Leptin repletion restores depressed {beta}-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 beta-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-1 day-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 beta-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 Gsalpha-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 beta-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.

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.

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.

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.

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.

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.

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.