Interaction between age and obesity on cardiomyocyte contractile function: role of leptin and stress signaling.

OBJECTIVES: This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca2+ properties.
METHODS: Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (+/- dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR(90)), intracellular Ca2+ levels and decay. O2(-) levels were measured by dihydroethidium fluorescence.
RESULTS: Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, +/- dL/dt, intracellular Ca2+ rise, prolonged TR(90) and intracellular Ca2+ decay, enhanced O2(-) production and p(47phox) expression without an additive effect of the two, with the exception of intracellular Ca2+ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIkappaB. Low levels of leptin reconciled contractile, intracellular Ca2+ and cell signaling defects as well as O2(-) production and p(47phox) upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca2+ response as well as O2(-) production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response.
CONCLUSIONS: Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O2(-) release.

Introduction

Obesity is a devastating health problem afflicting all ages, races and socioeconomic classes in both genders. Over the past decade, only modest success has been achieved in combating the escalating prevalence of obesity and metabolic syndrome [1], [2]. The current obesity epidemic may be attributed to many factors including environmental (e.g., caloric and nutrient intake), genetic and even evolutionary (e.g., interaction between human biology and human culture over the long period of human evolution) [1], [3]. With today's prolonged human lifespan, aging has also been considered as an obesogenic factor given the increased visceral fat associated with aging [4]. Paradoxically, visceral fat accumulation may in turn influence longevity, thus prompting the speculation that obesity could be a condition of premature aging [4]. Although effective physiological adjustments are present to counterbalance the potentially detrimental health outcome of obesity such as altered respiratory mechanical/muscular function peculiar to the aging condition [5], a number of obesity-associated comorbidities such as cancer, endocrine, cardiovascular and immune disorders may ultimately contribute to premature aging and the shortened lifespan. Therefore, the concept of health promotion, especially on nutrition and life style, has become an important part of health care in older adults [6].

Among a wide array of comorbidities associated with obesity including type 2 diabetes, hypertension, cancer and sleep apnea [1], heart disease, which is mainly manifested by cardiac hypertrophy and compromised ventricular function, may lead to heart failure or premature death [7]–[9]. The pathophysiological alterations associated with establishing and perpetuating obesity-induced heart disease are complex but are becoming more clear, including the interaction of sympathetic overactivation and endothelial dysfunction [10]. In an effort to better understand the pathophysiology of human obesity, several rodent models of obesity have been developed and implemented including high fat diet feeding and spontaneous mutants of the 16 KD obesity gene product leptin or its receptor such as ob/ob and db/db mice. A common feature of these obese animal models is the overtly compromised cardiac contractile function associated with a marked increase in visceral fat and hyperinsulinemia [11]–[14], similar to human obesity. Accumulating evidence has also implicated a role of the obese gene product leptin, which regulates food intake and energy expenditure, in the regulation of cardiac function, while the disruption of which contributes to obesity-associated cardiac contractile and morphometric defects [15], [16]. Human circulating leptin levels are elevated in obesity, vascular and coronary heart diseases, favoring a contemporary perception of hyperleptinemia being an independent risk factor for cardiovascular diseases [16], [17]. This notion is further supported by the experimental evidence that leptin may contribute to cardiac hypertrophy, atherosclerosis and thrombosis possibly through accumulation of reactive oxygen species [16], [18], [19]. Elevated leptin level or hyperleptinemia is correlated with hyperphagia, insulin resistance, hyperlipidemia and hypertension, independent of total adiposity [16]. Data from our lab revealed that leptin directly suppresses cardiomyocyte contraction and intracellular Ca2+ handling through mechanism(s) related to endothelial nitric oxide synthase (eNOS), superoxide (O2 −) production, activation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and stress signaling pathways including Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase [20]–[22]. Further evidence from our lab as well as others also indicated that leptin deficiency paradoxically triggers cardiac hypertrophy and contractile dysfunction in ob/ob obese mice with a mutant leptin gene, the effect of which is reconciled by leptin supplementation [12], [15]. Both hyperleptinemia and leptin-deficiency have been shown to be associated with increased apoptosis, DNA damage and mortality, suggesting a potential association between leptin signaling and aging-related DNA damage and premature death [23]. Nevertheless, the interaction between obesity and aging on cardiac function, with a focus on leptin signaling, has not been elaborated. Given the prevalence of metabolic syndrome in older adults and the detrimental impact of metabolic syndrome especially obesity on life expectancy and comorbidity in the elderly [24], the present study was designed to evaluate the influence of aging on basal and leptin-elicited cardiac contractile response in the leptin-deficient ob/ob mice. Expression and activation of the leptin receptor Ob-R and post-receptor signaling STAT-3, O2 − producing enzyme NADPH oxidase (p 47 subunit) [25], Akt, eNOS, AMP-activated kinase (AMPK) and the stress signaling molecules p38 MAP kinase, JNK, extracellular signaling regulated kinase (ERK) and NFκB were also examined in young and aging lean C57 and ob/ob leptin deficient obese mice. Twelve- and eighteen-month-old mice were chosen for the aging group largely due to the reduced lifespan and high mortality seen after one year of age in ob/ob mice [26]. Cardiomyocyte contractile function was also examined in the high fat diet-induced and the leptin receptor mutant hyperleptinemic db/db obesity models for comparison.

Materials and Methods

Experimental animals and high fat diet feeding

All animal procedures were conducted in accordance with humane animal care standards outlined in NIH Guide for the Care and Use of Experimental and were approved by the University of Wyoming and University of North Dakota Animal Care and Use Committees. In brief, young (4-month-old) and aging (12- or 18-month-old) male homozygous B6.V-lep/J leptin deficient ob/ob and B6.Cg-m +/+ Leprdb/J leptin receptor mutant db/db obese mice were housed in our institutional animal facilities. Age- and gender-matched wild-type C57BL/6J mice were used as lean controls. All animals were allowed free access to standard lab chow and tap water. For high fat diet-induced obesity model, 4- and 12-month-old male C57BL/6J mice (4 per group) were randomly assigned to a low fat (10% of total calorie) or a high fat (45% of total calorie) diet (Research Diets Inc., New Brunswick, NJ, USA) for 16 weeks [13]. Blood glucose was monitored with a glucometer (Accu-ChekII, model 792, Boehringer Mannheim Diagnostics, Indianapolis, IN, USA). All mice used for lifespan analysis (the Kaplan-Meier survival curve and log-rank test) were assigned to a longevity cohort at birth and were not used for any biochemical, immunoblotting or mechanical function tests. Only male mice were used for this study.

Body fat composition measurement

Body composition was measured using Dual Energy X-ray Absorptiometry (DEXA), which is a clinical measure of lean tissue mass, adipose tissue mass, and bone mineral mass and density. A low level pencil-beam x-ray moved transversely from the head to the tail across the sedated mouse. Difference in absorbance of the X-ray was detected according to tissue density. Percent fat was calculated using fat and body mass [27].

Cardiomyocyte isolation and in vitro leptin treatment

Mouse hearts were removed under anesthesia (ketamine/xylazine at 3∶1, 1.32 mg/kg) and were perfused with oxygenated (5% CO2–95% O2) Krebs-Henseleit bicarbonate (KHB) buffer containing (in mmol/L) 118 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 10 HEPES, and 11.1 glucose. Hearts were perfused with a Ca2+-free KHB containing Liberase Blendzyme 4 (Hoffmann-La Roche Inc., Indianapolis, IN, USA) for 20 min. After perfusion, left ventricles were removed and minced to disperse cardiomyocytes in Ca2+-free KHB buffer. Extracellular Ca2+ was added incrementally back up to 1.25 mM [12]. Myocyte yield was ∼75% which was not affected by obesity or age. Cohorts of cardiomyocytes were incubated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in a serum-free defined medium consisting of Medium 199 (Sigma) with Earle's salts. The concentrations of leptin were chosen to cover physiological (0.5 and 1.0 nM) as well as pharmacological levels [16], [22], [28]. Cardiomyocytes with obvious sarcolemmal blebs or spontaneous contractions were not used for mechanical recording.

Cell shortening/relengthening

Mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam® system (IonOptix Corporation, Milton, MA, USA) [12]. In brief, cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope (Olympus Incorporation, Model IX-70, Tokyo, Japan) and superfused at 25°C with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. The cells were field stimulated with suprathreshold voltage (150% of the threshold voltage of cell contraction) at a frequency of 0.5 Hz, 3 msec duration, using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE, USA). The myocyte being studied was displayed on a computer monitor using an IonOptix MyoCam camera. An IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), the amplitude myocytes shortened upon electrical stimulation, an indicative of peak ventricular contractility; time-to-PS (TPS), the duration of myocyte shortening, an indicative of systolic duration; time-to-90% relengthening (TR90), the duration to reach 90% relengthening, an indicative of diastolic duration (90% rather 100% relengthening was used to avoid noisy signal at baseline level); and maximal velocities of shortening/relengthening, maximal slope (derivative) of shortening and relengthening phases, indicatives of maximal velocities of ventricular pressure increase/decrease.

Intracellular Ca2+ transient measurement

Intracellular Ca2+ was measured using a dual-excitation, single-emission photomultiplier system (IonOptix) in myocytes loaded with Fura-2-AM (0.5 µM). Myocytes were placed on an inverted microscope and imaged through an Olympus (IX-70) Fluor ×40 oil objective. Myocytes were exposed to light emitted by a 75-W halogen lamp through either a 360- or 380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after initial illumination at 360 nm for 0.5 s and then at 380 nm for the duration of the recording protocol. The 360-nm excitation reading was repeated at the end of the protocol. Qualitative evaluation of intracellular Ca2+ was inferred from fluorescence intensity changes. Myocyte shortening was also evaluated in a cohort of the fura-2-loaded ventricular myocytes simultaneously to compare their temporal relationship with the fluorescence signal. However, their mechanical properties were not used for data summary due to the apparent Ca2+ buffering effect of fura-2 [12].

Western blot analysis

Following leptin treatment, cardiomyocytes from young and aging C57 and ob/ob mice were collected and sonicated in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS and protease inhibitor cocktail. The protein concentration of the supernatant was evaluated using the protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal-amounts (30 µg) of protein and prestained molecular weight marker (GIBCO, Gaithersburg, MD, USA) were loaded onto 7%∼10% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad), separated, and transferred to nitrocellulose membranes (0.2 µm pore size, Bio-Rad). Membranes were incubated for 1 hr in a blocking solution containing 5% nonfat milk in TBS-T before being washed in TBS-T. Membranes were then incubated overnight at 4°C with anti-p47 (1∶1000, kindly provided by Dr. Mark T. Quinn from Montana State University, Bozeman, MT), anti-p38 MAP kinase and anti-phospho-p38 MAP kinase (pp38, 1∶1,000), anti-ERK (1∶1,000;), anti-phospho-ERK (pERK; 1∶1,000), anti-SAPK/JNK (1∶1,000), anti-phospho-SAPK/JNK (pJNK, 1∶1,000), anti-IκB (1∶1,000), anti-phospho-IκB (pIκB, 1∶1,000), anti-Akt (1∶1,000), anti-phospho-Akt (pAkt, 1∶1,000), anti-eNOS, anti-phospho-eNOS (peNOS, 1∶1000), anti-STAT3 (1∶1,000), anti-phospho-STAT3 (pSTAT3, 1∶1,000), anti-Ob-R (long form isoform Ob-Rb, 1∶1000), anti-AMPK (1∶1000) and anti-phospho-AMPK (pAMPK,1∶1000) antibodies. GAPDH was used as the internal loading control (1∶5,000). All antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA) unless otherwise specified. After incubation with the primary antibodies, blots were incubated with either anti-mouse or anti-rabbit IgG horseradish peroxidase-linked antibodies at a dilution of 1∶5,000 for 60 min at room temperature. Immunoreactive bands were detected using the Super Signal West Dura Extended Duration Substrate (Pierce, Milwaukee, WI). Intensity of the bands was measured with a scanning densitometer (model GS-800; Bio-Rad) coupled with Bio-Rad personal computer analysis software.

Intracellular fluorescence measurement of superoxide (O2 −)

Intracellular superoxide were monitored by changes in fluorescence intensity resulting from intracellular probe oxidation according to a previously described method [22]. Following leptin (0.5, 1.0 and 50 nM) treatment, cardiomyocytes from young and aging C57 lean and ob/ob mice were loaded with 5 µM dihydroethidium (DHE) (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C and washed with PBS buffer. Cells were sampled randomly using an Olympus BX-51 microscope equipped with an Olympus MagnaFire™ SP digital camera and ImagePro image analysis software (Media Cybernetics, Silver Spring, MD). Fluorescence was calibrated with InSpeck microspheres (Molecular Probes). More than 150 cells per group were evaluated using the grid crossing method for cell selection in more than 15 visual fields per experiment.

Statistical analysis

Data are presented as mean ± SEM. Statistical significance (p<0.05) was determined by a one- or two-way analysis of variance (ANOVA) followed by a Tukey's post hoc analysis.

Results

General features of experimental animals

As expected, young ob/ob mice displayed significantly greater body, heart, liver and kidney weights compared with age-matched young C57 mice. The organ size (when normalized to tibial length) was also significantly greater in young ob/ob mice compared with the young C57 group. Body and organ (except kidney in aging ob/ob mice) weights were significantly heavier in aging (12-month-old) C57 or ob/ob mice compared with respective young groups, as expected. Consistently, the organ size (normalized to tibial length) was significantly greater in the 12-month-old aging C57 mice compared with young C57 mice. Nonetheless, liver size but not that of heart and kidney was significantly enhanced with aging in ob/ob mice. Body fat composition was significantly elevated with aging and obesity, with an additive effect between the two. There were no significant differences in tibial length and fasting glucose levels among the four mouse groups, excluding growth-related factor and the presence of full-blown diabetes mellitus (Table 1). The 18-month-old C57 and ob/ob mice displayed a comparable biometric profile somewhat similar to their 12-month-old counterparts (data not shown). The Kaplan-Meier survival curve comparison depicts that ob/ob mice display significantly reduced survival rates when compared to C57 lean mice. The median lifespan was 27.0 and 18.0 months for C57 and ob/ob mice, respectively (p = 0.0007). Survival curves of the two mouse groups began to separate after ∼6 months of age with ob/ob mice exhibiting a greater mortality rate (Fig. 1).

Table 1

General features of young (4-month-old) or aging (12-month-old) lean C57 and ob/ob mice.

	C57-young	C57-aging	ob/ob-young	ob/ob-aging
Body Weight (g)	24.0±0.5	29.0±0.5*	54.3±0.9**	63.6±2.9*,**
Heart Weight (mg)	144±3	178±3*	316±5**	350±8*,**
Tibial Length (mm)	16.6±0.2	17.0±0.2	16.5±0.2	17.1±0.3
HW/TL (mg/mm)	8.66±0.19	10.4±0.2*	19.1±0.3**	20.5±0.5**
Liver Weight (g)	1.28±0.08	1.58±0.07*	3.12±0.13**	4.58±0.17*,**
LW/TL (mg/mm)	77.2±4.8	92.5±4.2*	189.3±7.6**	267.8±9.9*,**
Kidney Weight (g)	0.27±0.02	0.37±0.02*	0.46±0.02**	0.49±0.02**
KW/TL (mg/mm)	16.1±0.6	21.8±1.1*	28.1±1.0**	28.9±0.9**
Body Fat Composition (%)	18.2±1.2	26.5±1.2*	69.1±2.1**	78.6±1.7*,**
Blood Glucose (mM)	5.50±0.12	5.60±0.21	5.79±0.13	5.79±0.20

HW  =  heart weight; LW  =  liver weight; KW  =  kidney weight; TL  =  tibial length; Mean ± SEM, * p<0.05 vs. corresponding young group, ** p<0.05 vs. corresponding C57 group, n = 13 and 14 mice for C57 and ob/ob groups, respectively.

Figure 1

Cumulative survival curve (Kaplan-Meier survival plot) of male C57 lean and ob/ob obese mice.

The cumulative survival rate was plotted against age in months. The Log rank test was performed to compare the two mouse groups (p = 0.0007). n = 26 and 16 mice for C57 and ob/ob mice, respectively.

Mechanical and intracellular Ca2+ properties of cardiomyocytes in ob/ob obesity

Neither obesity nor aging significantly affected the myocyte yield or overall appearance (Fig. 2). The resting cell length was significantly greater in young ob/ob and aging (both 12 and 18 months of age) C57 mice compared with young C57 mice. Obesity further augmented aging-elicited elongation in cardiomyocyte resting cell length at both 12 and 18 months of age. Short-term leptin treatment did not affect resting cell length in either young or aging C57 and ob/ob mice (Fig. 3A). Both obesity and aging (12 and 18 months) significantly reduced peak shortening (PS) amplitude and maximal velocity of shortening/relengthening (± dL/dt), prolonged time-to-90% relengthening (TR90) without affecting time-to-peak shortening (TPS). There was little difference in the aging-induced change in mechanical parameters between 12 and 18 months of age. In addition, there was no discernible synergistic effect between obesity and age on these mechanical indices. Leptin supplementation at physiological levels (0.5 and 1.0 nM) effectively nullified obesity-induced mechanical deficiencies (PS, ± dL/dt and TR90) in young but not aging (12-month) mouse groups. However, leptin treatment (0.5 and 1.0 nM) did not alter aging-induced mechanical changes in PS, ± dL/dt and TR90 (Fig. 3B-3F). Consistent with our previous report [22], pharmacological level of leptin (50 nM) overtly impaired cardiomyocyte mechanical function including depressed PS and ± dL/dt as well as prolonged TPS and TR90 in both young and aging C57 or ob/ob mouse groups (Fig. 3A–3F). To explore the possible role of intracellular Ca2+ homeostasis in obesity and/or aging-induced mechanical responses, we evaluated intracellular Ca2+ transients using the Fura-2 fluorescence measurement. Our results indicated that both obesity and aging enhanced resting intracellular Ca2+ levels without any additive effects. The rise of intracellular Ca2+ in response to electrical stimuli was significantly reduced by either obesity or aging (12- and 18-month) with an overt additive effective between the two. Both obesity and aging reduced the intracellular Ca2+ clearing rate (single and bi-exponential decay) with no additive effect. Consistent with its effect on cardiomyocyte shortening, there was little difference in the aging-induced change in intracellular Ca2+ property between 12 and 18 months of age. Furthermore, short-term leptin supplementation at physiological levels (0.5 and 1.0 nM) significantly attenuated or ablated intracellular Ca2+ abnormalities in young but not aging ob/ob mice. Consistent with its response in cardiomyocyte shortening, short-term leptin treatment at physiological levels (0.5 and 1.0 nM) failed to affect aging-induced changes in intracellular Ca2+ handling although pharmacological level of leptin (50 nM) drastically interrupted cardiomyocyte intracellular Ca2+ homeostasis including elevated resting intracellular Ca2+ levels, depressed intracellular Ca2+ rise in response to electrical stimuli and prolonged intracellular Ca2+ decay in both young and aging C57 or ob/ob mouse groups (Fig. 4). Given that 12 and 18 months of age produced reminiscent mechanical changes in C57 lean and ob/ob mice, 12 months of age was chosen as the only aging group the remaining of ob/ob study.

Figure 2

Light microscopic images of cardiomyocytes freshly isolated from young (4-month-old) and aging (12- or 18-month-old) lean (C57) and ob/ob mice.

200x, scale bar  = 100 µm.

Figure 3

Contractile properties of cardiomyocytes freshly isolated from young (4-month-old) and aging (12- or 18-month-old) lean (C57) and ob/ob mice treated with or without leptin (0.5, 1.0 and 50 nM) for 4 hrs.

A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS); F: Time-to-90% relengthening (TR90); Mean ± SEM, n = 50–53 cells from 3 mice per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Figure 4

Intracellular Ca2+ transient properties of cardiomyocytes freshly isolated from young (4-month-old) and aging (12- or 18-month-old) lean (C57) and ob/ob mice treated with or without leptin (0.5, 1.0 and 50 nM) for 4 hrs.

A: Resting intracellular Ca2+ fluorescence intensity; B: Rise in intracellular Ca2+ fluorescence intensity in response to electrical stimuli; C: Single-exponential Ca2+ transient decay rate and D: Bi-exponential Ca2+ transient decay rate. Mean ± SEM, n = 36–38 cells from 3 mice per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Influence of age and ob/ob obesity on O2 − production and NADPH oxidase (p subunit) expression

Depending on the level of exposure, leptin is known to elicit a paradoxical effect on cardiomyocyte contractile function through either inhibition or stimulation of O2 − production [22,22]. To determine whether O2 − production plays a role in the disparate leptin effects between young and aging ob/ob mice, we evaluated O2 − production and expression of the rate-limiting enzyme for O2 − production NADPH oxidase (p 47 subunit) [25] using DHE fluorescence and Western blot analysis, respectively. Our data suggested that obesity and aging (12-month) significantly enhanced O2 − production and upregulated expression of p 47 NADPH oxidase without an additive effect of the two. Leptin supplementation at physiological levels (0.5 and 1.0 nM) ablated obesity-induced O2 − production and p 47 NADPH oxidase expression in young but not aging ob/ob mice. Nonetheless, leptin treatment at 0.5 and 1.0 nM failed to reconcile aging-induced effects on O2 − production and p 47 NADPH oxidase expression. Consistent with the mechanical and intracellular Ca2+ response, pharmacological level of leptin (50 nM) directly enhanced O2 − production and upregulated expression of p 47 NADPH oxidase in both young and aging C57 or ob/ob mouse groups (Fig. 5).

Figure 5

O2 − production (Panel A) and p 47 NADPH oxidase subunit expression (Panel B) measured by DHE fluorescence and immunoblotting, respectively, in cardiomyocytes freshly isolated from young (4-month-old) or aging (12-month-old) lean (C57) and mice treated with or without leptin (0.5, 1.0 and 50 nM) for 4 hrs.

Insets: Representative gel blots of p 47 NADPH oxidase subunit using specific anti-p 47 antibody. GAPDH was used as the loading control. Mean ± SEM, n = 12–14 (Panel A) and 9–11 (Panel B) per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Influence of age and ob/ob obesity on leptin receptor (Ob-R) and STAT3 activation

To determine the potential involvement of leptin signaling in obesity and/or age-induced effects on cardiomyocyte contractile function, intracellular Ca2+ homeostasis and O2 − production, we evaluated the leptin receptor Ob-R, the post-leptin receptor signaling molecule STAT-3 and STAT-3 phosphorylation. Our results shown in Fig. 6 revealed that obesity, but not aging (12-month), significantly reduced Ob-R protein expression and its post-receptor signaling STAT-3 phosphorylation without affecting the total STAT-3 expression. Interestingly, short-term leptin supplementation at both 1.0 nM and 50 nM significantly upregulated Ob-R expression in young but not aging ob/ob mice and stimulated STAT-3 phosphorylation in both young and aging ob/ob groups. Leptin treatment at 1.0 and 50 nM did not alter the expression of Ob-R and STAT-3 in lean mice although the high but not the low concentration of leptin directly stimulated STAT-3 phosphorylation in lean mice.

Figure 6

The leptin receptor Ob-R expression (Panel A) and phosphorylation of the leptin receptor downstream signaling molecule STAT-3 (pSTAT3, Panel B) in cardiomyocytes freshly isolated from young (4-month-old) or aging (12-month-old) lean (C57) and ob/ob mice treated with or without leptin (1.0 and 50 nM) for 4 hrs.

Protein expression of Ob-R and pSTAT-3 was normalized to the loading control GAPDH or total STAT-3, respectively. Insets: Representative gel blots of Ob-R, pSTAT-3 and STAT-3 proteins using specific antibodies. Mean ± SEM, n = 3 – 6 isolations, * p<0.05 vs. respective C57 group, # p<0.05 vs. respective ob/ob group.

Influence of age and ob/ob obesity on Akt, eNOS and AMPK

A number of signaling molecules have been shown to participate in obesity and aging-induced biological responses and regulation of cardiac function, including Akt, the Akt downstream signal eNOS and the cellular fuel AMPK [29], [30]. We examined the expression of Akt, eNOS, AMPK and their phosphorylation in young and aging (12-month) C57 lean and ob/ob mouse cardiomyocytes. Our results revealed that either obesity or age independently dampened the phosphorylation of Akt and its downstream signaling molecule eNOS without affecting expression of Akt and eNOS. There was no interaction between obesity and age on the phosphorylation of Akt and eNOS. Short-term leptin treatment at physiological level (1.0 nM) reconciled the reduced phosphorylation of Akt and eNOS in young ob/ob, but not the aging mice. While obesity and age alone failed to affect AMPK and its phosphorylation, the combination of the two significantly attenuated AMPK phosphorylation but not total AMPK expression. Short-term leptin treatment at physiological level (1.0 nM) reduced AMPK phosphorylation in young ob/ob mice but not other groups. Short-term leptin treatment at physiological level did not affect the expression of non-phosphorylated Akt, eNOS and AMPK. Interestingly, short-term treatment of leptin at pharmacological level (50 nM) significantly suppressed the phosphorylation of Akt, eNOS and AMPK in all mouse groups (with the exception of AMPK phosphorylation in aging ob/ob group) without affecting the expression of non-phosphorylated Akt, eNOS and AMPK (Fig. 7).

Figure 7

Panel A: Representative gel blots of total and phosphorylated Akt, eNOS and AMPK in cardiomyocytes freshly isolated from young (4-month-old) or aging (12-month-old) lean (C57) and ob/ob mice treated with or without leptin (1.0 and 50 nM) for 4 hrs using specific antibodies; Panel B: Phosphorylation of Akt expressed as pAkt-to-Akt ratio; Panel C: Phosphorylation of eNOS expressed as peNOS-to-eNOS ratio; and Panel D: Phosphorylation of AMPK expressed as pAMPK-to-AMPK ratio.

Mean ± SEM, n = 4 – 6 isolations, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Influence of age and ob/ob obesity on p38 MAP kinase, JNK, ERK and IκB

To further examine the possible role of stress signaling pathways in obesity, aging and leptin-induced cardiac responses, expression of p38 MAP kinase, JNK, ERK and the NFκB inhibitor IκB as well as their phosphorylation were examined in young and aging C57 lean and ob/ob mouse cardiomyocytes. Our results revealed that both obesity and age significantly inhibited and stimulated phosphorylation of p38 MAP kinase and JNK, respectively, without affecting expression of total p38 MAP kinase or JNK. There was no additive effect between obesity and age on the phosphorylation of p38 MAP kinase and JNK. Short-term leptin supplementation at physiological level (1.0 nM) restored obesity-induced changes in the phosphorylation of p38 MAP kinase and JNK in young mice without affecting that in aging mice. Neither obesity nor age affected expression of total and phosphorylated ERK, although the combination of the two significantly reduced ERK phosphorylation. Leptin at 1.0 nM reconciled the reduced ERK phosphorylation in aging ob/ob mice without affecting any other mouse groups. Expression of non-phosphorylated ERK was unaffected by short-term leptin treatment at 1.0 nM. Our data further revealed that either obesity or aging significantly enhanced phosphorylation of IκB with no additive effect between the two. IκB is an inhibitor of NFκB where enhanced IκB phosphorylation removes its inhibition on NFκB). Similar to its effect on other stress signaling molecules, short-term leptin treatment at physiological level (1.0 nM) removed obesity-induced phosphorylation of IκB in young but not aging mice. Last but not the least, short-term treatment of leptin at pharmacological level (50 nM) significantly activated the stress signaling molecules p38, JNK, ERK and NFκB (via enhanced phosphorylation of IκB) in all mouse groups without affecting the expression of non-phosphorylated proteins (Fig. 8).

Figure 8

Total and phosphorylated protein expression of p38 MAP kinase, JNK, ERK and IκB in cardiomyocytes isolated from young (4-month-old) or aging (12-month-old) lean (C57) and ob/ob mice treated with or without leptin (1.0 and 50 nM) for 4 hrs.

Panel A: Phosphorylation of p38 expressed as pp38-to-p38 ratio; Panel B: Phosphorylation of JNK expressed as pJNK-to-JNK ratio; Panel C: Phosphorylation of ERK expressed as pERK-to-ERK ratio; and Panel D: Phosphorylation of IκB expressed as p IκB-to-IκB ratio. Insets: Representative gel blots of total and phosphorylated p38, JNK, ERK and IκB proteins using specific antibodies. Mean ± SEM, n = 4–8 isolations, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Influence of age and obesity on cardiomyocyte function in high fat-induced and db/db obesity

To further elucidate the interaction between aging and obesity on cardiac contractile function, we went on to examine the high fat diet-induced and the leptin receptor mutant db/db obesity models. A 16-week high fat diet feeding regimen was applied to young (4-month-old) and aging (12-month-old) C57 mice. Both young and aging mice were euglycemic (data not shown) and displayed a comparable separation in body weight in response to low and high fat diet feeding (Young: Low fat: 26.6±0.6 g vs. High fat: 30.6±0.6 g; Aging: Low fat: 28.8±0.4 g vs. High fat: 32.7±0.4 g, n = 4 mice per group). The resting cell length was significantly greater in high fat diet and aging groups without any additive effect between the two. Short-term leptin treatment (1.0 nM) failed to affect resting cell length in either low or high fat diet groups at both ages. Both high fat diet feeding and aging independently and significantly reduced PS and ± dL/dt, prolonged TR90 without affecting TPS. There was no additive or synergistic effect between the two on the mechanical responses. Leptin supplementation (1.0 nM) failed to reconcile high fat diet or aging-induced mechanical alteration in PS, ± dL/dt and TR90. Furthermore, leptin treatment (1.0 nM) did not alter any of the mechanical indices tested (Fig. 9). Our further study using the db/db obese model revealed a somewhat comparable euglycemic (data not shown) body weight gain between C57 (4-month: 24.0±0.8 g; 12-month: 28.0±0.9 g; 18-month: 30.2±0.8 g) and db/db (4-month: 48.2±2.0; 12-month: 56.7±2.3; 18-month: 59.9±2.9%, n = 4 mice per group, p<0.05 vs. corresponding C57 group) mice. The resting cell length was significantly greater in db/db and aging groups without any additive effect between the two. Both db/db obesity and aging significantly reduced PS and ± dL/dt, prolonged TR90 without affecting TPS. Interestingly, aging and db/db obesity exerted an additive inhibitory effect on PS and ± dL/dt without affecting TR90 at 18 but not 12 months of age (Fig. 10).

Figure 9

Contractile properties of cardiomyocytes isolated from young (4-month-old) and aging (12-month-old) male C57 mice fed a low (10%) or high (45%) fat diet for 16 weeks.

Cohorts of cardiomyocytes were treated with or without leptin (1.0 nM) for 4 hrs prior to mechanical study. A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS); F: Time-to-90% relengthening (TR90); Mean ± SEM, n = 50–51 cells from 3 mice per group, * p<0.05 vs. respective low fat group, ** p<0.05 vs. young low fat group.

Figure 10

Contractile properties of cardiomyocytes isolated from young (4-month-old) and aging (12- or 18-month-old) male C57 lean and the leptin receptor-deficient db/db obese mice.

A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS); F: Time-to-90% relengthening (TR90); Mean ± SEM, n = 102–103 cells from 3 mice per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 (4-month) group, # p<0.05 vs. young db/db (4-month) group.

Discussion

The major findings of our current study revealed that increased age mimicked leptin-deficient ob/ob obesity-induced changes in cardiomyocyte contractile function, intracellular Ca2+ homeostasis, NADPH oxidase expression, O2 − accumulation, Akt/eNOS and stress signaling (p38, JNK and NFκB). Little additive or synergistic actions were noted between aging and ob/ob obesity on the above-mentioned parameters, with the exception of a rise in intracellular Ca2+. Short-term treatment of leptin at physiological levels (0.5 and 1.0 nM) elicited a beneficial effect on cardiomyocyte contractile and intracellular Ca2+ responses in young but not aging ob/ob mice whereas pharmacological level of leptin (50 nM) compromised cardiomyocyte contractile function, intracellular Ca2+ handling, NADPH oxidase expression, O2 − accumulation, Akt/eNOS and stress signaling. The disparity between young and aging mice in physiological leptin level-induced mechanical responses was closely mirrored by an accumulation of O2 − and expression of NADPH oxidase (p 47), the enzyme responsible for O2 − production. Further scrutiny depicted that aging and obesity independently decreased the phosphorylation of Akt and its downstream signaling molecule eNOS, stimulated JNK and IκB phosphorylation as well as inhibited p38 phosphorylation without overt interactions between the two. Consistent with its responsiveness to mechanical function, O2 − production and p 47 expression, physiological levels of leptin effectively restored leptin deficiency-induced changes in the phosphorylation of Akt, eNOS, JNK, IκB and p38 in young but not aging ob/ob mice. These data favor a role of post-insulin receptor signaling and stress signaling in obesity-associated cardiac mechanical defects and O2 − accumulation. Our data did not favor a major role for the leptin receptor (Ob-R), its post-receptor signal STAT-3, ERK or AMPK in leptin-elicited beneficial effects in ob/ob obese mice. Given that leptin (at physiological levels) failed to reconcile aging-induced detrimental effects in cardiomyocytes, it appears that aging may produce cardiac contractile and intracellular Ca2+ defects associated with O2 − accumulation reminiscent of leptin-deficient obesity through a mechanism(s) independent of interrupted leptin signaling.

Development of obesity and its associated complications may be attributed to multiple factors including genetic, dietary, environmental and evolutionary components, although pinpointing each specific influence has been rather difficult [1], [3]. Although human obesity is usually accompanied by hyperleptinemia [16], both hypo- and hyper-leptinemia have been shown to induce obesity due to interrupted leptin signaling and energy expenditure [31]. Sustained obesity (diet-induced or genetically predisposed) impairs cardiac contractile function in a manner reminiscent of pre-diabetic insulin resistance and full-blown diabetes [32]–[35], indicating a role for insulin resistance in obesity-induced cardiac contractile dysfunction. This is supported by our current observation of dephosphorylated Akt and eNOS in young and aging ob/ob mice. In our study, the leptin deficient ob/ob mice were euglycemic at both ages, thus excluding possible contribution from full-blown diabetes to the cardiac anomalies of the ob/ob mice. Our data revealed a somewhat similar tibial length among young and aging C57 or ob/ob mice, excluding the possible contribution of disparate growth and development in these mice. These data are in line with the notion that tibial length reached plateau when body growth slowed down after postnatal day 70 in mice [36]. Nonetheless, these ob/ob mice were hyperinsulinemic, hypertriglyceridemic and glucose intolerant based on our earlier studies [12], [14], indicating the presence of insulin resistance. More importantly, our DEXA study depicted an age-dependent increase in body fat composition in both C57 lean and ob/ob obese mice, favoring aging itself as an independent obesogenic factor [4]. Data from our study indicated that aging itself produced a cascade of cardiomyocyte mechanical defects reminiscent of young ob/ob or db/db as well as high fat diet-induced obese mice. In all three murine obesity models used in our study, both aging and obesity independently triggered an elongation in resting cell length, depression in peak shortening (PS) amplitude and maximal velocity of shortening/relengthening amplitude (± dL/dt), as well as prolongation in relengthening duration (TR90) but not duration of shortening (TPS). These data are consistent with our previous observations from aged or obese mice [14], [37], [38]. Interestingly, there was little interaction between aging and obesity on cardiomyocyte contractile parameters with the exception of further depressed PS and ± dL/dt in 18-month-old db/db mice. These data seem to favor the notion that aging and obesity may share somewhat similar cellular mechanisms en route to cardiomyocyte mechanical dysfunction. The apparent disparity between ob/ob and db/db mice on the synergistic effect of aging (18 months) and obesity depicts presence of an overt age-related difference between the two leptin mutant murine obesity models. Thus caution should be taken to derive experimental conclusions using various rodent obesity models. Given our further observation that physiological leptin treatment failed to reconcile high fat diet- or age-induced detrimental effects in cardiomyocytes, the convergence between aging and obesity in cardiac contractile and intracellular Ca2+ defects as well as O2 − accumulation likely occurs at a point downstream of or independent of leptin signaling.

The Kaplan-Meier survival curve (Fig. 1) revealed a greatly elevated mortality in ob/ob mice, supporting the hypothesis that obesity may be considered a status of premature aging [4]. It is worth mentioning that the 12 or 18 months of age selected for our “aging” mice was not as old as other studies have used. However the mortality rate of ob/ob mice after one year of life is much higher than other mouse types [26], [38]. Although limited information is available for the precise cause of death for these ob/ob obese mice, it may be speculated that obesity-associated tumorigenesis (e.g., colon and skin cancer), chronic inflammation, immune deficiency and cardiovascular complications are among the leading causes of death in these mice [39]. In our study, the young and aging ob/ob mice exhibited significantly greater fat composition, heavier body and heart weights compared with the age-matched lean control group. Moreover, the aging ob/ob mice displayed an additional increase in body weight compared with the young ob/ob mice. Considering the comparable cardiomyocyte functional profiles between young and aging ob/ob mice, it appears that the extra body weight gain and body fat composition in aging ob/ob mice had little effect on cardiac dysfunction associated with obesity. Nonetheless, the additional increase in body and fat mass was mirrored by a drop in the rise of intracellular Ca2+ seen with aging. The greater cardiomyocyte cell length in ob/ob mice at both ages was not affected by short-term physiological leptin treatment, likely due to the fact that cardiac hypertrophy resulting from interrupted leptin signaling in ob/ob mice is a chronic process [23], [40]. During the chronic cardiac remodeling process with interrupted leptin signaling, the heart transforms from compensated to decompensated states accompanied by deteriorated cardiac function.

Our study revealed that both aging and obesity impaired intracellular Ca2+ handling shown as delayed intracellular Ca2+ clearance and reduced intracellular Ca2+ rise, consistent with our previous studies [14], [37], [38]. Unlike the observation from cardiomyocyte mechanical assessment, the obesity-induced decline in intracellular Ca2+ release was further accentuated with aging, indicating a possible change in myofilament Ca2+ sensitivity in the aging ob/ob murine cardiomyocytes. These observations favor the idea that dysregulated intracellular Ca2+ regulation may contribute to cardiomyocyte contractile dysfunction (prolonged TR90, reduced PS and ± dL/dt) under aging, obesity or both. Our data further revealed that physiological leptin reconciled intracellular Ca2+ mishandling in young but not aging ob/ob mice, indicating that intracellular Ca2+ handling may contribute to the beneficial mechanical response of leptin in young ob/ob mice. Our observation of elevated O2 - production and upregulated p 47 subunit of NADPH oxidase in both aging and obese groups (without interaction between the two) suggests a likely role of NADPH oxidase-dependent O2 − release in aging and/or obesity-elicited cardiomyocyte intracellular Ca2+ handling and contractile dysfunction. The NADPH oxidase-dependent O2 − production and other reactive oxygen species are known to cause cardiomyocyte mechanical dysfunction [22], [25]. The fact that physiological leptin alleviated obesity-elicited increases in O2 − production and p 47 expression in young but not aging ob/ob mice favors a role for NADPH oxidase-dependent O2 − production in the disparate cardiac response of short-term leptin treatment. Our further observation in Ob-R expression and STAT-3 phosphorylation depicted reduced Ob-R expression and STAT-3 phosphorylation in both ob/ob age groups. To our surprise, unlike its effect on cardiomyocyte contractile function, intracellular Ca2+ homeostasis and O2 − production, leptin treatment restored downregulated Ob-R expression and STAT-3 activation in both ob/ob age groups without any effect in lean groups. These data indicate that the likely mechanism responsible for the age-dependent disparity of cardiac leptin responses may not reside at the levels of the Ob-R or STAT-3. This notion received further support from our observation that the pharmacological concentration of leptin (50 nM) promoted Ob-R/STAT-3 signaling while compromising cardiomyocyte contractile function, intracellular Ca2+ handling, NADPH oxidase expression, O2 − accumulation, Akt/eNOS and stress signaling. Data from our previous study revealed that pharmacological levels of leptin (50 and 100 nM) compromised cardiac contractile function and intracellular Ca2+ homeostasis through an ET-1 receptor-/NADPH oxidase-dependent accumulation of reactive oxygen species [22]. Our current data revealed unchanged Ob-R (long form) expression and reduced STAT-3 activation in murine hearts at 12 months of age. Limited information is available with regards to aging-induced changes in Ob-R expression and STAT-3 activation. The leptin-induced STAT-3 phosphorylation was found to be higher along with an upregulated hypothalamic expression of the Ob-R at 14 - 18 months of mouse age compared with 2 months of age [41], indicating increased leptin sensitivity with aging in the mouse brain. However, little information is available on the heart with regards to the impact of aging on leptin sensitivity.

Our results showed that aging and obesity independently depressed the phosphorylation of Akt and eNOS, stimulated JNK and IκB phosphorylation as well as inhibited p38 phosphorylation without overt interaction between the two. Meanwhile, leptin supplementation at physiological levels rescued the dampened Akt/eNOS/p38 phosphorylation in young ob/ob mice, the effect of which was obliterated by aging. These data are consistent with the basal and leptin-elicited responses on cardiomyocyte contractile function, intracellular Ca2+ handling, O2 − production and NADPH oxidase expression. Under-activation of the key cardiac survival factor Akt and its downstream signaling molecule eNOS has been demonstrated in various models of cardiac dysfunction and heart failure [14], [34], suggesting a crucial role of Akt/eNOS in the maintenance of cardiac function. It is noteworthy that the dampened Akt/eNOS phosphorylation observed in our ob/ob mice may contribute to enhanced cardiac oxidative stress and compromised cardiac function since the Akt-eNOS cascade is known for its role in cardiac survival, glucose uptake and maintenance of cardiac contractile function [30]. In our hands, both obesity and aging independently decreased the phosphorylation of Akt and eNOS without any additive effects between the two, consistent with our data on mechanical, intracellular Ca2+ and O2 − production. These observations favor a key role for Akt/eNOS signaling in leptin-deficient obesity and age-induced cardiac dysfunction. Our data revealed reduced p38 MAP kinase phosphorylation in aged and obese mice, while leptin effectively restored p38 phosphorylation in young ob/ob but not aging mice. These results favor a beneficial role of p38 phosphorylation in the maintenance of cardiomyocyte function, which is supported by the previous finding that inhibition of p38 MAP kinase reduces insulin sensitivity and glucose uptake in human myotubes [42]. This is also in line with the finding that leptin directly stimulates p38 MAP kinase phosphorylation [43], [44]. JNK and NFκB may be turned on by pro-inflammatory cytokines and free fatty acids in aging and obesity, resulting in interrupted insulin signaling and development of cardiac dysfunction [45]. Our observation of enhanced phosphorylation of JNK and IκB (which removes its inhibition on NFκB) in aging and obesity are consistent with the reduced phosphorylation of Akt and eNOS. In addition, our results revealed that neither obesity nor age significantly affected the total expression and phosphorylation of ERK and AMPK. Interestingly, combining obesity and age significantly attenuated phosphorylation of ERK and AMPK, the effects of which were ablated and unaffected, respectively, by leptin treatment. Although we are unable to offer any precise explanation for the combined effect between age and obesity on ERK and AMPK at this time, our data do not favor a significant role for ERK and AMPK in the reminiscent cardiac defects between aging and obesity as well as the disparity in the leptin cardiac responsiveness. Further study is warranted to better address the interplay among various cell signaling pathways such as sirtuin, a key signaling molecule in longevity and lifespan [46], or RAGE, which plays a key role in aging-associated cardiomyocyte dysfunction via NFκB activation [47], in the aging- and obesity-associated cardiac contractile dysfunction.

Experimental limitations: Although our study provided a likely causal relationship among cardiac mechanical function, intracellular Ca2+ homeostasis, NADPH oxidase, O2 − accumulation, Akt/eNOS and stress signaling activation between aging and obesity, caution should be taken for the interpretation of the precise interaction between aging and obesity in cardiac dysfunction in the human setting. First and foremost, the short-term in vitro leptin incubation used in our study may not best represent the in vivo longer term effect of leptin on phenotypic changes in obesity. Oxidative modification of intracellular Ca2+ handling proteins is known to contribute to altered cardiomyocyte mechanics such as prolonged relaxation in obesity [37]. It may be speculated that short-term incubation of physiological levels of leptin may interrupt the oxidative processes (i.e. scavenging reactive oxygen species) thus shifting the redox balance towards reducing processes and promoting reactions to temporarily reverse oxidative modification of Ca2+ handling proteins. Nonetheless, this may not truly reflect the physiological setting in vivo. In our study, only male mice were used which ignored the important gender disparity in obesity and aging [2]. In our cell isolation procedure, butanedione monoxime was used to uncouple cardiomyocyte contractile elements and maintain cell viability for a prolonged period of time, which may unevenly alter the true in vivo cardiomyocyte mechanics and thus bias cardiomyocyte function from lean and ob/ob groups. Measurement of contractile performance in isolated cardiomyocytes has been established to provide a fundamental assessment of cardiac contractile function in pathological states. However, as in any study of this nature, caution needs to be taken when correlating our present cellular findings to whole heart function, as the latter is composed of heterogeneous cell types, including nerve terminals and fibroblasts, as well as the connective tissue alluded to above. Furthermore, cardiomyocytes beat at a high frequency in vivo as opposed to the non-physiological slow pace (0.5 Hz) used in our study despite the fact that a low frequency contraction is deemed as a “slow motion” to maximally reveal the cell mechanical defect. Last but not least, the long–form Ob-Rb receptor monoclonal antibody used in our study may cross-act with the short-form leptin receptors although the latter cannot turn on the full JAK-STAT leptin signaling due to the absence of the essential box-2 motif.

In summary, data from our present study suggested that aging and the leptin deficient ob/ob obesity compromise cardiac contractile function and intracellular Ca2+ homeostasis via comparable mechanisms involving NADPH oxidase-dependent O2 − production, phosphorylation of Akt, eNOS as well as the stress signaling molecules p38, JNK and NFκB. Our study further revealed an age-associated disparity in physiological leptin level-elicited responsiveness in cardiomyocyte contraction, intracellular Ca2+ handling and O2 − production. Collectively, these data favor a role for NADPH oxidase, O2 − generation, Akt, eNOS and the stress signaling molecules p38, JNK and NFκB, rather than Ob-R and STAT-3, in the basal and leptin-elicited cardiac response during aging and obesity. Our data further revealed both similarity and disparity in aging-associated cardiomyocyte mechanical response between ob/ob obesity and high fat diet-induced or the hyperleptinemic db/db obesity. Given the lack of knowledge of aging-induced changes in adiposity and leptin signaling, the precise interplay between aging and obesity, and contribution of leptin signaling and downstream stress signaling activation, if any, to the cardiac contractile dysfunction in the state of concurrent aging and obesity warrant further research.