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Cardiovascular Research 1997 36(3):337-346; doi:10.1016/S0008-6363(97)00187-9
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Differential roles of myocardial Ca2+ channels and Na+/Ca2+ exchange in myocardial reperfusion injury in open chest dogs: relative roles during ischemia and reperfusion

Steven C Smart*, Kiran B Sagar and David C Warltier

Division of Cardiology/Hypertension, Department of Medicine and the Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

* Corresponding author. Medical College of Wisconsin, Department of Medicine, Division of Cardiovascular Medicine, 9200 W. Wisconsin Ave., Milwaukee, WI 53226. Tel. (414) 2576697, Fax (414) 2577291, E-mail ssmart@post.its.mcw.edu

Received 11 November 1996; accepted 7 July 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Compare the roles of Ca2+ channels and Na+/Ca2+ exchange in reperfusion injury (reperfusion ventricular fibrillation and myocardial stunning). Methods: Open chest dogs undergoing 15 minutes of left anterior descending coronary artery occlusion and 3 hours of reperfusion were randomized to controls or intracoronary infusions of the respective antagonists, nifedipine (50 µg/min) or amiloride (5 mg/min), according to five protocols: (A) 40 minutes before occlusion to 30 minutes after reperfusion; (B) 2 minutes before to 5 minutes after reperfusion; (C) 10 minutes before to 10 minutes after reperfusion (two step infusion for nifedipine only 5 µg/min during occlusion and 50 µg/min after reperfusion); and (D) 0 to 30 minutes after reperfusion. The role of Ca2+ channels was further investigated by infusing the agonist, Bay K 8644 (50 µg/min), alone or simultaneously with any protocol B, C, or D infusions altering both reperfusion ventricular fibrillation and myocardial stunning. Results: Effects of the agents on injury did not result from hemodynamic effects or alterations in blood flow. Amiloride had no effect on ventricular fibrillation. Only protocol A infusion of amiloride prevented myocardial stunning. In contrast, protocol A and B infusions of nifedipine prevented both myocardial stunning (p = ns vs. baseline, p<0.01 vs. control) and ventricular fibrillation (0%, p<0.01). Protocol C prevented reperfusion ventricular fibrillation, but not stunning (p = ns vs. control). Protocol D did not alter injury. Bay K 8644 co-treatment reversed the effects of Protocol B infusion of nifedipine. Ventricular fibrillation was common and postischemic function worst in dogs treated with Bay K 8644 alone (protocol B). Conclusion: Myocardial Ca2+ channels contribute to both reperfusion ventricular fibrillation and stunning, whereas Na+/Ca2+ exchange contributes only to stunning. Inhibitors of myocardial Ca2+ channels are protective when infused in high doses just before reperfusion, whereas the efficacy of Na+/Ca2+ exchange inhibitors is dependent on pretreatment.

KEYWORDS Dogs; Amiloride; Nifedipine; Myocardial contraction; Myocardial ischemia; Reperfusion arrhythmias; Reperfusion injury; Stunned myocardium


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Reperfusion after transient ischemia may exacerbate myocardial injury by the induction of transient intracellular Ca2+ overload or oxygen free radicals [1–4]. Manifestations of this injury are reperfusion ventricular fibrillation and myocardial stunning (reversible postischemic contractile dysfunction). The sarcoplasmic reticulum accounts for >90% of the Ca2+ transient in mammals [5, 6]. Sarcolemmal Ca2+ channels regulate Ca2+ release by the sarcoplasmic reticulum and Ca2+ content [6–8]. The sarcolemmal Na+/Ca2+ exchanger mediates Ca2+ efflux during diastole and also regulates Ca2+ content in the sarcoplasmic reticulum by its competition with the Ca2+ pump (ATPase) of the sarcoplasmic reticulum [5, 6]. Na+/H+ exchange contributes to pH balance in myocytes [9].

Reactivation of myocardial Ca2+ channels and reversal of Na+/Ca2+ exchange may mediate transient Ca2+ overload during early reperfusion after transient ischemia [1–4, 10–12]. All are inhibited by low pH and anions during ischemia, but rapidly recover after reperfusion and earlier than the sarcoplasmic reticulum Ca2+ pump [6, 9, 13]. Intracellular Na+ increases due to acidosis, Na+/H+ exchange and reduced Na+/K+ ATPase activity. High intracellular Na+ may then induce Ca2+ influx by reversal of Na+/Ca2+ exchange [4]. Reactivation of myocardial Ca2+ channels may induce Ca2+ release by the sarcoplasmic reticulum before pump activity is restored [8]. Previous studies have not directly compared the roles of these mechanisms in reperfusion ventricular fibrillation and myocardial stunning [14–31].

Our hypothesis is that both myocardial Ca2+ channels and Na+/Ca2+ exchange contribute to reperfusion injury. To directly compare their relative roles in reperfusion injury, open chest dogs undergoing 15 minutes of left anterior descending artery occlusion and 3 hours of reperfusion were treated with intracoronary infusions of the Ca2+ channel inhibitor, nifedipine, or the Na+/H+ and Na+/Ca2+ exchange inhibitor, amiloride. The roles in reperfusion injury were investigated by varying the dose, start and end of the infusions relative to occlusion and reperfusion. The Ca2+ channel agonist, Bay K 8644, was then utilized to investigate if protective effects against reperfusion injury reverse with simultaneous Ca2+ channel activator treatment [32].


   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 blood gases monitored. Arterial and left ventricular pressures were measured by high fidelity double-tipped catheter (Millar PC 771, Dallas, TX) and electronically differentiated on-line. 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 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 silastic catheter. The tip was advanced into the left anterior descending artery and position confirmed by injection of food coloring. A silastic 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 zone) and left circumflex coronary distributions (nonischemic zone) were identified 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. End diastole was the onset of the rise in left ventricular pressure and end systole 20 ms before peak negative dP/dt [33]. 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 to baseline values [33].

2.3 Regional myocardial blood flow
Myocardial blood flow was measured by radiolabeled microspheres at (see Fig. 1): (a) 10 minutes before occlusion; (b) 10 minutes after occlusion; (c) 30 minutes after reperfusion; and (d) 3 hours after reperfusion [34]. Carbonized plastic microspheres (41Ce, Formula , 15±2 µm diameter, New England Nuclear, Boston, MA) in 10% Dextran and 0.01% Tween 80 were ultrasonicated and vortexed. Twenty µCi (3 to 4x106 microspheres) were injected in the left atrium and reference blood samples collected from the femoral artery at 7 ml/min for 130 seconds.


Figure 1
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  		Fig. 1 Experimental protocol. The protocol consisted of a 45 minute pretreatment period, 15 minutes of left anterior descending coronary artery occlusion (Occ), and 3 hours of reperfusion. Protocol A infused drug from 40 minutes before occlusion (–40 min) to 30 minutes after reperfusion (45 min) followed by a 150 minute washout period (45 to 195 min). Protocol B infused drug only from 13 minutes after occlusion (13 min) to 5 minutes after reperfusion (20 min) followed by a 175 minute washout period (20 to 195 min). Protocol C infused drug from 5 minutes after occlusion (5 min) to 10 minutes after reperfusion (25 min) followed by a 170 minute washout period. Protocol C infused: (i) nifedipine at 5 µg/min (LDN) from 5–15 minutes of occlusion (5–15 min) and then 50 µg/min (HDN) from 0–10 minutes after reperfusion (15–25 min) and (ii) amiloride at a constant 5 mg/min. Protocol D infused drug from the onset of reperfusion (15 min) until 30 minutes after reperfusion (45 min) followed by a 150 minute washout period (45 to 195 min). During the washout period, 0.9% saline was infused at 1 ml/min in all groups. Regional myocardial blood flow was assessed with radiolabeled microspheres before occlusion (–10 min), during occlusion (10 min), and after reperfusion (45 min and 195 min). Occ, occlusion.

 
After each experiment, the LAD was occluded and India ink injected to delineate area at risk. The heart was electrically fibrillated, removed, and fixed in 10% formalin. Ischemic and nonischemic zones were separated and weighed. Tissue samples (0.8–1.0 g) from the central ischemic (five samples) and nonischemic (three samples) regions were divided into subendo-, mid-, 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 the previously described protocol [31]. Blood flows were averaged and dogs excluded for subendocardial blood flow >20 ml/min/100 g during occlusion [35].

2.4 Sham studies
Three dogs underwent dose response studies with intracoronary nifedipine at 10–100 µg/min, Bay K 8644 at 10–100 µg/min, and amiloride at 1–10 mg/min separated by washout periods. The regional inotropic effects of nifedipine, Bay K 8644, and amiloride were maximal at 50 µg/min, 50 µg/min, and 5 mg/min, respectively. Higher doses altered hemodynamics. Drug effects were maximal by 2–3 minutes after starting the infusion and not influenced by resting coronary blood flow (25–45 ml/min). Nifedipine increased blood flow by 170±20% and reduced systolic shortening by 88±6%. Amiloride increased systolic shortening by 56±5%, reduced heart rate by 20±5%, and increased myocardial blood flow by 180±15%. Bay K 8644 increased systolic shortening by 80±5% without altering blood flow, reversed the negative inotropy of nifedipine, and potentiated the positive inotropy of amiloride. The effects of nifedipine resolved by 15 minutes after stopping the infusion and Bay K 8644 and amiloride by 30 minutes.

2.5 Treatment and stunning protocols
Experiments were performed with the room lights off. Syringes and tubing were covered with aluminum foil. Dogs were randomized to intracoronary control infusions [0.9% saline, 5% dextrose, or 0.9% saline with 4% ethanol and 2% polyethylene glycol 400 (vehicle); 1 ml/min], nifedipine [5 mg in 100 ml of 0.9% saline with 4% ethanol and 2% polyethylene glycol 400 (140 µM); 50 µg/min], or amiloride [500 mg in 100 ml 5% dextrose (16.7 mM); 5 mg/min], according to protocols A, B, C, or D (see Fig. 1). Protocol A infused drug from 40 minutes before occlusion until 30 minutes after reperfusion followed by a 150 minute washout period. Protocol B infused drug only from 13 minutes after occlusion to 5 minutes after reperfusion followed by a 175 minute washout period. Protocol C infused drug from 5 minutes of occlusion to 10 minutes after reperfusion followed by a 170 minute washout period. The protocol C nifedipine infusion was modified to a two step protocol of 5 µg/min from 5–15 minutes of occlusion, then 50 µg/min from 0–10 minutes after reperfusion to evaluate the role of high concentrations during occlusion. The protocol C amiloride infusion was constant at 5 mg/min to maximize Na+/Ca2+ exchange inhibition. Protocol D infused drug from 0 to 30 minutes after reperfusion followed by a 150 minute washout period. During the washout periods, 0.9% saline was infused at 1 ml/min in all groups. Any nifedipine or amiloride infusion(s) before or after reperfusion (protocols B, C, or D) preventing myocardial stunning were compared to combined treatment with Bay K 8644 [5 mg in 100 ml of vehicle (150 µM); 50 µg/min] and Bay K 8644 alone. Estimated drug concentrations were derived from the infusion rate and concentration and ischemic zone or coronary blood flow.

After instrumentation and baseline measurements, intracoronary infusion was started (see Fig. 1) and the coronary artery occluded 40 minutes later. The ligature was released after 15 minutes and the dogs monitored for 180 minutes. 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 Joules). A prior study revealed no prolonged effects on function [31].

2.6 Statistical analysis
All reported values were expressed as mean±standard error (SEM). Continuous data were compared by repeated measures or multi-way analysis of variance (ANOVA) with the Bonferroni t-test and animal exclusion by {chi}2 analysis. A two tailed p<0.05 was significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Animal data
One hundred twenty-one dogs were enrolled in the study. Three dogs underwent the sham studies. One hundred eighteen dogs were randomized to the 11 experimental groups. All dogs had resting left anterior descending blood flow of 20–45 ml/min. Thirty-five dogs were excluded due to high collateral blood flow during occlusion (4 dogs) or refractory ventricular fibrillation (31 dogs). Ventricular fibrillation occurred in 59 dogs (50%). Ventricular fibrillation occurred in 50% (14/28) of controls (6/12 saline, 5/10 dextrose, 3/6 vehicle), 81% (13/16) of protocol A amiloride dogs, 67% (8/12) of protocol B amiloride dogs, 86% (6/7) of protocol C amiloride dogs, 40% (4/10) of protocol D amiloride dogs, and 38% (3/8) of protocol D nifedipine dogs. Only protocol A, B, and C infusions of nifedipine prevented reperfusion ventricular fibrillation (0%, 0/9, 0/8, and 0/5, respectively, p<0.01 vs. control).

Since protocol B, C, and D amiloride infusions had no effect on injury and only protocol B infusion of nifedipine prevented both stunning and ventricular fibrillation, Bay K 8644 was only infused according to protocol B alone and with nifedipine. Ventricular fibrillation occurred in 63% (5/8) of protocol B Bay K 8644 and nifedipine, 75% (6/8) of protocol B Bay K 8644 dogs.

Defibrillation was unsuccessful in 31 (26%) including all 10 dogs that fibrillated during occlusion. In contrast, defibrillation was successful in 28 (57%) of the 49 that fibrillated after reperfusion. Defibrillation was successful in three control dogs, five protocol A amiloride dogs, four protocol B amiloride dogs, four protocol C amiloride dogs, one protocol D amiloride dog, two protocol D nifedipine dogs, four protocol B Bay K 8644 and nifedipine dogs, and five protocol B Bay K 8644 dogs.

Eight-four dogs completed the protocol. There were 16 control dogs (6 saline, 6 dextrose, and 4 vehicle), eight in protocol A and B amiloride and nifedipine groups, seven in protocol B Bay K 8644 and protocol B Bay K 8644 and nifedipine groups, five in the protocol C nifedipine and amiloride groups, and six in protocol D amiloride and nifedipine groups.

3.2 Systemic hemodynamics
Hemodynamics were equivalent in control and vehicle dogs and were combined for analysis. Heart rate in all nifedipine, Bay K 8644 and amiloride groups was similar to controls (128±5 bpm) throughout the protocol. Mean arterial pressure and peak positive dP/dt in all groups were similar to controls (96±5 mmHg and 1890±100 mmHg/s, respectively) after instrumentation (baseline) and remained similar during the experimental protocol with the following exceptions (see Table 1). Protocol A nifedipine infusion lowered mean arterial pressure before and during occlusion and the first 30 minutes of reperfusion, whereas protocols B, C, and D lowered mean arterial pressure only during the first 10, 20, and 40 minutes of reperfusion, respectively. Protocol A amiloride infusion modestly reduced mean arterial pressure during the first 30 minutes of reperfusion, but increased dP/dt throughout drug infusion. In contrast, protocols B, C, and D reduced mean arterial pressure only during the first 30 to 45 minutes of reperfusion and did not alter dP/dt. Protocol B Bay K 8644 infusion increased mean arterial pressure and dP/dt during drug infusion, but the effects washed out within 10 minutes. Combined nifedipine/Bay K 8644 infusion had no hemodynamic effects. Washout resolved all differences.


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  		Table 1 Hemodynamics

 
3.3 Myocardial blood flow
Myocardial blood flow in ischemic zone is summarized in Table 2. Protocol A nifedipine and amiloride infusions increased ischemic zone blood flow before occlusion. Nonischemic zone blood flow also increased in protocol A nifedipine (238±45 vs. 103±13 ml/min/100 g, p<0.05), but not amiloride dogs (121±10). Blood flow in both zones of protocols B, C, and D amiloride and nifedipine dogs was similar to controls before occlusion. Coronary blood flow was 75±10 ml/min and 78±9 ml/min in protocol A nifedipine and amiloride dogs, respectively.


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  		Table 2 Regional myocardial blood flow (ml/min/100 g)

 
Coronary artery occlusion severely reduced ischemic zone blood flow in all groups, especially in the subendocardium. After reperfusion, blood flow was higher (p<0.01) than controls at 30 minutes in all infusions of nifedipine and A, C, and D infusions of amiloride. Blood flow in dogs treated with protocol B infusion of Bay K 8644 alone or in combination with nifedipine was similar to controls throughout reperfusion. All differences resolved during the washout periods. Based on ischemic zone mass and microsphere data during occlusion, transmural ischemic zone collateral blood flow was 1.5–4.5 ml/min in all dogs. Finally, left anterior descending coronary blood flow ranged from 60–170 ml/min during the first few minutes of reperfusion in all groups.

Blood flow to the nonischemic zone in all amiloride groups and protocol B and C nifedipine dogs was similar to controls (103±13) throughout the protocol. Protocol A nifedipine infusion increased blood flow to the nonischemic zone during occlusion and at 30 minutes after reperfusion (223±48 and 226±33, respectively). Protocol D also increased nonischemic zone blood flow at 30 minutes after reperfusion (198±18). Bay K 8644 alone or with nifedipine had no effect on nonischemic zone blood flow. All differences resolved by 3 hours after reperfusion.

3.4 Area at risk
The area subjected to ischemia (range 30±3 to 37±2 g), left ventricular mass (range 105±5 to 120±7 g), and area at risk (range 28±2 to 33±2%) were similar in all groups.

3.5 Regional myocardial function
Systolic shortening in the ischemic zone is plotted in Figs. 2–5GoGoGo. Fig. 2 is a plot of systolic shortening from controls and dogs treated with 85 minute protocol A infusions of amiloride and nifedipine before occlusion, during occlusion, and early reperfusion. Amiloride increased systolic shortening from 17±2 to 26±2% before occlusion, whereas nifedipine reduced systolic shortening from 16±1 to 3±2% before occlusion. During coronary occlusion, systolic lengthening was similar in all groups. After reperfusion, systolic shortening did not recover in controls. In contrast, amiloride caused systolic shortening to steadily recover after reperfusion. Systolic shortening was similar to baseline by 30 minutes, completely recovered by 60 minutes, and did not deteriorate thereafter. Nifedipine also caused systolic shortening to recover after reperfusion, but the pattern was different. Systolic shortening returned to pretreatment values during the first 30 minutes, then completely recovered within 30 minutes after stopping the infusion (60 minutes after reperfusion) and did not deteriorate thereafter.


Figure 2
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  		Fig. 2 Systolic shortening in the ischemic zone (SS-IZ) of dogs treated with nifedipine and amiloride according to protocol A compared to controls. Amiloride and nifedipine increased and reduced SS, respectively, before occlusion. During occlusion, similar systolic lengthening occurred in all groups. Postischemic SS never recovered in controls. In contrast, postischemic SS completely recovered by 30–60 minutes in amiloride dogs did not deteriorate during washout. Postischemic SS also recovered in nifedipine dogs, but the pattern was different. Recovery occurred during washout (30–60 minutes after reperfusion). BL, baseline; Drug, drug infusion before occlusion; Occ, occlusion; 30R, 30 minutes after reperfusion, 180R, 180 minutes after reperfusion. **p<0.01 vs. controls, #p<0.01 vs. amiloride dogs.

 

Figure 3
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  		Fig. 3 Systolic shortening in the ischemic zone of protocol B and C nifedipine and amiloride dogs compared to controls. SS in all groups was similar to controls before occlusion. During occlusion (Occ), similar systolic lengthening occurred in all groups. Postischemic SS never recovered in protocol B or C amiloride dogs. In contrast, postischemic SS completely recovered by 60 minutes in protocol B nifedipine dogs. Postischemic SS transiently recovered during the first 15 minutes of reperfusion in protocol C nifedipine dogs, but rapidly deteriorated during washout. See Fig. 2 legend for other abbreviations and symbols. 5R, 5 minutes after reperfusion. *p<0.05 vs. controls.

 

Figure 4
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  		Fig. 4 Systolic shortening in the ischemic zone of protocol D nifedipine and amiloride dogs compared to controls. SS in amiloride and nifedipine dogs was similar to controls before occlusion. During occlusion, similar systolic lengthening occurred in all groups. Postischemic SS never recovered in either group. See legends of Figs. 2 and 3Go for abbreviations.

 

Figure 5
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  		Fig. 5 Systolic shortening in the ischemic zone of Bay K 8644 and combined Bay K 8644 and nifedipine dogs (protocol B) compared to controls and protocol B nifedipine dogs. SS in all groups was similar to controls before occlusion. During occlusion (Occ), similar systolic lengthening occurred in all groups. Bay K 8644 alone caused postischemic SS to rapidly increased to values greater than baseline during drug infusion, but SS rapidly deteriorated to control dysfunction during washout. Bay K 8644 also antagonized the effect of nifedipine on reperfusion injury. The combination of Bay K 8644 and nifedipine (Nif/Bay K 8644) modestly increased postischemic SS during the infusion, but SS again rapidly deteriorated during washout. See legends of Figs. 2 and 3Go for other abbreviations and symbols.

 
Fig. 3 compares ischemic zone systolic shortening of controls to dogs treated with protocol B infusions of amiloride and nifedipine from just before to just after reperfusion and the protocol C infusions of nifedipine and amiloride from 10 minutes before to 10 minutes after reperfusion. Systolic shortening was similar in all groups before occlusion. During occlusion, systolic lengthening was similar. After reperfusion, systolic shortening did not recover in either amiloride group (p = ns vs. controls). In contrast, protocol B infusion of nifedipine caused systolic shortening to steadily and completely recover by 60 minutes (p = ns vs. baseline) and not deteriorate thereafter. Protocol C infusion of nifedipine temporarily improved systolic shortening during the first 30 minutes after reperfusion, but deteriorated thereafter.

Fig. 4 compares ischemic zone systolic shortening of controls to dogs treated with the 30 minute protocol D infusions of amiloride and nifedipine beginning at reperfusion. Systolic shortening in amiloride and nifedipine dogs was similar to controls before occlusion. During occlusion, systolic lengthening was similar. After reperfusion, systolic shortening did not recover in amiloride or nifedipine dogs (p = ns vs. controls).

Fig. 5 compares ischemic zone systolic shortening in controls to dogs treated with protocol B infusions of nifedipine, Bay K 8644, and the combination. The effect of Bay K 8644 was opposite of nifedipine. Systolic shortening markedly increased to hyperdynamic values during the first 5–10 minutes of reperfusion, but rapidly deteriorated to values similar to controls after stopping the infusion. The combination of nifedipine and Bay K 8644 caused only a modest increase in systolic shortening during the infusion. Systolic shortening again rapidly deteriorated after stopping the infusion and remained similar to controls thereafter.

End diastolic segment length in the ischemic zone increased during coronary occlusion and then recovered by 2 hours after reperfusion in all groups of dogs. Only protocol A infusion of nifedipine caused end diastolic segment length to increase during pretreatment indicative of myocardial stretch. In all other groups, end diastolic segment length remained at baseline throughout the period before occlusion and followed the same time course after reperfusion.

In the nonischemic zone, protocol A infusions of both amiloride and nifedipine increased systolic shortening (p<0.05 vs. baseline and controls) during pretreatment (see Fig. 6). Left anterior descending coronary artery occlusion increased systolic shortening in all groups. After reperfusion, systolic shortening returned to baseline in all groups within 2 hours.


Figure 6
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  		Fig. 6 Systolic shortening in the nonischemic zone (SS-IZ) of protocol A–D nifedipine (top) and amiloride (bottom) dogs compared to controls. Protocol A infusions of both amiloride and nifedipine increased systolic shortening during pretreatment. Left anterior descending artery occlusion increased systolic shortening in all groups. After reperfusion, systolic shortening returned to baseline in all groups within 2 hours. See legends of Figs. 2 and 3Go for other abbreviations and symbols.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Previous investigations
Increased intracellular Ca2+ may contribute to myocardial stunning and ventricular fibrillation [1–4, 11, 28]. Intracellular Ca2+ increases during ischemia due to acidosis, depletion of ATP, inactivity of the sarcoplasmic reticulum Ca2+ pump, and/or Na+/H+ and Na+/Ca2+ exchange reversal and inactivation [4]. Ca2+ increases further early after reperfusion due to: (i) reactivation and reversal of Na+/H+ and Na+/Ca2+ exchange or (ii) reactivation of Ca2+ channels and Ca2+ release by the sarcoplasmic reticulum before re-uptake has been restored [4, 8, 11].

In vitro and in vivo studies have shown that of Ca2+ channels and Na+/Ca2+ exchange contribute to stunning, but did not define the timing of their contribution [14–31, 36, 37]. Ca2+ channel antagonists in vitro and in vivo often had no effect on postischemic dysfunction when infused at reperfusion so increased energy stores or alterations in hemodynamics, collateral or reperfusion blood flow were hypothesized as mechanisms [14–21, 31]. In vivo studies infused doses without effects on contractility, so myocardial channels were not inhibited. Vasodilating doses of intracoronary nifedipine (5 µg/min) were ineffective even if started before occlusion [31]. Antagonists of Na+/H+ and Na+/Ca2+ exchange also had minimal effect on stunning in vitro and in vivo when infused at reperfusion so nonspecific effects were again implicated [21–31, 36, 37]. Amiloride was most effective at concentrations with the positively inotropic and negatively chronotropic effects of Na+/Ca2+ exchange inhibition [38]. Specific Na+/H+ exchange inhibitors, HOE 694 or hexamethylene amiloride, were less effective or ineffective [31, 37].

The roles of these channels and exchangers have not been defined in reperfusion ventricular fibrillation. Amiloride was protective in vitro, but not in dogs [26–31, 36]. Na+/H+ exchange inhibitors produced equivocal results [31, 37]. Nifedipine has not been studied.

Several important issues have not been investigated [14–31, 36, 37]. Inhibitors are not effective for several minutes after initiation, so infusions at reperfusion may not be effective during the critical first few minutes. Most studies did not assess postischemic function after drug washout. Effects of antagonists have not been shown to reverse with agonist co-treatment.

4.2 The present study
The roles of myocardial Ca2+ channels and Na+/H+ and Na+/Ca2+ exchange in reperfusion injury were directly compared in open chest dogs by varying the dose, start and end of intracoronary infusions of the antagonists (nifedipine and amiloride) and the Ca2+ channel agonist, Bay K 8644 relative to occlusion and reperfusion. Dose-response studies showed that drug effects were not altered by coronary blood flow up to 100 ml/min or ischemic zone mass. The intracoronary route localized the negatively inotropic effects of nifedipine and the positively inotropic effects of amiloride and Bay K 8644 to the vascular territory of infusion [31, 32, 39, 40]. The data demonstrated the importance of high concentrations of nifedipine during occlusion and showed that injury results from changes during ischemia and after reperfusion.

The control data documented no effect of solvent on postischemic function. Hemodynamic and vascular alterations did not influence injury. Protocol A amiloride and nifedipine infusion increased myocardial blood flow and lowered blood pressure before occlusion and after reperfusion. Protocol B infusion of nifedipine around reperfusion was protective despite short-term alterations only during early reperfusion, whereas the more prolonged changes induced by protocols C and D infusions had no effect on stunning. Protocols B, C and D infusions of amiloride also did not reduce injury despite alterations similar to protocol A after reperfusion.

Induced mechanical quiescence and myocardial stretch did mediate the protection by nifedipine [41–43]. Nonischemic zone function was hyperdynamic before occlusion in protocol A dogs and during occlusion and early reperfusion in all dogs. Protocol A increased EDL and induced mechanical quiescence for 40 minutes before occlusion and the first 45 minutes after reperfusion, but protocol B was as protective without any induced quiescence or stretch. Protocols C and D did not reduce injury despite 25–45 minute quiescent periods after reperfusion.

Other studies by this laboratory confirmed that negative inotropy alone does not prevent reperfusion injury. Agents with different mechanisms have differential effects. Butadione monoxime may be Ca2+ buffering agent [41]. Intracoronary infusion during coronary occlusion alone reduced injury. In contrast, ryanodine and thapsigargin activate and inhibit sarcoplasmic reticulum Ca2+ release channels and the Ca2+ ATPase, respectively. Both agents were tested in 10 dogs (unpublished data). None survived reperfusion due to refractory ventricular fibrillation.

Positive inotropy alone did not mediate the improved postischemic function of protocol A infusion of amiloride [1–3]. Postischemic function did not deteriorate after drug washout and protocol B, C, and D infusions after reperfusion did not reverse postischemic dysfunction.

Nifedipine may also inhibit K+ or Na+ channels at >10 µM concentrations, but this effect did not mediate its protection [39]. K+ channel inhibition exacerbates, rather than attenuates, reperfusion injury [46]. Na+ channel inhibition has equivocal effects on reperfusion injury [47, 48]. The negatively inotropic effect of protocol A infusion occurred at estimated concentrations of 1.6–2.2 µM. Estimated concentrations of 25–50 µM were produced by protocol A and B during occlusion, but persisted for only the last minute in protocol B. The two step protocol C infusion induced only 2–4 µM estimated concentrations. Protocol A was protective rather than injurious. Protocol B was also protective despite only transient high concentrations. Protocol C still prevented ventricular fibrillation despite lower concentrations.

Likewise, nonspecific effects against K+ and Na+ channels cannot account for amiloride's prevention of stunning. Inhibition of Na+ channels also occurs at 0.2–1.0 mM concentrations, but mM concentrations are only minimally active against K+ channels [40]. Protocol A induced estimated myocardial concentrations of 0.19–0.28 mM before occlusion. Positively inotropic and negatively chronotropic effects were indicative of Na+/Ca2+ exchange inhibition, rather than the negatively inotropic effect of K+ or Na+ channel inhibition in dogs [49]. All amiloride infusions initiated during occlusion (A, B, and C) induced estimated concentrations of 3.0–6.5 mM during occlusion and 0.10–0.28 mM during early reperfusion, but only protocol A prevented stunning.

Thus, the results show that myocardial Ca2+ channels contribute to reperfusion ventricular fibrillation and myocardial stunning, but Na+/Ca2+ exchange contributes only to stunning. These channels and exchangers are inhibited within the first 5–10 minutes of ischemia by ionic changes so their contribution to injury can only occur early during occlusion or after reperfusion [9, 12, 50]. The results show that the several minute delay in the onset of the inhibitory effects of nifedipine and amiloride must be considered in the analysis of the effects [14–19, 21].

The results implied that the initial burst of Ca2+ influx with reactivation of Ca2+ channels at the onset of reperfusion mediated reperfusion ventricular fibrillation. The dysrrhythmia was prevented by nifedipine infusion starting before occlusion (Protocol A), early or late during coronary occlusion (Protocols B and C), but not at reperfusion (protocol D). Bay K 8644 co-infusion reversed nifedipine's protection. Based on blood flow data and pharmacokinetics, the only time when myocardial drug concentrations were lower in protocol D than protocols A, B and C dogs was the critical first 30 seconds of reperfusion when ventricular fibrillation occurred. Finally, Bay K 8644 alone and amiloride tended to exacerbate the dysrrhythmia.

More sustained effects of reactivation of Ca2+ channels early after reperfusion also mediated myocardial stunning. Stunning was prevented by high dose nifedipine infusion starting before occlusion or just before reperfusion, but not by low dose during occlusion with high dose at reperfusion or high dose only at reperfusion. Thus, high concentrations during occlusion were necessary to sustain Ca2+ channel inhibition during the first 5 minutes of hyperemic reperfusion (100–170 ml/min) when estimated plasma concentrations were <1 µM in many dogs.

In contrast, Na+/Ca2+ exchange reversal contributed to stunning early during occlusion, rather than after reperfusion. Amiloride in the present study was only protective when started before and infused throughout occlusion. The prevention of stunning correlated with exchange inhibition during the first 5–7 minutes of occlusion. Inhibition after 5–7 minutes of occlusion and during reperfusion only had no effect on stunning. The data connote a mechanism similar to that of the in vitro hypoxic injury myocyte model showing that Na+/Ca2+ exchange reversal during hypoxia mediates Ca2+ loading as ATP is depleted and intracellular Na+ increases and that inhibition during hypoxia, rather than after reoxygenation prevents injury [51, 52]. Delayed onset of effects due to antagonistic effects of increased K+ and Na+ during ischemia did not account for amiloride's ineffectiveness when started after occlusion or during reperfusion because infusions starting early (protocol C) and late (protocol B) during occlusion were similarly ineffective [40].

4.3 Limitations
The roles of myocardial Ca2+ channels and Na+/H+ and Na+/Ca2+ exchangers in reperfusion injury were deduced indirectly. Myocardial Ca2+ cannot be measured in vivo [2]. Plasma drug concentrations were not directly measured, but the coronary and collateral blood flow data permitted estimation. The inotropic effects confirm efficacy of the respective infusions [39, 40].

Nifedipine may also nonspecifically inhibit T-type Ca2+, but L-type channels predominate in cardiac muscle [53]. Amiloride also has other nonspecific effects [40], but its effects result mainly from Na+/Ca2+ exchange inhibition and it remains the best available agent to assess its role [31, 38]. Positively inotropic effects ruled out myocardial Ca2+ channel inhibition [39, 40]. It was shown to be more effective against injury than specific Na+/H+ exchange inhibitors and other Na+/Ca2+ exchange inhibitors [25–31, 36, 37]. Amiloride derivatives and bepridil have greater potency against the Na+/Ca2+ exchanger, but are even more active against Ca2+, Na+, and K+ channels [54, 55]. The use of these potent inhibitors would have produced even more complicated results.

Oxyradical production was not measured in the present study [1, 2]. Nifedipine and amiloride scavenge oxyradicals only at mM concentrations [1, 2, 44, 45]. Scavenger treatment at reperfusion has been shown to reduce injury [1, 2]. The data from the present study are inconsistent with these studies and estimated plasma concentrations during early reperfusion were too low (0.8–1.4 µM and 0.10–0.28 mM, respectively) to scavenge free radicals.

4.4 Conclusions
Reperfusion ventricular fibrillation is a manifestation of reperfusion injury, whereas myocardial stunning results from changes during ischemia and reperfusion. Reactivation of myocardial Ca2+ channels at reperfusion contributes to both reperfusion ventricular fibrillation and myocardial stunning. In contrast, reversal of Na+/Ca2+ exchange contributes to myocardial stunning early during ischemia, rather than after reperfusion, and has no role in reperfusion ventricular fibrillation. Thus, the strategy of inhibiting myocardial Ca2+ channels may be more effective than inhibiting Na+/Ca2+ exchange for reducing myocardial reperfusion injury.

Time for primary review 52 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, Wisconsin Affiliate, Milwaukee, WI.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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