Introduction

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Although early restoration of blood flow after coronary occlusion improves heart function and reduces infarct size (Kloner et al., 1983), delayed reperfusion after an episode of ischemia is invariably accompanied by acute deleterious effects that include cardiac contractile dysfunction, ultrastructural damage, and changes in energy metabolism (Dhalla et al., 1988). The ischemia-reperfusion (I/R)-induced injury is thought to be mediated by the occurrence of intracellular Ca²⁺ overload (Marban et al., 1989; Dhalla et al., 1996), which in turn causes derangement of cellular processes leading to a depletion of high energy phosphate content (Dhalla et al., 1995). Increased activity of the sympathetic system (Schömig and Richardt, 1990) as well as a 100- to 1000-fold increase in the release of norepinephrine from the nerve endings within 20 to 40 min of ischemia (Schömig, 1990) has also been reported to mediate myocardial cell damage. It should be pointed out that -adrenoceptor (-AR) blockers have been shown to provide protection against some of the abnormalities, including a reduction of the infarct size (Hammerman et al., 1984), an attenuation of arrhythmias (Lubbe et al., 1992), an improvement in the ventricular function, and an overall decrease in mortality (ISIS-1, 1986) in patients with ischemic heart disease. -AR blockers have also been used as an alternative to cardioplegic arrest during the coronary artery bypass surgery to reduce ischemic damage (Mehlhorn, 1997). Although the exact mechanisms for the cardioprotective action of these agents are not yet fully understood, -AR blockers are considered to exert beneficial effects on the ischemic heart by lowering myocardial oxygen consumption as a consequence of reduced contractility and heart rate, increasing oxygen delivery due to coronary artery dilation (Gross et al., 1982; Strangeland et al., 1984); as well as due to their antioxidant (Mak and Weglicki, 1988; Kramer et al., 1991) and membrane-stabilizing (Rochette et al., 1984) properties. These findings have formed the basis for developing therapeutic strategies in which -AR blockers are considered to be beneficial for the treatment of I/R-induced injury.

The sarcoplasmic reticulum (SR) is known to play a crucial role in the regulation of intracellular Ca²⁺ on a beat-to-beat basis. Ca²⁺ uptake in the SR occurs via an ATP-dependent Ca²⁺ pump ATPase (SERCA2a), which is regulated by its interaction with phospholamban (PLB) (Davis et al., 1983; Sasaki et al., 1992), whereas SR Ca²⁺ release occurs through the ryanodine receptor (RyR) (Coronado et al., 1994). Phosphorylation of SR proteins has been shown to affect the Ca²⁺ uptake and release activities (Xu et al., 1993; Hawkins et al., 1994; Li et al., 1997). We have recently reported that cardiac dysfunction due to I/R is associated with changes in Ca²⁺ uptake and release activities (Osada et al., 1998). Defects in SR gene expression (Temsah et al., 1999) and changes in SR function by protein phosphorylation (Osada et al., 1998; Netticadan et al., 1999) due to I/R have also been observed. In this study we investigated the effects of -AR blockade by atenolol, a ₁-specific blocker, and propranolol, a nonspecific -blocker, on changes in SR Ca²⁺ uptake, SR Ca²⁺ release, protein contents, and SR phosphorylation activities as well as SR gene expression in hearts subjected to I/R.

	Materials and Methods

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Heart Perfusion and Experimental Protocol. Male Sprague-Dawley rats weighing 230 to 330 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: 120 mM NaCl, 25 mM NaHCO₃, 11 mM glucose, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, and 1.25 mM CaCl₂. The hearts were electrically stimulated at a rate of 300 beats/min (Phipps and Bird Inc., Richmond, VA) and the perfusion rate was maintained at 10 ml/min. A water-filled latex balloon was inserted into the left ventricle and connected to a pressure transducer (model 1050BP; BIOPAC SYSTEM INC., Goleta, CA) for the left ventricular systolic and diastolic pressure measurements; the left ventricular developed pressure (LVDP) was the difference between systolic and diastolic pressures. The left ventricular end diastolic pressure (LVEDP) was adjusted at 10 mm Hg at the beginning of the experiment, and the left ventricular pressures were differentiated to estimate the rate of ventricular pressure development (+dP/dt) and the rate of ventricular pressure decline (dP/dt) using the Acknowledge 3.03 software for Windows (BIOPAC SYSTEM INC., Goleta, CA). All hearts were stabilized for a period of 30 min before use and were maintained at a constant temperature (37°C) throughout the experiments. The hearts were then randomly divided into four groups: control, I/R, atenolol-treated, and propranolol-treated hearts. The hearts were made globally ischemic by stopping the coronary flow for 30 min and then reperfusing for a period of 60 min. Atenolol and propranolol each at a final concentration of 10 µM (unless otherwise indicated in the text) were infused just above the perfusion cannula for 10 min before inducing ischemia as well as for 60 min during I/R (Fuller et al., 1990). Atenolol and propranolol were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). The selection of 10-µM concentrations of both atenolol and propranolol for use in this study was based on the work of other investigators (Richardt et al., 1990; Du et al., 1993). In another set of experiments, hearts were treated with atenolol or propranolol in the absence or presence of different concentrations of isoproterenol. In some of the experiments, hearts were also treated (in a similar manner as described above) with different concentrations of propranolol as well as phentolamine for studying their effects on the I/R-induced changes.

SR Preparation. SR membranes were prepared by a method used previously (Osada et al., 1998) with slight modifications. Briefly, ventricular tissue was homogenized in a mixture of: 10 mM NaHCO₃, 5 mM NaN₃, and 15 mM Tris-HCl, pH 6.8 (10 ml/g tissue) with a Polytron homogenizer (Brinkmann, Westbury, NY). The homogenate buffer also contained protease inhibitors: 1 µM leupeptin, 1 µM pepstatin, and 100 µM phenylmethylsulfonyl fluoride. The homogenate was then centrifuged for 20 min at 9500 rpm (JA 20.0; Beckman) and the supernatant was further centrifuged for 45 min at 19,000 rpm (JA 20.0; Beckman). The pellet was suspended in 8 ml of a mixture of 0.6 M KCl, 20 mM Tris-HCl, pH 6.8, and centrifuged for 45 min at 19,000 rpm. The final pellet was suspended in 1 ml of 250 mM sucrose and 10 mM histidine, pH 7.0. The purity of the membrane preparation was determined by measuring the activities of marker enzymes such as ouabain-sensitive Na⁺-K⁺-ATPase (sarcolemmal marker), cytochrome c oxidase (mitochondrial marker), glucose-6-phosphatase (SR marker), and rotenone-insensitive NADPH cytochrome c reductase (SR marker) according to methods described earlier (Osada et al., 1998). These marker studies revealed that SR preparations from control and experimental hearts contained negligible (2-4%), but to an equal extent, cross-contamination by other subcellular organelles. The protein concentration of the SR preparations was measured as indicated earlier (Osada et al., 1998).

Measurement of Ca²⁺ Uptake. Calcium uptake activity of SR vesicles was measured by a procedure described previously (Osada et al., 1998). In brief, a total volume of 250 µl contained: 50 mM Tris-maleate (pH 6.8), 5 mM NaN₃, 5 mM ATP, 5 mM MgCl₂, 120 mM KCl, 5 mM potassium oxalate, 0.1 mM EGTA, 0.1 mM ⁴⁵CaCl₂ (20 mCi/liter), and 25 µM ruthenium red. Ruthenium red was added as an inhibitor of the Ca²⁺-release channel under the assay conditions used here. The reaction was initiated by adding SR vesicles (10 µg protein) and terminated after 1 min by filtering a 200-µl aliquot of the incubation mixture through a 0.45-µm Millipore filter. The latter was washed with 5 ml of washing buffer, dried at 60°C for 1 h, and then counted in a liquid scintillation counter.

Measurement of EGTA-Induced Ca²⁺ Release. The Ca²⁺ release activity of SR vesicles was measured by a procedure described earlier (Osada et al., 1998). In brief, the SR fraction (62.5 µg protein) was suspended in 625 µl of the loading buffer containing: 100 mM KCl, 5 mM MgCl₂, 5 mM potassium oxalate, 5 mM NaN₃, and 20 mM Tris-HCl (pH 6.8). After incubation with 10 µM ⁴⁵CaCl₂ (20 mCi/liter) and 5 mM ATP for 45 min at room temperature, EGTA-induced Ca²⁺ release was carried out by adding 1 mM EGTA to the reaction mixture. The reaction was terminated at 15 s by the Millipore filtration technique. Radioactivity in the filter was counted in 10 ml of scintillation fluid.

Measurement of Phosphorylation by Endogenous Ca²⁺/Calmodulin-Dependent Protein Kinase (CaMK) and Exogenous cAMP-Dependent Protein Kinase (PKA). For the phosphorylation experiments, the SR was isolated in the presence of phosphatase inhibitors to prevent any dephosphorylation during the SR isolation procedure. The homogenization buffer contained 10 nM microcystin-LR and 1 mM sodium pyrophosphate for inhibiting the endogenous phosphatase activity. SR protein phosphorylation by CaMK was determined according to the procedure described by Osada et al. (1998). The assay medium (total volume 50 µl) for phosphorylation by endogenous CaMK contained: 50 mM HEPES (pH 7.4), 10 mM MgCl₂, 0.1 mM CaCl₂, 0.1 mM EGTA, 0.002 mM calmodulin, 0.8 mM [-³²P]ATP (specific activity 200-300 cpm/pmol), and SR (30 µg of protein). Phosphatase inhibitors, microcystin-LR (10 nM) and sodium pyrophosphate (1 mM), were also added to the reaction mixture. The initial concentration of free Ca²⁺, as determined by the computer program of Fabiato (1988), was 3.7 µM. The Ca²⁺/calmodulin dependence of phosphorylation was monitored in parallel assays lacking Ca²⁺ (1 mM EGTA was present) and calmodulin in the assay medium. The assay medium (50 µl) for phosphorylation by PKA contained: 50 mM HEPES (pH 7.4), 10 mM MgCl₂, 0.8 mM [-³²P]ATP (specific activity 200-300 cpm/pmol), SR (30 µg of protein), and PKA (catalytic subunit from the bovine heart; 5.6 µg). PKA dependence of phosphorylation was monitored in parallel assays lacking the PKA catalytic subunit. The phosphorylation reaction was initiated by the addition of [-³²P]ATP after preincubation of the assay medium for 3 min at 37°C. Reactions were terminated after 2 min by adding 15 µl of sample buffer, and the samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 4 to 18% gradient slab gels. The gels were stained with Coomassie Brilliant Blue, dried, and autoradiographed. The intensity of each phosphorylated band was scanned by an Imaging Densitometer (Bio-Rad Ltd., Hercules, CA).

Western Blot Analysis. The SR protein contents of SERCA2a, RyR, PLB, and calsequestrin (CQS) were determined according to methods described earlier (Temsah et al., 1999). Protein samples (20 µg of total protein/lane) were separated by electrophoresis through a 10% mini SDS-PAGE in 5% (for RyR), 10% (for SERCA2a), 12% (for CQS), and 15% (for PLB) gels. Samples for SERCA2a, PLB, and CQS were transferred to polyvinylidene difluoride membranes whereas that for RyR was transferred to nitrocellulose membrane. The membranes were probed with monoclonal anti-SERCA2a (1:1400; Affinity Bioreagents Inc., Golden, CO), monoclonal anti-RyR (1:1400), monoclonal anti-PLB (1:2000), or polyclonal anti-CQS (1:2000) antibodies. The antibodies for RyR, PLB, and CQS were purchased from Upstate Biotechnology (Lake Placid, NY). For SERCA2a and PLB, a peroxidase-linked anti-mouse IgG was used as a secondary antibody (1:5000), whereas biotinylated anti-mouse IgG antibody (1:2500; Amersham Life Science, Oakville, ON, Canada) was used for RyR and CQS. The membranes for RyR and CQS were incubated with streptavidin-conjugated horseradish peroxidase (1:5000; Amersham Life Science, Oakville, ON, Canada). Antibody-antigen complexes in all membranes were detected by the chemiluminescence ECL kit (Amersham Life Science). Protein bands were then visualized on Hyperfilm-ECL. An Imaging Densitometer model GS-670 (Bio-Rad Ltd., Hercules, CA) was used to scan the protein bands; these were quantified using the Image Analysis Software Version 1.3. It is pointed out that a linear relationship between the density of blots and protein load was observed when 5-, 10-, 20-, and 30-µg membrane protein was used per lane.

RNA Isolation and Northern Blot Analysis. Total RNA was extracted from the ventricular tissue by the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987). Samples normalized to 20 µg of total RNA were denatured with formaldehyde and electrophoresed in a 1.2% agarose/formaldehyde gel. The fractionated mRNA transcripts were transferred to a charge-modified nylon filter (NYTRAN Maximum Strength Plus; Schleicher & Schuell, Keene, NH) for 24 h. The membrane was then UV cross-linked (UV Stratalinker 2400; Stratagene). Blots were prehybridized at 42°C overnight using an INNOVA 4080 incubator (New Brunswick Scientific Inc., Edison, NJ) oscillating at a rate of 65 rpm. Labeled probes were added to the prehybridization solution and left overnight under the same conditions. The hybridized blots were exposed to X-ray film (Kodak-X-OMAT). The radiolabeled mRNA bands were scanned using a densitometer and quantified with the Image Analysis Software. Inserts were separated from recombinant plasmids and used as probes: 1) SR SERCA2a was a 0.762-kb cDNA fragment; 2) RyR was a 2.2-kb cDNA fragment; 3) PLB was a 0.503-kb cDNA fragment from the rabbit heart; 4) CQS was a 2.5-kb cDNA fragment from the rabbit heart; 5) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was a 1.2-kb cDNA of the human (American Type Culture Collection, Rockville, MD); 6) -actin was a 1.1-kb cDNA of the human (American Type Culture Collection); 7) -myosin heavy chain (-MHC) was a 39-bp (5'-GGGATAGCAACAGCGAGGCTCTTTCTGCTGGACAGGTTA-3'; 8) G_i2 was a 1.365-kb cDNA of the mouse (American Type Culture Collection). 18S was a 24-base oligonucleotide probe of the rat ribosomal RNA that was used as an internal standard. The cDNA used to hybridize specific mRNA transcripts was prepared and autoradiographed using a Random Primer DNA Labeling System radiolabeled with [-³²P]dCTP (New England Nuclear, Boston, MA).

Statistical Analysis. Results are expressed as mean ± S.E. and evaluated statistically by one-way ANOVA test followed by Student's t test. A level of P < .05 was considered the threshold for statistical significance between the control and experimental groups.

	Results

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Effect of -AR Blockers on Cardiac Performance and SR Function. Cardiac performance was evaluated by measuring LVDP, LVEDP, +dP/dt, and dP/dt for the control and experimental groups (Fig. 1 and Table 1). Both atenolol and propranolol at 10-µM concentrations showed no effect on cardiac performance in the control hearts (Table 1). Hearts subjected to 30 min of ischemia lost their contractile function completely. Reperfusion for 60 min after ischemia partially improved the contractile function as reflected by 28% recovery in LVDP, 19% recovery in +dP/dt, and 19% recovery in dP/dt. On the other hand, LVEDP in the I/R hearts increased by 7.7-fold over the control value (Fig. 1 and Table 1). I/R hearts treated with 10 µM atenolol demonstrated a significant increase in recovery of the cardiac performance as reflected by 45% recovery in LVDP, 44% recovery in the +dP/dt, and 64% recovery in dP/dt; the LVEDP was significantly lower by this treatment but was still 5.2-fold higher than the control (Fig. 1, A-D). Treatment with 10 µM propranolol also showed a significant improvement in cardiac function as the recovery in LVDP, +dP/dt, and dP/dt was 67, 57, and 79% in comparison with preischemic values, respectively (Fig. 1, A-C). Although the level of LVEDP in propranolol-treated hearts was 3.6-fold higher than the control, it was significantly less than that in the I/R group (Fig. 1B).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effect of -blockade on cardiac performance due to I/R in the isolated rat heart. Hearts were made ischemic by occluding the coronary flow for 30 min; the flow was restored for 60 min for inducing reperfusion. LVDP (A), LVEDP (B), +dP/dt (C), and dP/dt (D) in the control-untreated (C), I/R-untreated, and I/R hearts treated with 10 µM atenolol (A) and 10 µM propranolol (P). Values of LVDP, LVEDP, +dP/dt, and dP/dt for I/R hearts with or without drug treatments are represented as percentage of the respective preischemic values, whereas those values for control hearts are represented as percentage of values at the end of the stabilization period. *P < .05 versus control; , versus I/R; #, versus A. Each value is a mean ± S.E. of six hearts in each of the four groups.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effect of -blockade on SR function due to I/R in the isolated rat heart. Ca2+ uptake (A) and Ca2+ release (B) observed in the control-untreated (C), I/R-untreated, and I/R hearts treated with 10 µM atenolol (A) and 10 µM propranolol (P). The experimental protocol is the same as in the legend for Fig. 1. *P < .05 versus control; , versus I/R. Each value is a mean ± S.E. of six hearts in each of the four groups.

Phosphorylation of SR Proteins by Endogenous CaMK and Exogenous PKA. CaMK phosphorylation of SR proteins was determined by autoradiography (Fig. 3A) and the values are presented as percentage of control (Fig. 3B). I/R resulted in a marked decrease in CaMK phosphorylation of RyR, SERCA2a, and PLB [at both high (H) and low (L) molecular weights] by 73, 59, and 70% when compared with the control values, respectively (Fig. 3B). Treatment with atenolol significantly improved CaMK phosphorylation of RyR by 51%, SERCA2a by 23%, and PLB by 47% (Fig. 3B) in comparison to I/R. The recovery of phosphorylation levels by propranolol was 57% for RyR, 39% for SERCA2a, and 66% for PLB when compared with the I/R group. PKA-mediated phosphorylation of the high (H)- and low (L)-molecular-weight forms of PLB is depicted in Fig. 4 (A and B) and the values are presented as percentage of control (Fig. 4B). I/R reduced PKA phosphorylation of total PLB by 52% of the control value, whereas treatment with both atenolol and propranolol resulted in a significant improvement in PLB phosphorylation by 19 and 38% when compared with I/R values, respectively.

View larger version (52K): [in this window] [in a new window]   Fig. 3.   The endogenous CaMK-mediated phosphorylation of RyR, SERCA2a, high molecular weight PLB, PLB (H), and low molecular weight PLB, PLB (L), in the control-untreated (C), I/R-untreated, and I/R hearts treated with 10 µM atenolol (A) and 10 µM propranolol (P) is shown in A. The analysis of CaMK phosphorylation of RyR, SERCA2a, and PLB is shown in B. PLB phosphorylation was the sum of PLB (H) and PLB (L) phosphorylations. The cardiac SR was phosphorylated in the presence and absence of Ca2+ and calmodulin and subjected to 4 to 18% SDS-PAGE as described in Materials and Methods. The experimental protocol is the same as in Fig. 1. The results are mean ± S.E. of four hearts in each of the four groups. *P < .05 versus control; , versus I/R; #, versus A.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   The exogenous PKA-mediated phosphorylation of PLB (H) and PLB (L) in the control-untreated (C), I/R-untreated, and I/R hearts treated with 10 µM atenolol (A) and 10 µM propranolol (P) is shown in A; the analysis of PKA phosphorylation of PLB is shown in B. PLB phosphorylation was the sum of PLB (H) and PLB (L) phosphorylations. The cardiac SR was phosphorylated in the presence and absence of exogenous PKA and subjected to 4 to 18% SDS-PAGE as described in Materials and Methods. The results are mean ± S.E. of four hearts in each of the four groups. *P < .05 versus control; , versus I/R; #, versus A.

SR Protein Contents. To examine the mechanisms underlying the depressed SR function, we estimated the SR protein levels using Western blot analysis. In I/R hearts, the protein levels of SERCA2a, RyR, and PLB were depressed by 70, 55, and 15% from the control levels, respectively (Fig. 5, A-D). Hearts treated with atenolol showed a slight, but significant, protection only in SERCA2a protein levels (by 15%) when compared with I/R-induced changes in protein levels. On the other hand, propranolol-treated hearts showed a significant protection in SERCA2a (by 60%), RyR (by 15%), and PLB (by 15%) from I/R levels (Fig. 5, A-D). The CQS protein content was significantly high in I/R hearts (by 30% from control level), whereas atenolol and propranolol treatment significantly reduced the levels of CQS (Fig. 5E).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Autoradiograms (A) and analysis of SERCA2a (B), RyR (C), PLB (D), and CQS (E) of SR protein contents for control-untreated (C), hearts subjected to I/R-untreated, I/R hearts treated with 10 µM atenolol (A) or 10 µM propranolol (P). Protein samples were solubilized, separated on SDS-polyacrylamide gel, transferred to membranes, and incubated with respective antibodies. The results are mean ± S.E. of four hearts in each of the four groups. *P < .05 versus control; , versus I/R; #, versus A.

Gene Expression of SR Proteins. To determine the possibility of -blockers protection against I/R changes, gene expression of the SR proteins was examined using Northern blots (Fig. 6). The analysis of the autoradiograms revealed that I/R significantly decreased the levels of mRNA for SERCA2a by 61%, RyR by 89%, PLB by 58%, and CQS by 48% when compared with the control (Fig. 7, A-D). Treatment with atenolol showed improvement over I/R with respect to mRNA levels for SERCA2a, RyR, PLB, and CQS by 25, 26, 20, and 24%, respectively (Fig. 7, A-D). Propranolol also significantly improved the mRNA levels for SERCA2a by 39%, RyR by 40%, PLB by 23%, and CQS by 29% when compared with I/R group (Fig. 7, A-D).

View larger version (57K): [in this window] [in a new window]   Fig. 6.   Autoradiogram of mRNA expression of SERCA2a, RyR, PLB, CQS, GAPDH, -actin, -MHC, and Gi in isolated perfused rat heart treated with -blockers. 1, control-untreated; 2, I/R-untreated; 3, I/R hearts treated with 10 µM atenolol; and 4, I/R hearts treated with 10 µM propranolol. Total RNA (20 µg) was extracted from the heart tissue and assayed for the mRNA with respective probes (see Materials and Methods). 18S mRNA band was used as an internal standard to account for differences in nucleic acid loading and/or transfer. Bottom, the ethidium bromide-stained agarose gel before transfer.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Northern blot analysis of SR gene expression: SERCA2a (A); RyR (B); PLB (C); and CQS (D) in the control-untreated (C), I/R-untreated, and I/R hearts treated with 10 µM atenolol (A) and 10 µM propranolol (P). Values are mean ± S.E. of four hearts in each of the four groups. *P < .05 versus control; , versus I/R.

View this table: [in this window] [in a new window]   TABLE 3 Effect of I/R with and without -blocker treatment on the gene expression of non-SR genes The results are mean ± S.E. of six hearts in each of the four groups.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have found that I/R under acute conditions may induce cardiac dysfunction and SR abnormalities as reflected by a depression in both Ca²⁺ uptake and release activities. The depressed cardiac performance as well as SR Ca²⁺ uptake and Ca²⁺ release activities due to I/R, as observed in this study, are in agreement with our previous reports (Osada et al., 1998; Netticadan et al., 1999; Temsah et al., 1999). Furthermore, treatment of the hearts with either atenolol, a ₁-specific blocker, or propranolol, a nonspecific -blocker, significantly protected against the I/R-induced changes in the cardiac performance and SR function. The recovery of cardiac performance attained with propranolol is in agreement with other studies (Lu et al., 1990; Toleikis and Tomlinson, 1997). Although some investigators have reported no mechanical recovery with atenolol (Lu et al., 1990; Vandeplassche et al., 1991), our results indicate a significant recovery with atenolol. This may be due to differences in experimental models, species, and concentrations of the drug used. Nonetheless, the beneficial effects of -adrenergic blockers in I/R-induced injury are well documented (Hammerman et al., 1984; Schömig and Richardt, 1990; Thandroyen et al., 1990). Because SR Ca²⁺ release and SR Ca²⁺ uptake activities are known to play a major role in the handling of intracellular Ca²⁺ and subsequent cardiac contraction and relaxation processes (Dhalla et al., 1996), it appears that the I/R-induced changes in cardiac performance as well as the beneficial effects of -AR blockers may be mediated through corresponding changes in SR function.

In view of the fact that SR Ca²⁺ uptake and Ca²⁺ release activities are stimulated by CaMK- and PKA-mediated phosphorylations (Sasaki et al., 1992; Netticadan et al., 1999; Osada et al., 1998), it is possible that the observed changes in SR function may be due to abnormalities in CaMK- and PKA-mediated protein phosphorylations. Because the phosphorylation of RyR by the endogenous CaMK has been reported to increase the Ca²⁺ release channel activity (Li et al., 1997), the observed depression in RyR phosphorylation may account for the impaired SR Ca²⁺ release due to I/R (Osada et al., 1998). Furthermore, CaMK phosphorylation has been reported to increase ATP hydrolysis and Ca²⁺ transport in SR (Toyofuku et al., 1994), and thus the depression observed in SERCA2a phosphorylation may explain the I/R-induced decrease in SR Ca²⁺ uptake. Because -blocker treatments were found to improve the phosphorylation of RyR, SERCA2a, and PLB by CaMK and the phosphorylation of PLB by PKA, the observed improvements of the I/R-induced changes in SR Ca²⁺ uptake and release functions on treating the hearts with atenolol and propranolol may be related to the protection of both CaMK and PKA regulatory mechanisms. However, it should be pointed out that the RyR, SERCA2a, and PLB protein contents in the SR membrane are decreased in the I/R hearts, and these changes are ameliorated by treatments with -AR blockers. Thus the role of changes in SR proteins in explaining the observed alterations in SR Ca²⁺ uptake and Ca²⁺ release activities in the I/R hearts with or without drug treatments seems evident. Because proteases are activated in I/R (Yoshida et al., 1993) and have been reported to cause SR protein degradation (Yoshida et al., 1990), it is possible that the observed changes in SR protein in I/R hearts are due to proteolysis whereas the beneficial effects of -blockers are due to their action on proteolysis. It is also noteworthy that we have recently shown that the depression in the endogenous CaMK activity due to I/R may partly contribute to SR dysfunction (Netticadan et al., 1999) and thus the protection of SR regulatory mechanisms by -blocker treatments cannot be ruled out. Accordingly, it appears that the I/R-induced alterations in SR Ca²⁺ uptake and Ca²⁺ release activities and their attenuations by treatments with -AR blockers may be due to corresponding change in both SR proteins and regulatory phosphorylation activities.

The beneficial effects of -AR blockers on the I/R-induced changes in cardiac performance, SR function, and regulatory mechanisms may be attributed to their -blocking properties because the concentrations of both atenolol and propranolol used here prevented the positive inotropic effect of isoproterenol completely. Because an excessive amount of catecholamines released during I/R may alter SR Ca²⁺ transport mechanisms (Dhalla et al., 1996) resulting in intracellular Ca²⁺ overload (Dhalla et al., 1988), -AR blockade may attenuate these deleterious effects and render cardioprotection. However, it may be noted that the beneficial effects of propranolol were evident at low concentrations (1 µM) whereas a high concentration of propranolol (30 µM), which exerted a generalized cardiodepressant effect, showed no protection of I/R-induced changes in LVDP and SR Ca²⁺ transport. Furthermore, the improvement observed in cardiac performance as well as SR Ca²⁺ uptake, Ca²⁺ release, protein contents, and phosphorylation in hearts treated with 10 µM propranolol was significantly higher than in the hearts treated with 10 µM atenolol. This difference may be attributed to the properties, such as higher lipophilicity, membrane-stabilizing activity (Kramer et al., 1991), antiperoxidative activity (Mak and Weglicki, 1988), and antiradical effect (Khaper et al., 1997) of propranolol in comparison with those of atenolol. Furthermore, propranolol, unlike atenolol, has been reported to prevent the I/R-induced release of norepinephrine from the sympathetic nerve endings in the heart (Richardt et al., 1990; Du et al., 1993) and thus other actions of -AR blockers cannot be ruled out. Accordingly, it is suggested that -AR blockade as well as the ancillary properties of propranolol may contribute toward its cardioprotective effects in I/R hearts.

I/R was found to cause a decrease in the mRNA abundance of RyR, SERCA2a, PLB, and CQS, and this observation is in agreement with our previous report (Temsah et al., 1999). The protective action of -blockade on I/R-induced changes in the levels of mRNA for SR proteins is consistent with the beneficial effects of -AR blockers. Such changes in mRNA levels specific for some of the SR proteins can be considered to explain the alterations observed in the SR protein contents in the I/R hearts treated with or without -AR blockers. However, it can be argued that the relationship between changes in mRNA levels and SR protein contents is of questionable significance because of the short duration of the I/R conditions used in this study and the time that may be required for a message to be translated into functional proteins. Furthermore, a decrease in mRNA level for CQS was associated with an increase in the CQS protein content in SR in the I/R hearts and treatment with -blockers increased the mRNA level and decreased the SR protein content for CQS. Alterations in CQS protein content, unlike other SR proteins, may be due to changes in the immunoreactivity or translocation of this protein as a consequence of I/R. Although the decrease in the SR gene expression in I/R appears to be a general deterioration because other non-SR genes such as GAPDH, -actin, -MHC, and G_i were also depressed, it is pointed out that each change was of varying magnitude and thus it is possible that different genes may have different susceptibilities to cardiac stress due to as yet unidentified causes. Because the G_i gene expression, unlike others, did not recover with both -AR blocker treatment, there appears to be some degree of specificity with respect to the beneficial effects of treatment on the gene expression of SR proteins. It is likely that alterations in the gene expression may occur during or after cardiac dysfunction due to I/R as Temsah et al. (1999) have observed no changes in the PLB gene expression after 30 min, whereas the myocardial function ceased after 1 min of ischemia. Nonetheless, in view of the importance of cardiac gene expression in maintaining the function of cardiac proteins, the observed changes in mRNA levels for SR proteins due to I/R may reflect delay in the recovery of SR function and cardiac performance in the ischemic hearts subsequent to establishing reflow. In addition, the beneficial effect of -blockade in attenuating the I/R-induced changes in mRNA levels for SR proteins can also be seen to support the view regarding cardiac gene expression as a molecular site for the cardioprotective action of -blocking agents. Because the protection by -AR antagonists with respect to I/R-induced changes in SR function and contractile performance were partial in nature, this study does not exclude the participation of other factors in the genesis of I/R injury.