Although disruption of leptin signaling is associated with obesity as well as cardiac lipid accumulation and dysfunction, it has been difficult to separate the direct effects of leptin on the heart from those associated with the effects of leptin on body weight and fat mass. Using Cre-loxP recombinase technology, we developed tamoxifen-inducible, cardiomyocyte-specific leptin receptor-deficient mice to assess the role of leptin in regulating cardiac function. Cre recombinase activation in the heart resulted in transient reduction in left ventricular systolic function which recovered to normal levels by day 10. However, when cardiomyocyte leptin receptors were deleted in the setting of Cre recombinase-induced left ventricular dysfunction, irreversible lethal heart failure was observed in less than 10 days in all mice. Heart failure after leptin receptor deletion was associated with marked decreases of cardiac mitochondrial ATP, phosphorylated mammalian target of rapamycin (mTOR), and AMP-activated kinase (pAMPK). Our results demonstrate that specific deletion of cardiomyocyte leptin receptors, in the presence of increased Cre recombinase expression, causes lethal heart failure associated with decreased cardiac energy production. These observations indicate that leptin plays an important role in regulating cardiac function in the setting of cardiac stress caused by Cre-recombinase expression, likely through actions on cardiomyocyte energy metabolism.
- heart failure
- mammalian target of rapamycin
- AMP-activated kinase
- arterial pressure
the adipokine leptin acts on the hypothalamus to regulate body weight by reducing appetite and increasing metabolic rate through sympathetic nervous system activation. Leptin also stimulates glucose utilization and fatty acid metabolism in peripheral tissues such as skeletal muscle (44, 45).
Leptin has also been suggested to play a role in regulating cardiac function. For example, disruption of leptin signaling is associated with altered cardiac metabolism, cardiac lipid accumulation, myocardial apoptosis, and cardiac dysfunction (3, 11, 12, 30, 46). However, it has been difficult to separate the direct effects of leptin on the heart from those associated with the effects of obesity and increased fat mass that also occur with impaired leptin signaling in the brain.
To directly assess the role of leptin in regulating cardiac function, we generated a model in which temporally regulated cardiomyocyte-specific knockout of leptin receptors was achieved using Cre-loxP recombinase technology. This model is advantageous because it allows direct examination of the role of leptin in regulating cardiac function in the absence of obesity. However, studies from our lab as well as others revealed that Cre recombinase, when expressed at high levels in the heart, results in transient systolic dysfunction, although these mice are able to recover normal systolic function within 10 days (17, 21). Mice with increased Cre recombinase had significant reductions in mitochondrial ATP levels, although total cardiac ATP levels were unchanged, compared with values measured in control mice. Systolic dysfunction and reduced ATP levels in these mice were associated with markers of impaired oxidative metabolism and enhanced glycolytic metabolism, features commonly observed in myocardial ischemia and heart failure. Although mice with enhanced expression of Cre recombinase developed transient cardiac dysfunction, they were able to recover normal cardiac contractile function by enhancing glycolytic metabolism. Thus Cre recombinase-mediated heart failure is associated with transient cardiac systolic dysfunction that, in the absence of further metabolic derangements or additional cardiac stressors, can be recovered without the progression into overt heart failure.
We discovered in preliminary studies that acute loss of cardiomyocyte leptin receptors in the setting of Cre recombinase cardiotoxicity resulted in irreversible dilated cardiomyopathy and lethal heart failure within 10 days after loss of cardiac leptin receptors. Therefore, the present studies were designed to more fully assess the effects of cardiomyocyte-specific deletion of leptin receptors on myocardial structure and function, including cardiac mitochondrial ATP levels and other indicators of cardiac energetics, using a temporally controlled method of inducing Cre recombinase.
All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. Experiments were performed on male and female mice between 12 and 40 wk of age. Breeding pairs of floxed leptin receptor mice (LepRflox) mice were obtained from Dr. Jeffrey M. Friedman, Rockefeller University, and offspring were maintained on a C57BL/6 background. These mice contain loxP sites that flank exon 1 of the LR gene (7). Since this exon contains the signal sequence, Cre-mediated deletion of this exon results in the loss of all LR splice variants (7). Breeding pairs of cardiomyocyte-specific inducible Cre mice (Myh6-MerCreMer) were obtained from Dr. Jeffrey Molkentin and bred onto a C57BL/6J background for at least six generations before use. Myh6-MerCreMer and LepRflox mice were bred to create Myh6-MerCreMer/LepRflox (LepR KO) mice. Four different groups of mice were utilized in the present study. Control mice were mice homozygous for the floxed leptin receptor allele (LepRflox) but lacking Myh6-MerCreMer. In addition, two other groups were studied as controls for the effects of Cre recombinase expression in the heart; Myh6-MerCreMer mice with wild-type LepR alleles (LepRWT) and Myh6-MerCreMer mice that contained one copy of the wild-type LepR allele and one copy of the LepRflox (heterozygous mice). LepR KO mice contained both the Mhy6-MerCreMer and were homozygous for the LepRflox allele. All mice were fed a standard diet containing 0.29% NaCl and provided water ad libitum and were treated with tamoxifen. Tamoxifen (100 μg) was prepared in ethanol and diluted with corn oil and administered by intraperitoneal injection (100 μl) on 5 consecutive days. Mice were euthanized 9–10 days after tamoxifen administration.
PCR was performed on small cardiac biopsy samples. Genomic DNA was isolated from the biopsy samples using a modified proteinase K digestion method (DirectPCR, Viagen, Los Angeles, CA). PCR for leptin deletion was performed on 1 μl of cardiac biopsy using primers specifically designed to detect the deletion of exon 1 as previously described (7). Integrity of the DNA present in the lysates was determined using a specific set of primers designed to amplify both the LepRWT as well as the LepRflox allele as previously described (7). The sequences of the primers used for amplification of the deletion product were as follows: LepR1:5′-GTCACCTAGGTTAATGTTATTC-3′; LepR3:5′-GCAATTCATATCAAAACGCC-3′. The LepRWT as well as the LepRflox allele were amplified with the LepR1 primer used in conjunction with the following primer LepR2:5′-TCTAGCCCTCCAGCACTGGAC-3′. Amplification of the LepRflox allele yields a slightly larger PCR product compared with the LepRWT allele.
Assessment of cardiac function by echocardiography was conducted using a Vevo 770 High Resolution In Vivo Imaging System (Visualsonics, Toronto, ON, Canada). Measurements were made with a 710B RMV scanhead with a center frequency of 25 MHz and a frequency band ranging from 12.5 to 37.5 MHz. Baseline echocardiography sessions were conducted on different days on each mouse before induction of the cardiomyocyte-specific leptin receptor deletion or cardiac-specific Cre recombinase expression with tamoxifen injections. After initiation of the leptin receptor deletion or expression of Cre recombinase, echocardiography was performed on each mouse every other day until the end of the protocol. All echocardiographic data were obtained within 20 min. All measurements were made according to the American Society of Echocardiography Guidelines using the leading edge technique (23). Mice were anesthetized with 1.5% isofluorane gas and placed on a prewarmed pad. Heart rate was monitored via an electrocardiogram transducer pad throughout each echocardiography session. Two-dimensional (2-D) B-Mode parasternal long-axis views were obtained first to visualize the aortic and mitral valves. The transducer was then rotated clockwise 90 degrees to obtain the parasternal short-axis view. At least three 2-D guided M-Mode images were obtained at the midpapillary muscle level from this view. Ejection fraction (%), fractional shortening (%), cardiac output (ml/min), and stroke volume (μl) were analyzed and calculated using the Visualsonics Advanced Cardiovascular Measurements Package using the mean of 12–15 cardiac cycles derived from three separate M-Mode images during each session.
Arterial blood pressure.
Arterial blood pressures were measured in conscious, freely moving mice by radiotelemetry (Data Sciences International, St. Paul, MN). Pressure-sensing catheters were implanted into the left common carotid, and the transmitter was secured subcutaneously along the left flank of the mouse as previously described (5). Mice were given 7 days to recover from surgery after which time heart rates as well as systolic, diastolic, and mean arterial pressures were continuously recorded (sampling every 15 min for 10-s intervals, 24 h/day) throughout the entire experimental protocol. Baseline blood pressures were collected for 5 consecutive days after which time tamoxifen was administered for 5 days. Data were collected and stored using the Dataquest ART data acquisition system (Data Sciences International, St. Paul, MN) and imported into Microsoft excel for analysis.
Western blots were performed on lysates prepared from whole hearts collected at the end of the experiments. Samples of 50 μg of protein were boiled in Laemmli sample buffer (Bio-Rad, Hercules, CA) for 5 min and electrophoresed on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membrane. Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. Membranes were incubated with either Alex 680 (Molecular Probes, Eugene, OR) or IRDye 800 (Rockland, Gilbertsville, PA) secondary antibodies for 1 h at room temperature. Membranes were visualized using an Odyssey infrared imager (Li-COR), which allows for the simultaneous detection of two proteins. Densitometry analysis was performed using Odyssey software (LI-COR). Antibodies for Western blots were as follows: leptin receptor [ObRb; Ob-R(H-300), sc-8325, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA], total AMP-activated protein kinase-α (tAMPK-α; no. 2532, 1:500, Cell-Signaling Technology, Danvers, MA), phosphorylated-AMPK-α (pAMPK; no. 2531, 1:500 dilution, Cell-Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab8245, 1:10,000, Abcam, Cambridge, MA), phosphorylated mTOR (p-MTOR; no. 2971S, 1:1,000 Cell-Signaling Technology).
Measurement of cardiac mitochondrial ATP levels.
Hearts were collected from control and knockout (KO) mice between days 5 and 9 posttamoxifen injection and homogenized in a ice-cold homogenization solution (0.22 mM mannitol, 70 mM sucrose, 0.5 mM EGTA, 1 M HEPES, and 0.1% BSA), and mitochondria were isolated by differential centrifugation as previously described (13). Cardiac mitochondrial ATP levels were measured using a luciferase-based ATP determination kit (Molecular Probes). ATP assays were performed on 10 μl of resuspended mitochondria, and each sample was measured in triplicate. ATP levels were expressed as nanomoles per milligram of protein.
Mean values ± SE are presented. Significant differences between mean values were determined using an unpaired t-test or with the use of an ANOVA followed by a post hoc test (Dunnett's) for multiple comparisons of group means with the mean of a control group to maintain an experiment-wise α error rate of <5%. A P < 0.05 was considered to be significant.
Inducible deletion of cardiac leptin receptors was achieved using a cardiomyocyte-specific, ligand-activated Cre recombinase. Deletion of leptin receptors was observed only in the hearts of transgenic mice after administration of the estrogen receptor agonist, tamoxifen, and not in other organs examined (Fig. 1A). To test the extent of leptin receptor deletion in the heart, Western blot analysis for the leptin receptor was performed on cardiac tissue lysates from transgenic and nontransgenic mice treated with tamoxifen. A complete loss of leptin receptors at 170 kDa was observed in LepR KO mice compared with mice lacking the Cre recombinase (Fig. 1B) (15).
In our initial studies, we observed that all of the LepR KO mice died within 10 days after administration of tamoxifen, whereas none of the LepRflox control mice injected with tamoxifen died or showed evidence of cardiac dysfunction. Myh6-MerCreMer/LepRWT, and heterozygous mice exhibited a transient decrease in systolic function that recovered back to normal levels. None of these mice died after tamoxifen injection or exhibited any signs of heart failure. Postmortem analysis of the LepR KO mice revealed significant cardiac enlargement compared with control LepRflox mice injected with tamoxifen (Fig. 2A). The hearts from the LepR KO mice also displayed significant thinning of the ventricles compared with control mice (Fig. 2A). Histological analysis of the heart revealed significant myocyte elongation and myofibril disarray as evidenced by the large gaps between myocytes (Fig. 2B).
Cardiac-specific deletion of leptin receptors results in heart failure.
Cardiac function was evaluated by echocardiography under basal conditions, during and after treatment with tamoxifen for 5 consecutive days. No differences in basal cardiac function were observed between control and Myh6-MerCreMer/LepRflox mice before tamoxifen administration and deletion of the leptin receptors. However, heart failure was observed in LepR KO mice starting at day 4 of tamoxifen administration and lasting through the duration of the study (Figs. 3 and 4, and see supplemental videos 1–4).1 Ventricular M-mode imaging revealed marked attenuation of the contractile peaks in LepR KO compared with control mice (Fig. 3, A and B, and supplemental videos 1–4). 2-D B-mode imaging of the parasternal short axis of the left ventricles showed that LepR KO mice exhibited wall thinning and severely impaired systolic function compared with control mice (Fig. 3, C and D). The LepR KO mice exhibited a marked decrease in EF from 60 to 10% by day 7 posttamoxifen administration (Fig. 4). All of the LepR KO mice exhibited clinical signs of heart failure (labored breathing, limited physical activity) and either died or had to be euthanized before the end of the 9-day experimental protocol. Tamoxifen treatment had no significant effect on ejection fraction in control LepRflox mice (Fig. 4), and none of these mice developed any clinical signs of heart failure. Cardiomyocyte-specific deletion of the leptin receptor also resulted in significant decreases in fractional shortening, cardiac output, and stroke volume on days 5 and 7 (Table 1).
We also determined the effects of cardiomyocyte-specific leptin receptor deletion on blood pressure in LepR KO mice under basal conditions and in response to administration of tamoxifen. Blood pressure was recorded continuously 24 h/day by telemetry and averaged 102 ± 1.5 mmHg over a 5-day baseline period. A significant decrease in blood pressure was observed starting on day 5 of tamoxifen treatment with blood pressure averaging 85 ± 7 mmHg (Fig. 5).
Cardiac-specific deletion of leptin receptors decreased mitochondrial ATP levels and altered cardiac cellular energy status.
To investigate potential mechanisms for the development of rapid heart failure in cardiomyocyte-specific LepR KO mice, we measured ATP levels in mitochondria purified from the hearts of control and KO mice. Cardiac mitochondrial ATP levels averaged 4.3 ± 1.4 nmol/mg protein in control mice and decreased significantly in KO mice, averaging 1.4 ± 0.3 nmol/mg protein (Fig. 6). We also determined the levels of phosphorylated mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) in the hearts of control and cardiomyocyte-specific LepR KO mice. Cardiomyocyte-specific LepR KO mice exhibited significantly decreased levels of phosphorylated mTOR and AMPK compared with control mice (Fig. 7, A and B).
Inducible activation of Cre recombinase transiently decreased cardiac systolic function.
To determine the contribution of Cre recombinase activation to the development of heart failure in LepR KO mice, we also studied cardiac function in two additional control groups: heterozygous Mhy6-MerCreMer/LepRflox/WT and in Mhy6-MerCreMer/LepRWT. Treatment of heterozygous mice with tamoxifen significantly decreased ejection fraction from 61.6 ± 3.8% on day 1 to 24.1 ± 4.7% on day 7 posttamoxifen treatment (Fig. 8A). However, ejection fraction returned to day 1 levels by day 10 posttamoxifen treatment averaging 49.1 ± 4.8% (Fig. 8A). Treatment of Mhy6-MerCreMer mice with tamoxifen also significantly decreased ejection fraction by day 6 but ejection fraction recovered to control levels by the end of the experimental protocol (Fig. 8B). Neither the heterozygous Myh6-MerCreMer/LepRflox/WT nor the Myh6-MerCreMer mice exhibited any overt signs of heart failure as observed in the homozygous LepR KO mice. Heart weight-to-body weight ratio was significantly elevated in Lepr KO compared with control mice and averaged 7.4 ± 0.4 vs. 5.6 ± 0.2 mg/g. While heart weight-to-body weight ratio tended to increase in both heterozygous (6.1 ± 0.5) and Mhy6-MerCreMer (7.0 ± 0.4) compared with control mice, these changes did not achieve statistical significance.
Our results demonstrate that cardiomyocyte-specific deletion of leptin receptors in the presence of Cre recombinase-induced cardiotoxicity severely impairs the heart's ability to maintain adequate myocardial energy metabolism and systolic function. The lack of intact leptin signaling in the heart during cardiac stress associated with increased Cre recombinase activation resulted in lethal heart failure, whereas Cre recombinase activation by itself caused only transient systolic dysfunction and reductions in ejection fraction that recovered to normal levels by 10 days.
Although the effects of Cre recombinase per se on cardiac function have not been widely studied, two previous reports suggest that high level of constitutive expression of Cre recombinase may cause cardiac apoptosis and dysfunction (4, 25). Several previous studies have utilized the same cardiomyocyte-specific inducible Cre mice (Mhy6-MerCreMer) used in the present study for cardiac selective deletion of other genes besides leptin (8, 24, 35, 38, 43, 48). Some of these models exhibit cardiac dysfunction similar to that observed in heterozygous flox mice and Myh6-MerCreMer mice in the current study using an identical tamoxifen activation protocol (8, 35, 48). However, none of these studies included measurements from either heterozygous flox mice or Myh6-MerCreMer mice as control groups to examine the direct effect of Cre activation per se on cardiac function. Therefore, it has been difficult in these previous studies to separate the effects of Cre activation per se from the effects of inactivating the various genes that were investigated.
In the present study we observed a significant decrease in systolic function for 6–7 days after inducible activation of cardiac Cre recombinase in by tamoxifen administration in mice that contained either 1 or no floxed alleles. Unlike what we observed in homozygous floxed leptin receptor mice, the mice with only one or no floxed alleles recovered normal ejection fraction and other indicators of cardiac function on days 8–10 after tamoxifen administration. The decrease in cardiac mitochondrial ATP levels in LepR KO mice is most likely due to the inability of the hearts of these mice to switch substrate preference from fatty acids to glucose in response to Cre-mediated cardiotoxicity. This hypothesis is supported by our previous observations in tamoxifen-treated Myh6-MerCreMer mice in which the levels of PGC-1α in the heart were significantly decreased even after recovery of systolic function (17). PGC-1α decreases expression of genes involved in glycolytic pathways while increasing genes required for mitochondrial biogenesis and metabolism of fatty acids. Thus suppression of PGC-1α would enhance expression of genes involved in glycolytic pathway helping the heart switch substrates to enhance cardiac energy production. Further support for the important role of cardiac leptin signaling in the enhancement of the glycolytic pathway in the hearts of tamoxifen-treated Myh6-MerCreMer mice is a decrease in the levels of pyruvate dehydrogenase kinase-4 (PDK-4) protein (17). Glycolysis and glucose oxidation are both enhanced in association with decreases in PDK-4 levels. The results from the current study and our previous study (17) suggest that Cre-mediated cardiotoxicity alters cardiac energetics via disruptions in oxidative phosphorylation, which may be compensated for through enhanced energy production via the glycolytic pathway.
An important new finding of the current study is the cardiac-specific LepR deletion in the setting of altered cardiac oxidative phosphorylation (similar to that seen with moderate myocardial ischemia) caused by transient Cre recombinase expression leads to lethal heart failure associated with marked reductions in ATP production. The present observations offer new insights into the role of leptin in influencing cardiac function. The effects of LepR deletion are revealed when the heart is stressed by Cre-mediated cardiotoxicity. One might consider this analogous to the role of insulin in cardioprotection, which is most evident when the heart is stressed by ischemia or other insults that interfere with normal mitochondrial function. We also studied heterozygous floxed LepR/Myh6-MerCreMer mice with one copy of the targeted loxP LepR allele and demonstrated that the impact is intermediate to the effects of tamoxifen-induced Cre expression alone and the effects of complete LepR deletion in the setting of Cre-mediated cardiotoxicity.
In contrast to mice in which enhanced Cre recombinase activation resulted in transient systolic impairment without heart failure, mice with cardiac leptin receptor deletion in the setting of cardiac stress caused by Cre recombinase activation developed severe, lethal heart failure. The specific molecular mechanisms by which cardiomyocyte-specific leptin receptor deletion exacerbates cardiac dysfunction caused by Cre recombinase activation and leads to lethal heart failure are uncertain, although it is clear from our studies that induced deletion of leptin receptors impairs cardiac ATP production.
Leptin signals through three major pathways, including Jak2-IRS-phosphatidylinositol 3-kinase(PI3K), SH2-containing protein tyrosine phosphatase 2 (Shp2)-mitogen-activated protein (MAP) kinase, and signal transducer and activator of transcription protein 3 (STAT3). The importance of these individual pathways in mediating leptin signaling in the heart has not been widely studied. Deletion of STAT3 in the hearts of mice leads to a progressive dilated cardiomyopathy over a period of months (19), whereas cardiac-specific deletion of Shp2 results in a dilated cardiomyopathy over a period of weeks (22). However, it is still unclear what role disruption of leptin signaling versus other factors that activate these signaling pathways plays in the cardiac dysfunction associated with STAT3 or Shp2 deletion. Also, as discussed previously, it is unclear what role increased Cre recombinase may have contributed to cardiac dysfunction in these studies since these studies did not control for the effects of increased Cre recombinase activation per se. Nevertheless, these findings, when considered in the context of our present results, are consistent with the possibility that leptin-mediated STAT3 and Shp2 signaling may play a major role in regulating cardiac function. Leptin signaling through the PI3K pathway may also play an important role in the recovery from Cre-mediated toxicity. Loss of cardiac leptin receptors results in decrease PI3K activity and an inability to switch from fatty acid to glucose metabolism following acute myocardial infarction (47). However, stimulation of the this pathway as well as the STAT3 and Akt pathways with ciliary neurotrophic factor (CNTF) was able to restore glucose metabolism after acute myocardial infarction in cardiac leptin receptor-deficient mice (47). The results of this study indicate that leptin may play a similar role in the ability of the heart to switch from fatty acid to glucose metabolism in response to Cre-mediated cardiotoxicity.
A recent study by McGaffin et al. (29) investigated the effects of leptin receptor deletion on ischemic heart failure caused by coronary artery ligation. In this study, cardiac leptin deficiency did not cause lethal heart failure as observed in the present study. However, a lower dose of tamoxifen (20 mg/day) was utilized to induced Cre activity, which may have resulted in less Cre activation. This dose of tamoxifen (20 mg/day) also resulted in an incomplete deletion of cardiac leptin receptors, as evidenced by significant residual leptin receptor mRNA and protein levels in the hearts of the treated mice (29). This is in contrast to our study in which we found no detectable leptin receptor protein in the hearts of LepR KO mice treated with the higher tamoxifen dose. As demonstrated in our current study, complete leptin receptor deletion in the setting of the cardiac insult caused by Cre-recombinase activation led to lethal heart failure. It is also clear the even incomplete loss of the cardiac leptin receptor results in more severe heart failure and worsens survival after coronary artery ligation (28). The results of this study as well as our current study indicate that cardiac leptin receptor activation plays an important protective role in the heart in response to cardiac stress.
In the present study leptin receptor deletion in the setting of Cre-mediated cardiotoxicity led to marked decreases in cardiac mitochondrial ATP levels that were associated with reduced pmTOR and pAMPK. Previous studies suggest that leptin increases fatty acid metabolism in several tissues including the heart (26, 37, 42). Impaired leptin receptor activation, especially in circumstances associated with other cardiac stresses, may result in decreased fatty acid delivery to mitochondria, which then leads to decreased ATP levels. Leptin receptor deletion may also impair glucose utilization by the heart. Although some in vitro studies with cultured cardiomyocytes as well as whole animal studies have failed to demonstrate an increase in cardiac glucose uptake in response to leptin (20, 37), studies in isolated Langendorff-perfused hearts have demonstrated that leptin increases glucose uptake (16). Therefore, loss of cardiac leptin receptor function may not only decrease fatty acid metabolism and delivery to the mitochondria but may also diminish the ability of the heart to increase glucose utilization to compensate for decreased fatty acid metabolism. Given the unclear role of leptin in cardiac glucose utilization, this issue should be examined in greater detail in future studies.
The mTOR and pAMPK pathways are involved with intracellular nutrient sensing and are important targets of leptin in the hypothalamus and in macrophages (1, 27). Leptin is believed to activate mTOR through the PI3K pathway (18). Activation of mTOR in the heart occurs in pressure overload forms of cardiac hypertrophy and inhibition of mTOR reduces hypertrophy in these models (14, 31). Our finding of decreased levels of phosphorylated mTOR in the heart of leptin receptor knockout mice could be due to loss of leptin signaling and severe decrease in energy status of the cardiomyocytes as reflected by decreased levels of mitochondrial ATP. Studies in transformed HEK293 cells have shown that mTOR is directly regulated by cellular ATP levels (10). Another potential mechanism for decreased phosphorylated mTOR in the hearts of knockout mice is through decreased glucose metabolism as previous studies have demonstrated that glucose metabolism is required for mTOR signaling in the heart (41). Loss of mTOR signaling in the hearts of leptin knockout mice could also result in further decreases in fatty acid metabolism contributing to further reductions in ATP levels (9).
Our finding of decreased phosphorylated AMPK in the hearts of leptin receptor knockout mice may seem surprising given that phosphorylation of AMPK is inversely related to the level of ATP in the cell (6). However, the cardiac energy levels of the LepR KO mice may be below those sustainable for phosphorylation of AMPK to occur or there may not be sufficient AMP present in the heart to increase levels of phosphorylated AMPK levels. AMPK activation has been demonstrated to increase glucose uptake in the heart (34). Loss of AMPK signaling in this model could contribute to the inability of the heart to increase glucose metabolism to compensate for lack of fatty acid metabolism as a result of defective leptin signaling in the hearts of knockout mice. In another study, cardiac leptin receptor deletion increased apoptosis, resulted in systolic dysfunction, and worsened survival following coronary arterial ligation, and these changes were associated with decreased active AMPK and impaired glycolytic metabolism (29). Treatment of these mice with aminoimidazole carboxamide ribonucleotide, an AMPK activator, improved glucose metabolism as well as cardiac function. Leptin increases phosphorylation and activation of the α2 catalytic subunit of AMPK (α2 AMPK) in skeletal muscle (32, 33). Acute leptin treatment in Langendorff-perfused hearts, however, was not associated with increases in AMPK phosphorylation (2). Whether leptin can chronically regulate cardiac AMPK phosphorylation is not known and merits further investigation.
Although Cre recombinase-mediated cardiotoxicity is not a pathological cause of human heart failure, it appears to mimic other toxic cardiomyopathies such as doxorubicin-induced cardiomyopathy (36). Systolic heart failure is generally associated with reductions in myocardial energy production and a switch in myocardial metabolism from primarily fatty acid oxidation to utilization of glucose. For reasons that are not completely understood, circulating leptin levels are elevated independent of body weight in patients with systolic heart failure (39). Furthermore, levels of leptin and other adipokines are elevated in patients with acute decompensated heart failure and decreased as patients became more compensated (40). These clinical observations, when taken into context with our findings and others such as McGaffin et al. (29), suggest that augmented cardiac leptin signaling may help the heart maintain ATP formation and systolic function during myocardial stress.
In summary, we found that acute activation of Cre recombinase resulted in transient impairment of cardiac systolic function that recovered within a few days with subsequent cardiac hypertrophy and normalization of left ventricular systolic function. However, when activation of Cre recombinase was coupled with abrupt loss of leptin signaling in the heart, this led to irreversible heart failure, and 100% lethality. These cardiac phenotypes are accompanied by significant decreases in mitochondrial ATP levels as well as decreases in energy-sensing proteins mTOR and AMPK. Thus our model has revealed a previously unknown role for leptin in the regulating cardiac function and energy metabolism, and that these effects become especially important in the setting of cardiac stress caused by Cre-recombinase expression.
This work was supported by the National Heart, Lung, and Blood Institute Grant PO1HL-51971, as well as a seed grant from the American Medical Association (to M. E. Hall).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: M.E.H., J.E.H., and D.E.S. conception and design of research; M.E.H. and G.S. performed experiments; M.E.H., G.S., and D.E.S. analyzed data; M.E.H., G.S., J.E.H., and D.E.S. interpreted results of experiments; M.E.H. and D.E.S. prepared figures; M.E.H., J.E.H., and D.E.S. drafted manuscript; M.E.H., G.S., J.E.H., and D.E.S. edited and revised manuscript; M.E.H., G.S., J.E.H., and D.E.S. approved final version of manuscript.
We thank Dr. Jeffrey D. Molkentin, Cincinnati Children's Hospital, for the Myh6-MerCreMer transgenic mice and Dr. Jeffrey M. Friedman, Rockefeller University, for the LepRflox mice.
↵1 The online version of this article contains supplemental material.
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