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(Circulation. 2001;103:140.)
© 2001 American Heart Association, Inc.

Basic Science Reports

A Ca²⁺-Dependent Transgenic Model of Cardiac Hypertrophy

A Role for Protein Kinase C

James N. Muth, BA; Ilona Bodi, PhD; William Lewis, MD; Gyula Varadi, PhD; Arnold Schwartz, PhD

From the Institute of Molecular Pharmacology and Biophysics (J.N.M., I.B., G.V., A.S.) and the Department of Cell Biology, Neurobiology, and Anatomy (J.N.M., G.V.), University of Cincinnati Medical Center, Cincinnati, Ohio; and the Department of Pathology (W.L.), Emory University, School of Medicine, Atlanta, Ga.

Correspondence to Arnold Schwartz, Institute of Molecular Pharmacology and Biophysics, University of Cincinnati Medical Center, 231 Bethesda Ave, Mail Location 0828, Cincinnati, OH 45267-0828. E-mail schwara{at}email.uc.edu

	Abstract

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Background—Calcium imbalances have been implicated as an underlying mechanism of human cardiac dysfunction. The voltage-dependent calcium channel plays a critical role in calcium regulation in the heart. Thus, aberrant calcium signaling arising from this channel could initiate the calcium imbalances observed in heart failure. In the present study, we used a transgenic mouse with an increased number of L-type calcium channels to identify the role of an increased, sustained ingress of calcium as an initiator of hypertrophy.

Methods and Results—Whole-heart histology and electrophysiology in isolated cardiomyocytes identified calcium-channel overexpression in the hearts of transgenic mice. Calcium-channel density was increased in 2-, 4-, and 8-month-old transgenic cardiomyocytes. Ventricular fibrosis, damage, and remodeling became more pronounced as the transgenic mice aged. Apoptosis was also present in transgenic hearts at 8 months of age. Increased protein kinase C activation was elevated before the development of hypertrophy and failure.

Conclusions—Transgenic mice developed hypertrophy and severe cardiomyopathy as a function of age, thus confirming that changes in channel density are sufficient to induce disease. The small, sustained increase in the ingress of Ca²⁺ through the calcium channel elevated protein kinase C before the development of hypertrophy, suggesting that protein kinase C plays an important role in triggering hypertrophy.

Key Words: calcium • ion channels • heart failure

	Introduction

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The L-type voltage-dependent calcium channel (L-VDCC) serves as a voltage-regulated "gate" between extracellular calcium and contraction. On membrane depolarization, Ca²⁺ enters through the channel, causing an amplified rise in intracellular Ca²⁺ (calcium-induced calcium release¹ ) and subsequent contraction. Diastole occurs by pumping Ca²⁺ into the sarcoplasmic reticulum, where it is catalyzed by the sarcoplasmic reticulum Ca²⁺-ATPase, thus effectively reloading internal stores for the next contraction. Ca²⁺ also exits the cell via a sarcolemmal ATPase and the Na⁺/Ca²⁺ exchanger.² The orchestration of Ca²⁺ cycling is finely tuned and tightly regulated to insure proper contraction and relaxation, which are direct determinants of pump function.

Small, sustained increases of Ca²⁺ ingress result in an increase in systolic Ca²⁺ that is sufficient to change Ca²⁺ homeostasis. Fleckenstein³ first implicated Ca²⁺ imbalance as one possible underlying mechanism of cardiac dysfunction in both humans and animal models. Elevated intracellular Ca²⁺ has been observed with increased loads on isolated working hearts and in response to myocardial stretch.⁴ Additionally, long-term treatment with angiotensin II,⁵ phenylephrine,⁶ and isoproterenol⁶ elevates intracellular Ca²⁺, resulting in cardiac hypertrophy. Results from studies with genetic models of cardiac dysfunction, such as the cardiomyopathic Syrian hamster, also underscore the importance of calcium.⁷

The role of the L-VDCC in human heart failure remains controversial. Changes in the expression of the L-VDCC, either directly or indirectly, could contribute to heart failure because of the essential role that calcium channels play in the initiation and regulation of excitation-contraction coupling. Increased L-VDCC density has been reported in animal models with congestive heart failure^{7 8} and in patients with hypertrophic cardiomyopathy.^{9 10} Other reports, including one from our laboratory,¹¹ describe no change in L-VDCC density. In one report,¹² a putative decrease in L-channel density in human end-stage heart failure was described. Evidence is compelling that increased calcium ingress is a characteristic of certain cases of human cardiac hypertrophy in which a prolongation of the action potential¹³ or an increased availability and open probability of single L-type calcium channels¹⁴ were measured.

Cardiac-specific transgenesis has provided an effective means to address this complex problem. The genetic manipulation of selected proteins that result in decreased cytosolic Ca²⁺ does not cause myocardial damage or hypertrophy.^{15 16} In contrast, genetic manipulation that results in increased cytosolic Ca²⁺ does result in hypertrophy and failure.^{17 18} In the case of the phospholamban knockout mouse, which shows enhanced contractility and increased sarcoplasmic reticulum Ca²⁺, no hypertrophy or failure develops. The increased Ca²⁺ transient is primarily on the falling phase, and there is no change in either intracellular diastolic Ca²⁺ or in Ca²⁺ current density and voltage-dependence.^{19 20} Therefore, it seems from limited experiments that diastolic levels (ie, resting levels) of intracellular Ca²⁺ coordinate or perhaps signal the initiation of the hypertrophic gene program. One biochemical characteristic observed in human heart failure and in some models of hypertrophy and failure is an increased activation of protein kinase C (PKC).^{19 21}

We previously reported that 8-week-old transgenic mice with L-VDCC ₁-subunit overexpression show an increase in Ca²⁺ current, basal contractility, and defects in the ß-adrenergic signaling pathway.²² The defects in the signaling pathway occur before the development of hypertrophy. The present study investigated the consequences of a small, sustained Ca²⁺ increase through the channel on ventricular remodeling as a function of mouse age. Our results indicate a progression of myocardial disease, with the severity corresponding to the age of the transgenic mice, thus implicating the L-VDCC as an important contributor to heart failure. Furthermore, our biochemical data implicate PKC early in the disease process. This model seems to resemble the characteristics of some cases of slowly progressing human cardiac disease, and it may be a valuable addition to our armamentarium for studying mechanisms of cardiac hypertrophy.

	Methods

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_1C-Subunit Transgenic Mice
Transgenic mice that cardiac-specifically overexpress the L-VDCC _1C-subunit were generated as previously described.²² Transgenic mice began to die, presumably from pump failure, at the age of 8 to 10 months. Very few animals survived >1 year.

Cardiomyocyte Isolation and Electrophysiological Recordings
Single cardiomyocytes were isolated from 2-, 4-, and 8-month-old transgenic and nontransgenic hearts, as was previously reported.²² Ca²⁺ channel currents were recorded using the whole-cell mode of the patch-clamp method.²³ External and pipette solutions provided isolation of L-VDCC currents from Na⁺ and K⁺ channel currents and Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger.

Histological Examination
Transgenic and nontransgenic hearts were removed and prepared as previously described.²⁴ The fixed hearts were dehydrated, embedded in paraffin, sectioned (5 µm), and stained with either hematoxylin and eosin, Masson’s trichrome, or Von Kossa’s stain.

Detection of Apoptotic Cells
Terminal deoxyribonucleotide transferase–mediated dUTP nick-end labeling (TUNEL) assays were completed on transgenic and nontransgenic tissues according to the manufacturer’s recommendations (Promega). DAPI staining was completed on deparaffinized tissues for 10 minutes at room temperature in an environment protected from light.

Isolation of Particulate and Cytosolic Fractions and Western Analysis of PKC
Separation of particulate and cytosolic fractions was performed as previously described.²⁵ Particulate and cytosolic fractions (15 µg) were separated on a 4% to 20% SDS-PAGE gel and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked with 5% nonfat milk overnight at 4°C. Blots were incubated with PKC and PKC primary antibodies (Santa Cruz) at room temperature for 2 hours. Calsequestrin (Upstate Biotechnology) identification was completed to indicate equal loading. Horseradish peroxidase–conjugated secondary antibody was added for 1 hour at room temperature and visualized by the enhanced chemiluminescence procedure (Amersham Life Science). Quantitation of subcellular PKC localization was completed using ImageQuant Software (Molecular Dynamics).

Statistical Analysis
Data are reported as mean±SEM A Student’s t test was used for statistical comparison between heart weight/body weight values and PKC levels for transgenic and nontransgenic specimens. Single-cell data were analyzed by 2-way ANOVA to account for differences between individual animals and individual cells. P<0.05 was considered statistically significant.

	Results

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Ca²⁺ Channel Density in Cardiomyocytes
To investigate the number of functional Ca²⁺ channels in the mice with L-VDCC ₁-subunit overexpression, a detailed, age-dependent study of channel properties was completed. We previously demonstrated that the overexpression of the L-VDCC ₁-subunit resulted in an increase in mRNA expression and peak Ca²⁺ current.²² Examination of transgenic cardiomyocytes from 2-, 4-, and 8-month-old animals showed a significant increase in Ca²⁺ channel density (Figure 1A). Ca²⁺ channel density was increased by 44% in 2-month-old transgenic cardiomyocytes (12.04±0.56 pA/pF, n=26) compared with nontransgenic cells (8.35±0.60 pA/pF, n=24, P<0.001; Figure 1B); it was increased by 52% in 4-month-old cardiomyocytes (12.49±0.69 pA/pF, n=24; nontransgenic: 8.21±0.72 pA/pF, n=22, P<0.001; Figure 1C). Current density was increased in 8-month-old transgenic cardiomyocytes by 29% (10.10±0.44 pA/pF, n=37; nontransgenic: 7.83±0.38 pA/pF, n=48, P<0.001; Figure 1D).

View larger version (21K): [in this window] [in a new window]   Figure 1. Manifestation of increased Ca2+ ingress in transgenic cardiomyocytes. A, Ca2+ channel density in transgenic (Tg) and nontransgenic (Ntg) cardiomyocytes. Current densities were calculated at peak Ca2+ current and found to be significantly increased in 2-, 4-, and 8-month-old transgenic myocytes. B, Current-voltage relationship of 2-month-old animals. A marked increase in Ca2+ channel density in transgenic cells was identified. C, Current-voltage relationship in 4-month-old transgenic and nontransgenic cardiomyocytes. Transgenic cells also showed a significant increase in Ca2+ channel density. D, Current-voltage relationship in cardiomyocytes from 8-month animals. Transgenic cells showed a significant increase in Ca2+ channel density. n indicates number of cells measured from 4 to 7 animals. *P<0.05.

Induction of Cardiac Hypertrophy In Vivo by L-VDCC Overexpression
Beginning at 8 months of age, transgenic mice began to display overt symptoms of heart failure, which were hallmarked by lethargic movement, labored breathing, ruffled fur, hunched posture, peripheral edema, ascites, hepatomegaly, and edematous lungs. Gross examination of the heart revealed severe cardiomegaly in all 4 cardiac chambers (Figure 2A). White gelatinous zones appeared in the atria, suggesting organized thrombi (Figure 2A). Four-month-old transgenic mice displayed a significant increase in the heart weight/body weight ratio (4.57±0.13 versus 5.27±0.29, n=7, P<0.05). This index of cardiomegaly was even more pronounced in 8-month-old transgenic mice (4.89±0.12 versus 10.29±2.14, n=6, P<0.05; Figure 2B). The majority of transgenic mice died from heart failure within 1 year.

View larger version (22K): [in this window] [in a new window]   Figure 2. Cardiomyopathy in transgenic animals. A, Explanted hearts from 8-month-old nontransgenic (left) and transgenic (right) mice. Transgenic hearts display clear cardiomegaly and white granules in atria. B, Age dependence of myocardial hypertrophy in transgenic animals. Transgenic (open bars) and nontransgenic (solid bars) littermates were anesthetized and weighed and their hearts were removed. The hearts were then trimmed and weighed. Heart weight-to-body weight ratios (HW/BW) were significant in 4- and 8-month-old transgenic animals. Measurements were completed in a blinded fashion. *P<0.05.

Histological analysis of hearts from 8-month-old transgenic mice showed signs of hypertrophic myopathy (ie, 4-chamber dilation with mild thickening of the intraventricular septum; Figure 3B) compared with nontransgenic littermates (Figure 3A). In contrast to the characteristic, well-organized pattern of ventricular myocyte organization in nontransgenic animals (Figure 3C), cardiomyocytes from L-VDCC overexpression mice were poorly organized, and extensive interstitial fibrosis separated groups of myocytes (Figure 3D). Fibrosis and repair were present in 4- and 2-month-old hearts (Figures 3C through 3F).

View larger version (93K): [in this window] [in a new window]   Figure 3. Hypertrophy and tissue damage in L-VDCC overexpression transgenic hearts. Hearts from 8-month-old nontransgenic (A) and transgenic (B) mice were longitudinally sectioned and stained with hematoxylin and eosin. Hypertrophy and thrombosis were extensive in transgenic hearts. Hearts from 2-month-old nontransgenic (C) and transgenic (D) mice were stained with Masson’s trichrome (to reveal fibrosis) and visualized at magnifications of 200x and 1000x (insets), respectively. Myocardial damage was minimal. Hearts from 4-month-old nontransgenic (E) and transgenic (F) mice were stained with Masson’s trichrome, visualized, and photomicrographed. Myocardial damage seemed greater at 4 months than at 2 months in transgenic mice. Hearts from 8-month-old nontransgenic (G) and transgenic (H) mice were sectioned and stained with Masson’s trichrome. Extensive interstitial fibrosis surrounds cardiomyocytes. Magnification of A and B, 25x; C through H, 200x; insets, 1000x.

The modest but sustained increase ingress of Ca²⁺ beat-to-beat could trigger hypertrophy and ultimate heart failure. To investigate this possibility on a pathological basis, tissue sections were stained with Von Kossa’s stain. Hearts from 8-month-old transgenic mice revealed the presence of focal and densely stained myocytes (Figures 4C and 4F), which were absent in nontransgenic mice (Figures 4A and 4C). Calcium-laden cells occurred at a low frequency, but they could be easily identified in cardiac sections from 8-month-old transgenic mice. Interestingly, Von Kossa staining was not observed in the heart sections of 2- and 4-month-old transgenic mice (data not shown).

View larger version (108K): [in this window] [in a new window]   Figure 4. Calcium precipitates in transgenic mouse hearts. Hearts from nontransgenic (A) and transgenic (B) mice at 8 months of age were sectioned, processed, and stained with Von Kossa’s stain. Transgenic mouse heart sections revealed focal cells with dense calcium precipitates (arrow). C and D, Higher magnification of precipitates in A and B, respectively. Precipitates were absent in nontransgenic mouse samples (D). Magnification of A and B, 400x; C and D, 1200x.

Apoptosis Detection in Aged Transgenic Myocardium
Accumulating evidence in both human and animal models indicates the important role of apoptosis in cell death during heart failure. TUNEL assays in transgenic myocardial sections identified the presence of apoptosis in 8-month-old hearts (Figure 5A). Counterstaining the section with propidium iodide (Figure 5B) stained all nuclei of the section, and the overlay (Figure 5C) identified the apoptotic nuclei. In addition to DNA cleavage, apoptotic cells can be identified by DNA condensation and nuclear breakdown. Hoescht 33258 or DAPI staining identified sections with these morphometric changes in 8-month-old transgenic mice (Figure 5C) but not in nontransgenic mice (Figure 5D), thus providing additional evidence of the presence of apoptosis. Apoptotic cells were not detected in 2- and 4-month-old transgenic hearts.

View larger version (61K): [in this window] [in a new window]   Figure 5. Apoptosis in transgenic hearts. A, TUNEL labeling of an 8-month-old transgenic heart. Labeling of a tissue section identified cells undergoing apoptosis (arrows). B, Counterstaining with propidium iodide stained all nuclei of the section. C, Overlay of A and B demonstrated a colocalization of staining, thus confirming free 3'-OH groups (arrows). Nontransgenic (D) and transgenic (E) heart sections were stained with DAPI stain. Nuclei remained intact in nontransgenic sections, with a brightened staining of nucleolus. Nucleus breakdown and nuclear condensation and fragmentation were present in transgenic cardiomyocytes (arrows).

PKC and PKC Expression
We investigated the possible involvement of 2 PKC isoforms in cardiac hypertrophy in L-VDCC transgenic mice. Membrane translocation of PKC, a Ca²⁺-dependent isoform, was significantly increased in 2- and 4-month-old transgenic mice (1.8-fold and 3.3-fold, respectively; n=4 animals; Figures 6A and 6C). Interestingly, translocation of PKC in 8-month-old transgenic mice was mildly reduced from the levels observed in nontransgenic animals (Figures 6A and 6C). In contrast to activated PKC, activation of PKC was not significantly altered in any of the investigated age groups (Figure 6B).

View larger version (54K): [in this window] [in a new window]   Figure 6. Translocation of PKC isoforms in transgenic (Tg) and nontransgenic (Ntg) mice. Cytosolic and membrane fractions were prepared from 2-, 4-, and 8-month-old nontransgenic and transgenic hearts and separated on a 10% to 20% SDS-PAGE gel. A, Immunoblot analysis for PKC. Transgenic preparations showed an increased PKC translocation to membrane fraction (M) in 2- and 4-month-old animals. No significant difference was observed in membrane fractions in 8-month animals, but a decrease in cytosolic content (C) was observed. Blot containing 4-month-old samples was stripped and probed with calsequestrin antibody to identify equal loading. B, Immunoblot analysis of PKC. No statistical difference in PKC translocation was observed between transgenic and nontransgenic mice. C, PKC expression histogram. PKC blots were scanned and analyzed with ImageQuant software. Membrane-localized PKC was significantly increased in 2 and 4-month-old transgenic hearts compared with nontransgenic hearts. The 8-month-old transgenic hearts had a small but significant decrease in PKC membrane levels. n=4 animals; *P<0.05.

	Discussion

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Fleckenstein³ first suggested the calcium hypothesis ("Ca²⁺ sclerosis") of heart failure. Experiments from many laboratories provided a plethora of supporting data on a variety of experimental animal models ranging from surgical interventions to stress-related, parathyroid hormone injected, or genetically linked models. Typically, to provide "proof" of the Ca²⁺ hypothesis, organic Ca²⁺ antagonist drugs were administered; these drugs frequently prevented or attenuated the disease process in the animal models.³ From these and other approaches, biochemical and pathological assessments of animal and human diseased myocardium have implicated increased Ca²⁺ as a key player in the disease process.

The issue of cause and effect, however, remains a polemic, and interest in this concept has fallen since the 1980s. The small, sustained increases of Ca²⁺ observed in our transgenic model recapitulates, in a sense, the experiment cited above, in which an increased concentration of cellular calcium accompanied the failing process in acute, chronic, and human heart disease. Loss of Ca²⁺ homeostasis has been reported in failing human myocardium, with a multitude of potential molecular alterations responsible for the disease.²⁶ To find a "natural" or a genetically linked model, cardiomyopathic Syrian hamsters were developed.^{7 11} Although excess calcium in failing hearts from such models reproduced the acute and chronic heart failure–induced animal models cited above, the issue of the L-VDCC as a major source of the excess calcium became, and remains, controversial. Wagner and colleagues⁷ reported that the L-VDCC has increased density in human heart failure. Some contributions from other laboratories regarding human heart failure seemed to endorse these data, whereas others described no change in L-VDCC in Syrian hamster models¹¹ and in one case described a decrease in the "Dihydropyridine receptor" in severe end-stage human heart failure.¹²

In the present study, we approached the original problem by producing a transgenic mouse in which a modest increase in the density of the L-VDCC ₁-subunit was created, which approximated that which has been reported in human cardiomyopathy.¹⁰ This model has proven to be an interesting paradigm that closely resembles the characteristics of human cardiac hypertrophy in its slow progression. Our data provide evidence that the sustained, increased ingress of Ca²⁺ initiates the hypertrophic program with associated fibrosis, alterations in gene transcription, and apoptosis, as well as an elevation of PKC. Thus, the Ca²⁺ channel overexpression mouse model provides a useful tool to define the prominent intracellular signaling pathways influenced by aberrant Ca²⁺ signaling in heart failure and to emphasize a central role for Ca²⁺ in hypertrophy and, possibly, heart failure. With regard to the latter, we are not addressing the issue of calcium and heart failure in this article per se, but our evidence to date indicates that these animals died of heart failure within 1 year of age. The clinical signs and the pathological data strongly support this cause of death. In animals aged 8 to 10 months, attempts to measure physiological parameters on the isolated heart were only partially successful because the grossly enlarged hearts did not develop enough tension to make quantitative measurements.

The presence of apoptosis has been described in diseased human hearts²⁷ and in transgenic animal models of heart failure.^{28 29} The contribution of apoptosis to heart failure is unclear. However, the identification of apoptotic cells in only 8-month-old L-VDCC overexpression transgenic mice in heart failure suggests that it is a late event in the failing process. The calcineurin overexpression mouse develops ventricular dilation, hypertrophy, and failure without evidence of apoptosis.³⁰ Nevertheless, Ca²⁺ may play a role in triggering apoptosis in various cells and tissues.³¹ It is probable that elevated Ca²⁺ levels could initiate apoptosis in the cardiovascular system.

The role of cardiac PKC isoforms in the normal and failing myocardium is the subject of intense investigation.^{17 21 25 29} Emphasis has been placed on the -isoform of PKC because it is elevated in human heart failure.²¹ We found a significant activation of PKC in L-VDCC overexpression mice, particularly at 2 months, which is before the development of frank hypertrophy, suggesting an important " trigger" role for PKC in the induction of the hypertrophic program. A "cross-talk" of hypertrophic signaling pathways involving calcium, calcineurin, and PKC was recently reported.¹⁷ Thus, it is likely that PKC, which is Ca²⁺-dependent, is activated by the sustained increase of the Ca²⁺ current early on in our transgenic mice and serves as the initiator of the hypertrophic gene program. Additional PKC substrates include the ß-adrenergic receptor (ßAR)³² and a key ß-receptor modulator ßAR kinase (ßARK).³³ Phosphorylation of these substrates via PKC decreases ßAR signaling. Because our 2-month-old transgenic animals showed a striking loss of the inotropic response to isoproterenol,²² it is likely that PKC also underlies the blunting of the ß-adrenergic signaling pathway observed in our calcium channel overexpression mice. Furthermore, ß-adrenergic desensitization and elevation of ßARK1 levels precede the development of heart failure in both transgenic mouse models³⁴ and infarcted rabbits.³⁵ Thus, the web of signaling pathways including Ca²⁺, PKC, ßARK, and the ßAR are intimate players in the disease development of this model.

Our results indicate that overexpression of the L-VDCC initiates slowly developing hypertrophy and sets the stage for late-onset heart failure similar to that in humans. We want to emphasize that our hypothesis of increased calcium does not exclude mechanisms other than the Ca²⁺ channel as the source. We think that it is the increased intracellular calcium that serves as the initiator of the disease process. In the model described, a sustained elevation of intracellular Ca²⁺ secondary to an increase in Ca²⁺ channel density does result in activation of PKC, which leads to changes in ß-adrenergic signaling and gene transcription.

	Acknowledgments

 
This work was supported by Program Project Grant HL-22619 (to A.S.) and Training Grant HL-07382 (to A.S. and J.N.M.) from the National Institutes of Health.

Received September 11, 2000; revision received September 28, 2000; accepted October 2, 2000.

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