Leptin repletion restores depressed β-adrenergic contractility in ob/ob mice independently of cardiac hypertrophy

Abstract

Impaired leptin signalling in obesity is increasingly implicated in cardiovascular pathophysiology. To explore mechanisms for leptin activity in the heart, we hypothesized that physiological leptin signalling participates in maintaining cardiac β-adrenergic regulation of excitation–contraction coupling. We studied 10-week-old (before development of cardiac hypertrophy) leptin-deficient (ob/ob, n = 12) and C57Bl/6 (wild-type (WT), n = 15) mice at baseline and after recombinant leptin infusion (0.3 mg kg⁻¹day⁻¹ for 28 days, n = 6 in each group). Ob/ob-isolated myocytes had attenuated sarcomere shortening and calcium transients ([Ca²⁺]_i) versus WT (P < 0.01 for both) following stimulation of the β-receptor (with isoproterenol (isoprenaline)) or at the post-receptor level (with forskolin and dibutryl-cAMP). In addition, sarcoplasmic reticulum (SR) Ca²⁺ stores were depressed. Leptin replenishment in ob/ob mice restored each of these abnormalities towards normal without affecting gross (wall thickness) or microscopic (cell size) measures of cardiac architecture. Immunoblots revealed alterations of several proteins involved in excitation–contraction coupling in the ob/ob mice, including decreased abundance of G_sα-52 kDa, as well as alterations in the expression of Ca²⁺ cycling proteins (increased SR Ca²⁺-ATPase, and depressed phosphorylated phospholamban). In addition, protein kinase A (PKA) activity in ob/ob mice was depressed at baseline and correctable towards the activity found in WT with leptin repletion, a finding that could account for impaired β-adrenergic responsiveness. Taken together, these data reveal a novel link between the leptin signalling pathway and normal cardiac function and suggest a mechanism by which leptin deficiency or resistance may lead to cardiac depression.

Obesity has substantial health implications, predisposing individuals to cardiovascular diseases such as hypertension, hyperlipidemia, atherosclerosis and congestive heart failure (Eckel et al. 2002). An emerging theme in obesity-related cardiovascular pathophysiology is that neurohormonal pathways that regulate adipose homeostasis also have cardiovascular activity, disruption of which may contribute to cardiovascular dysfunction (Alpert, 2001; Barouch et al. 2003; Sader et al. 2003). Recent epidemiological studies support increased cardiovascular risk in obese subjects independent of blood pressure, left ventricular hypertrophy (LVH), diabetes mellitus or underlying organic heart disease (Kenchaiah et al. 2002). Thus, underlying signalling derangements may be the proximate cause of cardiac dysfunction, not obesity and its haemodynamic consequences, per se.

In this regard, there is growing interest in the cardiovascular activity of the leptin signalling pathway (Nickola et al. 2000; Illiano et al. 2002; Barouch et al. 2003; Rajapurohitam et al. 2003) because leptin deficiency or resistance, both causes of obesity (Considine et al. 1996; Bray & York, 1997; Montague et al. 1997), may also participate in cardiovascular disease (Pladevall et al. 2003; Sader et al. 2003). Leptin, a 167 amino acid polypeptide synthesized predominantly in adipose tissue, has widely distributed receptors (Sweeney, 2002). Indeed, cardiac myocytes also express leptin receptors, which are coupled to signalling pathways that influence both myocardial contractility (Nickola et al. 2000; Wold et al. 2002) and cellular growth (Barouch et al. 2003; Rajapurohitam et al. 2003; Xu et al. 2004). We have recently shown that mice lacking leptin (ob/ob) or its receptor (db/db) develop cardiac hypertrophy, reversible by leptin repletion (in the case of ob/ob), but not weight loss alone, strongly supporting a role for leptin in maintaining normal cardiac architecture (Barouch et al. 2003).

Another key cardiovascular phenotype which leptin has the potential to influence is β-adrenergic signal transduction. In adipose tissue, leptin regulates β-adrenergic signalling which in turn mediates lipolysis and thermogenesis (Collins et al. 1994; Collins & Surwit, 2001; Bachman et al. 2002). As depressed β-adrenergic contractility is a hallmark phenotype of the failing myocardium (Bristow et al. 1990), we tested whether leptin influences cardiac β-adrenergic inotropic responses.

Methods

Animals

We studied ob/ob mice and their wild-type (C57Bl/6 J, WT) controls (Jackson Laboratory, Bar Harbour, ME, USA). The ob/ob mice were backcrossed on a C57Bl/6 background greater than 30 generations, resulting in obese mice that are statistically 99.9% similar to WT at all unlinked loci. Animals were housed under diurnal lighting conditions and allowed food and tap water ad libitum. Animals were killed by cervical dislocation after deep anaesthesia with isoflurane by inhalation. The Institutional Animal Care and Use Committee of The Johns Hopkins University School of Medicine approved all protocols and experimental procedures.

Exogenous leptin infusion

Effects of leptin treatment were evaluated in both ob/ob and WT mice. Six-week-old mice were anaesthetized with 2% isoflurane for pump implantation. Leptin was infused for 28 days using 100 μl osmotic minipumps (replaced after 14 days), implanted subcutaneously in the interscapular area (Alzet, Palo Alto, CA, USA), which delivered continuous recombinant mouse leptin (Amgen, Thousand Oaks, CA, USA; 0.3 mg kg⁻¹ day⁻¹) (Breslow et al. 1999; Barouch et al. 2003).

Echocardiography

Echocardiographic assessments were performed in ob/ob, WT and leptin-treated mice at 6 and 10 weeks of age. Mice were anaesthetized with 1–2% isoflurane. Studies were performed using a Sonos 5500 Echocardiogram (Agilent) with a 15 MHz linear transducer. Anterior wall thickness (AWT), posterior wall thickness (PWT), end-diastolic (EDD) and end-systolic (ESD) left ventricular dimensions were recorded from M-mode images using averaged measurements from three to five consecutive cardiac cycles. Relative wall thickness (RWT) was calculated using RWT = (AWT + PWT)/EDD.

Isolated myocyte isolation

Cardiac myocytes were prepared from 10-week-old WT and ob/ob mice, as previously described by Khan et al. (2003). Hearts were perfused with Ca²⁺-free bicarbonate buffer containing (mmol/l): 120 NaCl, 5.4 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5.6 glucose, 20 NaHCO₃, 10 2,3-butanedione monoxime (BDM; Sigma), and 5 taurine (Sigma), gassed with 95% O₂–5% CO₂, and digested with collagenase type 2 (1 mg ml⁻¹; Worthington Biochemicals, Lakewood, NJ, USA) and protease type IV (0.1 mg ml⁻¹; Sigma). Myocytes were obtained by mechanically disrupting digested hearts, followed by filtration, centrifugation and suspension in 0.125 Ca²⁺ Tyrode solution containing (mmol/l): 144 NaCl, 1MgCl₂, 10 Hepes, 5.6 glucose, 5 KCl adjusted to a pH of 7.4 with NaOH. Myocytes were resuspended first in 0.250 mmol l⁻¹ Ca²⁺ Tyrode solution, then in 0.5 mmol l⁻¹ Ca²⁺ Tyrode solution, and finally stored in Tyrode solution containing 0.5 mmol l⁻¹ probenecid and 1.8 mmol l⁻¹ Ca²⁺. Mechanical studies were completed within 6 h after isolation at room temperature.

Cell shortening and Ca²⁺ transient measurements

Mechanical properties of myocytes were assessed using a video-based myocyte sarcomere spacing acquisition system (IonOptix, Milton, MA, USA). Myocytes were incubated with 5 μmol l⁻¹ fura-2 AM (Molecular Probes) for 10 min then transferred to a lucite chamber on the stage of an inverted microscope (Nikon TE 200), continuously superfused with Tyrode solution containing 1.8 mmol l⁻¹ Ca²⁺ and 0.5 mmol l⁻¹ probenecid, stimulated at 1 Hz. Myocyte length and width, and sarcomere length (SL) were recorded with an IonOptix iCCD camera. Change in average SL was determined by fast Fourier transform of the Z-line density trace to the frequency domain and SL shortening was calculated as (diastolic SL − systolic SL)/diastolic SL.

Intracellular Ca²⁺ concentrations [Ca²⁺]_i were measured using the Ca²⁺-sensitive dye fura-2 and a dual-excitation spectrofluorometer (IonOptix), alternately excited with a xenon lamp at wavelengths of 365 and 380 nm. The emission fluorescence was reflected through a barrier filter (510 ± 15 nm) to a photomultiplier tube. The ratio of the photon live count detected by excitation at 365 nm compared with 380 nm represents the fura-2 fluorescence ratio. Intracellular Ca²⁺ concentrations were compared at baseline and peak contraction to determine the calcium transient.

Myocyte stimulation

Myocyte contractile function was assessed with (a) isoproterenol (Sigma, 10⁻⁸–10⁻⁶ mol l⁻¹), a non-selective β-receptor agonist; (b) forskolin (Sigma, 10⁻⁸–10⁻⁶ mol l⁻¹), a direct activator of adenylyl cyclase; and (c) dibutryl cAMP (Sigma, 10⁻³ mol l⁻¹), a phosphodiesterase-resistant cAMP analog. The myocytes were continuously perfused, at approximately 2 ml min⁻¹, with increasing doses of the activators until they reached a steady state. Caffeine (10 mmol l⁻¹) was administered rapidly, after a 10 s pause, to estimate sarcoplasmic reticulum (SR) calcium stores.

Membrane preparation

Pieces of ventricular tissue (∼20 mg) from 10-week-old WT − leptin, WT + leptin (n = 6 each), ob/ob− leptin, and ob/ob+ leptin (n = 4 each) mice were thawed (from −80°C storage) in buffer A (5 mmol l⁻¹ Tris, pH 7.4, 0.25 mol l⁻¹ sucrose, 1 mmol l⁻¹ MgCl₂, 1 mmol l⁻¹ EDTA, 10 μmol l⁻¹ PMSF (phenylmethylsulphonyl fluoride)) and homogenized using high speed cutting blades (Tissuemizer, Tekmar Co., Cincinnati, OH, USA) on top speed for 45 s. Homogenates were subjected to centrifugation at 1000 g for 10 min at 4°C. The supernatant was recovered and subjected to centrifugation at 45 000 g for 25 min at 4°C. The pellet was resuspended in 5 ml of buffer B (50 mm Tris, pH 7.4, 10 mmol l⁻¹ MgCl₂, 1 mmol l⁻¹ EDTA, 10 μmol l⁻¹ PMSF) and centrifuged again at 45 000 g for 25 min at 4°C. The pellet was washed an additional time in 25 ml buffer B. The final pellet was resuspended in 0.5 ml buffer B and used immediately in adenylyl cyclase assays.

Adenylyl cyclase assays

Triplicate samples were incubated for 10 min at 37°C and contained the indicated effector along with 10–20 μg of membrane protein, 25 mmol l⁻¹ Tris, pH 7.5, 5 mmol l⁻¹ MgCl₂, 0.5 mmol l⁻¹ EDTA, 1 mmol l⁻¹ cAMP, 1 mmol l⁻¹ ATP, ³²P-α-ATP (0.5–1.5 μCi tube⁻¹, 800 Ci mmol⁻¹), 5 μmol l⁻¹ PMSF, 7 mmol l⁻¹ creatine phosphate, 50 μg ml⁻¹ creatine phosphokinase and 0.25 mg ml⁻¹ BSA in a final volume of 100 μl. Adenylyl cyclase activity was measured under basal conditions (no added effectors) or in the presence of 1 μmol l⁻¹ forskolin. Reactions were terminated by the addition of 100 μl of stop buffer (50 mmol l⁻¹ Hepes, pH 7.5, 2 mmol l⁻¹ ATP, 0.5 mmol l⁻¹ cAMP, 2% SDS, 1 μCi ml⁻¹³H-cAMP (37 Ci mmol⁻¹)). Newly synthesized ³²P-cAMP was separated from the precursor ³²P-α-ATP by sequential column chromatography over dowex (Bio-Rad Laboratories) and alumina (Sigma-Adlrich) as described previously (Salomon et al. 1974), using recovery of ³H-cAMP to monitor individual column efficiency (Emala, 1997). Eluted radioactivity was quantified by liquid scintillation.

Protein kinase A (PKA) activity

PKA activity was measured using a non-radioactive fluorescent detection assay (Promega Corporation). In 10-week-old WT − leptin, WT + leptin (n = 6 each), ob/ob− leptin, and ob/ob+ leptin (n = 4 each) mice, hearts were removed rapidly after killing, rinsed several times in ice-cold 1 × PBS and connective tissue, aorta and atria removed. The left ventricle was frozen in liquid nitrogen and stored at −80°C. On the day of the experiment, left ventricular tissue was homogenized with a Polytron (3 × 15 s) in 1 × cell lysis buffer (Cell Signalling Tech.). The homogenization buffer also contained 1 mm PMSF, and Protease Inhibitor Cocktail (Roche Diagnostics GmbH). The homogenate was centrifuged at 4°C at 14 000 g for 30 min. The supernatants were recovered and their concentrations were determined using BCA reagent (Pierce Biotechnology) and bovine serum as a standard.

A portion of the cAMP-dependent protein kinase catalytic subunit was diluted to 2 μg ml⁻¹ in PKA dilution buffer (350 mm K₃PO₄, pH 7.5 and 0.1 mm DTT) and was used as positive control. For each sample, PKA reaction 5 × buffer, PKA-specific peptide substrate (PepTag^® A1 peptide Promega or Poration), PKA activator 5 × solution with peptide protection solution, and water were mixed and kept on ice. The samples were incubated at room temperature for 30 min, loaded onto a 0.8% agarose gel (prepared in 50 mm Tris-HCl, pH 8.0), and run at 135 V for 30–40 min. The bands were visualized under ultraviolet light, quantified by spectrophotometry, and their absorbance read at 570 nm. Finally, the PKA activity was calculated using Beer's Law in accordance with the manufacturer's instructions.

Western blots

As previously described (Khan et al. 2003), Western blot analysis was performed on total protein lysate prepared from 10-week-old WT − leptin, WT + leptin, ob/ob− leptin, and ob/ob+ leptin (n = 5 in each group) mice for all proteins tested except β₂-adrenergic receptor, for which microsomal preparations were used (centrifugation of total lysate at 100 000 g for 1 h). Protein concentration was assayed for loading with bicinchoninic acid (Pierce Biotechnology, Rockford, IL, USA) and equal amounts were resolved with polyacrylamide gels (Invitrogen Life Technologies, Carlsbad, CA, USA). Proteins were then transferred to nitrocellulose or polyvinylidene difluoride membrane. The following antibodies were used in 1: 500–1: 1000 dilutions: sarcoplasmic reticulum (SR) Ca²⁺-ATPase, phospholamban (PLB), G_iα, and β₁-adrenergic receptor (Affinity Bioreagents Inc.), phospho-phospholamban (P-PLB) (Upstate biotechnology), β₂-adrenergic receptor (Santa Cruz Biotechnology, Inc.), and G_sα (United States Biological). Polyclonal anti-p38 MAP kinase antibody (1: 1000 dilutions, Santa Cruz Biotechnology, Inc.) was used to normalize protein quantity. Bands were visualized by chemiluminescence (SuperSignal Substrate kit, Pierce) and quantified using the NIH Imaging software.

Plasma measurements

Plasma glucose, leptin, insulin and triglycerides levels were measured by radioimmunoassay (Linco Diagnostic Services).

Statistical analysis

Data are reported as mean ± s.e.m. Statistical significance was determined by one-way or two-way ANOVA where appropriate and Student-Newman-Keuls post hoc test (GraphPad Instat, STATA, and SAS statistical software). P values less than 0.05 were considered significant.

Results

Cardiac structure in young ob/ob mice

In order to separate the effects of leptin signalling on β-adrenergic inotropic responses from cardiac hypertrophy, we studied young ob/ob mice at an age preceding development of LVH (Barouch et al. 2003). Overall, cardiac structure and isolated myocyte size were similar at 10 weeks of age in WT − leptin (n = 6) and ob/ob− leptin (n = 6) mice (Table 1). Importantly, leptin repletion (WT + leptin; n = 11 and ob/ob+ leptin; n = 7) did not affect either gross (wall thickness, Fig. 1) or microscopic (n = 50–60 cells per strain) measures of cardiac architecture. Taken together, these findings establish the young ob/ob mice as a valid model to study impaired leptin signalling without the confounding influence of cardiac hypertrophy.

Table 1.

Ventricular parameters and cell size in 10-week-old mice

	WT − leptin	WT + leptin	ob/ob − leptin	ob/ob + leptin
Mice (n)	6	11	6	7
Body weight (g)	21.4 ± 1.5	20.3 ± 1.0	49.2 ± 1.3*	23.6 ± 1.3
AWT (mm)	0.67 ± 0.04	0.62 ± 0.03	0.69 ± 0.05	0.66 ± 0.03
RWT (mm)	0.49 ± 0.04	0.50 ± 0.03	0.55 ± 0.08	0.45 ± 0.03
EDD (mm)	2.87 ± 0.12	2.69 ± 0.10	2.92 ± 0.14	3.08 ± 0.11
Myocyte length (μm)	132.9 ± 2.4	129.4 ± 2.0	131.7 ± 2.8	131.3 ± 2.0
Myocyte width (μm)	27.4 ± 0.7	27.6 ± 0.7	28.1 ± 0.7	27.8 ± 0.8

^*P < 0.01 versus WT − leptin, WT + leptin, and ob/ob+ leptin. AWT, anterior wall thickness; PWT, posterior wall thickness; EDD, end-diastolic diameter. Relative wall thickness (RWT) was calculated using RWT = (AWT + PWT)/EDD.

Figure 1. Change in wall thickness after leptin infusion.

Leptin was infused in young (6 week old) ob/ob (n = 7) and WT mice (n = 11) for 4 weeks. At the end of treatment, there was no appreciable change in anterior wall thickness (AWT) or relative wall thickness (RWT) in either group.

Isoproterenol response

Based on observations that leptin maintains β-AR signalling in adipose tissue (Collins et al. 1994), we hypothesized that leptin deficiency could also lead to β-adrenergic inotropic hyporesponsiveness in the heart, and investigated inotropic responses to isoproterenol in isolated cardiac myocytes. Baseline characteristics were similar between myocytes from ob/ob and WT mice at 1 Hz (Table 2). Consistent with prior studies (Barouch et al. 2002), isoproterenol (10⁻⁸–10⁻⁶ mol l⁻¹), a non-selective β-AR agonist, stimulated myocyte contraction in WT myocytes (10⁻⁶ mol l⁻¹; SL shortening 399 ± 89%; [Ca²⁺]_i 126 ± 16%; Fig. 2). In contrast, this positive inotropic effect was markedly attenuated in the ob/ob myocytes with parallel decreases in SL shortening (10⁻⁶ mol l⁻¹; 243 ± 46%) and [Ca²⁺]_i (88 ± 20%, both P < 0.01 versus WT).

Table 2.

Baseline myocyte characteristics at 1 Hz

	WT − leptin	WT + leptin	ob/ob− leptin	ob/ob+ leptin
Mice (n)*	14	7	10	6
Diastolic SL (μm)	1.72 ± 0.01	1.74 ± 0.01	1.72 ± 0.01	1.74 ± 0.01
SL shortening (%)	2.9 ± 0.2	2.6 ± 0.2	3.1 ± 0.2	2.7 ± 0.2
[Ca2+]i (%)	22.0 ± 1.4	24.9 ± 1.1	21.0 ± 1.3	17.9 ± 1.4

^*5–7 cells were studied per heart. Sacromere shortening (SL shortening), (diastolic SL – systolic SL)/diastolic SL; [Ca²⁺]_i, change in the ratio of the photon live count detected by excitation at 365 nm compared with 380 nm during contraction.

Figure 2. Impact of leptin repletion on β-adrenergic inotropic responses in obese (ob/ob) and C57Bl6 (WT) isolated myocytes.

Depicted are sarcomere shortening (SL shortening) and calcium transients ([Ca²⁺]_i) in isolated cardiac myocytes exposed to isoproterenol. A, sample transients illustrate similar baseline (continuous line) SL shortening and [Ca²⁺]_i in isolated myocytes from both WT and ob/ob mice, but suppressed response to isoproterenol (10⁻⁶m, dashed line) in ob/ob mice. B, concentration–effect response to isoproterenol (10⁻⁸–10⁻⁶ mol l⁻¹) demonstrates potent isoproterenol responses in SL shortening and [Ca²⁺]_i in WT (WT − leptin; n = 9 mice, 3–4 cells per heart), and an attenuated response in ob/ob (ob/ob− leptin; n = 6). Leptin repletion of ob/ob (ob/ob+ leptin; n = 6) restores the inotropic response to the level of WT, but does not alter the response in WT (WT + leptin; n = 6). *P < 0.01 for concentration–effect responses within groups. †P < 0.01 versus WT − leptin, WT + leptin, and ob/ob+ leptin groups by 2-way ANOVA. bl, baseline.

We next assessed the impact of leptin treatment in both ob/ob and WT mice. Leptin administration for 4 weeks to WT mice did not change the response in SL shortening or [Ca²⁺]_i (Fig. 2B). On the other hand, leptin treatment of ob/ob mice augmented β-AR-mediated inotropic responses towards levels indistinguishable from WT controls (10⁻⁶ mol l⁻¹; SL shortening 365 ± 54% and [Ca²⁺]_i 145 ± 30%).

Adenylyl cyclase (AC) activity and cAMP response

We next sought to examine downstream sites of β-adrenergic inotropic signalling. Direct AC activation with forskolin (10⁻⁸ mol l⁻¹ to 10⁻⁶ mol l⁻¹) also led to a depressed contractile response in ob/ob compared with WT mice (Fig. 3A). Specifically, forskolin (10⁻⁶ mol l⁻¹) led to peak SL shortening of 154 ± 34% (with [Ca²⁺]_i of 39 ± 10%) in ob/ob compared with peak SL shortening of 416 ± 79% (with [Ca²⁺]_i of 107 ± 33%) in WT (P < 0.001 for both). As before, leptin treatment of the ob/ob mice fully restored these inotropic responses towards normal, but did not affect WT contractility. Similarly, additional post-adenylyl cyclase defects were also evident by depressed contractile responses to dibutryl cAMP (10⁻³m), a phosphodiesterase-resistant cAMP analogue (Fig. 3B). Here too, depressed SL shortening and [Ca²⁺]_i in ob/ob were restored to the level of WT responses by leptin. To explore underlying biochemical mechanisms for suppressed inotropic responses, we initially tested adenylyl cyclase activity in vitro. However, we found no changes in the forskolin-stimulated (1–300 units) AC activity across WT and ob/ob mice with and without leptin repletion (P = NS between groups, Fig. 3C).

Figure 3. Impact of forskolin and dibutryl cAMP on inotropic responses, and adenylyl cycalse (AC) activity.

A, SL shortening and [Ca²⁺]_i were attenuated in myocytes from ob/ob (ob/ob− leptin; n = 5 mice, 3–4 cells per heart) compared with WT (WT − leptin; n = 5) controls in response to forskolin (10⁻⁶ mol l⁻¹). Leptin treatment of ob/ob mice (ob/ob+ leptin; n = 3) reverses this abnormality (*P < 0.001 versus baseline, and †P < 0.001 versus WT − leptin, WT + leptin and ob/ob+ leptin), but does not affect the WT response (WT + leptin; n = 3). B, responses to dibutryl cAMP were suppressed in ob/ob mice and restored to WT levels by leptin. *P < 0.01 versus aseline, and †P < 0.05 versus WT − leptin, WT + leptin and ob/ob+ leptin. C, basal and forskolin-stimulated AC activity was unchanged in hearts from WT (n = 6) and ob/ob (n = 4) mice, and was unaffected with leptin repletion: WT + leptin (n = 6) and ob/ob+ leptin (n = 4) (P = NS between groups). *P < 0.05 versus basal for dose–effect responses within all groups.

Protein kinase A (PKA) activity

We next examined PKA activity; it was markedly suppressed in ob/ob (15.07 ± 5.41 units ml⁻¹) as compared with WT mice (41.23 ± 4.11 units ml⁻¹, P < 0.05 versusob/ob) (Fig. 4). Leptin repletion restored it towards normal in ob/ob mice (31.76 ± 6.55 units ml⁻¹, P < 0.05 versusob/ob and P = NS versus WT) without affecting activity in WT mice (37.45 ± 4.49 units ml⁻¹).

Figure 4. Protein kinase A (PKA) activity.

Another downstream site of β-adrenergic inotropic signalling, the PKA activity was suppressed in ob/ob (15.07 ± 5.41 units ml⁻¹– the number of units of kinase activity per 1 ml) as compared with WT mice (41.23 ± 4.11 units ml⁻¹). Leptin repletion restored it towards normal in ob/ob mice (31.76 ± 6.55 units ml⁻¹) without affecting activity in WT (37.45 ± 4.49 units ml⁻¹). *P < 0.05 versus WT − leptin, WT + leptin, and ob/ob+ leptin.

Calcium stores

One of the key downstream organelles in β-adrenergically stimulated cardiac excitation–contraction coupling is the sarcoplasmic reticulum (SR). SR Ca²⁺ stores, measured by caffeine-induced Ca²⁺ release, were reduced in the ob/ob (%[Ca²⁺]_i, 64 ± 10) compared with the WT (100 ± 8%, P < 0.05) myocytes (Fig. 5). As with the inotropic responses and PKA activity, leptin treatment of ob/ob mice restored SR Ca²⁺ stores towards normal (111 ± 17%, P < 0.05 versusob/ob), but did not change stores in WT mice (112 ± 4%).

Figure 5. Sarcoplasmic reticulum (SR) Ca²⁺ stores.

Isolated cardiac myocytes stimulated at 1 Hz were rapidly exposed to caffeine (10 mmol l⁻¹) after a brief pause. SR Ca²⁺ reserves were measured as percentage change from baseline [Ca²⁺]_i, and are presented normalized to WT − leptin. The ob/ob (ob/ob− leptin; n = 3 mice, 3–4 cells per heart) mice had reduced SR Ca²⁺ stores compared with controls (WT − leptin; n = 5). Leptin treatment (ob/ob+ leptin; n = 3) restored stores towards normal but did not affect them in WT (WT + leptin). *P < 0.01 versus baseline, and †P < 0.05 versus WT − leptin, WT + leptin and ob/ob+ leptin.

Western blot analysis

To establish additional molecular correlates of the depressed inotropic responses, we performed immunoblot quantification of a large panel of proteins involved in β-adrenergic inotropic responses (see Fig. 6 and Table 3). Ob/ob mice had abnormalities at both PKA and SR levels correlating with the observed functional defects at these levels. In terms of G-proteins, there were parallel decreases in G_sα (52 kDa) and G_iα, and leptin repletion restored the levels of G_sα (52 kDa), but not G_iα, to normal in ob/ob mice. At the level of SR reuptake proteins, we found that P-PLB was decreased, consistent with depressed PKA activity. In addition, SR Ca²⁺-ATPase (SERCA2a) abundance was found to be increased. Here, leptin infusion restored P-PLB towards normal and augmented levels of SERCA2a in both ob/ob and WT hearts. Finally, the abundance of the β-adrenergic receptors was not affected in ob/ob mice.

Figure 6. Western blot analysis of myocardial tissue extracts from ob/ob and WT mice without (−) and with 4 weeks of leptin treatment (+).

Representative Western blots depict expression of key proteins in β-adrenergic regulation of excitation–contraction coupling. Notable findings include down-regulation of G_sα (52 kDa), G_iα, and P-PLB and up-regulation of SERCA2a in ob/ob mice. Leptin repletion restored levels of P-PLB and G_sα (52 kDa), but not G_iα, to normal in ob/ob mice. In addition, leptin infusion augmented levels of SERCA2a in both ob/ob and WT hearts. See Table 3 for normalized values.

Table 3.

Cardiac protein abundance

	WT − leptin	WT + leptin	ob/ob − leptin	ob/ob + leptin
Mice (n)	5	5	5	5
β1-AR	1.00 ± 0.10	1.03 ± 0.10	0.99 ± 0.09	1.06 ± 0.03
β2-AR	1.00 ± 0.45	1.21 ± 0.61	1.11 ± 0.50	1.12 ± 0.50
Gsα (52 kDa)	1.00 ± 0.14	0.85 ± 0.10	0.44 ± 0.03*‡	0.95 ± 0.21†
Gsα (45 kDa)	1.00 ± 0.09	1.39 ± 0.47	1.40 ± 0.43	1.81 ± 0.31
Giα	1.00 ± 0.11	0.66 ± 0.20	0.40 ± 0.07*	0.46 ± 0.20*
SERCA2a	1.00 ± 0.27	2.94 ± 0.74*	4.25 ± 0.50*	6.30 ± 0.58*†‡
PLB	1.00 ± 0.24	1.19 ± 0.23	0.98 ± 0.17	1.00 ± 0.20
P-PLB	1.00 ± 0.16	1.06 ± 0.36	0.42 ± 0.09*‡	0.77 ± 0.11†

^*P < 0.05 versus WT − leptin;

^†P < 0.05 versusob/ob− leptin;

^‡P < 0.05 versus WT + leptin. Values are normalized to WT − leptin.

Plasma measurements

We confirmed adequate leptin repletion with our leptin infusion protocol (ob/ob+ leptin 12.6 ± 5.4 ng ml⁻¹; n = 5 vs. WT − leptin 7.5 ± 1.6 ng ml⁻¹, n = 8, P = NS) (Table 4). Additionally, other plasma indices, including glucose (mg dl⁻¹), insulin (ng ml⁻¹) and triglycerides (mg dl⁻¹) which were elevated in ob/ob− leptin were restored towards normal with leptin repletion. These parameters remained unchanged in WT animals with leptin repletion (Table 4).

Table 4.

Plasma measurements

	WT − leptin	WT + leptin	ob/ob − leptin	ob/ob + leptin
Mice (n)*	8	11	5	7
Glucose (mg dl−1)	203 ± 11	202 ± 9	347 ± 23*	192 ± 6
Leptin (ng ml−1)	4.8 ± 0.7	7.5 ± 1.6	1.7 ± 0.2*	12.6 ± 5.4
Insulin (ng ml−1)	0.47 ± 0.06	0.41 ± 0.09	4.15 ± 1.88*	0.35 ± 0.11
Triglycerides (mg dl−1)	69 ± 7	54 ± 6	124 ± 6*	48 ± 5

^*P < 0.05 versus WT − leptin, WT + leptin, and ob/ob+ leptin.

Discussion

The major new findings of this study are that leptin deficiency impairs cardiac β-adrenergic inotropic responses by a mechanism involving protein kinase A activity and sarcoplasmic reticulum Ca²⁺ reuptake. β-Adrenergic inotropic responses are depressed in ob/ob mice at a young age before the development of LVH, in a manner completely reversible by leptin repletion. Ob/ob mice exhibit depressed protein kinase A activity with a concomitant depression in phosphorylated phospholamban and reduced SR Ca²⁺ stores, all of which are restored towards normal with leptin repletion. Together, these results suggest novel mechanism(s) by which leptin deficiency or resistance may contribute to heart failure and offer insights into cardiac dysfunction in obesity.

The young ob/ob mouse model offers unique insight into the mechanisms underlying obesity–heart failure pathophysiology. Notably, the model is normotensive and has not developed LVH (Barouch et al. 2003). Unlike other models of obesity (Hohl et al. 1993; Carroll et al. 2002), β-adrenergic signalling and molecular findings in the young ob/ob mice are not confounded by cardiac hypertrophy, a crucial point when considering that cardiac contractility and reserve are depressed in human obesity prior to the development of LVH (Licata et al. 1992, 1995; Ferraro et al. 1996). In addition, disrupted leptin signalling is almost universal in obesity (Considine et al. 1996) and leptin signalling is linked to many effector molecules, allowing leptin to have wide ranging effects (Cohen et al. 1996; Nickola et al. 2000; Wold et al. 2002). The majority of human obesity is associated with hyperleptinaemia and leptin resistance (Considine et al. 1996; Ren, 2004). However, it is important to note that the observed derangements are due to leptin deficiency, whether actual or perceived (leptin resistance). Thus, identifying the key mechanisms of pathophysiology in ob/ob mice has the potential to offer insight into the pathophysiology of the deranged signalling pathways in human obesity. In this regard, we demonstrate that the ob/ob mice exhibit disrupted β-adrenergic signalling mediated by leptin deficiency and have localized the defect to the levels of PKA activity and Ca²⁺ cycling.

β-Adrenergic receptor and adenylyl cyclase (AC)

The manner in which obesity affects β-adrenergic signalling remains controversial, as the few studies that have addressed basic mechanisms have had conflicting results. Initially, decreased β-receptor density (Bass & Ritter, 1985; Strassheim et al. 1992) was implicated in impaired contractility; however, more recent studies indicate that attenuated inotropy occurs independently of changes in β₁ or β₂ receptor expression (Hohl et al. 1993; Ernsberger et al. 1994; Carroll et al. 2002). Our results are in agreement with the concept that distal signalling transduction defects play a major role in depressed inotropic responses, as we show unchanged β₁ and β₂ protein expression in ob/ob hearts. Notably, we demonstrate that leptin deficiency alters both G_s and G_i proteins. Interestingly, leptin repletion only restores depressed G_sα-52 kDa to normal without affecting either G_i or G_sα-45 kDa, suggesting specificity of the impact of leptin restoration. Of the G proteins that stimulate AC, the long splice variant (G_sα-52 kDa) has been shown to have greater potency and more constitutive basal activity than the short splice variant (G_sα-45 kDa) (Seifert et al. 1998). These observed alterations could be an adjunct mechanism by which leptin deficiency mediates disruption of β-adrenergic signal transduction. Furthermore, our demonstration of unchanged AC activity in obesity consistent with previous reports (Carroll et al. 2002), in conjunction with a suppressed response to exogenous dibutryl cAMP suggests downstream leptin-deficiency-mediated defects in the β-adrenergic pathway.

PKA and sarcoplasmic recticulum

Cardiac β-adrenergic-mediated inotropy is greatly dependent on SR Ca²⁺ availability (Bers, 2002). Accordingly, we demonstrate that leptin deficiency impairs SR Ca²⁺ cycling as ob/ob myocytes have reduced SR Ca²⁺ stores and diminished levels of P-PLB, a key component modulating SR reuptake (Bers, 2002). Additionally, our findings of depressed PKA activity in ob/ob mice is pivotal as β-adrenergic activity increases contractility, at least in part, by cAMP-mediated phosphorylation of PLB, which removes the tonic inhibition of PLB on SERCA2a, allowing for increased Ca²⁺ reuptake (Bers, 2002). Consequently, reduced amounts of P-PLB, as seen in ob/ob, would disrupt β-adrenergic reserve. Reductions of P-PLB may be secondary to diminished PKA activity, but may also result from a direct action of leptin on PLB. Another potential pathway of this interaction may involve leptin's action on protein kinase C (PKC) (Ookuma et al. 1998), which has recently been shown to play a role in modulating PLB (Watanuki et al. 2004; Braz et al. 2004). However, our findings of depressed PKA activity, lowered P-PLB expression, and depressed Ca²⁺ stores associated with leptin deficiency, all of which are restored to normal with leptin repletion, strongly suggest that leptin mediates phosphorylation of PLB probably due in large part to PKA activity. It is noteworthy that the depressed SR Ca²⁺ stores in ob/ob are not overcome by what appears to be a compensatory increase in SERCA2a abundance. Interestingly, we previously observed a similar compensatory up-regulation of SERCA2a in NOS1 knockout mice which also have reduced SR Ca²⁺ stores (Khan et al. 2003). Leptin infusion increases SERCA2a in both WT and ob/ob mice indicating that leptin may also have non-specific effects on abundance of some (the minority) of the proteins.

Leptin signalling

Disrupted leptin signalling can attenuate cardiovascular function through several potential pathways. PKC (Ookuma et al. 1998) along with nitric oxide (Fruhbeck, 1999; Nickola et al. 2000), the Janus family tyrosine kinase and signal transducers and activators of transcription (Wold et al. 2002), mitogen-activated protein kinase (Wold et al. 2002; Rajapurohitam et al. 2003), phosphatidylinositol 3-kinase (Cohen et al. 1996), type 3 phosphodiesterase (Zhao et al. 1998), extracellular regulated kinase, phospholipase C, insulin receptor substrates protein, and protein kinase B (Szanto & Kahn, 2000) (see Sweeney, 2002 for review), are just some of the many effectors coupled to leptin signalling. These intermediate effectors influence key components of excitation–contraction coupling (Hare, 2003; Braz et al. 2004; McDowell et al. 2004; Hare & Stanler 2005). To the best of our knowledge, for the first time, we demonstrate the role of PKA as an intermediate effector molecule in leptin signalling, and thus offer a novel mechanism by which leptin attenuates β-adrenergic signal transduction in the heart.

Clinical implications

In contrast to leptin deficiency characteristic of ob/ob mice, human obesity is associated with leptin resistance and circulating leptin excess (Considine et al. 1996). Hyperleptinaemia has been described as the key driver of obesity-related cardiovascular dysfunctions, with the observed pathology attributed to leptin resistance (see Ren, 2004 for review). The main findings of this study are leptin-deficiency-mediated disruption of β-adrenergic signal transduction and consequent depression of myocyte contractility in ob/ob mice, defects that are corrected with leptin repletion. A key point is that there is relative leptin deficiency downstream of the receptor in both leptin-deficient mice and leptin-‘insensitive’ obese humans (Sader et al. 2003). Therefore, the underlying leptin signalling derangements are the same regardless of the reason behind its unavailability, whether it is leptin deficiency or resistance. Thus, to the extent that there is ‘relative leptin deficiency’ downstream of the receptor in the deficient state, it is attractive to speculate that the signalling abnormalities in the ob/ob mice are clinically relevant to the development of obesity-associated cardiac dysfunction. We have already extended the value of this murine model by inducing weight loss by either re-infusing exogenous leptin or by calorific restriction (Barouch et al. 2003). From a physiological standpoint these interventions are extremely valuable in the recapitulation of the cardiovascular benefits that could result in humans. Specifically, it should be noted that leptin infusion to ob/ob mice is the physiological correlate of weight loss in leptin-resistant obesity, a situation in which leptin signalling is restored (Sader et al. 2003).

Limitations

Although impaired leptin signalling disrupts cardiac β-adrenergic activity, whether this occurs entirely due to direct effects of leptin on the heart or is partially a consequence of sympathetic nervous system regulation by leptin is still unclear. Peripheral effects of leptin on ventricular myocytes have been demonstrated (Nickola et al. 2000) and may be responsible for cardiac changes. At the same time, central nervous system-mediated cardiac modulation cannot be excluded as leptin administered intracerebroventricularly has been shown to modify the sympathetic outflow to peripheral tissues (Dunbar et al. 1997; Haynes et al. 1997). Similarly, the impact of other hormones associated with obesity, and possibly leptin, such as cortisol, thyroxine and testosterone, cannot be ignored (Cohen et al. 2001). Changes in insulin sensitivity may also contribute to β-adrenergic hyporesponsiveness, as we (Table 4) and others (Hunt et al. 1976) have shown that both ob/ob mice and obese humans are hyperglycaemic and hyperinsulinaemic. In fact, the insulin and leptin hormonal signalling axes are closely interrelated (Segal et al. 1996). However, Hintz and colleagues have recently found that insulin resistance does not alter leptin responsiveness in cardiac myocytes (Hintz et al. 2003). Additionally, insulin treatment of diabetic animals did not affect PKA-mediated phosphorylation and activity (Netticadan et al. 2001). Thus, it is unlikely that altered insulin levels account for impaired β-adrenergic signalling and our key finding of depressed PKA activity and P-PLB levels in ob/ob mice.

Another limiting factor might be that all the functional effects of leptin are shown in isolated myocytes, whereas the molecular biology data are derived from whole myocardium. Nonetheless, our findings are consistent between molecular and functional levels.

Conclusion

Impaired leptin signalling disrupts multiple sites in the β-adrenergic signal transduction pathway independent of hypertrophy. Our findings localize the defect to the level of PKA activity with consequent impact on sarcoplasmic reticulum Ca²⁺ cycling. Although leptin abnormalities are increasingly described in obesity–heart failure pathophysiology, the mechanisms of leptin signalling in the heart remain poorly understood. The current findings offer novel insights into mechanisms by which leptin deficiency or resistance contributes to cardiac signal transduction and contractile regulation.