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(Circulation Research. 2001;88:9.)
© 2001 American Heart Association, Inc.

Reports

Reduction of [Ca²⁺]_i Restores Uncoupled ß-Adrenergic Signaling in Isolated Perfused Transgenic Mouse Hearts

Vladimir B. Serikov, Natalia N. Petrashevskaya, Amy M. Canning, Arnold Schwartz

From the Institute of Molecular Pharmacology and Biophysics (V.B.S., N.N.P., A.S.) and the Division of Cardiovascular Medicine, Department of Internal Medicine (A.M.C.), University of Cincinnati, College of Medicine, Cincinnati, Ohio.

Correspondence to Dr A. Schwartz, Director, Institute of Molecular Pharmacology and Biophysics, Cardiovascular Research Center, University of Cincinnati, 231 Bethesda Ave, Cincinnati, OH 45267-0828.

	Abstract

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The effects of alterations in calcium in the perfusion media were studied on ß-adrenergic coupling in isolated hearts from 3 different transgenic mice: cardiac-specific overexpressed ₁ subunit of L-type calcium channel, overexpressed G_q, and phospholamban knockout. Isolated hearts from all 3 models, when studied at [Ca²⁺] of 2 mmol/L in the perfusate, showed the usual blunted or no response to ß-adrenergic stimulation. Lowering [Ca²⁺] to 0.75 to 1.5 mmol/L unloaded the hearts of calcium and restored to nearly normal the responsiveness to ß-agonist stimulation.

Key Words: calcium • adenylyl cyclase • cardiac hypertrophy

	Introduction

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Isolated heart perfusion using rats remains a popular approach to study mechanisms of regulation of myocardial contraction and drug action.^{1 2} Adult rat heart has always behaved in a peculiar way, showing resistance to digitalis, "negative treppe," and a short action potential.^{1 3 4} The mouse heart has been shown to exhibit the same characteristics as the rat.^{4 5} The uniqueness of the rat and mouse hearts with regard to calcium handling has been recently emphasized,^{4 5} and caution has been advised in interpreting phenotypes.⁵

Several transgenic murine models of cardiac hypertrophy and failure display diminished responsiveness to ß-adrenergic (ß-AR) stimulation.^{2 6 7 8} These models share the commonality of an increased [Ca²⁺]_i. It is possible, therefore, that [Ca²⁺]_i may be of considerable importance in the ß-AR response.⁹ The initial goal of this study was to investigate the effects of varying [Ca²⁺] on ß-AR stimulation in a model in which cardiac-specific overexpression of the ₁ subunit of the L-type voltage-dependent calcium channel (L-VDCC) results in a phenotype that exhibits an early uncoupling of the ß-AR, baseline hypercontractility, and subsequent cardiac hypertrophy.⁷ We included 2 other mouse models, the cardiac-specific overexpression of G_q⁶ and the phospholamban knockout (PLB-KO),² to determine whether [Ca²⁺] was an important general variable in the blunted or loss of ß-AR responsiveness. The phenotype of the G_q includes normal basal contractility, uncoupling of the ß-AR, and cardiac hypertrophy.⁶ The phenotype of the PLB-KO mouse displays a very high basal contractility and uncoupling of the ß-AR but no hypertrophy or failure.² Animal protocols were approved by the University of Cincinnati Animal Care Committee and met Guiding Principles for Research Involving Animals.

	Materials and Methods

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Mice were anesthetized with pentobarbital sodium (30 mg/kg ip) and heparinized (500 U/kg). Hearts were removed, and the aortas were cannulated. Retrograde or Langendorff (L-mode) and, subsequently, antegrade or working heart (W-mode) perfusion with Krebs-Henseleit solution (KHS) at 37.7°C was carried out as described,² with a slight modification. Temperature was maintained by submerging the heart in a bath with KHS. Hemodynamic variables were recorded using the Biobench data acquisition board and software package (National Instruments). Contractility and relaxation were assessed as maximal (+dP/dt max) or minimal (-dP/dt min) derivatives of intraventricular pressure with the same software. Perfusions were then performed in separate experiments with KHS with [Ca²⁺] of 2 mmol/L or lower.

We compared the ß-AR response to isoproterenol (ISO) at different [Ca²⁺] between genetically similar animals perfused in exactly the same mode. Control was normal [Ca²⁺] of 2 mmol/L, and experiment was reduced [Ca²⁺]. Nontransgenic (NTG) littermates of all models performed well at 2 mmol/L [Ca²⁺] in the W- and L-modes and were not different in responses to ISO. Some hearts of transgenic animals worked well at low [Ca²⁺] in the W-mode (PLB-KO and L-VDCC), whereas others (G_q) could sustain contraction only in L-mode at low [Ca²⁺]. The PLB-KO hearts functioned well in the W-mode at [Ca²⁺] down to 1 mmol/L, but the ß-AR response was still blunted, probably because of the continued hypercontractility reflecting a high [Ca²⁺]_i. To study the pharmacology and physiology of this model, it was necessary to lower the perfusion [Ca²⁺] to 0.5 to 0.75 mmol/L. These hearts functioned very well and in a sustained manner in the L-mode. Thus, we adjusted our perfusion mode to maintain a similar and sustained basal contractility for all of the models. All animals were studied at age 12 to 16 weeks, and there was no decompensation or heart failure. ISO (Sigma) was added to the KHS using microperfusion pumps. Statistical analysis was performed using paired (different ISO concentration) and nonpaired (different [Ca²⁺]) Student’s t test, with P<0.05 being significant.

	Results and Discussion

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In all animal groups (Table), as expected, a decrease in [Ca²⁺] resulted in a decrease in baseline contraction. All NTGs showed a normal ß-AR response, which was in fact enhanced at low [Ca²⁺]. In all transgenic animals, ß-AR signaling at [Ca²⁺] of 2 mmol/L was significantly blunted. The ß-AR activity was restored at [Ca²⁺] of 1.5 mmol/L in the L-VDCC and G_q models (Figure and Table). In the PLB-KO mice, a nearly complete restoration of the response was observed at [Ca²⁺] of 0.75 mmol/L. In the 3 transgenic models, the phenotypes exhibited increased [Ca²⁺]_i attributable to altered pathways of calcium metabolism in the cells, as described.^{2 6 7 11} We suggest that studies performed in vivo and ex vivo under conditions that are generally regarded as normal or physiological for a nontransgenic animal should be considered abnormal in terms of the [Ca²⁺] internal milieu for these transgenic animals. By lowering [Ca²⁺], we returned these cells to normal conditions of [Ca²⁺] handling and thus restored ß-AR response.

View this table: [in this window] [in a new window]   Table 1. ISO Effect on Contraction (mm Hg/s) and Relaxation (-mm Hg/s) in Isolated Mouse Heart

View larger version (17K): [in this window] [in a new window]   Figure 1. Contractility (circles) and relaxation (squares) dose-response curve to ISO. Open symbols [Ca2+]=2 mmol/L; closed symbols [Ca2+]=1.5 mmol/L. Data are shown as percent change from baseline (mean±SE). A, 1 Subunit of L-type calcium channel–overexpressing mice. B, Gq-overexpressing mice.

As is well-known, the positive inotropic effect of ß-AR stimulation occurs through a signaling pathway that includes an activation of adenylyl cyclase (AC).^{8 9} The body of evidence chiefly from the laboratory of Cooper et al¹⁰ reinforces the importance of calcium in activating and inhibiting AC, acting at the Mg²⁺/Ca²⁺ site on the enzyme. Thus, a cycling or oscillation of cAMP in the heart dependent on the levels of [Ca²⁺]_i seems logical. Murine and rat hearts are substantially different from other mammals in levels, mechanisms, and pathways of [Ca²⁺] cycling.^{3 4} Our data demonstrate that altering the level of [Ca²⁺] allows a complete restoration of ß-AR contractile response in transgenic models in which none or very low reactivity to ß-agonist stimulation has been reported. In the G_q mouse, molecular mechanisms are thought to involve increased expression of G_i, diminished activity of AC,⁶ a much slower relaxation time of the Ca²⁺ transient, and a reduced level of Ca²⁺ uptake by the sarcoplasmic reticulum.¹¹ The latter would result in a higher diastolic [Ca²⁺]_i. In the L-VDCC model, myocardial cells display increased cytoplasmic [Ca²⁺]_i.⁷ In the PLB-KO model, the primary substrate for phosphorylation by AC, PLB is absent and cells exhibit a high level of basal contraction, thereby reflecting increased [Ca²⁺] distribution.² From the present data, it is clear that other proteins (troponin I and L-VDCC) can substitute for the absent PLB in ß-AR effects on contraction and relaxation. In all 3 models examined, it is important that the common thread is an increased [Ca²⁺]_i or distribution of [Ca²⁺]_i. Under conditions of low [Ca²⁺], isolated rat and mouse cardiac muscle reacts to various pharmacological and physiological stimuli in the same way as other mammalian species.^{1 3} The regulation of cAMP requires systems to elevate and degrade cAMP and increase and decrease [Ca²⁺]_i.¹⁰ The simplest explanation for the loss of ß-AR responses in the 3 models is [Ca²⁺]-induced inhibition of AC. In addition, high [Ca²⁺]_i might alter the activity of a phosphodiesterase.¹⁰ In the 3 models, it is clear that the balance among these systems has been disturbed, and unloading the heart of calcium allows for a rapid and complete restoration of a response to ß-AR stimulation.

	Acknowledgments

 
The L-VDCC mouse was from the Institute of Molecular Pharmacology and Biophysics (supported by National Institutes of Health [NIH] grant P021 HL22619). The G_q-overexpressing mouse was a gift from Dr G.W. Dorn II, University of Cincinnati, College of Medicine, Cincinnati, Ohio (supported by NIH grant R01 HL58010). The PLB KO mouse was a gift from Dr E.G. Kranias, University of Cincinnati, College of Medicine, Cincinnati, Ohio (supported by NIH grant 5 P40 RR12358). V.B.S. and N.N.P. are postdoctoral fellows supported by NIH grant 5 T32 HL07382.

	Footnotes

 
Original received October 23, 2000; revision received November 30, 2000; accepted November 30, 2000.

	References

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