Calcium antagonists, diltiazem and nifedipine, protect broilers against low temperature-induced pulmonary hypertension and pulmonary vascular remodeling

INTRODUCTION

Ascites syndrome (also known as pulmonary hypertension syndrome), one of the common end points of a number of pathogenies, is a metabolic disease in broilers characterized by pulmonary hypertension (PH)-induced right ventricular hypertrophy and failure (Huchzermeyer & De Ruyck 1986; Wideman et al. 1995; Currie 1999). These pathogenies may be due to a high basal metabolic rate originating from intensive genetic selection of broilers for rapid growth, long-term exposure to extreme environmental conditions and nutritional levels, such as low temperature, high altitude or high energy diets, which lead to an increased requirement for oxygen at tissue and cell levels. The resultant hypoxia in turn triggers an increased cardiac output and a consequential elevation in pulmonary arterial pressure, leading to right ventricular hypertrophy and pressure overload in the right ventricle, and sequentially results in right ventricular failure, liver congestion and ultimate ascites (Currie 1999). Regardless of the etiology, prolonged pulmonary vasoconstriction has been suggested to be an important pathogenetic factor in all forms of PH. Therefore, pulmonary vasodilation is a major strategy for the treatment of this disease, and vasodilator drugs have been proposed as an alternative or an adjunct approach to oxygen supplementation to attenuate the PH and prevent ascites in broilers for years (Yang et al. 2005, 2007). Evidence indicated that alteration of calcium homeostasis contributed to the development and perpetuation of PH through destroying the balance between vasodilating and vasoconstrictive forces, and proliferative and anti-proliferative forces (Enkvetchakul et al. 1995; Wideman et al. 1995; Currie 1999).

Application of calcium antagonists alone, or in combination with other agents is one of the effective ways to understand whether the calcium signaling is involved in these pathophysiological phenomena, such as various types of hypertension and cardiovascular diseases in mammals including humans. Attention was initially drawn to calcium antagonists because of their vasodilating properties, which might prevent vascular smooth muscle cells, cardiac muscle cells and conductive tissues from proliferating by inhibiting their trans-membrane influx of calcium ions (Cubeddu et al. 1986; Armas-Padilla et al. 2000). Calcium antagonists have been extensively investigated and are being used in treatment for hypertension, angina, and certain cardiac diseases in humans; however, the pharmacological reversal of hypoxic PH with a calcium antagonist has not been comprehensively evaluated in poultry affected with chronic pulmonary arterial hypertension.

In this study, nifedipine and diltiazem, two L-type Ca²⁺ channel antagonists were used in chicks to understand the actions of calcium signaling in the development of pulmonary arterial hypertension and to characterize their preventive efficacy on hemodynamics and pulmonary artery function of broilers with pulmonary hypertension syndrome induced by low temperature.

MATERIALS AND METHODS

RESULTS

Pulmonary artery remodeling

The relative median thickness and relative median area of pulmonary arterioles are shown in Table 2. There was no difference in external diameters of pulmonary arteries between groups, either ranging 20 ≤Φ≤ 50, 50 ≤Φ≤ 100 µm or 100 ≤Φ≤ 200 µm on days 29, 36 and 43. The relative median thicknesses and relative median areas were significantly greater in the two non-pharmaceutically treated groups at low temperature than that at normal temperature on days 36 and 43, respectively (P_all < 0.05) (Table 2, Fig. 1A,C). No significant difference on relative median thickness and relative median area was observed between diltiazem-treated birds and that of controls, whereas the two parameters were significantly greater in nifedipine-treated groups at low temperature than at normal temperature within the three ranges on days 36 and 43, respectively (P_all < 0.05).

Table 2. Relative medial thickness (RMT) and relative medial area (RMA) of broilers with pulmonary arteriol external diameter ranging from 20 to 50, 50–100 µm and 100–200 µm in the different groups and investigated intervals. Values are mean of 15 birds with 5–10 arteriols each and expressed as mean ± SD

Treatment			Normal temperature		Low temperature
Item	Φ(µm)	Age (day)	Control (n = 15)	Placebo (n = 15)	Control (n = 15)	Placebo (n = 15)	Nifedipine (5mg/kg/day) (n = 15)	Diltiazem (15mg/kg/day) (n = 15)
RMT (%)	20∼50	29	30.6 ± 4.23	31.5 ± 4.17	34.6 ± 4.46	35.5 ± 2.51	31.5 ± 2.47	29.5 ± 3.27
		36	33.3 ± 3.92a	32.7 ± 3.69a	38.5 ± 2.59b	38.3 ± 3.79b	35.2 ± 4.31ab	32.8 ± 3.83a
		43	32.5 ± 4.08a	31.9 ± 4.36a	40.3 ± 4.26b	41.2 ± 5.15b	37.6 ± 3.84c	33.5 ± 4.21a
	50∼100	29	24.4 ± 3.02	24.9 ± 2.13	26.7 ± 2.98	27.6 ± 2.41	24.8 ± 1.90	24.1 ± 1.74
		36	25.7 ± 2.69a	26.2 ± 1.88a	30.0 ± 2.87b	30.7 ± 3.02b	30.8 ± 2.64b	25.4 ± 2.13a
		43	26.9 ± 2.95a	27.1 ± 2.98a	32.2 ± 4.11b	33.7 ± 3.85b	31.9 ± 3.08b	25.9 ± 1.89a
	100∼200	29	18.8 ± 1.42	19.1 ± 1.76	20.5 ± 1.57	21.7 ± 3.11	21.7 ± 3.28	18.9 ± 1.61
		36	19.1 ± 2.48a	19.4 ± 2.11a	23.4 ± 3.65b	22.6 ± 3.14ab	23.7 ± 2.28b	20.4 ± 3.95a
		43	19.7 ± 1.91a	20.5 ± 2.33ac	24.6 ± 2.58b	23.1 ± 2.08b	24.5 ± 3.04bc	20.8 ± 1.79a
RMA (%)	20∼50	29	49.2 ± 5.62	48.1 ± 4.48	53.7 ± 6.43	55.6 ± 4.95	52.4 ± 6.45	50.5 ± 4.57
		36	50.4 ± 3.25a	49.4 ± 3.57a	56.6 ± 5.38b	57.3 ± 6.36b	53.6 ± 4.31a	48.8 ± 4.58a
		43	48.7 ± 3.67a	50.3 ± 4.33a	57.7 ± 6.25b	57.6 ± 4.69b	56.6 ± 5.34b	48.6 ± 3.36a
	50∼100	29	51.6 ± 4.95	52.3 ± 5.32	57.6 ± 5.67	56.4 ± 5.14	55.1 ± 4.78	50.5 ± 4.69a
		36	54.8 ± 5.11a	54.1 ± 5.32a	66.1 ± 5.71b	66.5 ± 6.28b	64.9 ± 5.74b	53.9 ± 4.61a
		43	53.7 ± 4.61a	54.3 ± 5.14a	66.7 ± 4.85b	67.5 ± 5.29b	64.1 ± 5.66b	52.8 ± 4.47a
	100∼200	29	35.1 ± 2.78	36.3 ± 2.29	38.7 ± 3.19	39.5 ± 3.86	39.1 ± 3.07	35.4 ± 2.32
		36	35.4 ± 2.66a	36.4 ± 2.35a	44.8 ± 2.96b	43.9 ± 3.46b	40.5 ± 3.73b	34.2 ± 3.08a
		43	36.8 ± 3.21a	37.5 ± 2.18a	46.4 ± 3.56b	47.7 ± 4.69b	41.6 ± 3.79b	34.6 ± 3.38a

• ^a,b,c means values within the same row with the different letters in their superscripts differs significantly (P < 0.05).

DISCUSSION

The method of low temperature-induced pulmonary hypertension symptoms in broilers has been confirmed (Wideman et al. 1995). As expected, in this study, low temperature did cause the development of the pathologic process, which is represented by PH, increased PCV and right ventricular hypertrophy, as well as the thickening of the pulmonary small artery smooth muscle layer (Enkvetchakul et al. 1995; Wideman et al. 1995). Under a low temperature environment, broilers' demands for oxygen were physiologically increased to produce more energy and heat to maintain body temperature at a normal level, leading to a relative hypoxia status. Hypoxia has been confirmed as an important pathophysiological stimulus for erythrocyte number increment accompanied by PH (Maxwell et al. 1987). Additionally, with long-term hypoxia and the development of PH, right ventricular compensatory hypertrophy forms, which explains the increase in AHI (Wideman & Tackett 2000).

The main finding of the study is that, except for a slight decrease in femoral arterial pressure by nifedipine on day 43, no difference was observed in other parameters between diltiazem-treated broilers (15.0 mg/kg BW/12 h) and those of controls throughout the experiment. This result showed that diltiazem prevented PH and PCV from increasing, induced lower temperature, and arrested right ventricular hypertrophy and pulmonary arterial vascular remodeling. Although nifedipine prevented pulmonary arterial pressure from increasing during the early stage of the experiment (29 and 36 days old), it did not suppress the development of PH and pulmonary artery remodeling during the later stage (43 days old), and did not keep HR, PCV and AHI at the same levels as those of controls. It should be noted that nifedipine and diltiazem, as well as verapamil which was used in our previous study (Yang et al. 2007), all belong to L-type Ca²⁺ channel blockers. They display differences in preventing low temperature-induced pulmonary arterial hypertension in broilers. Diltiazem and verapamil attenuate pulmonary hypertension, whereas the nifedipine has little effect on pulmonary hypertension and displays some side-effects on the growth and performance of broilers. This is in consistent with the observations obtained from humans, and suggests that this may be due to the differences in molecular mechanisms. Further study is needed to explain these observations.

The predominant viewpoint on pulmonary hypertension are based on the principle that hypoxia inhibits an outward potassium current, leading to membrane depolarization and calcium entry through the voltage-dependent calcium channels (Post et al. 1992; Hong et al. 2004). The elevation of intracellular Ca²⁺ concentration ([Ca²⁺]i) in pulmonary small artery smooth muscle cells activates the contractile system in the smooth muscle, causing cell contraction and proliferation, consequently resulting in the thickening of the pulmonary small artery smooth muscle layer and eventually PH (Platoshyn et al. 2000; Wamhoff et al. 2006). It has been proven that the voltage-dependent L-type Ca²⁺ channel antagonists could inhibit the rise of [Ca²⁺]i. Two decades after their first introduction in treatment for primary PH (Camerini et al. 1980; Kambara et al. 1981), diltiazem and nifedipine have been reported to be effective in various experimental models and human clinics (Gassner et al. 1990; Antezana et al. 1998; Islam et al. 1999; Cockrill et al. 2001).

In the present study, oral nifedipine with 5.0 mg/kg BW/12 h increased the broilers' HR and depressed PH at the same time. However, in our preliminary experiment, several doses of nifedipine were tested and the results showed that those doses lower than 5.0 mg/kg BW/12 h did not prevent PH, while doses higher than 5.0 mg/kg BW/12 h prevented PH, but it depressed FAP and boosted HR greatly. In addition, we found that nifedipine could not arrest the pulmonary artery smooth muscle remodeling at several doses (2.5, 5.0, 8.0, 10.0 mg/kg BW/12 h, data not shown). These data indicated that although nifedipine may be a useful agent to reduce pulmonary resistance in PH, this effect was less pronounced on PAP than FAP in broilers. Several other studies also demonstrated that the effects of nifedipine were often partial, temporary and sometimes complicated by changes in cardiac output (Clozel et al. 1987; Agostoni et al. 1989; Davidson et al. 1989). It is suggested that nifedipine may be particularly useful in low temperature-induced PH in broilers when vasoconstriction is the main determinant factor of PH as in primary PH in humans, and nifedipine cannot arrest the PH caused by pulmonary artery wall remodeling developed in the later stage (Rich & Brundage 1987).

Diltiazem was reported to reduce pulmonary arterial pressure in recurrent PH associated with pulmonary hypoplasia (Islam et al. 1999) and two clinical studies had also shown a reduction of PH in patients after diltiazem administration (Kambara et al. 1981; Crevey et al. 1982). Our results on diltiazem confirmed that the appropriate dose of diltiazem might depress PH in broilers, maintain HR at normal levels (no HR reduction), arrest right ventricular hypertrophy (expressed by AHI) and prevent pulmonary artery wall remodeling in the later stages (Fig. 1C). No mortality or serious morbidity was associated with the drug testing. Moreover, the results also provided another beneficial action of diltiazem in preventing PH syndrome, which is represented by a higher erythrocyte packed cell volume and right ventricular hypertrophy (Julian 1993). In this case, diltiazem is preferable to nifedipine in preventing low temperature-induced PH in broilers as it has no systemic side-effects, and is more selective for the pulmonary vasculature than for the systemic one.

The mechanism of calcium antagonists preventing PH in broilers is the same as in mammals, that is, calcium antagonists block the intracellular signal pathway, which interdicts the activation of the contractile action on pulmonary vessels, keeping normal pulmonary function (Mohanty et al. 1987; Wang et al. 2005). Therefore, calcium antagonists prevent PH and avoid increasing erythrocyte production pathologically. The preventive effects of L-type Ca²⁺ channel antagonists suggest that calcium signals are associated with PH in broilers, which leads to the development of ascites. Calcium antagonists, such as diltiazem, when administered at appropriate doses, may cause substantial reductions in pulmonary arterial pressure and prevent the occurrence of pulmonary arterial hypertension syndrome in broilers.