QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.089672v1 315/3/1203    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, Z.-S. Articles by Cheng, C.-P. Search for Related Content PubMed PubMed Citation Articles by Zhang, Z.-S. Articles by Cheng, C.-P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Heart Failure

CARDIOVASCULAR

Enhanced Inhibition of L-type Ca²⁺ Current by ₃-Adrenergic Stimulation in Failing Rat Heart

Wake Forest University School of Medicine, Winston-Salem, North Carolina

Received May 16, 2005; accepted August 30, 2005.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
₃-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). ₃-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca²⁺ channels in normal hearts; however, the effects of ₃-AR activation on Ca²⁺ channel in HF remain unknown. We compared the effects of ₃-AR activation on L-type Ca²⁺ current (I_Ca,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). I_Ca,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a ₃-AR agonist, caused a dose-dependent decrease in I_Ca,L with maximal inhibition (21%, 1.1 ± 0.2 versus 1.4 ± 0.1 nA) (p < 0.01) at 10^–7 M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in I_Ca,L (1.1 ± 0.2 versus 1.6 ± 0.2 nA) (p < 0.05). A similar inhibition of I_Ca,L was also observed with ISO (10^–7 M) in the presence of a ₁- and ₂-AR antagonist, nadolol (10^–5 M). Inhibition was abolished by the ₃-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10^–6 M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N^G-nitro-L-arginine methyl ester (10^–4 M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 µg/ml, 36°C, 6 h). In conclusion, ₃-AR activation inhibits L-type Ca²⁺ channel in both normal and HF myocytes. In HF, ₃-AR stimulation-induced inhibition of Ca²⁺ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.

Recently, several studies have reported that ₃-ARs are up-regulated in the failing human heart (Moniotte et al., 2001), in the canine models of HF (Cheng et al., 2001), as well as in diabetic rat hearts (Dincer et al., 2001). HF is associated with selective down-regulation of ₁-AR and a marked increase in G_i protein (Cheng et al., 2001; Moniotte et al., 2001). The exaggerated ₃-AR/G_i signaling may cause alteration in the regulation of Ca²⁺ channel, thus contributing to contractile dysfunction. However, the role and mechanism of ₃-AR activation on I_Ca,L in HF have not been defined.

Accordingly, the purpose of this study was to compare the effects of ₃-AR stimulation on cardiac I_Ca,L in LV myocytes of normal rats and rats infused with ISO as a model of HF and to determine the underlying cellular mechanism. Our results indicate that ₃-AR activation inhibits L-type Ca²⁺ channel in both normal and HF myocytes. In HF, ₃-AR stimulation-induced inhibition of I_Ca,L was enhanced. These effects are likely coupled with a pertussis toxin (PTX)-sensitive G_i protein and partially mediated through a NOS-dependent pathway. The current findings extend our knowledge regarding the impaired -adrenergic regulation of L-type Ca²⁺ channel in HF and provide valuable new insight into the cellular mechanism of the progression of functional impairment in HF.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Experimental Heart Failure Model. This investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH Publication No 85-23, revised 1985).

As previously described (Teerlink et al., 1994; Suzuki et al., 1998; Kong et al., 2004), HF in the rat model was induced by ISO injections with some modification (4 months after 340 mg/kg s.c., for 2 days). Briefly, male Sprague-Dawley rats (200–250 g) received two subcutaneous injections of 340 mg/kg ISO-HCl at 24-h intervals. The mortality rate was approximately 40 to 48% within 48 h. The control group of rats received the same amount of sterile saline. Animals were housed and fed under identical conditions.

Four months after the injection protocol, survivors (n = 16) and sham-injected rats (n = 20) were lightly anesthetized with intraperitoneal Ketamine HCl (50 mg/kg) and Xylazine (10 mg/kg). Then, hemodynamic measurements were obtained using a micro-tip pressure transducer (Millar Instruments, Houston, TX), inserted into the LV through the carotid artery to verify the presence of HF in the ISO-treated rats. Heart and lung weight was obtained after the study. Consistent with previous reports (Rona et al., 1959; Teerlink et al., 1994), the hearts of ISO-injected rats displayed large infarct-like necrosis involving more than one-third to half of the LV extending to the adjacent area of the interventricular septum and right ventricle. Diffuse subendocardial necrosis was also observed. To obtain a high yield of viable isolated myocytes, as previously described, with some modification (Rona et al., 1959; Pfeffer et al., 1979), the gross lesions of infarct-like necrosis area were measured. Briefly, after Langendorff perfusion with an enzymatic buffer, the heart was weighed, and LV was separated. Then the infarct-like necrosis area was carefully excised from the LV and weighed separately. Calculation of the ratio of the weights between the necrosis areas to LV was used as an approximate estimation of infarct size.

Isolation of LV Myocytes. Myocytes were enzymatically dissociated by Langendorff perfusion as previously described (Suzuki et al., 1998). With our well established technique, more than an 80% yield of viable myocytes was obtained from both control and ISO-treated rats. 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 as previously described (Hamill et al., 1981; Zhang et al., 2001). An 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 a 5-kHz low-pass filter and digitized at 5 kHz.

After stabilization, a drop of cell pellet containing the isolated myocytes was placed in a perfusion chamber (0.5-ml volume) mounted on the stage of an inverted microscope (IMT 2-F3; Olympus, Herndon, VA) and continuously superfused at a constant rate of 2 ml/min. Only quiescent rod-shaped cells with clear cross-striations were studied. Borosilicate glass micropipette (O.D. 1.6 mm) was pulled with a two-step puller (Model PP-83; Narishige, Tokyo, Japan) and heat-polished with a microfuge (MT-83; Narishige). The tip resistances were 1.5 to 2.5 M when filled with pipette (internal) solution.

Liquid junction potentials (<5 mV) were corrected before the pipette touched the cell. After formation of G-seal, the electrode capacitance was compensated electronically. Then the cell membrane was ruptured by gentle suction to establish whole-cell configuration.

In a subgroup, a perforated-patch recording technique was used (Horn and Marty, 1988) in which nystatin stock was added to the internal solution (final nystatin concentration of 100–200 µg/ml). The development of electrical access was monitored by the appearance of a capacitive current. The access resistance was <20 M.

The membrane capacitance and series resistance were compensated to minimize the duration of the capacitive transient. The membrane capacitance was measured before compensation with a 10-mV depolarizing step from a holding potential of –80 mV to –70 mV and integrated the area under the current transient calculated with the following formula: C_m = _cI₀/V_m[1 – (I/I₀)], where C_m is the membrane capacitance, _c is the decay time constant of membrane capacitance, I₀ is the maximum capacitance current value, V_m is the amplitude of the voltage step, and I is the amplitude of steady-state current. The membrane capacitance was used as an index to normalize I_Ca,L for cell size. The series resistance was calculated as R_s = V_m/I₀, where R_s is the series resistance, and compensated at about 80%.

Myocytes were voltage clamped at –80 mV. Ca²⁺ currents were elicited by stepping up the membrane voltage from a holding potential to 0 mV testing potential for 200 ms at 12-s intervals. To avoid contamination by fast sodium channel activation and to reduce the run-down of I_Ca,L, a brief prepulse (60-ms duration) was applied to –40 mV before approaching the test potential. The average peak I_Ca,L run-down was about 10 to 20% for 30 min after initial measurement. Most (80%) of the run-down occurred within the initial 8 to 10 min. Thus, the window of time between 10 and 30 min after the initial recording was chosen to measure I_Ca,L with respect to drug effects (Xiao and Lakatta, 1993). I_Ca,L was measured by the standard method as the difference between peak inward current and the current at the end of a 200-ms pulse. For current-voltage relations, test potentials were from –35 to +60 mV at 5-mV increments and 0.1 Hz.

Solutions. The compositions of the pipette solution and recording bath solution were chosen to allow isolation of ion flow through the Ca²⁺ channel 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 20–22°C. After formation of giga- seal, superfusion buffer was changed to a patch-recording bath solution, i.e., Na⁺-K⁺-free Tyrode's solution, in which NaCl was substituted by tetraethylammonium chloride, and 50 µM tetrodotoxin was added to eliminate sodium current and KCl 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).

For perforated patch recording, nystatin stock solution (10 mg/ml in acidified methanol) was prepared on each day and added to the internal solution at a final concentration of 100 to 200 µg/ml (Horn and Marty, 1988). The pipette was dipped in nystatin-free internal solution for 2 s and then back-filled with nystatin internal solution.

Drugs. BRL 37,344 was obtained from Tocris (Ballwin, MO). L-748,337 was a gift from Merck Research Laboratories (Rahway, NJ). Nadolol, isoproterenol, ICI-118,551 (ICI), L-NAME, nifedipine, and PTX were obtained from Sigma-Aldrich (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) 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 to model cooperativity between multiple receptor sites on each cardiomyocyte with respect to ₃-AR agonist (BRL) binding. Thus, data were fitted with the Hill equation. The best fit by the Hill equation was also compared with a fit by the one-site competition equation.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Verification of Experimental HF. The general hemodynamic and I_Ca,L features in the ISO-treated rats are presented in Table 1. LV end-diastolic pressure increased 5-fold, and LV dP/dt_max and LV dP/dt_min were significantly decreased. The rate of LV relaxation slowed as indicated by a significant increase in the time constant of isovolumic LV pressure decay (, 185%) (p < 0.05).

All ISO-treated animals had clear evidence of HF (anorexia, edema, and pulmonary congestion). In the ISO-injected rats, the total infarction area was about 43 ± 3%. There was no significant change in body weight (596 ± 8 versus 601 ± 9 g) (p = N.S.), whereas the heart weight (2.25 ± 0.03 versus 1.68 ± 0.03 g) (p < 0.05), calculated ratio of LV to body weight (2.53 ± 0.04 versus 1.86 ± 0.04 g/kg) (p < 0.05), and calculated ratio of wet lung to body weight (4.85 ± 0.09 versus 2.89 ± 0.08 g/kg) (p < 0.05) were all significantly increased in ISO-injected rats. In the HF rat myocytes, the membrane capacitance was significantly increased, whereas 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 -AR stimulation in HF myocytes was significantly attenuated. As shown in Fig. 1, in the normal myocytes, in response to the exposure to ISO (10^–7 M), I_Ca,L was doubled (119 ± 9%) (p < 0.01, n = 11). However, in HF myocytes, ISO only caused half the increase in I_Ca,L (55 ± 4%) (p < 0.05, n = 11). The current was blocked by nifedipine (5 x 10^–6 M), a Ca²⁺ channel blocker, consistent with the characteristics of I_Ca,L. These findings demonstrated the existence of established HF in this model.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Group means (± S.E.M.) of isoproterenol (ISO)-induced changes of peak ICa,L in myocyte of normal and HF rats. Compared with normal myocytes, an ISO (10–7 M)-induced increase in ICa,L was markedly attenuated in HF myocytes (55 ± 4 versus 119 ± 9%, respectively) (p < 0.01). In contrast, in the presence of a 1- and 2-antagonist, nadolol (Nad, 10–5 M), ISO caused an enhanced inhibition of ICa,L in HF myocytes (29 ± 2 versus 20 ± 2%, respectively) (p < 0.01).

Increased Inhibition of I_Ca,L to ₃-AR Stimulation in HF Myocytes. The effects of direct ₃-AR stimulation with BRL, the most potent ₃-AR agonist in rats (Gauthier et al., 1999), on I_Ca,L in normal and HF myocytes are summarized in Table 2 and displayed in Fig. 2. Superfusion of BRL (10^–7 M) caused significant decreases in peak I_Ca,L in normal myocytes (21%, 1.1 ± 0.1 versus 1.4 ± 0.1 nA, p < 0.01, n = 7). In HF myocytes, BRL caused a greater relative and absolute decrease in I_Ca,L (31%, 1.1 ± 0.2 versus 1.6 ± 0.2 nA, p < 0.05, n = 7). The absolute decrease in I_Ca,L was greater in HF myocytes (0.3 ± 0.03 nA) versus (0.5 ± 0.06 nA) (p < 0.05). In the HF myocytes, the membrane capacitance was significantly increased. After normalization of the membrane capacitance, the BRL-induced decreases in I_Ca,L remained statistically different in both normal myocytes (21%, 5.9 ± 0.4 versus 7.5 ± 0.5 pA/pF) (p < 0.01, n = 7) and HF myocytes (31%, 2.9 ± 0.4 versus 4.1 ± 0.3 pA/pF) (p < 0.05, n = 7). Figure 2, C and D demonstrates current-voltage relations for the response of I_Ca,L to BRL in normal and HF myocytes. BRL caused no change in the voltage dependence of peak I_Ca,L amplitude in normal and HF myocytes.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Effects of BRL-37,344 (BRL, 10–7 M) on ICa,L. A, a ventricular myocyte from normal rat was initially superfused with external solution. During the period indicated by the horizontal line, the cell was exposed to BRL, then to nifedipine (NIF, 5 x 10–6 M). The superimposed current tracings were recorded before (a) and after (b) exposure to BRL and exposure to NIF (c). ICa,L was elicited by depolarization pulses from a holding potential of –80 mV to 0 mV for 200 ms with a brief prepulse to –40 mV (60 ms) (inset) at 12-s intervals. B, same experiment as in A in a HF myocyte. C, current-voltage relations of ICa,L in the absence (open symbols) and presence (filled symbols) of BRL in six normal myocytes. An asterisk 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 five HF cells, measured under the same experimental conditions as in C. E, average inhibition of ICa,L caused by BRL (10–7 M). Values are mean ± S.E.M. for n = 7 experiments. An asterisk indicates that the difference between normal and HF myocytes is statistically significant (p < 0.05). The results showed an enhanced effect of BRL on ICa,L in HF myocytes.

The inhibitory effects of BRL persisted after washout. To eliminate the influence of "run-down", in a subgroup, we used nystatin perforated patch recording to examine the effect of BRL. A similar result was observed (data not shown). This is consistent with the findings of Au and Kwan (2002).

In a subgroup, we further examined the I_Ca,L response to ISO (10^–7 M) in the presence of a ₁- and ₂-AR antagonist, nadolol (Nad, 10^–5 M). In contrast to ISO alone, we observed an inhibition of I_Ca,L in both normal and HF myocytes. Compared with normal myocytes, this inhibition was enhanced in HF myocytes (29 ± 2 versus 20 ± 2%) (p < 0.05, n = 5).

Concentration-Dependent Inhibition of I_Ca,L by BRL. Concentration-response curves of I_Ca,L to BRL in normal and HF rat myocytes are compared in Fig. 3. The maximal response of I_Ca,L to BRL was significantly enhanced in HF myocytes. The dose-response curve was shifted downward. The half-maximal inhibition concentration (IC₅₀) was 1.1 nM for HF myocytes and 1.2 nM for normal myocytes.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Concentration-dependent inhibition of ICa,L with BRL. Average percentage of decrease of ICa,L was plotted against BRL concentrations for normal (open symbols) and HF (filled symbols) myocytes. The cell numbers for each data point are indicated in parentheses. Smooth curves were obtained by fitting the data with the Hill equation. Half-maximal inhibition concentration was 1.2 nM for normal and 1.1 nM for HF myocytes.

As shown in Fig. 4, after using Nad to block ₁- and ₂-AR, BRL-induced decreases in I_Ca,L still persisted [(5.5 ± 0.5 versus 7.0 ± 0.6 pA/pF) (p < 0.05, n = 3) and (3.3 ± 0.4 versus 4.5 ± 0.4 pA/pF) (p < 0.05, n = 3)] in normal and HF myocytes, respectively. In a subgroup of normal rats, after preincubation with the ₂-AR antagonist ICI, the BRL-induced reductions in I_Ca,L remained unaffected (6.5 ± 0.5 versus 8.1 ± 0.6 pA/pF) (p < 0.05, n = 4). In contrast, as shown in Fig. 5, after preincubation with a ₃-AR blocker, L-748,337, BRL-induced inhibition in I_Ca,L was abolished [(7.2 ± 0.5 versus 7.4 ± 0.4 pA/pF) (n = 4) and (4.6 ± 0.6 versus 4.7 ± 0.6 pA/pF) (n = 4, p = N.S.)] in normal and HF cells, respectively, indicating that BRL inhibited I_Ca,L through ₃-AR, not ₁- and ₂-AR.

View larger version (21K): [in this window] [in a new window]   Fig. 4. The effect of BRL on ICa,L is not prevented with 1- and 2-AR blockade in normal and HF myocytes. The myocytes were preincubated with Nad, a 1- and 2-AR antagonist (10–5 M), for 20 min. A, superimposed current tracings recorded before and at 5 min after exposure to BRL (10–7 M) in the presence of Nad 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 a HF myocyte. The results showed the effects of BRL persisted in the presence of 1-and 2-AR blocker (Nad). C, average effects of 1- and 2-AR blockade on BRL-induced ICa,L responses. Values are mean ± S.E.M. for n = 3 experiments. An asterisk indicates p < 0.05 between control and BRL.

View larger version (21K): [in this window] [in a new window]   Fig. 5. The effect of BRL on ICa,L is prevented by 3-AR blockade in normal and HF myocytes. The myocytes were preincubated with L-748,337 (3-AR antagonist, 10–6 M) for 20 min. A, superimposed current tracings recorded before and 5 min after exposure to BRL (10–7 M) in the presence of L-748,337 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 a HF myocyte. C, average effects of 3-AR blockade on BRL-induced ICa,L responses. Values are mean ± S.E.M. for n = 4 experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 6. The effect of BRL on ICa,L is prevented by PTX in normal and HF myocytes. The myocytes were preincubated with PTX (2 µg/ml at 36°C for 6 h). A, superimposed current tracings recorded before and 5 min after exposure to BRL (10–7 M) 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 a HF myocyte. C, average effects of PTX on BRL-induced ICa,L responses. Values are mean ± S.E.M. for n = 4 experiments. The results showed the effects of BRL were prevented by PTX pretreatment, suggesting a PTX-sensitive G protein mechanism involved in BRL-induced ICa,L response of ventricular myocytes.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Effect of L-NAME on BRL-induced ICa,L responses of normal and HF myocytes. A, superimposed current tracings recorded before and 5 min after exposure to BRL (10–7 M) in the presence of L-NAME (10–4 M) 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 a HF myocyte. C, average effects of L-NAME on BRL-induced ICa,L responses. Values are mean ± S.E.M. for n = 4 to 5 experiments. The results showed the effects of BRL were attenuated in the presence of L-NAME. *, p < 0.05 between L-NAME control and L-NAME + BRL.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study demonstrates that ₃-AR stimulation with BRL causes an enhanced inhibition of I_Ca,L in LV myocytes of rats with HF. These effects were coupled with PTX-sensitive inhibitory G_i protein and partly mediated through a NOS-dependent mechanism.

Effects and Possible Mechanism of BRL on I_Ca,L. -AR activation modulating cardiac I_Ca,L plays an important role in the positive inotropic response to -AR stimulation. ₁- and ₂-AR stimulation of L-type Ca²⁺ channel are mediated by a cAMP/protein kinase A-signaling mechanism and coupled with G_s proteins (Skeberdis et al., 1997; Xiao et al., 1999; Zhang et al., 2001). ₂-ARs also have the ability to activate nonclassical signaling pathways and link to inhibitory G proteins (G_i), suggesting a function distinct from the ₁-AR subtype (Zhang et al., 2001; Xiao et al., 2003). Recent observations indicate a more complex -AR-mediated regulation of myocardial inotropism by catecholamines. In addition to ₁- and ₂-AR, a third -AR, ₃-AR, initially found to be widely expressed in fat tissues (Krief et al., 1993), was also found to exist in mammalian hearts and to modulate cardiac contractile function (Gauthier et al., 1996; Kitamura et al., 2000; Cheng et al., 2001; Dincer et al., 2001). Stimulation of ₃-AR negatively modulates cardiac function through the G_i protein pathway and is coupled with the NOS system (Gauthier et al., 1996, 1998; Seppet, 2003). The negative inotropic effect of ₃-AR stimulation was associated with alterations of myocardial electrophysiology and Ca²⁺ signaling (Gauthier et al., 1996; Kitamura et al., 2000; Cheng et al., 2001).

In the present study, using freshly isolated cardiomyocytes, we found that stimulation of ₃-AR with BRL resulted in a dose-dependent inhibition in I_Ca,L with maximum inhibition (21%) at a concentration of 10^–7 M in normal cells. This effect was completely abolished by a highly-selective ₃-AR antagonist but not by the ₁- and ₂-AR blockade or ₂-AR blockade, indicating that the alteration of I_Ca,L following BRL superfusion was due to ₃-AR stimulation but not mediated by ₁-AR or ₂-AR activation.

Furthermore, as in previous studies (Gauthier et al., 1998; Kitamura et al., 2000; Varghese et al., 2000), we found that in the presence of L-NAME, the BRL-induced negative response of I_Ca,L was significantly reduced, indicating involvement of the NO pathway in ₃-AR-mediated action. However, our current observation indicates that the NO pathway may not be fully responsible for the altered LV myocyte I_Ca,L response to BRL since we found that the I_Ca,L response to BRL was only partially inhibited by pretreatment myocytes with a NOS inhibitor. In the presence of L-NAME, myocyte I_Ca,L remained significantly reduced, indicating ₃-AR activation is not mediated exclusively through the NO pathway.

Our study indicates that ₃-AR-induced inhibition of I_Ca,L is mediated by G_i, since pretreatment of PTX completely prevented BRL-induced I_Ca,L responses in both normal and HF myocytes. However, this is not consistent with the past report of Gauthier et al. (1996). In their study, it was shown that in human ventricular strips treated with PTX, the effect of BRL on contractility was attenuated but not completely suppressed. This discrepancy may be due to species difference or incomplete blockade of G_i by PTX since a lower level of exposure to PTX (0.5 µg/ml for 2 h) was used in that study. It has been reported that PTX treatment for 3 to 5 h at 5 µg/ml is required to completely inactivate human cardiac G_i (Brown and Harding, 1992). Therefore, our current study indicated that the enhanced I_Ca,L response to ₃-AR stimulation in rat CHF myocytes may be coupled to G_i through both NO-dependent and NO-independent mechanisms.

These results are consistent with previous observations of cardiac functional response to ₃-AR stimulation made in normal hearts and cardiomyocytes of several species, including that of the human, dog, and guinea pig (Gauthier et al., 1996; Kitamura et al., 2000; Cheng et al., 2001). Gauthier and colleagues first demonstrated that in the human heart, stimulation of ₃-AR with a BRL resulted in dose-dependent, negative inotropic effects, which were associated with decreased action potential amplitude and reduced action potential duration (Gauthier et al., 1996). However, in their study, ₃-AR transcripts were not detected in the rat ventricular strips by using a reverse transcription-polymerase chain reaction assay. The failure to detect functional cardiac ₃-AR expression may be attributed to the use of nonspecific primers for rat ₃-AR mRNA (Gauthier et al., 1999). Recently, by using reverse transcription-polymerase chain reaction, polyacrylamide gel electrophoresis, and Western blot analysis, Dincer et al. (2001) clearly demonstrated the presence of functional ₃-AR in rat hearts and showed significantly increased ₃-AR mRNA and protein levels in streptozotocin-induced diabetic rat hearts. Barbier and colleagues also detected transcripts and cell surface expression of ₃-AR in rat hearts. They further found that LV ₃-AR density significantly increased in female rats with treadmill training for 8 weeks (Barbier et al., 2004).

A novel finding in the present study is that the inhibition of I_Ca,L with ₃-AR was enhanced after HF. In HF myocytes, the basal I_Ca,L was significantly decreased, and its response to -AR stimulation (ISO 10^–7 M) was also markedly blunted (55 versus 119% increment) (p < 0.05). However, the inhibitory response to BRL (10^–7 M) was significantly enhanced (31 versus 21%).

This is consistent with our past observation made in a pacing-induced canine HF model (Cheng et al., 2001). In that study, we assessed the direct effects of BRL on cardiomyocytes isolated from the dogs before and after pacing-induced advanced HF. In these studies, we removed the effects of extracardiac factors. We clearly demonstrated that compared with normal myocytes, BRL caused a much greater decrease in I_Ca,L in HF myocytes. We further found that ₃-AR stimulation produces direct inhibition of myocyte contraction, relaxation, and [Ca²⁺]_i transient_. In pacing-induced HF, these inhibitions were increased. These responses were coupled to G_i protein. However, our observations do not agree with the findings by Moniotte et al. (2001), who reported a similar up-regulation of ₃-AR expression but blunted negative inotropic response to BRL in failing human myocardium. This discrepancy might result from several factors, such as species difference in ₃-AR (Gauthier et al., 1999), variation in severity of HF, or the effect of pharmacotherapy on the failing human heart.

There is a documented different specificity for ₃-AR across species and even tissues. It is possible that the intracellular coupling of ₃-AR may be different in the failing rat and human heart. It is also possible that the severity of HF may contribute to the different finding. Our rat model of HF was associated with a 40 to 48% mortality, which is much higher than would be expected in a population of human HF patients. The model of HF is also different. We examined a single LV myocyte obtained from ISO-induced failing rat hearts, whereas Moniotte et al. (2001) studied ventricular strips obtained from dilated or ischemia-caused failing human hearts. The use of ventricular strips of multiple cell preparation versus single LV myocyte preparation may induce quantitatively different results due to the additional variable of extracellular matrix and remodeling, although qualitative differences are less likely. We feel that the most likely explanation for the discrepancy in our finding is that the human group received multiple cardiovascular specific medications for HF. A standard regimen for HF patients consists of significant neurohormonal blockade with -AR blockers, angiotensin-converting enzyme inhibitors, and aldosterone antagonists. It would not be surprising if this regimen alters ₃-AR expression or function. Chronic treatment with cardiovascular specific medications (such as -AR blockers and angiotensin-converting enzyme inhibitors) may alter the primary defect in the contractile properties of the myocyte itself as well as the abnormalities of extracardiac factors in HF, thus, further modifying LV and cardiomyocyte functional response to ₃-AR stimulation in HF (Spinale et al., 1995, 1998; Gunja-Smith et al., 1996; Bristow, 2000; Cohn et al., 2000; Lohse et al., 2003). It is possible that the intracellular coupling of ₃-AR may be also altered by these cardiovascular specific medications in the failing human hearts. Although we studied a rat model of HF (ISO-induced cardiomyopathy) that reproduces many of the functional and neurohormonal features of clinical HF, we cannot be certain that our results apply generally to HF of other causes. Nevertheless, the findings of Moniotte et al. (2001) and our past reports all demonstrated a similar, potentially detrimental, functional consequence with ₃-AR activation in HF.

The mechanism(s) for the BRL-induced enhanced decrease in I_Ca,L for HF cells versus normal cells are unclear. We speculate that an increase in ₃-AR density on the membrane of HF cells may contribute to our current findings.

Since ₃-ARs are activated at higher catecholamine concentrations than ₁- and ₂-ARs (Lafontan, 1994), and ₃-ARs are relatively resistant to chronic, agonist-induced desensitization processes (Liggett et al., 1993), they could be involved in HF. Although the ₃-AR density per myocyte has not been measured in HF previously, it is possible that the enhanced inhibition of I_Ca,L in LV myocytes of rats with HF may reflect the presence of an increase in the number of ₃-ARs per cell. Further studies are currently underway to elucidate this point.

The enhanced response to ₃-AR stimulation in HF may also be related to an altered signal transduction. Although, the intracellular pathway coupling ₃-AR stimulation is incompletely characterized, it has been reported that ₃-AR stimulation decreases cardiac contractility through activation of the NOS pathway (Kitamura et al., 2000; Varghese et al., 2000). In HF, the NO-cGMP signaling may be altered (Mohan et al., 1996), thereby altering HF myocyte response to ₃-AR stimulation. Consistent with previous studies, we found that in the presence of L-NAME, BRL-induced inhibition with I_Ca,L was largely attenuated, indicating an involvement of the NO pathway in ₃-AR-mediated action. In addition, we also found that the enhanced I_Ca,L response to ₃-AR stimulation in HF myocytes couples to G_i. Thus, an up-regulation of G_i in HF may also contribute to the enhancement of the inhibitory effect of ₃-AR. The activation of G_i also has the potential to couple ₃-AR to other important signaling pathways such as the mitogen-activated protein kinase (Soeder et al., 1999). Clearly, up-regulation of cardiac ₃-AR-mediated inhibitory pathways is responsible for the enhanced BRL-induced inhibition of I_Ca,L in HF. However, the exact contribution of up-regulation of cardiac ₃-AR versus increased levels of G_i is unclear. Further studies are needed to fully characterize the intracellular pathway coupling ₃-AR stimulation.

In conclusion, we found that ₃-AR activation inhibits L-type Ca²⁺ channel and decreases I_Ca,L in both normal and HF myocytes. In rats with ISO-induced HF, ₃-AR activation-induced inhibition of I_Ca,L was enhanced. These effects are likely to be partially mediated through a NOS-dependent mechanism and coupled with a PTX-sensitive inhibitory G protein. This finding is possibly an underlying mechanism for the impaired inotropic response to -AR stimulation in the failing heart.

	Acknowledgements

 
We acknowledge the expertise and assistance of Drs. J. Mu and R. L. Wu with regard to the patch-clamp technique, the computer programming of Dr. Ping Tan, the technical assistance of Ellen Tommasi and Michael Cross, and the editorial review and administrative support of Amanda Burnette. We thank Merck Research Laboratories (Rahway, NJ) for providing L-748,337.

	Footnotes

 
This study was supported in part by grants from the National Institutes of Health (HL45258 and HL53541) and the American Heart Association (9640189N).

doi:10.1124/jpet.105.089672.

ABBREVIATIONS: AR, adrenergic receptor; NOS, nitric-oxide synthase; BRL, 4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino]propyl]phenoxyacetate (BRL-37,344); I_Ca,L, L-type Ca²⁺ current; L-NAME, N^G-nitro-L-arginine methyl ester; HF, heart failure; LV, left ventricular; ISO, isoproterenol; PTX, pertussis toxin; L-748,337, (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide; ICI, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI-118,551); Nad, nadolol; NO, nitric oxide.

¹ Current affiliation: First Department of Internal Medicine, Mie University School of Medicine, Tsu City, Japan.

² Current affiliation: Department of Internal Medicine and Pathophysiology, Nagoya City University Graduate School of Medical Sciences, Mizuho-cho, Mizuho-ku, Japan.

Address correspondence to. Dr. Che-Ping Cheng, Cardiology Section, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1045. E-mail: ccheng{at}wfubmc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Au ALS and Kwan YW (2002) Modulation of L-type Ca²⁺ channels by beta₃-adrenoceptor activation and the involvement of nitric oxide. J Cardiovasc Surg 17: 465–469.

Barbier J, Rannou-Bekono F, Marchais J, Berthon PM, Delamarche P, and Carre F (2004) Effect of training on beta₁, beta₂, beta₃ adrenergic and M₂ muscarinic receptors in rat heart. Med Sci Sports Exerc 36: 949–954.[CrossRef][Medline]

Bristow MR (2000) [beta]-Adrenergic receptor blockade in chronic heart failure. Circulation 101: 558–569.[Free Full Text]

Brown LA and Harding SE (1992) The effect of pertussis toxin on beta-adrenoceptor responses in isolated cardiac myocytes from noradrenaline-treated guinea pigs and patients with cardiac failure. Br J Pharmacol 106: 115–122.[Medline]

Cheng HJ, Zhang ZS, Onishi K, Ukai T, Sane DC, and Cheng CP (2001) Upregulation of functional beta₃-adrenergic receptor in the failing canine myocardium. Circ Res 89: 599–606.[Abstract/Free Full Text]

Cohn JN, Ferrari R, and Sharpe N (2000) Cardiac remodeling: concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. J Am Coll Cardiol 35: 569–582.[Abstract/Free Full Text]

Dincer UD, Bidasee KR, Guner S, Tay A, Ozcelikay T, and Altan VM (2001) The effect of diabetes on expression of beta₁-, beta₂- and beta₃-adrenoreceptors in rat hearts. Diabetes 50: 455–461.[Abstract/Free Full Text]

Gauthier C, Leblais V, Kobzik L, Trochu JN, Khandoudi N, Bril A, Balligand JL, and Marec HL (1998) The negative inotropic effect of beta₃-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle. J Clin Investig 102: 1377–1384.[Medline]

Gauthier C, Tavernier G, Charpentier F, Langin D, and Le Marec H (1996) Functional beta₃-adrenoceptor in the human heart. J Clin Investig 98: 556–562.[Medline]

Gauthier C, Tavernier G, Trochu JN, Leblais V, Laurent K, Langin D, Escande D, and Le Marec H (1999) Interspecies differences in the cardiac negative inotropic effects of beta₃ adrenoceptor agonists. J Pharmacol Exp Ther 290: 687–693.[Abstract/Free Full Text]

Gunja-Smith Z, Morales AR, Romanelli R, and Woessner JF (1996) Remodeling of human myocardial collagen in idiopathic dilated cardiomyopathy: role of metalloproteinases and pyridinoline cross-links. Am J Pathol 148: 1639–1648.[Abstract]

Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflueg Arch Eur J Physiol 391: 85–100.[CrossRef][Medline]

Horn R and Marty A (1988) Muscarinic activation of ion currents measured by a new whole-cell recording method. J Gen Physiol 92: 145–159.[Abstract/Free Full Text]

Kitamura T, Onishi K, Dohi K, Okinaka T, Isaka N, and Nakano T (2000) The negative inotropic effect of beta₃-adrenoceptor stimulation in the beating guinea pig heart. J Cardiovasc Pharmacol 35: 786–790.[CrossRef][Medline]

Kong YH, Li WM, and Tian Y (2004) Effect of beta₃-adrenoreceptors agonist on beta₃-adrenoreceptors expression and myocyte apoptosis in a rat model of heart failure. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 16: 142–147.[Medline]

Krief S, Lonnqvist F, Raimbault S, Baude B, Van Spronsen A, Arner P, Strosberg AD, Ricquier D, and Emorine LJ (1993) Tissue distribution of beta₃-adrenergic receptor mRNA in man. J Clin Investig 91: 344–349.

Lafontan M (1994) Differential recruitment and differential regulation by physiological amines of fat cell beta₁-, beta₂- and beta₃-adrenergic receptors expressed in native fat cells and in transfected cell lines. Cell Signal 6: 363–392.[CrossRef][Medline]

Lands AM, Luduena FP, and Buzzo HJ (1967) Differentiation of receptors responsive to isoproterenol. Life Sci 6: 2241–2249.[CrossRef][Medline]

Liggett SB, Freedman NJ, Schwinn DA, and Lefkowitz RJ (1993) Structural basis for receptor subtype-specific regulation revealed by a chimeric beta₃-/beta₂-adrenergic receptor. Proc Natl Acad Sci USA 90: 3665–3669.[Abstract/Free Full Text]

Lohse MJ, Engelhardt S, and Eschenhagen T (2003) What is the role of beta-adrenergic signaling in heart failure? Circ Res 93: 896–906.[Abstract/Free Full Text]

Mohan P, Brutsaert DL, Paulus WJ, and Sys SU (1996) Myocardial contractile response to nitric oxide and cGMP. Circulation 93: 1223–1229.[Abstract/Free Full Text]

Moniotte S, Kobzik L, Feron O, Trochu JN, Gauthier C, and Balligand JL (2001) Upregulation of beta₃-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation 103: 1649–1655.[Abstract/Free Full Text]

Pfeffer MA, Pfeffer JM, Fishbein MC, Fletcher PJ, Spadaro J, Kloner RA, and Braunwald E (1979) Myocardial infarct size and ventricular function in rats. Circ Res 44: 503–512.[Abstract/Free Full Text]

Robberecht P, Delhaye M, Taton G, De Neef P, Waelbroeck M, De Smet JM, Leclerc JL, Chatelain P, and Christophe J (1983) The human heart beta-adrenergic receptors. Heterogeneity of the binding sites: presence of 50% beta₁- and 50% beta₂-adrenergic receptors. Mol Pharmacol 24: 169–173.[Abstract]

Rona G, Chappel CI, Balazs T, and Gaudry R (1959) An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. AMA Arch Pathol 67: 443–455.[Medline]

Seppet EK (2003) Negative inotropy starts with the beta₃-adrenoceptor. Cardiovasc Res 59: 262–265.[Free Full Text]

Skeberdis VA, Jurevicius J, and Fischmeister R (1997) Beta₂ adrenergic activation of L-type Ca²⁺ current in cardiac myocytes. J Pharmacol Exp Ther 283: 452–461.[Abstract/Free Full Text]

Soeder KJ, Snedden SK, Cao W, Della Rocca GJ, Daniel KW, Luttrell LM, and Collins S (1999) The beta₃-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism. J Biol Chem 274: 12017–12022.[Abstract/Free Full Text]

Spinale FG, Coker ML, Thomas CV, Walker JD, Mukherjee R, and Hebbar L (1998) Time-dependent changes in matrix metalloproteinase activity and expression during the progression of congestive heart failure. Circ Res 82: 482–495.[Abstract/Free Full Text]

Spinale FG, Holzgrefe HH, Mukherjee R, Hird RB, Walker JD, Arnim-Barker A, Powell JR, and Koster WH (1995) Angiotensin-converting enzyme inhibition and the progression of congestive cardiomyopathy: effects on left ventricular and myocyte structure and function. Circulation 92: 562–578.[Abstract/Free Full Text]

Suzuki M, Ohte N, Wang ZM, Williams DL Jr, Little WC, and Cheng CP (1998) Altered inotropic response of endothelin-1 in cardiomyocytes from rats with isoproterenol-induced cardiomyopathy. Cardiovasc Res 39: 589–599.[Abstract/Free Full Text]

Teerlink JR, Pfeffer JM, and Pfeffer MA (1994) Progressive ventricular remodeling in response to diffuse isoproterenol-induced myocardial necrosis in rats. Circ Res 75: 105–113.[Abstract/Free Full Text]

Varghese P, Harrison RW, Lofthouse RA, Georgakopoulos D, Berkowitz DE, and Hare JM (2000) Beta₃-adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility. J Clin Investig 106: 697–703.[Medline]

Xiao RP, Cheng H, Zhou YY, Kuschel M, and Lakatta EG (1999) Recent advances in cardiac beta₂-adrenergic signal transduction. Circ Res 85: 1092–1100.[Abstract/Free Full Text]

Xiao RP and Lakatta EG (1993) Beta₁-adrenoceptor stimulation and beta₂-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca²⁺ and Ca²⁺ current in single rat ventricular cells. Circ Res 73: 286–300.[Abstract/Free Full Text]

Xiao RP, Zhang SJ, Chakir K, Avdonin P, Zhu W, Bond RA, Balke W, Lakatta EG, and Cheng H (2003) Enhanced G_i signaling selectively negates beta₂-adrenergic receptor (AR)-but not beta₁-AR-mediated positive inotropic effect in myocytes from failing rat hearts. Circulation 108: 1633–1639.[Abstract/Free Full Text]

Zhang ZS, Cheng HJ, Ukai T, Tachibana H, and Cheng CP (2001) Enhanced cardiac L-type calcium current response to beta₂-adrenergic stimulation in heart failure. J Pharmacol Exp Ther 298: 188–196.[Abstract/Free Full Text]

This article has been cited by other articles:

				U. Siedlecka, M. Arora, T. Kolettis, G. K. R. Soppa, J. Lee, M. A. Stagg, S. E. Harding, M. H. Yacoub, and C. M. N. Terracciano Effects of clenbuterol on contractility and Ca2+ homeostasis of isolated rat ventricular myocytes Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H1917 - H1926. [Abstract] [Full Text] [PDF]

				J. R. Somers, P. L. Beck, J. P. Lees-Miller, D. Roach, Y. Li, J. Guo, S. Loken, S. Zhan, L. Semeniuk, and H. J. Duff iNOS in cardiac myocytes plays a critical role in death in a murine model of hypertrophy induced by calcineurin Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1122 - H1131. [Abstract] [Full Text] [PDF]

				H. Wang, M. J. Kohr, D. G. Wheeler, and M. T. Ziolo Endothelial nitric oxide synthase decreases {beta}-adrenergic responsiveness via inhibition of the L-type Ca2+ current Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1473 - H1480. [Abstract] [Full Text] [PDF]

				S. Nattel, A. Maguy, S. Le Bouter, and Y.-H. Yeh Arrhythmogenic Ion-Channel Remodeling in the Heart: Heart Failure, Myocardial Infarction, and Atrial Fibrillation Physiol Rev, April 1, 2007; 87(2): 425 - 456. [Abstract] [Full Text] [PDF]

S. Imbrogno, T. Angelone, C. Adamo, E. Pulera, B. Tota, and M. C. Cerra Beta3-Adrenoceptor in the eel (Anguilla anguill