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J Thorac Cardiovasc Surg 1998;115:190-199
© 1998 Mosby, Inc.

CARDIOPULMONARY SUPPORT AND PHYSIOLOGY

Downstream defects in ß-adrenergic signaling and relation to myocyte contractility after cardioplegic arrest

From the Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, S.C.

Address for reprints: Francis G. Spinale, MD, PhD, CardiothoracicSurgery, Room 418 CSB, Medical University of South Carolina, Charleston, SC29425.

Abstract

Objective: Transientleft ventricular dysfunction can occur after hypothermic, hyperkalemic cardioplegicarrest and is associated with decreased ß-adrenergic receptor responsiveness.Occupancy of the ß-adrenergic receptor activates adenylate cyclase, whichphosphorylates the L-type Ca²⁺ channel–enhancing myocytecontractility. The goal of this study was to identify potential mechanismsthat contribute to the defects in the ß-adrenergic receptor signalingcascade after cardioplegic arrest.
Methods:Isolated left ventricular porcine myocytes were assigned to one of two treatmentgroups: (1) cardioplegic arrest (24 mEq/L K⁺, 4° C x2 hours, then 5 minutes in 37° C cell media; n =130) or (2) normothermic control (cell media, 37° C x 2 hours; n = 222). Myocyte contractility was assessed atbaseline and after either ß-adrenergic receptor occupancy (25 nmol/Lisoproterenol [INN: isoprenaline]), activation of adenylate cyclase (0.5 µmolforskolin), or direct activation of the L-type Ca²⁺-channel (10nmol/L or 100 nmol/L (–)BayK 8644).
Results:Myocyte velocity of shortening (µm/sec) was increased with ß-adrenergicreceptor occupancy or direct adenylate cyclase stimulation compared with baselinein the normothermic group (187.3 ± 6.9, 181.7 ± 10.2,and 73.9 ± 2.9, respectively; p <0.0001) and after cardioplegic arrest (128.6 ± 8.9, 124.3 ±9.4, and 46.1 ± 2.6, respectively; p <0.0001). However, the response after cardioplegic arrest was significantlyreduced compared with normothermic values under all conditions (p = 0.012). Direct activation of the L-type Ca²-channel, which eliminates ß-adrenergic receptor–dependentevents, increased myocyte contractility in the normothermic group (161.90 ±12.0, p < 0.0001) and after cardioplegicarrest (92.78  ± 6.8, p <0.0001), but the positive inotropic response appeared reduced compared withnormothermic control values (p = 0.003).
Conclusion: These findings suggest that contributorymechanisms for the reduced ß-adrenergic receptor–mediated responseafter hypothermic, hyperkalemic cardioplegic arrest lie downstream from thesespecific components of the transduction pathway and likely include defectsin Ca²⁺ homeostasis, myofilament Ca²⁺ sensitivity, orboth.

Hypothermic, hyperkalemic cardioplegic arrest is a common means of providinga quiescent heart during cardiac surgery. However, after reperfusion and rewarming,transient left ventricular (LV) dysfunction can occur. ^1,2 ß-Adrenergicagonists are commonly administered to improve LV pump function in the earlyreperfusion-rewarming period. Stimulation of the ß-adrenergic receptor(ß-AR) activates adenylate cyclase with resultant production of cyclicadenosine monophosphate (cAMP). One of the intracellular events after cAMPproduction is phosphorylation of the L-type Ca²⁺ channel. ^3,4Phosphorylation of this channel augments Ca²⁺current during theexcitation-contraction process, thereby enhancing myocyte contractility. However,diminished ß-adrenergic responsiveness after cardioplegic arrest hasbeen previously described. ^5–7 Schwinnand colleagues ⁶ attributedthis decrease to a desensitization of the ß-AR with uncoupling of the ß-ARfrom the G-protein complex after cardioplegic arrest. However, specific intracellulardefects that contribute to this blunted ß-adrenergic response after cardioplegicarrest and rewarming remain unclear.

Accordingly, the overall goal of this study was to examine the specificdownstream intracellular defect(s) in the ß-adrenergic signaling cascadeafter cardioplegic arrest. Well-documented changes in systemic loading conditionsand neurohormonal system activity occur after cardioplegic arrest and separationfrom cardiopulmonary bypass. ^1,2,5–9 For example, a twofold increase in plasmanorepinephrine has been described to occur in the early period after cardiopulmonarybypass. ⁹ Thus defining therelationship between changes in contractile performance and specific defectsin ß-AR transduction processes in vivo can be problematic. This laboratoryhas previously described an isolated LV myocyte system that simulates cardioplegicarrest and rewarming. ¹⁰ Byuse of this in vitro system, past studies have demonstrated that the contractileresponse to ß-AR stimulation is reduced after cardioplegic arrest, consistentwith clinical observations. ^5–7,10 Accordingly, this study used this myocyte model of cardioplegicarrest to examine the relationship between activation of specific componentsof the ß-AR signaling pathway and contractile performance.

Methods

The overall goal of this study was to examine the specific intracellulardefect(s) in the ß-adrenergic signaling pathway after cardioplegic arrest.This was accomplished through pharmacologic dissection of the ß-adrenergictransduction pathway. Specifically, the contractile response of isolated myocyteswas measured in the presence of either isoproterenol, a ß-AR agonist,the naturally occurring diterpene, forskolin, shown to directly stimulateadenylate cyclase, ^11,12 or the L-type Ca²⁺ channel agonist,(–)BayK 8644 ^13,14 under normothermic conditions and after cardioplegicarrest.

Myocyte isolation.
Six age- and weight-matched Yorkshire swine were the source of myocytesin the study. All animals were treated and cared for in accordance with theNational Institutes of Health "Guide for the Care and Use of LaboratoryAnimals" (National Research Council, Washington, D.C., 1996). On theday of the study, the individual animal was anesthetized with a combinationof diazepam (Valium) (10 mg) and 2.5% isoflurane in oxygen, and a sternotomywas performed. The heart was then quickly excised and placed in cold (4°C) oxygenated Krebs solution. The LV free wall encompassing the left circumflexcoronary artery (5 x 5 cm) was dissected free and the artery wascannulated. This was then used to perfuse the tissue with a modified Krebssolution containing collagenase (0.5 mg/ml, type II; 273 U/mg; WorthingtonBiochemical Corp, Freehold, N.J.) for 20 minutes. The tissue was then minced,added to an oxygenated solution containing bovine serum albumin (2%, SigmaChemical Co., St. Louis, Mo.), deoxyribonuclease (51 Kunitz units/ml, typeIV, Sigma), 400 µmol CaCl₂, and collagenase (0.5 mg/ml) andgently agitated. After 15 minutes, the supernatant was removed, filtered,and the cells allowed to settle. The liberated myocytes were then resuspendedin standard culture medium (2 mmol/L Ca²⁺; medium M199, Gibco Laboratories,Grand Island, N.Y.) and plated on coverslips previously coated with a laminin/fibronectinmatrix (Matrigel, Collaborative Research, Bedford, Mass.).

Isolated myocyte function and analysis.
Isolated myocytes were placed into a thermostatically controlled chamberat 37° C and imaged with an inverted microscope (World Precision Instruments,Sarasota, Fla.). Myocyte contractions were elicited by field stimulation ofthe chamber at 1 Hz (S11, Grass Instruments, Quincy, Mass.) with a 5 msecpulse width. Contraction data for each myocyte were recorded from a minimumof 20 consecutive contractions. Myocyte contractions were imaged by use ofa charge-coupled device, digitized, and input into a computer (80486; ZeosInternational, Minneapolis, Minn.) for subsequent analysis, as previouslydescribed. ^10,15 Parameters computed from the digitized contraction profiles includedpercent shortening (%), velocity of shortening (µm/sec), velocity ofrelengthening (µm/sec), total contraction duration (msec), and timeto peak contraction (msec). All parameters were calculated for each contraction,and the results averaged for all contractions were observed. In past reportsit has been demonstrated that changes in steady state isolated myocyte contractilefunction directly reflect changes in the intrinsic capacity of the LV myocardiumto function against a given load. ¹⁶ By means of this isolated myocyte system, this laboratory has demonstratedthat myocytes respond in a predictable manner to incrementally increased externalload in both normal and failing preparations. ¹⁷ Thus, although the isolated myocyte function studies described inthis study were performed under equivalent unloaded conditions, it is likelythat these findings can be translated to intrinsic myocardial contractilecapacity.

L-type Ca²⁺ channel activation with (–)BayK 8644-dose–dependenteffects on myocyte function.
Although the effects of the L-type Ca²⁺ channel agonist (–)BayK8644 (Research Biochemicals International, Natick, Mass.) with respect toaugmentation of myocyte Ca²⁺ current have been well described, ^13,14 the concentration-dependent effects on myocyte contractile performanceremained undefined. Thus initial dose-response studies were performed to determineappropriate concentrations of (–)BayK 8644 to be used in the subsequentcardioplegic arrest studies. After baseline measurements of contractile function,measurements were repeated in the presence of (–)BayK 8644 concentrationsranging from 0.1 nmol/L to 1 µmol/L. To avoid L-type Ca²⁺channelrundown, myocytes were exposed to only one concentration of (–)BayK8644, and a minimum of 15 myocytes were examined at each concentration. Thedose-response relation for increasing concentrations of (–)BayK 8644with respect to myocyte shortening velocity is presented in Fig. 1. These dose-response data were subjectedto log-linear regression analysis to determine the effective concentrationof (–) BayK 8644, which elicited 50% of maximal response (EC₅₀) and a maximal or 100% response (EC₁₀₀). These computationsrevealed an EC₅₀ and EC₁₀₀ for (–)BayK 8644 withrespect to myocyte velocity of shortening of 10 nmol/L and 100 nmol/L, respectively.Accordingly, these concentrations of the L-type Ca²⁺ channel agonistwere used in the experimental protocols described in the following section.

View larger version (12K): [in this window] [in a new window]   Figure 1. The concentration-dependenteffects of the L-type Ca+2 channel agonist, (–)BayK 8644,were determined with respect to changes in isolated myocyte velocity of shortening.After steady state measurements, myocyte function was measured in the presenceof (–)BayK 8644 using concentrations of 0.1 nmol/L to 1 µmol/L.At concentrations of 1 to 100 nmol/L (–)BayK 8644, a concentration-dependenteffect was observed and subjected to regression analysis. The computed effectiveconcentrations of (–)BayK 8644 that yielded a 50% maximal response (EC50) and a maximal or 100% response (EC100) were 10 nmol/Land 100 nmol/L, respectively. The effects of these concentrations of (–)BayK8644 on myocyte contractile performance under normothermic conditions andafter simulated cardioplegic arrest are shown in Table I.

Data analysis.
In this study, two main treatment effects were used: cardioplegic arrestand drug treatment. Accordingly, the initial data analysis was performed usinga two-way analysis of variance (ANOVA) to identify the main treatment effectsand any potential interaction between the main effects with respect to myocytecontractility. A multiway ANOVA was then carried out to identify the specifictreatment interventions (i.e., ß-adrenergic occupancy, adenylate cyclasestimulation) on contractile performance under both normothermic control conditionsand after simulated cardioplegic arrest. If the multiway ANOVA revealed significantdifferences, pairwise tests of individual group means were compared usingBonferroni's probabilities. All statistical analyses were performed usingstandard statistical software programs (BMDP Statistical Software, Inc., LosAngeles, Calif.). Results are presented as mean ± standard errorof the mean. Values p < 0.05 wereconsidered statistically significant.

Results

Indices of steady state myocyte contractile function under normothermiccontrol conditions and after ß-AR occupancy, adenylate cyclase stimulation,or L-type Ca²⁺ channel activation are summarized in Table I. Myocyte contractilitywas increased after ß-AR occupancy with isoproterenol and after directadenylate cyclase stimulation with forskolin. For example, myocyte velocityof shortening was increased by 153% after ß-AR occupancy and by 146%after adenylate cyclase stimulation. Direct activation of the L-type Ca²⁺ channel with (–)BayK 8644 increased myocyte contractility.For example, velocity of shortening was increased by 62% with 10 nmol/L and120% with 100 nmol/L, reflecting the EC₅₀ and EC₁₀₀concentrations, respectively. In myocytes maintained in hypothermic cell culturemedia for 2 hours (n = 59), baseline contractile function was unchangedafter rewarming compared with normothermic control values. For example, myocytevelocity of shortening was 79.5 ± 4.1 µm/sec in the hypothermiccontrol group compared with 81.8 ± 2.1 µm/sec in the normothermiccontrol group (p = 0.6237). Therefore it did not appear that hypothermicconditions significantly affected myocyte contractility. These results areconsistent with a past report from this laboratory, which demonstrated thatprolonged hypothermia alone had minimal effects on myocyte contractile function. ¹⁰

Cardioplegic arrest and rewarming group.
Steady state myocyte contractile function after cardioplegic arrestand rewarming and after ß-AR occupancy, adenylate cyclase stimulation,or L-type Ca²⁺ channel activation is summarized in Table I. Baseline myocyte contractile function was reduced after cardioplegicarrest. For example, myocyte velocity of shortening was reduced by 38% comparedwith normothermic control values. The two-way ANOVA revealed that cardioplegicarrest was a significant main effect with respect to contractile function.For example, the F values for myocyte percent shortening and velocity of shorteningwere significant for the main treatment effect of cardioplegia (F = 132.57, F =108.35, p < 0.0001, respectively).The two-way ANOVA also demonstrated that the drug treatment interventionsrepresented a main effect with respect to contractile performance. For example,the F values for myocyte percent shortening and velocity of shortening weresignificant (F = 118.93, F = 108.88, p < 0.0001,respectively). More important, this analysis identified a significant interactionbetween these two main effects; specifically, Fvalues of 2.87 and 3.82 were obtained for myocyte percent shortening and velocityof shortening (p = 0.0225 and p = 0.0044, respectively). In light of the significantinteraction between the main treatment effects, individual pairwise comparisonsfor indices of myocyte contractility were performed using Bonferroni confidenceintervals constructed from the mean square error obtained from a multiwayANOVA. The results from these pairwise contrasts and the individual p values obtained are shown in Table I.Myocyte contractility increased after ß-AR occupancy or adenylate cyclasestimulation, but these effects appeared reduced compared with normothermiccontrol values. For example, myocyte velocity of shortening was reduced bymore than 30% after either ß-AR occupancy or adenylate cyclase stimulationcompared with respective normothermic control values. Direct activation ofthe L-type Ca²⁺ channel increased myocyte contractility after cardioplegicarrest but was reduced when compared with the normothermic control values.For example, myocyte velocity of shortening was reduced by 37% and 43% comparedwith the normothermic control values at the 10 nmol/L and 100 nmol/L (–)BayK8644 concentrations, respectively.

In light of the significant differences in baseline contractile functionbetween the normothermic control myocytes and myocytes after cardioplegicarrest and rewarming, the effect of ß-adrenergic occupancy, adenylatecyclase stimulation, or direct L-type Ca²⁺ channel activation onmyocyte contractile function may be difficult to interpret. Therefore theabsolute change from baseline for velocity of shortening was computed in eachmyocyte after each treatment (Fig. 2). Cardioplegic arrest and rewarming wereassociated with a significant decrease in absolute change from baseline inmyocyte velocity of shortening after ß-adrenergic occupancy, adenylatecyclase stimulation, or direct L-type Ca²⁺ channel activation.Another approach for expressing the relative contractile response to the individualdrug interventions would be as a percent change from steady state basal values.By means of this strategy, myocyte velocity of shortening was increased by231% and 304% with ß-AR occupancy, by 110% and 109% with adenylate cyclasestimulation, and by 90% and 85% with Ca²⁺ channel activation (100nmol/L BayK) in the normothermic control and hypothermic cardioplegia groups,respectively. Both analyses revealed that the myocytes retained the capacityto respond to an inotropic stimulus after hypothermic cardioplegic arrest.Nevertheless, the absolute value for indices of contractility remained significantlyblunted in myocytes after cardioplegic arrest compared with controls afterany of the three drug interventions.

View larger version (32K): [in this window] [in a new window]   Figure 2. Myocyte contractile functionin both the normothermic state and after cardioplegic arrest was measuredby absolute change in myocyte shortening velocity after ß-AR (B-AR) occupancywith 25 nmol/L isoproterenol, stimulation of adenylate cyclase with 0.5 µmol/Lforskolin (AC), or direct activation of theL-type Ca2+ channel with both 10 nmol/L and 100 nmol/L (–)BayK8644. The absolute change in shortening velocity after ß-receptor occupancy,stimulation of adenylate cyclase, or direct activation of the L-type Ca2+ channel after cardioplegic arrest was reduced compared with normothermicvalues (p = 0.012, p = 0.005, p = 0.0006,respectively). The response to 100 nmol/L (–)BayK 8644 was greater thanthat to 10 nmol/L (–)BayK 8644 in the normothermic group (p = 0.02) and after cardioplegic arrest (p = 0.0015) as previously predicted by the dose-responsestudy (*p < 0.05 vs normothermic control, #p < 0.05 vs 10 mol/L (–)BayK8644; actual p values provided in text).

Transient LV dysfunction can occur after hypothermic, hyperkalemic cardioplegicarrest and is associated with decreased ß-adrenergic responsiveness.The overall goal of this study was to define the contributory intracellularmechanism(s) potentially responsible for the defects in the ß-adrenergicsignaling cascade that occur after cardioplegic arrest. Under normal conditions,occupancy of the ß-adrenergic receptor stimulates adenylate cyclase withsubsequent activation of the L-type Ca²⁺ channel. Activation ofthis channel increases Ca²⁺ current thereby enhancing myocyte contractility.Accordingly, this study examined myocyte contractile performance after ß-ARoccupancy, direct stimulation of adenylate cyclase, or direct activation ofthe L-type Ca²⁺ channel under normothermic conditions and aftersimulated cardioplegic arrest. The significant findings of this study weretwofold. First, ß-AR occupancy or direct adenylate cyclase stimulationincreased myocyte contractility; but the effect of either was reduced aftersimulated cardioplegic arrest. Second, direct activation of the L-type Ca²⁺channel, which eliminates ß-AR dependent events, increased myocytecontractile performance under both conditions. However, the myocyte inotropicresponse to L-type Ca²⁺ channel activation was reduced after cardioplegicarrest. These findings suggest that contributory mechanisms for the reduced ß-adrenergicresponse after cardioplegic arrest are downstream from this transduction pathway.

A schematic representation of the ß-adrenergic signaling cascadeis shown in Fig. 3, with emphasis on the rationale for the experimental designused in this study. Under normal conditions, occupancy ofthe ß-AR initiates a signaling cascade involving a G-protein complex,through which adenylate cyclase is stimulated to increase production of cAMP.Increased intracellular cAMP activates protein kinase A (PKA), which mediatesthree important intracellular events. First, PKA phosphorylates the L-typeCa²⁺ channel, augmenting Ca²⁺ flux across the sarcolemmaand thereby enhancing the release of intracellular Ca²⁺ storesfrom the sarcoplasmic reticulum (SR). ^4,19,20 Second, PKA phosphorylates the SR-bound regulatory protein phospholamban,which augments intracellular Ca²⁺ resequestration by the SR. ^4,19,20 Third, PKA phosphorylates troponin-I, whichdirectly influences myofilament Ca²⁺ sensitivity and thereforecross-bridge formation. ^4,21 In this study, myocyte contractile response to ß-adrenergicstimulation was reduced after cardioplegic arrest. Additionally, the inotropicresponse failed to return to normal levels with either direct stimulationof adenylate cyclase or direct activation of the L-type Ca²⁺ channel.These findings suggest that the decreased myocyte inotropic response to ß-ARstimulation after cardioplegic arrest may be the result of three intracellulardefects. First, diminished PKA activation may occur with subsequent defectsin phosphorylation of the L-type Ca²⁺ channel, phospholamban, andtroponin-I. Second, the intracellular homeostatic mechanisms responsible forrelease and resequestration of SR Ca²⁺ stores may be altered. Third,a decrease in the sensitivity of the contractile apparatus to Ca²⁺witha subsequent reduction in cross-bridge formation may occur. Using this isolatedmyocyte model of cardioplegic arrest, future studies that more carefully focuson these potential intracellular defects with emphasis on PKA-mediated eventsare warranted.

View larger version (32K): [in this window] [in a new window]   Figure 3. A schematic representationof the ß-adrenergic signaling cascade, with emphasis on the rationalefor the experimental design used in this study. Under normal conditions, occupancyof the ß-AR initiates a signaling cascade involving a G-protein complex,through which adenylate cyclase is stimulated to increase production of cAMP.Increased intracellular cAMP activates protein kinase A, which mediates threeimportant intracellular events: (A) phosphorylationof the L-type Ca2+ channel, which augments Ca2+ fluxacross the sarcolemma, enhancing the release of intracellular Ca2+stores from the SR; (B) phosphorylation oftroponin-I, which directly influences myofilament Ca2+ sensitivityand thereby cross-bridge formation; and (C)phosphorylation of the SR-bound regulatory protein phospholamban, which enhancesintracellular Ca2+ resequestration by the SR. In this study the ß-adrenergicsignaling cascade was examined by pharmacologic dissection at three specificpoints. First, the ß-AR was stimulated with isoproterenol. Second, adenylatecyclase was stimulated with forskolin. Third, the L-type Ca2+ channelwas activated with (–)BayK 8644. After simulated cardioplegic arrest,significant blunting of the inotropic response to all three treatments comparedwith normothermic control values was demonstrated. These results suggest thatdefects in intracellular Ca2+ homeostasis, myofilament Ca2+ sensitivity, or both contribute to the diminished inotropic responseafter cardioplegic arrest.

Forskolin, a naturally occurring diterpene, has been shown to directlystimulate adenylate cyclase, which increases intracellular cAMP, ^11,12 effectivelybypassing the ß-AR and G-protein complex of the ß-adrenergic signalingcascade. Several past studies have demonstrated that forskolin produces apositive inotropic effect in myocardial preparations. ^11,12 For example,Bristow and colleagues ¹¹ demonstratedthat forskolin increased adenylate cyclase activity and produced a potentpositive inotropic response in human right ventricular papillary muscle. Hearseand colleagues, ¹² using anisolated rat heart model of cardioplegic arrest, demonstrated that while contractilefunction was improved and adenylate cyclase activity was increased in thepresence of forskolin, contractile performance remained lower than normalcontrol values. Consistent with these past observations, this study foundthat the inotropic response to forskolin was blunted after cardioplegic arrest.Other past studies have demonstrated that phosphodiesterase inhibitors, whichprevent the degradation of cAMP, have a positive inotropic effect after cardiopulmonarybypass with cardioplegic arrest. ²³ However, this laboratory has previously demonstrated that althoughphosphodiesterase inhibition increased isolated myocyte contractility aftercardioplegic arrest, the inotropic response was blunted compared with normothermiccontrol values. ²⁴ Taken togetherthe findings of this study and evidence from past reports suggest that thedefect(s) resulting in reduced responsiveness to ß-adrenergic stimulationafter cardioplegic arrest occur downstream from cAMP production in the signalingcascade. Because one of the important events after adenylate cyclase activationincludes phosphorylation of the L-type Ca²⁺ channel, this studynext examined the effect of direct activation of this channel on myocyte contractilefunction under normothermic conditions and after simulated cardioplegic arrest.

The dihydropyridine derivative, (–)BayK 8644, binds to the alphasubunit of the L-type Ca²⁺ channel, induces prolonged channel opening,and thereby increases Ca²⁺ flux through the channel. ^4,14 Past studieshave demonstrated that (–)BayK 8644 increased contractile function inmyocardial preparations. ^13,14 However, to our knowledge, this study is thefirst to examine the direct effects of (–)BayK 8644 on the contractileperformance of the isolated myocyte under normothermic conditions and aftersimulated cardioplegic arrest. In this study, (–)BayK 8644 eliciteda dose-dependent positive inotropic response consistent with specific activationof the L-type Ca²⁺ channel in isolated myocytes under normothermicconditions. After simulated cardioplegic arrest, an apparent similar dose-dependentincrease in myocyte contractility was noted. Under both normothermic conditionsand after simulated cardioplegic arrest, total contraction duration was increasedby more than 25%. A likely contributory mechanism for the increased contractionduration in the presence of (–)BayK 8644 was alterations in Ca²⁺ homeostatic processes. (–)BayK 8644 increases intracellularCa²⁺ levels without elevating cAMP levels. ¹⁴ Thus, while intracellular Ca²⁺ levelsrise, basal phospholamban activity and rate of Ca²⁺ resequestrationby the SR remain unchanged. This net effect would potentially result in increasedcross-bridge release time and prolonged elevation in the cytoplasmic Ca²⁺ concentration that would be translated as an increase in total contractionduration. Although direct activation of the L-type Ca²⁺ channelelicited a positive inotropic response after cardioplegic arrest, this responsewas significantly blunted compared with normothermic control values. A recentstudy by Lopez and colleagues ²⁵ demonstrated that a hyperkalemic environment increased resting intracellularCa²⁺ levels in isolated myocytes. A past study from this laboratorydemonstrated that the positive inotropic response to increased extracellularCa²⁺ in the isolated myocyte is diminished after cardioplegic arrest. ²⁶ Taken together, results from ourstudy and past reports suggest that the decrease in myocyte inotropic responseafter cardioplegic arrest may involve defects in intracellular Ca²⁺handling,use, or both. Increased intracellular Ca²⁺ accumulation has beenshown to contribute to reoxygenation injury and decreased recovery of contractileperformance with reperfusion. ²⁷ Alterations in intracellular Ca²⁺ compartmentalization andsequestration may occur during and immediately after prolonged periods ofcardioplegic arrest. These persistent defects in Ca²⁺ homeostaticprocesses within the myocyte after cardioplegic arrest may contribute to thediminished contractile responsiveness with direct L-type Ca²⁺ channelactivation. The specific abnormalities in intracellular Ca²⁺ compartmentalizationafter cardioplegic arrest, particularly with respect to the function of theSR warrant further study.

Although the isolated myocyte system used in this study provided a meansto pharmacologically dissect the ß-adrenergic signaling cascade to examinespecific defects in contractile mechanisms, several limitations to this invitro system exist. The model excludes environmental factors present in vivo,including changes in systemic loading conditions and neurohormonal influences.This model of cardioplegic arrest differs with respect to in vivo conditionsin that the myocytes are not subject to variations in temperature and arecontinuously exposed to the hyperkalemic environment. This model of simulatedcardioplegic arrest causes a decrease in myocyte contractility. However, thisdecrease in contractile performance is a transient phenomenon. Specifically,30 minutes after reperfusion/rewarming, indices of myocyte contractile functionapproach normothermic values. ²⁸ Although this is a limitation of the model, it does appear to recapitulatethe transient LV dysfunction that may be encountered after cardioplegic arrestin the clinical setting. Our study makes the assumption that the responseelicited by (–)BayK 8644 after cardioplegic arrest arises from a functionallyintact L-type Ca²⁺ channel. Whether cardioplegic arrest causesdirect negative effects to the L-type Ca²⁺ channel with respectto functional state is unknown. Future studies measuring the Ca²⁺current through the L-type Ca²⁺ channel in the presence and absenceof (–)BayK 8644 in the normothermic state and after cardioplegic arrestwould be necessary to address this issue. It should also be understood thatthis study was conducted using asanguineous cardioplegia and cell media andthat results in a blood-perfused in vivo preparation may result in differentialfindings with respect to the ß-adrenergic signaling cascade.

The findings of this study open avenues for future studies aimed atidentifying other possible sites in the ß-adrenergic signaling cascadethat may be affected by cardioplegic arrest. Specifically, future studiesexamining the role of PKA-mediated intracellular events on myocyte contractileperformance after cardioplegic arrest are warranted. An increasing numberof patients are presenting for cardiac surgery with preexisting LV dysfunction, ²⁹ a condition that has been associatedwith decreased sensitivity and inotropic response to ß-adrenergic stimulation. ³⁰ Thus direct activation of the L-typeCa²⁺ channel may prove beneficial as a therapeutic modality inthis subset of patients. However, future in vitro and in vivo studies wouldbe required to examine this hypothesis. Nevertheless, the findings of thisstudy support the conclusion that defect(s) contributing to decreased inotropicresponsiveness in isolated myocytes after cardioplegic arrest may includedefects in the ß-AR signaling cascade, disturbances in intracellularCa²⁺ homeostasis, changes in myofilament Ca²⁺ sensitivity,or any combination of the above.

Footnotes

Supported by National Institutes of Health grants 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 Institutional Research Funds of 1996-97. F.G.S is an Established Investigator of the American Heart Association.

Received for publication Jan. 27, 1997; revisions requested May 14, 1997; revisions received August 8, 1997; accepted for publication August 13, 1997.

From the Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, S.C.

References

1. Phillips HR, Carter JE, Okada RD, LevineFH, Boucher CA, Osbakken M, et al. Serial changes in left ventricular ejectionfraction in the early hours after aortocoronary bypass grafting. Chest 1983;83:28-34.[Abstract/Free Full Text]
   
2. Roberts AJ, Spies SM, Sanders JH, Moran JM,Wilkinson CJ, Lichtenthal PR, et al. Serial assessment of left ventricularperformance following coronary artery bypass grafting. J Thorac CardiovascSurg 1981;81:69-84.[Abstract]
   
3. Hartzell HC. Regulation of cardiac ion channelsby catecholamines, acetylcholine and second messenger systems. Prog BiophysMol Biol 1988;52:165-247.
   
4. Katz AM. Physiology of the heart. 2nd ed.New York: Raven Press; 1992.
   
5. Fremes SE, Weisel RD, Mickle DA, Ivanov J,Madonik MM, Seawright SJ, et al. Myocardial metabolism and ventricular functionfollowing cold potassium cardioplegia. J Thorac Cardiovasc Surg 1985;89:531-46.[Abstract]
   
6. Schwinn DA, Leone BJ, Spahn DR, Chesnut LC,Page SO, McRae RL, et al. Desensitization of myocardial ß-adrenergicreceptors during cardiopulmonary bypass: evidence for early uncoupling andlate downregulation. Circulation 1991;84:2559-67.[Abstract/Free Full Text]
   
7. Schranz DS, Droege A, Broede A, BrodermannG, Schafer E, Oelert H, et al. Uncoupling of human cardiac ß-adrenoceptorsduring cardiopulmonary bypass with cardioplegic cardiac arrest. Circulation 1993;87:422-6.[Abstract/Free Full Text]
   
8. Vatner DE, Vatner SF, Nejima J, Uemura N,Susanni EE, Hintze TH, et al. Chronic norepinephrine elicits desensitizationby uncoupling the ß-receptor. J Clin Invest 1989;84:1741-8.
   
9. Reves JG, Karp RB, Buttner EE, Tosone S,Smith LR, Samuelson PN, et al. Neuronal and adrenomedullary catecholaminerelease in response to cardiopulmonary bypass in man. Circulation 1982;66:49-55.[Abstract/Free Full Text]
   
10. Handy JR, Spinale FG, Mukherjee R, CrawfordFA. Hypothermic potassium cardioplegia impairs myocyte recovery of contractilityand inotropy. J Thorac Cardiovasc Surg 1994;107:1050-8.
    
11. Bristow MR, Ginsburg R, Strosberg A, MontgomeryW, Minobe W. Pharmacology and inotropic potential of forskolin in the humanheart. J Clin Invest 1984;74:212-23.
    
12. Hearse DJ, Zucchi R, Buschmans E, ManningAS. Forskolin and myocardial function in the normal, ischemic, and reperfusedrat heart. Can J Cardiol 1986;2:303-12.[Medline]
    
13. Hess P, Lansman JB, Tsien RW. Differentmodes of Ca channel gating behaviour favoured by dihydropyridine Ca agonistsand antagonists. Nature 1984;311:538-44.[Medline]
    
14. McCall E, Hryshko LV, Stiffel VM, ChristensenDM, Bers DM. Possible functional linkage between the cardiac dihydropyridineand ryanodine receptor: acceleration of rest decay by BayK 8644. J Mol CellCardiol 1996;28:79-93.
    
15. Mukherjee R, Spinale FG, Crawford FA. Measurementof dynamic cellular and sarcomere contractile properties from the same cardiocyte.Proc IEEE Med Biol 1992;7803:392-4.
    
16. Tsutsui H, Spinale FG, Nagatsu M, NagatsuM, Schmid PG, Ishihara K, et al. The effects of chronic ß-adrenergicblockade on the left ventricular and cardiocyte abnormalities of chronic mitralregurgitation. J Clin Invest 1994;93:2639-48.
    
17. Wang Z, Lam CF, Mukherjee R, Hebbar L, WangY, Spinale FG. Relation between external load and isolated myocyte contractilefunction. Am J Physiol 1997;42:H183-91.
    
18. Tanaka R, Fulbright BM, Mukherjee R, BurchellSA, Crawford FA, Zile MR, et al. The cellular basis for the blunted responseto ß-adrenergic stimulation in supraventricular tachycardia-induced cardiomyopathy.J Mol Cell Cardiol 1993;25:1215-33.[Medline]
    
19. Bassani JWM, Yuan W, Bers DM. FractionalSR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes.Am  J Physiol 1995;268:C1313-9.[Abstract/Free Full Text]
    
20. Sipido KR, Callewaert G, Carmeliet E. Inhibitionand rapid recovery of Ca²⁺ current during Ca²⁺ releasefrom sarcoplasmic reticulum in guinea pig ventricular myocytes. Circ Res 1995;176:102-9.
    
21. Winegrad S. Regulation of cardiac contractileproteins: correlations between physiology and biochemistry. Circ Res 1984;55:565-74.[Abstract/Free Full Text]
    
22. O'Brien DJ, Alexander JA. Postoperativemanagement of the cardiac surgical patient. In: Civetta JM, Taylor RW, KirbyRR. Critical care. 2nd ed. Philadelphia: JB Lippincott; 1992. p. 693-706.
    
23. Gilbert EM, Hershberger RE, Wiechmann RJ,Movsesian, Bristow MR. Pharmacologic and hemodynamic effects of combined ß-agoniststimulation and phosphodiesterase inhibition in the failing human heart. Chest 1995;105:1524-32.
    
24. Dorman BH, Cavallo MJ, Roy RC, Spinale FG.The direct and interactive effects of phosphodiesterase inhibition and ß-adrenergicstimulation on myocyte contractile function after hypothermic cardioplegicarrest. Anesth Analg 1995;81:925-31.[Abstract]
    
25. Lopez JR, Jahangir R, Jahangir A, Shen WK,Terzic A. Potassium channel openers prevent potassium-induced calcium loadingof cardiac cells: possible implications in cardioplegia. J Thorac CardiovascSurg 1996;112:820-31.[Abstract/Free Full Text]
    
26. Cavallo MJ, Dorman BH, Spinale FG, Roy RC.Myocyte contractile responsiveness after hypothermic, hyperkalemic cardioplegicarrest: disparity between exogenous calcium and ß-adrenergic stimulation.Anesthesia 1985;82:926-39.
    
27. Steenbergen C, Murphy E, London RE. Elevationin cytosolic free Ca²⁺ concentration early in myocardial ischemiain perfused rat heart. Circ Res 1987;60:700-7.[Abstract/Free Full Text]
    
28. Hebbar L, Dorman BH, Roy RC, Spinale FG.The direct effects of propofol on myocyte contractile function after hypothermiccardioplegic arrest. Anesth Analg 1996;83:949-57.[Abstract]
    
29. Weintrub WS, Wenger NK, Jones EL, CraverJM, Guyton RA. Changing clinical characteristics of coronary surgery patients:differences between men and women. Circulation 1993;88:79-86.
    
30. Bristow MR, Ginsburg R, Minobe W, CubicciottiRS, Sageman S, Lurie K, et al. Decreased catecholamine sensitivity and ß-adrenergicreceptor density in failing human hearts. N Engl J Med 1982;307:205-11. [Abstract]
    

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