Effect of apelin on the cardiac hemodynamics in hypertensive rats with heart failure

Abstract

It is known that apelin has definite protective effects on various cardiovascular diseases; however, the mechanism through which hypertension with heart failure (H-HF) is affected by pyroglutamylated apelin-13 (Pyr-AP13) remain unclear. Thus, in the present study, we investigated the effects of apelin on the cardiac hemodynamics in rats with hypertension and heart failure. In our study, cardiac function, dimensions and histological determination of the fibrosis of rats with two-kidney, one-clip induced hypertension and sham-operated rats were assessed using an echocardiography system and Masson's trichrome. The infusion of either 5% glucose injection (GS) alone or 5% GS containing Pyr-AP13 as a dose, time-matched design on the cardiac hemodynamics in H-HF rats and sham-operated rats was recorded. For the determination of the effects of potential related proteins on cardiac hemodynamics in the H-HF rats, the animals were divided into 5 groups: i) the sham-operated group (n=8); ii) H-HF (n=8); iii) H-HF with infusion of 0.1 µg dose of Pyr-AP13 (n=8) or 5% glucose (GS) (n=8); iv) H-HF with infusion of 1 µg dose of Pyr-AP13 (n=8) or 5% GS (n=8); and v) H-HF with infusion of 10 µg dose of Pyr-AP13 (n=8) or 5% GS (n=8). The concentration of cyclic adenosine 3',5'-monophosphate (cAMP) was determined by ELISA. The expression of membrane and cytosolic proteins was evaluated by western blot analysis. Significant cardiac and perivascular fibrosis was observed in the H-HF rats. Following the infusion of Pyr-AP13, the systolic and diastolic function was significantly improved in the cardiac hemodynamic parameters in the H-HF rats treated with Pyr-AP13. The apelin receptor (APJ), which was activated by the exogenous infusion of Pyr-AP13, was partially recycled from the cytoplasm back to the plasma membrane; however, membrane APJ was eventually downregulated in the H-HF rats treated with Pyr-AP13 compared with the sham-operated group rats. Our findings suggested that a complex was formed after Pyr-AP13 combined with cellular membrane APJ receptor. However, the endogenous downregulation of the APJ receptor results in benefits from the exogenous administration of apelin.

Figure 1

Diagram showing the bolus dose at different time points (0, 10, 20 and 30 min). i) 0.1 μg: the rats with hypertension and heart failure (H-HF) were infused with 0.1 μg dose of pyroglutamylated apelin-13 (Pyr-AP13) (n=8) or injected with 5% glucose injection (GS) (n=8) at 0 min, and sacrificed at 10 min; ii) 1 μg: the H-HF rats were infused with Pyr-AP13 (n=8) or injected with 5% GS (n=8) in incremental doses of 0.1 and 1 μg at 10-min intervals, and sacrificed at 20 min; iii) 10 μg: the H-HF rats were infused with Pyr-AP13 (n=8) or injected with 5% GS (n=8) in incremental doses of 0.1, 1 and 10 μg at 10-min intervals and sacrificed at 30 min.

Figure 2

Significant cardiac fibrosis (CF) and perivascular fibrosis (PF) was observed in the rats with hypertension and heart failure (H-HF). (A) Upper panels, staining with hematoxylin and eosin. Lower panels, staining with Masson’s trichrome. Images were acquired at x200 magnification. Quantitative analysis of blue stained area to the total area was used to assess (B) cardiac fibrosis and (C) perivascular fibrosis in the sham-operated rats (n=8) and the H-HF rats (n=8). ^*P<0.05 vs. sham-operated rats.

Figure 3

Following the infustion of pyroglutamylated apelin-13 (Pyr-AP13; 0.1, 1 and 10 μg), hemodynamic changes, including heart rate (HR), mean left ventricular systolic pressure (mLVSP), mean left ventricular diastolic pressure (mLVDP) and highest increase/decrease in maximum rate of LV pressure (± dP/dtmax) in the sham-operated rats and the rats with hypertension and heart failure (H-HF). ^*P<0.05 vs. injection of 5% glucose injection (GS).

Figure 4

Isolated left ventricular myocytes of the rats with hypertension and heart failure (H-HF) at week 17 after clipping. (A) Low contrast bright field image (magnification, x200). (B) Hematoxylin and eosin whole cell staining (magnification, x200).

Figure 5

Cyclic adenosine 3′,5′-monophosphate (cAMP) levels. (A) Decreased cAMP level was documented in H-HF group. (B) Pyroglutamylated apelin-13 (Pyr-AP13) or 5% glucose injection (GS) was administrated in incremental doses as i.v. boli injections of 0.1, 1 and 10 μg at 10-min intervals. Upregulation of cAMP level was observed in H-HF group after treating with 1 and 10 μg Pyr-AP13. ^#P<0.05 compared with sham group. ^*P<0.05 compared with the animals received injection of 5% GS.

Figure 6

Protein expression of p-Akt, p-ERK1/2, APJ receptor in the cellular membrane (APJ-CM) and APJ receptor in the cytoplasm (APJ-CP) in isolated left ventricular myocytes of rats with hypertension and heart failure (H-HF). (A) p-Akt, p-ERK1/2, APJ-CM and APJ-CP were expresson was detected by western blot analysis. (B) The intensity of each band on the blot was quantified by densitometric scanning and all values are expressed as the means ± SD. ^*P<0.05 compared with sham-operated rats.

Figure 7

Protein expression of (A) p-Akt, (B) p-ERK1/2, (C) APJ receptor in the cellular membrane (APJ-CM) and (D) APJ receptor in the cytoplasm (APJ-CP) in isolated left ventricular myocytes of rats with hypertension and heart failure (H-HF) treated with 5% glucose injection (GS) or pyroglutamylated apelin-13 (Pyr-AP13). All values are expressed as the means ± SD. ^*P<0.05 vs. injection of 5% glucose injection (GS).