Introduction

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-Adrenergic receptor (AR) stimulation modulating cardiac L-type Ca²⁺ current (I_Ca,L) plays an important role in the positive inotropic response to -AR stimulation, and it has been shown to be impaired in heart failure (HF) myocytes (Richard et al., 1998). Besides ₁-AR stimulation of L-type Ca²⁺ channels, which is mediated by a cAMP/PKA-signaling mechanism, the ₂-AR-mediated increase in cardiac I_Ca,L has been documented in normal subjects (Xiao and Lakatta, 1993; Cerbai et al., 1995; Xiao et al., 1995; Skeberdis et al., 1997). It is proposed that cardiac ₂-AR couples with both G_s and G_i protein (Xiao et al., 1999). The ₂-AR-coupled G_i activation functionally localizes the G_s-mediated adenylyl cyclase-cAMP/PKA signaling (Zhou et al., 1997), thereby negatively regulating the G_s-mediated I_Ca,L and contractile response in the heart (Xiao et al., 1995). Chronic HF is associated with a marked increase in G_i protein and a selective down-regulation of ₁-AR (higher ₂/₁). The exaggerated ₂-AR/G_i signaling may contribute to the HF-associated dysfunction of -AR stimulation (Xiao et al., 1999). Previously, Bristow et al. (1989) reported a diminished ₂-AR-mediated cardiac response by documenting a decreased adenylate cyclase stimulation. However, we and other investigators have observed an increased positive inotropic responsiveness to ₂-AR stimulation in dogs with pacing-induced HF (Altschuld et al., 1995; Cheng et al., 1998) and in left ventricular (LV) myocytes from rats with infarction-induced HF (Ukai et al., 1999). This effect may be due to a ₂-AR stimulation-induced alteration in the regulation of the Ca²⁺ channel. However, the role and mechanism of ₂-AR activation on I_Ca,L in HF have not been previously assessed.

Accordingly, the purpose of this study was to compare the effect of ₂-AR stimulation on cardiac I_Ca,L in LV myocytes of normal rats and rats with HF and to determine the underlying cellular mechanism. Our results indicate that the ₂-AR activation-induced augmentation of I_Ca,L was enhanced in the HF myocytes. These effects are likely to be mediated through a cAMP-dependent mechanism and coupled with both G_s and PTX-sensitive G_i protein.

	Experimental Procedures

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Experimental Heart Failure Model. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, revised 1985).

Isolation of LV Myocytes. Myocytes were enzymatically disassociated by Langendorff perfusion as we previously described (Suzuki et al., 1998). Care was taken to excise the infarct scar in the heart of each rat with MI before the final enzymatic digestion step. With this technique, the yield of viable myocytes was greater than 80% from both control rats and rats with MI. The cells were used within 10 h.

Electrophysiological Measurement. Membrane calcium current was recorded at 22 to 23°C with the whole-cell patch-clamp technique (Hamill et al., 1981). Axopatch 200A amplifier (Axon Instruments, Foster City, CA) was interfaced with a 12-bit A/D-D/A converter (Digidata 1200; Axon Instruments). PClamp software (PClamp 6.02; Axon Instruments) was used for data acquisition and analysis. Data were filtered by means of a 5-kHz low-pass filter and digitized at 5 kHz.

Solutions. The composition of the pipette solution and that of the recording bath solution were chosen to allow isolation of ion flow through Ca²⁺ channels by blocking other ionic currents. Initially, the myocytes were superfused with a modified Tyrode's solution containing 137 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO₄, 15 mM glucose, 10 mM HEPES, and 1.5 mM CaCl₂. The pH was adjusted to 7.4, with NaOH at 20 to 22°C. After formation of a G seal, the perfusion buffer was changed to a patch recording bath solution, i.e., Na⁺- K⁺-free Tyrode's solution in which tetraethylammonium chloride was substituted for NaCl, 50 µM tetrodotoxin was added to eliminate sodium current, and KCl was replaced by CsCl and 3 mM 4-aminopyridine to abort the potassium current. The solution was gassed with 100% O₂. The internal solution for the pipette contained 140 mM cesium aspartate, 1.0 mM MgCl₂, 3 mM Na₂ATP, 0.4 mM GTP, 10 mM EGTA, and 5 mM HEPES. The pH was adjusted to 7.2 (with titrated CsOH).

Materials. Zinterol {MJ-9184-1; N-(5(2-[1,1 dimethyl-2-phenyl-ethyl] amino)-1-hydroxyethyl)-2-hydroxyphenyl]-monohydrochloride} was provided by Bristol-Myers Squibb Co. (Princeton, NJ); ICI-118,551 and CGP-20712A (CGP) was obtained from RBI/Sigma, Natick, MA; and Rp-cAMPS was from Biology Life Science Institute, La Jolla, CA. Other agents were obtained from Sigma, St. Louis, MO.

Statistical Analysis. Data are presented as mean ± S.E.M. Statistical comparisons were performed with Student's t test or analysis of variance. A p value of <0.05 was considered significant. Prism 3.0 (GraphPad software; GraphPad, San Diego, CA) was used for the concentration-I_Ca,L relationship nonlinear regression analysis. As previously described by Robberecht et al. (1983) and Lands et al. (1967), the Hill equation may allow us modeling cooperatively between multiple receptor sites on each cardiomyocyte with respect to ₂-AR agonist (ZIN) binding. Thus, data were fitted with the Hill equation. The best fit by the Hill equation was also compared with a fit by one-site competition equation.

	Results

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Verification of Experimental HF

The general hemodynamic and I_Ca,L features in the rats with MI are presented in Table 1. LV end-diastolic pressure was increased 5-fold; left ventricular systolic pressure, LV dp/dt_max, and LV dp/dt_min were significantly decreased. The rate of LV relaxation was slowed, as indicated by a significant increase in the time constant of isovolumic LV pressure decay (, 196%, p < 0.05).

All of the animals with MI had clear evidence of HF (anorexia, edema, and pulmonary congestion). There were no significant changes in body weight (593 ± 9 versus 598 ± 7 g, p = N.S.), whereas the heart weight (2.21 ± 0.03 versus 1.62 ± 0.02 g, p < 0.05), the calculated ratio of LV-to-body weight (2.50 ± 0.04 versus 1.85 ± 0.03 g/kg, p < 0.05), and the wet lung-to-body weight ratio (4.83 ± 0.10 versus 2.87 ± 0.07 g/kg, p < 0.05) were all significantly increased in the rats with MI. In the myocytes of rats with HF, the membrane capacitance was significantly increased (66%), indicating an existence of hypertrophy of the myocytes. After normalized by the membrane capacitance, the current density was significantly lower than that of the normal myocytes (62%, p < 0.01), indicating an absolute reduction of I_Ca,L.

In addition, the response of I_Ca,L to -adrenergic receptor stimulation in HF myocytes was significantly attenuated. The exposure to isoproterenol (ISO, 10⁷ M) increased the I_Ca,L by 120 ± 14% (p < 0.05, n = 10) in the normal myocytes. However, in the myocytes of rats with HF, the increase in I_Ca,L with ISO was only half (56 ± 8%, p < 0.05, n = 9). The current was blocked by nifedipine (5 × 10⁶ M), a Ca²⁺ channel blocker, consistent with the characteristics of I_Ca,L. We previously showed that in rats with MI, the contractility of the myocytes was also markedly impaired (Ukai et al., 1999). These findings demonstrated the existence of an established HF in this model.

Increased Responses of I_Ca,L to ₂-AR Stimulation in HF Myocytes

Figure 1 shows typical I_Ca,L responses to ₂-AR stimulation in normal (Fig. 1A) and HF myocytes (Fig. 1B). Superfusion of ZIN, a highly selective ₂-AR agonist (10⁵ M), caused significant increases in peak I_Ca,L in normal myocytes (21%, 2.13 ± 0.19 versus 1.75 ± 0.15 nA, p < 0.05, n = 21). In HF myocytes, ZIN caused a greater increase in I_Ca,L (30%, 2.18 ± 0.14 versus 1.66 ± 0.10 nA, p < 0.01, n = 24). The ZIN-induced absolute increases in I_Ca,L between normal (0.38 ± 0.05 nA) and HF cells (0.52 ± 0.06 nA) are statistically significant (p < 0.05). After normalized by the membrane capacitance, the ZIN-induced increases in I_Ca,L remained statistically different in both normal (9.21 ± 0.24 versus 7.59 ± 0.20 pA/pF) and HF cells (6.20 ± 0.24 versus 4.75 ± 0.17 pA/pF). This current was blocked by nifedipine (5 × 10⁶ M). Figure 1, C and D, shows the current-voltage relations for the response of I_Ca,L to ZIN in normal myocytes and in those of rats with HF. The stimulatory effects of ZIN on I_Ca,L were not accompanied by a significant change in the voltage dependence of peak I_Ca,L amplitude in normal and HF myocytes.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effects of ZIN (105 M) on ICa,L. A, a ventricular myocyte from a normal rat was initially superfused with external solution. During the period indicated by the horizontal line, the cell was exposed to ZIN and then to nifedipine (NIF, 5 × 106 M). The superimposed current tracings were recorded before (a), and 10 min after (b) exposure to ZIN, and exposed to NIF (c). ICa,L were elicited by depolarization pulses from a holding potential of 80 to 0 mV for 200 ms with a brief prepulse to 40 mV (60 ms) (inset) at 0.1 Hz. B, same experiment as in A in an HF myocyte. C, current-voltage relations of ICa,L in the absence (open symbols) and presence (filled symbols) of ZIN in eight normal myocytes. *, indicates that the difference between pre- and postdrug is statistically significant (p < 0.05). The cells were depolarized from a holding potential of 40 mV to test potentials from 35 to +60 mV in 5-mV increments. D, current-voltage plots for seven HF cells, measured under the same experimental conditions as in C. E, average increments of ICa,L induced by ZIN (105 M). Values are mean ± S.E.M. for n = 21 to 24 experiments. *, indicates that the difference between normal and HF myocytes is statistically significant (p < 0.05). The results showed an enhanced effect of ZIN on ICa,L in HF myocytes.

Concentration-Dependent Activation of I_Ca,L by ZIN

Concentration-response curves of I_Ca,L to ZIN of myocytes from normal rats and rats with HF are compared in Fig. 2. Smooth curves were obtained by fitting the data with the Hill equation. The dose-response curve was shifted upward, and the half-maximal activation concentration (EC₅₀) of the HF myocytes was lower than that of the normal myocytes (168 versus 688 nM). The Hill coefficient of ZIN (0.83 in normal and 0.88 in HF cells) was similar as reported by Robberecht et al. (1983). Similarly, with one-site competition equation, the half-maximal activation concentration was 733 nM for normal and 174 nM for HF myocytes. There were no significant differences between the best fits achieved by both Hill and one-site competition equation.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent activation of ICa,L by ZIN. Average percentage of increase of ICa,L was plotted against ZIN concentrations for normal (open symbols) and HF (filled symbols) 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 688 nM for normal and 168 nM for HF myocytes, and the Hill coefficient was 0.83 and 0.88, respectively.

Effects of ₁- and ₂-AR Blockade on ZIN-Induced Increase in I_Ca,L

The affinity of ZIN for ₂-AR is approximately 50 times over ₁-AR (disassociation constant is 20 versus 1000 nM) (Minneman et al., 1979). ZIN at 10⁵ M has been shown to induce maximal response in cell contraction [Ca²⁺]_i transient and I_Ca,L in rat cardiomyocyte (Xiao and Lakatta, 1993; Xiao et al., 1995). This high concentration of ZIN may also stimulate ₁-AR or ₁-AR or work through nonadrenergic receptor system (Edwards and Whitaker-Azmitia, 1987; Parfitt and Bickford-Wimer, 1990). To determine the potential mechanism, we preincubated the myocytes with CGP (a ₁-AR antagonist, 3 × 10⁷ M) or ICI-118551 (ICI, a ₂-AR antagonist, 10⁷ M) for 20 min, and ZIN was given in the presence of CGP or ICI. As shown in Fig. 3, after using CGP to block the ₁-AR, the ZIN-induced increase in I_Ca,L persisted in both normal (7.51 ± 0.40 versus 6.20 ± 0.30 pA/pF, p < 0.05, n = 4) and HF cells (5.16 ± 0.30 versus 4.03 ± 0.20 pA/pF, p < 0.05, n = 4). In contrast, as displayed in Fig. 4, after preincubation with the ₂-AR blocker, ICI, the ZIN-induced increase in I_Ca,L was abolished in both normal (7.22 ± 0.30 versus 7.07 ± 0.30 pA/pF, p = N.S., n = 4) and HF cells (4.52 ± 0.35 versus 4.51 ± 0.38 pA/pF, p = N.S., n = 4), indicating that ZIN increased I_Ca,L through ₂-AR, not ₁-AR, ₁-AR, or other nonadrenergic system.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of ZIN on ICa,L is not prevented by 1-AR blockade in normal and HF myocytes. The myocytes were preincubated with CGP (a 1-AR antagonist, 3 × 107 M) for 20 min. A, superimposed current tracings recorded before and 5 min after exposure to ZIN (105 M) in the presence of CGP in a normal myocyte. ICa,L was elicited by depolarization pulses from a holding potential of 80 to 0 mV for 200 ms with a brief prepulse to 40 mV (60 ms). B, same experiment as in A in an HF myocyte. The results showed that the effect of ZIN persisted in the presence of the 1-AR blocker (CGP 20712A). C, average effects of 1-AR blockade on ZIN-induced ICa,L responses. Values are mean ± S.E.M. for n = 4 experiments. *p < 0.05 between control and ZIN.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of ZIN on ICa,L is prevented by 2-AR blockade in normal and HF myocytes. The myocytes were preincubated with ICI (a 2-AR antagonist, 107 M) for 20 min. A, superimposed current tracings recorded before and 5 min after exposure to ZIN (105 M) in the presence of ICI in a normal myocyte. ICa,L were elicited by depolarization pulses from a holding potential of 80 to 0 mV for 200 ms with a brief prepulse to 40 mV (60 ms). B, same experiment as in A in an HF myocyte. C, average effects of 2-AR blockade on ZIN-induced ICa,L responses. Values are mean ± S.E.M. for n = 4 experiments.

Effect of G_i Protein Blockade-Induced Increase in I_Ca,L

Distinct from ₁-AR in signal transduction, ₂-AR couples with both G_s and G_i proteins (Xiao et al., 1999). To define the role of G_i protein in ₂-AR stimulation of I_Ca,L, we pretreated the myocytes with PTX (2 µg/ml, 36°C, 6 h). The adequacy of the complete blockage of inhibitory G_i protein in PTX-treated cells was routinely verified by the loss of the ability of acetylcholine (ACh, 10⁵ M) to reverse the stimulatory effect of ISO on I_Ca,L.

Effect of ACh on Response to ISO. ACh is generally thought to activate PTX-sensitive G_i protein and counteract the G_s-activating effect by -AR stimulation. PTX-treated myocytes were compared with myocytes that had been kept at 36°C in the absence of PTX for an equal time. In the normal cells, ISO (10⁷ M) increased I_Ca,L by 120 ± 14%, and further application of ACh (10⁵ M) decreased the ISO-induced augmentation in I_Ca,L to 84 ± 10% (p < 0.05, n = 4). With PTX pretreatment, in the normal cells, the reduction of ISO response on I_Ca,L caused by ACh was prevented (an increase of 116 ± 12%, p = N.S., n = 4). This is consistent with the observations of Lauer et al. (1992). In HF cells, the increase of I_Ca,L by ISO was markedly attenuated (56 ± 8%), and the addition of ACh almost prevented the ISO-induced increase in I_Ca,L (10 ± 8%, p = N.S., n = 4). Thus, compared with normal cells, the inhibition effect of ACh on ISO response in HF cells was significantly augmented (82 versus 30%, p < 0.05, n = 4). In the PTX-treated HF cells, the reduction of ISO response caused by ACh was totally abolished (increases of 93 ± 14 versus 95 ± 10%, p = N.S., n = 4). This observation is in line with the phenomenon of PTX-restoring ISO response of contraction in cardiomyocytes from patients with HF reported by Brown and Harding (1992).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of ZIN on ICa,L is augmented by PTX in normal and HF myocytes. The myocytes were preincubated with PTX (2 µg/ml, 36°C, 6 h). A, superimposed current tracings recorded before and 5 min after exposure to ZIN (105 M) in a normal myocyte. ICa,L were elicited by depolarization pulses from a holding potential of 80 to 0 mV for 200 ms with a brief prepulse to 40 mV (60 ms). B, same experiment as in A in an HF myocyte. C, average effects of PTX on ZIN induced ICa,L responses. Values are mean ± S.E.M. for n = 9 to 10 experiments. *p < 0.05 between control and ZIN. The results showed that the effects of ZIN were enhanced by PTX pretreatment, suggesting a PTX-sensitive G protein mechanism involved in the ZIN-induced ICa,L response of ventricular myocytes.

Effects of ZIN on Response to a ₁-AR Agonist. In a subgroup of animals, we examined the effect of ZIN on I_Ca,L response after a ₁-AR stimulation with norepinephrine (NE, 10⁷ M). We found that in normal myocytes, NE caused a significant increase in I_Ca,L (78%, 14.05 ± 1.50 versus 8.17 ± 1.00 pA/pF, p < 0.05, n = 4). The addition of ZIN (10⁵ M) caused an attenuation (43%) in NE-induced I_Ca,L increase (10.53 ± 1.30 versus 8.17 ± 1.00 pA/pF, p < 0.05, n = 4). In contrast, in HF myocytes, compared with normal myocytes, NE caused a much smaller increase in I_Ca,L (17%, 5.39 ± 1.20 versus 4.62 ± 0.90 pA/pF, p < 0.05). The application of ZIN caused an additive increase in I_Ca,L (approximately 8%, from 5.39 ± 1.20 to 5.75 ± 1.30 pA/pF, p < 0.05, n = 4). Thus, it appears that in normal cardiomyocytes, ZIN had an inhibitory effect when applied after the effect of ₁-AR stimulation had developed; but in HF cells, ZIN exhibited a stimulatory action on I_Ca,L, suggesting an altered interaction between ₁- and ₂-AR stimulation in HF cells.

Effect of G_s-cAMP Pathway Blockade on ZIN-Induced Increase in I_Ca,L

Since accumulating evidence indicates that the effect of -AR stimulation on cardiac I_Ca,L is mediated by a cAMP-dependent mechanism (Zhou et al., 1997), we further examined the role of cAMP-dependent PKA activation in ₂-AR stimulation of I_Ca,L by including an inhibitory cAMP analog Rp-cAMPS (10⁴ M) in pipette solution to block the stimulatory G_s protein-cAMP pathway. As shown in Fig. 6, with intracellular application of Rp-cAMPS, basal I_Ca,L was decreased about 20%, which is consistent with previous observations (Zhou et al., 1997). However, under this condition, ZIN-induced I_Ca,L increase was prevented in both normal (6.06 ± 0.32 versus 6.17 ± 0.25 pA/pF, p = N.S., n = 6) and HF (3.78 ± 0.06 versus 3.88 ± 0.10 pA/pF, p = N.S., n = 4) myocytes.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of Rp-cAMPS on ZIN induced ICa,L responses of normal and HF myocytes. A, superimposed current tracings recorded before and 5 min after exposure to ZIN (105 M) in the presence of Rp-cAMPS (104 M) in pipette solution in a normal myocyte. ICa,L were elicited by depolarization pulses from a holding potential of 80 to 0 mV for 200 ms with a brief prepulse to 40 mV (60 ms). B, same experiment as in A in an HF myocyte. C, average effects of Rp-cAMPS on ZIN induced ICa,L responses. Values are mean ± S.E.M. for n = 4 to 6 experiments. The results showed that the effects of ZIN were abolished in the presence of Rp-cAMPS in pipette solution.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The present study demonstrated, for the first time, that ₂-AR stimulation of I_Ca,L by ZIN was enhanced in LV myocytes of rats with infarction-induced heart failure. These effects were mediated through a cAMP-dependent mechanism and coupled with both stimulatory G_s protein and PTX-sensitive inhibitory G_i protein.

Effects and Possible Mechanism of ZIN on I_Ca,L. It has been increasingly recognized that ₁- and ₂-AR coexist in the heart. In large mammal hearts, ₂-AR may account for approximately 40% of the total -AR number, while in small animals, cardiac ₂-ARs are less abundant. The reported ratio of ₁-/₂-AR was 92-80/8-20 in adult rat hearts (Cerbai et al., 1995; Kuznetsov et al., 1995). -AR stimulation modulating cardiac I_Ca,L plays an important role in the positive inotropic response to -AR stimulation.

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