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Ann Thorac Surg 1996;61:558-564
© 1996 The Society of Thoracic Surgeons

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Original Article: Cardiovascular

Beneficial Effects of Myocyte Preconditioning on Contractile Processes After Cardioplegic Arrest

Division of Cardiothoracic Surgery and Department of Anesthesiology, Medical University of South Carolina, Charleston, South Carolina

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Myocardial preconditioning, which can be achieved through short intervals of ischemia or hypoxia followed by reperfusion, protects the myocardium with subsequent prolonged periods of ischemia. Accordingly, the present study tested the hypothesis that hypoxic preconditioning before cardioplegic arrest would have direct and beneficial effects on myocyte contractile processes with reperfusion.

Methods. Left ventricular porcine myocytes (n = 335) were randomly assigned to one of three treatments: normothermia, maintained in cell media (37°C, 2 hours); cardioplegia, hyperkalemic arrest (24 mEq K⁺, 4°C, 2 hours) followed by normothermic reperfusion; preconditioning, hypoxia (20 minutes) and reperfusion (20 minutes), and then followed by cardioplegic arrest and rewarming. Myocyte velocity of shortening was measured using computer-assisted videomicroscopy at baseline and with ß-adrenergic receptor stimulation with isoproterenol (25 nmol/L).

Results. In the cardioplegia group, myocyte function was reduced at baseline (22 ± 1 versus 57 ± 2 µm/s) and with ß-adrenergic receptor stimulation (81 ± 5 versus 156 ± 7 µm/s) compared to normothermic controls (p < 0.05). Preconditioning improved myocyte function at baseline (38 ± 2 µm/s) and with ß-adrenergic receptor stimulation (130 ± 6 µm/s) compared to the cardioplegic alone group (p < 0.05).

Conclusions. The important findings from this study are twofold. First, preconditioning can be induced directly at the level of the myocyte, independent of nonmyocyte populations and extracellular influences. Second, myocyte preconditioning provides protective effects on myocyte function and ß-adrenergic responsiveness after cardioplegic arrest and rewarming. These findings suggest that preconditioning may provide a novel approach in protecting myocyte contractile processes during cardioplegic arrest.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
See also page 564.

Preconditioning describes a phenomenon in which myocardium made transiently ischemic becomes more tolerant to a subsequent and prolonged period of ischemia [1, 2]. Murry and colleagues [1] reported that myocardium subjected to a brief period of coronary occlusion preceding a more prolonged ischemic interval significantly decreased the size of myocardial infarction compared to hearts subjected to a single prolonged ischemic episode. Transient left ventricular dysfunction may be encountered after hypothermic cardioplegic arrest and rewarming [3, 4]. Efforts at reducing this dysfunction have centered on optimizing cardioplegic solutions and manipulating reperfusion conditions [5–7]. However, there has been little focus on preemptive strategies to protect the myocardium before the initiation of cardioplegic arrest. Thus, the induction of a potent endogenous protective mechanism against myocardial ischemia and reperfusion injury, or preconditioning, may have significant applications in cardiac surgical procedures. In a recent study, Illes and colleagues [8] demonstrated that preconditioning in the rabbit heart improved left ventricular pump function after hypothermic cardioplegic arrest and rewarming. This study suggests that preconditioning may have beneficial effects on left ventricular pump performance in the setting of cardiac operation. However, two fundamental questions remain unanswered: (1) Does preconditioning directly improve myocyte contractile function after hypothermic cardioplegic arrest and rewarming?; (2) Does preconditioning impart a protective mechanism that depends on interaction between the nonmyocyte and myocyte cell populations? This laboratory has previously demonstrated that contractile function and ß-adrenergic responsiveness are reduced after hyperkalemic hypothermic cardioplegic arrest and rewarming [9, 10]. Therefore, the present investigation used this isolated myocyte system to test the hypothesis that preconditioning would have a direct and beneficial effect on myocyte contractile function.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Myocyte Isolation and Contractile Function Measurement
Five Yorkshire pigs (25 to 30 kg) were the source of left ventricular myocytes for the study. All animals were cared for and treated in accordance with the ``Guide for the Care and Use of Laboratory Animals'' published by the National Institutes of Health (NIH publication 85-23, revised 1985). On the day of study, the animals were anesthetized with isoflurane (0.5%/1.5 L/min) and ventilated through a nonrecirculating anesthesia circuit. A sternotomy was then performed, the heart was extirpated and placed in cold oxygenated Krebs solution. The region of the left ventricular free wall comprising the left circumflex coronary artery (5 x 5 cm) was dissected free, the artery cannulated, and the tissue prepared for myocyte isolation, as described previously [9–12]. Briefly, oxygenated modified Krebs solution containing aerobic substrates and collagenase (0.5 mg/mL, type II; 146 U/mg; Au: manufacturer?Worthington, PA) was perfused and recirculated through the cannulated circumflex artery for 20 minutes. The tissue was then minced into 2-mm sections and added to an oxygenated solution containing 400 µmol/L CaCl₂ and collagenase (0.5 mg/mL). At 15-minute intervals, the supernatant was removed and filtered, and the cells were allowed to settle. The isolated myocytes were then suspended in standard culture media (Media 199; GIBCO-BRL, Grand Island, NY). A 2-mL aliquot of the isolated myocyte suspension was plated onto coverslips previously coated with a laminin/fibronectin matrix (Matrigel, Collaborative Research, Inc, Bedford, MA) and incubated at 37°C for 1 hour in the presence of 95% oxygen and 5% carbon dioxide. The myocytes were quiescent and did not exhibit spontaneous contractions in this preparation.

Myocyte contractile function was examined using video-assisted microscopy techniques described previously [11, 12]. Myocytes were imaged on an inverted microscope (Axiovert IM35; Zeiss Inc, Oberkochen, Germany) in a 2.5-mL tissue chamber with a thermoregulator to maintain the media temperature at 37°C. Myocytes were stimulated at 1 Hz and contractions were imaged using a charge-coupled device (GPCD60; Panasonic, Secaucus, NJ). Myocyte motion signals were input through an edge detector system (Crescent Electronics, Sandy, UT), converted into a voltage signal, digitized, and input to a computer (80286;ZBV2526, Zenith Data Systems, St. Joseph, MI) for subsequent analysis. Stimulated myocytes were allowed a 5-minute stabilization period after which contraction data for each myocyte was recorded from a minimum of 20 consecutive contractions. Parameters computed from the digitized contraction profiles included resting length (in micrometers), percent shortening (percent), peak velocity of shortening (micrometers per second), peak velocity of relengthening (micrometers per second), total contraction duration (milliseconds), time to peak contraction (milliseconds), and time to 50% relaxation (milliseconds). Contractile measurements were obtained only on those myocytes that maintained a long axis orientation perpendicular to the microscope objective throughout the contraction profile. After baseline contractile performance measurements, myocytes were exposed to the ß-adrenergic agonist isoproterenol (25 nmol/L, Sigma Chemical Co, St. Louis, MO) and contractile function measurements repeated. This concentration of isoproterenol was previously determined to be the effective dose for maximum response for this isolated myocyte preparation [11].

Experimental Design
PROJECT ONE: ISOLATED MYOCYTE PRECONDITIONING.
The first phase of this study was performed to develop a preconditioning protocol for this isolated myocyte system. To simulate ischemic conditions, isolated myocytes were exposed to a hypoxic environment. Specifically, isolated myocytes were suspended in a phosphate buffer solution (pH 7.4) and incubated in a hypoxia chamber (Radnoti Glass Technology, Inc, Monrovia, CA) that was continuously flushed with 100% nitrogen gas and kept at 37°C. During this hypoxic interval, the percent oxygen was continuously monitored using an oxygen selective microelectrode system (OM-4, Microelectrodes Inc, Londonderry, NH). The percent oxygen within the hypoxia chamber never exceeded 2% and the percent oxygen of the phosphate buffer solution, while in the chamber, was always less than 5%. In preliminary studies, the interval of hypoxia was varied from 10 to 60 minutes. After each specific period of hypoxia, the myocytes were resuspended in oxygenated normothermic cell culture media for 20 minutes. After this resuspension period, myocyte viability and contractile function was examined. Myocyte viability was determined as the ratio of the number of rod-shaped myocytes to total cells per high powered field (x100). In these studies, no observable effect on myocyte contractile performance was observed for periods of hypoxia shorter than 15 minutes. More prolonged periods of hypoxia (more than 30 minutes), followed by a 20-minute resuspension period, caused a significant reduction in the number of viable myocytes. Therefore, a 20-minute period of hypoxia followed by a 20-minute resuspension period in oxygenated normothermic cell culture media was selected as the preconditioning protocol for this isolated myocyte system. Contractile function and ß-adrenergic responsiveness was then examined in isolated myocytes maintained under normothermic conditions at baseline and after hypoxic preconditioning using the protocol described above. After this protocol, myocyte viability was again determined and the cells discarded.

PROJECT TWO: HYPOTHERMIC HYPERKALEMIC CARDIOPLEGIC ARREST AND REWARMING: EFFECTS OF PRECONDITIONING.
The objective of this project was to examine the effect of preconditioning on isolated myocyte contractile performance after hypothermic hyperkalemic cardioplegic arrest and rewarming. Myocytes were randomly assigned to one of three treatment groups: (1) normothermic control group: isolated myocytes were incubated in oxygenated normothermic cell culture media and then stored for 2 hours at 37°C in a 95% oxygen environment; (2) cardioplegia group: isolated myocytes were incubated with 4°C Ringers solution containing 20 mEq/L potassium and 5 mEq/L HCO₃^- then stored at 4°C for 2 hours and subsequently rewarmed in normothermic oxygenated (oxygen partial pressure, more than 300 mm Hg) cell culture media; (3) preconditioning and cardioplegia group: isolated myocytes were preconditioned using the protocol described in the previous section and subjected to the identical hypothermic, hyperkalemic cardioplegic arrest, and rewarming protocol as the cardioplegia group. After the specific experimental protocol, steady-state myocyte contractile function and ß-adrenergic responsiveness was examined.

Data Analysis
Indices of myocyte contractile function for the treatment groups shown in Table 1 were compared using two-way analysis of variancetab 1. If the analysis of variance detected significant differences with respect to treatment, mean separation was performed using Bonferroni bounds [13]. In a similar manner, for the ß-adrenergic response studies, myocyte contractile function at baseline and after ß-adrenergic stimulation was directly compared using analysis of variance. All statistical procedures were performed using the BMDP statistical software package (BMDP Statistical Software Inc, Los Angeles, CA). Results are presented as mean ± standard error of the mean. Values of p less than 0.05 were considered to be statistically significant.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

Hypothermic Hyperkalemic Cardioplegic Arrest and Rewarming: Effects of Preconditioning
The effects of hypothermic hyperkalemic cardioplegic arrest and rewarming with and without preconditioning on steady-state myocyte contractile function was next examined. Results from this portion of the experiment are summarized in Table 2 and representative contraction profiles for each group are shown in Figure 1. Myocyte contractile function was significantly decreased after cardioplegic arrest and rewarming when compared with normothermic controls. For example, the percent and velocity of myocyte shortening were decreased by 59% and 61%, respectively, after cardioplegia when compared with the normothermic control group. In marked contrast, preconditioning of isolated myocytes before the induction of cardioplegic arrest resulted in a significant improvement in myocyte contractile processes with subsequent rewarming. Specifically, in the preconditioning group the percent and velocity of shortening were increased by 77% and 73%, respectively, compared with the untreated myocytes after cardioplegic arrest and rewarming. Moreover, the total duration of contraction and time to 50% relaxation were not different in the preconditioning and cardioplegia group compared with controls. There was no change in the number of viable myocytes in either cardioplegia protocol when compared with normothermic control conditions.

View larger version (20K): [in this window] [in a new window]   Fig 1. . Representative contraction profiles for a control myocyte, a myocyte after cardioplegic arrest and rewarming, and a myocyte after preconditioning and cardioplegic arrest and rewarming. Steady-state contractile function was significantly lower after cardioplegic arrest and rewarming compared with control. Preconditioned myocyte contractile function after cardioplegic arrest and rewarming was significantly increased compared with the cardioplegia only group. Indices of myocyte contractile function are found in Table 2.

View larger version (54K): [in this window] [in a new window]   Fig 2. . Absolute change in myocyte velocity of shortening after ß-adrenergic receptor stimulation with isoproterenol (25 nmol/L). Preconditioning improved myocyte contractile performance after cardioplegic arrest and rewarming. More important, preconditioning normalized ß-adrenergic responsiveness after cardioplegic arrest and rewarming. (*p < 0.05 versus control; p < 0.05 versus cardioplegia.)

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

Ischemic or hypoxic preconditioning is a novel method of inducing endogenous cardioprotection from the ischemia/reperfusion injury that may occur after hypothermic cardioplegic arrest and rewarming. Ischemic preconditioning is a process by which a brief ischemic episode confers a state of myocardial protection against a subsequent more prolonged ischemic/reperfusion injury [1]. Brief hypoxic perfusion can substitute for ischemia as a preconditioning stimulus and the benefits of preconditioning with respect to the ischemic myocardium has been well described in animals and humans [1, 2, 14–17]. However, there have been few studies that have examined the effects of preconditioning with cardioplegic arrest and rewarming [8, 16, 17]. Illes and associates [8] observed that indices of left ventricular pump function were significantly increased in preconditioned hearts after hypothermic cardioplegic arrest and rewarming compared with controls. In these experiments using isolated whole heart preparations, preconditioning had a beneficial effect on left ventricular performance after cardioplegic arrest and rewarming. Previous investigations in this laboratory have demonstrated that a significant decrease in myocyte contractile function occurs after cardioplegic arrest and rewarming [9, 10]. However, the cellular basis for the observed effects of preconditioning in the setting of cardioplegic arrest and rewarming remained unclear. The findings of the current study demonstrated for the first time a beneficial effect of preconditioning on myocyte contractile function after cardioplegic arrest and rewarming. Therefore, the present study builds on past reports by demonstrating that the beneficial effects of preconditioning can be manifested in isolated myocytes after cardioplegic arrest and rewarming.

The initial goal of the present study was to develop a protocol for hypoxic preconditioning that would induce a measurable physiologic response in this isolated myocyte system without causing permanent injury or cell death. The current study demonstrated that a period of hypoxia followed by oxygenated reperfusion yielded viable myocytes that had diminished steady-state contractile performance. However, the preconditioning protocol did not significantly affect the ability of the myocyte to respond to an inotropic stimulus as evidenced by similar ß-adrenergic responsiveness in the preconditioning and control groups. The findings from this portion of the study demonstrated that preconditioning could be induced in this isolated myocyte system without causing a permanent effect on myocyte contractile processes. Past investigations have suggested that nonmyocyte cell populations play a contributory role in the mechanism of preconditioning [18, 19]. Specifically, both neutrophils and endothelial cells have been implicated as important components of the preconditioning phenomenon [18, 19]. The present study demonstrated that preconditioning could be induced in an isolated myocyte system in the absence of nonmyocyte influences. Therefore, the present study provides evidence to suggest that the preconditioning phenomenon may occur independent of nonmyocyte cell populations.

After cardioplegic arrest and rewarming, ß-adrenergic receptor agonists are commonly used to augment left ventricular pump performance. However, cardiopulmonary bypass with cardioplegic arrest and rewarming has been associated with alterations in the ß-adrenergic receptor system that may blunt the response to ß-adrenergic agonist therapy. For example, Schwinn and colleagues [20] demonstrated a decrease in ß-adrenergic receptor density in dogs after cardiopulmonary bypass. Therefore, the present study examined the direct effects of preconditioning on ß-adrenergic responsiveness after hypothermic cardioplegic arrest and rewarming. In untreated myocytes, ß-adrenergic responsiveness was significantly decreased after cardioplegic arrest and rewarming. In contrast, the present study demonstrated that preconditioning of myocytes preserved ß-adrenergic responsiveness after cardioplegic arrest and rewarming.

Although the preconditioning phenomenon remains an area of active investigation, potential contributory mechanisms for the preconditioning phenomenon include: (1) adenosine A₁-receptor stimulation; (2) ATP-sensitive potassium channels; and (3) altered energy metabolism of the myocyte. A study by Liu and colleagues [21] demonstrated that adenosine was equivalent to preconditioning in limiting infarct size in isolated rabbit hearts. Gross and Auchampach [22] have shown that blockade of the ATP-sensitive potassium channel prevented preconditioning in dogs. Furthermore, infusion of the potassium channel opener, aprikalim, produced a significant reduction in infarct size, similar to the effect of preconditioning [22]. Currently, there is evidence that the role of adenosine and the ATP-sensitive potassium channel are closely integrated in the preconditioning phenomenon [2]. Although the present study demonstrated that preconditioning could be manifested in an isolated myocyte system, whether any or all of the mechanisms outlined above is operable remains speculative. On the basis of the results from the present investigation in which preconditioning had direct and beneficial effects on myocyte contractile function after cardioplegic arrest and rewarming, further studies to determine the basic mechanisms for these effects seem warranted.

There are limitations to the present study that must be recognized. First, an isolated myocyte system was used in which the extracellular influence and effects of nonmyocyte cell populations were absent. Therefore, the protective effects of preconditioning in the intact myocardium, with respect to cardioplegic arrest and rewarming, could not be addressed directly in the present study. On the basis of the finding of the current study in which preconditioning could be induced in isolated myocytes, investigations using coculture experiments of endothelial cells and other nonmyocyte cell populations could be expanded in future studies. The preconditioning protocol used in the present study caused a decrease in myocyte contractile performance and suggests that stunning may have occurred. However, it is controversial whether stunning is a necessary component to induce the cardioprotective effects of preconditioning [23]. Recent studies have described preconditioning protocols that improved left ventricular pump performance after prolonged cardioplegic arrest and rewarming in the absence of stunning [8, 17]. Future experiments using this isolated myocyte system could examine whether stunning is a necessary component of the preconditioning phenomenon.

It is estimated that 10% of patients undergoing elective coronary bypass procedures have significant left ventricular dysfunction in the early period after cardioplegic arrest and rewarming requiring inotropic agent and/or intraaortic balloon pump support [24, 25]. Mortality in this group of patients may reach as high as of 15% [24]. The induction of potent endogenous protective mechanisms against myocardial injury is attractive and would have significant applications in cardiac operations. The present study suggests that preconditioning may provide direct and beneficial effects on myocyte contractile function, and therefore, may improve left ventricular pump performance after cardioplegic arrest and rewarming. Thus, preconditioning may have novel clinical applications in achieving myocardial protection during cardioplegic arrest for cardiac operations.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This study was supported by National Institutes of Health grant HL-45024, a Basic Research Grant from Pfizer Inc, and a Grant-in-Aid from the American Heart Association. Doctor Spinale is an Established Investigator of the American Heart Association.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Presented at the Forty-second Annual Meeting of the Southern Thoracic Surgical Association, San Antonio, TX, Nov 9–11, 1995.

Address reprint requests to Dr Spinale, Cardiothoracic Surgery, Medical University of South Carolina, Rm 418 CSB, 171 Ashley Ave, Charleston, SC 29425.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract 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): James L. Zellner Fred A. Crawford, Jr Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zellner, J. L. Articles by Spinale, F. G. Search for Related Content PubMed PubMed Citation Articles by Zellner, J. L. Articles by Spinale, F. G.