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(Circulation. 1998;97:892-899.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Expression of G_q and PLC-ß in Scar and Border Tissue in Heart Failure Due to Myocardial Infarction

Haisong Ju, MD; Shufang Zhao, MD, PhD; Paramjit S. Tappia, PhD; Vincenzo Panagia, MD, PhD; ; Ian M. C. Dixon, PhD

From the Laboratories of Molecular Cardiology (H.J., S.Z., I.M.C.D.) and Membrane Biology (P.S.T., V.P.), Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.

Correspondence to Ian M.C. Dixon, PhD, Institute of Cardiovascular Sciences, St Boniface General Hospital Research Centre, University of Manitoba, 351 Tache Ave, Winnipeg, Manitoba, Canada R2H 2A6. E-mail iand{at}sbrc.umanitoba.ca

	Abstract

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Background—Large transmural myocardial infarction (MI) leads to maladaptive cardiac remodeling and places patients at increased risk of congestive heart failure. Angiotensin II, endothelin, and ₁-adrenergic receptor agonists are implicated in the development of cardiac hypertrophy, interstitial fibrosis, and heart failure after MI. Because these agonists are coupled to and activate G_q protein in the heart, the aim of the present study was to investigate G_q expression and function in cardiac remodeling and heart failure after MI.

Methods and Results—MI was produced in rats by ligation of the left coronary artery, and G_q protein concentration, localization, and mRNA abundance were noted in surviving left ventricle remote from the infarct and in border and scar tissues from 8-week post-MI hearts with moderate heart failure. Immunohistochemical staining localized elevated G_q expression in the scar and border tissues. Western analysis confirmed significant upregulation of G_q proteins in these regions versus controls. Furthermore, Northern analysis revealed that the ratios of G_q/GAPDH mRNA abundance in both scar and viable tissues from experimental hearts were significantly increased versus controls. Increased expression of phospholipase C (PLC)-ß₁ and PLC-ß₃ proteins was apparent in the scar and viable tissues after MI versus controls and is associated with increased PLC-ß₁ activity in experimental hearts. Furthermore, inositol 1,4,5-tris-phosphate is significantly increased in the border and scar tissues compared with control values.

Conclusions—Upregulation of the G_q/PLC-ß pathway was observed in the viable, border, and scar tissues in post-MI hearts. G_q and PLC-ß may play important roles in scar remodeling as well as cardiac hypertrophy and fibrosis of the surviving tissue in post-MI rat heart. It is suggested that the G_q/PLC-ß pathway may provide a possible novel target for altering postinfarct remodeling.

Key Words: proteins • phospholipase • myocardial infarction • heart failure • remodeling

	Introduction

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Myocardial infarction is characterized by early infarct expansion in which the infarct region thins and elongates, and it is followed by discrete scar formation via the wound-healing response.^{1 2} The effect of scar formation is contingent on the size of the MI insofar as decreased net contractile force associated with a relatively small MI is sufficiently compensated by the viable myocardium, and thereby ventricular performance and geometry are maintained. After a large MI, ventricular chamber dilatation and sphericalization are attended by cardiac hypertrophy and interstitial fibrosis, leading to the loss of normal cardiac function.³ An understanding of the molecular mechanisms that underlie processes at the site of infarct healing as well as those during the development of interstitial cardiac fibrosis and hypertrophy is warranted.^{2 4}

Although the signaling properties of G_i2 and G_s in heart failure secondary to MI and hypertension have been investigated,^{5 6} specific information addressing the status of G_q expression and function in heart failure is lacking. The G_q protein is necessary and sufficient for the induction of cardiac myocyte hypertrophy mediated by phenylephrine in cultured cardiac myocytes.⁷ Ang II, ET, and ₁-adrenergic receptor agonists have been implicated in the development of maladaptive cardiac hypertrophy and fibrosis.^{8 9 10} Stimulation of AT₁,^{11 12} ET,^{13 14} or ₁-adrenergic receptors⁷ has been demonstrated to induce myocyte hypertrophy, which is mediated by G_q. Activated G_q protein is known to stimulate PLC-ß,¹⁵ which hydrolyzes phosphatidylinositol 4,5-bis-phosphate to release IP₃ and sn-1,2-diacylglycerol. Both IP₃ and 1,2-diacylglycerol are involved in proliferation of cardiac fibroblasts and myocyte hypertrophy mediated by further downstream signaling mechanisms.¹² It should be noted that Ang II concentration is increased at the site of infarct healing (scar) after induction of MI.¹⁶ Furthermore, increased expression of AT₁ receptors in both viable and scar tissues of post-MI rat hearts have been reported,^{17 18 19} suggesting that Ang II may be involved in wound healing of scar tissues as well as in myocyte hypertrophy. Similarly, increased ET and ET receptor density has been demonstrated in post-MI rat heart.⁸ Furthermore, increased cardiac ₁-adrenergic receptor density is present in rats with chronic heart failure.²⁰

It is conceivable that G_q is upregulated in post-MI hearts and is involved in the ongoing alteration of scar tissue as well as in the development of myocyte hypertrophy and fibrosis of surviving myocardium. The present study was conducted to examine myocardial G_q protein quantity and localization in the ventricular myocardium remote from the site of infarction as well as in border and scar regions of failing hearts subsequent to MI; in addition, cardiac G_q mRNA abundance was investigated. To examine the functional significance of G_q in post-MI hearts, we addressed the expression of downstream PLC-ß₁ and PLC-ß₃ proteins as well as PLC-ß₁ activity and IP₃ accumulation in experimental hearts.

	Methods

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Experimental Model
All experimental protocols for animal studies were approved by the Animal Care Committee of the University of Manitoba, Canada, following guidelines established by the Medical Research Council of Canada. MI was produced in male Sprague-Dawley rats (weighing 200 to 250 g) by surgical occlusion of the left coronary artery, as described previously, with minor modifications.²¹ In short, after isoflurane anesthesia, the chest was opened by cutting of the third and fourth ribs, and the heart was extruded through the intercostal space. The left coronary artery was ligated 2 to 3 mm from the origin with a suture (6–0 silk), and the heart was repositioned in the chest. The wound was closed with a purse-string suture. Throughout the operation, ventilation of the lungs was maintained by positive-pressure inhalation of 95% O₂/5% CO₂ mixed with isoflurane. Sham-operated animals were treated similarly, except that the coronary suture was not tied. The mortality of all animals operated on in this fashion was 45% within 48 hours. Eight weeks after surgery, the animals underwent cardiac function assessment and infarct size determination, and subsequently the viable LV (noninfarcted LV free wall remote from infarct and septum), border tissue (2 mm viable tissue and 2 mm scar tissue), and scar were used to assess G_q mRNA abundance as well as G_q protein concentration and localization. Furthermore, cardiac PLC-ß concentration and activity as well as IP₃ levels were investigated in the present study.

Hemodynamic Measurements
LV function and blood pressure of control and MI animals were measured 8 weeks after induction of MI, as described previously.²¹ Briefly, rats were anesthetized by injection of ketamine/xylazine (100:10 mg/kg IP). A micromanometer-tipped catheter (2–0) (Millar SPR-249) was inserted into the right carotid artery. The catheter was advanced into the aorta to measure blood pressure and then advanced into the LV to record LV systolic pressure, LVEDP, the maximum rate of isovolumic pressure development (+dP/dt_max), and the maximum rate of isovolumic pressure decay (-dP/dt_max). Hemodynamic data were computed instantaneously and displayed on a computer data acquisition workstation (Biopac, Harvard Apparatus Canada). A total of 18 rats were included in the function measurement.

Infarct Size
After heart function recordings, the LV was fixed by immersion in 10% formalin and embedded in paraffin. Six transverse slices were cut from the apex to the base. Serial 5-µm sections were made and mounted. The percentage of infarcted LV was estimated at 8 weeks after coronary ligation by planimetric techniques as described previously.²² Animals (n=16) with large infarcts (40% of the LV free wall) were used in this study.

Determination of Cardiac Total Collagen
Samples from sham-operated and MI groups were ground into powder in liquid nitrogen. Then 100 mg (wet wt) cardiac tissue was dried to constant weight. Tissue samples were digested in 6 mol/L HCl (0.12 mL/mg dry wt) for 16 hours at 105°C. Hydroxyproline was measured according to the method of Chiariello et al.²³ A stock solution containing 40 mmol/L of 4-hydroxyproline in 1 mmol/L HCl was used as a standard. Collagen concentration was calculated by multiplying hydroxyproline levels by a factor of 7.46, assuming that interstitial collagen contains an average of 13.4% hydroxyproline.²³ The data were expressed as µg collagen/mg dry tissue.

Immunofluorescence
A total of 9 rats after surgery were used in this assay: 4 sham, 5 post-MI. After anesthesia with ketamine/xylazine, animals were killed by decapitation. Hearts were rapidly excised and immersed immediately in PBS solution, pH 7.4. The viable LV remote from the infarct and scar were immersed in OCT compound (Miles Inc) and stored at -80°C. Serial cryostat sections, 7 µm thick, of the ventricular tissues were mounted on gelatin-coated slides, prefixed in 4% paraformaldehyde, and allowed to air-dry. A minimum of six sections from each ventricle of each group were processed, and representative sections were chosen. Immunohistochemical staining was performed by the indirect immunofluorescence technique described in detail previously.²² Rabbit polyclonal anti-G_q subunit (Calbiochem-Novabiochem International) at 0.4 mg/mL were diluted 1:500 with 1% BSA in PBS and applied as the primary antibody. After incubation overnight at 4°C, the sections were washed in PBS and incubated with biotinylated anti-goat IgG secondary antibody and subsequently incubated with FITC-labeled streptavidin (Amersham Life Sciences Inc Canada) for 90 minutes. Finally, the slides were mounted and coverslipped. The tissue sections were examined under a Nikon Labophot microscope equipped with epifluorescence optics and appropriate filters. The results were recorded by photography on Kodak TMAX 400 black-and-white film.

Western Blot
G_q, PLC-ß₁, and PLC-ß₃ were detected by Western blot analysis. Cardiac tissues from sham-operated LV, viable LV, border area, and scar were homogenized in 100 mmol/L Tris (pH 7.4) containing 1 mmol/L EDTA, 1 mmol/L PMSF, 4 µmol/L leupeptin, 1 µmol/L pepstatin A, and 0.3 µmol/L aprotinin. Samples were sonicated for 3x5 seconds. Crude membrane and cytosolic fraction was isolated according to the method of Gettys et al.²⁴ Briefly, samples were centrifuged at 3000g at 4°C for 10 minutes to remove unbroken cells and nuclei. The supernatant was further subjected to centrifugation at 48 000g for 20 minutes at 4°C. The subsequent crude membrane pellet was resuspended in the homogenizing buffer. Total protein concentration of membrane fractions was measured by the bicinchoninic acid method.²⁵ Prestained high-molecular-weight marker (Bio-Rad) and 20-µg proteins from samples were separated on 10% (G_q) and 6% (PLC-ß₁ and PLC-ß₃) SDS-PAGE. Separated proteins were transferred onto 0.45-µm polyvinylidene difluoride membrane. This membrane was blocked overnight at 4°C in TBS-T containing 5% skim milk and probed with primary antibodies G_q (Calbiochem-Novabiochem International), PLC-ß₁, and PLC-ß₃ (Santa Cruz Biotechnology, Inc). Primary antibodies were diluted in TBS-T (G_q in 1:1000, PLC-ß₁ and PLC-ß₃ in 1:250). Horseradish peroxidase–labeled anti-rabbit IgG was diluted in 1:10 000 in TBS-T and used as secondary antibody. The G_q and PLC-ß were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Life Science Inc Canada). Autoradiographs from the Western blot were quantified with a CCD camera imaging densitometer (Bio-Rad GS 670).

RNA Extraction and Northern Blot Analysis
Total RNA was isolated from sham-operated, viable LV, border, and scar 8 weeks after operation by the method of Chomczynski and Sacchi²⁶ as described previously.²⁷ A total of 12 animals were included in this assay. Recovered RNA was dissolved in diethyl pyrocarbonate–treated water, and the concentration of nucleic acid was calculated from the absorbance at 260 nm before size fractionation. Total RNA (20 µg) was electrophoresed in a 1.2% agarose/formaldehyde gel, and the fractionated RNA was transferred to a 0.45-µm positive charge–modified nylon membrane (NYTRAN Plus, Schleicher & Schuell). The RNA was covalently cross-linked to the membrane with UV radiation (UV Stratalinker 2400, Stratagene). Blots were prehybridized at 42°C for 16 hours. Each membrane was hybridized with cDNA probes labeled with ³² P by a random primer labeling kit (specific activity, >10⁹ cpm/µg DNA) at 42°C for 16 to 20 hours. After washing, the membranes were exposed to x-ray film (Kodak X-OMAT) at -80°C with intensifying screens. The cDNA fragments for human G_q and GAPDH were obtained from the American Type Culture Collection. Results of autoradiographs from Northern blot analysis were quantified by densitometry (Bio-Rad, GS 670). The signals of specific mRNAs were normalized to those of GAPDH to normalize for differences in loading and/or transfer of mRNA.

Immunoprecipitation of PLC-ß₁ and Assay for PLC-ß₁ Activity
Crude membrane proteins were extracted with buffer containing 1% wt/vol Na-cholate, 50 mmol/L HEPES (pH 7.2), 200 mmol/L NaCl, 2 mmol/L EDTA, 10 µg/mL PMSF, and 10 µg/mL leupeptin by rotation for 2 hours at 4°C. The samples (n=18) were then centrifuged (280 000g for 25 minutes), and the supernatant was recovered as the solubilized membrane fraction. The membrane extract was incubated overnight at 4°C (rotation) with mixed monoclonal antibodies to PLC-ß₁ (5 µg antibody to 350 µg membrane extract, ie, a ratio of 1:70 µg/µg). The immunocomplex was captured by addition of 100 µL washed protein G sepharose bead slurry (50 µL packed beads) at 4°C by rotation for 2 hours. The agarose beads were collected by pulse centrifugation (5 seconds) at 10 000g and assayed for PLC-ß₁ activity. The hydrolysis of PtdIns 4,5-P₂ was measured according to the method described by Wahl et al with minor modification.²⁸ Briefly, the reaction was performed in the presence of 30 mmol/L HEPES (pH 6.8), 70 mmol/L KCl, 100 mmol/L NaCl, 0.8 mmol/L EGTA, 0.8 mmol/L CaCl₂, and 20 µmol/L [³H]PtdIns 4,5-P₂ dissolved in 14 mmol/L Na-cholate overnight and an aliquot (10 µL) of immunoprecipitate suspension. The reaction was carried out at 37°C for 2.5 minutes, after which the reaction was stopped by trichloroacetic acid precipitation. Precipitates were removed by centrifugation at 11 000g for 5 minutes, and the supernatant was collected for quantification of inositol phosphates by liquid scintillation counting.

Measurement of Cardiac IP₃ Accumulation
Cardiac tissues from sham, viable, and scar+border regions were used for the measurement of IP₃ with the Biotrak radioimmunoassay kit (Amersham Life Science Inc Canada). Briefly, the cytosolic fraction from different regions of post-MI heart was prepared the same way as in the Western blot analysis.²⁴ All other procedures followed the manufacturer's instructions modified according to the method of Chilvers et al.²⁹ Unlabeled IP₃ in the samples competes with fixed amount of [³H]-labeled IP₃ for a limited number of bovine adrenal IP₃ binding proteins. Bound IP₃ is then separated from the free IP₃ by centrifugation. D-myo-Inositol 1,4,5-trisphosphate was used as standard. Results were expressed as pmol/mg protein.

Statistical Analysis
All values are expressed as mean±SEM. One-way ANOVA followed by Bonferroni's test was used for comparing the differences among multiple groups (SigmaStat). Significant differences among groups were defined by a value of P<.05.

	Results

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General Observations: LV Cardiac Hypertrophy, Fibrosis, and Heart Failure
Experimental animals in this study were characterized by the presence of large MI, comparable to values reported earlier (Table). Hearts of experimental animals underwent significant cardiac hypertrophy, which was reflected by an increase in the mass of viable LV weight and also by the increased ratio of LV weight to body weight in 8-week experimental animals compared with control values. The incidence and magnitude of LV hypertrophy noted in this study were comparable to our previous findings,²¹ as was the averaged transmural scar weight (one measure of the extent of MI) from experimental animals. Animals were assessed for LV function at 8 weeks after MI, and these data revealed an increase in LVEDP and a decrease in ±dP/dt_max relative to their controls. Lung congestion was noted by the ratio of wet weight to dry weight (Table). Collagen concentration in myocardium remote from the site of infarct (47.0±3.2 µg/mg dry wt) and border+scar tissues (110.4±12.4 µg/mg dry wt) were both significantly higher than that of the sham control value (22.4±2.4 µg/mg dry wt).

View this table: [in this window] [in a new window]   Table 1. General and Hemodynamic Characteristics of Sham and Experimental Rats 8 Weeks After Induction of MI

Localization of Cardiac G_q
G_q protein distribution in 8-week experimental and age-matched control tissues was localized by immunofluorescence techniques. In the representative photograph (Fig 1), the staining pattern of immunoreactive G_q is marked by bright clusters along the myocyte cell membranes. The results demonstrate that relatively strong staining of G_q was localized in scar tissue proper and in hypertrophied cardiac myocytes that bordered on scar tissue; comparatively less immunoreactive protein was visualized in surviving (viable) tissues and in control myocardium (Fig 1).

View larger version (71K): [in this window] [in a new window]   Figure 1. Immunohistochemically stained sections showing Gq in sham hearts and viable, border, and scar tissues from post-MI (8 weeks) animals. Immunoactive Gq protein appear as brightly stained material (arrows). Magnification x400.

Changes in Cardiac G_q Protein Abundance in Hearts With MI
Quantitative assessment of cardiac membrane G_q-protein expression in control and LV tissues of 8-week post-MI rats was carried out by Western blot techniques. Fig 2A provides a representative autoradiograph illustrating the presence of a characteristic 42-kD band for G_q protein. These data indicated that G_q was increased by 2.0- and 2.5-fold in border and scar tissue, respectively, compared with band intensity from control animals. There was no significant alteration of the G_q band intensity in samples from viable LV versus controls (Fig 2B).

View larger version (48K): [in this window] [in a new window]   Figure 2. Western blot for Gq in sham, viable, border, and scar tissues from 8-week experimental animals. A, Representative Western blot showing specific band for 42-kD Gq. Lanes 1 and 2 are sham, lanes 3 and 4 are viable LV, lanes 5 and 6 represent border tissue, and lanes 7 and 8 are scar. B, Quantified data of Gq protein concentration in sham, viable, border, and scar tissue. Control group is sham-operated rats age-matched to the 8-week post-MI experimental group. Data are mean±SEM of six experiments. *P<.05 and +P<.05 vs sham and viable sample values, respectively.

Alteration of Steady-State mRNA Abundance of Cardiac G_q
We addressed mRNA abundance of the cardiac G_q gene in tissues taken from various LV regions of rats 8 weeks after MI. Fig 3A shows a representative Northern blot with autoradiographic bands for G_q and GAPDH mRNAs from LV samples of sham, viable, and border+scar tissues. The transcription of the G_q gene is variably processed, as reported by others³⁰ ; we found the presence of three different G_q transcripts of 7.5, 6, and 5 kb in our blots (Fig 3A). Estimation of G_q mRNA abundance was calculated by the ratio of G_q to GAPDH signal; this ratio was significantly increased in both the viable (1.4-fold) and border+scar tissue (3-fold) regions of the LV versus controls (Fig 3B). Furthermore, mRNA signal ratios from border+scar tissue samples were significantly increased (2.2-fold) in G_q/GAPDH mRNA values versus those obtained from surviving viable LV.

View larger version (43K): [in this window] [in a new window]   Figure 3. A, Representative autoradiograph from Northern blot analysis showing Gq bands of 7.5, 6, and 5 kb in sham (lanes 1 to 6), viable (lanes 7 to 12), and border and scar tissues (lanes 13 to 18) from hearts of 8-week post-MI rats. Hybridization of fractionated total RNA with cDNA probes for Gq and GAPDH indicates relative steady-state mRNA levels for each gene tested. B, Quantified data of Gq/GAPDH in sham, viable, and border and scar tissue. Data are mean±SEM of six experiments. *P<.05 and +P<.05 vs sham and viable sample values, respectively.

Alteration of Cardiac PLC-ß Protein Expression and Activity
Western analysis was used to determine immunoreactive PLC-ß protein bands. Fig 4A and 4B depicts a representative blot with bands corresponding to PLC-ß₁ (150- and 140-kD) and PLC-ß₃ (152-kD) proteins, respectively. Densitometric analysis of band intensity revealed a significant increase in both PLC-ß₁ (Fig 4C) and PLC-ß₃ (Fig 4D) protein abundance in viable, border, and scar tissues compared with scanned samples from control hearts. To determine whether actual PLC-ß₁ activity was altered in the surviving (viable) and scar tissue from experimental hearts, PLC-ß₁ proteins were immunoprecipitated from solubilized membrane extracts of the aforementioned tissues (Fig 5). These experiments revealed that PtdIns 4,5-P₂ hydrolysis by PLC-ß₁ was significantly increased in surviving (viable) myocardium from experimental hearts versus age-matched controls. Furthermore, PLC-ß₁–mediated PtdIns 4,5-P₂ hydrolysis activity from scar lysates was significantly elevated compared with values from both control and viable groups.

View larger version (24K): [in this window] [in a new window]   Figure 4. Western blot for PLC-ß in sham, viable, border, and scar tissue in post-MI (8-week) cardiac tissues. A, Representative Western blot showing 150- and 140-kD PLC-ß1. Lanes 1 through 3 are sham, lanes 4 through 6 are viable LV, and lanes 7 through 9 represent border tissue and scar. B, Representative Western blot showing 152-kD PLC-ß3. Lanes 1 through 3 are sham, lanes 4 through 6 are viable LV, and lanes 7 through 9 represent border tissue and scar. C, Quantified data of PLC-ß1 protein concentration in sham, viable, border, and scar tissues. D, Quantified data of PLC-ß3 protein concentration in sham, viable, border, and scar tissues. Data are mean±SEM of six experiments. *P<.05 and +P<.05 vs sham and viable sample values, respectively.

View larger version (31K): [in this window] [in a new window]   Figure 5. Cardiac PLC-ß1 activity in membrane fraction isolated from sham, viable, and border and scar tissues in post-MI (8-week) animals. PLC-ß1 activity is expressed as pmol · min-1 · mg-1. Data are mean±SEM of six experiments. *P<.05 and +P<.05 vs sham and viable sample values, respectively.

Alteration of Cardiac IP₃
IP₃ is a downstream signal molecule generated by PLC activity, and its concentration was detected in various tissues from post-MI and control hearts. IP₃ concentration was markedly increased in border+scar tissue compared with values derived from assays of viable and sham control samples (Fig 6).

View larger version (30K): [in this window] [in a new window]   Figure 6. Cardiac IP3 concentration in cytosolic fraction isolated from sham, viable, and border and scar tissues in post-MI (8-week) animals. IP3 concentration is expressed as pmol/mg protein. Data are mean±SEM of four to six experiments. *P<.05 vs sham and viable sample values, respectively.

	Discussion

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MI and Heart Failure
Loss of normal LV systolic pressure and elevated LVEDP, decreased ±dP/dt_max, and the presence of pulmonary congestion were noted in the 8-week experimental animals. Although these cardiac functional abnormalities were clearly influencing systemic tissues, these animals did not display overt dyspnea, cyanosis, or marked lethargy and thus were considered to be in a stage of "moderate heart failure." This classification matched our previous observation that the development of post-MI heart failure in rats with relatively large MI (40% LV free wall) is time-dependent and was in agreement with our previous arbitrary classification system established to facilitate the comparison of differently timed experimental groups.²¹ The incidence of heart failure with clinical signs was established in the present study to provide a basis for objective comparison of cardiac dysfunction with changes in ventricular expression and function of target genes and gene products, respectively.

Role of G_q Expression and Function in Failing Hearts: Experimental and Clinical Studies
The neurohormonal activation of the sympathetic and renin-angiotensin axes and the stimulation of various growth factors is important for the development of failure.^{10 31} Among these factors, Ang II, norepinephrine, and ET are major players in the regulation of cardiac fibrosis and myocyte hypertrophy, and G_q is known to serve as the common signal coupler for these factors.⁶ In a variety of clinical and experimental studies, altered G_i and G_s expression and function have been suggested as a mechanism mediating the development of heart failure.⁶ Heart failure subsequent to MI has been reported to be associated with altered bioactivity and/or expression of G_s and G_i.^{5 32} Although it has been reported that both G_q and PLC-ß are expressed in the heart,³³ the alteration of this signaling pathway in heart disease is unknown. The present study demonstrates for the first time that G_q expression (both mRNAs and protein) is increased at the site of infarct healing and in myocardium bordering the scar. Upregulated expression of the G_q and PLC-ß pathway was evidenced in scar itself and in myocardium bordering the scar, as well as in viable tissue from post-MI heart. G_q expression in tissues from post-MI hearts positively correlated with increased expression of its downstream effectors (PLC-ß₁ and PLC-ß₃) as well as increased PLC-ß₁ activity. The latter findings together with increased IP₃ accumulation suggest that the signal amplification function of G_q is increased in scar tissue and border myocardium in experimental hearts. Translocation of cardiac protein kinase C is known to occur in association with G_q activation; this parameter remains to be demonstrated in our experimental hearts. The high level of G_q and PLC-ß activity and expression in the relatively hypocellular scar may be explained by the presence of myofibroblasts at this site, which are distinguished from other fibroblasts by their expression of -actin.^{34 35} These cells have been localized in the scar 90 days after MI in rat heart³⁴ and are known to express Ang II, ACE, and Ang II receptors.^{19 36} We have observed the presence of a substantial number of these cells in the scar from 8-week experimental animals (data not shown). Furthermore, scar from post-MI patients are known to be populated by myofibroblasts and to persist in these hearts for years.³⁷ The high level of G_q and PLC-ß expression may reflect the hyperfunctionality of these myofibroblasts in ongoing scar remodeling. Our data provide strong evidence of a correlation between the enhanced expression and function of the G_q/PLC-ß pathway at the site of infarct healing as well as in the development of post-MI cardiac hypertrophy and heart failure. This hypothesis is supported by a recent study demonstrating that overexpression of G_q induces cardiac hypertrophy and heart failure.³⁸

Increased G_q Expression: Molecular Mechanisms
The precise molecular mechanisms for increased G_q expression in scar and border regions in infarcted heart are unknown. Upstream receptors (Ang II, ET, etc) for G_q activation are characteristically upregulated in surviving LV myocardium in experimental animals, and these alterations occur in relatively early stages of healing after MI.^{8 17} Administration of losartan, an AT₁ receptor antagonist, has been associated with the attenuation of cardiac hypertrophy and fibrosis in post-MI hearts.³⁹ Recently, the application of an ET receptor blocker (BQ-123) was associated with improved heart function and reduced mortality after MI.⁸ Other data indicate that the acute phase of MI (1 week) is not associated with alteration of cardiac G_q protein content in myocytes isolated from viable tissues.⁴⁰ We suggest that changes in the receptor density of multiple neurohormonal factors may lead to increased downstream G_q expression, and further experiments are necessary to test this hypothesis. Our data provide new evidence that expression and function of the common molecular pathway for these receptors are augmented in scar and surviving post-MI myocardium.

It is well known that G_q selectively activates PLC-ß₁ but not PLC-₁ or PLC- isoforms^{15 41} and that the activation of PLC-ß isoforms may occur in the following order: PLC-ß₁PLC-ß₃PLC-ß₂.⁴² Thus, increased PLC-ß activity in experimental hearts may be mediated mainly through the activation of G_q. The significance of the elevated PLC-ß₁ activity is as yet unclear. We suggest that activation of PLC-ß is associated with incidence of wound healing at the site of infarction as well as in cardiac hypertrophy and fibrosis in viable tissue, on the basis of its regulatory role in cell growth and differentiation. Because multiple hormonal systems have been implicated in the pathogenesis of heart failure, abrogation of a single system may be insufficient to prevent the development of subsequent hypertrophy and fibrosis in heart failure. However, the specific modulation of the G_q/PLC-ß pathway may provide a new therapeutic approach for prevention and treatment of heart failure.

Ongoing Remodeling of Scar Tissue: A Case for Chronic Wound Healing
Heart failure due to MI is characterized not only by cardiac hypertrophy but also by fibrosis of scar and myocardium remote from infarcted tissue both in patients and in the rat experimental model.¹⁰ Although interstitial fibrosis and attendant decreased compliance of the surviving myocardium is believed to contribute to the occurrence of cardiac dysfunction,⁴³ it has become clear that the size of the scar is a reliable marker for the development of heart failure after MI.⁴⁴ Recently, examination of collagen architecture from scars of post-MI rats has revealed that scars develop as highly anisotropic tissues, allowing the scar to resist circumferential stretching while maintaining longitudinal deformation compatibility with adjacent noninfarcted myocardium.⁴⁵ This finding supports a specific role for the healing scar in preservation of function of the infarcted ventricle. In this regard, it has been suggested that progressive regional cardiac remodeling of the infarcted ventricle may depend on scar size, transmurality, scar wall thickness, and collagen content of the healed scar.⁴⁶ Although gross morphological examination of experimental hearts has indicated that scar formation is complete 3 weeks after MI,⁴⁷ our findings suggest that the scar is not quiescent even later than 8 weeks after MI, as indicated by the activation of G_q/PLC-ß in scars. Our finding agrees with recent work showing that Ang II receptors are highly expressed in scar during the chronic phase of post-MI wound healing.^{19 48 49} We suggest that altered G_q/PLC-ß expression/function occurs beyond the classically defined period of infarct healing. It is possible that enhanced G_q/PLC-ß expression is involved in the ongoing remodeling of scar morphology. Therefore, the mechanisms that are activated during the wound healing of the infarct per se may not be terminated within a brief defined period.

In conclusion, the present study has demonstrated that the cardiac G_q/PLC-ß pathway was activated in heart failure subsequent to MI. Therefore, it is suggested that G_q and PLC-ß may play an important role in the evolution of scar remodeling, cardiac hypertrophy, and fibrosis of the surviving tissue in post-MI rat heart and that these events may be linked to the development of heart failure. Thus, pharmacological modulation of the G_q/PLC-ß pathway may provide a possible novel target for altering postinfarct remodeling.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1 = Ang II type 1 receptor DAG = sn-1,2-diacylglycerol ET = endothelin IP3 = inositol 1,4,5-tris-phosphate LV = left ventricle, left ventricular LVEDP = left ventricular end-diastolic pressure MI = myocardial infarction PLC = phospholipase C PtdIns 4,5-P2 = phosphatidylinositol 4,5-bis-phosphate TBS-T = Tris-buffered saline with 0.1% Tween-20

	Acknowledgments

 
This study was supported by funding from the Heart and Stroke Foundation of Manitoba (Dr Dixon). Dr Dixon is a scholar of the Medical Research Council of Canada/PMAC health program with funding provided by Astra Pharma, Inc (Canada). H. Ju is a recipient of a Manitoba Health Research Council Studentship. We would like to thank Tracy Scammell-La Fleur for the excellence of her technical assistance.

Received September 2, 1997; revision received October 7, 1997; accepted October 10, 1997.

	References

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