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Ann Thorac Surg 1998;65:1279-1283
© 1998 The Society of Thoracic Surgeons

Normothermic Versus Hypothermic Hyperkalemic Cardioplegia: Effects on Myocyte Contractility

^a Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, South Carolina, USA

Accepted for publication December 11, 1997.

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

	Abstract

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Background. This study was designed to determine the effects of prolonged hyperkalemic cardioplegic arrest under normothermic or hypothermic conditions with respect to left ventricular myocyte contractile performance and ß-adrenergic responsiveness.

Methods. Isolated left ventricular porcine myocytes were randomly assigned to one of three groups: (group 1) normothermic control, (group 2) hypothermic cardioplegic arrest, or (group 3) normothermic cardioplegic arrest. Myocyte contractility was evaluated by high-speed video microscopy at baseline and after ß-adrenergic stimulation with isoproterenol (25 nmol/L).

Results. Myocyte velocity of shortening was decreased after both hypothermic and normothermic cardioplegic arrest (68 ± 2 and 69 ± 2 µm/s, respectively) compared with normothermic control values (96 ± 2 µm/s; p < 0.05). This relative reduction in baseline contractile function was equivalent in both cardioplegia groups (p = 0.5356). With ß-adrenergic stimulation, myocyte velocity of shortening was 186 ± 4 µm/s in the hypothermic and 176 ± 3 µm/s in the normothermic cardioplegia groups (p = 0.0563). However, myocyte contractility with ß-adrenergic stimulation was reduced in both cardioplegia groups compared with normothermic controls (205 ± 4 µm/s; p < 0.05, respectively).

Conclusions. Hyperkalemic cardioplegic arrest under either normothermic or hypothermic conditions resulted in an equivalent reduction in baseline myocyte contractile function with reperfusion/rewarming. Hypothermic cardioplegic arrest may have provided mild protective effects on ß-adrenergic responsiveness. Nevertheless, these results suggest that an important contributory factor for diminished myocyte contractility after simulated cardioplegic arrest was prolonged exposure to a hyperkalemic environment.

	Introduction

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Hypothermia has been historically considered to be a fundamental requirement for both myocardial and systemic protection during cardioplegic arrest. However, hypothermia is reported to be associated with several potentially deleterious effects, which include activation of cold agglutinins, microvascular sludging, and cellular edema [1, 2]. Furthermore, after hypothermic, hyperkalemic cardioplegic arrest, a transient reduction in global left ventricular (LV) function can occur [3]. Several clinical studies have maintained normothermic conditions during cardioplegic arrest and demonstrated similar protective effects to that of hypothermic conditions [4–7]. For example, Lichtenstein and colleagues [4] reported that clinical outcomes were not different for those patients maintained at normothermic arrest and perfusion temperatures compared with those maintained under hypothermic conditions. The present study used an in vitro model of cardioplegic arrest [8, 9] to examine the effects of hypothermic versus normothermic cardioplegia on overall contractile function in the myocyte, the fundamental contractile unit of the heart.

	Material and methods

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Myocyte isolation and analysis
Yorkshire swine were the source of myocytes in the study. All animals were treated and cared for in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication no. 85-23, revised 1985). The pigs were anesthetized with 3% isoflurane in oxygen and a sternotomy was performed. The heart was then quickly extirpated and placed in cold (4°C) oxygenated Krebs solution. The LV free wall encompassing the left circumflex coronary artery (5 by 5 cm) was dissected free and the artery cannulated. This cannula was then used to perfuse the tissue with a modified Krebs solution containing collagenase (0.5 mg/mL, type II; 273 U/mg; Worthington Biochemical Corp, Freehold, NJ) for 20 minutes. The tissue was then minced and added to an oxygenated solution containing bovine serum albumin (2%, Sigma Chemical Co, St. Louis, MO), deoxyribonuclease (51 Kunitz units/mL, type IV, Sigma), 400 µmol/L CaCl₂, and collagenase (0.5 mg/mL) and gently agitated. After 15 minutes, the supernatant was removed, filtered and the cells allowed to settle. The liberated myocytes were resuspended in standard culture medium (2 mmol/L Ca²⁺; Medium M199, Gibco Laboratories, Grand Island, NY) and then plated onto coverslips previously coated with a laminin/fibronectin matrix (Matrigel, Collaborative Research, Bedford, MA).

Isolated myocytes were placed into a thermostatically controlled chamber (2.5 mL, 37°C) filled with oxygenated cell medium. Imaging of the myocytes was performed on an inverted microscope (World Precision Instruments, PIM, Sarasota, FL). Myocyte contractions were elicited by field stimulation of the chamber at 1 Hz (S11; Grass Instruments, Quincy, MA) with a 5-millisecond pulse width. Contraction data for each myocyte was recorded from a minimum of 20 consecutive contractions using a charge-coupled device (GPCD60, Panasonic, Secaucus, NJ), digitized, and analyzed as previously described [8]. Parameters computed from the digitized contraction profiles included percent shortening, velocity of shortening (micrometers per second), velocity of relengthening (micrometers per second), total contraction duration (milliseconds), and time to peak contraction (milliseconds).

Experimental protocol
Myocytes were randomly assigned to one of three groups: (1) normothermic control, (2) hypothermic, hyperkalemic cardioplegic arrest, or (3) normothermic, hyperkalemic cardioplegic arrest. The simulated hypothermic cardioplegic arrest protocol that has been previously described [9] was performed by placing myocytes in an oxygenated, conventional hyperkalemic crystalloid cardioplegic solution (lactated Ringers’, 24 mEq/L K⁺, 30 mEq/L HCO₃^-) for 2 hours at 4°C. The pH, partial pressure of carbon dioxide, and partial pressure of oxygen were measured at the beginning and end of each experimental protocol and were not significantly different for any of the treatment groups (pH, 7.45 to 7.50; partial pressure of carbon dioxide, 30 to 35 mm Hg; partial pressure of oxygen, 150 to 200 mm Hg; p > 0.45). The osmolarity of the crystalloid cardioplegia solution was 280 mOsm. The volume of the experimental chambers for the cardioplegic arrest studies was 2.5 mL. The myocytes were then placed in 37°C cell media for a 5-minute rewarming period before study. Normothermic cardioplegic arrest was simulated by placing myocytes in an identical cardioplegic solution for 2 hours at 37°C. Normothermic control myocytes not undergoing cardioplegic arrest were maintained in 37°C cell media and used for comparison. Myocyte contractile function was measured at baseline and then in the presence of isoproterenol (25 nmol/L, Sigma), which in previous dose response studies has been demonstrated to elicit maximal effects on myocyte contractility under control conditions [10].

Data analysis
Isolated myocytes were randomly assigned to each subplot of the experimental design and the effects on myocyte contractile function with respect to each treatment factor and the interaction between treatments was determined using an analysis of variance based on a 2 by 2 factorial design. The response variables to be measured included steady-state myocyte contractile function and contractile response after activation of the ß-adrenergic transduction pathway. In light of the fact that the randomization scheme resulted in unequal sample sizes in each treatment plot, then the sum of squares obtained from the analysis of variance was adjusted for the actual degrees of freedom in each treatment plot. Repeated measurements were not performed with respect to the main treatment effects. If the analysis of variance revealed that significant differences existed with respect to the main treatment effects, then pairwise tests of individual group means were compared using Bonferroni’s probabilities. All statistical analysis was performed using standard statistical software programs (BMDP Statistical Software, Inc, Los Angeles, CA). Results are presented as mean ± standard error of the mean. Probability values less than 0.05 were considered to be statistically significant.

	Results

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Indices of steady-state myocyte contractile function and response to ß-adrenergic stimulation with isoproterenol under normothermic conditions, after hypothermic, hyperkalemic cardioplegic arrest, and after normothermic, hyperkalemic cardioplegic arrest are summarized in Table 1. The analysis of variance revealed a significant difference existed between the main treatment effect of hyperkalemic cardioplegic arrest, irrespective of temperature (F = 546; p < 0.0001). Myocyte baseline contractile function was decreased in both the hypothermic and normothermic cardioplegia groups when compared with normothermic control values. For example, myocyte velocity of shortening was reduced by 29% and 27% in the hypothermic and normothermic cardioplegia group, respectively, compared with normothermic control values. The reduction in contractility was equivalent in both the hypothermic and normothermic cardioplegia groups (p = 0.5356).

	Comment

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Hypothermia has been historically considered to be a fundamental requirement for both myocardial and systemic protection during cardioplegic arrest. However, hypothermia has been demonstrated to have several potentially detrimental effects at the cellular level such as abnormalities in tissue volume regulatory mechanisms [1, 2]. Furthermore, after hypothermic, hyperkalemic cardioplegic arrest, a transient reduction in global LV function can occur, which is associated with a decrease in ß-adrenergic responsiveness [3]. Recent clinical reports have suggested that infusion of a continuous normothermic cardioplegia solution provides equivalent myocardial protection to that of hypothermic conditions [4–7]. However, whether and to what degree hypothermia contributes to LV dysfunction after prolonged hyperkalemic arrest remains poorly defined. Accordingly, the present study examined myocyte contractility after prolonged hyperkalemic cardioplegic arrest under normothermic or hypothermic conditions with respect to LV myocyte contractile performance. The important findings of the present study were twofold. First, baseline myocyte contractile function was decreased to an equivalent degree after normothermic or hypothermic, hyperkalemic cardioplegic arrest compared with normothermic control values. Second, both normothermic and hypothermic cardioplegic arrest were associated with a reduced ß-adrenergic response compared with the normothermic control response. After hypothermic cardioplegic arrest, specific indices of contractile function, namely velocity of shortening and time to peak contraction, revealed a trend toward a greater response in the hypothermic group compared with the normothermic cardioplegia group. Nevertheless, the findings of the present study suggest that an important contributory factor for diminished myocyte contractility with simulated cardioplegic arrest was prolonged exposure to a hyperkalemic environment.

Reports comparing normothermic and hypothermic cardioplegic arrest in both experimental models and clinical studies have suggested that potential differences exist between the two techniques with regard to LV myocardial metabolism [4–7, 10–15]. However, these reported dissimilarities have not translated into differences in overall clinical outcomes [4–7]. For example, a large, randomized, multicenter clinical trial using either normothermic or hypothermic cardioplegic arrest failed to demonstrate any differences in overall clinical outcomes between the two techniques [5]. However, secondary end points such as release of creatine kinase MB were lower in the normothermic cardioplegic group when compared with the hypothermic cardioplegic group [5]. Furthermore, a previous clinical study demonstrated that delivery of intermittent antegrade warm blood cardioplegia was associated with a reduction in the release of troponin T and creatine kinase MB when compared with hypothermic blood cardioplegic delivery [15]. The results of the present study suggest that a prolonged hyperkalemic environment contributes to myocyte contractile dysfunction in the early reperfusion and rewarming period. However, there are other factors that may contribute to the contractile dysfunction that may occur after cardioplegic arrest in vivo. For example, the type and duration of cardioplegic arrest, as well as the mode of reperfusion and rewarming may all affect myocardial contractile performance in the immediate postcardioplegia setting. Furthermore, the LV myocardial dysfunction that may exist before the induction of cardioplegic arrest will likely be translated into poor contractile function after cardioplegic arrest [16]. These additional factors would be appropriate to include in future experimental designs using this isolated myocyte system of simulated cardioplegic arrest.

It has been reported that hypothermic, hyperkalemic cardioplegic arrest is associated with a reduction in ß-adrenergic responsiveness in experimental models as well as in the clinical setting [10, 11, 17, 18]. The findings of the present study are consistent with these previous reports in that myocyte ß-adrenergic responsiveness was reduced after cardioplegic arrest. However, myocytes maintained under hypothermic conditions demonstrated a trend toward improved preservation of ß-adrenergic responsiveness compared with those maintained under normothermic conditions. One possible explanation for this observation is that by further reducing the intracellular metabolic processes that are ongoing during the cardioplegic arrest period, hypothermia imparts additional protective effects with respect to ß-adrenergic activity compared with cardioplegic arrest alone. Qiu and colleagues [13], in a blood-perfused rat heart model of cardioplegic arrest demonstrated a greater reduction in tissue adenosine triphosphate levels in normothermic compared with hypothermic cardioplegia groups. Yau and associates [7] demonstrated that in patients undergoing normothermic cardioplegia, there was a greater decrease in total adenine nucleotides compared with a group undergoing hypothermic cardioplegic arrest. These high-energy nucleotides are used by adenylate cyclase in the production of cyclic adenosine monophosphate, a key step in the ß-adrenergic signaling pathway. Decreased high energy substrates after normothermic cardioplegic arrest may account for the differences in myocyte velocity of shortening with ß-adrenergic stimulation. However, this issue remains speculative and warrants further study.

The results of the present study add to past clinical and experimental evidence that a contributory factor in contractile dysfunction after cardioplegic arrest is prolonged exposure to the hyperkalemic environment of conventional cardioplegia solutions [19, 20]. Potassium-induced (hyperkalemic) cardioplegic arrest results in sustained depolarization with a subsequent eflux of sodium and influx of calcium ions. This prolonged depolarization may cause what has been termed calcium loading and has been speculated to be a contributory mechanism for the contractile dysfunction observed after hyperkalemic cardioplegic arrest [19, 20]. Cohen and coworkers [21], using an isolated rabbit heart model, demonstrated that hyperpolarized arrest, achieved through activation of adenosine triphosphate-sensitive potassium channels, resulted in improved functional recovery compared with hyperkalemic-induced depolarized arrest. Together with evidence from previous reports, the findings of the present study suggest that prolonged exposure to a hyperkalemic environment is likely an important contributory factor in the reduction of contractile performance that can occur after conventional hyperkalemic cardioplegic arrest.

Although the isolated myocyte system used in the present study provided a means to examine the specific differences encountered in myocyte contractile function comparing normothermic versus hypothermic cardioplegia, there are limitations to this in vitro system. For example, this myocyte model of cardioplegic arrest excludes environmental factors present in vivo such as changes in systemic loading conditions and neurohormonal influences. The translation of results obtained from this in vitro study to the clinical condition can be problematic. In a large clinical trial in which warm cardioplegia was compared with hypothermic cardioplegia, a reduction in low-output syndrome was observed in the warm cardioplegia group [5]. In the present study, LV myocyte contractility was measured at 5 minutes after reperfusion and rewarming. This laboratory has demonstrated previously that by 30 minutes after reperfusion, myocyte contractility returns to approximately normothermic, control values [22]. Thus, whether and to what degree differences may exist between normothermic and hypothermic cardioplegia with respect to recovery patterns in this model of simulated cardioplegic arrest remains unclear. Nevertheless, the results of the present study demonstrate that hyperkalemic cardioplegic arrest under either normothermic or hypothermic conditions resulted in an equivalent reduction of baseline myocyte contractile function. This finding suggests that an important contributory mechanism for transient LV dysfunction after cardioplegic arrest may be prolonged exposure to a hyperkalemic environment.

	Acknowledgments

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This study was supported by National Institutes of Health grant HL-45024 and HL-56603, a grant-in-aid from the South Carolina Heart Association, a grant-in-aid from the American Heart Association, and the Medical University of South Carolina’s Institutional Research Funds of 1996–97. Francis G. Spinale, MD, PhD, is an Established Investigator of the American Heart Association.

	References

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