Differences in Tail Vascular Bed Reactivity in Rats with and without Heart Failure following Myocardial Infarction

Coronary artery ligation in rats has been used as a model of chronic left ventricular failure that closely mimics human condition (Hodsman et al., 1988). Indeed, compromised cardiac function (De Felice et al., 1989; Solomon et al., 1999) and increased peripheral resistance (Drexler and Lu, 1992; Schrier and Abraham, 1999) are found in this model. Systemic vasoconstriction can result from many compensatory mechanisms, including activation of the renin-angiotensin system, activation of the sympathetic nervous system, and alterations in the synthesis of local vascular factors (Zelis and Flaim, 1982; Gschwend et al., 2003).

Much has been reported regarding endothelial dysfunction in postinfarction myocardial failure (Teerlink et al., 1993, 1994; Didion et al., 1997; Bauersachs et al., 1999; Indik et al., 2001; Gschwend et al., 2003), but there is no consensus regarding vascular function in heart failure due to heterogeneity of alterations in vascular reactivity. These alterations may depend on the duration of the disease and the type of artery studied (Stassen et al., 1997a). Despite much research on vascular reactivity in postinfarction heart failure, little is known concerning vascular reactivity in a chronic phase of myocardial infarction (MI), when heart failure is absent.

The purpose of this study was, in the first place, to show that there are two animal models with similar infarction areas produced by coronary ligation: chronic MI without heart failure and with heart failure and, secondly, to demonstrate that these two models have different mechanisms involved in the control of vascular tone.

Materials and Methods

Myocardial Infarction Model. Male Wistar rats (120; 220-240 g) were housed with free access to food and water. Ninety of these rats were randomly selected to undergo MI and 30 to undergo sham surgery. MI was induced by ligation of the left coronary artery as described previously (Pfeffer et al., 1979). The anesthesia was induced by halothane. After recovery, the animals were kept in collective cages at the animal facility.

From the total number of 90 rats that were MI operated, 27 rats died early after the operation (<24 h). Sixty-three of the 90 rats (70%) survived the entire 4-week period and were included for analysis. All the 30 animals survived the sham surgery procedure. The experimental procedures were performed in accordance with the National Institutes of Health guidelines.

Heart failure was considered when three criteria were met: lung/body weight (bw) ratio greater than lung/bw_sham approximately 2-fold (Francis et al., 2001), right ventricle (RV) hypertrophy (RV/bw) proportional to lung/bw (Davidoff et al., 2004), and the left ventricle end diastolic pressure (LVEDP) greater than 15 mm Hg (Teerlink et al., 1993). Thirty of the 63 MI rats met the criteria for inclusion into the InfHF group, and the remaining 33 were used in the Inf without heart failure group.

The presence of infarction was confirmed when the animals were sacrificed. Hypertrophy was evaluated by the ratios of left ventricle (LV) and RV to bw. Tetrazolium chloride was used to discriminate between viable and nonviable myocardium. The infarct area was calculated as described previously (Mill et al., 1990).

Hemodynamic Measurements and LV Function. Rats were anesthetized with chloral hydrate (0.3 g/kg i.p.). The measurements performed were: systolic and diastolic blood pressure, left ventricular systolic pressure (LVSP), LVEDP, heart rate, and positive (+dP/dt) and negative (-dP/dt) rates of pressure development.

In Vitro Preparation of Rat Tail Vascular Bed. Isolated tail vascular bed preparations were obtained from rats 4 weeks after MI and anesthetized with sodium pentobarbital (60 mg/kg i.p.), as previously described (França et al., 1997). Briefly, after anesthesia, the tail artery was cannulated, and the tail was immediately severed from the body. The tail vascular bed was perfused with Krebs' solution (36°C, 95% O₂, and 5% CO₂) at a constant flow of 2.5 ml/min. Approximately 1 cm of the tail extremity was removed to prevent the return of the Krebs' solution through the venous system. After passing through the vascular bed, the perfusate was artificially drained out from the preparation. After a 45-min stabilization period, the experimental protocol was initiated. Mean perfusion pressure (MPP) was measured; as a result of maintenance of a constant flow, changes in the MPP represented changes in vascular resistance.

To assess the contribution of nitric oxide on vasodilatation evoked by acetylcholine (ACh; 10^-10-10^-3 M), the tail vascular bed was constricted with potassium chloride (KCl, 65 mM) added to the perfusion fluid containing indomethacin (10^-5 M). Dilatory responses to the endothelium-independent vasodilator sodium nitroprusside (SNP; 10^-9-10^-2 M) were determined. The contractile responses evoked by KCl before ACh were taken as 100%.

The vasoconstrictor response to stimulation of α-adrenoceptors by phenylephrine (0.001-300 μg) was analyzed. The role of endothelium on the changes induced by MI was investigated by comparing doseresponse curves for phenylephrine generated in arteries with intact endothelium and with endothelial damage (induced by CHAPS; 8 mg in 80 μl). To analyze the involvement of nitric oxide and the cyclo-oxygenase prostanoids derived on the response to phenylephrine, the preparations were perfused for 30 min with the nitric oxide synthase inhibitor l-NAME (10^-4 M) and indomethacin (10^-5 M), respectively. Basal nitric oxide release was evaluated by comparing the areas under the dose-response curves for phenylephrine obtained both in the absence and presence of l-NAME (Rossoni et al., 2002).

Solutions and Drugs. The Krebs bicarbonate solution was freshly prepared daily with the following composition: 119 mM NaCl, 5 mM KCl, 1.25 mM CaCl₂·2H₂O, 1.2 mM MgSO₄, 1 mM NaH₂PO₄, 27.2 mM NaHCO₃, 11 mM glucose, and 0.03 mM EDTA, all purchased from Merck (Darmstadt, Germany). All other compounds were purchased from Sigma-Aldrich (St. Louis, MO).

Data Analysis. Dilator responses are given as percentage dilation relative to the preconstriction level. Values are expressed as means ± S.E.M. For each dose-response curve, the maximum effect (E_max) and the dose of agonist that produced one-half of E_max (ED₅₀) were estimated using nonlinear regression analysis (GraphPad Software Inc., San Diego, CA). The sensitivity of the agonists is expressed as pED₅₀. One- and two-way ANOVA were followed by a Bonferroni t test, or paired or unpaired Student's t tests, as appropriate, were used for statistical analyses. P < 0.05 was considered statistically significant.

Results

Rat Characteristics. From the MI rats (n = 63), 45% (n = 30) developed HF. Infarct size did not differ between Inf and InfHF groups. The RV/bw and lung/bw ratios were increased in the InfHF group when compared with Inf and Sham (n = 30) groups. Body weight did not differ among groups (Table 1). There were no correlations between infarct size and RV/bw (r = 0.02, P > 0.05) or infarct size and lung/bw ratios (r = 0.08, P > 0.05).

The Inf group (n = 33) presented changes in all measured hemodynamic variables. Systolic, diastolic, and mean aortic blood pressures were increased. Heart rate increased about 20% compared with Sham rats. In contrast, there were no changes in the variables for left ventricular function: LVSP, +dP/dt, -dP/dt, and LVEDP (Table 1).

In InfHF rats, LV function was affected, with a reduction of the LVSP, systolic dP/dt, and diastolic dP/dt, and elevation of the LVEDP. Arterial systolic blood pressure in rats with heart failure was reduced (Table 1).

Vascular Reactivity. No differences were found in the basal perfusion pressures in preparations obtained from all groups. The respective values for the Sham (n = 24), Inf (n = 27), and InfHF (n = 24) groups were 86 ± 3, 85 ± 3, and 78 ± 3 mm Hg. The constrictor response evoked by KCl was similar among groups (Sham, 182.5 ± 8; Inf, 202.3 ± 10.7; InfHF, 197.3 ± 7.7 mm Hg).

Table 2 summarizes the results of the curve fitting to obtain the parameters for maximal response and sensitivity for the vasodilator actions of ACh and SNP.

Figure 1 shows the dose-response curves for endothelium-dependent relaxation produced by ACh. Ach induced a dose-dependent relaxation that was attenuated in the Inf rats (Table 2). The E_max to SNP was not altered, whereas the sensitivity of the InfHF group was increased compared with the Inf group (Table 2).

Dose-response curves for phenylephrine were constructed in three different series of experiments in which the effects of endothelial damage, l-NAME, or indomethacin were determined. The rank order of the E_max values were Inf > Sham > InfHF and each was significantly different from the others (P < 0.01). There were no significant differences in the potency for phenylephrine (Table 3).

Figure 2 shows the dose curves for the constrictor actions of phenylephrine on the tail artery bed generated in the presence of an intact endothelium, where the E_max value was increased in preparations obtained from the Inf group (Table 3). In the InfHF group, the E_max value for phenylephrine was reduced. Following endothelial damage (Fig. 2), the sensitivity and E_max values for phenylephrine were increased in all three groups without changing the rank order found in the presence of the endothelium (Table 3).

The effects of l-NAME (Table 3) were similar to those of endothelial damage in that the sensitivity and E_max value for phenylephrine increased in each group. The increase in the E_max for phenylephrine in the InfHF group was relatively larger than that in the other groups. Namely, in the presence of l-NAME, the E_max value was not significantly different from that of the Sham group in the presence of l-NAME. The differences of area under the curves from Sham, Inf, and InfHF were, respectively, 32.3 ± 1.87, 37.5 ± 1.8, and 42.2 ± 2.14% (P < 0.05, InfHF versus Sham). This suggests that in relation to control, there is a higher basal release of nitric oxide in tail artery preparations following the development of postinfarction heart failure.

The putative role of prostanoids in modulating vascular responses was investigated by determining the effects of indomethacin (10^-5 M) on dose-response curves for phenylephrine. When compared with its respective group control value, the E_max for phenylephrine increased in the Inf group and remained unchanged in the other two groups in the presence of indomethacin (see Table 3). The maximal responses for phenylephrine prior to indomethacin showed the same rank order as the control values for those experiments described in Table 3. This effect of indomethacin suggests that the modulation of the response to phenylephrine by a vasodilator prostanoid is greater in the Inf group than in other groups. In addition, the difference observed in the E_max between the Sham and InfHF groups before indomethacin perfusion was abolished, suggesting a little modulation of the response to phenylephrine by a vasodilator prostanoid in this group. The effects of indomethacin on the dose-response curves for phenylephrine are shown in Table 3.

Discussion

The primary finding in this study is the fact that, despite a similar infarct size, 4 weeks after coronary ligation, we found animals with and without heart failure. The heart failure rats presented a depressed LV function, which is consistent with heart failure (De Felice et al., 1989; Francis et al., 2001). Additionally, this study demonstrated that there is no correlation between infarct size and the development of heart failure, although there are studies demonstrating a rapid development of heart failure after large infarcts in rats (Norman and Coers, 1960; Anversa et al., 1984; Novaes et al., 1996).

As to vascular function, we observed that the changes in tail vascular bed reactivity differed between animals that developed heart failure from those that did not.

In agreement with other studies (Baggia et al., 1997; Bauersachs et al., 1999, 2002; Ceiler et al., 1999; Schäfer et al., 2003), we found no impairment of the endothelium-independent relaxation produced by SNP following infarction. However, we found impairment in the endothelium-dependent relaxation mediated by ACh in the tail vascular beds in Inf rats. Impairments in endothelium-dependent relaxation have also been reported in patients with cardiovascular disease and chronic heart failure (Belardinelli, 2001; Annuk et al., 2003; Linke et al., 2003). Studies in different vessels using the rat infarction model for heart failure show either reductions or no changes in endothelial dependent relaxation, at different times after infarction (Teerlink et al., 1993; Baggia et al., 1997; Bauersachs et al., 1999, 2002; Ceiler et al., 1999; Indik et al., 2001; Annuk et al., 2003). Possibly, the endothelial dysfunction observed in heart failure may depend on its stage and the type of vessels studied (Fang and Marwick, 2002).

In human subjects with heart failure and in the rat postinfarction model for heart failure (Stassen et al., 1997a,b; Fang and Marwick, 2002; Annuk et al., 2003) reductions in the contractile activity of α-adrenoceptor agonists have been reported. This reduced contractile activity might be due to a down-regulation of α-adrenoceptors because of high and prolonged sympathetic stimulation during heart failure. Although we did not evaluate the neurohumoral activation, the InfHF rats presented hyporeactivity to the α-agonist phenylephrine. In contrast with a down-regulation of α-adrenoceptors found in rat mesenteric arteries after MI, Feng et al. (1996) and Stassen et al. (1997b) found no changes in the density of α₁-adrenoceptors in those vascular beds that displayed a decreased responsiveness to phenylephrine. However, absence of changes in the density of α₁-adrenoceptors does not rule out changes in the receptor signaling pathway that may lead to a reduction in responsiveness. In addition, the decrease in reactivity to phenylephrine is paralleled by an increased nitric oxide basal release, which can contribute to this hyporeactivity.

The increase in responsiveness to phenylephrine seen in the Inf group is paralleled by a decrease in responsiveness to acetylcholine, which suggests that the reduced endothelium-dependent relaxation is a common factor.

These changes in phenylephrine responsiveness occurred in the absence of any changes in the contractile response to KCl, suggesting that there is no defect in the contractile apparatus per se in the tail arterial bed.

The effects of endothelial damage and l-NAME were similar in that both treatments increased the E_max and pED₅₀ values for phenylephrine in all groups. Endothelial damage decreased the difference between the Sham and InfHF groups and further accentuated the increase in the contractile actions of phenylephrine in the Inf group. This suggests an involvement of a muscular component on the increased response to phenylephrine in the Inf group.

When the contribution of nitric oxide to the negative modulation of contractile agents was evaluated, an increased nitric oxide basal release was observed in the InfHF group. The nitric oxide basal production is reported as having increased (Habib et al., 1994), decreased (Annuk et al., 2003), or unaltered (Drexler and Lu, 1992) in experimental models for postinfarction heart failure. The results obtained in the present study suggest that an increase in the basal production and the availability of nitric oxide contributed to the decreased responsiveness to phenylephrine in the tail artery bed in animals with heart failure.

Additionally, our findings suggest that the actions of another endothelial-derived negative modulator of contractility were enhanced in the Inf group. The contribution of a vasodilator prostanoid was examined by the inhibition of cyclo-oxygenase by indomethacin. In the presence of indomethacin only, the E_max to phenylephrine in the Inf group was significantly altered. There was an increase in the E_max in this group that was accompanied by a change in the sensitivity to phenylephrine. These results suggest that a vasodilator prostanoid plays a modulatory role in the tail bed vascular contractility in the Inf group. This is likely to be a compensatory adaptation in response to the increased contractility observed in the Inf group.

In summary, this study demonstrated that despite similar extension of infarct, two animal models can be found after coronary ligation: one chronic MI without heart failure and another with heart failure. Comparing these models, we found differences in the vascular reactivity of the tail vascular bed. Interestingly, the vascular responses to phenylephrine and acetylcholine differed between the groups. Compromised cardiac function in rats with heart failure was related to a hyporeactivity to phenylephrine, to an increased nitric oxide basal release, and to a normal endothelium-dependent relaxation. In the MI rats without heart failure, the cardiac function was preserved, and there were an impaired endothelium-dependent relaxation and an increased responsiveness to phenylephrine regardless of the endothelial integrity. This hyperreactivity occurred despite a normal nitric oxide basal release and despite an increased production of a vasodilator prostanoid. Moreover, our results suggest that this hyperreactivity was due, in part, to nonendothelial changes.