Introduction

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Use of the sympathetic system is a major adaptation mechanism by which the heart can augment its performance during exercise and acute stress. Stimulation of -adrenergic receptors has a positive inotropic effect on normal cardiac muscle. Inotropic response to -adrenergic stimulation is generally found to be depressed in hypertrophied and failing hearts of both humans and experimental animals (Gende et al., 1985; Brown et al., 1986; Foster et al., 1991; Taquini et al., 1991; Harding et al., 1992, 1994; Atkins et al., 1995; Moravec et al., 1995). One of the major defects responsible for the impaired inotropic response is reduced -adrenergic responsiveness of the sarcolemmal L-type Ca²⁺ channels in hypertrophied myocytes. The maximal increase in I_Ca,L amplitude stimulated by -adrenergic agonist (±)-isoproterenol is significantly diminished in hypertrophied left ventricular myocytes in rats with aortic stenosis (Scamps et al., 1990). Blunted response of ventricular I_Ca,L amplitude to (±)-isoproterenol stimulation also was also found in spontaneous hypertensive rats (Habuchi et al., 1995; Nakata et al., 1995) and in the infarcted hearts (Zhang et al., 1995; Aggarwal and Boyden, 1996). Atrial and ventricular myocytes from failing human hearts also show less I_Ca,L amplitude increase in response to (±)-isoproterenol stimulation than those from nonfailing human hearts (Quadid et al., 1995; Cheng et al., 1996).

The desensitization of the -adrenergic signaling system in hypertrophied heart is usually due to elevated sympathetic activity. It is known that elevated level of angiotensin II in renovascular hypertension contributes to the stimulation of peripheral sympathetic nervous system. Chronic use of captopril, an angiotensin-converting enzyme (ACE) inhibitor that inhibits the formation of angiotensin II, has been found to cause regression of left ventricular hypertrophy (LVH) and to correct certain electrophysiologic abnormalities associated with LVH (Rials et al., 1997). Chronic treatment with captopril started 2 to 3 days before myocardial infarction also has been shown to preserve normal inotropic responsiveness to -adrenergic stimulation in infarcted rat hearts (van Wijngaarden et al., 1992).

Because I_Ca,L is an important mediator of the positive inotropic response to -adrenergic stimulation and its modulation by -adrenergic stimulation has been shown to be impaired in hypertrophied myocytes, it is important to find out whether chronic treatment with ACE inhibitors can normalize functional responsiveness of L-type Ca²⁺ channel of ventricular myocytes to -adrenergic stimulation. The goal of this study was to investigate the role of chronic captopril treatment of renovascular hypertension in regulating the -adrenergic responsiveness of L-type Ca²⁺ channels in ventricular myocytes from rabbits.

	Materials and Methods

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Experimental Animals. Unilateral renovascular band and contralateral nephrectomy were performed in 2-month-old New Zealand White rabbits (1.8-2.2 kg). The surgical procedures have been described in Rials et al. (1997). Three months after surgery when LVH had developed, banded rabbits were either used for experiments or randomized to the untreated group or to the regress group. For the regress group, captopril was given orally (5 mg/kg/day) for 3 months and the last dose was given ~24 h before sacrifice. Five groups of rabbits were used in the study: rabbits 3 months after renal artery banding (LVH 3-month group) and age-matched controls (control 3-month group); rabbits 6 months after renal artery banding without drug treatment (LVH 6-month group) or with a 3-month captopril treatment beginning at 3 months after banding (regress group) and age-matched controls (control 6-month group).

Membrane Current Recording. Single myocytes were enzymatically isolated from left ventricular free wall of rabbits as described previously (Rials et al., 1997). Cells were randomly selected for recording as long as they looked healthy. Membrane currents were recorded at 23 ± 0.5°C with the whole-cell patch-clamp technique. AXOPATCH-1C (Axon Instruments Inc., Burlingame, CA) was interfaced with a personal computer through a TL-1 DMA interface (Axon Instruments Inc.). pClamp software was used for data acquisition and analysis. Electrodes had a resistance of 0.5 to 1.0 M when filled with the pipette solution. Series resistances (<3 M) were compensated electronically to the maximal extent before oscillation occurred. Currents were filtered at 1 kHz and sampled at 2.5 kHz.

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Statistical Analysis. Data are presented as means ± S.E. Statistical comparisons were performed with Student's t test or ANOVA. A P value of <.05 was considered significant.

	Results

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LVH and Regression. Heart weight (HW), body weight (BW), heart weight-to-body weight ratio (HW/BW), and cell membrane capacitance (C_m) of five groups of rabbits are summarized in Table 1. HW/BW and C_m increased significantly in LVH 3-month group and LVH 6-month group compared with the corresponding control group. These parameters were normalized in the regress group, which confirmed our previous findings (Rials et al., 1997).

Reduced Responses of I_Ca,L to -Adrenergic Stimulation in Hypertrophied Myocytes. I_Ca,L of hypertrophied myocytes showed smaller-amplitude increases in response to (±)-isoproterenol stimulation than that of control myocytes. Figure 1 illustrates the average I_Ca,L density/voltage relation before and after bath application of 1 µM (±)-isoproterenol for control 3-month group (left) and LVH 3-month group (right). In the absence of the drug, hypertrophied cells and control cells had similar I_Ca,L density over a broad range of membrane potentials. In the presence of 1 µM (±)-isoproterenol, I_Ca,L density was smaller in hypertrophied cells than in control cells. We calculated percentages of increase of I_Ca,L amplitude in response to 1 µM (±)-isoproterenol at membrane potentials of 10, 0, and 10 mV (Fig. 2). (±)-Isoproterenol induced significantly smaller percentages of increase of I_Ca,L in the myocytes isolated from LVH 3-month group than those from control 3-month group.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Comparison of ICa,L density/voltage relation between control (left; n = 13) and hypertrophied myocytes (right; n = 16) in the absence (open symbols) and presence of 1 µM (±)-isoproterenol (filled symbols). *, indicates that the difference between pre- and postdrug is statistically significant.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   (±)-Isoproterenol induced smaller percentages of increase of ICa,L in hypertrophied myocytes. A, example ICa,L traces induced by test pulses to 10, 0, and 10 mV from a holding potential of 40 mV before (solid line) and after bath application of 1 µM (±)-isoproterenol (dashed line) in a cell from a control 3-month rabbit (top) and from a LVH 3-month rabbit (bottom). B, average percentages of increase of ICa,L amplitude induced by 1 µM (±)-isoproterenol at membrane potentials of 10, 0, and 10 mV in myocytes isolated from control 3-month group (; n = 13) and from LVH 3-month group (; n = 18). *, indicates that the difference between LVH 3-month group and control 3-month group is statistically significant.

Concentration-Dependent Activation of I_Ca,L by (±)-Isoproterenol. Concentration-response curves of I_Ca,L to (±)-isoproterenol of control and LVH rabbits are compared in Fig. 3. Although the maximal response of I_Ca,L to (±)-isoproterenol was significantly depressed in hypertrophied myocytes, the half-maximal activation concentration (IC₅₀) was about the same between control and hypertrophied myocytes (13.2 versus 12.9 nM).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent activation of ICa,L by (±)-isoproterenol was similar between control and hypertrophied myocytes. Average percentage of increase of ICa,L was plotted against (±)-isoproterenol concentrations for control () and hypertrophied () myocytes. The cell number for each data point is indicated in parentheses. Smooth curves were obtained by fitting the data with the Hill equation. Half-maximal activation concentration was 13.2 nM for control and 12.9 nM for hypertrophied myocytes, and the Hill coefficient was 0.92 and 0.91, respectively.

Stimulating Effects of Forskolin on I_Ca,L. To determine whether the depressed -adrenergic response of I_Ca,L in the hypertrophied myocytes is due to defects at receptor level or at a more distal point in the signal transduction pathway, we compared the responses of I_Ca,L to forskolin (a direct activator of adenylate cyclase) in hypertrophied and control myocytes. Forskolin at 10 µM has been shown to induce maximal I_Ca,L amplitude increases in cardiac myocytes (Osaka and Joyner, 1992; Zhang et al., 1995). The response of I_Ca,L to 10 µM forskolin at 10, 0, and 10 mV was not statistically different between the LVH 6-month and the control 6-month group (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Forskolin induced similar increase of ICa,L in control and hypertrophied myocytes. Average percentages of increase of ICa,L at 10, 0, and 10 mV in response to 10 µM forskolin was not significantly different between control (n = 12) and LVH rabbits (n = 11).

Effects of Chronic Captopril Treatment on -Adrenergic Modulation of I_Ca,L. (±)-Isoproterenol (1 µM) induced much smaller percentages of increase of I_Ca,L amplitude at 10, 0, and 10 mV in the myocytes isolated from LVH 6-month group than in those from age-matched controls (Fig. 5). However, the maximal response of I_Ca,L to 1 µM (±)-isoproterenol was fully restored in the regress group.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Treatment of renovascular hypertensive rabbits with captopril-restored normal response of ICa,L to (±)-isoproterenol. Percentage of increase of ICa,L induced by 1 µM (±)-isoproterenol was significantly smaller in LVH 6-month rabbits (n = 14) than in age-matched controls (n = 16). Treatment of hypertensive rabbits with captopril restored normal response of ICa,L to (±)-isoproterenol (n = 17). *, indicates that the difference between LVH 6-month group and control 6-month group is significant.

	Discussion

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This study demonstrated that in rabbits 3 months after renal artery banding, the response of I_Ca,L to -adrenergic stimulation was seriously depressed as reported for many ventricular hypertrophy models (Scamps et al., 1990; Habuchi et al., 1995; Nakata et al., 1995; Ouadid et al., 1995; Cheng et al., 1996). Although the maximal percentages of increase of I_Ca,L induced by (±)-isoproterenol were significantly reduced in hypertrophied myocytes, the concentration-response curve was not shifted. The half-maximal activation concentration of (±)-isoproterenol was 13.2 nM in control myocytes versus 12.9 nM in hypertrophied myocytes. This result was consistent with the findings in rats with aortic stenosis-induced LVH (Scamps et al., 1990) and in hypertrophied atrial myocytes from failing human hearts (Ouadid et al., 1995). However, reduced half-maximal activation concentration of (±)-isoproterenol in ventricular myocytes has been found in spontaneously hypertensive rats (Habuchi et al., 1995). The reason for different modulation of concentration-response curve of (±)-isoproterenol by LVH in different animal models is unknown. Direct activation of adenylate cyclase by forskolin produced normal maximal response of I_Ca,L in hypertrophied myocytes from rabbits with induced renovascular hypertension compared with control myocytes, suggesting that defects at receptor level are responsible for the impaired -adrenergic responses of I_Ca,L. This result is consistent with the findings in LVH rats with aortic stenosis (Scamps et al., 1990), in infarct left ventricular myocardium (Zhang et al., 1995), and in atrial myocytes from failing hearts (Cheng et al., 1996). In many hypertrophy- and heart-failure models, -adrenergic receptors have been found down-regulated (Bouanani et al., 1991; Gopalakrishnan et al., 1991; Gengo et al., 1992; Hammond et al., 1992; Harding et al., 1994) with a few exceptions (Atkins et al., 1995; Gende et al., 1985). Increased sympathetic nerve activity and increased catecholamine levels found in renovascular hypertension seem a likely cause of cardiac -adrenergic receptor down-regulation.

It is widely appreciated that chronic ACE inhibition improves survival in patients with heart failure of various etiologies. It is possible that restoration of normal inotropic responsiveness to -adrenergic stimulation in ventricular myocytes contributes to the beneficial effects of chronic ACE inhibition. We have previously demonstrated the beneficial effects of chronic captopril treatment of renovascular hypertension in a rabbit model (Rials et al., 1997). Rabbits at 3 months after renal artery banding had higher mean arterial blood pressure, increased HW/BW, reduced ventricular fibrillation threshold, and prolonged action potential duration compared with control rabbits. These parameters remained abnormal in untreated rabbits compared with age-matched controls, but were normalized by treatment of rabbits with captopril for 3 months beginning 3 months after renal artery banding (Rials et al., 1997). Action potential duration and transient outward potassium current abnormalities also could be prevented by captopril treatment of spontaneously hypertensive rats (Yokoshiki et al., 1997).

ACE inhibitors have been found to resensitize the depressed -adrenergic signaling system in hypertrophied and failing hearts (Bohm et al., 1998). The reduced density of -adrenergic receptors and the depressed -adrenergic-stimulated adenylate cyclase activity in spontaneously hypertensive rats were partially normalized by chronic treatment of ACE inhibitor fosinopril or captopril (Bohm et al., 1995a,b; Castellano et al., 1995). Captopril treatment of cardiac hypertrophy induced by long-term catecholamine exposure results in up-regulation of cardiac -adrenergic receptor (Maisel et al., 1989). However, there is no information available about the functional restoration of I_Ca,L response to -adrenergic stimulation by ACE inhibitor treatment. In renovascular hypertensive rabbits, we found the responsiveness of I_Ca,L to -adrenergic stimulation remained depressed if the rabbits were left untreated but fully recovered in the rabbits treated with captopril. Our results demonstrated, for the first time, that impaired modulation of I_Ca,L by -adrenergic stimulation in hypertrophied ventricular myocytes can be corrected by chronic treatment with captopril. The restoration of normal response of I_Ca,L to -adrenergic stimulation is likely to be the underlying mechanism for the improved inotropic responsiveness in hypertrophied and failing hearts treated with ACE inhibitors.