Beneficial effects of leptin treatment in a setting of cardiac dysfunction induced by transverse aortic constriction in mouse

Abstract

Key points: Leptin, is a 16 kDa pleiotropic peptide not only primarily secreted by adipocytes, but also produced by other tissues, including the heart. Controversy exists regarding the adverse and beneficial effects of leptin on the heart We analysed the effect of a non-hypertensive dose of leptin on cardiac function, [Ca²⁺ ]_i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure-overload by transverse aorta constriction. We find that leptin activates mechanisms that contribute to cardiac dysfunction under physiological conditions. However, after the establishment of pressure overload, an increase in leptin levels has protective cardiac effects with respect to rescuing the cellular heart failure phenotype. These beneficial effects of leptin involve restoration of action potential duration via normalization of transient outward potassium current and sarcoplasmic reticulum Ca²⁺ content via rescue of control sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase levels and ryanodine receptor function modulation, leading to normalization of Ca²⁺ handling parameters.

Abstract: Leptin, is a 16 kDa pleiotropic peptide not only primary secreted by adipocytes, but also produced by other tissues, including the heart. Evidence indicates that leptin may have either adverse or beneficial effects on the heart. To obtain further insights, in the present study, we analysed the effect of leptin treatment on cardiac function, [Ca²⁺ ]_i handling and cellular electrophysiology, which participate in the genesis of pump failure and related arrhythmias, both in control mice and in mice subjected to chronic pressure-overload by transverse aorta constriction (TAC). Three weeks after surgery, animals received either leptin (0.36 mg kg^-1 day^-1 ) or vehicle via osmotic minipumps for 3 weeks. Echocardiographic measurements showed that, although leptin treatment was deleterious on cardiac function in sham, leptin had a cardioprotective effect following TAC. [Ca²⁺ ]_i transient in cardiomyocytes followed similar pattern. Patch clamp experiments showed prolongation of action potential duration (APD) in TAC and leptin-treated sham animals, whereas, following TAC, leptin reduced the APD towards control values. APD variations were associated with decreased transient outward potassium current and Kv4.2 and KChIP2 protein expression. TAC myocytes showed a higher incidence of triggered activities and spontaneous Ca²⁺ waves. These proarrhythmic manifestations, related to Ca²⁺ /calmodulin-dependent protein kinase II and ryanodine receptor phosphorylation, were reduced by leptin. The results of the present study demonstrate that, although leptin treatment was deleterious on cardiac function in control animals, leptin had a cardioprotective effect following TAC, normalizing cardiac function and reducing arrhythmogeneity at the cellular level.

Keywords: heart failure; intracellular calcium; leptin.

Figure 1. Chronic leptin treatment impairs cardiac function in control but improves cardiac outcomes after TAC mice

A, representative M‐mode echocardiographic images of sham and TAC mice treated either with vehicle or with leptin. B and C, average ejection fraction (B) and shortening fraction (C), measured by M‐mode echocardiography. Data are the mean ± SEM (n = 6–8 mice). ^* P < 0.05 and ^*** P < 0.001 vs. sham vehicle; ^† P < 0.05 and ^††† P < 0.001 vs. TAC vehicle; ^# P < 0.05 and ^## P < 0.01 vs. sham leptin.

Figure 2. Leptin treatment depresses cellular [Ca²⁺]_i transients and contraction in sham but restores it in TAC cardiomyocytes

A, representative line‐scan confocal images of [Ca²⁺]_i transients obtained in Fluo‐3 loaded cardiomyocytes from sham and TAC mice treated either with vehicle or with leptin. B, average [Ca²⁺]_i transient amplitude measured as peak F/F ₀, where F is the fluorescence trace and F ₀ is the fluorescence in the diastolic period in each experimental group. C, cellular shortening expressed in percentage of cell length in each experimental group. D, representative line‐scan images of cardiac myocytes from each experimental group during field stimulation at 2 Hz and rapid 10 mmol L⁻¹ caffeine application. E, average SR content. F, representative immunoblots (top) and average ratio of protein levels (bottom) expressed as a percentage of SERCA normalized by GAPDH. G, representative immunoblots (top) and average ratio of protein levels (bottom) expressed as a percentage of p‐PLB (Ser16) of the total PLB and (H) p‐PLB (Thr17) normalized by the total PLB. Data are the mean ± SEM [n = 4–6 mice; n = 50–70 cells in (B) and (C) and n = 20–40 cells in (D)]. ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001 vs. sham vehicle; ^† P < 0.05 and ^††† P < 0.001 vs. TAC vehicle; ^# P < 0.05 and ^### P < 0.001 vs. sham leptin.

Figure 3. Leptin treatment prolonged APD in control mice but reverted it in mice that underwent TAC

A, representative AP recorded in cardiomyocytes from sham (left) and TAC (right) mice treated either with vehicle or with leptin. B, average APD measured at 20% (left), 50% (middle) and 90% (right) of the repolarization. Data are the mean ± SEM (n = 4–6 mice; n = 18–25 cells). ^*** P < 0.001 vs. sham vehicle; ^††† P < 0.001 vs. TAC vehicle; ^### P < 0.001 vs. sham leptin.

Figure 4. TAC‐induced alterations of K⁺ currents and Kv4.2 and KChIP2 expression are rescued by Leptin

Representative current traces (left) and I–V relationships (right) of I _to (A), I _Kur (B) and I _ss (C), recorded in isolated cardiomyocytes from sham (left) and TAC (right) mice treated either with vehicle or with leptin. The current density and times scales are the same for sham and TAC groups. Data, normalized to cell capacitance, are presented as the mean ± SEM (n = 4–6 mice; n = 10–24 cells). ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001 vs. vehicle treated group. For statistical comparisons between sham and TAC, see text. Representative immunoblots (top) and average protein levels normalized by GAPDH and expressed as a percentage of sham vehicle (bottom) for Kv4.2 (D) and KChIP2 (E) in cardiac tissue from sham and TAC mice treated either with vehicle or with leptin. Data are the mean ± SEM (n = 3 or 4 mice). ^** P < 0.01 and ^*** P < 0.001 vs. sham vehicle; ^†† P < 0.01 vs. TAC vehicle; ^# P < 0.05 and ^## P < 0.01 vs. sham leptin.

Figure 5. Leptin treatment reduced the occurrence of trigger activities and spontaneous Ca²⁺ waves induced by TAC

A, representative example of DAD and spontaneous AP (TA, triggered activity) recorded in ventricular cardiomyocytes isolated from TAC mouse. B, percentage of cells eliciting TA (n = 4–6 mice; n = 19–28 cells). The number of cells with TA/total analysed cells is noted above each bar. C, representative example of spontaneous Ca²⁺ wave recorded in TAC cardiomyocyte at rest. D, percentage of cardiomyocytes presenting Ca²⁺ waves (n = 4–6 mice; n = 46–67 cells). The number of cells with Ca²⁺ waves/total analysed cells is noted above each bar. E, representative immunoblots (top) and average ratio of protein levels (bottom) expressed as a percentage of NCX normalized by GAPDH. Data are presented as a percentage. ^** P < 0.01 and ^*** P < 0.001 vs. sham vehicle; ^††† P < 0.001 vs. TAC vehicle; ^### P < 0.001 vs. sham leptin.

Figure 6. Chronic leptin treatment reduces the TAC‐induced CaMKII activation and RyR2 phosphorylation

A and B, representative immunoblots (top) and average ratio of protein levels (bottom), expressed as a percentage of p‐CaMKII (Thr286) of the total CaMKII in (A) and p‐RyR2 (Ser2814) normalized by the total RyR2 (B) in hearts (n = 3 or 4 mice) from sham and TAC mice treated either with vehicle or with leptin. C, representative line‐scan images presenting Ca²⁺ sparks recorded in cardiomyocytes from sham (left two panels) and TAC (right two panels) mice treated either with vehicle or with leptin, as indicated in the labels below each image. Average Ca²⁺ spark frequency per second and per 100 μm (D), their amplitude as peak F/F ₀ (E) and Ca²⁺ spark frequency per second and per 100 μm normalized to SR load measured as F/F ₀ (F) in each group studied (n = 4–6 mice; n = 50–70 cells). Data are the mean ± SEM. ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001 vs. sham vehicle; ^† P < 0.05 and ^††† P < 0.001 vs. TAC vehicle; ^# P < 0.05 and ^### P < 0.001 vs. sham leptin.

Figure 7. Ca²⁺ spark duration and width

Average Ca²⁺ spark duration (A) and width (B) in each studied group (N = 4–6 mice; n = 50–70 cells). Data are presented as the mean ± SEM. ^*** P < 0.001 vs. sham vehicle; ^† P < 0.05 and ^††† P < 0.001 vs. TAC vehicle; ^## P < 0.01 and ^### P < 0.001 vs. sham leptin.