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Am J Physiol Heart Circ Physiol 279: H139-H148, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 1, H139-H148, July 2000

Changes in ionic currents and beta -adrenergic receptor signaling in hypertrophied myocytes overexpressing Galpha q

Sayaka Mitarai, Thomas D. Reed, and Atsuko Yatani

Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transgenic overexpression of Galpha q causes cardiac hypertrophy and depressed contractile responses to beta -adrenergic receptor agonists. The electrophysiological basis of the altered myocardial function was examined in left ventricular myocytes isolated from transgenic (Galpha q) mice. Action potential duration was significantly prolonged in Galpha q compared with nontransgenic (NTG) myocytes. The densities of inward rectifier K+ currents, transient outward K+ currents (Ito), and Na+/Ca2+ exchange currents were reduced in Galpha q myocytes. Consistent with functional measurements, Na+/Ca2+ exchanger gene expression was reduced in Galpha q hearts. Kinetics or sensitivity of Ito to 4-aminopyridine was unchanged, but 4-aminopyridine prolonged the action potential more in Galpha q myocytes. Isoproterenol increased L-type Ca2+ currents (ICa) in both groups, with a similar EC50, but the maximal response in Galpha q myocytes was ~24% of that in NTG myocytes. In NTG myocytes, the maximal increase of ICa with isoproterenol or forskolin was similar. In Galpha q myocytes, forskolin was more effective and enhanced ICa up to ~55% of that in NTG myocytes. These results indicate that the changes in ionic currents and multiple defects in the beta -adrenergic receptor/Ca2+ channel signaling pathway contribute to altered ventricular function in this model of cardiac hypertrophy.

action potential; potassium currents; sodium-calcium exchanger; calcium currents; heart failure; transgenic model


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MYOCARDIAL HYPERTROPHY is an initial adaptive process to a variety of physiological and pathological conditions associated with increased cardiac work. The hypertrophic response initially normalizes wall stress and maintains ventricular function. However, decompensated congestive heart failure occurs when the adaptive process fails. The molecular mechanisms that trigger cardiac hypertrophy and regulate the progression to heart failure have not been well elucidated (7). Results from recent studies in transgenic mice indicate that stimulation of the signal transduction pathways mediated by heterotrimeric Gq protein is closely related to the induction of cardiac hypertrophy and its progression to heart failure (1, 2, 8, 20, 23).

In ventricular myocytes isolated from failing human hearts, the magnitude of contraction is diminished and relaxation is prolonged. These changes are associated with changes in electrophysiological properties and abnormal intracellular Ca2+ handling (3, 4, 11). Prolongation of the action potential is the most consistently observed electrophysiological abnormality in failing human hearts. Downregulation of the repolarizing K+ currents, the inward rectifier K+ currents (IK1) and the transient outward K+ currents (Ito), has been postulated to underlie the observed action potential prolongation (5). Molecular and biochemical studies in failing human hearts indicate that altered expression of the sarcoplasmic reticulum (SR) Ca2+ regulatory proteins, such as SR Ca2+-ATPase and phospholamban, may contribute to altered intracellular Ca2+ transients (10, 12, 16, 21). Upregulation of the sarcolemmal Na+/Ca2+ exchanger has also been suggested as a compensatory mechanism for decreased SR Ca2+ uptake (9, 22).

Another hallmark of human heart failure is depressed contractile responsiveness to catecholamines. Multiple changes in the beta -adrenergic receptor (beta -AR) signaling pathway, including changes in beta -AR expression (a selective loss of beta 1-ARs) and G protein activity levels [inhibitory G protein (Gi) and/or stimulatory G protein (Gs)], increased beta -AR kinase levels, impaired beta -AR/adenylyl cyclase coupling, and decreased adenylyl cyclase activity, occur in the failing human heart (13). However, most studies in human hearts are limited to a single time point, usually in hearts with severe hypertrophy or terminal heart failure, and the relationships between the degree of ventricular dysfunction and cellular abnormalities are not well characterized.

In this regard, a transgenic mouse model, overexpressing the alpha -subunit of Gq in the heart, which reproduces many biochemical and hemodynamic changes seen in human heart disease, may provide an informative model system to investigate the cellular mechanisms of cardiac hypertrophy and failure (8, 23). For example, at higher levels of Galpha q overexpression (5-fold over endogenous levels), frank cardiac decompensation occurs, with development of biventricular failure, pulmonary congestion, and death between 11 and 14 wk of age (8). Twofold overexpression of Galpha q shows no detectable effects on cardiac gene expression, function, or hypertrophy, suggesting that a threshold level of Galpha q expression is necessary to transduce these effects. By contrast, relatively modest (~4-fold) overexpression of Galpha q (Galpha q-25) results in moderate cardiac hypertrophy and increased hypertrophy-associated gene expression. Echocardiography and in vivo cardiac hemodynamic studies revealed significantly impaired intrinsic contractility and responses to beta -AR agonists. Nevertheless, Galpha q-25 mice exhibit relatively compensated left ventricular function. These mice have a normal life span and do not develop overt heart failure. Thus Galpha q-25 mice provide an intermediate-phase cardiac hypertrophy model, neither fully compensated nor decompensated, that has been designated previously as "compromised" heart (23).

Recently, we reported that left ventricular myocytes isolated from transgenic Galpha q-25 (Galpha q) mice exhibit prolonged contractions and Ca2+ transients associated with reductions in Ca2+ uptake rates and the apparent affinity of SR Ca2+-ATPase for Ca2+ (30). There was no change in L-type Ca2+ current (ICa) density. If impaired Ca2+ uptake by the SR were the only cellular abnormality, then significantly decreased amplitude of contractions and Ca2+ transients should be observed in Galpha q myocytes. However, we found no such differences, suggesting that other Ca2+ regulatory mechanisms may be involved in the abnormal Ca2+ signaling observed in Galpha q myocytes.

The most consistent electrophysiological change in a variety of experimental models as well as in human heart failure is action potential prolongation. Prolongation of the action potential may have a profound effect on cellular excitation-contraction coupling. Because a normal action potential is the result of an orderly sequence of changes in the permeability of the membrane inward and outward ionic currents, the delayed repolarization can occur secondary to an increase in the inward currents, a decrease in the outward currents, and/or more complex changes generated by the sarcolemmal Na+/Ca2+ exchanger. Accordingly, we examined electrophysiological parameters such as action potentials, K+ channel currents, and Na+/Ca2+ exchange currents in left ventricular myocytes isolated from Galpha q and nontransgenic (NTG) control mice. In addition, because the cardiac Ca2+ channel is a well-characterized physiological effector of beta -AR signal transduction, we examined the effects of isoproterenol (Iso) to identify cellular mechanisms related to beta -AR dysfunction.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of transgenic mice. Transgenic (Galpha q) mice with a nearly fourfold increase of the murine alpha -subunit of Gq in the heart (Galpha q-25) were generated as previously described (8). The mice overexpressing moderate levels of Galpha q do not exhibit cardiomyocyte replacement or apoptotic heart failure at the age (12-15 wk) used in the present studies (1).

Preparation of myocytes. Cardiac myocytes from 14 Galpha q mice and 14 control NTG littermates were used for electrophysiolgical measurements. Single left ventricular myocytes were isolated from the hearts of NTG and Galpha q mice by a method described previously (19). Briefly, the heart was perfused with Ca2+-free Tyrode solution containing collagenase (type II, 0.5 mg/ml; Worthington) and BSA (1 mg/ml) for 10-20 min by the Langendorff method at 37°C. At the end of the perfusion period, the heart was removed, myocytes were prepared from the apical two-thirds of the left ventricle, and these myocytes were passed through 200-µm nylon mesh. In some experiments, atrial myocytes were prepared (18) and used to test the ability of pertussis toxin (PTX) to block muscarinic receptor-mediated activation of K+ channel currents. The isolated cells were stored in low-Cl-, high-K+ medium, and all experiments were performed at room temperature (20-22°C).

The amount of myocyte hypertrophy as estimated by the cell membrane capacitance in the present experiments was comparable to that reported in previous studies of Galpha q myocytes (30). Cell capacitance was 129.0 ± 2.7 pF (n = 120) in NTG and 146.1 ± 3.9 pF (n = 131) in Galpha q myocytes (P < 0.01).

Electrophysiology. Whole cell currents were recorded using patch-clamp techniques, as previously described (19). Patch electrodes had 1.5- to 2.5-MOmega tip resistance for whole cell current recordings and 10- to 15-MOmega tip resistance for action potential measurements. Membrane capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of -50 mV. The experimental chamber (0.2 ml) was placed on a microscope stage, and external solution changes were made rapidly using a modified Y-tube technique (29).

For action potential and K+ current recordings, myocytes were perfused with normal Tyrode solution composed of (mM) 135 NaCl, 1.8 CaCl2, 1 MgCl2, 5.4 KCl, 10 glucose, and 10 HEPES (pH 7.3). No Ca2+-chelating buffer was included in the pipette solution to avoid interference with intracellular Ca2+ signaling. The "physiological" pipette filling solution for action potential recordings consisted of (mM) 140 KCl, 2 MgCl2, 10 NaCl, 2 ATP, and 5 HEPES (pH 7.3). In some experiments, 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was included in the pipette solution to minimize Ca2+-induced inactivation.

For the measurement of K+ currents, 1 µM nifedipine was added to block ICa, and the patch pipette solution contained (mM) 110 potassium aspartate, 20 KCl, 2 MgCl2, 2 ATP, 0.5 GTP, 5 EGTA, and 5 HEPES (pH 7.3). In the initial series of experiments, using varying amounts of EGTA (0, 0.1, and 2 mM), we also dialyzed the myocytes with lower concentrations of Ca2+ buffer to minimize interference with Ca2+ signaling. However, under these conditions, activation of cell contraction occurred frequently in the initial periods of cell dialysis, rendering stable recordings difficult. We therefore performed all K+ current measurements in the presence of 5 mM EGTA. The voltage dependence of Ito inactivation was determined by applying 500-ms depolarizing pulses to different potentials (-100 and 0 mV) from a holding potential of -80 mV. The extent of activation was quantified by the peak current measured at +40 mV. The prepulse inactivation curves were normalized to the maximum current fitted with a Boltzmann equation: I/Imax = 1/{1 + exp[(Vm - V0.5)/k]}, where I is current, Imax is maximum current, Vm is membrane potential, V0.5 is midpotential, and k is the slope factor.

Ca2+ currents were recorded using an external solution containing (mM) 2 CaCl2, 1 MgCl2, 135 tetraethylammonium chloride, 5 4-aminopyridine (4-AP), 10 glucose, and 10 HEPES (pH 7.3). To determine responses to beta -AR stimulation, myocytes were dialyzed with the pipette solution containing (mM) 100 cesium aspartate, 20 CsCl, 1 MgCl2, 2 MgATP, 0.5 GTP, 10 BAPTA, and 5 HEPES (pH 7.3). These solutions isolated ICa from other membrane currents such as Na+ and K+ channel currents and also Ca2+ flux through the Na+/Ca2+ exchanger. As we previously showed, beta -AR regulation of the Ca2+ channel can be reliably measured under these experimental conditions (25). The voltage dependence of activation was determined using an interactive nonlinear regression fitting procedure to the Boltzmann equation: Gmax = 1/{1 + exp[(V0.5 - Vm)/k]}, where Gmax is maximum conductance.

For measurements of the Na+/Ca2+ exchanger currents, the external solution contained (mM) 150 NaCl, 2 CsCl, 2 MgCl2, 1 CaCl2, 0.001 nifedipine, 0.02 ouabain, and 5 HEPES (pH 7.3). The pipette solution contained (mM) 20 NaOH, 110 CsOH, 50 aspartic acid, 1 MgCl2, 2 MgATP, 42 EGTA, and 5 HEPES (pH 7.4). The concentration of free internal Ca2+ was adjusted to 67 nM by addition of CaCl2 (15). To activate Na+/Ca2+ exchange currents, the cells were held at -40 mV, and the external solution was rapidly switched to one in which equimolar LiCl was substituted for NaCl (15, 17). Exposure of the myocytes to Na+-free solution produced outward Na+ extrusion through the Na+/Ca2+ exchanger.

RNase protection assay. Total RNA was isolated from the hearts of five Galpha q mice and five NTG littermates by use of the ULTRASPEC-II RNA Isolation System (Biotecx Laboratories, Houston, TX). The MAXIscript In Vitro Transcription Kit (Ambion, Austin, TX) was used to synthesize radiolabeled cRNA probes from linearized DNA riboprobe templates. The mouse Na+/Ca2+ exchanger (NCX-1) riboprobe template was prepared by subcloning an RT-PCR-generated cDNA fragment (spanning nucleotides -31 to +118 bp relative to the AUG translational start codon) into pBluescript SK(+). Gene expression levels were determined from total RNA samples by use of the RNase Protection Assay (RPA) Kit (Ambion). The RPA gel was dried and then exposed to BIOMAX-MR X-ray film (Kodak, New Haven, CT). Overall NCX-1 levels were determined by 1) exposing the RPA gel to a Kodak phosphor screen, 2) scanning signal levels with a PhosphorImager (Molecular Dynamics, Wayzata, MN), and 3) analyzing the data with NIH Image Analysis software.

Values are means ± SE. Comparisons between conditions were evaluated using the Student's t-test, with significance imparted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Action potential. Action potentials (Fig. 1) were recorded in control NTG and Galpha q left ventricular myocytes in Tyrode solution with physiological pipette solution (see MATERIALS AND METHODS). NTG myocytes displayed a brief action potential with a rapid initial phase of repolarization without a discernible plateau phase. The shape and duration of the action potential recorded in NTG myocytes were similar to those observed in adult mouse ventricular myocytes reported previously (27, 31). In contrast, the action potential recorded in Galpha q myocytes showed a relatively small initial notch followed by a clear plateau phase. Action potential duration quantified at 50 and 70% repolarization (APD50 and APD70) was significantly prolonged in Galpha q myocytes (Table 1). There was no significant difference in the resting membrane potentials between the two groups.


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  		Fig. 1.   Typical action potentials recorded in ventricular myocytes isolated from hearts of nontransgenic (NTG, A) and Galpha q (B) mice. Membrane potential was recorded in the current-clamp mode with a patch electrode filled with K+-rich internal solution. The external solution was normal Tyrode solution. Myocytes were stimulated at 0.2 Hz through the patch pipette.


                              
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  		Table 1.   Action potential characteristics recorded from NTG and Galpha q myocytes

Action potential shape and duration are species specific because of differences in the underlying inward and outward currents. In adult mouse ventricular myocytes, the 4-AP-sensitive Ito has been described to play an important role in the early and middle phase of the action potential (27, 31), suggesting that changes in Ito may contribute to the action potential prolongation observed in Galpha q myocytes.

K+ channel currents. Figure 2 illustrates typical outward currents recorded in NTG (A) and Galpha q (B) myocytes. In both groups, depolarization positive to -30 mV activated outward currents, which then decayed slowly to a sustained outward current at the end of a 300-ms voltage step. Details of electrophysiological characteristics of the outward currents in adult mouse ventricular myocytes that exhibit a sum of fast and slow components have been described elsewhere (31). In the present study, we refer to the total K+ current components simply as Ito. Myocytes isolated from Galpha q hearts showed a smaller current amplitude than those from NTG hearts.


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  		Fig. 2.   Transient outward currents (Ito) recorded in NTG (A) and Galpha q (B) myocytes. Representative families of currents elicited by voltage steps from -60 to +60 mV in 20-mV increments from a holding potential of -80 mV are shown. C: current-voltage (I-V) relationships in NTG and Galpha q myocytes. Current amplitude at the peak (open symbols) and the end of 300-ms pulses (filled symbols) were normalized to the cell capacitance to give current densities. Values are means ± SE of 52 NTG and 46 Galpha q cells. D: average voltage dependence of inactivation of Ito. Data were fitted (solid lines) to a Boltzmann function. Values are means ± SE of 10 NTG and 7 Galpha q cells.

When the time course of inactivation was fit to a sum of the two (fast and slow) exponentials, fast (tau f) and slow (tau s) time constants and the relative amplitude [Af/(Af + As)] in Galpha q myocytes were comparable to those in NTG myocytes. The tau f, tau s, and Af/(Af + As) at +60 mV were 13.8 ± 1.1 ms, 99.1 ± 8.2 ms, and 0.48 ± 0.02 for NTG (n = 10) and 15.3 ± 1.5 ms, 102.5 ± 8.8 ms, and 0.46 ± 0.02 for Galpha q (n = 21) myocytes, respectively. The current-voltage (I-V) relationships of peak and sustained current amplitudes, normalized relative to cell capacitance (pA/pF), are plotted in Fig. 2C. The average current densities of peak and sustained components were significantly decreased in Galpha q myocytes. At +60 mV, the peak and sustained currents were 25.4 ± 1.1 and 13.0 ± 0.8 pA/pF (n = 52) for NTG and 17.8 ± 1.4 and 10.0 ± 0.8 pA/pF (n = 46) for Galpha q myocytes. The voltage dependence of Ito inactivation was similar between the two groups (Fig. 2D). The mean values of V0.5 and k were -41.9 ± 0.9 mV and 6.4 ± 1.1 mV for NTG myocytes (n = 10) and -44.9 ± 2.2 mV and 8.0 ± 1.8 mV for Galpha q myocytes (n = 7), respectively.

Peak and sustained currents were blocked by 4-AP (Fig. 3, A and B). Cumulative concentration-response relationships revealed that IC50 was similar between the two groups (Fig. 3C).


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  		Fig. 3.   Effects of 4-aminopyridine (4-AP) on Ito recorded in NTG (A) and Galpha q (B) myocytes. Traces show superimposed currents before and after application of 5 mM 4-AP. Currents were elicited from a holding potential of -80 to +60 mV. C: concentration-dependent effects of 4-AP on Ito in NTG and Galpha q myocytes. The inhibition of the current amplitude relative to the effects of 5 mM 4-AP was plotted. The solid lines were fitted to a 1:1 binding model with IC50 = 163.8 and 221.1 µM for NTG and Galpha q myocytes, respectively. Values are means ± SE of 30 NTG and 33 Galpha q cells.

To measure IK1, hyperpolarizing pulses were applied from a holding potential of -40 mV to test potentials between -50 and -100 mV. Figure 4 shows representative IK1 recorded from NTG (A) and Galpha q (B) myocytes. The current was blocked by external application of 0.5 mM Ba2+ (5). Galpha q myocytes showed a significant reduction of the current density (Fig. 4C). The mean current density at -100 mV was -12.1 ± 0.9 and -7.2 ± 0.9 pA/pF for NTG (n = 22) and Galpha q (n = 32) myocytes, respectively.


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  		Fig. 4.   Inward rectifier currents (IK1) and effects of 0.5 mM Ba2+ recorded in NTG (A) and Galpha q (B) myocytes. A family of currents elicited from a holding potential of -40 mV by voltage steps from -50 to -100 mV in 10-mV increments is shown before (top) and after (bottom) the application of Ba2+. C: I-V relationships of IK1 in NTG and Galpha q myocytes. Peak inward current amplitudes were normalized to the cell capacitance to give current densities. Values are means ± SE of 22 NTG and 32 Galpha q cells.

Na+/Ca2+ exchange currents. The Na+/Ca2+ exchanger is the principal mechanism for Ca2+ extrusion from the cell. However, it has been proposed that inward Na+ flux in exchange for Ca2+ efflux via the Na+/Ca2+ exchanger can contribute to depolarizing current during the action potential plateau (6). Therefore, Na+/Ca2+ exchange currents were compared between NTG and Galpha q myocytes. To measure Na+/Ca2+ exchange activity, the cells were held at -40 mV, and the external solution was rapidly switched from Na+-containing solution to Na+-free solution in which LiCl was substituted for NaCl. Figure 5 shows typical examples of Na+/Ca2+ exchange currents recorded from NTG (A) and Galpha q myocytes (B). When the external Na+ was changed to Li+, the membrane current shifted to an outward direction in both groups; however, peak current amplitude was significantly smaller in Galpha q myocytes. Under our experimental conditions, the average Na+/Ca2+ exchange current density in NTG and Galpha q myocytes was 0.76 ± 0.1 (n = 15) and 0.38 ± 0.04 pA/pF (n = 22), respectively (Fig. 5C).


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  		Fig. 5.   Representative Na+/Ca2+ exchange currents recorded from NTG (A) and Galpha q (B) myocytes. Na+/Ca2+ exchange currents were induced by a rapid solution change from 150 mM Na+ to 150 mM Li+ at a holding potential of -40 mV. C: average Na+/Ca2+ exchange current density in NTG and Galpha q myocytes. Values are means ± SE of 22 NTG and 15 Galpha q cells. * Significantly different (P < 0.01) from NTG myocytes.

Na+/Ca2+ exchanger gene expression. To determine whether the functional changes were associated with alterations in the Na+/Ca2+ exchanger gene expression, we used an RPA to quantify NCX-1 mRNA levels (Fig. 6). Total RNA from the hearts of Galpha q mice and NTG littermates was hybridized with a riboprobe specific for the mouse NCX-1 transcript. The hybridization reactions were subjected to RNase treatment, electrophoresed, and visualized by autoradiography (Fig. 6A). Overall NCX-1 expression levels were determined by exposing the RPA gel to a phosphor plate, scanning the plate with a PhosphorImager, and analyzing the results with NIH Image Analysis software. As summarized in Fig. 6C, NCX-1 mRNA levels were reduced by 40% in Galpha q hearts compared with NTG hearts. These changes could not be accounted for by changes in loading conditions (Fig. 6B).


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  		Fig. 6.   Determination of Na+/Ca2+ exchanger (NCX-1) gene transcript levels in NTG and Galpha q hearts. Total RNA was isolated from hearts of Galpha q (G) mice (n = 5) and NTG (N) littermates (n = 5). A portion (5 µg) of each RNA sample was hybridized with a riboprobe specific for the mouse NCX-1 gene. The reaction mixture was treated with RNase, electrophoresed on a 5% denaturing gel, and visualized after a 24-h exposure to X-ray film (A). C: quantification of NCX-1 levels. * Significantly different (P < 0.01) from NTG hearts. To demonstrate that equivalent levels of RNA were analyzed, 2-µg aliquots of the Galpha q and NTG cardiac RNA were electrophoresed and visualized (B).

Contribution of Ito to action potential prolongation. In an earlier study, we found that the time course of ICa inactivation was significantly slower in Galpha q than in NTG myocytes, although ICa density was not altered (30). It is therefore possible that slower ICa inactivation may also contribute to action potential prolongation in Galpha q myocytes. The possible contribution of this effect to action potential prolongation was tested with BAPTA (10 mM) used as an intracellular Ca2+ buffer to minimize Ca2+-dependent ICa inactivation (19, 24, 25). In NTG myocytes, action potential duration was significantly longer in the presence of BAPTA than without an intracellular Ca2+ buffer. The APD50 and APD70 were 27.8 ± 1.4 and 37.7 ± 2.1 ms (n = 11), respectively.

We next tested whether a reduction of Ito alone is sufficient to account for the changes in action potential duration, then pharmacological inhibition of Ito by 4-AP in NTG myocytes should reproduce the action potential phenotype observed in Galpha q myocytes. To address this question, we measured APD50 and APD70 in NTG myocytes after application of 4-AP (100 µM) to reduce Ito by ~40% (Fig. 3C). Although 4-AP prolonged action potential duration in NTG myocytes, action potential duration was still significantly shorter than in Galpha q myocytes: APD50 before and after 4-AP was 13.5 ± 1.0 and 18.5 ± 2.0 ms, and APD70 before and after 4-AP was 16.7 ± 1.9 and 24.8 ± 3.3 ms (n = 7), respectively.

Furthermore, when action potentials were compared in the presence of a higher concentration of 4-AP (2 mM), action potential duration became significantly longer in Galpha q than in NTG myocytes (Fig. 7). These measures of APD50 and APD70 are summarized in Table 2. Taken together, these data indicate that a decreased Ito expression may contribute to the action potential prolongation observed in Galpha q myocytes. However, additional changes, including a slower ICa inactivation, may also participate in the overall changes in action potential duration.


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  		Fig. 7.   Effects of 2 mM 4-AP on action potential duration recorded in NTG (A) and Galpha q (B) myocytes. The cell was stimulated at 0.2 Hz through the patch pipette. The relative increase in action potential duration produced by 4-AP is significantly larger in Galpha q myocytes.


                              
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  		Table 2.   Effects of 4-AP on action potential repolarization in NTG and Galpha q myocytes

beta -AR signaling: effects of Iso and forskolin on ICa. To minimize Ca2+-dependent inactivation and subsequent negative beta -AR regulation of the Ca2+ channels, myocytes were dialyzed with BAPTA (24, 25). As in our previous study (30), the peak ICa densities in NTG and Galpha q myocytes were similar: 13.4 ± 0.7 (n = 36) and 12.3 ± 0.8 pA/pF (n = 32), respectively. When ICa inactivation was measured in myocytes dialyzed with EGTA (30), the time to half-decay was significantly slower in Galpha q myocytes (30.9 ± 1.6 ms, n = 30) than in NTG myocytes (18.5 ± 1.8 ms, n = 29). However, with BAPTA, the mean times to half-decay were similar: 44.2 ± 2.4 (n = 10) and 44.4 ± 3.4 ms (n = 8) for NTG and Galpha q myocytes, respectively.

The traces in Fig. 8 show ICa activated at different membrane potentials in the absence and presence of Iso (1 µM). The application of Iso increased ICa amplitude in both groups; however, the effect of Iso was significantly less in Galpha q myocytes. In these experiments, Iso increased peak ICa by 160% of basal value in NTG myocytes (Fig. 8A) but only by 15% in Galpha q myocytes (Fig. 8B). A negative shift of the I-V relationships was also observed in both groups. When data were fitted to a Boltzmann relationship (see MATERIALS AND METHODS), the shift in the I-V relationship was -13.1 ± 1.0 and -4.8 ± 0.7 mV for NTG (n = 15) and Galpha q myocytes (n = 18), respectively. Figure 9A summarizes the cumulative dose-response effects of Iso on peak ICa. Although EC50 was similar in both groups (28.9 and 27.4 nM for NTG and Galpha q, respectively), Iso was significantly less effective in Galpha q myocytes. The increase of peak ICa by Iso (1 µM) was 124 ± 13% for NTG and 30 ± 5% for Galpha q myocytes.


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  		Fig. 8.   Effects of 1 µM isoproterenol (Iso) on Ca2+ current (ICa) recorded in NTG (A) and Galpha q (B) myocytes. Top: current traces recorded from a holding potential of -50 mV to the indicated test potentials in the absence (open circle ) and presence of Iso (). Bottom: voltage dependence of peak ICa before and after the application of Iso.



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  		Fig. 9.   Effects of Iso and forskolin. A: concentration-dependent effects of Iso on ICa in NTG and Galpha q myocytes. Percent increase of peak current amplitude is plotted against Iso concentrations. Solid lines were fitted to a 1:1 binding model with EC50 = 28.9 and 27.4 nM for NTG and Galpha q myocytes, respectively. Values are means ± SE of 19 NTG and 23 Galpha q cells. B: summarized data of the effects of 1 µM Iso and 5 µM forskolin on ICa in NTG and Galpha q myocytes. Values are means ± SE. Numbers above bars correspond to total number of cells tested. * Significantly different (P < 0.01) from NTG myocytes.

The attenuated response of Galpha q myocytes to Iso could be due to changes at several levels in the beta -AR signaling cascade (13). To test the possibility that loss of response to Iso was caused by reduced adenylyl cyclase activity, we examined the effects of an adenylyl cyclase activator, forskolin. In NTG myocytes, the maximal responses of ICa to forskolin (5 µM) and Iso (1 µM) were similar. Furthermore, forskolin did not lead to a further increase of ICa after maximum ICa induced by Iso. In contrast, we found that subsequent forskolin application further enhanced ICa in Galpha q myocytes. However, the combined effect was still significantly less than the effect of Iso in NTG myocytes. Thus the attenuated responsiveness of the Galpha q myocytes to Iso occurs not only at the receptor level but also at or below the adenylyl cyclase level. To test this hypothesis, we compared the effects of forskolin (5 µM) on ICa in NTG and Galpha q myocytes. As shown in Fig. 9B, forskolin increased ICa by 125 ± 21% in NTG myocytes (n = 17), whereas the stimulatory effect was significantly less in Galpha q myocytes (67 ± 6%, n = 25).

Previous studies in animal models of hypertrophy as well as those in human heart failure indicate a change in G protein expression levels (13). To test possible involvement of Gi in beta -AR regulation of ICa, we measured effects of Iso and forskolin in Galpha q myocytes after incubation with PTX (2 µg/ml for 4-6 h). The effect of PTX treatment was evaluated by testing the effects of carbachol (10 µM) on the activation of muscarinic K+ currents in atrial myocytes. Figure 10A shows, as expected, carbachol-activated K+ currents in an untreated atrial myocyte. In contrast, the activation was completely abolished in cells treated with PTX. Despite such a pretreatment, Galpha q myocytes continue to show significantly reduced responses to Iso (Fig. 10B). Maximal Iso (1 µM)-stimulated increases in ICa in PTX-treated myocytes were 122 ± 17 and 17 ± 8% over baseline in NTG and Galpha q myocytes, respectively.


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  		Fig. 10.   Effects of Iso on PTX-treated cells. A: effects of carbachol (Carb) on K+ currents in mouse atrial myocytes with and without pertussis toxin (PTX) treatment. Top: current traces were recorded from a holding potential of -40 to -100 mV in the absence (open circle ) and presence of 10 µM carbachol (). Bottom: current-voltage relationships before and after the application of carbachol. Myocytes were bathed in Tyrode solution, and currents were recorded with K+ current-recording patch solution. B: effects of 1 µM Iso on ICa in PTX-pretreated NTG and Galpha q myocytes. Values are means ± SE. Numbers above bars correspond to total number of cells tested. * Significantly different (P < 0.01) from NTG myocytes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we found that myocytes isolated from Galpha q hearts exhibit prolonged action potentials and decreased densities of the repolarizing K+ currents (Ito and IK1). These observations suggest that a common pattern of electrophysiological changes, associated with human heart failure, occurs in this model of cardiac hypertrophy. The data also support the hypothesis that enhanced Ca2+ influx during the prolonged action potential in Galpha q myocytes may compensate for the reduced SR Ca2+ loading to support peak contractions or Ca2+ transients that are otherwise significantly reduced as a result of diminished SR function (30). Additionally, our data indicate that multiple changes, including a decrease in beta -AR coupling to adenylyl cyclase and a reduction in the activity of adenylyl cyclase, contribute to the depressed responses of ICa to Iso. It is possible that catecholamines released by tonic sympathetic activation of cardiac nerves or in the circulation could stimulate beta -AR, even under basal conditions, and attenuated responsiveness of ICa to beta -AR stimulation in Galpha q myocytes may contribute to depressed ventricular dysfunction observed in vivo.

Our experiments demonstrate that action potential duration was significantly prolonged in Galpha q myocytes. APD50 and APD70 were prolonged, and these changes were associated with reductions in Ito, IK1, and Na+/Ca2+ exchange current densities. In cardiac myocytes, Ca2+ influx through the L-type Ca2+ channel is the primary pathway to trigger Ca2+ release from the SR. However, prolongation of the action potential may result in an enhanced inward Ca2+ influx and SR Ca2+ loading to support contractility in Galpha q myocytes.

The most prominent electrophysiological abnormality found in a variety of experimental models of heart failure as well as human heart failure is action potential prolongation. The duration of the cardiac action potential is controlled by a balance of inward and outward currents. As in human ventricular myocytes, Ito are present in adult ventricular myocytes and are responsible for the repolarization phase of the action potential (27, 31). It is therefore likely that a decrease in Ito contributes to changes in the action potential in Galpha q myocytes. Consistent with this notion, 4-AP prolonged the action potential duration in NTG cells, but 4-AP prolonged the action potential more in Galpha q than in NTG myocytes. These results indicate that a change in Ito alone is not sufficient to account for the action potential profile observed in Galpha q myocytes. Similar results, i.e., that 4-AP prolongs action potential more dramatically in myocytes from failing hearts than in normal myocytes, have been reported previously (14).

Because IK1 is responsible for the terminal phase of repolarization, it is possible that a decrease in IK1 may also contribute to some of the prolongation of the terminal phase of the action potential (14, 28). Many factors affect the Na+/Ca2+ exchange activity, including membrane potential and internal and external Na+ and Ca2+ (6). It is therefore difficult to predict the extent to which action potential duration is altered by decreasing Na+/Ca2+ exchange in Galpha q myocytes. However, it is unlikely that the reduction of this current played a significant role in the action potential prolongation found in Galpha q myocytes. An alternative explanation for the prolonged action potential would be a reduced Ca2+-dependent inactivation. We previously demonstrated that Galpha q myocytes exhibit significantly slower ICa inactivation due to impaired SR function and/or a defective ICa-induced SR Ca2+ release process (30). It is therefore likely that slower ICa inactivation associated with altered cellular Ca2+ handling contributes to the action potential prolongation in Galpha q myocytes. A modulatory role of increased Ca2+ influx as a result of reduced Ca2+-dependent inactivation in the plateau phase of the action potential has been reported in the failing heart by computer model analysis (28).

It has been reported that Na+/Ca2+ exchange activity is increased in failing hearts (17, 22). In failing hearts with significantly impaired SR Ca2+ removal, enhanced Na+/Ca2+ exchange activity during the Ca2+ transients may be an important compensatory mechanism. However, in our study, which uses a relatively compensated form of hypertrophy (23), we found that Na+/Ca2+ exchange current activity and expression of the NCX-1 gene were reduced. In Galpha q hearts the SR Ca2+ uptake rate was reduced by ~30% (30). Therefore, the decrease in NCX-1 expression cannot serve to compensate for defective SR Ca2+ uptake. The cause of this difference is not known. It is possible that the decrease in NCX-1 may serve to compensate for a different Ca2+ pathway or result from a noncompensatory inhibition of NCX-1 gene transcript by a Galpha q-mediated signaling pathway. Future studies of a direct comparison of Na+/Ca2+ exchanger function at various stages of cardiac hypertrophy and failure in the same model would permit establishment of the functional roles mediated by changes in Na+/Ca2+ exchanger activity.

Our data show that Iso increased ICa in NTG and Galpha q myocytes with similar affinity, but the magnitude of the response to the drug was significantly reduced in Galpha q myocytes. beta -AR-mediated increases in ICa depend on the beta -AR signaling cascade. Because beta -AR density was found to be normal in the Galpha q heart (8), the reduced response to Iso in Galpha q myocytes suggests potential changes in beta -AR and Ca2+ channel coupling. In NTG myocytes, maximal activation of ICa with Iso or forskolin was similar, and such activation was not additive. In contrast, although the relative increase in ICa with forskolin was still significantly less in Galpha q than in NTG myocytes, forskolin was capable of activating ICa more potently than Iso. Pretreatment of cells with PTX did not alter the ICa responses to Iso or forskolin. We previously demonstrated that the responses of ICa to dihydropyridine drugs and a membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP, were not altered in Galpha q myocytes, suggesting that modulation of the Ca2+ channel by cAMP-dependent protein kinase A activation is normal (30). Taken together, our data suggest that impaired beta -AR-adenylyl cyclase coupling and reduced adenylyl cyclase activity are involved in the reduced responsiveness of ICa to Iso. The electrophysiological observations are consistent with biochemical observations that basal and forskolin-activated adenylyl cyclase activities in Galpha q hearts were reduced by ~45% compared with NTG hearts (26).

In summary, we have studied electrophysiological properties that may contribute to altered Ca2+ handling and beta -AR regulation of ICa in a genetic model of cardiac hypertrophy. The hypertrophy-associated action potential prolongation is a prominent feature of Galpha q myocytes. Our data suggest that multiple changes in the beta -AR signaling pathway that regulates ICa occur in Galpha q myocytes. Because the signal transduction pathway mediated by Galpha q is closely associated with the induction of cardiac hypertrophy and the progression of heart failure (1, 2, 8, 20, 23), this transgenic model may serve as an informative model system for understanding the cellular mechanisms of heart failure in humans.


    ACKNOWLEDGEMENTS

The authors thank Dr. G. W. Dorn for providing the transgenic mice, Dr. R. Millard for helpful comments on the manuscript, and Drs. M. Periasamy and G. J. Babu for guidance during the study. This work was supported by the American Heart Association, Ohio Valley Affiliate, and National Institutes of Health Grants GM-54169 and HL-61476 and Training Grant HL-07382.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Yatani, Dept. of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575 (E-mail: Yatania{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 18 October 1999; accepted in final form 10 January 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(1):H139-H148
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