J Biol Chem, Vol. 274, Issue 31, 21503-21506, July 30, 1999

COMMUNICATION
Cardiac-specific Overexpression of the ₁ Subunit of the L-type Voltage-dependent Ca²⁺ Channel in Transgenic Mice
LOSS OF ISOPROTERENOL-INDUCED CONTRACTION^*

James N. Muth^§, Hiroshi Yamaguchi, Gabor Mikala, Ingrid L. Grupp^¶, William Lewis, Heping Cheng^**, Long-Sheng Song^**, Edward G. Lakatta^**, Gyula Varadi^§, and Arnold Schwartz

From the  Institute of Molecular Pharmacology and Biophysics, Departments of ^§ Cell Biology, Neurobiology, and Anatomy,  Pathology and Laboratory Medicine, ^¶ Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 and the ^** Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224

	ABSTRACT

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The L-type voltage-dependent calcium channel (L-VDCC) regulates calcium influx in cardiac myocytes. Activation of the -adrenergic receptor (AR) pathway causes phosphorylation of the L-VDCC and that in turn increases Ca²⁺ influx. Targeted expression of the L-VDCC ₁ subunit in transgenic (Tg) mouse ventricles resulted in marked blunting of the AR pathway. Inotropic and lusitropic responses to isoproterenol and forskolin in Tg hearts were significantly reduced. Likewise, Ca²⁺ current augmentation induced by iso- proterenol and forskolin was markedly depressed in Tg cardiomyocytes. Despite no change in AR number, isoproterenol-stimulated adenylyl cyclase activity was absent in Tg membranes and NaF and forskolin responses were reduced. We postulate an important pathway for regulation of the AR by Ca²⁺ channels.

	INTRODUCTION

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The cardiac L-type voltage-dependent Ca²⁺ channel (L-VDCC)¹ is a large glycoprotein complex consisting of ₁, , and ₂/ subunits. The ₁ subunit serves as the Ca²⁺ conducting pore, while the  and ₂/ subunits are auxiliary and modulate the activity of the ₁ subunit (1, 2). The level of intracellular Ca²⁺ is a determinant of cardiac function. Calcium is not only essential for contraction but also for various enzymatic reactions including activation of proteases, phosphatases, kinases, signal transduction cascades, and regulation of gene transcription (3-6).

-Adrenergic receptor (₁AR, ₂AR) activation regulates the L-VDCC by phosphorylating the ₁ subunit, thereby causing an increase in Ca²⁺ influx (7). This forward signal is via cAMP-dependent protein kinase A phosphorylation of the channel (7). Other non-cAMP-dependent protein kinase-dependent mechanisms have also been proposed (8). However, there is little or no information regarding possible Ca²⁺-dependent regulation of the AR signal transduction pathway via the L-VDCC.

In order to investigate possible reciprocal regulation between these pathways in vivo, we overexpressed the ₁ subunit of the Ca²⁺ channel in hearts of Tg mice and studied channel activity and the AR-G protein cascade by physiological and biochemical techniques. We found a major change in cardiac function regulated by the AR system and conclude that Ca²⁺ derived from channel influx modules AR activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Generation of Transgenic Mice-- The overexpression construct was generated by ligating the full-length human L-VDCC _1C subunit (9) coding sequence to the -myosin heavy chain (-MHC) promoter (clone 26) (10) and completed with a bovine growth hormone poly(A)-adenylation signal (Fig. 1A). The -MHC-L-VDCC construct was cleaved with NotI and the -MHC-L-VDCC fusion cDNA fragment was purified and eluted in oocyte injection buffer (5 mM Tris-HCl, 0.2 mM EDTA, pH 7.4). This construct (20 µg) was then microinjected into the male pronucleus of fertilized zygotes from superovulated FVB/n mice and the surviving zygotes implanted into pseudopregnant foster mothers. Transgenic founder mice were identified with genomic DNA utilizing polymerase chain reactions and confirmed by restricted Southern blotting. Polymerase chain reaction was carried out with a sense primer (5'-cactcttagcaaacctcagg-3') specific for the -MHC gene (bp 859) and an antisense primer (5'-caatgcgaccatctccacagtc-3') located at bp 1530 of the human ₁ subunit yielding a 375-bp product from mice expressing the transgene. For Southern blots, genomic DNA was extracted from tail clips of 18-day-old pups, digested with EcoRI, and separated on a 0.7% agarose gel. The -MHC-L-VDCC construct (100 pg, 500 pg, and 1 ng) was cleaved with EcoRI and loaded on the gel for quantitation. Following transfer to supported nitrocellulose (Hybond-C extra, Amersham Pharmacia Biotech), the DNA was probed with a 2295-bp EcoRI fragment from the fusion construct (Fig. 1A). The fragment contained ~1.5 kilobase pairs of the -MHC promoter and 0.75 kilobase pair of the L-VDCC _1C cDNA. Radioactive bands were quantitated using PhosphorImager and ImageQuant software (Molecular Dynamics).

RNA Dot Blots of L-VDCC Subunits and Hypertrophic Markers-- Total RNA was isolated from frozen heart samples of Tg and nontransgenic (Ntg) littermate controls using TriZol (Life Technologies, Inc.) according to the manufacturer's recommendations. Two µg of RNA was loaded onto Hybond N⁺ membranes (Amersham Pharmacia Biotech) using a dot blot apparatus. Probing for the ₁ subunit was completed with a 1350-bp fragment isolated by cleaving the -MHC-L-VDCC vector with EcoRI (Fig. 1A). This fragment corresponds to bases 2098-3448 of the human heart ₁ cDNA. The full-length L-VDCC _2a cDNA was used to probe for gene expression levels of the  subunit. The ₂/ subunit expression was analyzed using a 1550-bp fragment probe generated by cleaving the cDNA with HincII. Probing of the hypertrophic markers (atrial natriuretic factor, -MHC, -MHC, cardiac -actin, skeletal -actin, sarco-endoplasmic reticulum ATPase, and phospholamban) using gene-specific antisense oligonucleotides were completed as described previously (11). All probes were ³²P-labeled by the random priming technique. Radiolabeled dot blots were quantitated using a PhosphorImager and ImageQuant software (Molecular Dynamics) and normalized to the signal from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), since evidence from other mouse models has demonstrated no change in gene expression of GAPDH in Tg and Ntg littermates (11, 12).

Work Performing Hearts-- Eight- to ten-week-old Tg and Ntg littermates of either sex were anesthetized with sodium pentobarbital (intraperitoneal 30 mg/kg), protected with heparin, and placed in an isolated working heart mode as described (13). Following base line establishment, the heart was exposed to cumulative isoproterenol or forskolin concentrations and the inotropic, lusitropic (relaxation), and chronotropic responses measured.

Echocardiography-- Analysis of left ventricular function and mass was assessed using an ATL HDI3000 broadband digital echo-Doppler machine as described previously (12). Sedated, spontaneously breathing, mice were studied using two-dimensional-guided M-mode echocardiography to estimate left ventricular dimensions and wall thickness. Fractional shortening was calculated as (EDD-ESD)/EDD, where EDD is end diastolic dimension and ESD is end systolic dimension. Echocardiographic measurements were recorded and analyzed blinded.

Isolation of Single Cardiomyocytes and Electrophysiological Recording-- Single ventricular cells were isolated from the hearts of 8-10-week-old Tg and Ntg mice by enzymatic dissociation protocol as described previously (14) using Type I collagenase (Worthington). L-type Ca²⁺ channel currents were recorded using the whole-cell mode of the patch clamp method (15).

-Adrenergic Receptor Density, Adenylyl Cyclase Assays, and cAMP-- Measurement of adenylyl cyclase activity using a cAMP ¹²⁵I radioimmunoassay kit (NEN Life Science Products) and radioligand binding of the AR were carried out as described (16). Powdered tissue for cAMP measurements was prepared by homogenizing in 20 mM Tris-HCl, pH 7.5, 250 mM sucrose, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride in a glass homogenizer. Membranes were pelleted by centrifugation at 100,000 × g for 1 h at 4 °C, resuspended in 10 mM Tris-HCl, pH 7.5, and stored at 70 °C. The particulate fraction (50 µg/ml of protein) was incubated for 10 min at 30 °C with reagents as described (17), in a final volume of 0.5 ml of cAMP reaction buffer. cAMP measurements were performed using a competitive protein-binding cAMP ³H assay kit (Amersham Pharmacia Biotech) according to the manufacturer's recommendations. Proteins were solubilized by adding 0.5 ml of 1 N NaOH to the trichloroacetic acid-extracted tubes and quantified using Bio-Rad assay.

Statistical Analysis-- Data are reported as means ± S.E. n values are equivalent to the number of mice tested except for the patch clamp experiments, which indicates the number of cardiomyocytes used. A Student's t test was used for statistical comparisons between Tg and Ntg hearts with a two-tail p value of <0.05 considered significant.

	RESULTS

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Using the -MHC promoter, the human L-VDCC _1C subunit was overexpressed in mouse ventricles (Fig. 1A). The -MHC promoter becomes active within a few days after birth and reaches steady state maximal activity around day 60 (18). We therefore used 8-10-week-old mice for our studies. A total of five founder lines were identified, classified as M1-M5. Two of these transgenic lines died within 8 weeks of age. Pathological examination of these two lines revealed severe myocardial hypertrophy and dilatation. Other transgenic lines were maintained at heterozygosity and compared with Ntg littermates for controls. The M1 and M5 lines had similar ₁ subunit protein levels. Experiments were predominantly carried out in the M1 line except as noted.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Molecular characterization of 1 subunit overexpression mice. A, schematic representation of the -MHC-L-VDCC fusion construct used for the generation of the Tg mice. Also included are important EcoRI sites used for generating Southern (2295 bp) and dot blot (1350 bp) probes. E1 and E2 represent the first two noncoding exons of the -MHC gene. B, Southern blot of two Ntg and two M1 Tg mice. 20 µg of genomic DNA and the indicated amount of standard fusion construct were cleaved, blotted, and hybridized. C, total RNA was isolated from cardiac homogenates using TriZol reagent (Life Technologies, Inc.). 2 µg of total RNA was transferred to Hybond N+ (Amersham Pharmacia Biotech) using a dot blot apparatus and hybridized. The mRNA levels of L-VDCC subunits were estimated using the probes: 1 subunit, a 1350-bp EcoRI fragment;  subunit, full-length 2a cDNA; 2/ subunit, a 1550-bp HincII fragment. Values were normalized to the hybridization signal of a GAPDH probe. Bars show the mRNA expression level of the L-VDCC subunits.

Transgenic mice had no overt phenotype by observation. Additionally, litter sizes and pup survivals were similar to Ntg littermates. Loading known amounts of plasmid construct, the M1 line was determined to carry eight copies of the transgene (Fig. 1B). A quantitative RNA dot blot showed a 2.8-fold increase in the ₁ subunit and no change in the  and ₂/ subunits (Fig. 1C, Table I). In order to determine whether the increased gene product was translated into corresponding ₁ subunit protein, homogenates were probed with specific antisera that does not distinguish between endogenous and Tg ₁ protein. Western analysis revealed a clear increase in ₁ subunit protein (data not shown). Assays of 8-week-old ventricular RNA for potentially regulated cardiac genes showed a significant 6.6-fold increase in ANF mRNA levels, but no significant differences in -MHC, -MHC, cardiac -actin, skeletal -actin, sarco-endoplasmic reticulum ATPase, and phospholamban gene expression (Table I).

View this table: [in this window] [in a new window]   Table I mRNA transcripts for hypertrophic markers and L-VDCC subunits Abbreviations: du, densitometric units;  SK actin,  skeletal actin; SERCA2, sarco-endoplasmic reticulum ATPase 2; PLB, phospholamban.

We hypothesized that the increased ₁ subunit in the transgenics should result in augmented contractility. Indeed, basal contractility and relaxation (expressed as +dP/dt and dP/dt) were significantly higher for Tg hearts (4760 ± 46 mmHg/s and 3935 ± 56 mmHg/s, n = 5) compared with Ntg hearts (4094 ± 44 and 3160 ± 56, n = 4, p < 0.05). Infusion of the -adrenergic receptor agonist, isoproterenol, did not elicit the expected inotropic and lusitropic (relaxation, diastole) increases observed in Ntg animals (Fig. 2). Heart rate increases were, however, comparable and normal for isoproterenol effects in both sets of mice (data not shown). Additionally, increases in contractility induced by forskolin (direct AC activator) were significantly decreased in Tg hearts compared with the Ntgs, suggesting that the defect in the AR signaling pathway is not limited to an "uncoupling" of the receptor (Table II).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of isoproterenol on transgenic and nontransgenic isolated perfused hearts. Whole hearts were antegradely perfused with increasing concentrations of isoproterenol. Increases in contractility (+dP/dt) and relaxation (dP/dt) were observed at concentrations from 4 × 109 M and continued to increase until reaching toxicity at 8 × 107 M in Ntg hearts; Tg hearts did not respond to isoproterenol at any concentration. [M], molar concentration; R, resistance.

Echocardiographic measurements indicated that left ventricular end-diastolic dimension and posterior wall thickness were minimally increased in Tg as compared with Ntg mice (Table III). Left ventricular mass in the transgenic hearts was clearly increased (23%) compared with Ntg hearts. However, no significant differences were found in left ventricular end-systolic dimension, septal wall thickness, or fractional shortening percentage (Table III). Heart-to-body weight differences were significantly increased in Tg animals compared with Ntg littermates (Table III). These findings are consistent with a mild hypertrophy without ventricular dysfunction. On the other hand, ex vivo studies of these hearts revealed increases in basal contractility and a dramatic loss of AR responsiveness.

To determine possible functional changes in the L-type Ca²⁺ channels, whole-cell voltage clamp recordings were carried out on isolated ventricular cardiomyocytes. Average cell capacitance was significantly larger in the Tg myocytes compared with Ntg myocytes (206.6 ± 16.5 pF, n = 9 versus 161.1 ± 7.1 pF, n = 7, p < 0.05), which indicates cellular hypertrophy. L-VDCC current amplitude was larger in the Tg myocytes compared with Ntg myocytes (Fig. 3, A and B). Average peak current amplitude was 1.8 ± 0.2 nA (n = 7) for Tg myocytes and 1.3 ± 0.1 nA (n = 9) for Ntg myocytes (p < 0.05, Fig. 3C). When the current amplitude was normalized for cell capacitance, there was no significant difference between Tg and Ntg cardiomyocytes (8.8 ± 0.6 pA/pF (n = 9) and 8.3 ± 0.6 pA/pF (n = 7)). There were no significant differences in the voltage dependence of activation or the activation/inactivation kinetics of the channels (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Whole-cell calcium current, isoproterenol responses, and adenylyl cyclase activity in transgenic and nontransgenic hearts. A and B, representative calcium current traces recorded with 1.8 mM Ca2+ as a charge carrier from Ntg (A) and Tg (B) mouse ventricular myocytes. Currents were elicited by a 500-ms depolarizing pulse from a holding potential of 60 mV to 30, 10, +10, and +30 mV. C and D, averaged current-voltage relationship (C) and current density-voltage relationships from Ntg and Tg mice. D, data points represent the means of 7 (Ntg) and 9 (Tg) experiments. E, increase in peak current amplitude in response to 107 M isoproterenol. Data are means of six (Ntg) and five (Tg) experiments. F, membrane adenylyl cyclase activity was carried out in the presence of vehicle, 105 M isoproterenol, 102 M NaF, and 104 M forskolin.

The L-VDCC has been identified as a downstream phosphorylation target of the -adrenergic receptor cascade (7). To determine whether this cascade remained intact in the Tg mice, isolated cells were stimulated with isoproterenol. When cells isolated from Ntg mice were superfused with 10⁷ M isoproterenol, peak Ca²⁺ channel currents were augmented by 72.9 ± 21.6% (n = 6), but only 19.4 ± 11.4% (n = 5) for the transgenics (p < 0.05) (Fig. 3E). Consistent with the results in the intact heart experiments, forskolin produced only a small increase in the peak Ca²⁺ current in the Tg hearts compared with the robust increases observed in the Ntgs (Table II).

To examine the blunted isoproterenol and forskolin responses found in whole heart and single cells, the AR signaling pathway was investigated using isolated membranes. No change in total AR receptor density was found (Tg, 19.2 ± 1.2 fmol/mg; Ntg, 19.6 ± 0.6 fmol/mg; n = 3). Isoproterenol-stimulated activities were 150% of basal in Ntg mice (i.e. a ~50% increase over basal). In marked contrast, isoproterenol failed to stimulate AC activity in membranes from the Tg mice (indeed levels were slightly below basal) (Fig. 3F). Basal AC levels were 44.8 ± 17 pmol/min/mg for Ntg and 52.7 ± 11 pmol/min/mg for Tg extracts. As predicted NaF and forskolin-stimulated activities were depressed compared with Ntg, but the values did not reach statistical significance (Fig. 3F). Direct cAMP measurements further supported the loss of the AR responsiveness. As observed in the AC assays, Tg membranes did not respond to isoproterenol nor was forskolin able to restore cAMP levels to the Ntg levels (Table II).

	DISCUSSION

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We constructed a transgenic mouse in which the L-type voltage-dependent Ca²⁺ channel was overexpressed. The transgene is defined as an increased VDCC by four criteria, Southern analysis, RNA analysis, electrophysiological identification of an increased Ca²⁺ current, and a higher basal contractile state in the Tg animals compared with the Ntgs. Thus, these characteristics reflect a probable small, but sustained, increase in Ca²⁺ influx in Tg myocytes that explains the significant increase in basal contraction and relaxation we observed. Accompanying the latter, we found a surprising and striking loss of the usual effects of a well known _1,2 adrenergic agonist, isoproterenol, on myocardial contraction. In contrast, the heart rate increases secondary to isoproterenol administration were normal, which defines an interesting separation of -agonist actions on contractility and heart rate. This differentiation implies that downstream effectors and/or intermediates in the G protein pathway for -agonist action are different for contractility and heart rate. It seems possible that for heart rate regulation Ca²⁺ derived from the L-VDCC may not be limiting as it is for contraction. Consistent with the loss of contractile action was a blunting of isoproterenol and forskolin stimulation of Ca²⁺ current in isolated myocytes. Thus, the effects found on the whole heart were for the most part duplicated on single cells for both isoproterenol and forskolin.

We refer to this loss of -agonist contraction effect as a defect or a "loose coupling" in the AR signaling pathways. The present data provide convincing evidence for a novel "reciprocal regulation" of AR signaling by an increased influx of Ca²⁺ provided by the L-VDCC. A possible mechanism to consider is protein kinase C activation by Ca²⁺ (19) that in turn activates AR kinase leading to phosphorylation of the AR resulting in a decrease in signaling activity (20). Other mechanisms possibly responsible for the loss of AR signaling include an inhibition of AC activity by Ca²⁺ (21-23) and an indirect up-regulated phosphatase activity via calcium-calmodulin-dependent protein kinase (24). The latter is supported by recent data implicating calcineurin in cardiac hypertrophy (5). Further possibilities include up-regulation of G_i, changes in the ratio of ₁AR:₂AR isoforms, etc. (20). Consistent with the suggestion of a Ca²⁺-dependent "cascade" involving a phosphatase is the slow development of hypertrophy and subsequent cardiac failure in these animals.

Our results suggest an interesting "cross-talk" between the L-VDCC and the AR in vivo. A modest increase in the ₁ subunit of the L-VDCC in cardiomyocytes results in a remarkable decrease in -adrenergic signaling that affects contractility but interestingly does not alter the usual responses of heart rate to AR agonists. The Ca²⁺ channel ₁ overexpression also produces a setting in which late stage ventricular remodeling occurs. This is surprising, since the cardiac cell has a remarkable network of sarcoplasmic reticulum and mitochondria that one would think would be poised to sequester any increase in calcium. In all experimental models of hypertrophy in which "Ca²⁺ overload" was produced in no case was a paradigm used in which a small but sustained Ca²⁺ increase through the L-VDCC was provided. Clearly the present experiments show that the L-VDCC in heart is a highly sensitive conduit for Ca²⁺ as the link between excitation and contraction. Furthermore, it is likely that the increased calcium is sequestered in a pool that is linked to a growth program. A very recent publication has revealed a pathway to gene expression and growth that requires a very low concentration of calcium (25). These Tg mice represent the first animal model with an increase in voltage-dependent Ca²⁺ channels specifically in the heart that provides a paradigm for studies of the role of Ca²⁺ in growth adaptation (25), maladaptation, and receptor/channel regulation.

	ACKNOWLEDGEMENTS

We thank Dr. J. Robbins and J. Gulick for providing us with clone 26 containing the murine -MHC promoter. We are very grateful to Dr. G. Dorn for echocardiography and hypertrophic marker experiments and to Dr. S. Liggett and N. Tepe for AC assays. A special thanks to Drs. R.-P. Xiao and D.-J. Wang for cAMP assays. We also thank G. Newman, T. Jackson, and M. Neyland for technical assistance and J. C. Neumann in the Transgenic Core Facility of the University of Cincinnati, College of Medicine for the pronuclear injections.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants P01 HL22619 (to A. S.), RO1 R37HL 43231, and T32 HL 07382 (to J. M. and A. S.).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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of Cincinnati College of Medicine, Institute of Molecular Pharmacology and Biophysics, P. O. Box 670828, Cincinnati, OH 45267-0828. Tel.: 513-558-2200; Fax: 513-558-1778; E-mail: schwara@email.uc.edu.

	ABBREVIATIONS

The abbreviations used are: L-VDCC, L-type voltage-dependent calcium channel; AR, -adrenergic receptor; Tg, transgenic; Ntg, nontransgenic; AC, adenylyl cyclase; MHC, myosin heavy chain; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EDD, end diastolic dimension; ESD, end systolic dimension; F, farad.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES