Cardiovascular Research

Cardiovascular Research 1997 36(2):174-184; doi:10.1016/S0008-6363(97)00175-2
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Injury to the Ca²⁺ ATPase of the sarcoplasmic reticulum in anesthetized dogs contributes to myocardial reperfusion injury

Steven C Smart^a,*, Kiran B Sagar^a, Jo El Schultz^a, David C Warltier^a and Larry R Jones^b

^aDivision of Cardiovascular Medicine, Department of Medicine and the Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA
^bThe Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

^* Corresponding author. Tel. (+1-414) 257-6697; Fax (+1-414) 2577291; E-mail: ssmart@post.its.mcw.edu

Received 19 November 1996; accepted 24 June 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Sarcoplasmic reticulum dysfunction may contribute to calcium (Ca²⁺) overload during myocardial reperfusion. The aim of this study was to investigate its role in reperfusion injury. Methods: Open chest dogs undergoing 15 min of left anterior descending coronary artery occlusion and 3 h of reperfusion were randomized to intracoronary infusions of 0.9% saline, vehicle, or the Ca²⁺ channel antagonist, nifedipine (50 µg/min from 2 minutes before to 5 minutes after reperfusion). After each experiment, transmural myocardial biopsies were removed from ischemic/reperfused and nonischemic myocardium in the beating state and analyzed for (i) sarcoplasmic reticulum protein content (Ca²⁺ ATPase, phospholamban, and calsequestrin) by immunoblotting and (ii) Ca²⁺ uptake by sarcoplasmic reticulum vesicles with and without 300 micromolar ryanodine or the Ca²⁺ ATPase activator, antiphospholamban (2D12) antibody. Results: Contractile function did not recover in controls and vehicle-treated dogs after ischemia and reperfusion (mean systolic shortening, –2±2%), but completely recovered in nifedipine-treated dogs (17±2%, p = NS vs. baseline, p<0.01 vs. control). Ventricular fibrillation occurred in 50% of controls and vehicle dogs and 0% of nifedipine-treated dogs (p<0.01). Ca²⁺ uptake by the sarcoplasmic reticulum vesicles was severely reduced in ischemic/reperfused myocardium of controls and vehicle dogs (p<0.01 vs. nonischemic). Ryanodine and the 2D12 antibody improved, but did not reverse the low Ca²⁺ uptake. Protein content was similar in ischemic/reperfused and nonischemic myocardium. In contrast, Ca²⁺ uptake and the responses to ryanodine and 2D12 antibody were normal in ischemic/reperfused myocardium from nifedipine-treated dogs. Conclusion: Dysfunction of the sarcoplasmic reticulum Ca²⁺ ATPase pump correlates with reperfusion injury. Reactivation of Ca²⁺ channels at reperfusion contributed to Ca²⁺ pump dysfunction. Ca²⁺ pump injury may be a critical event in myocardial reperfusion injury.

KEYWORDS Nifedipine; Calcium channel antagonist; Myocardial ischemia; Calcium, intracellular concentration; Protein analysis; Stunning arrhythmias; Ventricular fibrillation; Reperfusion; Dog, anesthetized

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Myocardial reperfusion injury is probably mediated by oxygen free radicals and transient calcium (Ca²⁺) overload [1–7]. Targets of oxyradicals and Ca²⁺ activated enzymes include the sarcolemma, sarcoplasmic reticulum, and contractile proteins [2, 7]. Injury to the sarcoplasmic reticulum may exacerbate Ca²⁺ overload during early reperfusion and setup the myocardium for reperfusion injury (contractile dysfunction and ventricular fibrillation) [8, 9].

The sarcoplasmic reticulum has a major role in myocardial Ca²⁺ homeostasis and accounts for >90% of the Ca²⁺ transient in mammals [2, 7, 8, 10, 11]. Release is triggered by Ca²⁺ influx through sarcolemmal Ca²⁺ channels [11]. Uptake is mediated by a Ca²⁺ pump (ATPase) regulated by phospholamban [11]. Ca²⁺ is concentrated in the sarcoplasmic reticulum by the binding protein, calsequestrin [11]. Ca²⁺ content is determined by the competition between the sarcoplasmic reticulum Ca²⁺ pump and efflux by the sarcolemmal Na⁺/Ca²⁺ exchanger [11].

Reperfusion injury correlates with a secondary increase in Ca²⁺ after reperfusion [10]. Reactivation of myocardial Ca²⁺ channels and Ca²⁺ release by the sarcoplasmic reticulum at the onset of reperfusion may have a major role in this secondary increase [12, 13], but prior studies have not investigated how resumption of this normal trigger for contraction can mediate injury.

The hypotheses tested in this study are: (i) the sarcoplasmic reticulum is injured by transient ischemia and reperfusion and (ii) the resulting dysfunction in Ca²⁺ homeostasis sets up the myocardium for reperfusion injury. Our aims were to: (i) compare sarcoplasmic reticulum function in ischemic/reperfused myocardium from saline and vehicle treated dogs with control nonischemic myocardium; (ii) determine protein content by immunoblotting, and (iii) compare these findings with sarcoplasmic reticulum function from ischemic/reperfused myocardium of dogs treated with intracoronary nifedipine at the onset of reperfusion. Thereby, open chest dogs undergoing 15 min of left anterior descending occlusion and 3 h of reperfusion were randomized to intracoronary saline, vehicle, or nifedipine (50 µg/min from 2 minutes before until 5 minutes after reperfusion). Transmural biopsies were taken from the ischemic/reperfused and nonischemic zones and analyzed for sarcoplasmic reticulum Ca²⁺ uptake and protein content.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 General preparation
Mongrel dogs (15–30 kg) were anesthetized with intravenous sodium pentobarbital (25 mg/kg) and barbital (200 mg/kg) and ventilated. Saline (0.9%) was infused at 300 ml/hour and body temperature and arterial blood gases maintained at physiological levels. Arterial and left ventricular pressures and dP/dt were measured by a high-fidelity, double-tipped pressure transducer catheter (Millar PC 771, Dallas, TX) inserted from the left carotid artery with electronic differentiation. Silastic catheters were inserted in the right femoral artery and vein.

A thoracotomy was performed in the left fifth intercostal space and the heart suspended in a pericardial cradle. A segment of the left anterior descending coronary artery was isolated proximal to the first major diagonal for an electromagnetic flow probe (Statham SP7515) and a silk ligature. A small distal branch was cannulated retrogradely with a heparin filled 27 gauge catheter. The tip was advanced into the left anterior descending coronary artery and position confirmed by injection of food coloring. A catheter was placed in the left atrial appendage for microsphere injection. Dogs were demand atrial paced at 120 bpm via the left atrial appendage. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2 Regional myocardial function
The left anterior descending (ischemic/reperfused zone) and left circumflex coronary distributions (nonischemic zone) were identified by the injection of food coloring and pairs of 5.0 MHz sonomicrometers implanted in the subendocardium in the circumferential plane in both zones 2 cm away from the intracoronary catheter. Segment length was monitored by ultrasonic transit time (Triton Inc., San Diego, CA). End diastole was the onset of the rapid rise in left ventricular pressure and end systole 20 ms before peak negative dP/dt [14]. Systolic shortening (SS) was the change (%) in segment length from end diastole (EDL) to end systole (ESL): SS=((EDL–ESL)/EDL)x100. End diastolic segment lengths were normalized baseline [14].

2.3 Regional myocardial blood flow
Myocardial blood flow was measured by radiolabeled microspheres at: (a) 10 min before occlusion; (b) 10 min after occlusion; (c) 30 min after reperfusion; and (d) 3 h after reperfusion [15]. Carbonized microspheres (⁴¹Ce, , , or , 15±2 µm, New England Nuclear, Boston, MA) in 10% Dextran and 0.01% Tween 80 were ultrasonicated and vortexed. Twenty µCi were injected in the left atrium. Reference samples were collected from the right femoral artery at 7 ml/min for 130 s starting just before microsphere injection.

After myocardial biopsies, the left anterior descending artery was occluded and India ink injected to delineate the ischemic/reperfused zone. The heart was electrically fibrillated, removed, and fixed in 10% formalin. Ischemic/reperfused and nonischemic zones were separated and weighed. Tissue samples (0.8–1.0 g) from the ischemic/reperfused (five) and nonischemic (three) zones were divided into subendo-, mid myo-, and subepi-cardial layers and weighed. Net activity of each isotope in tissue and reference blood samples was measured by a gamma counter (Packard Series 5000, Meriden, CT). Tissue blood flow was calculated by: Q_m=(Q_rxC_r/C_m)x100. Q_m was tissue blood flow (ml/min/100 g), Q_r the withdrawal rate of reference blood sample, C_r the reference sample activity (cpm), and C_m the tissue sample activity (cpm). Blood flows were averaged and dogs excluded for subendocardial flow >20 ml/min/100 g during occlusion [16].

2.4 Treatment and ischemia/reperfusion protocols
Dogs were randomized to intracoronary infusions of 0.9% saline (control; 1 ml/min), vehicle (0.9% saline with 4% ethanol and 2% polyethylene glycol 400), or nifedipine (5 mg in 100 ml of 0.9% saline with 4% ethanol and 2% polyethylene glycol 400; 50 µg/min) at the onset of reperfusion. Nifedipine was infused from 2 min before reperfusion until 5 min after reperfusion (total infusion time 7 min) to ensure maximal effects at the onset of reperfusion in the ischemic/reperfused zone [13]. Before and after nifedipine or vehicle, 0.9% saline was infused at 1 ml/min.

After instrumentation and baseline measurements, intracoronary infusion was started and the coronary artery occluded 40 min later. The ligature was released after 15 min and the dogs monitored for 180 min. If ventricular fibrillation occurred, dogs were defibrillated with pads on the lateral left ventricle and the right ventricle with up to 3 shocks (20, 30, and 30 J). A prior study revealed no prolonged effects on function [17].

2.5 Biopsy protocol and tissue preparation
Biopsies were taken from ischemic/reperfused and nonischemic myocardium of control, vehicle, and nifedipine-treated dogs. At the end of each experiment, purse string sutures were placed in the ischemic/reperfused and nonischemic zones. Eleven mm transmural core biopsies were removed in the beating state in random order, flash frozen in liquid nitrogen and stored at –70°C until analysis.

2.6 Isolation of sarcoplasmic reticulum vesicles
Each pair of ischemic/reperfused and nonischemic biopsy samples (0.5–1.0 g) were homogenized at 4°C in a Polytron PT-10-35 (Brinkmann Instruments) for 90 s in 10 ml (v/w) of 10 mM NaHCO₃ [18]. Sarcoplasmic reticulum vesicles were isolated by the method of Jones and Cala [19]. Homogenates were centrifuged at 14 000 g_max at 4°C for 20 min to remove large membrane particles and organelles. The supernatant was then centrifuged at 45 000g_max at 4°C for 30 min to yield membrane vesicles enriched in sarcoplasmic reticulum. The vesicles were washed once with 0.6 M KCl, 30 mM histidine (pH 7.0) to extract loosely bound proteins. The vesicles were then resuspended in 0.25 M sucrose and 30 mM histidine (pH 7.0) and stored frozen at –40°C. Protein content was measured by the Lowry method [20].

2.7 uptake studies
Ca²⁺ uptake and the interaction of the Ca²⁺ pump (ATPase) with phospholamban were studied as previously described [21, 22]. Studies with the monoclonal antiphospholamban 2D12 antibody were performed at low ionized Ca²⁺ (0.05 µM) because the antibody only stimulates Ca²⁺ transport at low concentrations [21]. ATP dependent uptake studies at low ionized Ca²⁺ were performed at pH 7.0 and 37°C in 1 ml of uptake medium containing (millimolar): 50 MOPS, 3 MgCl₂, 100 KCl, 10 potassium oxalate, 3 Na₂ATP, 5 NaN₃, 1 ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 0.2 CaCl₂ with tracer [21]. Reactions were started by adding sarcoplasmic reticulum vesicles to the uptake medium after preincubation in the presence and absence of the 2D12 antibody [21, 22]. At the selected time points in the studies, 100 µl samples were removed for Ca²⁺ uptake analysis [19, 22].

The role of the Ca²⁺ release channel was studied by determining ryanodine effects on Ca²⁺ uptake at high ionized Ca²⁺ (1.0 µM) to maximize its stimulation of Ca²⁺ uptake [23]. Studies were done in an uptake medium containing (millimolar): 50 histidine (pH 7.0), 3 MgCl₂, 100 KCl, 5 potassium oxalate, and 0.05 CaCl₂ with tracer [21]. Sarcoplasmic reticulum vesicles were preincubated in uptake medium for 10 min at 37°C with or without 300 µM ryanodine, prior to initiation of reactions by adding 3 mM ATP. At the same time points in the studies, 1600 µl samples were removed for Ca²⁺ uptake analysis [19, 22].

The suction filtration method for measuring Ca²⁺ uptake has been reported by this laboratory [19, 22]. The 100 and 1600 µl samples from the low (±2D12) and high ionized Ca²⁺ (±ryanodine) uptake solutions, respectively, were analyzed for protein content by a modified Lowry assay and ionized Ca²⁺ by spectrophotometry with 23 µM 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid as the Ca²⁺ indicator [19, 22]. Aliquots were suction filtered on a manifold through a filter (0.45 µm) and washed with 100 mM KCl, 20 mM imidazole (pH 7.0), 1 mM EGTA, and 5 mM MgCl₂. Radioactivity (cpm) of the filtered and filtrate fractions was counted and uptake determined (nmol of Ca²⁺/mg of protein) by the following formula:

2.8 Immunoblotting of sarcoplasmic reticulum proteins
The aliquot of the sarcoplasmic reticulum vesicle fraction was thawed and 300 µl added to 600 µl of 20% sodium dodecyl sulfate (SDS) [18]. The mixture was incubated at 25°C for 20 min and ultracentrifuged for 5 min at 100 000 rpm (Beckman TL-100.2 rotor). Supernatants were collected and protein content determined by the Lowry method [20].

SDS-polyacrylamide gel electrophoresis (PAGE) was performed with an 8% gel using a modified method of Porzio and Pearson [24]. Five and 15 µg of protein from each supernatant was electrophoresed per lane. For immunoblotting, protein samples were transferred to 0.2-µm pore size nitrocellulose membranes (Schleicher and Schuell) by electrophoresis at 3.0 amp for 90 min in 50 mM NaHPO₄ buffer (pH 7.5) and stained with amido black [25]. The nitrocellulose membranes were cut into horizontal strips corresponding to the mobility regions of Ca²⁺ ATPase (SERCA2), low and high molecular weights of phospholamban, and calsequestrin. Immunoblotting was performed using a 1:500 dilution of ascites fluid containing monoclonal antibody 2A7-A1, which is specific for the cardiac isoform of SERCA2; a 1:500 dilution of ascites fluid containing monoclonal antibody 2D12, which reacts with phospholamban; and polyclonal rabbit antibody to canine cardiac calsequestrin, affinity purified by incubation with canine cardiac calsequestrin bound to nitrocellulose membranes followed by acid extraction [18]. The transfer was blocked with 2% bovine serum albumin. Thereafter, the strips were incubated with the appropriate antibodies followed by incubation with -labeled protein A (New England Nuclear). Protein bands identified by autoradiography were excised and bound radioactivity quantified by gamma counting. Background counts (<10% of the total counts for each band) were subtracted from all measurements.

2.9 Statistical analysis
Values for Ca²⁺ ATPase (SERCA2), phospholamban, and calsequestrin were compared by the Student's t test for paired samples. Repeated-measures and multi (three to six)-way analysis of variance (ANOVA) with the Bonferroni t-test were used to identify changes in continuous data within and between groups, respectively. ² analysis was used to evaluate the effects of animal exclusion. Protein content and maximal (33 min after initiation of Ca²⁺ uptake) ryanodine and antiphospholamban antibody induced Ca²⁺ uptake of ischemic/reperfused myocardium were normalized by dividing the values by the respective values of each paired specimen of nonischemic myocardium. Linear regression analysis was used to determine whether normalized changes in protein content of the sarcoplasmic reticulum from ischemic/reperfused myocardium correlated with normalized changes in maximal ryanodine or antiphospholamban antibody induced Ca²⁺ uptake. A two tailed p<0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Animal data
Thirty-two dogs were randomized to the three experimental groups. Ventricular fibrillation occurred in 12 dogs (32%): 50% (8/16) of controls, 50% of vehicle (4/8) and 0% (0/8, p<0.05 vs. control/vehicle) of nifedipine dogs. Only 2 (17%) dogs were successfully defibrillated. One control was excluded due to high collateral blood flow during coronary occlusion. The control, vehicle, and nifedipine groups consisted of eight, five, and eight dogs, respectively.

3.2 Systemic hemodynamics
Hemodynamic data are summarized in Table 1. Hemodynamics in control and vehicle dogs were similar throughout the experimental protocol. Nifedipine infusion only reduced mean arterial pressure during drug infusion. Heart rate and peak positive dP/dt were unaffected and remained similar to the other groups throughout the protocol. Mean arterial pressure was similar to controls and vehicle dogs before and during coronary occlusion, lower during the first 5 minutes of reperfusion, but similar thereafter. Normalized end diastolic segment length increased during occlusion and recovered by 1–2 h after reperfusion, similar to controls and vehicle dogs.

View this table: [in this window] [in a new window]   Table 1 Hemodynamics

 
3.3 Myocardial blood flow
Table 2 summarizes myocardial blood flow in ischemic/reperfused and nonischemic zones. Blood flow before occlusion was similar in controls, vehicle and nifedipine dogs. Collateral blood flow to the ischemic/reperfused zone including the subendocardium was similar in all groups during occlusion. After reperfusion, blood flow to the ischemic/reperfused zone in controls and vehicle dogs was reduced at 30 min and returned to normal at 3 h. Ischemic/reperfused zone blood flow in nifedipine dogs was similar to baseline at 30 min (p<0.05 vs. controls) and 3 h after reperfusion. Nonischemic zone blood flow was similar in all groups at all timepoints.

View this table: [in this window] [in a new window]   Table 2 Regional myocardial blood flow (ml/min/100 g)

 
3.4 Regional myocardial function
Systolic shortening in the ischemic/reperfused and nonischemic zones is plotted in Fig. 1A and 1B, respectively. Systolic shortening before occlusion was similar in all groups. Coronary occlusion caused similar degrees of systolic lengthening. After reperfusion, systolic shortening remained depressed in controls and vehicle dogs, but completely recovered in nifedipine-treated dogs within the first hour. Nonischemic zone systolic shortening was similar in all groups at baseline and similarly increased during coronary occlusion and recovered after reperfusion.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Systolic shortening (SS) from ischemic/reperfused [IZ, Figure 1A)] and nonischemic zones (NZ, Figure 1B) of controls (N = 8) and dogs treated with vehicle (N = 5) and nifedipine (NIF, N = 8). Values are represented as mean±SEM. (A) Systolic shortening before occlusion was similar in all groups. During occlusion, systolic lengthening was similar in each group. After reperfusion, systolic shortening in controls and vehicle treated dogs did not recover and remained severely depressed. In contrast, systolic shortening in nifedipine dogs completely recovered by 60 min, and did not deteriorate thereafter. (B) Nonischemic zone systolic shortening was also similar at baseline, increased during left anterior descending coronary occlusion, and returned to baseline by 1-2 h after reperfusion. ** p<0.01 vs. control.

 
3.5 uptake studies
The yield of sarcoplasmic reticulum membrane protein was similar in nonischemic and ischemic/reperfused myocardium from control, vehicle, and nifedipine-treated dogs. The yields were 1.57±0.24 mg/g, 2.19±0.25 mg/g, 2.10±0.45 mg/g, and 2.02±0.61 mg/g, respectively.

Fig. 2A and 2B plot Ca²⁺ uptake at low ionized Ca²⁺ by sarcoplasmic reticulum vesicles from nonischemic and ischemic/reperfused myocardium of controls measured at low ionized Ca²⁺ with and without the 2D12 antiphospholamban antibody and at high ionized Ca²⁺ with and without 300 µM ryanodine, respectively. The antibody increased Ca²⁺ uptake of nonischemic myocardium at low ionized Ca²⁺ by 5–6 fold (Fig. 3A). Normalized Ca²⁺ uptake in ischemic/reperfused myocardium was markedly reduced (11±6%, p<0.01). Antibody caused a 4 fold increase in uptake, but normalized uptake remained depressed (22±5%, p<0.01). Ryanodine doubled Ca²⁺ uptake of nonischemic myocardium at high ionized Ca²⁺ (Fig. 3B). Again, normalized Ca²⁺ uptake in ischemic/reperfused myocardium was depressed (27±9%, p<0.01). Ryanodine doubled uptake, but normalized uptake remained depressed (27±6%, p<0.01).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Ca2+ uptake by sarcoplasmic reticulum vesicles from controls (N = 8) with and without preincubation with the antiphospholamban (2D12) antibody or 300 µM ryanodine (Ryn). Ca2+ uptake studies were performed at (A) low (0.05 µM) and (B) high ionized Ca2+ (1.0 µM), respectively. Values are mean±SEM. (A) Shows that preincubation of nonischemic myocardium (NZ) with 2D12 increased Ca2+ uptake by 5-6 fold. In contrast, Ca2+ uptake in ischemic/reperfused myocardium (IZ) was much less. Uptake responded to 2D12, but remained much less. (B) Shows that Ryn doubled Ca2+ uptake in nonischemic myocardium (NZ). Again, Ca2+ uptake in ischemic/reperfused myocardium was much less than nonischemic. Uptake responded to Ryn, but remained much less. * p<0.05 vs. NZ

 

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Ca2+ uptake by sarcoplasmic reticulum vesicles from vehicle treated dogs (N = 5) with and without preincubation with the antiphospholamban (2D12) antibody or 300 µM ryanodine (Ryn). Ca2+ uptake studies were performed at (A) low (0.05 µM) and (B) high ionized Ca2+ (1.0 µM), respectively. Values are mean±SEM. (A) and (B) show similar findings to saline treated controls. * p<0.05 vs. NZ

 
Fig. 3A and 3B plot Ca²⁺ uptake by vesicles from nonischemic and ischemic/reperfused myocardium of vehicle dogs measured at low ionized Ca²⁺ with and without the 2D12 antibody and at high ionized Ca²⁺ with and without ryanodine, respectively. Findings were similar to controls. Normalized Ca²⁺ uptake in ischemic/reperfused myocardium was depressed at low ionized Ca²⁺ (p<0.01) with and without the 2D12 antibody (10±5% and 18±4%, respectively) and at high ionized Ca²⁺ (p<0.01) with and without ryanodine (28±7% and 33±8%, respectively).

Fig. 4A and 4B plot Ca²⁺ uptake by vesicles from nonischemic and ischemic/reperfused myocardium of nifedipine-treated dogs at low ionized Ca²⁺ with and without 2D12 antibody and at high ionized Ca²⁺ with and without ryanodine, respectively. Normalized Ca²⁺ uptake in ischemic/reperfused myocardium was similar to nonischemic myocardium at low ionized Ca²⁺ with and without the 2D12 antibody (105±8% and 94±12%, respectively) and at high ionized Ca²⁺ with and without ryanodine (84±15% and 88±10%, respectively). Absolute Ca²⁺ uptake in ischemic/reperfused myocardium of these dogs at low ionized Ca²⁺ with and without 2D12 antibody was similar (p = NS) to control and vehicle nonischemic myocardium and higher (p<0.01) than control and vehicle ischemic/reperfused myocardium. Absolute Ca²⁺ uptake in nonischemic myocardium of these nifedipine-treated dogs at high ionized Ca²⁺ with ryanodine was mildly depressed compared to control dogs (see Fig. 2B and 4B). This effect was attributed to the vehicle, as a similar depression in Ca²⁺ uptake was observed using sarcoplasmic reticulum vesicles from nonischemic myocardium of vehicle-dogs.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Ca2+ uptake by sarcoplasmic reticulum vesicles from nifedipine treated dogs (N = 8) with and without preincubation with the antiphospholamban (2D12) antibody or 300 µM ryanodine (Ryn). Ca2+ uptake studies were performed at (A) low (0.05 µM) and (B) high ionized Ca2+ (1.0 µM). Values are mean±SEM. (A) Shows that Ca2+ uptake at low Ca2+ in ischemic/reperfused myocardium (IZ) was similar to nonischemic myocardium (NZ) and responded normally to 2D12. (B) Shows that similar findings at high Ca2+ in ischemic/reperfused myocardium and a normal response to Ryn. * p<0.05 vs. NZ

 
3.6 Immunoblotting of sarcoplasmic reticulum proteins
Fig. 5Fig. 6Fig. 7 demonstrate that the reduced Ca²⁺ uptake by the sarcoplasmic reticulum in dysfunctional myocardium after ischemia and reperfusion did not result from alterations in protein content. Fig. 5 is an autoradiograph of the immunoblot of paired ischemic/reperfused (IZ) and nonischemic (NZ) myocardial samples from 5 control dogs and 2 vehicle dogs. Antibodies to calsequestrin, phospholamban (2D12) and Ca²⁺ ATPase (2A7-A1) discretely bound to their expected bands. Gamma counting of bands from ischemic/reperfused and nonischemic myocardium (Fig. 6) revealed that the concentration of proteins (Ca²⁺ ATPase, calsequestrin, and phospholamban) was not reduced (p = NS) in ischemic/reperfused myocardium of either controls or vehicle dogs. Fig. 7 is a linear regression plot of protein content (Ca²⁺ ATPase, calsequestrin, and phospholamban) and maximal ryanodine and 2D12 antibody induced Ca²⁺ uptake in ischemic/reperfused myocardium normalized to each paired sample of nonischemic myocardium. The normalized contents of Ca²⁺ ATPase, calsequestrin, and phospholamban in ischemic/reperfused myocardium were 85±9% (range 60–136), 80±14% (46–120), and 89±20% (34–209), respectively, in controls and 100±11% (range 87–136), 93±21% (52–170), and 117±29% (47–210), respectively, in vehicle dogs. There was no correlation between the normalized changes in content of any protein and normalized maximal ryanodine or 2D12 antibody induced Ca²⁺ uptake in controls or vehicle dogs.

View larger version (74K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Composite autoradiograph of the immunoblot of paired specimens of ischemic/reperfused (IZ) and nonischemic (NZ) myocardium. Lanes 1-10 are paired immunoblots of ischemic/reperfused and nonischemic myocardium from control dogs using 15 µg of enriched sarcoplasmic reticulum fraction. Lanes 11-14 are the immunoblots from ischemic/reperfused and nonischemic myocardium from vehicle dogs. The top portion represents bound antibody to Ca2+ ATPase (2A7-A1), the middle to calsequestrin (CSQ), and the bottom to the high (H) and low (L) molecular weight forms of phospholamban. Migration of molecular weight standards (kd) is indicated at the right.

 

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Bar graphs of the gamma counts of the bands of sarcoplasmic reticulum proteins from the immunoblots of ischemic/reperfused (IZ) and nonischemic (NZ) myocardium of controls (N = 8) and vehicle dogs (N = 5). Values are represented as mean±standard error of mean. Gamma counting revealed that the concentrations of Ca2+ ATPase (SERCA2), calsequestrin (CSQ), and phospholamban (PLB) were similar in ischemic/reperfused (IZ) and nonischemic (NZ) myocardium of controls and vehicle dogs.

 

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Linear regression plots of the normalized content of Ca2+ ATPase (A), calsequestrin (CSQ) (B) and phospholamban (C) in ischemic/reperfused (IZ) myocardium from control (C, N = 8) and vehicle dogs (V, N = 5) and maximal normalized ryanodine (RYN) and 2D12 induced Ca2+ uptake. Maximal ryanodine or 2D12 induced Ca2+ uptake in controls or vehicle dogs did not correlate (r range 0.02-0.28) with the content of any protein.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Oxygen free radicals and transient Ca²⁺ overload probably mediate myocardial reperfusion injury (contractile dysfunction and ventricular fibrillation) [1–7]. Isolated heart studies have consistently demonstrated a transient, secondary increase in intracellular Ca²⁺ just after the onset of reperfusion [10]. Pretreatment with activators and inhibitors of Ca²⁺ release and inhibitors of the Ca²⁺ ATPase by sarcoplasmic reticulum have been shown to reduce injury in isolated hearts by depleting Ca²⁺ stores and inhibition of Ca²⁺ release [12, 26, 27]. Antagonists of Ca²⁺ influx also reduce injury in vivo and in vitro, but again were most effective with pretreatment [1–3, 9, 28–35].

Our laboratory has observed that intracoronary nifedipine prevents reperfusion injury [13], but has not investigated how resumption of this normal trigger for contraction could mediate injury. The sarcoplasmic reticulum Ca²⁺ ATPase is susceptible to injury by oxyradicals and Ca²⁺ activated proteases [2, 7, 25, 36]. Since it is the major mediator of Ca²⁺ homeostasis [11], dysfunction could set up the myocardium for induced protease or oxyradical induced injury.

Most studies regarding sarcoplasmic reticulum function after ischemia and reperfusion have been done in isolated myocytes and Langendorff perfused hearts. Increased Ca²⁺ release channel open probability and transport capacity correlate with infarction rather than reversible injury [7, 37]. Impaired Ca²⁺ pump function has also been found in myocardial infarction [7, 38–40], but reports of function with reperfusion after transient (<20 min) ischemia in isolated hearts have been inconsistent due to the mild dysfunction (>80% of baseline) [7, 41–45]. The only previous study in open chest dogs used repetitive 5 min occlusions and reperfusion to induce dysfunction (10% of baseline) [46]. Ca²⁺ pump activity was reduced by only 15–20%, but the injury differs in this model [1–3]. Intracellular Ca²⁺ does not secondarily increase and reperfusion ventricular fibrillation is uncommon [1–7, 10, 47].

Several other important aspects of sarcoplasmic reticulum function also have not been investigated in reversible regional reperfusion injury. Prior studies have not: (i) directly compared Ca²⁺ pump, release channel, and phospholamban function; (ii) quantitated sarcoplasmic reticulum protein content; or (iii) evaluated sarcoplasmic reticulum function in ischemic/reperfused myocardium from animals treated with inhibitors of Ca²⁺ influx.

To investigate these questions, this study documented function of Ca²⁺ release channels, phospholamban, and the Ca²⁺ pump of the sarcoplasmic reticulum in nonischemic myocardium and ischemic/reperfused myocardium from control, vehicle, and intracoronary nifedipine-treated dogs. Protein content was measured by immunoblotting. Injury was more severe than that reported from repetitive short-term occlusion and reperfusion [46]. Control and vehicle dogs demonstrated a high rate of reperfusion ventricular fibrillation and severe dysfunction. Nifedipine prevented both aspects of reperfusion injury.

Likewise, abnormalities in Ca²⁺ uptake were more severe than reported after repetitive ischemia and reperfusion [46]. Ca²⁺ release channel and phospholamban function were preserved, but the Ca²⁺ pump was impaired. Ca²⁺ uptake at low and high ionized Ca²⁺ was only 11 and 27% of control myocardium, respectively. Both the 2D12 antibody and ryanodine caused the same relative increase in uptake in nonischemic and ischemic/reperfused myocardium, but absolute Ca²⁺ uptake remained severely depressed in ischemic/reperfused myocardium from control and vehicle dogs.

The reduced Ca²⁺ uptake of ischemic/reperfused myocardium resulted from Ca²⁺ pump dysfunction. Sarcoplasmic reticulum protein yield and content in ischemic/reperfused myocardium were similar to that of nonischemic myocardium. Ca²⁺ uptake was severely reduced irrespective of protein content. The data are comparable to a dog study reporting preserved protein content after prolonged ischemia [48].

In contrast, sarcoplasmic reticulum function was preserved in ischemic/reperfused myocardium of nifedipine-treated dogs. Ca²⁺ uptake at low and high Ca²⁺ responded normally to the 2D12 antibody and ryanodine.

These data imply that reactivation of myocardial Ca²⁺ channels at the onset of reperfusion contributes to injury to the sarcoplasmic reticulum Ca²⁺ pump. The impaired Ca²⁺ uptake then sets up the myocardium for injury by escalating diastolic Ca²⁺ during early reperfusion. The preserved content of sarcoplasmic reticulum proteins and the absence of degradative products suggest free radicals rather than proteases probably mediated the injury [1–9].

4.1 Limitations
The role of impaired Ca²⁺ pump activity in reperfusion injury was derived indirectly. Myocardial Ca²⁺ transients were not measured because techniques have not been adapted in vivo [2]. Multiple studies have demonstrated the specificity of nifedipine against Ca²⁺ channels [34]and that the protective effects result from inhibition of myocardial Ca²⁺ channels [13, 18]. Nifedipine at concentrations up to 50 µM has no effects on Na⁺/Ca²⁺ exchange [34]. Furthermore, several studies have demonstrated that the myocardial manifestation of inhibition of Na⁺/Ca²⁺ exchange is positive inotropy [18, 49]. Dose response studies from this laboratory demonstrate that the dose of nifedipine infused in this study was very negatively inotropic [13].

The method of isolation of sarcoplasmic reticulum vesicles may alter Ca²⁺ uptake data from ischemic/reperfused myocardium, but the methods utilized in this study produce vesicles representative of the whole cell homogenate [48, 50]. The isolation technique produces a subfraction of sarcoplasmic reticulum vesicles without significant contamination by sarcolemma [19]. The effects of tissue deterioration were minimized by taking myocardial biopsies in the beating state and freezing the specimens in liquid nitrogen. The isolation was completed in <2 h at 4°C [19].

The function of the calcium release channels and phospholamban was derived indirectly, but prior work has validated the use of ryanodine and the 2D12 antibody to investigate their respective function and Ca²⁺ pump activity in sarcoplasmic reticulum vesicles [21–23]. Ryanodine at 300 µM stimulates Ca²⁺ uptake by completely inhibiting Ca²⁺ release at a pH of 7.2. The monoclonal 2D12 antibody has the same effect as phosphorylation of phospholamban, removing its inhibitory effect on the Ca²⁺ pump [21, 22]. Ryanodine maximally stimulates Ca²⁺ uptake at high ionized Ca²⁺ without altering Ca²⁺ pump activity [23], whereas the 2D12 antibody maximally stimulates Ca²⁺ uptake at low ionized Ca²⁺ by increasing Ca²⁺ pump activity [22].

Previous studies have also validated the immunoblotting technique with monoclonal or polyclonal antibodies for protein content [18]. These antibodies are specific for the canine isoforms of calsequestrin, phospholamban, and Ca²⁺ ATPase [18]. Sarcoplasmic reticulum Ca²⁺ release channel content (i.e., ryanodine binding) was not measured because it has previously been shown to be normal or only mildly reduced, rather than increased [7, 37].

Only the strips of the Western blots corresponding to the mobility regions of Ca²⁺ ATPase (SERCA2), low and high molecular weights of phospholamban, and calsequestrin were immunoblotted with monoclonal antibody 2A7-A1, monoclonal antibody 2D12, and polyclonal antibody calsequestrin, respectively. Thus, the protocol may not have detected degradation products of SERCA2 or calsequestrin smaller than the region encompassed by the respective strips.

4.2 Conclusions and clinical implications
Ca²⁺ uptake by the sarcoplasmic reticulum was markedly depressed in this model of severe reperfusion injury. The mechanism was depressed Ca²⁺ pump function rather than alterations in protein content or function of Ca²⁺ release channels and phospholamban. Resumption of Ca²⁺ influx at the onset of reperfusion contributed to injury to the Ca²⁺ pump. Thus, injury to the Ca²⁺ pump of the sarcoplasmic reticulum at the onset of reperfusion may be a critical step that accelerates the secondary increase in intracellular Ca²⁺ when Ca²⁺ influx resumes. Inhibition of myocardial Ca²⁺ channels at the onset of reperfusion may be an optimal target for the minimizing reperfusion injury by delaying resumption of Ca²⁺ influx and preventing injury to the Ca²⁺ pump of the sarcoplasmic reticulum.

Time for primary review 24 days.

	Acknowledgements

 
Supported in part by grants from the Kyle Company, Mequon, WI, the R.D. and Linda Peters Endowment, Milwaukee, WI, and the American Heart Association, Milwaukee, WI.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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