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Ann Thorac Surg 1998;65:1065-1070
© 1998 The Society of Thoracic Surgeons

Calcium Preconditioning in Human Myocardium

^a Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado, USA

Accepted for publication November 18, 1997.

Address reprint requests to Dr Cain, Department of Surgery, University of Colorado Health Sciences Center, C-320, 4200 East Ninth Ave, Denver, CO 80262

	Abstract

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Background. Ischemic stress and other protein kinase C (PKC)–linked receptor stimuli can induce rapid cardiac protection against ischemia-reperfusion injury. We and others have demonstrated that exogenous calcium (Ca²⁺) pretreatment confers PKC-mediated cardiac functional and infarct protection in animal models, but it remains unknown whether Ca²⁺ preconditioning confers similar postischemic functional protection in human myocardium, and, if so, whether the mechanism is mediated by PKC. We postulated that Ca²⁺ preconditioning confers ischemic tolerance to human myocardium by a PKC-dependent mechanism.

Methods. Human atrial trabeculae were suspended in organ baths and paced at 1 Hz, and force development was recorded. After 90 minutes of equilibration, all trabeculae were subjected to ischemia (45 minutes) and reperfusion (120 minutes). Exogenous CaCl₂ (3.0 mmol/L for 5 minutes) or vehicle (saline solution) was administered before simulated ischemia, with or without concurrent PKC inhibition (bisindolylmaleimide I, 150 nmol/L).

Results. Ischemia-reperfusion resulted in decreased postischemic developed force, Ca²⁺ preconditioning protected human myocardium against ischemia-reperfusion injury (p < 0.05 versus control ischemia-reperfusion), and concurrent PKC inhibition abolished the salutary effect of Ca²⁺ preconditioning in human myocardium (p < 0.05 versus Ca²⁺ preconditioning).

Conclusions. Preconditioning with Ca²⁺ represents a potent means of accessing PKC-mediated protection of the human myocardium against ischemia-reperfusion injury.

	Introduction

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Since Murry and co-workers [1] demonstrated that brief myocardial ischemia induced tolerance to a subsequent ischemic stress in dogs, investigators have reproduced this form of endogenous myocardial protection (ischemic preconditioning) in multiple other species [2]. Of interesting clinical relevance, patients who have experienced angina before myocardial infarction repeatedly have exhibited an improved postinfarction prognosis compared with those without antecedent angina [3]. Preconditioning-induced ischemic tolerance has been evaluated in humans during angioplasty [4] and before the initiation of cardiac bypass [5]. Mechanistic evaluation of cardiac preconditioning in the laboratory has revealed that ischemic stress induces protection through adenosine [6], norepinephrine [7], and calcium (Ca²⁺) [8–11]. Further studies have revealed that these signaling mechanisms induce myocardial protection against ischemia mediated by protein kinase C (PKC).

Inducing PKC-mediated myocardial protection with exogenous Ca²⁺ may be more clinically appealing than using ischemic preconditioning on an already impaired myocardium. We [8, 9] and others [10, 11] have demonstrated that exogenous Ca²⁺ pretreatment confers cardiac functional and infarct protection through PKC in animal models. It remains unknown, however, whether Ca²⁺ preconditioning confers similar postischemic functional protection in human myocardium. We postulated that exogenous Ca²⁺ preconditioning confers ischemic tolerance to human myocardium through mechanisms mediated by PKC.

	Material and methods

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Isolated atrial trabeculae
Right atrial appendages were obtained from patients undergoing cardiac operation. All trabeculae and appendages were obtained from patients with stable cardiac disease who were undergoing their first cardiac surgical procedure. The indication for operation in all patients evaluated in this study was ischemic coronary artery disease. Patients who required valve repair or replacement were not evaluated to decrease any potential confounding influence. No trabeculae were obtained from patients with diabetes who were taking oral sulfonylurea agents. Patients were excluded from the study if they had hemodynamic instability (mean arterial pressure, <80 mm Hg) within 48 hours of cardiopulmonary bypass, atrial dysrhythmias, or right atrial pressures greater than 10 mm Hg. Informed consent was obtained from all subjects, and the study was approved by the University of Colorado Health Sciences Center.

Each appendage was placed in oxygenated, modified Tyrode’s solution at 4°C. Three to four trabeculae (diameter, <1 mm; length, 4 to 7 mm) were obtained from each appendage and suspended vertically in an organ bath between two clips. The bottom clip was fixed and the top clip was attached to a force transducer. Each organ bath contained 30 mL of modified Tyrode’s solution that was bubbled (40 mL/min) with a 92.5% O₂ and 7.5% CO₂ gas mixture during normoxia. This mixture provided for an oxygen tension of greater than 360 mm Hg, a carbon dioxide tension of 38 to 42 mm Hg, and a buffer pH of 7.35 to 7.45, which were checked routinely with an automated blood gas analyzer (ABL Instruments, Vienna, Austria). The temperature in the organ bath was maintained at 37.5°C. During the simulated ischemic period, the gas mixture was switched to 92.5% N₂ and 7.5% CO₂, which produced an oxygen tension of less than 50 mm Hg, and the organ bath was covered to prevent atmospheric gas exchange. Except during the period of simulated ischemia, the Tyrode’s buffer was replaced at 20-minute intervals throughout the experiment.

Thirty minutes were allowed to pass after the suspension of each trabecula for recovery. After this time, the trabeculae were stretched gradually to a resting force of 1 g, which was determined to be the optimum length–tension relation for human atrial trabeculae in our laboratory, and then they were field-stimulated. Field stimulation was accomplished with platinum electrodes (Radnoti Glass, Inc, Monrovia, CA) at a frequency of 1 Hz. The platinum electrodes were positioned on each side of each trabecula and were driven with stimulators (Grass SD9 stimulator, Warwick, RI) with 5-ms pulses at a voltage of 10% above threshold. Isometric contractile responses were detected by force-displacement transducers (Grass FT03) and recorded with a computerized preamplifier/digitizer (MacLab 8; AD Instruments, Milford, MA) and a Macintosh computer (Apple Computer, Cupertino, CA). The indices of contractile function assessed were developed force (in grams) and resting force (in grams). Trabeculae that failed to generate at least 0.25 g of developed force were excluded from study. Baseline developed force data are shown in Table 1.

Experimental design
The isolated crystalloid-superfused human atrial trabeculae model was used as previously described [6, 12–15]. All trabeculae were subjected to a 90-minute equilibration period to allow for stabilization of developed force, and the experiments were conducted for 180 minutes. Control ischemia-reperfusion trabeculae were challenged with a 45-minute period of simulated ischemia, which consisted of hypoxic, substrate-free Tyrode’s solution with pacing at 3 Hz, followed by 120 minutes of reperfusion with normoxic Tyrode’s solution with pacing at 1 Hz. Ca²⁺-preconditioned trabeculae were exposed to a 50% increase in the Ca²⁺ bath concentration (3 mmol/L for 5 minutes) to simulate a clinically relevant Ca²⁺ bolus or vehicle with and without concurrent PKC inhibition (BIS hydrochloride, 150 nmol/L), followed by 10 minutes of standard superfusion (2 mmol/L) and then ischemia-reperfusion. Ca²⁺ dose-response data were determined in a separate set of experiments and indicated that 3 mmol/L represented approximately the inotropic midpoint (Fig 1), corresponding to half the maximum inotropic response to Ca²⁺. These Ca²⁺ dose-response experiments (n = 6) were performed by increasing the Ca²⁺ bath concentration in a stepwise fashion (by 0.5 mmol/L) and determining the change in baseline developed force. Each trabecula was used in only a single experiment and multiple trabeculae from the same patient were used in different protocols. The experimental protocols are depicted in Figure 2. In addition, control trabeculae were equilibrated routinely for 90 minutes and perfused with normoxic Tyrode’s solution with pacing at 1 Hz for 180 minutes to ensure model stability.

View larger version (11K): [in this window] [in a new window]   Fig 1. Calcium dose-response curve: percentage of baseline developed force, measured at end-equilibration, versus the Ca2+ concentration in the organ bath. A positive inotropic effect on human myocardium results from an increase in the Ca2+ concentration, reaching approximately one half the maximum inotropy at 3 mmol/L, which was the dose used in the subsequent studies.

View larger version (18K): [in this window] [in a new window]   Fig 2. Experimental protocols. All experiments were proceeded by a 90-minute period that allowed for the stabilization of developed force. Normoxic perfusion in oxygenated Tyrode’s solution with pacing at 1 Hz was used. Ca2+ refers to the calcium that was added to increase the bath calcium by 1 mmol/L. Simulated ischemia refers to the incubation of trabeculae in substrate-free hypoxic Tyrode’s solution while pacing at 3 Hz. Bisindolylmaleimide I (BIS, 150 nmol/L) was administered 3 minutes before Ca2+ administration or alone to block protein kinase C. (I/R = ischemia-reperfusion; PC = preconditioning.)

Presentation of data and statistical analysis
All values are reported as the mean plus or minus the standard error of the mean (n = 4 to 7 per group). Differences at the 95% confidence level were considered statistically significant. Functional performance (ie, percentage baseline developed force) was compared at the corresponding time points between groups using one-way analysis of variance with the post hoc Bonferroni/Dunn test (StatView 4.0; Abacus Concepts, Berkeley, CA).

	Results

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Effect of ischemia-reperfusion on functional recovery
To determine the functional recovery of human atrial trabeculae after ischemia-reperfusion, trabeculae (n = 7) were equilibrated for 90 minutes and then exposed to 45 minutes of simulated ischemia and 120 minutes of reoxygenation. Ischemia-reperfusion decreased postischemic developed force to 18.1% ± 2.5% baseline developed force, demonstrating postischemic impairment of human myocardial contractile function (Fig 3).

View larger version (16K): [in this window] [in a new window]   Fig 3. Effect of ischemia-reperfusion (I/R) and Ca2+ preconditioning (PC) on functional recovery. Ischemia-reperfusion resulted in decreased postischemic developed force. Ca2+ preconditioning resulted in increased postischemic developed force. (*p < 0.05 versus control ischemia-reperfusion.)

Effect of Ca²⁺ preconditioning with concurrent protein kinase c inhibition on functional recovery
To determine whether Ca²⁺ preconditioning confers protection against ischemia through a mechanism mediated by PKC, a PKC-selective inhibitor (BIS) was administered for 3 minutes before the administration of a Ca²⁺ bolus. Protein kinase C inhibition by BIS (n = 6) abolished the improvement in developed force conferred by Ca²⁺ preconditioning (17.2% ± 3.1% baseline developed force; p < 0.05 versus Ca²⁺ preconditioning) (Fig 4). To determine whether BIS alone was responsible for the decreased developed force when it was administered concurrently with Ca²⁺ preconditioning, BIS alone (n = 4) was administered, followed by the ischemia-reperfusion protocol. Bisindolylmaleimide I alone did not decrease functional recovery after ischemia-reperfusion (p > 0.05 versus control ischemia-reperfusion) (Fig 5).

View larger version (17K): [in this window] [in a new window]   Fig 4. Effect of Ca2+ preconditioning (PC) with protein kinase C inhibition on functional recovery. Whereas Ca2+ preconditioning resulted in increased postischemic developed force, protein kinase C inhibition by bisindolylmaleimide I (BIS, 150 nmol/L) abolished the salutary effects of Ca2+ preconditioning (p < 0.05 versus Ca2+ preconditioning.)

View larger version (15K): [in this window] [in a new window]   Fig 5. Effect of protein kinase C inhibition on developed force. Bisindolylmaleimide I (BIS) alone was administered before ischemia-reperfusion (I/R) to determine whether protein kinase C inhibition alone was responsible for the decreased developed force demonstrated with Ca2+ preconditioning with protein kinase C inhibition. Bisindolylmaleimide I alone did not depress developed force after ischemia-reperfusion (p > 0.05 versus control ischemia-reperfusion).

	Comment

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Ischemic preconditioning is now recognized as a means to induce an endogenous program of myocardial protection after ischemia-reperfusion injury [2]. Multiple stimuli leading to ischemic protection, such as antecedent ischemic stress, adenosine, norepinephrine, and Ca²⁺, act through mechanisms mediated by PKC [8–13]. It is paradoxical that, whereas increased [Ca²⁺]_i may result from ischemia-reperfusion, substrate deprivation, or even cell death [16], preischemic elevation of [Ca²⁺]_i induces protection against subsequent ischemia-reperfusion. Some insight into this seeming paradox is that many ischemic stimuli, such as transient ischemia or ₁-receptor stimulation, result in the elevation of [Ca²⁺]_i [17]. Although endogenous Ca²⁺ is known to be a potent stimulator of PKC [18], it remained unknown whether exogenous Ca²⁺ itself could activate PKC and thereby confer ischemic protection in the human heart.

Protein kinase C is a ubiquitous serine-threonine kinase, and evidence indicates that PKC is involved in the modulation of myocardial contraction by the phosphorylation of multiple intramyocardial targets [18–20]. In the present study, Ca²⁺ administration resulted in inotropy in human myocardial trabeculae during the 5-minute Ca²⁺ bolus(Figs 3, 4), corroborating basic and clinical observations indicating a positive inotropic effect of Ca²⁺ on myocardium [15, 21]. The inotropic effects of extracellular Ca²⁺ derive predominantly from its effects as a "Ca²⁺ trigger" of intracellular sarcoplasmic reticulum Ca²⁺ release during depolarization, resulting in Ca²⁺-induced Ca²⁺ release [16]. Increased intracellular Ca²⁺ enhances Ca²⁺ binding to troponin C, permitting the conformational change required for actin-myosin interaction and resulting in inotropy [16]. In addition to having direct effects on the contractile apparatus, Ca²⁺ also acts as an intracellular second messenger and participates in signal transduction that activates numerous intracellular enzymes, including PKC [16]. Protein kinase C is recognized as an important Ca²⁺-sensitive myocardial regulatory enzyme [2, 7, 18–20]. The inhibitory subunit of troponin I and the tropomyosin-binding subunit of troponin T both are phosphorylated by PKC [19]. The tropomyosin-binding subunit of troponin T modulates Ca²⁺ sensitivity of force production in myocardium, whereas phosphorylation of the inhibitory subunit of troponin I decreases its inhibitory effects [20]. Protein kinase C activation also phosphorylates and activates the Na⁺-H⁺ antiporter, which promotes intracellular alkalinization and thereby increases myofilament responsiveness to Ca²⁺ [20]. Other inotropes, such as the ₁-adrenergic agonists, which stimulate PKC activity through diacylglycerol- and inositol triphosphate-induced sarcoplasmic reticulum Ca²⁺ release, result in myocardial inotropy that also may involve PKC [7, 18]. Thus, many effectors downstream from PKC participate in the inotropic effects of Ca²⁺ on the myocardium.

The administration of CaCl₂ in combination with a PKC inhibitor (BIS) abolished Ca²⁺ preconditioning. Bisindolylmaleimide I has been shown to inhibit effectively all PKC isoforms at the concentration used in this study [22]. We interpret our results to indicate that PKC mediates the preconditioning effect of exogenous Ca²⁺ on human myocardium. Our finding that Ca²⁺ preconditioning is operative in human myocardium is in concert with previous work from our laboratory [8, 9] and the laboratories of other investigators [10, 11], which has demonstrated that stimulated release of endogenous Ca²⁺ or exogenous Ca²⁺ conferred ischemic protection through PKC to rat myocardium. Although the role of Ca²⁺ in preconditioning had been speculated on [16, 23], Ashraf and colleagues [10] investigated the role of Ca²⁺ depletion and repletion in preventing the Ca²⁺ paradox. The Ca²⁺ paradox results in myocardial injury that approximates that caused by ischemia-reperfusion. Therefore, Ca²⁺ stress hypothetically could prevent or attenuate ischemia-reperfusion injury. Subsequent investigations confirmed this hypothesis [8, 9, 11]. However, it has remained unknown whether PKC-mediated Ca²⁺ preconditioning is operative in human myocardium. As a point of reference to evaluate the efficacy of Ca²⁺ preconditioning, we previously have reported that ischemic preconditioning increased postischemic developed force to approximately 50% of the baseline developed force through mechanisms mediated by PKC [13, 14]. Preconditioning stimuli are species-specific [2] and therefore must be evaluated using human myocardium to determine their clinical utility.

This study should be interpreted with several important caveats. First, the use of human atrial tissue as a representative surrogate for the myocardial response to ischemia-reperfusion may lead to a different set of conclusions from that using ventricular samples. We chose atrial tissue because it is routinely available during coronary artery bypass grafting, allowing a wide variety of patients with generally healthy atria to be examined. Atrial tissue appears to be relatively free of ischemic, restrictive, or myopathic disease, in contrast to ventricular tissue. Using atrial tissue avoids the ethical considerations of evaluating experimental agents in vivo and avoids drawing conclusions about the response of healthy human myocardium using explanted cardiomyopathic hearts subjected to generous inotropic therapy. Indeed, we previously have demonstrated that human ventricular tissue can be functionally preconditioned [12] and that the protection is qualitatively similar to atrial preconditioning [13, 14], implying that basic myocardial mechanisms of excitation-contraction coupling are conserved between the two anatomic regions of the human heart. In this study, we did not measure tissue creatine kinase levels. We previously have used creatine kinase levels as markers of tissue viability and determined that they correlate directly with functional recovery in our model [6, 13]. Hypoxia (simulated ischemia) was used in the present study because ischemia (lack of blood flow) would not be an accurate term in regard to the superfused atrial trabeculae model. Whether the effects of Ca²⁺ preconditioning are limited to protection against hypoxia versus ischemia remains to be determined.

The exact mechanism by which PKC provides myocardial functional protection is unknown. It is possible that PKC upregulates the cellular machinery required to adapt to the subsequent ischemia-reperfusion insult. For example, PKC may activate the cellular machinery required to prepare the heart to handle the ion gradient dyshomeostasis that is present after ischemia-reperfusion. In particular, PKC can activate the adenosine triphosphate–sensitive potassium (K_ATP) channel, which shortens the action potential and thereby reduces ischemic Ca²⁺ overload [24]. Further, the mechanism by which exogenous Ca²⁺ activates PKC remains unknown. Possibly, increased extracellular Ca²⁺ increases intracellular Ca²⁺ by Ca²⁺-induced Ca²⁺ release or voltage-gated channels, resulting in sufficient stimulus to activate PKC [16]. This, however, remains to be determined. The present study was not designed to measure PKC activity because results from these assays are difficult to interpret in regard to the bioactivity of the enzyme reactions. Instead, we chose to determine the functional consequences of PKC blockade. The link between preconditioning and PKC may be only an association, and although PKC may mediate some forms of preconditioning, other kinases may be operative after other preconditioning stimuli.

Another unanswered question is whether the supraphysiologic Ca²⁺ concentration required to induce PKC activation will prove arrhythmogenic. High extracellular Ca²⁺ levels may result in inotropy (as was demonstrated in this and other experiments [15, 21]) that exceeds the myocardial energy supply, resulting in an inability of membrane ionic pumps to function normally and resulting in decreased [K⁺]_i. Myocyte membrane potential then would drift toward threshold, resulting in myocardial hyperexcitability [25]. Further, hypercalcemia causes spontaneous oscillations in myocardial membrane potential [25]. Throughout these paced experiments using human tissues, we did not detect spontaneous contractile activity during exposure to supranormal Ca²⁺ concentrations.

The ultimate benefit of preischemic induction of endogenous myocardial functional protection relates to its clinical applicability. Transient ischemia, which is known to be a potent protective stimulus in animals, has limited clinical appeal. Because CaCl₂ is both clinically accessible and acceptable, stimulation of human myocardial PKC-mediated functional protection with preischemic CaCl₂ infusion may provide a potent means of enhancing human cardiac function after coronary angioplasty, cardiac bypass operations, or heart transplantation.

	Acknowledgments

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This work was supported in part by National Institutes of Health grants HL-43696, HL-44186, and GM-08315. Daniel R. Meldrum, MD, is a recipient of the National Research Service Award. We thank the cardiothoracic surgical teams for their help in obtaining the atrial samples, specifically, Drs Fred L. Grover, James M. Brown, David N. Campbell, Salim Aziz, Mary M. Wollmering, Max B. Mitchell, and Irving Shen, who made this study possible.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Brian S. Cain Daniel R. Meldrum Alden H. Harken Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cain, B. S. Articles by Harken, A. H. Search for Related Content PubMed PubMed Citation Articles by Cain, B. S. Articles by Harken, A. H.