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Cardiovasc Res. Jan 1, 2011; 89(1): 60–71.
Published online Sep 9, 2010. doi:  10.1093/cvr/cvq288
PMCID: PMC3002875

Cardiac-specific leptin receptor deletion exacerbates ischaemic heart failure in mice



The obesity-related adipokine, leptin, has multiple actions on peripheral organs, including the mitigation of adverse cardiovascular outcomes after myocardial infarction (MI). Although we recently demonstrated that leptin, its receptor, and downstream signalling are up-regulated in the heart after MI, the significance of intact cardiomyoctye leptin signalling is unknown. Therefore, our objective was to generate a cardiomyocyte-specific leptin receptor knock-out (ObRKO) mouse to determine whether worse cardiac outcomes after MI result from impaired leptin signalling in cardiomyocytes.

Methods and results

Tamoxifen-inducible ObRKO mice were subjected to experimental MI or sham surgeries and studied after 1 month. After MI, ObRKO mice displayed a loss of cardiac signal transducer and activator of transcription (STAT) 3 and adenosine monophosphate-activated protein kinase (AMPK) signalling. Worse survival and cardiac morbidity were also seen in the ObRKO mouse post-MI, including decreased contractile function and glycolytic metabolism, and increased left ventricular dilation, hypertrophy, collagen deposition, matrix metalloproteinase activity, apoptosis, and inflammation. Treatment of ObRKO mice post-MI with an ObR-independent AMPK activator improved cardiac function and restored many of these maladaptive processes to wild-type levels.


These data indicate that leptin signalling mitigates cardiac injury in the post-MI failing heart by acting directly on cardiomyocytes to increase STAT3 and AMPK activation, to decrease cardiac hypertrophy, apoptosis, and inflammation, and to limit deleterious changes in cardiac structure, function, and glycolytic metabolism.

Keywords: Leptin, Heart failure, Cre-lox, Apoptosis, Metabolism

1. Introduction

Adipose tissue produces and secretes a number of substances that act centrally to regulate appetite, metabolism, and cellular physiology.1 Many of these ‘adipokines’ also mediate effects directly in peripheral tissues through membrane bound receptors. Within the heart, the adipokine leptin and its receptor (ObR) are abundantly expressed in cardiomyocytes.2,3 Leptin can regulate the baseline physiology of the heart, including myocyte contractility,4 hypertrophy,5 apoptosis,6 and metabolism.7,8 During pathological states of myocardial infarction (MI)9 and heart failure,10 circulating leptin is increased, most likely from fat-derived sources. However, we have recently shown that increased expression of leptin and its receptor can occur locally in the failing human heart.3 Moreover, we demonstrated in an experimental murine model of MI that whole-body leptin deficiency results in impaired cardiac structure, function, and survival2,11 and that the phenotype can be rescued by the systemic repletion of leptin. However, the question remains as to whether the beneficial effects of leptin repletion in the setting of MI are dependent on leptin signalling in cardiomyocytes or secondary to the multiple effects of leptin peripheral to the heart.

Thus, the first goal of our study was to determine whether or not the cardiac-specific disruption of ObR expression, through the development of a mouse with inducible excision of the ObR gene, impacted on cardiac outcomes after MI. We hypothesized that a loss of cardiomyocyte-specific ObR expression would lead to greater decrements in cardiac structure and function, as well as increased myocardial apoptosis, inflammation, and hypertrophy, post-MI. The second goal of our study was to establish whether or not the disruption of cardiac ObR expression also impairs activation of downstream signalling pathways that are known to provide cardioprotective benefit in MI, including signal transducer and activator of transcription (STAT) 3 and adenosine monophosphate-activated protein kinase (AMPK). In particular, since it is known that AMPK is a key regulator of cardiac substrate metabolism,12 we further hypothesized that the ability of the ischaemic, failing heart to anaerobically metabolize glucose is dependent on an intact ObR-AMPK signalling axis.

2. Methods

2.1. Animals

All animal use conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Previously characterized mice harbouring the floxed ObR gene13 were a gift of Dr Jeff Friedman (Rockefeller University, NY, USA) and were bred into a pure C57BL/6J background for 8+ generations prior to subsequent breeding with mice homozygous for the α-myosin heavy chain promoter driven MerCreMer transgene (Jackson Laboratories, USA, B6129SF1/J background, stock #005650). Subsequent inbreeding generated double homozygous (+/+) mice for the MerCreMer transgene and the ObR floxed allele. To induce cardiac-specific ObR excision, 8-week-old male MerCreMer+/+ ObR floxed+/+ (ObRKO) transgenic mice were treated with tamoxifen [tam; 20 mg/kg intraperitoneal (ip)] daily for 7 days, whereas vehicle (oil)-treated control mice received an equivalent volume of 95% peanut oil/5% ethanol. One week after oil or tam treatment, mice were subjected to open thoracotomy and a suture was placed around the left anterior descending coronary artery and left loose (sham procedure) or tightly tied (CAL procedure) as previously described2 and detailed in the Supplementary material online. All surviving mice were studied at 1-month post-surgery by echocardiography and by an in vivo conductance catheter, followed by either tissue collection for biochemical studies, or ex vivo perfusion of the heart for glycolytic measurements. For histological and biochemical analyses, seven groups of mice were examined and included two groups of sham mice: (i) vehicle (n = 16)- and (ii) tam (n = 16)-treated ObRKO; and five groups of CAL mice: (iii) vehicle-treated ObRKO (n = 16), (iv) tam-treated ObRKO (n = 14), (v) tam-treated parental MerCreMer+/+ (n = 16), and (vi) tam-treated parental ObR floxed+/+ (n = 15) and tam-treated ObRKO administered the leptin-independent AMPK activator, aminoimidazole carboxamide ribonucleotide (AICAR), at a dose of 0.5 mg/g ip daily starting at 3 days prior to animal sacrifice (n = 9). In a separate set of experiments, an additional five (for sham groups) or eight (for CAL groups) mice were examined per group for cardiac glycolytic metabolism. Finally, an additional set of ObRKO mice were treated with oil (n = 10) or tam (n = 10), subjected to CAL, and administered leptin (n = 5 per tam/oil group at a dose of 0.3 mg/kg ip) or equal volume saline (n = 5 per tam/oil group) after 1 month, followed by animal sacrifice 30 min later.

2.2. Genotyping

DNA extraction from tissue was accomplished using the Extract-N-Amp kit (Sigma Aldrich, USA) per manufacturer's instructions. Primers and PCR conditions used are detailed in the Supplementary material online. PCR products were visualized using agarose gel electrophoresis.

2.3. Echocardiography

Parasternal short-axis B- and m-mode images were obtained on mice under 1–2% isoflurane anaesthesia using a Visualsonics echocardiography machine as previously described2 and detailed in the Supplementary material online.

2.4. In vivo pressure–volume loops

At 1-month post-CAL/sham surgeries, left ventricular pressure–volume loops were recorded using Chart and Scope software version 5.4.2 (AD Instruments, USA), an MPVS-400 system (Millar Instruments, USA), and a 1.4 F PV catheter (Millar Instruments). A minimum of five time points per animal (6–10 loops/time point) were analysed and averaged during steady state using PVAN version 3.6 software (Millar) as previously described14 and detailed in the Supplementary material online.

2.5. Ex vivo cardiac perfusion

Hearts were quickly excised, aortas cannulated, and subjected to perfusion with a modified Krebs buffer containing 3H-glucose. 3H-water in collected perfusate samples was separated from 3H-glucose by ion exchange as described by Lopaschuk and Barr,15 and steady-state calculations were adjusted for the volume of perfusate collected per unit time to determine glycolytic rate. Buffer composition and additional experimental details are provided in the Supplementary material online.

2.6. Quantitative real-time RT–PCR analysis

Total RNA was isolated from whole heart homogenates and subjected to quantitative PCR, as previously described,2 and detailed in the Supplementary material online.

2.7. Western blotting/zymography

Protein was extracted from whole heart homogenates in RIPA buffer and subjected to SDS–PAGE in equal amounts as described previously.2 Antibodies and incubation conditions used are detailed in the Supplementary material online.

2.8. Immunofluorescence/histology

Mouse cardiac sections were used for ObR, TUNEL, CD45, α-actinin, picrosirius-red, phalloidin, and H&E stains as previously described2 and detailed in the Supplementary material online. The determination of apoptotic and inflammatory indices, as well as collagen fractional area, was accomplished using the image analysis software Metamorph v7.5 (Molecular Devices, USA).

2.9. Determination of infarct size, cardiomyocyte area, and width

Mouse cardiac sections were stained with FITC-linked wheat germ agglutinin (to visualize cell membranes) and DAPI (to visualize nuclei) or H&E, and the determination of infarct area and cardiomyocyte dimensions was accomplished using Image J software (NIH), all as previously described,2 and detailed in the Supplementary material online.

2.10. In vitro caspase-3 activity assay

Caspase-3 activity was determined using whole heart homogenates and the caspase-3/CPP32 Colorimetric Assay kit (Biovision, USA) as previously described11 and detailed in the Supplementary material online.

2.11. Statistics

Data presented are mean ± standard error of the mean (SEM). Statistical significance of mean changes was determined by ANOVA with post hoc comparisons between means using Bonferrroni's simultaneous tests. Mathematical calculations, determination of P values, Student's t-tests, ANOVAs, and the Kaplan–Meier log-rank survival statistics and survival graph were generated using the computer program Statistical Package for the Social Sciences (SPSS) v17 (SPSS, USA).

3. Results

3.1. Tam administration to homozygous MerCreMer ObR floxed mice results in cardiac-specific ObR gene excision

After 7 days of tam treatment, mice homozygous for both MerCreMer+/+ and ObR floxed+/+ alleles (ObRKO mice) demonstrated excision of the floxed ObR gene in cardiac tissue only (Figure 1A). This was accompanied by a reduction in cardiac ObR mRNA and protein expression to 14 ± 3 and 13 ± 1% of oil-treated control levels, respectively (Figure 1B). Immunofluorescent examination of cardiac sections showed a marked reduction in ObR expression in cardiomyocytes (see Supplementary material online, Figure S1A). All mice maintained a lean phenotype and demonstrated no confounding tam effects16 on cardiac structure and function after tam treatment, with mean end-diastolic dimensions (EDDs), end-diastolic volumes (EDVs), average wall thicknesses, left ventricular ejection fractions, fractional shortenings (FSs), and developed pressures unchanged in sham mice after 7 days of tam treatment relative to oil treatment (Tables 1 and 22; see Supplementary material online, Figure S1B).

Table 1
Mean ± SEM weight and echocardiographic measurements on oil-treated and tam-induced ObRKO sham and CAL mice, and on tam-treated parental MerCreMer+/+ and ObR flox+/+ CAL mice, at 1-month post-surgery
Table 2
Mean ± SEM cardiac functional data derived from in vivo pressure–volume loops on oil-treated and tam-induced ObRKO sham and CAL mice, and on tam-treated parental MerCreMer+/+ and ObR floxed+/+ CAL mice at 1-month post-surgery
Figure 1
Cardiac-specific ObR deletion and reduction in ObR expression are seen in ObRKO mice after tam treatment. (A) Representative agarose gel electrophoresis demonstrating PCR amplification of DNA from the tail, skeletal muscle, heart, liver, and fat from ...

3.2. Cardiac-specific reduction in ObR expression results in a loss of cardiac STAT3 and AMPK signalling at 1-month post-MI

We have previously established that cardiac ObR expression and STAT3 signalling are increased in wild-type mice at 1-month post-MI.2 Here, we again utilized STAT3 phosphorylation as a read-out of leptin signalling17 and also examined the phosphorylation state of AMPK. The cardiac expression of total (t) and phosphorylated (p) STAT3 (Figure 2A) and AMPK (Figure 2B) was unchanged in tam-treated sham mice relative to oil-treated controls. All groups of CAL mice demonstrated an approximately two-fold increase in t-STAT3 and t-AMPK expression; however, only vehicle-treated ObRKO mice, and their corresponding tam-treated parental control mice, demonstrated a significant increase in the ratio of p/t-STAT3 and -AMPK post-MI. With cardiac-specific reduction in ObR expression, tam-treated ObRKO mice demonstrated a significant reduction in p/t-STAT3 and -AMPK relative to all other groups post-MI. Moreover, acute administration of exogenous leptin at 1-month post-MI activated cardiac STAT3 (see Supplementary material online, Figure S2A) and AMPK (see Supplementary material online, Figure S2B) only in oil-treated ObRKO mice. In control experiments, both total and p/t-STAT3 and -AMPK in tam-treated parental MerCreMer+/+ and ObR floxed+/+ mice were not significantly different from oil-treated ObRKO mice post-MI (Figure 2A and B).

Figure 2
Tam-induced ObRKO mice demonstrate a loss of cardiac STAT3 and AMPK activation post-MI. *P < 0.05 vs. shams, P < 0.05 vs. all CAL groups. (A) Mean ± SEM fold change in cardiac p/t-STAT3 (middle panel) and t-STAT3/GAPDH ...

3.3. Cardiac-specific reduction in ObR expression exacerbates indices of heart failure that are rescued with AMPK activation at 1-month post-MI

At one-month post-surgery, survival in sham mice was 100% (32/32). With CAL, 80% survival (16/20) was seen in oil-treated ObRKO mice, which was not different than survival in tam-treated MerCreMer+/+ (16/20) and ObR floxed+/+ (15/18) parental mice, but was significantly better than the 46% survival (14/30) seen in tam-treated ObRKO mice (Figure 3E). Despite equal infarct areas in oil- and tam-treated ObRKO mice (6.35 ± 0.26 vs. 6.31 ± 0.28 mm2, P > 0.05; see Supplementary material online, Figure S3), both heart and lung weights of tam-treated ObRKO mice were significantly increased, and tam-treated ObRKO mice subjected to CAL demonstrated the greatest increase in EDD and end-systolic dimension, and the greatest decrease in per cent FS, at 3-day, 1-, 2-, and 3-week, and 1-month post-surgery (see Supplementary material online, Figure S4 and Table S1; Table 1 and Figure 3A). These exacerbated indices of heart failure in the tam-treated ObRKO mouse post-MI were consistent with pressure–volume data at 1-month post-MI, which demonstrated significant increases in EDV and end-systolic volume, and significant decreases in cardiac output, ejection fraction, and developed pressures (Table 2 and Figure 3B). With each measured outcome, tam-treated homozygous (+/+) MerCreMer (Cre+/+) and floxed (flox+/+) parental mice demonstrated changes in cardiac structure and function at 1-month post-MI that were no different than oil-treated ObRKO mice post-MI (Tables 1 and 22 and Figure 3A and B). Consistent with the literature,18,19 AICAR administration resulted in the activation of cardiac AMPK (Figure 3C), and significant improvements in both echocardiographic- and pressure–volume-determined indices of heart failure were observed (Figure 3D, and Tables 1 and 22).

Figure 3
Tam-induced ObRKO mice demonstrate worse survival and greater decrements in cardiac structure and function post-MI. *P < 0.05 vs. shams and P < 0.05 vs. all CAL groups. (A) Representative m-mode echocardiographic images from oil-treated ...

3.4. Cardiac-specific reduction in ObR expression results in greater indices of biochemical remodelling that are rescued with AMPK activation at 1-month post-MI

The cardiac mRNA expression of atrial naturetic peptide (ANP), brain naturetic peptide (BNP), β-myosin heavy chain (βMHC), interleukin (IL)-1β, and tumour necrosis factor α (TNFα) were all increased post-MI in tam-treated ObRKO mice, whereas IL10 mRNA was decreased (Figure 4A, see Supplementary material online, Table S2). Further, both myocyte width and area (Figure 4B; see Supplementary material online, Figure S5), as well as echocardiographic determination of wall thicknesses (Figure 3A and Table 1), were increased post-MI in tam relative to oil-treated ObRKO mice, or tam-treated homozygous (+/+) MerCreMer (Cre+/+) and floxed (flox+/+) parental mice. Moreover, increased deposition of collagen was observed in cardiac sections from tam-treated ObRKO mice (Figure 4C; see Supplementary material online, Figure S6) and correlated with increased zymographically determined matrix metalloproteinase (MMP) 2 and 9 activity (Figure 4D). With AICAR administration, tam-treated ObRKO mice demonstrated significant reductions in foetal gene and inflammatory cytokine mRNA expression, myocyte hypertrophy, collagen deposition, and MMP activity (Figure 4A–D, Table 1; see Supplementary material online, Figures S5 and S6).

Figure 4
Tam-induced ObRKO mice demonstrate greater biochemical and histological measures of cardiac remodelling post-MI. *P < 0.05 vs. shams and P < 0.05 vs. all CAL groups. (A) Mean ± SEM fold change in cardiac ANP, BNP, βMHC, ...

3.5. Cardiac-specific reduction in ObR expression results in greater cardiomyocyte apoptosis, inflammation, and caspase-3 activity that are rescued with AMPK activation at 1-month post-MI

Hearts from both oil- and tam-treated ObRKO mice subjected to sham procedure demonstrated comparable rates of total apoptosis (~0.09%), inflammation (i.e. the per cent of nuclei that are CD45+, ~4%), and TUNEL/α-actinin-positive cells (~0.05%; Figure 5A–F). Additionally, in control experiments, rates of apoptosis, inflammation, and TUNEL/α-actinin-positive cells in hearts from homozygous (+/+) MerCreMer (Cre+/+) and floxed (flox+/+) parental lines of mice treated with tam and subjected to CAL were not significantly different from each other, or oil-treated ObRKO mice (all P > 0.05; Figure 5A–F). However, increased indices of inflammation and apoptosis were seen in both remote and infarcted myocardium from ObRKO mice with cardiomyocyte-specific reduction in ObR expression (Figure 5A–F). Specifically, relative to oil-treated mice, remote tissue from tam-treated ObRKO mice subjected to CAL demonstrated significantly greater levels of total TUNEL-positive (~0.58 vs. ~0.16%; P < 0.05), CD45-positive (~26 vs. ~20%; P < 0.05), and TUNEL/α-actinin positive (~0.29 vs. ~0.07%; P < 0.05) cells. Similarly, infarcted tissue from tam-treated ObRKO mice subjected to CAL demonstrated significantly greater levels of total TUNEL-positive (~3 vs. ~0.48%; P < 0.05), CD45-positive (~50 vs. ~32%; P < 0.05), and TUNEL/α-actinin-positive cells (~1.8 vs. ~0.22%; P < 0.05). Moreover, and consistent with histological data, a ~1.7-fold increase in caspase-3 activity was seen in oil-treated ObRKO mice subjected to CAL relative to shams (P < 0.05), with an additional increase to approximately three-fold in tam-treated ObRKO mice post-CAL (P < 0.05; Figure 5G; see Supplementary material online, Figure S7). In these in vitro experiments, caspase-3 activities in tam-treated homozygous (+/+) MerCreMer (Cre+/+) and floxed (flox+/+) parental mice subjected to CAL were not significantly different from vehicle-treated ObRKO mice (Figure 5G). With AICAR administration, significant reductions in total apoptosis, inflammation, TUNEL/α-actinin-positive cells, and caspase-3 activity were seen in tam-treated ObRKO mice at 1-month post-MI (Figure 5A–G).

Figure 5
Tam-induced ObRKO mice demonstrate increased cardiac apoptosis, inflammation, and caspase-3 activity post-MI in both remote and infarcted myocardium. *P < 0.05 vs. shams, P < 0.05 vs. shams and all remote CAL groups, and ...

3.6. Cardiac-specific reduction in ObR expression results in impaired glycolytic metabolism that is rescued with AMPK activation at 1-month post-MI

Post-MI, cardiac glycolysis was increased in oil-treated ObRKO mice and tam-treated homozygous (+/+) MerCreMer (Cre+/+) and floxed (flox+/+) parental mice, relative to shams (Figure 6). In tam-treated ObRKO mice, glycolytic rate was significantly impaired in both sham and infarcted hearts, and complete rescue was seen in CAL mice after AICAR administration (Figure 6).

Figure 6
Tam-induced ObRKO mice demonstrate reduced steady-state rates of glycolysis that can be rescued with AICAR. Mean ± SEM steady-state rate of glycolysis (in µmol/min/g dry heart weight) in oil-treated and tam-induced sham and CAL ObRKO mice, ...

4. Discussion

Here, we establish that leptin has a direct effect on the heart by demonstrating that cardiomyocyte-restricted loss of leptin signalling results in worse cardiac structure, function, and survival relative to wild-type mice after experimental MI, consistent with our previous observations in the whole-body leptin-deficient Ob/Ob mouse.2,11 We go on to demonstrate that intact cardiac leptin signalling in ischaemic heart failure is important for the maintenance of glycolytic metabolism, involves the activation of STAT3 and AMPK, and is associated with attenuated indices of inflammation, apoptosis, and adverse cardiac remodelling post-MI. By demonstrating improvement in many exacerbated indices of cardiac structure and function that occur in the tam-treated ObRKO mouse post-MI with AICAR, we mechanistically link the cardioprotective benefit of leptin signalling to AMPK. Taken together, these results provide potential mechanisms by which intact leptin signalling improves outcomes in ischaemic heart failure.

In the present study, experiments using the ObRKO mouse demonstrate the relative importance of cardiac vs. non-cardiac leptin signalling in the ischaemic failing heart and suggest a compensatory role for the increased ObR expression that occurs in wild-type mice post-MI2,20 and in humans with heart failure.3 Specifically, in ObRKO mice with reduced cardiac ObR expression, a corresponding loss of STAT3 and AMPK activation is seen in the failing heart. The importance of this finding is that STAT3 and AMPK activation mitigates cardiac ischaemic injury. For example, in mice with a loss of STAT3 activation, greater adverse left ventricular remodelling 21 and increased apoptosis22 occur post-MI. Similarly, in mice expressing the kinase inactive form of AMPK, ischaemic glucose uptake and glycolysis are impaired, and post-ischaemic cardiac function and apoptosis are exacerbated.23 Our results using the ObRKO mouse are consistent with these2123 published reports and demonstrate that a loss of cardiac STAT3 and AMPK activation in the ischaemic failing heart is linked to increased levels of adverse structural, functional, biochemical, and metabolic remodelling.

Our data demonstrate that excision of the ObR gene has no effect on the basal activation state of STAT3 or AMPK, or on myocardial structure or contractile performance during a short (4 week) period of observation. Indeed, data from Ob/Ob and Db/Db mice suggest that longer perturbation of ObR signalling is required to see changes in cardiac structure and function under basal conditions5 and that ischaemic injury2,24 accelerates this process. Combined, these in vivo data2,5,24 are in contrast to several,2527 but not all,28 in vitro studies reporting that leptin can induce hypertrophy in cultured rat neonatal or human paediatric cardiomyocytes. Reasons for these varied in vivo vs. in vitro responses may be due to a number of factors, including the reported lack of long-form ObR expression in rat neonatal cardiomyocytes,26 the lack of non-cardiomyocyte cells in culture preparations that secrete factors such as angiotensin-(1–7)29 and interferon-γ30 that mediate anti-hypertrophic effects, the loss of extracellular matrix (ECM) that provides and/or alleviates varied degrees of wall stress in vivo,31 and the different culture conditions under which experiments were performed. The acute effect of leptin administration may also be different than the chronic effect of deficiency. In particular, it has been proposed that the age-related hypertrophy observed in Ob/Ob and Db/Db mice is a consequence of many deranged physiological parameters that occur in these mice over time and is not a direct consequence of leptin deficiency per se.25 Although our data do not directly address the issue of age-related hypertrophy in states of leptin deficiency, we do demonstrate using our cardiac-specific ObR knock-out mouse that the mitigating effects of leptin on post-MI hypertrophy are specific to cardiac tissue and not dependent on any systemic actions of leptin. Thus, although final conclusions regarding leptin-mediated cardiomyocyte hypertrophy based on in vitro data remain to be clarified, our data suggest that impaired cardiac leptin signalling post-MI results in a decrease in AMPK activity and an increase in cardiomyocyte hypertrophy.

Cardiomyocyte hypertrophy can develop as a compensatory response to increases in cardiac pressure and/or volume. However, with time, hypertrophy leads to cardiac failure, characterized by maladaptive remodelling of the ECM, reduced contractile performance, and ventricular dilation. Inflammation plays a role in regulating ECM remodelling by contributing to alterations in cardiac MMP activity and collagen deposition.31 Specifically, mast cells within the myocardium release a variety of factors, including TNFα, that increase MMP expression and activation,32 and evidence suggests that both neutrophils and macrophages secrete and activate MMPs in the post-MI heart.33 Consistent with these observations, we show increased cardiac MMP activity, collagen deposition, inflammatory cytokine expression (including TNFa), and increased inflammatory cell infiltrates in tam-treated ObRKO mice post-MI. Further, we demonstrate a reduction in these measures with AICAR administration, suggesting the involvement of AMPK. This is consistent with other reports34,35 demonstrating that AMPK modulates inflammatory responses. In particular, AMPK not only suppresses pro-inflammatory responses in macrophages,34 but it also inhibits the production and activity of pro-inflammatory cytokines such as TNFa.35 Thus, impaired cardiac leptin signalling post-MI is linked to decreased AMPK activation and increased maladaptive cardiac remodelling characterized by exacerbated inflammation, collagen deposition, and MMP activity.

In addition to increased inflammation, we also show increased cardiomyocyte apoptosis post-MI in tam-treated ObRKO mice that is rescued with AICAR. Our findings complement reported rates of cardiac apoptosis seen in Ob/Ob and Db/Db mice with ageing6 and extend the results of our previous study which demonstrated increased cardiac apoptosis post-MI in both lean and obese Ob/Ob mice.11 Specifically, we now not only show the increased apoptotic cell number in mice with impaired cardiac leptin signalling post-MI, but also demonstrate that the majority of cells undergoing apoptosis in both remote and infarcted regions stain with the cardiomyocyte-specific marker α-actinin. Mechanistically, our experiments with AICAR demonstrate the involvement of AMPK and are consistent with studies showing AMPK-mediated protection from hypoxia/reoxygenation-,36 TNFα-,37 and ischaemia/reperfusion23-induced cardiac apoptosis. We speculate that complete normalization of apoptotic rates was not achieved in our study, however, due to either the relatively short duration of AICAR administration, and/or the involvement of additional leptin-dependent, but AMPK-independent, anti-apoptotic factors and pathways. For example, in addition to AMPK,36 evidence suggests that leptin attenuates apoptosis in cardiac cells through the activation of p38 mitogen-activated kinase,36 phosphoinositide 3-kinase,38 and STAT3.11 Nevertheless, our experiments using AICAR confirm a mechanistic link and demonstrate a unique AMPK-dependent anti-apoptotic role for leptin in the ischaemic failing heart.

Our data demonstrate that leptin impacts hypertrophy, collagen deposition, MMP activity, inflammation, and apoptosis in the ischaemic failing heart and thereby plays an important role in mitigating adverse structural remodelling post-MI. Another major finding in this study is that leptin also mediates metabolic remodelling in ischaemic heart failure. Specifically, we demonstrate increased glycolysis post-MI only in hearts with intact leptin signalling and reduced glycolytic rates in mice with impaired leptin signalling that can be rescued with AICAR. These results are in agreement with findings in Ob/Ob and Db/Db mice that show a reduction in cardiac glucose utilization and contractile function that precede the development of obesity and hyperglycaemia.7,39 Given these observations, it is likely that the reduction in anaerobic metabolism we see in hearts with impaired leptin signalling is a major contributing factor to the accentuated impairments we see in LV systolic function, cardiac output, stroke work, and contractility (dP/dtmax) in the tam-treated ObRKO mouse post-MI. Indeed, independent of leptin, studies consistently demonstrate a significant reduction in cardiac mechanical efficiency when free fatty acids are used preferentially over glucose for energy production in the failing heart.40 Our data demonstrating restoration of glycolysis and significant improvements in cardiac function with AICAR treatment suggest a direct link between leptin-mediated glycolytic metabolism and cardiac function post-MI, and highlight the dependence on AMPK in the process.

In conclusion, our data show that leptin mitigates heart failure post-MI by acting directly on the cardiomyocyte to increase ObR signalling and decrease cardiac hypertrophy, apoptosis, and inflammation. Further, our results demonstrate that intact cardiac leptin signalling is an important mediator of anaerobic metabolism which occurs in the ischaemic failing heart and is associated with less severe decrements in cardiac function and apoptosis post-MI. Together, these observations complement studies reporting increased circulating leptin acutely after MI9 and increased cardiac ObR expression3 and circulating leptin10 in human heart failure, by establishing a cardiomyocyte-specific role for leptin to improve outcomes in cardiac ischaemic injury.


This work was supported by the National Institutes of Health grants (HL-087009 to K.R.M., DK-072162 to R.M.O., and HL-077785 to C.P.O.), the Great Rivers Affiliate of the American Heart Association Beginning Grant-in-Aid (0865432D to K.R.M.), and the Cardiovascular Institute at the University of Pittsburgh Medical Center.

Supplementary Material

Supplementary Data:


We acknowledge the assistance of the CBI at the University of Pittsburgh in providing access to microscopy equipment.

Conflict of interest: none declared.


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