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Eur J Cardiothorac Surg 2003;24:974-985
© 2003 Elsevier Science NL

Effect of low [CaCl2] and high [MgCl2] cardioplegia and moderate hypothermic ischemia on myoplasmic [Ca2+] and cardiac function in intact hearts

Jianzhong Ana, Amadou K.S. Camaraa, Qun Chena, David F. Stowea,b,c,d,e*

a Anesthesiology Research Laboratory, Department of Anesthesiology, M4280, 8701 Watertown Plank Road, Medical College of Wisconsin, Milwaukee Regional Medical Center, Milwaukee, WI 53226, USA
b Department of Physiology, Medical College of Wisconsin, Milwaukee Regional Medical Center, Milwaukee, WI 53226, USA
c Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee Regional Medical Center, Milwaukee, WI 53226, USA
d Department of Biomedical Engineering, Marquette University, Milwaukee, WI 53233, USA
e Research Service, Veterans Affairs Medical Center, Milwaukee, WI 53295, USA

Received 14 November 2002; received in revised form 13 June 2003; accepted 16 June 2003.

* Corresponding author. Tel.: +1-414-456-5722; fax: +1-414-456-6507
e-mail: dfstowe{at}mcw.edu


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A
 References
 
Objective: Cold cardioplegia (CP) protects against ischemic damage in part by reducing [Ca2+]i overload on reperfusion. Hyperkalemic cardioplegic solutions are widely used in coronary artery bypass procedures, and the specific ionic composition of these solutions may contribute to their variable myocardial protective effects secondary to reduced Ca2+i loading. We reported previously that CP decreased the rise in cardiac diastolic (dia) [Ca2+]i observed during 4 h cold storage at 3 °C in Krebs–Ringer's (KR) solution and decreased dia[Ca2+]i and increased systolic (sys) [Ca2+]i and function on reperfusion after cold storage. Our aim here was to determine if low Ca2+o and high Mg2+o adds to the protective effects of high K+o by decreasing [Ca2+]i during ischemia and reperfusion. Methods: We compared effects of 4.5 mM K+o, 2.5 mM Ca2+o and 2.4 mM Mg2+o KR solution with a higher K+o (18 mM), a lower Ca2+o (1.25 mM) and/or higher Mg2+o (7.2 mM) CP solutions on cardiac mechanic function and sys and dia[Ca2+]i during and after moderate hypothermic global ischemia (17 °C for 4 h) in guinea pig intact hearts isolated by the Langendorff technique. Isovolumetric left ventricular pressure (LVP) was measured with a transducer connected to a fluid-filled balloon placed in the LV and [Ca2+]i was measured using indo-1 fluorescence and a fiberoptic cable placed on the LV free wall. Results: For all CP groups compared to the KR control group after 60 min reperfusion, we observed significant lowering of dia[Ca2+]i by 47%, left ventricular diastolic pressure (diaLVP) by 55%, and infarct size by 43%. We also found significant elevation of sys[Ca2+]i by 25%, d[Ca2+]i/dtmax and d[Ca2+]i/dtmin by 33 and 34%, sys–diaLVP by 55%, dLVP/dtmax and dLVP/dtmin by 34 and 40%, coronary flow by 31%, cardiac efficiency by 21%, and MVO2 by 25%. These results indicate that CP reduces myoplasmic Ca2+ loading and improves mechanical and metabolic function on warm reperfusion compared to KR. However, there were no differences in these indices of Ca2+i cardiac function or metabolism among any CP group after warm reperfusion with KR solution. Conclusion: Increasing K+o to produce cardiac arrest was the most cardioprotective effect of CP against ischemia reperfusion injury; lowering Ca2+o or raising Mg2+o did not add to this protective effect or additionally alter [Ca2+]i.

Key Words: Magnesium • Calcium • Potassium • Perfusion solutions • Myocardium • Guinea pig


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A
 References
 
Cardioplegia (CP), i.e. a solution with a high depolarizing [K+]o, is an important adjunct to hypothermia in that it can provide significant added protection to the ischemic myocardium during cardiac surgery. Contraction and relaxation of cardiac myofilaments is highly dependent on the free intracellular Ca2+ concentration ([Ca2+]i). But hypothermia inhibits the Na/K ATPase (Na pump) which in turn promotes Ca2+ loading by Na+/Ca2+ exchange (NCE) [1,2]. Hypothermia may also alter sarcolemmal Ca2+ flux via Ca2+ channels and other ion channels and ion exchangers to cause a change in Ca2+-induced Ca2+ release by the sarcoplasmic reticulum (SR) [3]. Ca2+ homeostasis can be disrupted by slowed release or attenuated reuptake of Ca2+ by the SR during hypothermia. A rise in diastolic [Ca2+]i (dia[Ca2+]i) and attenuated myofilament ATP hydrolysis during hypothermia contribute to a dissociation of contractility from [Ca2+]i and can cause diastolic contracture [4]. Thus although very cardioprotective, hypothermia has deleterious side effects.

Cold CP is generally used to rapidly induce cardiac arrest and reduce the risk of ischemic myocardial injury. Hearse [5] and Hearse et al. [6] long ago reviewed the properties of an ideal CP solution. St. Thomas' Hospital cardioplegic solution is a widely used solution that contains either 20 mM K+o, 16 mM Mg2+o and 2.2 mM Ca2+o (type 1), or 16 mM K+o, 16 mM Mg2+o and 1.2 mM Ca2+o (type 2) [6]. In later refinements of CP solution constituents, Robinson and Harwood suggested in their work [7] that the optimal ionized [Ca2+]o of St. Thomas solution was approximately 0.6 mM, whereas Yamamoto et al. [8] arrived at 1.2 mM [Ca2+]o as ideal. Hearse et al. [6] found that the [Mg2+]o was also an important factor in protection. A higher extracellular Mg2+ than normal is believed to reduce deleterious effects of normal Ca2+o on Ca2+i-induced energy depletion and impairment of functional recovery [9].

Another cardioplegic solution, containing 0.25 mM Ca2+o and 1 mM Mg2+o, better improved post-ischemic function than either a 1 or 9 mM Mg2+o, 1 mM Ca2+o solution containing citrate to chelate Ca2+o [10]. A consensus on optimal [Mg2+]o and [Ca2+]o has not been reached possibly because an optimal solution depend on the varying conditions of ischemia time and temperature, [H+]o, [K+]o, glucose, buffers, chelators, and other factors. Loss of cell Mg2+ may contribute to Ca2+i overload, but the relationship between altered [Mg2+]o and [Ca2+]o in cold CP solutions on altering systolic and diastolic [Ca2+]i (sys and dia[Ca2+]i) in the intact heart has not been described.

Recent fluorescent techniques using the Ca2+i indicator indo-1 allow measurement of myoplasmic [Ca2+]i in beating, perfused hearts [4,11–13]. We showed that CP decreased the rise in cardiac dia[Ca2+]i observed during cold storage (3 °C for 4 h) in KR solution. Decreased dia[Ca2+]i and increased sys[Ca2+]i after CP improved function on reperfusion in part because of reduced Ca2+ loading during and immediately after cold CP storage [4,12]. Our hypothesis was that elevating [Mg2+]o while lowering [Ca2+]o during hypothermic ischemia would further reduce Ca2+i loading and be more protective on reperfusion than a solution in which [Mg2+]o was not increased.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A
 References
 
2.1. Langendorff isolated heart preparation and measurements
The investigation conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health (NIH No. 85-23, revised 1996). Prior approval was obtained from the Medical College of Wisconsin Animal Studies Committee. Our methods have been described in detail previously [4,12,14]. Ketamine (30 mg) and heparin (1000 units) were injected intraperitoneally into 48 albino English short-haired guinea pigs (250–300 g) 15 min before the animals were decapitated when unresponsive to noxious stimulation. After thoracotomy, inferior and superior vena cavas were cut and the aorta was cannulated distal to the aortic valve. Each heart was immediately perfused via the aortic root at 55 mmHg with a cold oxygenated KR solution (equilibrated with 97% O2 and 3% CO2; pH 7.39±0.01, PO2 630±10 mmHg) and was rapidly excised.

Each solution contained the following in mM: 137 NaCl, 15.5 HCO3-, 1.2 H2PO4-, 11.5 glucose, 2 pyruvate, 16 mannitol, 0.05 EDTA, 0.1 probenecid, and 5 units/l insulin; concentrations of K+o, Ca2+o, and Mg2+o in the KR and CP solutions are given in Table 1. Left ventricular pressure (LVP) was measured isovolumetrically with a transducer connected to a saline-filled latex balloon inserted into the LV through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain an end-diastolic LVP (diaLVP) of 0 mmHg during the initial control period so that any increase in diaLVP reflected an increase in left ventricular wall stiffness or diastolic contracture. Pairs of bipolar electrodes were placed in the right atrial appendage and right ventricular free wall to monitor spontaneous heart rate (HR) and atrial-ventricular (AV) conduction time. If ventricular fibrillation (VF) occurred and did not convert spontaneously to sinus rhythm after 30 s, 10 µg lidocaine was injected into the aortic cannula. Coronary flow (aortic inflow, CF) was measured at constant temperature and constant perfusion pressure (55 mmHg) by a self-calibrating, in-line, ultrasonic flowmeter (Transonic T106X, Ithaca, NY) placed directly into the aortic inflow line.


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  		Table 1. Composition of normal guinea pig Krebs–Ringer (KR) perfusate and four cardioplegic (CP) perfusates

 
Coronary arterial and coronary effluent Na+, K+, Ca2+, PO2, pH, and PCO2 were measured during each condition or at least hourly off-line with an intermittently self-calibrating gas analyzer system. Coronary sinus effluent was collected by placing a small catheter into the right ventricle through the pulmonary artery after ligating both vena cavas. Coronary sinus venous PO2 tension was also measured continuously on-line with an O2 Clark type electrode. Percent O2 extraction (%O2E) was calculated as 100(PO2a-PO2v/PO2a (where P=partial pressure in mmHg, a=coronary artery and v=coronary sinus); myocardial O2 consumption (MVO2) as CF (ml/g) (PO2a-PO2v) O2 solubility at 760 mmHg; O2 solubility is 24 µl at 37 °C and 33 µl at 17 °C. Cardiac efficiency (CE) was calculated as sys–diaLVPxHR/MVO2. Our methods to measure [Ca2+]i in intact hearts using the fluorescent probe indo-1 and a fiberoptic cable placed on the LV free wall have been previously published [4,12,13,15]; details are given in the Appendix A.

2.2. Protocol
The experimental protocol is shown in Fig. 1 . Perfusate and bath were maintained at 37±0.2 °C before and after cold ischemia by a hearted water circulator and at 17±0.4°C during global ischemia by a parallel, refrigerated water circulator. Cardiac functional and Ca2+ loading effects of the one normal and four CP solutions were compared. Between 0 and 80 min, hearts were stabilized and loaded with indo-1 for 20–30 min; after wash out of excess indo-1, a 37 °C control period was established (80–90 min). Eight guinea pig hearts comprised each group; hearts were randomized to a group before each experiment. Each heart was exposed to a CP solution at the onset of cooling (time 90 min). An ischemic time of 4 h at 17 °C was chosen because preliminary experiments showed this to produce about a 40–50% decrease in contractility and perfusion below the pre-ischemic values. It took 15 min (time 90–105 min) for hearts to cool down from 37 °C to 17 °C before ischemia, and 10 min (time 350–360 min) to warm up from 17 °C to 37 °C after ischemia. Cytosolic [Ca2+]i and LVP were recorded continuously at all temperatures to 17 °C and back to 37 °C.



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  		Fig. 1. Experimental schema. All hearts were perfused with Krebs–Ringer's (KR) solution at 37 °C during a 30 min stabilization period and then loaded for 30 min (at 60 min) with indo-1 AM or its vehicle (auto-fluorescence background); this was followed by washout of residual indo-1 AM and vehicle with KR for 30 min (at 90 min, baseline) at 37 °C. Hearts were then perfused (37 °C) for 10 min (at 80–90 min) with KR. At 90 min, cooling from 37 °C to 17 °C (90–110 min) was initiated with CP solution. At 17 °C, flow was stopped (ischemia) for 4 h (at 110–350) and on rewarming and reperfusion, hearts were reperfused with the respective treatment solution for 10 min (at 350–360 min); this was followed by KR for 50 min reperfusion ( at 360–410 min).

 
2.3. Measurement of infarct size
The 2,3,5-triphenltetrazolium chloride (TTC) staining technique was used to determine infarct size in hearts harvested after 60 min reperfusion (time 410 min). Fresh TTC was prepared daily in 0.1 M of phosphate buffer adjusted to pH 7.4 and warmed to 37 °C for 30 min before incubation of hearts. After each experiment, the heart was weighted and transferred to a microtome for slicing into six to seven transverse sections. The sections were immersed in 1% TTC solution and incubated for 25–30 min at 37 °C. TTC stained the non-infarcted myocardium a bright red color that is caused by reduction of TTC by dehydrogenases present in viable tissue. Hearts were labeled and stored in 10% formaldehyde; 48 h later, the atria were removed and discarded. Left and right ventricles were carefully dissected in blind fashion for assessment of infarcted tissue (unstained) and non-infarcted tissue (red stained) and weighted so that percent infarct size was expressed as a percentage of total ventricular weight. Average heart weight was 1.5±0.1 g after 60 min reperfusion; there were no significant differences in total heart weight among the groups.

2.4. Statistical analysis
All data were expressed as mean±SEM. One-way analysis of variance (ANOVA) for repeated measures (Super ANOVA 1.11® software for Macintosh® from Abacus Concepts, Inc, Berkeley, CA) was used to assess within group differences over time. Among groups data were compared at discrete time points before cooling at 37 °C (baseline, at 90 min), after 20 min of cooling (105 min), after 4 h cold ischemia (at 350 min), and during rewarming and reperfusion (at 352, 370, 410 min). Two-way ANOVA was used to assess among group differences at these time points. If F values for the ANOVA were significant, Tukey multiple-comparison post hoc tests were used to differentiate within or among group differences. Differences among means were considered significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A
 References
 
Table 2 displays HR, CF, %O2 extraction, MVO2, and CE data before and after 4 h 17 °C hypothermia. No differences in baseline values were observed among the groups. All data, except HR, were significantly lower in the KR group compared to baseline values and in CP groups after 60 min reperfusion. CF returned to 80% of baseline values in CP groups; this was significantly higher than the 58% return in the KR group. Baseline sys[Ca2+]i (nM) for each group was 230±6 for KR, 225±3 for CP1, 240±4 for CP2, 238±4 for CP3, and 223±6 for CP4; baseline dia[Ca2+]i (nM) values were 158±3 for KR, 150±3 for CP1, 168±4 for CP2, 159±4 for CP3, and 149±5 for CP4. These values were not different among groups.


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  		Table 2. Changes in metabolic variables in KR and CP groups before CP (baseline, time line 90 min) and ischemia and 60 min after warm reperfusion (time line 410 min)

 
Fig. 2 displays the interval change in sys[Ca2+]i (panel A) and systolic LVP (sysLVP) (panel B) before, during and after 4 h 17 °C ischemia in the KR and four CP groups. Sys[Ca2+]i and sysLVP increased significantly during cooling from 37 °C to 17 °C in the KR group. Sys[Ca2+]i markedly decreased during cooling in the CP groups and sysLVP increased slightly due to mild contracture as shown by the same rise in diaLVP. At 20 °C (time 106 min), sys[Ca2+]i and sysLVP peaked highest in the KR group, then fell during cooling to 17 °C at the first hour of ischemia. Dia[Ca2+]i gradually increased in all groups during the 4 h cold ischemic period; since there was no phasic Ca2+i, sys[Ca2+]i also increased concomitantly. Dia and sys[Ca2+]i remained higher in the KR than in each CP group throughout ischemia (P<0.01). On initial reperfusion and rewarming, sys[Ca2+]i and sysLVP increased in all groups but more so in the KR group. On continued reperfusion, sys[Ca2+] remained significantly higher and sysLVP lower, in the KR group than in the CP groups throughout reperfusion. There were no difference in sys[Ca2+]i and sysLVP among CP groups.



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  		Fig. 2. Change in systolic [Ca2+]i (A) and sysLVP (B) before, during and after 4 h 17 °C ischemia in KR (control) and four CP groups. Note that [Ca2+]i and LVP rose markedly during cooling from 37 °C to 17 °C in the KR but not in the CP groups. Systolic [Ca2+]i gradually increased in all groups during 4 h 17 °C ischemia; this occurred to the same degree for dia[Ca2+]i. Systolic [Ca2+]i was different (P<0.01) between KR and CP groups. On initial reperfusion and rewarming, systolic [Ca2+]i increased in all groups but more so in the KR group. [Ca2+]i remained significantly higher in the KR group but decreased gradually toward baseline values in each CP group during reperfusion. Systolic LVP increased slightly during initial reperfusion and rewarming and remained significantly depressed in the KR group but increased gradually toward baseline values in each CP groups at 60 min reperfusion.

 
Fig. 3 shows that dia[Ca2+]i (panel A) increased during cooling and continued increasing during the 4 h cold ischemia period in all groups, but dia[Ca2+]i was lower in each CP group compared to the control group (P<0.01). At 2 min reperfusion (time 352 min), dia[Ca2+]i peaked highest in the KR group compared to each CP group. DiaLVP (panel B) increased markedly in each group during cooling and decreased during the ischemic period but remained elevated in the KR group. The apparent decrease in diastolic LVP below zero is an artifact of setting balloon volume initially to end-diastolic LVP in the beating heart. During reperfusion, diaLVP remained highest in the KR group but returned to the baseline in each CP group. DiaLVP increased significantly in the KR groups after 60 min reperfusion compared to the CP groups. There were no differences in dia[Ca2+]i and diaLVP among the CP groups at 60 min reperfusion.



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  		Fig. 3. Change in diastolic [Ca2+] (A) and diastolic LVP (B) before, during and after 4 h 17 °C ischemia in KR and four CP groups. Note the greater increase in both diastolic [Ca2+] and diastolic LVP in the KR and CP groups during cooling. Diastolic [Ca2+], same as sys[Ca2+], continued to increase in each group during 4 h 17 °C ischemia in the KR group; this was greater than the increase in the CP groups. On initial reperfusion and rewarming, diastolic [Ca2+] peaked in the KR group to a greater value compared to the CP groups. Diastolic LVP increased more in the KR groups compared to each CP group.

 
Fig. 4 shows that d[Ca2+]/dtmax (panel A) increased during initial cooling in the KR group due to continued phasic changes in [Ca2+]i; this was abolished in each CP group due to the high [K+]o. After 1 h in the KR group, the remaining d[Ca2+]/dtmax signal was due only to noise as phasic Ca2+i ceased. During reperfusion, d[Ca2+]/dtmax was greater in the CP groups and returned to the baseline levels but not in the KR group. dLVP/dtmax (panel B) decreased during cooling and was zero during ischemia in all groups. On reperfusion, dLVP/dtmax returned to baseline levels in the CP groups, but not in the KR group.



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  		Fig. 4. Change in d[Ca2+]/dtmax (A) and dLVP/dtmax (B) before, during and after 4 h 17 °C in KR and four CP groups. d[Ca2+]/dtmax and dLVP/dtmax were nil in all CP groups during cooling, ischemia and rewarming. d[Ca2+]/dtmax and dLVP/dtmax were greater in the CP groups than in KR group during reperfusion and similar among the CP groups.

 
Fig. 5 shows inversely that d[Ca2+]/dtmin (panel A) increased during initial cooling in the KR group but was abolished in each CP group. After 1 h in the KR and CP groups, the small, but apparent, rise in both d[Ca2+]/dtmax and d[Ca2+]/dtmin was due to increased noise of the indo signal during the cold period as there was no obvious phasic change in [Ca2+]. During reperfusion, d[Ca2+]/dtmin was greater in each CP group and returned to the baseline levels except in the KR group. dLVP/dtmin (panel B) decreased during cooling and was zero during ischemia in all groups. On reperfusion, dLVP/dtmin returned to baseline levels in the CP groups, but not in the KR group. A comparison of Figs. 4 and 5 shows that the rates of Ca2+ entry and exit, and the rates of contraction and relaxation, are closely related throughout the phases of cooling, ischemia, and reperfusion.



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  		Fig. 5. Change in d[Ca2+]/dtmin (A) and dLVP/dtmin (B) before, during and after 4 h 17 °C in KR and four CP groups. d[Ca2+]/dtmin and dLVP/dtmin were nil in all CP groups during cooling, ischemia and rewarming. d[Ca2+]/dtmin and dLVP/dtmin were greater in the CP groups than in KR group during reperfusion and similar among the CP groups. The results are nearly inverse of those shown in Fig. 4.

 
Fig. 6 displays infarct size as a percentage of total heart weight after 60 min reperfusion. Infarct size was significantly larger in the KR group than in the CP groups, and infarct size was not different among the CP groups.



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  		Fig. 6. Infarct size as percent whole heart weight after 4 h 17 °C ischemia and 70 min reperfusion. Treatment with each CP significantly decreased infarct size compared to the KR (control) group; there were no differences among the CP groups.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A
 References
 
This study demonstrates in intact hearts (a) the temporal association between cardiac function and sys and dia[Ca2+]i before, during and after 4 h 17 °C ischemia and (b) the relative lack of effect of either a higher concentration MgCl2 or a lower concentration of CaCl2 to further attenuate Ca2+ loading or improve cardiac function over that obtained with the high KCl CP solution. Each CP solution similarly reduced myoplasmic dia[Ca2+]i and increased sys[Ca2+]i, dCa2+/dtmax and dCa2+/dtmin, developed (systolic–diastolic) LVP, and dLVP/dtmax and dLVP/dtmin during reperfusion compared to the KR solution alone. CF, MVO2, and CE were also improved similarly by each CP solution. This study suggests that the primary protection afforded by altering the ionic composition of the coronary perfusate before, during and after long-term cold ischemia is cardiac arrest by high KCl; altering CaCl2 or MgCl2 in the presence of higher KCl had little additional effect to benefit [Ca2+]i homeostasis or cardiac function in this model of 4 h, 17 °C global ischemia.

4.1. Moderate hypothermic cardioprotection
Hypothermia protects against cardiac ischemia by prolonging the time to stunning or permanent damage [12–14]. Increasing hypothermia is key to protection because it causes a step-wise reduction in metabolism and delays degradation of intracellular enzymes. Enzymatic activity decreases approximately 50% for each 10 °C fall in temperature, so even at 17 °C, MVO2 is about 25% of that at 37 °C. Reperfusion with an oxygenated, normal-ionic solution after storage triggers rapid ATP regeneration and partially or completely restores ion pump activities, action potentials (AP), and mechanical function. But hypothermia, per se, can also induce deleterious effects; one is Ca2+i overloading during cold ischemia that can lead to reduced diastolic ventricular compliance and contractility on rewarming and reperfusion [4]. Increasing the duration of cold ischemia to 3–5 h leads to worsened mechanical and metabolic function on reperfusion.

Hypothermia is known to induce membrane lipid phase transitions resulting in a less fluid state of the lipid bilayer [16]. This change with cooling influences the activity of membrane-bound processes such as enzymes and receptors.

Some studies suggest that hypothermia decreases Ca2+i inflow through the slow channel [17]. With these changes, a lower [CaCl2] may be beneficial because of a less fluid, less permeable, and thus more stable membrane during cold ischemia. In a lower [CaCl2] solution, there is a less energy dependent Ca2+-induced contraction; inversely, contractions may be reduced in a high [MgCl2] solution. The effectiveness of Ca2+ channel blocking agents is temperature dependent [17]; this is caused in part by cell membrane lipoprotein phase transitions with declining temperature [16].

4.2. Hypothermia, Ca2+ overload and myocardial dysfunction
Ca2+ overload is considered to be a primary contributor to ischemia–reperfusion injury. Cardiac SR, a major regulator of [Ca2+]i under normal conditions, is a target for ischemic myocardial injury. Reduced SR Ca2+ uptake during infusion of cold cardioplegic solution may contribute to myocardial dysfunction after hypothermic global ischemia [3]. Exposure to a low temperature with alterations of Ca2+ uptake and Ca2+ ATPase activity may disrupt Ca2+ homeostasis, thereby interrupting excitation–contraction coupling and relaxation. Fukumoto et al. [18] showed that cold storage increased sarcolemmal Ca2+ uptake and decreased Ca ATPase activity on reperfusion. Labow et al. [3] examined the effect of temperature on Ca2+ ATPase and Ca2+ uptake activities over the temperature range to which a donor heart is usually exposed (4 °C–37 °C) and found that the activity of Ca2+ uptake and Ca2+ ATPase showed highest sensitivity at assay temperatures below 22 °C; after 4 h 4 °C storage, Ca2+ uptake activity was reduced by 50% in the SR when compared to non-storage controls. On the other hand, Ca2+ ATPase was not affected until 24 h of storage, when the activity was also decreased to less than 50%.

In our 4 h 17 °C storage experiments, we found that net Ca2+ flux began to fall at 20 °C during cooling before cold storage (Figs. 4A and 5A). After 60 min reperfusion, dCa2+/dtmax and dCa2+/dtmin (rate of Ca2+ entry and exit) approached the baseline control level in each CP group but not in the KR group. This was associated with improved contractility and relaxation with each beat in the CP groups. The reduced contractility on reperfusion with KR was associated with a lesser Ca2+i overload compared to before ischemia, particularly in the KR group (Fig. 2A). This mismatch of [Ca2+]i and function is likely caused by inhibited dissociation of the troponinC–Ca2+ complex by excess [Ca2+]i so that relaxation, and consequently, contractility become impaired [4]. Injury could result from impaired mitochondrial metabolic effects mediated via mitochondrial Ca2+ overloading on reperfusion as observed after normothermic ischemia. The larger infarct size after cold ischemia alone in the KR group was associated with greater Ca2+i loading during cold ischemia and initial reperfusion because the CP groups had less Ca2+i loading and smaller infarct sizes. We have shown that use of CP reduced myoplasmic Ca2+ loading on cooling, storage, and rewarming after 4 h 3 °C ischemia, and improved function on reperfusion better than after storage in a non-CP, i.e. a KR solution [12]; on reperfusion after cold storage in the CP solution, systolic and sys–dia[Ca2+]i were higher and dia[Ca2+]i was lower. In the present 4 h 17 °C ischemia study, on reperfusion after CP, systolic and sys–dia[Ca2+]i and dia[Ca2+]i were all lower compared to the KR group.

4.3. Ca2+ and Mg2+ in cardioplegia solutions
Throughout development of cardioplegic protective solutions for cardiac surgery, controversy has surrounded the formulation of the optimal ionic solution. The cations of greatest interest have been K+, Ca2+ and Mg2+. Mg2+ and Ca2+ ions are very important in myocardial metabolism because they are the most abundant intracellular cations, excluding K+, and are critical to almost all intracellular reactions. Ca2+i is the primary regulatory factor of the myocardial contraction apparatus and Mg2+ is a cofactor of the Mg2+-dependent ATPase system, which provides energy for myocardial contraction and allows the Na+–K+ and Ca2+ pumps to maintain intracellular homeostasis [1,19]. During cardiac surgery, hyperkalemia can induce Ca2+ entry into cells [20]; this may promote increased myocardial tension with a subsequent increase in ATP consumption. For this reason, increasing MgCl2 in a cardioplegic solution is thought to antagonize effects of Ca2+i and to prevent loss of ATP due to contraction [21–23].

Other than a supra-normal concentration of K+, which can cause unwanted coronary constriction, the ideal concentrations of CaCl2 and MgCl2 have generated the most discussion. Accumulation of cell Ca2+ is thought to cause contractile dysfunction and myocardial injury by inducing activation of ATPase, activation of the ATP-sensitive K+ channel, inhibition of Na+–K+ pump [2], destruction of lipid membrane phospholipids and dysfunction of mitochondria. Various strategies have been advocated to reduce Ca2+ overload during ischemia or reperfusion. In addition to Ca2+ channel blockers [17], other drugs added to CP solutions that enhance the protection by basic CP solutions include Na+/H+ exchange inhibitors [10,13,14], adenosine [20], and KATP channel openers [24]. Another method is to simply lower [CaCl2]; but by how much is not clear [7,8]. The amount of CaCl2 lowering may be dependent on the degree and length of hypothermia. Also, the benefit of lowering [CaCl2] must be weighed against how much [KCl] and [MgCl2] are increased.

Mg2+ is recognized as a ‘physiological Ca2+ inhibitor’ because at high concentrations, it might inhibit Ca2+i overload [25]. Mg2+ may compete with Ca2+ in the Ca2+ channel and suppress the inflow of Ca2+ to the cells. Cell Mg2+ activates Ca2+ ATPase and promotes active transport of Ca2+ out of cells via a Ca2+ pump. Furthermore, high Mg2+ may inhibit Ca2+ outflow from SR and stimulate uptake of Ca2+ into the SR. Thus, Mg2+ is considered to play an important role in maintaining the homeostasis of the cell. In addition to the inter-ion protective effects of Mg2+ described above, Mg2+ is reported to be useful in preserving ATP in the ischemic state [21] and for relieving mitochondrial dysfunction [26]. For example, Kronon et al. [27,28] reported in neonatal piglets that 5–6 mM MgCl2, given with 0.2–0.4 mM CaCl2, gave a better return of function after 20 min of normothermic cardiopulmonary bypass than did a group given only a lower CaCl2 concentration. Fukuhiro et al. [10] reported that lowering [Ca2+]o in a CP solution was beneficial in MgCl2-containing CP but that high MgCl2 was detrimental in the presence of citrate to chelate Ca2+. Recently, Sharikabad et al. [29] reported that elevation of [MgCl2] to 5 mM led to reduced Ca2+ influx and cell accumulation during reoxygenation of hypoxic cardiomyocytes but did not alter Ca2+ efflux kinetics. We recently reported that normothermic ischemia with a high KCl, low CaCl2 solution reduced the elevation in [Ca2+]i and helped restore indices of contractility, relaxation and metabolism compared to warm reperfusion [15]; however, there was no added benefit of a higher [MgCl2]. In the present study, we tripled [MgCl2] in two CP groups but there was no difference in [Ca2+]i or cardiac functional recovery after 60 min reperfusion compared to the other CP groups with normal [MgCl2]; the higher [KCl] was the sole ionic factor responsible for the normalization of [Ca2+]i and improved function after long-term cold ischemia.

Limitations of this animal study are that the experimental conditions do not necessarily represent the timing, temperature, or duration of cardiac ischemia in the clinical setting. Blood products were not included in the perfusate and the model was one of global ischemia in non-ejecting hearts. It is possible that a wider range of cation concentrations under study would result in differences in function and infarct size. In summary, hypothermia is the most protective mechanism for protecting the heart under these experimental conditions; the next most powerful protective mechanism is elevated K+. The relative roles of changing [Ca2+]i and [Mg2+]i by altering external [CaCl2] and [MgCl2], appear to be small both for improving function and reducing Ca2+i loading in our model. It is possible that the optimal concentrations of [CaCl2] and [MgCl2], as well as [KCl], are highly dependent on the temperature, duration of ischemia, and conditions of cooling and reperfusion in a given model. Knowledge of specific time-dependent changes in myoplasmic [Ca2+] in normal and CP solutions before, during, and after hypothermic ischemia should aid in development of improved perfusion methods and cardioplegic formulations for heart preservation.


   Acknowledgments
 
The authors wish to thank Jim Heisner, Anita Tredeau, Steve Contney, and Samhita S. Rhodes for their contributions to this research.

This research was funded by the US National Institutes of Health (NHLB-058691) and the American Heart Association (0265181Z).


   Footnotes
 
This work was presented in part previously (FASEB J 15:A768, 2001, and Anesthesiology 93:A629, 2000 and 95:A678, 2001).


   Appendix A
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A
References
 
A.1. Measurement of Ca2+ transients in intact hearts
Experiments were carried out in a light-shielded Faraday cage. The distal end of a tri-furcated fiber silica fiber optic cable was placed against the LV epicardial surface through a hole in the bath and a rubber O ring was placed between the ferrule and the heart to reduce cardiac motion at the contact point of the fiber optic tip. Background auto-fluorescence (AF) was determined for each heart after initial perfusion and equilibration at 37 °C. Initial background (before indo-1 loading) measurements were obtained after 30 min of stabilization. Hearts were then loaded with indo-1 AM at 25±0.6 °C (to facilitate loading) for 20–30 min with 165 ml of a re-circulated, modified KR solution containing 6 µM indo-1 AM (Sigma Chemical, St. Louis, MO). Indo-1 AM was initially dissolved in 1 ml of dimethyl sulfoxide containing a cell permeant, 16% (w/v) Pluronic I-127 (Sigma Chemical), and diluted to 165 ml with modified KR solution. Loading was stopped when fluorescence (F) intensity of the 385 nm signal was increased 10-fold. Residual indo-1 AM was washed out by perfusing the heart with standard perfusate for at least another 20 min and then each heart was rewarmed to 37.2±0.1 °C before initiating the study. The perfusate contained 100 µM probenecid to retard leakage of indo-1.

The LV region of the heart was excited via one limb of the fiberoptic cable with light filtered through a 360±16 nm (bandwidth) monochromator. To avoid blanching of indo-1, the arc lamp shutter was opened only for 2.5 s recordings. F emissions were collected by fibers of the remaining two limbs of the fiber optic cable with one limb filtered at 385 nm (390±5 nm) and the other at 456 nm (460±5 nm). Photomultiplier tube output settings for F385 and F456 were set at 525 mV and 385 mV, respectively, to optimize recordings of physiological concentrations of Ca2+. F385 and F456 transients were recorded using a modified luminescence spectrophotometer ((SLM Aminco-Bowman II, Spectronic Instruments, Urbana, IL). At each sampling interval, F385, F456, F385/F456, and LVP were recorded digitally over 7–8 cardiac cycles every 8 ms for 2.5 s. Each experiment comprised 40–50 recordings. Data were stored (computer software OS/2 version 4, IBM, Armonk, NY) for background correction and conversion of F data to [Ca2+] off-line (Matlab® The Math Works, Natick, MA, and Excel®, Microsoft Corp., Redmond, WA). Using this ratiometric method applied to our model, F385 and F456 both decline over time but remain at least fivefold greater than background after 3 h at 37 °C; however, the F385/F456 ratio is unchanged over this time in the absence of ischemia.

Ca2+ transients were recorded every 1–5 min during normothermia, cooling, and rewarming, and once per hour during hypothermic storage. SysLVP (mmHg) before indo-1 loading was not different among groups: KR, 91±3; CP1, 89±5; CP2, 93±3; CP3, 92±3, and CP4, 88±3. SysLVP decreased by up to 25% after indo-1 loading and washout (Fig. 2B, 80 min) due to its vehicle and to [Ca2+]i buffering by indo-1, per se; there were no differences among groups on washout.

A.2. Correcting for background and non-cytosolic [Ca2+]
Initial control measurements (time 80 min) were obtained after the periods of stabilization, fluorescent dye or vehicle loading and washout. MnCl2 (100 µM) was infused at the end of each experiment to quench cytosolic Ca2+i transients. Quenching does not alter phasic LVP. In non-ischemic time controls, all functional data remained statistically unchanged for 410 min experimental period (data not shown). 100 µM385 and 100 µM456 indo-1 Ca2+i transients, LVP, and dLVP/dt were displayed simultaneously on a computer screen and stored digitally using proprietary software on an IBM OS/2 system. After correcting for tissue AF over time, with or without hypothermic perfusion, cold ischemia and warm reperfusion, and quenching of the cytosolic Ca2+ compartment to quantify the non-cytosolic Ca2+ compartment, the signals were calibrated to nM [Ca2+] using algorithms we developed. LVP and raw metabolic data were recorded (MacLab®, AD Instruments, Castle Hills, Australia), and together with the Ca2+i transient signals, were later analyzed together using programs we developed on Matlab® and Microsoft Excel® software.

Loss of membrane integrity in infarcted cells on reperfusion could result in the leakage of indo-1 and lower signal intensities; however, because this is a ratiometric determination of [Ca2+]i, both F signals degrade similarly so that the F ratio is relatively unchanged and remains no less than half the post-loaded signal strength and fivefold greater than the unloaded signal strength [4,12].

Each of these groups was backed by a number of calibration studies, time controls, and AF controls as detailed above. Background AF, but not indo-1 dye F, is influenced by tissue oxygenation state at these two isobestic wavelengths. It is necessary to measure the change in tissue fluorescence, primarily due to NADH that occurs during hypothermia, ischemia and reperfusion. In seven ancillary hearts, only the indo-1 vehicle was washed in and out after which AF was measured during the time course of warm ischemia and reperfusion; in six other hearts, this protocol was repeated in the presence of 4 h 17 °C ischemia and 37 °C reperfusion. The time related effect of changes in indo-1 F units and contractility using this model have been published [4,12]. In all experiments, the mean AF385 and AF456 background values were subtracted from the corresponding indo-1 F385 and F456 values at the same time point (t) and for the same experimental condition so that the F ratio R was calculated as:

(A1)

Calibration curves were derived according to previously published protocols by Brandes et al. [11] and used a modification of the standard equation for fluorescent indicators. AF corrected total (tot) intracellular [Ca2+] was calculated from the totF385/totF456 ratio (totR), Rmax=Sr/bH (for >100 µM Ca2+), Rmin=RmaxS385/S456 (for 0 Ca2+), S385=I385/I385 (at min/max Ca2+)=0.05, S456=I456/I456 (at min/max Ca2+)=2.4, and Kd, according to the equation:

(A2)
where S is the ratio of light intensities (I) at the same wavelength at min and max Ca2+, Sr=(1-S456)/(1-S385)=-1.48, and bH=the slope (b) of totF385 as a function of totF456=-0.25. Rmax was calculated as 6.0 and Rmin as 0.06. The dissociation constant Kd is inversely proportional to temperature. Free indo-1 reduces the fluorescence ratio F385/F456 in a nearly linear fashion by 0.30, 0.23, and 0.16 per 10 °C fall, so that Kd increased 28% at 27 °C (305 nM), 44% at 17 °C (354 nM), and 67% at 7 °C (385 nM) [4,12]. The linear relationship (y=mx+b) for temperature and Kd was Kd=-4.6 °C+423.8 (r2=0.99).

A.3. Measurement of non-cytosolic (mitochondrial) [Ca2+]
Non-cytosolic (primarily mitochondrial) m[Ca2+]m was calculated similarly:

(A3)
where mitoR was calculated as the ratio of the non-cytosolic F, mitoF385 and mitoF456, respectively. Non-cytosolic F was measured at the end of each indo-1 experiment by perfusing hearts with 100 µM MnCl2 to quench F derived from the cytosolic (cyto) compartment. mitoF385 and mitoF456 were calculated at each time point by multiplying the residual mitochondrial fluorescence (f) fractions (f385 and f456) by total end-diastolic fluorescence so that:

(A4)

Similar to Eqs. (A2) and (A3), cytosolic cyto[Ca2+] was calculated as:

(A5)
where cytoR was derived from the ratio of the cytosolic fluorescence, cytoF385 and cytoF456, respectively, calculated at each time point by effectively subtracting mitochondrial compartment Ca2+ ([Ca2+]m) from tot[Ca2+] and multiplying the remainder by total end-diastolic fluorescence (as in Eq. (A3)) so that:

(A6)

We have shown that the cellular indo-1 F ratio is not altered appreciably over time so that non-cytosolic Ca2+ does not also become quenched [12]. Non-stimulated endothelium does not contribute significantly to tot[Ca2+]i [30].


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A
 References
 

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J. An, A. K. S. Camara, S. S. Rhodes, M. L. Riess, and D. F. Stowe
Warm ischemic preconditioning improves mitochondrial redox balance during and after mild hypothermic ischemia in guinea pig isolated hearts
Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2620 - H2627.
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