KMUP-1 attenuates isoprenaline-induced cardiac hypertrophy in rats through NO/cGMP/PKG and ERK1/2/calcineurin A pathways

Abstract

Background and purpose: To determine whether KMUP-1, a novel xanthine-based derivative, attenuates isoprenaline (ISO)-induced cardiac hypertrophy in rats, and if so, whether the anti-hypertrophic effect is mediated by the nitric oxide (NO) pathway.

Experimental approach: In vivo, cardiac hypertrophy was induced by injection of ISO (5 mg.kg(-1).day(-1), s.c.) for 10 days in Wistar rats. In the treatment group, KMUP-1 was administered 1 h before ISO. After 10 days, effects of KMUP-1 on survival, cardiac hypertrophy and fibrosis, the NO/guanosine 3'5'-cyclic monophosphate (cGMP)/protein kinase G (PKG) and hypertrophy signalling pathways [calcineurin A and extracellular signal-regulated kinase (ERK)1/2] were examined. To investigate the role of nitric oxide synthase (NOS) in the effects of KMUP-1, a NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA) was co-administered with KMUP-1. In vitro, anti-hypertrophic effects of KMUP-1 were studied in ISO-induced hypertrophic neonatal rat cardiomyocytes.

Key results: In vivo, KMUP-1 pretreatment attenuated the cardiac hypertrophy and fibrosis and improved the survival of ISO-treated rats. Plasma NOx (nitrite and nitrate) and cardiac endothelial NOS, cGMP and PKG were all increased by KMUP-1. The activation of hypertrophic signalling by calcineurin A and ERK1/2 in ISO-treated rats was also attenuated by KMUP-1. All these effects of KMUP-1 were inhibited by simultaneous administration of L-NNA. Similarly, in vitro, KMUP-1 attenuated hypertrophic responses and signalling induced by ISO in neonatal rat cardiomyocytes.

Conclusions and implications: KMUP-1 attenuates the cardiac hypertrophy in rats induced by administration of ISO. These effects are mediated, at least in part, by NOS activation. This novel agent, which targets the NO/cGMP pathway, has a potential role in the prevention of cardiac hypertrophy.

Figure 1

Effects of KMUP-1 on the survival rate (A) and heart weight to body weight ratio (B) in rats. ^#P < 0.05 versus control group; **P < 0.01 versus ISO group; ⁺P < 0.05 versus ISO/KMUP-1 group. HW/BW, heart weight/body weight; ISO, isoprenaline; L-NNA, N^ω-nitro-L-arginine.

Figure 2

Effects of KMUP-1 on cardiac fibrosis. Representative photomicrographs of heart slices dyed with Masson's trichrome stain (original magnification ×100) were shown with white arrows indicating fibrotic tissue. Quantification of fibrotic area was shown in the bottom panel. Data are shown as mean ± SE. ^#P < 0.05 versus control group; **P < 0.01 versus ISO group; ⁺P < 0.05 versus ISO/KMUP-1 group. The size of the bar is 100 µm. ISO, isoprenaline; L-NNA, N^ω-nitro-L-arginine.

Figure 3

Effects of KMUP-1 on serum nitric oxide (NOx) content and protein expression of cardiac eNOS and iNOS. The NOx production was quantified by measurements of nitrite and nitrate in rat serum (A). Effects of KMUP-1 on eNOS (B) and iNOS (C) protein expression in rat hearts were determined by Western blot analysis and densitometry. ^#P < 0.05 versus control group; **P < 0.01 versus ISO group; ⁺ P < 0.05, ^‡P < 0.01 versus ISO/KMUP-1 group. eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; ISO, isoprenaline; L-NNA, N^ω-nitro-L-arginine.

Figure 4

Effects of KMUP-1 on levels of cGMP (A) and PKG (B) protein expression in rat hearts, determined by Western blot analysis and densitometry. ^#P < 0.05 versus control group; **P < 0.01 versus ISO group; ⁺P < 0.05, ^‡P < 0.01 versus ISO/KMUP-1 group. cGMP, guanosine 3′5′-cyclic monophosphate; ISO, isoprenaline; L-NNA, N^ω-nitro-L-arginine; PKG, protein kinase G.

Figure 5

Effects of KMUP-1 on the activation of calcineurin A (A) and ERK1/2 (B) protein expression in rat hearts determined by Western blot analysis and densitometry. ^#P < 0.05 versus control group; **P < 0.01 versus ISO group; ⁺P < 0.05, ^‡P < 0.01 versus ISO/KMUP-1 group. ERK, extracellular signal-regulated kinase; ISO, isoprenaline; L-NNA, N^ω-nitro-L-arginine.

Figure 6

Effect of KMUP-1 on isoprenaline (ISO)-induced hypertrophy of neonatal rat cardiomocytes. Cardiomyocytes were pretreated with KMUP-1 (0.1, 1 and 10 µmol·L⁻¹) for 1 h before addition of 1 µmol·L⁻¹ ISO for 48 h. After being washed with phosphate-buffered saline, adherent cells were fixed with 1% glutaraldehyde in phosphate-buffered saline for 30 min and stained with 0.1% crystal violet for 10 min. Cell surface area was determined by planimetry, and >100 cells were analysed per condition in six repeated experiments. ^#P < 0.05 versus serum-free (SF) group. *P < 0.05, **P < 0.01 versus ISO group. The size of the bar is 100 µm.

Figure 7

Effects of KMUP-1 on ISO-induced ERK1/2 phosphorylation and calcineurin A protein expression in neonatal rat cardiomocytes. Western blots show time courses of ISO-induced ERK1/2 phosphorylation (A) and calcineurin expression (C). Quiescent neonatal cardiomocytes were pretreated with different concentrations of KMUP-1, followed by incubation with ISO (1 µmol·L⁻¹). KMUP-1 attenuated ISO-induced ERK1/2 phosphorylation (B) and calcineurin A activation (D). Representative Western blots and densitometric quantification of all experiments (n = 5) for each group are shown. ^#P < 0.05 versus serum-free (SF) group. *P < 0.05, **P < 0.01 versus ISO group. ERK, extracellular signal-regulated kinase; ISO, isoprenaline.

Figure 8

Effect of KMUP-1 on calcineurin activity and NFATc3 translocation in cardiomyocytes. Neonatal rat cardiomocytes were treated for 1 h in the presence or absence of KMUP-1, followed by incubation with ISO (1 µmol·L⁻¹) for 24 h. (A) Cell lysates were subjected to calcineurin phosphatase activity assays (n = 3). (B) Immunoblotting studies on cytosolic and nuclear fractions of NFATc3 were performed separately. The quantifications of NFATc3 protein are shown in the bottom panels (n = 3). ^#P < 0.05 versus serum-free (SF) group. *P < 0.05, **P < 0.01 versus ISO group. ISO, isoprenaline; NFAT, nuclear factor of activated T cell.