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Am J Physiol Heart Circ Physiol 287: H2598-H2605, 2004. First published August 5, 2004; doi:10.1152/ajpheart.00506.2004
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Inhibition of PLC improves postischemic recovery in isolated rat heart

Girma Asemu,1 Naranjan S. Dhalla,2 and Paramjit S. Tappia1

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

Submitted 28 May 2004 ; accepted in final form 28 July 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Ca2+-dependent PLC converts phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Because these products modulate Ca2+ movements in the myocardium, PLC may also contribute to a self-perpetuating cycle that exacerbates cardiomyocyte Ca2+-overload and subsequent cardiac dysfunction in ischemia-reperfusion (I/R). Although we have reported that I/R-induced changes in PLC isozymes might contribute to cardiac dysfunction, the present study was undertaken to examine the beneficial effects of the PLC inhibitor, U-73122, as well as determining the role of Ca2+ on the I/R-induced changes in PLC isozymes. Isolated rat hearts were subjected to global ischemia 30 min, followed by 5 or 30 min of reperfusion. Pretreatment of hearts with U-73122 (0.5 µM) significantly inhibited DAG and Ins(1,4,5)P3 production in I/R and was associated with enhanced recovery of cardiac function as indicated by measurement of left ventricular (LV) end-diastolic pressure (EDP), LV diastolic pressure (LVDP), maximum rate of pressure development (+dP/dtmax), and maximum rate of LV pressure decay (–dP/dtmax). Verapamil (0.1 µM) partially prevented the increase in sarcolemmal (SL) PLC-{beta}1 activity in ischemia and the decrease in its activity during the reperfusion phase as well as elicited a partial protection of the depression in SL PLC-{delta}1 and PLC-{gamma}1 activities during the ischemic phase and attenuated the increase during the reperfusion period. Although these changes were associated with an improved myocardial recovery after I/R, verapamil was less effective than U-73122. Perfusion with high Ca2+ resulted in the activation of the PLC isozymes studied and was associated with a markedly increased LVEDP and reduced LVDP, +dP/dtmax, and –dP/dtmax. These results suggest that inhibition of PLC improves myocardial recovery after I/R.

sarcolemma; U-73122; verapamil; hemodynamics

THE PHOSPHOINOSITIDE-SPECIFIC PLC isozymes associated with the cardiac sarcolemmal (SL) membrane play an important role in activating intracellular signal transduction pathways for the regulation of various cell functions (4, 21, 26, 34, 35). PLC converts its substrate, phosphatidylinositol 4,5-bisphosphate, into two messenger molecules, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and 1,2-diacylglycerol (DAG). Whereas Ins(1,4,5)P3 may serve to enhance the sarcoplasmic reticulum (SR) Ca2+ release (13, 19), DAG functions as a potent activator of PKC isozymes which in turn phosphorylate several cardiac proteins (33) and influence Ca2+ movements into the cardiomyocyte (25, 33).

Ischemia-reperfusion (I/R) injury is known to occur during clinical procedures, such as coronary bypass surgery, angioplasty, thrombolytic therapy, and cardiac transplantation (3, 6), and has been shown to be associated with cardiac dysfunction and others in myocardial abnormalities (2, 17, 22, 23, 36, 39, 44). Given the role of PLC products in modulating Ca2+ movements in the myocardium and that the enzymatic activity of different PLC isozymes is dependent on Ca2+ (26, 35), it is conceivable that PLC may also contribute to a self-perpetuating cycle that exacerbates cardiomyocyte Ca2+ overload and subsequent cardiac dysfunction in I/R (7, 9).

Although we have earlier identified changes in PLC isozymes in I/R (1), the present study was undertaken for the following reasons: 1) to examine the effects of inhibition of PLC isozyme activities, with U-73122 on cardiac function after I/R; 2) to determine the role of Ca2+ in the I/R-induced changes in PLC isozyme activities by examining the effects of the L-type Ca2+ channel blocker verapamil on PLC activities; and 3) to further investigate the role of Ca2+ in PLC isozyme activities and cardiac dysfunction by perfusing hearts with low and high concentrations of Ca2+.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animal model and isolated perfused hearts. 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. Heart perfusion and assessment of cardiac performance were conducted as previously described (1). Briefly, male Sprague-Dawley rats weighing 250–300 g were anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The hearts were rapidly excised, cannulated to the Langendorff apparatus, and perfused with Krebs-Henseleit solution (37°C) gassed with a mixture of 95% O2 and 5% CO2, pH 7.4, containing (in mM) 120 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.25 CaCl2, and 11 glucose. The hearts were electrically stimulated at 300 beats/min (Phipps and Bird; Richmond, VA), and the perfusion rate was maintained 10 ml/min. For left ventricular (LV) pressure recordings, a water-filled small latex balloon by polythene cannula was inserted through left atrium into the LV and connected to the pressure transducer (model 1050BP; Biopac System) for the LV systolic and diastolic pressure measurements; the LV developed pressure (LVDP) was the difference between systolic and diastolic pressures. The LV end-diastolic pressure (LVEDP) was adjusted at 10 mmHg at the beginning of the experiment, and the LV pressures were differentiated to estimate the maximum rate of pressure development (+dP/dtmax) and the maximum rate of LV pressure decay (–dP/dtmax) with Acknowledge version 3.71 software for Windows (Biopac System). All hearts were stabilized for a period of 30 min before use in this study and were maintained at a constant temperature (37°C) throughout the experiment. The control hearts were perfused for a period of 60, 65, and 90 min, and because no significant difference (P > 0.05) with respect to each parameter was observed between these hearts, the values were grouped together. The hearts were perfused with Krebs-Henseleit solution containing either low Ca2+ (0.5 mM) or high Ca2+ (2.55 mM) for 15 min. In another set of experiments, the hearts were pretreated for 5 min with either 0.1 µM verapamil or 0.5 µM U-73122, before global ischemia, by stopping the coronary flow for 30 min, and reperfusion for 5 or 30 min in the presence of these agents. At the end of perfusion, the LV tissues were frozen immediately in liquid nitrogen, and stored at –80°C for further processing.

Preparation of cardiac SL membrane and cytosolic fractions. The ventricular tissue from four to five hearts was pooled to prepare SL membrane fractions. Briefly, the tissue was washed, minced, and homogenized in 3.5 ml of ice-cold 0.6 M sucrose-10 mM 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 (1 ml) of the resultant supernatant was centrifuged at 110,000 g (60 min, 4°C), and the resulting supernatant was frozen and stored (–80°C) as the cytosolic fraction. The rest of the first supernatant was diluted to 30 ml with 300 mM KCl, containing 20 mM MOPS/KOH, pH 7.4, to solubilize myofibrillar proteins, and further processed for the preparation of SL according to the method used previously (1, 41, 44). The final pellet was resuspended in 0.25 M sucrose and 10 mM histidine (pH 7.4), frozen in liquid N2, and stored at –80°C until assayed. All of the above steps were carried out at temperatures of 0–4°C. Protein concentrations were determined by the Lowry method, as described elsewhere (1, 42, 43).

Determination of cytosolic Ins(1,4,5)P3 and SL DAG contents. The cytosolic Ins(1,4,5)P3 concentration and the SL DAG amounts were measured using their respective Biotrak radioimmunoassay kit (Amersham Biosciences; Quebec, Canada) according to the manufacturer's instruction.

Determination of PLC isozymes activities. Measurement of SL PLC isozyme activities, by immunoprecipitation, was conducted as already reported (1, 42, 45). Briefly, solubilized membrane proteins were incubated overnight at 4°C with monoclonal antibodies to PLC-{beta}1, -{gamma}1, or -{delta}1 (5 µg of antibody to 350 µg membrane extract). The immunocomplex was captured by the addition of 100 µl (50 µl packed beads) of washed (three 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 and washed with HEPES buffer and then assayed for the activity of PLC isozymes by measuring the hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate, as described previously (1, 42, 45). For control experiments, immunoprecipitation and subsequent activity measurements were conducted with nonimmune mouse IgG. The immunoprecipitation of the specific PLC isozymes is complete under the condition described here (42, 45).

RNA isolation and semiquantitative PCR. Total RNA was isolated from LV tissue with the use of RNA isolation kit (Life Technologies, ON, Canada) 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 Technologies) as previously described (1). 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 was 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.

Western blot analysis of PLC isozymes. High-molecular-weight markers (Bio-Rad; Hercules, CA) and 20 µg of SL proteins were separated on SDS-PAGE as previously described (1, 42, 45). The separated proteins were transferred onto 0.45-µm polyvinylidene difluoride membrane. The polyvinylidene difluoride 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 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). 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, Quebec, Canada). Band intensities of the Western blot analysis were quantified using a charge-coupled device camera imaging densitometer (model GS 800, Bio-Rad).

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 test. A probability of 95% or more (P < 0.05) was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Effects of PLC inhibition on myocardial recovery after I/R. In the absence of U-73122 reperfusion of the ischemic heart for 30 min was associated with values for LVDP, +dP/dtmax, and –dP/dtmax of 25%, 24%, and 34% of control values, respectively (Table 1). Pretreatment of the hearts with U-73122 (0.5 µM) before I/R resulted in a 42%, 41%, and 32% recovery in LVDP, +dP/dtmax, and –dP/dtmax, respectively, after 5 min of reperfusion and 69%, 73%, and 54% recovery in these parameters, respectively, after 30-min reperfusion of the ischemic heart. Although a marked increase in LVEDP was seen in reperfusion, this was significantly attenuated with U-73122 pretreatment (Table 1). Analysis of the products of PLC activities, DAG, and Ins(1,4,5)P3 in the SL membrane and cytosolic compartments, respectively, revealed that the hemodynamic changes induced by I/R were associated with a decrease in the SL DAG (to 63% of control value) as well as a decrease in cytosolic Ins(1,4,5)P3 (to 79% of control value) level in the ischemic heart. On the other hand, increases in the SL DAG (to 182% of control value) and cytosolic Ins(1,4,5)P3 (to 162% of control value) at 5 min of reperfusion were observed. Pretreatment of the hearts with U-73122 markedly attenuated the changes in SL DAG and cytosolic Ins(1,4,5)P3 concentrations that occurred at 5 min of reperfusion of the ischemic heart (Fig. 1, A and B). To determine whether U-73122 exerted a selective inhibition of one or more of the PLC isozymes, PLC isozyme activities were measured in the absence and presence of U-73122 (0.5 µM) in SL fractions isolated from control hearts. The data shown in Fig. 2 revealed that U-73122, at this concentration, exerted a varying intensity of inhibition of PLC isozyme activities (PLC {beta}1 > PLC {gamma}1 > PLC {delta}1).


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  		Table 1. Effect of U73122 [GenBank] on left ventricular contractile function during ischemia-reperfusion in isolated rat heart

 


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  		Fig. 1. Sarcolemmal (SL) diacylglycerol (DAG; A) and cytosolic inositol 1,4,5-trisphosphate (IP3; B) contents in myocardial ischemia-reperfusion in the absence and presence of U-73122. Values are means ± SE of 5 experiments. Hearts were pretreated with U-73122 (0.5 µM) for 5 min before global ischemia (Isch) for 30 min, followed by 5 and 30 min of reperfusion, as described in MATERIALS AND METHODS. *P < 0.05 vs. corresponding control in the absence of U-73122. #P < 0.05 vs. corresponding control in the presence of U-73122. Control values for DAG and IP3 in the absence of U-73122 were 1.35 ± 0.23 (nmol/mg SL protein) and 4.83 ± 0.45 (pmol/mg cytosolic protein), respectively, and 0.69 ± 0.12 (nmol/mg SL protein) and 2.26 ± 0.18 in the presence of U-73122 (pmol/mg cytosolic protein).

 


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  		Fig. 2. Cardiac SL PLC isozyme activities in the absence and presence of U-73122. Values are means ± SE of 3 experiments. PLC isozyme activities were measured in the absence and presence of U-73122 (0.5 µM) after immunoprecipitation with specific monoclonal antibodies against PLC-{beta}1, -{gamma}1, and -{delta}1, as described in MATERIALS AND METHODS. InsPs is the sum of the inositol phosphates produced by PLC-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). *P < 0.05 vs. control.

 
Effects of verapamil on the I/R-induced changes in PLC isozymes. To examine whether the PLC isozyme activation in I/R is due to the occurrence of intracellular Ca2+ overload, the effects of verapamil, a known cardioprotective agent (11, 40, 43), on PLC isozyme activities was conducted. In agreement with our earlier report (1), PLC-{beta}1 activity, was increased in the ischemic heart, but was progressively recovered on reperfusion (Fig. 3A). Both PLC-{gamma}1 and -{delta}1 activities were decreased due to ischemia and increased due to reperfusion (Fig. 3, B and C). Hearts subjected to global ischemia for 30 min failed to generate LVDP, +dP/dtmax, and –dP/dtmax, but showed a marked increase in LVEDP; however, an attenuation of the elevation in LVEDP was observed in the verapamil-treated hearts (Table 2). Although reperfusion of the ischemic hearts recovered the contractile function, as represented by LVDP, +dP/dtmax, and –dP/dtmax, by 18–19% (after 5 min of reperfusion) and by 35–37% (after 30 min of reperfusion) of the respective preischemic values, LVEDP was increased further (Table 2). Although verapamil had no effect on the control PLC isozyme activities, a partial correction of the elevated PLC-{beta}1 activity in the ischemic heart and a partial normalization of the activated PLC-{gamma}1 and -{delta}1 activities (Fig. 3, A–C) was observed. These changes were associated with an enhanced the recovery of the contractile function of the ischemic heart during reperfusion as indicated by values for LVDP, +dP/dtmax, and –dP/dtmax, of 27–30% (after 5 min of reperfusion) and 55–63% (after 30 min of reperfusion) of the values in the absence of verapamil (Table 2).



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  		Fig. 3. Cardiac SL PLC isozyme activities in myocardial ischemia-reperfusion in the absence and presence of verapamil. Values are means ± SE of 5 experiments. Hearts were pretreated with verapamil (0.1 µM) for 5 min before global ischemia for 30 min, followed by 5 and 30 min of reperfusion. PLC isozyme activities were measured after immunoprecipitation with specific monoclonal antibodies against PLC-{beta}1 (A), PLC-{gamma}1 (B), and PLC-{delta}1 (C), as described in MATERIALS AND METHODS. InsPs is the sum of the inositol phosphates produced by PLC-dependent hydrolysis of PIP2. *P < 0.05 vs. control; #P < 0.05 vs. corresponding value in the absence of verapamil.

 

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  		Table 2. Effect of verapamil on LV contractile function during ischemia-reperfusion in isolated rat heart

 
PLC isozyme activities, protein levels, and mRNA abundance in hearts perfused with low and high Ca2+. The role of Ca2+ and PLC isozyme activation was further examined by perfusing hearts with low (0.5 mM) and high Ca2+ (2.55 mM). Although no changes in the SL PLC-{beta}1 and -{gamma}1 isozyme activities and protein contents (Figs. 4 and 5) and their mRNA levels (Fig. 6) were observed on perfusion with low Ca2+ for 15 min, a decrease in both SL PLC-{delta}1 activity and protein content was detected, with no change in its mRNA level. These changes were associated with a reduced generation of LVDP, +dP/dtmax, and –dP/dtmax and an almost 4.5-fold increase in LVEDP at the end of the 15-min perfusion period (Table 3). On the other hand, perfusion with high Ca2+ resulted in increases in PLC isozyme activities with concomitant increases in their SL protein content. No changes in PLC-{gamma}1 and -{delta}1 mRNA levels were detected; however, the PLC-{beta}1 mRNA level was reduced. Although an initial (after 2 min of perfusion) increase in LVDP and +dP/dtmax was seen, this was followed (at 15 min of perfusion) by a marked reduction in LVDP, +dP/dtmax, and –dP/dtmax (20%, 16%, and 27% of control values, respectively) and a sevenfold increase in LVEDP (Table 3).



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  		Fig. 4. SL PLC isozyme activities in hearts perfused with low and high calcium. Values are means ± SE of 5 experiments. Hearts were stabilized for 30 min, followed by perfusion with low (0.5 mM) and high Ca2+ (2.55 mM) for 15 min. PLC isozyme activities were measured after immunoprecipitation with specific monoclonal antibodies against PLC-{beta}1 (A), PLC-{gamma}1 (B), and PLC-{delta}1 (C), as described in the MATERIALS AND METHODS. InsPs is the sum of the inositol phosphates produced by PLC-dependent hydrolysis of PIP2. *P < 0.05 vs. control (1.25 mM Ca2+).

 


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  		Fig. 5. SL PLC isozyme protein contents in hearts perfused with low and high Ca2+. Shown are representative immunoblots and quantified data of PLC isozyme-{beta}1 (A), PLC-{gamma}1 (B), and PLC-{delta}1 (C) protein concentration. Hearts were stabilized for 30 min, followed by perfusion with low (0.5 mM) and high Ca2+ (2.55 mM) for 15 min, as described in MATERIALS AND METHODS. Values are means ± SE of 5 experiments. *P < 0.05 vs. control (1.25 mM Ca2+).

 


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  		Fig. 6. PLC isozyme mRNA levels in hearts perfused with low and high Ca2+. Values are ± SE of 5 experiments. The mRNA levels of PLC isozyme-{beta}1 (A), PLC-{gamma}1 (B), and PLC-{delta}1 (C) were determined by RT-PCR using gene-specific primers for PLC isozymes in hearts subjected to perfusion with low (0.5 mM) and high Ca2+ (2.55 mM) for 15 min. Representative blots (D) are shown. *P < 0.05 vs. control (1.25 mM Ca2+).

 

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  		Table 3. LV functional changes of the isolated rat heart due to perfusion with low and high Ca2+

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We (1) have previously reported that cardiac I/R induces differential changes in PLC isozyme activities, SL protein contents, and mRNA expression levels that might contribute to impairment of postischemic myocardial recovery. The present study was undertaken to elucidate some of the mechanisms responsible for these changes and whether inhibition of PLC either directly with U-73122 or indirectly with verapamil attenuates cardiac dysfunction in I/R. Because the enzymatic activity of all PLC isozymes is dependent on Ca2+, it was suggested that the occurrence of intracellular Ca2+ overload as a consequence of the abnormalities in Ca2+ handling by cardiomyocytes (7, 9) could contribute to the increase in PLC-{gamma}1 and PLC-{delta}1 activities in I/R. On the basis of our present findings, cardiomyocyte Ca2+ overload does play an important role in PLC activation in I/R and subsequent cardiac dysfunction and that inhibition of PLC isozymes with U-73122 improves recovery of cardiac function after I/R. Although a similar attenuation of I/R-induced cardiac dysfunction has been seen with the PLC inhibitor neomycin (28), here we have demonstrated that the generation of DAG and Ins(1,4,5)P3 is significantly diminished by the PLC inhibitor, U-73122, which could be related to a selective intense inhibition of PLC-{beta}1 and -{gamma}1 activities, and that these effects are associated with improved cardiac recovery after I/R.

Verapamil had no effect on the basal (control) PLC isozyme activities, but partially attenuated the PLC isozyme activation during the reperfusion period, and was associated with an improved recovery. Although this can be explained on the basis of attenuation of the increase in intracellular Ca2+ concentration ([Ca2+]i), verapamil also partially attenuated the increased PLC-{beta}1 activity in the ischemic heart. While the increase in this PLC isozyme activity may be due to an elevated {alpha}1-adrenoceptor signaling to PLC-{beta}1 due to increased release of catecholamines in the ischemic heart (16), as well as the enhanced sensitivity of the {alpha}1-adrenoceptor under ischemic conditions (10), the attenuation of the increase in PLC-{beta}1 activity in the ischemic heart by verapamil may be due to the reported inhibitory effects of verapamil on {alpha}1-adrenoceptors (38).

The significance of the role of Ca2+ and PLC activities was further addressed. Perfusion with an increased Ca2+ concentration (from 1.25 to 2.55 mM) resulted in an initial increase in inotropy; however, with longer perfusion time (15 min) a severe cardiac dysfunction occurred, indicating that this concentration of Ca2+ is damaging to the heart. Perfusion of hearts with high Ca2+ also resulted in the activation of all PLC isozymes (PLC-{delta}1 > PLC-{beta}1 > PLC-{gamma}1) and was associated with an increase in their SL protein abundance, suggesting that PLC activation, specifically PLC-{gamma}1 and -{delta}1, which occurs in I/R may be due to the increase in [Ca2+]i. Indeed, the intensity of the activation of PLC-{delta}1 by Ca2+ may be due to the reported higher sensitivity of PLC-{delta} isozymes to Ca2+ (34). Furthermore, in this regard, it is interesting to note that perfusion of the hearts with low Ca2+ revealed a specific decrease in the activity of PLC-{delta}1. Although these data provide some information on the role of Ca2+ on PLC isozymes, it should be noted that we are aware that the role of Ca2+ under basal conditions could be different from its role under I/R and in this regard the increase in PLC-{beta}1 activity seen in perfusion with high Ca2+ is in contrast to the profile of the activity during I/R. This discrepancy could be explained on the basis that PLC-{beta}1, compared with the other PLC isozymes, is more susceptible to free radical-mediated damage, which occurs during the early reperfusion phase (5, 7–9), rendering PLC-{beta}1 insensitive to Ca2+ or as a result of a selective degradation due to activation of proteases which occurs in I/R (12).

While some mechanisms associated with the functional damage to the stunned myocardium for a prolonged period of ischemia have been proposed (14, 15), they remain to be fully defined; however, the contribution of the changes in the SL PLC isozymes observed in our studies cannot be ignored, although the distinct functions of each PLC isozyme in the adult cardiomyocyte, and the extent of their overlap has yet to be completely established. Nonetheless, the changes in PLC isozyme activities observed in the ischemia and I/R may have functional relevance to PKC isozymes, which are activated specifically by PLC-derived DAG (18, 31), and reported to be associated with I/R injury (20, 32, 41), therefore implicating a key role of PLC in I/R injury. Indeed, it has been reported that enhanced protection of the heart can be achieved by administration of PKC-{delta} inhibitor at the beginning of reperfusion, whereas activation of PKC-epsilon before ischemia mimics ischemic preconditioning (20). Thus it is conceivable that the differential changes in PLC isozymes results in specific PKC isozyme activation and that prevention of the I/R-induced activation of specific PLC isozymes, directly with U-73122 and indirectly with verapamil, in turn precludes the PKC isozyme changes. In addition, both of these agents would inhibit the increase in the PLC-derived Ins(1,4,5)P3, which would otherwise enhance the release of Ca2+ from the SR. Although the deleterious effects of endogenously released catecholamines during ischemia are well established (37), the specific activation of PLC-{beta}1 in the ischemic heart may have implications for cardiac fibrosis, which occurs in I/R (30) and may contribute significantly to cardiac dysfunction in I/R. Indeed, we have previously proposed a role for the {alpha}1-adrenoceptor-G{alpha}q-PLC-{beta}1-signaling pathway in myocardial fibrosis (24). Furthermore, it is interesting to note that prazosin, an {alpha}1-adrenoceptor blocker, has been reported to attenuate myocardial injury in I/R (29).

While suppression of the increase in [Ca2+]i by verapamil is known to be cardioprotective due to the energy sparing effects at the level of contractile proteins and that the favorable effects of verapamil may also be due to its reported antioxidant properties (27), our findings have provided evidence that the beneficial effects of verapamil also extend to attenuation of the changes in PLC isozyme activities induced by I/R. Moreover, the enhanced myocardial recovery seen with U-73122, which was greater than with verapamil, confers pathophysiological relevance to PLC as a causative factor contributing to I/R injury. Indeed, given that the products of PLC activities play a role in activating Ca2+ transporting systems and that PLCs are activated by Ca2+, it is reasonable to assume that PLC may contribute to cardiomyocyte Ca2+ overload and subsequent cardiac dysfunction in I/R. Thus it is reasonable to speculate that modulation of the cardiomyocyte Ca2+ levels by inhibition of Ca2+ influx via the L-type Ca2+ channel and release of Ca2+ from internal stores via PLC may prove to be additive or synergistic and in turn provide greater cardioprotection. In conclusion, our findings suggest that PLC isozymes could be potential targets for the clinical management of ischemic heart disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a grant from the Canadian Institutes of Health Research-Regional Partnership Program with the St. Boniface Hospital Research Foundation. G. Asemu was supported by a Postdoctoral Fellowship from the Manitoba Health Research Council.


   ACKNOWLEDGMENTS
 
We thank Nina Aroutiounova for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. S. Tappia, Institute of Cardiovascular Sciences, Laboratory of Cardiac Membrane Biology, St. Boniface Hospital Research Centre (R3020), 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (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
 GRANTS
 REFERENCES
 

  1. Asemu G, Tappia PS, and Dhalla NS. Identification of the changes in phospholipase C isozymes in ischemic-reperfused rat heart. Arch Biochem Biophys 411: 174–182, 2003.[CrossRef][Web of Science][Medline]
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