Nifedipine inhibits cardiac hypertrophy and left ventricular dysfunction in response to pressure overload

Abstract

Pathological hypertrophy is commonly induced by activation of protein kinases phosphorylating class II histone deacetylases (HDACs) and desuppression of transcription factors, such as nuclear factor of activated T cell (NFAT). We hypothesized that nifedipine, an L-type Ca(2+) channel blocker, inhibits Ca(2+) calmodulin-dependent kinase II (CaMKII) and NFAT, thereby inhibiting pathological hypertrophy. Mice were subjected to sham operation or transverse aortic constriction (TAC) for 2 weeks with or without nifedipine (10 mg/kg/day). Nifedipine did not significantly alter blood pressure or the pressure gradient across the TAC. Nifedipine significantly suppressed TAC-induced increases in left ventricular (LV) weight/body weight (BW; 5.09 +/- 0.80 vs. 4.16 +/- 0.29 mg/g, TAC without and with nifedipine, n = 6,6, p < 0.05), myocyte cross-sectional area (1,681 +/- 285 vs. 1,434 +/- 197 arbitrary units, p < 0.05), and expression of fetal-type genes, including atrial natriuretic factor (35. 9 +/- 6.4 vs. 8.6 +/- 3.3 arbitrary units, p < 0.05). TAC-induced increases in lung weight/BW (7.7 +/- 0.9 vs. 5.5 +/- 0.5 mg/g, p < 0.05) and decreases in LV ejection fraction (65.5 +/- 3.1% vs. 75.7 +/- 3.3%, p < 0.05) were attenuated by nifedipine. Nifedipine caused significant inhibition of TAC-induced activation of NFAT-mediated transcription, which was accompanied by suppression of Thr 286 phosphorylation in CaMKII. Nifedipine inhibited activation of CaMKII and NFAT by phenylephrine, accompanied by suppression of Ser 632 phosphorylation and nuclear exit of HDAC4 in cardiac myocytes. These results suggest that a subpressor dose of nifedipine inhibits pathological hypertrophy in the heart by inhibiting activation of CaMKII and NFAT, a signaling mechanism commonly activated in pathological hypertrophy.

Figure 1. Nifedipine infusion does not affect blood pressure

Mice were subjected to either sham or transverse aortic constriction (TAC) operation in the presence of continuous infusion of nifedipine (10mg/kg/day) or vehicle by osmotic pump for two weeks. Post constriction mean arterial pressure (MAP) (A) and pressure gradient (B) were measured, using a catheter under anesthesia. Values are mean ± SEM. n=4 in each group. (C) Representative simultaneous recording of blood pressure at ascending aorta and abdominal aorta from animals subjected to TAC in the presence ((+) Nife) or absence ((−) Nife) of nifedipine.

Figure 2. Nifedipine inhibits TAC-induced cardiac hypertrophy

Mice were subjected to either sham or transverse aortic constriction (TAC) operation in the presence of continuous infusion of nifedipine (10mg/kg/day) or vehicle by osmotic pump for two weeks. Postmortem measurements of heart weight/body weight (HW/BW, mg/g) (A) and left ventricular weight/body weight (LVW/BW, mg/g) (B) are shown. (C) Representative wheat germ agglutinin (WGA)-Texas Red staining. Bar = 50 µm. (D) Myocyte cross-sectional area was determined from WGA-stained 1-µm-thick sections. The outline of 100–200 myocytes was traced in each section. The ImagePro system software was used to measure myocyte cross-sectional area. Values are mean ± SEM. n=6 in each group. * p<0.05.

Figure 3. Nifedipine attenuates TAC-induced upregulation of fetal type genes

Mice were subjected to either sham or transverse aortic constriction (TAC) operation in the presence of continuous infusion of nifedipine (10mg/kg/day) or vehicle by osmotic pump for two weeks. The apex portion of the LV was homogenized and total RNA was extracted. qRT-PCR analyses of ANF mRNA (A) and α-skeletal actin mRNA (B) were conducted. Values are mean ± SEM. n=6 in each group. *p<0.05.

Figure 4. Nifedipine inhibits TAC induced cardiac dysfunction

Mice were subjected to either sham or transverse aortic constriction (TAC) operation in the presence of continuous infusion of nifedipine (10mg/kg/day) or vehicle by osmotic pump for two weeks. (A) Postmortem measurement of lung weight/body weight (Lung W/BW, mg/g). (B) Echocardiographic analysis was conducted under anesthesia to evaluate ejection fraction (%), an index of LV systolic function. Values are mean ± SEM. n=6 in each group. *p<0.05.

Figure 5. Nifedipine inhibits TAC induced NFAT transcriptional activity

NFAT-Luciferase reporter mice were subjected to either sham or transverse aortic constriction (TAC) operation in the presence of continuous infusion of nifedipine (10mg/kg/day) or vehicle by osmotic pump for two weeks. (upper) A cartoon showing the NFAT-luciferase reporter construct used in transgenic mice. (lower) The luciferase activity in heart homogenates from NFAT-luciferase transgenic mice was measured and normalized by the protein content. Values are mean ± SEM. n=6 in each group. *p<0.05.

Figure 6. Nifedipine attenuates TAC-induced upregulation of calcium-related cardiac genes

Mice were subjected to either sham or transverse aortic constriction (TAC) operation in the presence of continuous infusion of nifedipine (10mg/kg/day) or vehicle by osmotic pump for two weeks. Heart homogenates were prepared for immunoblot analyses. Immunoblots shown are representative of 4 experiments.

Figure 7. Nifedipine attenuates phenylephrine-induced activation of NFAT transcription, nuclear exit of HDAC4, and Ser 632 phosphorylation of HDAC4 in cultured cardiac myocytes

(A) Cultured neonatal rat cardiac myocytes were subjected to transient transfection with NFAT-Luc reporter gene. Twenty-four hours after transfection, cardiac myocytes were treated with phenylephrine (PE) and/or nifedipine for 36–48 hours and harvested for the NFAT luciferase activity measurement. n=9 in each group. *p<0.05. (B) Myocytes were treated with phenylephrine in the presence or absence of nifedipine. After forty-eight hours, cell surface area was evaluated as described in the Method section. n=4 in each group. (C,D) Cultured cardiac myocytes were treated with PE in the presence or absence of nifedipine for 4 hours either for either immunostaining using anti-HDAC4 antibody (green), anti-α-actinin (red) and DAPI (blue) (C) or for immunoblotting with antibodies as indicated (D). The results are representative of 4 experiments. Bar = 50 µm.

Figure 8. Nifedipine inhibits cardiac hypertrophy

A cartoon demonstrating the pathways by which nifedipine inhibits cardiac hypertrophy. The current hypothesis is that nifedipine inhibits activation of Ca²⁺/calmodulin kinase (CaMK), thereby preventing nuclear exit of class II HDACs. Since recent evidence suggests that nucleo-cytoplasmic shuttling of class II HDACs is also regulated by other mechanisms, including oxidation of cysteine residues, it is possible that nifedipine induces nuclear localization of class II HDACs through unknown mechanisms.