AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 287: H719-H727, 2004. First published April 8, 2004; doi:10.1152/ajpheart.01107.2003
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Phospholipase C gene expression, protein content, and activities in cardiac hypertrophy and heart failure due to volume overload

Melissa R. Dent,1 Naranjan S. Dhalla,1 and Paramjit S. Tappia2

1Department of Physiology, Faculty of Medicine; and 2Department of Human Nutritional Sciences, Faculty of Human Ecology, Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada

Submitted 24 November 2003 ; accepted in final form 8 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Volume overload due to arteriovenous (AV) shunt results in cardiac hypertrophy followed by the progression to heart failure. The phosphoinositide phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP2) to 1,2-diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate (IP3), which are known to influence cardiac function. Therefore, we examined the time course of changes in DAG and IP3 as well as PLC isozyme gene expression, protein content, and activities in cardiac hypertrophy and heart failure induced by AV shunt in Sprague-Dawley rats by the needle technique. An increase in the left ventricle (LV)-to-body weight ratio demonstrated that LV hypertrophy was established at 4 wk after the induction of the shunt. PLC-{beta}1 activity was increased two- and sevenfold at 3 days and 1 and 2 wk after the induction of volume overload, respectively. These changes were associated with increases in the mRNA and sarcolemmal (SL) protein content; however, no changes in PLC-{beta}1 were detected at 4 wk. On the other hand, a significant increase in PLC-{gamma}1 activity as well as mRNA and SL protein was seen at 3 days and 4 wk. A progressive decrease in PLC-{delta}1 activity with concomitant reductions in the gene expression and SL protein abundance was detected during 1 to 4 wk. Activity of {gamma}1- and {delta}1-isozymes was significantly depressed during the 8- and 16-wk time points, whereas {beta}1-isozyme was increased significantly during these time points. A progressive decrease in the SL PIP2 content was observed during cardiac hypertrophy and heart failure. Our findings indicate that PLC isozyme signaling processes are increased in hypertrophy and decreased in heart failure due to volume overload.

cardiac sarcolemma; signal transduction

PHOSPHOINOSITIDE-SPECIFIC phospholipase C (PLC) isozymes play a central role in activating intracellular signal transduction pathways, during early key events in the regulation of various cell functions (6, 20, 22, 35). Its most common physiological substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), is converted into two messenger molecules, inositol (1,4,5)-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). These two products participate in many different physiological processes (14, 18, 24–25, 33–34), including myocyte hypertrophy via downstream signaling mechanisms and Ca2+ movements within the cardiomyocyte. In addition, PIP2 also serves as an anchor point for proteins containing pleckstrin-homology (PH) domains (51).

The role of PLC in the development of some types of cardiac hypertrophy has been documented; for example, in stroke-prone spontaneously hypertensive rats, the development of cardiac hypertrophy has been suggested to involve an increase in the PLC signaling pathway (23, 40). Other studies with the cardiomyopathic hamster (BIO 14.6) have shown that the development of cardiac hypertrophy is associated with an increase in PLC activity as a consequence of an enhanced responsiveness to ANG II (37). Lamers et al. (26) have reported that stimulation of cultured rat neonatal cardiomyocytes by endothelin-1 induces a rapid activation of PLC-{beta} isozymes and is accompanied by characteristic phenotypic changes related to the development of cardiac hypertrophy (26). In addition, recent studies in neonatal rat cardiomyocytes stimulated with different hypertrophic stimuli have shown an increased mRNA expression of PLC-{beta} isozymes (39). On the other hand, reductions in PLC isozyme activities and protein abundance at the cardiac sarcolemmal (SL) membrane have been reported in congestive heart failure (CHF) due to myocardial infarction (43) as well as in the failing heart of the cardiomyopathic hamster (UM-X7.1) (51).

Although these lines of evidence demonstrate an increase in PLC isozyme activity during the development of hypertrophy and specific decreases in PLC isozyme activities in the failing heart, no information is available in the literature regarding the status of the PLC signaling pathway in cardiac hypertrophy and heart failure due to volume overload. We have recently reported and perfected the needle technique to create an arteriovenous (AV) shunt or fistula for inducing volume overload that results in consistent hypertrophic growth, which develops within the first 2 wk after the induction of the AV shunt (47), followed by progression to moderate and chronic CHF at 8 and 16 wk, respectively, after the surgical procedure. Given the role of PLC and its products in influencing cardiac function, the present study was therefore undertaken to investigate the time course of changes in the level of cytosolic IP3 and SL DAG and how these relate to the PLC isozyme status during the development of cardiac hypertrophy and subsequent progression to heart failure due to volume overload. In addition, the SL amount of PIP2 was also assessed during this time course.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental model. All experimental protocols for animal studies were approved by the Animal Care Committee of the University of Manitoba, following the guidelines established by the Canadian Council on Animal Care. An arteriovenous (AV) shunt was performed in male Sprague-Dawley rats (weighing 150–200 g). The animals were anesthetized with 5% isoflurane with a flow rate of oxygen (2 l/min). After the abdominal fur was shaved, an abdominal laparotomy was then performed. After exposure of the abdominal aorta and inferior vena cava between the renal arteries and ileac bifurcation, the descending aorta and the ileac bifurcation will be temporarily occluded proximal to the intended puncture site. An 18-gauge needle will be inserted and withdrawn across the medial wall of the descending aorta three times to ensure the size and presence of the shunt and finally withdrawn. The puncture site will then be sealed immediately with a drop of isocyanate (Krazy glue). The creation of the shunt was visualized by the pulsatile flow of oxygenated blood into the vena cava from the abdominal aorta. Throughout the operative procedure, the rats were maintained on 2.5% isoflurane in 2 l/min of oxygen. Age-matched, sham-operated animals served as controls and were treated similarly, except that the puncture into the descending aorta was not performed. The animals were allowed to recover and were maintained on food and water ad libitum. The circulation system was only occluded for 25 s–1 min, and the entire procedure was finished within 10 min. It is pointed out the mortality rate of the control group was 0% and the mortality rate of the AV shunt animals operated on in this manner was <4%, 6 h after surgery. After this time point no mortality was seen in either group due to the surgical procedure.

Hemodynamic studies in vivo. The left ventricular (LV) function of animals from the each of the sham-operated and AV shunt groups was assessed. Rats were anesthetized by an injection of ketamine-xylazine (100:10 mg/kg ip). The right carotid artery was exposed, and a micromanometer-tipped catheter (2-0; Millar SPR-249) was inserted and advanced into the LV. The catheter was secured with a silk ligature around the artery, and after a 15-min stabilization of the heart function, LV pressures and maximum rates of isovolumic pressure development (+dP/dtmax) and decay (–dP/dtmax) were recorded. Hemodynamic data were computed instantaneously and displayed on a computer data-acquisition workstation (Biopac, Harvard Apparatus). It is pointed out that after hemodynamic assessment the LV tissue was frozen in liquid N2 and stored at –80°C.

Preparation of cardiac SL membrane. The LV tissue from four to five hearts was pooled to prepare a SL membrane fraction. Briefly, the tissue was washed, minced, and homogenized in 3.5 ml 0.6 mol/l sucrose, 10 mmol/l imidazole, pH 7.0/g tissue with a Polytron (6 x 10 s, setting 5). Large cellular particles were removed by centrifugation at 12,000 g (30 min, 4°C). A small aliquot of the first supernatant was centrifuged at 110,000 g (60 min, 4°C), and the resulting supernatant was frozen and stored (–80°C) as the soluble cytosolic fraction. The rest of the first supernatant was diluted with 300 mmol/l KCl containing 20 mmol/l MOPS/KOH, pH 7.4 to solubilize myofibrillar proteins and further processed for the preparation of SL according to the method used previously (3, 43). The final pellet was resuspended in 0.25 mol/l sucrose, 10 mmol/l histidine (pH 7.4), frozen in liquid N2, and stored at –80°C until assayed. All the above steps were carried out at 0–4°C. Protein concentrations were determined by the Lowry method as described elsewhere (43).

Determination of PLC isozyme activities. Measurement of SL PLC isozyme activities, by immunoprecipitation, was conducted as already reported (3). Briefly, solubilized membrane proteins were incubated overnight at 4°C with monoclonal antibodies to PLC-{beta}1, -{delta}1, and -{gamma}1 (5 µg of antibody to 350 µg membrane extract). The immunocomplex was captured by adding 100 µl (50 µl packed beads) of washed (3 times with 30 mM HEPES; pH 6.8) protein G-Sepharose bead slurry at 4°C by rotation for 2 h. The agarose beads were collected by pulse centrifugation (5 s) at 10,000 g, washed with HEPES buffer, and then assayed for the activity of PLC isoenzymes by measuring the hydrolysis of [3H]PIP2, as described previously (3). For control experiments, immunoprecipitation and subsequent activity measurements were conducted with nonimmune mouse IgG. It is pointed out that the immunoprecipitation of the specific PLC isozymes is complete under the condition described here.

Western blot analysis of PLC isozymes. High-molecular-weight markers (Bio-Rad, Hercules, CA) and 20 µg of sarcolemmal proteins were separated on SDS-PAGE as previously described (3). Separated proteins were transferred onto 0.45-µm polyvinylidene difluoride (PVDF) membrane. PVDF membrane was blocked overnight at 4°C in Tris-buffered saline (TBS) containing 5% skim milk and probed with mouse monoclonal primary PLC isozyme antibodies (Upstate Biotechnology). Primary antibodies were diluted in Tris-buffered saline with 0.1% (vol/vol) Tween 20 (TBS-T) (1:200 for PLC-{beta}1, 1:2,000 for PLC-{gamma}1, and 1:10,000 for PLC-{delta}1, according to the manufacturer's instructions). It should be noted that all the antibodies cross-react with their corresponding PLC isozyme but do not cross-react with the other two isozymes (3). Horseradish peroxidase-labeled anti-mouse IgG (Bio-Rad) was diluted 1:3,000 in TBS-T and used as secondary antibody. PLC-{beta}1, -{gamma}1, and -{delta}1 were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Boehringer Mannheim, Laval, PQ, Canada). Band intensities of the Western blot analysis were quantified using a CCD camera imaging densitometer (Bio-Rad GS 800). The linearity of the Western blot procedure used for the quantification of PLC isozymes has been previously determined (3). In subsequent blotting experiments 20 µg SL protein was used because it is in the linear range. Furthermore, the time of exposure used was 5 min. In some experiments, Western blotting with PLC-{gamma}1 was performed with immunoprecipitated SL phosphotyrosyl proteins [immunoprecipitation was performed with anti-phosphotyrosyl monoclonal antibodies (PY99, Santa Cruz Biotechnology); 5 µg of antibody to 855 µg membrane extract, solubilization procedures as described above].

RNA isolation and semiquantitative PCR. Total RNA was isolated from LV tissue using RNA isolation Kit (Life Technologies) according to the manufacturer's procedures. Reverse transcription (RT) was conducted for 45 min at 48°C using the Superscript Preamplification System for First Strand cDNA Synthesis (Life Technology) as previously described (3). Primers used for amplification were synthesized as follows: PLC-{beta}1, 5'-AATAAGGAGACGGAGCTGTTAG-3' (forward) and 5'-ATGGAAGACAAGCCTCTAGCG-3'(reverse); PLC-{gamma}1, 5'-CCTCTATGGAATGGAATTCCG-3' (forward) and 5'-CTAGGGAGGACTCGCTGGAGAACT-3' (reverse); and PLC-{delta}1, 5'-AGGATCGATGCTTCTCCATTGT-3' (forward) and 5'-TTATCAGCCTTTCGCAAGCA-3' (reverse). Amplification of cDNAs of PLC isozyme genes was performed using specific primers and the Superscript Preamplification System (Life Technology). Temperatures used for PCR were as follows: denaturation at 94°C for 30 s, annealing at 62°C for 60 s, and extension at 68°C for 120 s, with a final extension for 7 min; 25 amplification cycles for each individual primer sets were carried out. For the purpose of normalization of the data, GAPDH primers, 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' (forward) and 5'-GCATGTCAGATCCACAACGGATAC-3' (reverse), were used to amplify GAPDH gene as a multiplex with the target genes. The PCR products were analyzed by electrophoresis in 2% agarose gels. The intensity of the bands was photographed and quantified using a Molecular Dynamics STORM scanning system (Amersham Biosciences) as a ratio of a target gene over GAPDH.

Determination of cytosolic IP3 and SL DAG and PIP2 contents. The cytosolic IP3 concentration and SL DAG and PIP2 were measured using their respective Biotrak RIA kit (Amersham Biosciences) according to the manufacturer's instructions as previously described (43, 51). It should be noted that the sensitivities are 0.1 pmol for IP3 and PIP2 and 10 pmol for DAG. The RIA kits are reliable and generate accurate and reproducible values.

Statistical analysis. All values are expressed as means ± SE. The differences between two groups were evaluated by Student's t-test. The data from more than two groups were evaluated by one-way ANOVA, followed by Duncan's multiple-comparison tests. A probability of 95% or more (P < 0.05) was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
General characteristics and LV function. The AV shunt resulted in a consistent and reproducible occurrence of cardiac hypertrophy and subsequent heart failure. The time course of changes in the general characteristics of the rats with and without AV shunt is shown in Table 1. Although there was no significant difference in body weight between the sham-operated and AV shunt groups at each time interval, heart weight of the experimental group increased progressively during the 3-day to 16-wk time points. Accordingly, heart weight-to-body weight ratio was significantly increased at all time intervals in the AV shunt volume overload-induced groups.


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  		Table 1. General characteristics and LV function of rats with or without an AV shunt for different time intervals

 
Cardiac hypertrophy and failure led to significant alterations in cardiac performance to compensate for the increased workload due to the volume overload placed on the heart. The time course changes in the in vivo cardiac performance due to volume overload are shown in Fig. 1. A significant progressive elevation of LV end-diastolic pressure (LVEDP) was detected throughout the 16-wk observation period (Fig. 1A). On the other hand, the LV systolic pressure (LVSP) was decreased significantly throughout the 16-wk observation period; however, the results were biphasic in nature. The most significant decreases in LVSP occurred at 2, 8, and 16 wk, whereas the decrease in LVSP was reduced at the 3-day and 1- and 4-wk observation points. Also, no changes were detected for +dP/dt and –dP/dt at 1, 2, and 4 wk after the surgery, but progressive depressions were seen during the heart failure stages of 8 and 16 wk.



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  		Fig. 1. Time-dependent changes in left ventricular (LV) function of rats after arteriovenous (AV) shunt. Values are means ± SE of 16–20 experimental animals in each group. Sham, age-matched sham-operated controls; LVEDP, LV end-diastolic pressure (A); LVSP, LV systolic pressure (B); +dP/dt, rate of contraction (C); –dP/dt, rate of relaxation (D). * P < 0.05 vs. sham-operated control values.

 
Cytosolic IP3 and SL DAG content. To examine the role of PLC isozyme activity in the development of cardiac hypertrophy and heart failure due to volume overload, we examined products of PLC activity, IP3 mass, and DAG mass in the cytosolic and SL membrane compartments, respectively, of hypertrophied and failing hearts. As shown in Fig. 2, increases in both IP3 and DAG levels were seen during the development of hypertrophy (3 days to 2 wk) that remained elevated at 4 wk. Although at 8 wk (moderate CHF) the IP3 and DAG levels were significantly diminished, a further attenuation of the levels of these molecules was seen at 16 wk (chronic CHF). Such a profile strongly indicated that PLC activities are increased during cardiac hypertrophy and decreased during CHF. It should be noted a 12 and 9% loss in SL DAG and cytosolic IP3, respectively, due to two freeze-thaw cycles (LV tissue and SL membrane or cytosol) was measured (data not shown). These losses were taken into consideration when calculating the absolute SL DAG and cytosolic IP3 values.



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  		Fig. 2. Sarcolemmal (SL) 1,2-diacylglycerol (DAG; A) and cytosolic inositol (1,4,5)-trisphosphate (IP3) levels in volume overload-induced hypertrophied and failing hearts; B. Values are means ± SE of duplicate experiments performed with 4 different SL preparations. DAG and IP3 levels were determined using Biotrak RIA kits as described in MATERIALS AND METHODS. Measurements were conducted at 3 days and 1, 2, 4, 8, and 16 wk after the AV shunt. * P < 0.05 vs. sham-operated control values.

 
SL PLC isozyme activities in hypertrophied and failing hearts. To understand if the changes in the IP3 and DAG levels in hypertrophied and failing hearts are due to PLC, the activities of three predominant PLC isozymes ({beta}1, {gamma}1, and {delta}1) were determined at 3 days and 1, 2, 4, 8, and 16 wk after the induction of the AV shunt (Fig. 3). It can be seen that both PLC-{beta}1 and -{gamma}1 activities were significantly increased during the hypertrophic stages (Fig. 3, A and B). While a sixfold increase in PLC-{beta}1 activity was detected at 2 wk after surgery, the peak activation of PLC-{gamma}1 occurred at 3 days. Although PLC-{beta}1 protein and mRNA levels were elevated at 2 wk, a sixfold increase in PLC-{beta}1 activity was seen at 2 wk. In heart failure, although PLC-{gamma}1 activity was significantly depressed at 8 and 16 wk after the AV shunt, a second smaller peak of PLC-{beta}1 activity was seen at 8 wk, which was depressed at 16 wk (Fig. 3, A and B). The profile of the PLC-{gamma}1 activity at all the time points studied was consistent with the level of phosphorylation of its tyrosyl residues (Fig. 4). In fact, the biphasic nature of the PLC-{gamma}1 activity was correlated with the pattern of its phosphorylation. In contrast to the activity profiles of PLC-{beta}1 and -{gamma}1, a progressive decline in PLC-{delta}1 activity was observed throughout the time course, where heart failure was associated with approximately threefold and fivefold decreases in PLC-{delta}1 activity at 8 and 16 wk, respectively, after the induction of the AV shunt (Fig. 3C).



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  		Fig. 3. Changes in SL phospholipase C (PLC) isozyme activities in hearts of rats after the induction of volume overload. PLC isozyme activities were measured after immunoprecipitation with specific monoclonal antibodies against PLC-{beta}1 (A), -{gamma}1 (B), and -{delta}1 (C) as indicated in MATERIALS AND METHODS, at 3 days and 1, 2, 4, 8, and 16 wk after induction of volume overload. Inositol phosphates formed refers to the sum of the total inositol phosphates produced by PLC-dependent hydrolysis of PIP2. Values are means ± SE of quadruplicate experiments performed using 4 different SL preparations. * P < 0.05 vs. sham-operated control values.

 


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  		Fig. 4. Representative Western blots (A) and quantified data (B) of the tyrosyl phosphorylation of SL PLC-{gamma}1 in hearts of rats after induction of volume overload. Western blots shows tyrosyl-phosphorylated PLC-{gamma}1 at 3 days and 1, 2, 4, 8, and 16 wk after AV shunt. Values are means ± SE of 3 experiments performed with 3 different SL preparations. * P < 0.05 vs. sham-operated control values.

 
PLC isozyme protein abundance and mRNA levels in hypertrophied and failing hearts. To understand whether the changes in PLC isozyme activities are due to altered SL abundance and mRNA levels, Western blot analysis and RT-PCR were performed. Time-dependent increases in the SL PLC-{beta}1 protein content (Figs. 5A and 6) and mRNA levels (Figs. 7A and 8) were seen up to 8 wk after the induction of the AV shunt. Whereas at 16 wk the SL PLC-{beta}1 protein abundance was markedly diminished, the mRNA level remained elevated. A significant increase in SL PLC-{gamma}1 protein abundance was observed only at 4 wk after the AV shunt, while a progressive decline in the SL PLC-{delta}1 protein content was observed (Fig. 5, B and C). However, a strict correlation (increased mRNA translates to increased protein) of these isozyme protein changes with their respective mRNA levels (Figs. 5 and 7) was not revealed.



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  		Fig. 5. Quantified data of sarcolemmal PLC isozyme [PLC-{beta}1 (A), PLC-{gamma}1 (B), and PLC-{delta}1 (C)] protein concentration in hypertrophied and failing hearts due to volume overload assessed by Western blotting. Values are means ± SE of 3 experiments performed with 3 different SL preparations. * P < 0.05 vs. sham-operated control values.

 


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  		Fig. 6. Representative Western blots of SL PLC isozymes in hearts of rats after induction of volume overload. Western blots show (arrows) 150-kDa PLC-{beta}1, 135-kDa PLC-{gamma}1, and 85-kDa PLC-{delta}1 at 3 days and 1, 2, 4, 8, and 16 wk after AV shunt.

 


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  		Fig. 7. LV PLC isozyme [PLC-{beta}1 (A), PLC-{gamma}1 (B), and PLC-{delta}1 (C)] mRNA levels in hypertrophied and failing hearts after induction of volume overload. PLC isozyme mRNA levels in left ventricular tissue were determined by RT-PCR using gene-specific primers for PLC isozymes as described in MATERIALS AND METHODS. Values are means ± SE of 3 experiments performed with 3 different SL preparations. * P < 0.05 vs. sham-operated control values.

 


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  		Fig. 8. Representative blots of LV PLC isozyme mRNA levels in hearts of rats after induction of volume overload.

 
SL PIP2 content. PIP2 serves as a substrate for PLC for the generation of second messengers. Therefore, assessment of the SL amounts of this phospholipid was conducted to understand if the observed changes in IP3 and DAG concentrations levels as well as PLC isozymes were due to altered SL PIP2 content. In addition, knowledge of the SL PIP2 level would provide a mechanism to explain changes in the SL abundance of proteins containing PH domains such as PLC-{delta}1 (51). The data presented in Fig. 9 show that a progressive decrease from 100% at 3 days to 19% (of 3-day value) at 16 wk in the SL PIP2 level occurred. Of note significant sixfold and fourfold decreases in PIP2 levels were observed in the SL membrane fraction of the 8- and 16-wk failing hearts, respectively. The PIP2 loss during quantification due to the Dowex column was 13%, whereas in two freeze-thaw cycles (LV tissue and SL membrane) it was 8% (data not shown). Subsequently, the calculation for the absolute SL PIP2 concentration took these losses into consideration.



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  		Fig. 9. SL phosphatidylinositol 4,5-bisphosphate (PIP2) levels in hypertrophied and failing hearts due to volume overload. Values are means ± SE of 3 experiments performed with 3 different SL preparations. * P < 0.05 vs. sham-operated control values. The SL PIP2 content was measured at 3 days and 1, 2, 4, 8, and 16 wk after the AV shunt using the Biotrak RIA kit as described in MATERIALS AND METHODS.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The needle technique used in this study is a newly established technique for inducing volume overload; this method provides reproducible animal models of cardiac hypertrophy and heart failure. The volume overload induced by this method results in cardiac hypertrophy and heart failure resembling that occurring in humans during hyperthyroidism, anemia, and bundle branch block (47). In the present study we addressed the hypothesis that PLC-mediated signal transduction processes are increased during cardiac hypertrophy and decreased during heart failure. This is the first study to show that the elevation of cytosolic IP3 and SL DAG amount during cardiac hypertrophy is associated with specific increases in PLC-{beta}1 and -{gamma}1 isozyme activities, whereas the depressed production of IP3 and DAG in the failing hearts is linked to marked reductions in PLC-{delta}1 and -{gamma}1 isozyme activities.

Although PLC isozymes display differences in structure and activating enzymes (22, 35), the specific cardiac effects may depend on the type, quantity, and activity of the PLC isozyme present at the SL membrane. The increase in PLC-{beta}1 activity may be due to an enhanced SL compartmentalization as a result of elevated gene expression in combination with agonist-evoked recruitment of PLC-{beta}1 to the membrane. In fact, the high plasma level of norepinephrine reported in volume overload (48) may play a role. Because norepinephrine has been suggested to cause hypertrophic growth of cardiomyocytes via stimulation of {alpha}1-adrenoceptors (2, 46), such a response may be related to an augmentation of the {alpha}1/Gq{alpha}/PLC-{beta}1 signal transduction mechanisms (12). In addition, because ANG II also acts through the PLC-{beta} class (36) via ANG II type I (AT1) receptors (45), which have been reported to be increased during cardiac hypertrophy (21), the increase in the responsiveness of hypertrophic myocardial cells to ANG II in volume-overloaded hearts (27) could also be attributable to an increase in signaling via AT1/Gq{alpha}/PLC-{beta}1. Taken with the fact that ANG II may also mediate some of its cardiac effects by activation of PLC-{gamma}1 (22, 35), it can be suggested that the activation of PLC-{beta}1 and -{gamma}1 isozymes may contribute to the hypertrophic response after the induction of the AV shunt. It is pointed out that selective {beta}1-adrenoceptor stimulation causes hypertrophic growth of ventricular cardiomyocytes by a mechanism that is cAMP independent but dependent on tyrosine kinase (38). In fact, because PLC-{gamma} isoforms are activated by receptor (and nonreceptor) tyrosine kinases (22, 35–36) and tyrosine kinase activation has been implicated in cardiac hypertrophy (1, 7, 15), the increase in PLC-{gamma}1 activity seen during the hypertrophic stage may be due to an increase in the level of phosphorylation of the tyrosyl residues of PLC-{gamma}1. In addition, the biphasic activation of PLC-{gamma}1 (first peak at 3 days and a second peak at 4 wk) was consistent with its phosphorylation profile.

Among potential mediators of hypertrophy, one upstream signaling protein of importance is Gq{alpha}, a heterotrimeric G protein to which are coupled the heptahelical, serpentine receptors for multiple growth factors including ANG II and norepinephrine (46). Stimulation of signaling pathways via Gq{alpha} provokes cardiac hypertrophy in cultured cardiomyocytes and transgenic mouse models overexpressing Gq{alpha} (9–10, 29–30). It is therefore conceivable that although the amount and expression of PLC-{beta}1 were increased, in our studies, an elevation in SL Gq{alpha} expression and coupling to PLC-{beta}1 could have also occurred and thus contribute toward the sixfold increase in PLC-{beta}1 activity during hypertrophy, a possibility that warrants further investigation.

The primary step of the signal transduction pathway for the activation of PKC involves the stimulation of PLC. PKC isozymes, specifically PKC-{alpha} (4) and -{epsilon} (42), and DAG have been implicated in the regulation of hypertrophic growth of cardiomyocytes (4, 19, 42). Therefore, the observation of an increase in the total SL level of DAG during the hypertrophy in the present study is suggestive of a similar activation of PKC in cardiac hypertrophy due to volume overload and remains to be elucidated. Although DAG kinase is involved in the termination of PKC activation (41) and a decreased DAG kinase {epsilon} activity increases the PKC activity and may accelerate the cardiomyocyte hypertrophic response (32), a decrease in its activity, in our studies, cannot be excluded. On the other hand, because the changes in DAG were not very large, a compensatory increase in DAG kinase activity may have occurred. However, despite this possibility, the SL DAG levels were significantly elevated during cardiac hypertrophy. Similar changes in DAG lipase may also exist.

The progressive decrease in PLC-{delta}1 activity correlated to the reduction in the SL abundance of this isozyme. One of the reasons for this finding could be due to the NH2-terminal part of the PH domain of PLC-{delta}1, which possesses a critical region rich in basic amino acid residues that bind with high affinity to the polar head of PIP2 (49) that confers a unique capacity of PLC-{delta}1 to associate with the plasma membrane. Our finding that there is a time-dependent decrease in the SL PIP2 content provides a mechanism whereby the attachment of PLC-{delta}1 to the SL membrane decreases during the cardiac hypertrophy and its transition to heart failure, which could also account for the progressive decrease in SL PLC-{delta}1 activity. Also, the reduced PIP2 could contribute to the depressed PLC-{gamma}1 activity as a consequence of a decreased production of phosphatidylinositol 3,4,5-trisphosphate, which is known to activate PLC-{gamma}1 (35), via phosphatidylinositol 3-kinase phosphorylation of PIP2 (11). Furthermore, it is pointed out that the depressed PLC-{gamma}1 activity in the failing heart may also be related to the decreased level of phosphorylation of its tyrosyl residues. Therefore, taken together, it can be suggested that bioprocesses mediated by PLC-{gamma}1 and -{delta}1 may be severely impaired during CHF due to volume overload. In this regard, it is interesting to note that we have observed similar reductions in PLC-{gamma}1 and -{delta}1 activities in CHF subsequent to myocardial infarction, which were associated with a significant attenuation of the contractile responsiveness of the failing cardiomyocyte to phosphatidic acid (PA) (44), a known inotropic agent that mediates its effects via activation of PLC (8); therefore it is possible that such depressed responses to PA may also exist in cardiomyocytes of the failing heart due to volume overload. It is pointed out that although a second smaller peak of PLC-{beta}1 activity was seen at 8 wk, the activity remained elevated at 16 wk, but the extent was slightly reduced. It is possible that these elevations may be due to the fact that activation of the sympathetic and renin-angiotensin systems are also seen in heart failure (13, 31). Interestingly, we have previously reported that inhibition of angiotensin-converting enzyme by imidapril partially corrects the changes in SL PLC isozyme activity in the failing heart, indicating a pathophysiological significance of PLC isozymes (44).

The diminished amount of PIP2 in the SL membrane cannot be totally accounted for by the changes in PLC isozyme activities. Although the decreases detected at 1, 2, and 4 wk could be attributed, in part, to the increased PLC isozyme activities and consequent increased PIP2 hydrolysis, the diminished amount of SL PIP2 during the heart failure stage may not be explained on the basis of reduced PIP2 hydrolysis by PLC activities, given that the total PLC activities ({beta}1 + {gamma}1 + {delta}1) were depressed, but perhaps could be explained on the basis of its reduced synthesis. In this regard, we have previously reported a reduced SL PIP2 in CHF (subsequent to myocardial infarction) due to depressed phosphatidylinositol 4 and phosphatidylinositol 4-phosphate 5-kinase activities (43). Furthermore, the reduced SL PIP2 level could also be an additional factor contributing to attenuation of the PLC-dependent generation of IP3 and DAG in the failing heart. It is pointed out that independent of the effect of PIP2 on SL PLC activities, there are a number of biochemical events that are sensitive to the membrane level of PIP2 that could influence the contractile performance of the failing heart. For example, reduced PIP2 can cause a depression of the inward rectifier K+ channels (17), as well as SL Na+/Ca2+ exchanger and Ca2+ pump activities (5, 16). Also of interest, the reduced SL PIP2 levels in heart failure could affect the activities of other phospholipases known to modulate cardiac function and are sensitive to the membrane level of PIP2. For example, we have earlier reported that the activity and SL binding of the type IV phospholipase A2 as well as phospholipase D1 activity are markedly reduced in CHF subsequent to myocardial infarction (28, 50). It is possible that a similar situation may exist in heart failure due to volume overload.

In conclusion, our findings of increased PLC-{beta}1 and -{gamma}1 activities during cardiac hypertrophy and decreased PLC-{gamma}1 and -{delta}1 activities during heart failure suggest an important role of PLC isozymes in cardiac hypertrophy and heart failure induced by volume overload. In addition, the reduced PIP2 level may also contribute to the depressed contractile performance of the failing heart. Therefore modulation of elements within the PLC signal transduction pathway may constitute potential therapeutic strategies for the prevention of cardiac hypertrophy and heart failure.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from the Manitoba Health Research Council. N. S. Dhalla holds Canadian Institutes of Health Research/Pharmaceutical Research and Development Chair in Cardiovascular Research supported by Merck Frosst of Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. S. Tappia, Institute of Cardiovascular Sciences, Laboratory of Cardiac Membrane Biology, St. Boniface General Hospital Research Centre (R3020), 351 Tache Ave., Winnipeg, Manitoba R2H 2A6, Canada (E-mail: ptappia{at}sbrc.ca).

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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