IK1 Channel Agonist Zacopride Alleviates Cardiac Hypertrophy and Failure via Alterations in Calcium Dyshomeostasis and Electrical Remodeling in Rats

Abstract

Intracellular Ca²⁺ overload, prolongation of the action potential duration (APD), and downregulation of inward rectifier potassium (I_K1) channel are hallmarks of electrical remodeling in cardiac hypertrophy and heart failure (HF). We hypothesized that enhancement of I_K1 currents is a compensation for I_K1 deficit and a novel modulation for cardiac Ca²⁺ homeostasis and pathological remodeling. In adult Sprague-Dawley (SD) rats in vivo, cardiac hypertrophy was induced by isoproterenol (Iso) injection (i.p., 3 mg/kg/d) for 3, 10, and 30 days. Neonatal rat ventricular myocytes (NRVMs) were isolated from 1 to 3 days SD rat pups and treated with 1 μmol/L Iso for 24 h in vitro. The effects of zacopride, a selective I_K1/Kir2.1 channel agonist, on cardiac remodeling/hypertrophy were observed in the settings of 15 μg/kg in vivo and 1 μmol/L in vitro. After exposing to Iso for 3 days and 10 days, rat hearts showed distinct concentric hypertrophy and fibrosis and enhanced pumping function (P < 0.01 or P < 0.05), then progressed to dilatation and dysfunction post 30 days. Compared with the age-matched control, cardiomyocytes exhibited higher cytosolic Ca²⁺ (P < 0.01 or P < 0.05) and lower SR Ca²⁺ content (P < 0.01 or P < 0.05) all through 3, 10, and 30 days of Iso infusion. The expressions of Kir2.1 and SERCA2 were downregulated, while p-CaMKII, p-RyR2, and cleaved caspase-3 were upregulated. Iso-induced electrophysiological abnormalities were also manifested with resting potential (RP) depolarization (P < 0.01), APD prolongation (P < 0.01) in adult cardiomyocytes, and calcium overload in cultured NRVMs (P < 0.01). Zacopride treatment effectively retarded myocardial hypertrophy and fibrosis, preserved the expression of Kir2.1 and some key players in Ca²⁺ homeostasis, normalized the RP (P < 0.05), and abbreviated APD (P < 0.01), thus lowered cytosolic [Ca^{2 +}]_i (P < 0.01 or P < 0.05). I_K1channel blocker BaCl₂ or chloroquine largely reversed the cardioprotection of zacopride. We conclude that cardiac electrical remodeling is concurrent with structural remodeling. By enhancing cardiac I_K1, zacopride prevents Iso-induced electrical remodeling around intracellular Ca²⁺ overload, thereby attenuates cardiac structural disorder and dysfunction. Early electrical interventions may provide protection on cardiac remodeling.

Keywords: calcium overload; cardiac remodeling; inward rectifier potassium channel; isoproterenol; zacopride.

Figure 1

Schematic protocol of in vivo experiments. Iso, isoproterenol; Zac, zacopride; Chlo, chloroquine; RS23597, RS23597-190, an antagonist of 5-HT₄ receptor. m-CPBG, m-chlorophenylbiguanide, an agonist of 5-HT₃ receptor.

Figure 2

Cardiac remodeling induced by 10 days of Iso exposure in rats in vivo. (A) The gross morphology of the whole hearts. (B) Representative echocardiographic images from each of the corresponding hearts shown in (A). (C) HE staining of transverse LV sections (250×). Scale bars = 50 µm. (D) Masson’s trichrome staining showing collagen deposition in rat LV (100 ×). Cardiomyocytes and collagen fibers were stained as red and blue, respectively. Scale bars = 100 µm. (E) Statistical results of the cross sectional area of myofiber in different groups. N = 50 in each group. (F) Statistical results of fibrotic area which was expressed as the percentage of total area in each field, n = 5 in each group. (G) The heart/LV mass index. (H) The expression levels of Kir2.1 channel protein. Iso, isoproterenol; Zac, zacopride; Chlo, chloroquine; RS23597, RS23597-190; m-CPBG, m-chlorophenylbiguanide. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with control. ^## P < 0.01 compared with Iso+Zac.

Figure 3

Time courses of structural remodeling and electrical remodeling 3 days, 10 days, and 30 days post-Iso infusion. (A) Representative echocardiographic images of the corresponding hearts. (B) Time courses of IVSd, LVIDd, LVPWd, and EF changes post-Iso toxication. *P < 0.05, **P < 0.01 for Iso compared with age-matched control. ^#P < 0.05, ^##P < 0.01 for Iso compared with age-matched Iso+Zac group. (C) Cytosolic Ca²⁺ and SR Ca²⁺ fluorescences measured using laser scanning confocal microscopy. Upper row, Fluo-5N cellular distribution indicating SR Ca²⁺. Lower row, Fluo-4 cellular distribution indicating cytosolic Ca²⁺. Scale bars = 20 µm. (D) Statistical summary of cytosolic Ca²⁺ and SR Ca²⁺ levels of 3 days, 10 days, and 30 days post-Iso exposure, respectively. SR, sarcoplasmic reticulum. LVIDd, left ventricular dimension in end diastole. IVSd, interventricular septum end-diastolic thickness. LVPWd, LV posterior wall thickness at end diastole; EF, ejection fraction; Iso, isoproterenol; Zac, zacopride; Chlo, chloroquine; RS23597, RS23597-190; m-CPBG, m-chlorophenylbiguanide. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with age-matched control. ^#P < 0.05, ^##P < 0.01 compared with age-matched Iso+Zac.

Figure 4

Zacopride improved Iso-induced maladaptive protein alterations in rat hearts. (A) Representative western blotting images 3 days, 10 days, and 30 days after the onset of Iso infusion. (B) Dynamic changes of Kir2.1 protein expression levels. (C) Dynamic changes of p-CaMKII protein expression levels relative to total CaMKII. (D) Dynamic changes in p-RyR2 protein expression levels relative to total RyR2. (E) Dynamic changes in SERCA2 protein expression. (F) Dynamic changes of cleaved caspase 3 protein expression. All data were normalized to control. Iso, isoproterenol; Zac, zacopride; Chlo, chloroquine. Data were presented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01 compared with control. ^#P < 0.05, ^##P < 0.01 compared with Iso+Zac.

Figure 5

Zacopride restored RP depolarization and APD prolongation in isolated rat ventricular myocytes 10 days post-Iso infusion. These effects could be partially reversed by chloroquine. (A) Representative AP recording. (B) APD₅₀ and APD₉₀ in different groups. Iso, isoproterenol; Zac, zacopride; Chlo, chloroquine. N = 6 cells. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01 compared with control. ^##P < 0.01 compared with Iso+Zac.

Figure 6

Zacopride inhibited Iso-induced hypertrophy and intracellular calcium overload in cultured NRVMs. (A) Fluorescent images of Fluo-4/AM loaded neonatal cardiomyocytes. Bar = 50 μm. (B) Zacopride treatment normalized the cell size of NRVMs. (C) Zacopride treatment attenuated Iso-induced intracellular calcium overload. BaCl₂ or chloroquine reversed the effects of zacopride on cell sizes (B) and [Ca²⁺]_i(C). Zacopride had no effect on [Ca²⁺]_i in normal NRVMs. Iso, isoproterenol; Zac, zacopride; Chlo, chloroquine. N = 6 cells. Data were presented as mean ± SEM. **P < 0.01 compared with control; ^##P < 0.01 compared with Iso+Zac.

Figure 7

Apoptosis of NRVMs following exposure to 1 μmol/L Iso for 24 h. (A) Cells in the lower right (LR) quarter represents early apoptosis. (B) Zacopride protected cardiomyocytes from Iso-induced apoptosis. I_K1 channel blocker BaCl₂ or chloroquine reversed the effects of Zac. Iso, isoproterenol; Zac, zacopride; Chlo, chloroquine. N = 5 parallel samples in each group. Data were presented as means ± SEM. **P < 0.01, compared with control. ^#P < 0.05, ^##P < 0.01, compared with Iso+Zac.