes. Despite the fact that these cells have been used extensively to study cardiac hypertrophy they do exhibit phenotypic differences compared to adults, for example with respect to their increased dependence on glucose metabolism for ATP production as compared to preference for fatty acids in adult cells. Such differences in substrate utilization may affect the precise nature of the cellular response to leptin which need to be assessed in future studies.

Acknowledgments

This study was supported by the Canadian Institutes of Health Research. A. Zeidan is supported by the Heart and Stroke Foundation of Ontario Program in Heart Failure. M. Karmazyn holds a Canada Research Chair in Experimental Cardiology.

References

Author notes