Acute left ventricular dysfunction is commonly encountered after hypothermic, hyperkalemic cardioplegic arrest (HHCA) and often requires inotropic intervention for successful separation from cardiopulmonary bypass. However, the basic mechanisms involved in depressed left ventricular function and the cellular basis for the differential effects of inotropic drugs after HHCA are unknown. Accordingly, the goal of this study was to determine the effects of calcium (Ca2+) and beta-adrenergic receptor agonists (beta AR) stimulation on isolated myocyte contractile function after HHCA.
Myocytes were isolated from the left ventricle of nine pigs and randomly assigned to one of the following treatment groups: (1) normothermic, control: incubation in oxygenated cell culture media for 2 h at 37 degrees C; and (2) cardioplegia: incubation in 4 degrees C crystalloid cardioplegia for 2 h, followed by rewarming. Steady-state myocyte contractile function was measured after pulse stimulation at baseline, in the presence of extracellular Ca2+ (3-10 mM), and in the presence of the beta AR agonist isoproterenol (2-100 nM). Myocyte profile surface area was measured for both normothermic myocytes and myocytes after HHCA. In a separate set of experiments, myocyte contractile function also was documented after 2 h of hypoxic conditions with both normothermic incubation and HHCA, in the presence and absence of beta AR stimulation.
Baseline myocyte contractile function was significantly less in the cardioplegia group compared to control. Extracellular Ca2+ produced a dose-dependent significant increase in myocyte contractile function in the normothermic control group, whereas increased extracellular Ca2+ only minimally increased myocyte contractile function in the cardioplegia group. A dose-dependent, significant increase in myocyte contractile function was observed in both groups after beta AR stimulation by isoproterenol; however, myocyte contractile function in the cardioplegia group was decreased compared to the control group. Hypoxia under normothermic conditions significantly reduced myocyte contractile function, myocyte relaxation, and beta-adrenergic responsiveness. Hypoxia in combination with cardioplegic arrest compounded the negative effects on contractile processes but did not further impair beta-adrenergic responsiveness. Myocyte profile surface area was significantly increased after HHCA.
The minimal improvement in myocyte contractile function after HHCA with increased extracellular Ca2+ suggests that Ca2+ depletion is not the primary mechanism for depressed myocyte contractility after HHCA. On the other hand, because beta AR administration improved myocyte contractile function after HHCA, the cellular basis for the effects of beta AR stimulation after HHCA is probably not increased myocyte Ca2+ but rather alternative mechanisms, such as changes in myofilament sensitivity to Ca2+. These results also suggest that the abnormalities in left ventricular function after HHCA result from the direct effects of hyperkalemic induced electromechanical uncoupling as well as relative hypoxic conditions.
Key words: beta-Adrenergic receptor agonists, Calcium, Crystalloid cardioplegia, Myocyte.
THE most common method of myocardial protection during cardiopulmonary bypass (CPB) is hypothermic, hyperkalemic cardioplegic arrest (HHCA). On separation from bypass, acute left ventricular (LV) dysfunction is commonly encountered. beta-Adrenergic receptor agonists (beta AR) and calcium (Calcium2+) frequently are administered to patients after HHCA in an attempt to improve LV pump function. Calcium administration has been shown to increase LV contractile state in both human and animal studies. [1-5]However, the effects of Calcium2+ administration on LV function immediately after HHCA and rewarming remain unclear. Conflicting reports exist concerning the hemodynamic changes that occur after calcium administration in the post-CPB setting, in part due to different methodologies used to measure inotropy. Several studies have reported improved indexes of LV pump function after Calcium sup 2+ administration immediately after separation from bypass, [6-8]whereas other reports have failed to identify a beneficial effect of Calcium2+ on LV function when administered in the post-CPB setting. [9-11]Changes in extracellular Calcium2+ can cause alterations in systemic vascular resistance, LV filling pressure, [1-3]and heart rate, [1,2]all of which make direct measurement of LV contractile function after Calcium2+ administration difficult. Therefore, one aspect of the current study was to examine the direct effects of increased extracellular Calcium2+ on myocyte contractile function after cardioplegic arrest in an isolated myocyte preparation.
The changes in LV loading conditions, heart rate, and plasma catecholamine concentrations that occur in patients after HHCA and rewarming make it difficult to determine the direct effect of pharmacologic agents on LV contractile function. Examination of contractile properties of isolated myocytes has some distinct advantages over an in vivo preparation, including removal of loading conditions, an absence of neurohumoral influences, and the capacity to directly examine contractile function of myocytes independent of the effects of the extracellular matrix and alterations in coronary perfusion. [12-15]Furthermore, the extracellular milieu can be carefully controlled so that specific, direct influences of agents such as Calcium2+ and beta-adrenergic agents on contractility can be determined.
It has been reported by this laboratory that isolated myocyte contractile function is significantly depressed after HHCA and subsequent rewarming. However, the basic mechanisms responsible for depressed LV function after hypothermic cardioplegic arrest and rewarming remain unclear. The responsiveness to inotropy after HHCA is also not well understood. Therefore, an examination of the differential effects of potential inotropic agents such as Calcium2+ and beta AR after HHCA in an isolated myocyte system may provide valuable information both on the agents best suited to improve contractile function after cardioplegic arrest and on the mechanism of myocardial dysfunction in the immediate post-CPB setting.
Myocyte isolation and determination of myocyte function were performed using previously described methods. Nine Yorkshire swine (25-30 kg) were the source of myocytes for the study. All animals were cared for and treated in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals."* During surgical anesthesia, consisting of 1.5% isoflurane in oxygen, a sternotomy was performed, and the heart quickly extirpated and placed in an oxygenated Krebs solution. The great vessels were rapidly removed at the aortic and pulmonary valves, and a region of the LV free wall incorporating the circumflex artery (5 x 5 cm) was excised and used for myocyte isolation. This region was isolated to obtain myocytes strictly from the LV free wall, to ensure uniformity of the cell population. Myocytes from the right ventricle have a different response to beta-agonist stimulation and therefore, if included, could result in a myocyte population with a wide spectrum of contractile responses to inotropes. The left circumflex coronary artery was cannulated, distal branches ligated, and the tissue rinsed free of blood with 35 ml of a modified Krebs solution supplemented with 5 mM nitrilotriacetic acid and 0.1% salt-free bovine serum albumin (BSA). Collagenase (0.5 mg/ml, Worthington, type II; 146 U/mg) was then added to the solution and the tissue was perfused for 35 min. The tissue was then minced into 2 mm sections and added to an oxygenated solution containing 2% BSA, deoxyribonuclease II (DNase, 51 Kunitz units/ml, type IV, Sigma, St. Louis, MO), 300 micro Meter CaCl2and collagenase (0.5 mg/ml). The tissue and trituration solution was transferred to a centrifuge tube and gently agitated. After 15 min, the supernatant was removed, filtered and the cells allowed to settle. The myocyte pellet was then resuspended in standard culture medium (2 mM Calcium2+; Medium M199, Gibco Laboratories, Grand Island, NY). A 2 ml aliquot of the isolated myocyte suspension was then plated on coverslips previously coated with a laminin/fibronectin matrix (Matrigel, Collaborative Research, Bedford, MA) and incubated at 37 degrees Celsius for 1 h in the presence of 95% Oxygen2and 5% CO2, to ensure adequate oxygenation of myocytes. Spontaneous electrical activity did not occur in any of the myocytes included in the current study; adult mammalian myocytes have been demonstrated to remain quiescent in culture and not exhibit spontaneous contractions. [13,18]The yield of viable myocytes was greater than 80% from each preparation and did not significantly change after hypothermic cardioplegic arrest and rewarming. Viable myocytes included those that retained a rod shape, were calcium tolerant and excluded trypan blue.
Isolated Myocyte Function and Analysis
Isolated myocytes were placed in a thermostatically controlled chamber at 37 degrees Celsius for imaging on an inverted microscope (Axiovert IM35. Ziess, Oberkochen, Germany). The chamber volume was 2.5 cc and contained 2 stimulating platinum electrodes and a miniature thermocouple (CN7100; Omega Engineering, Stamford, CT). The myocytes were imaged using a 20x Hoffmann Modulation Contrast objective (Modulation Optics, Greenvale, NY) with a final magnification of x1,100). Myocyte contractions were elicited by field-stimulating the tissue chamber at 1 Hz (S11, Grass Instruments, Quincy, MA), using current pulses of 5 ms duration. A standard voltage, 3 volts, was delivered to the media for all myocytes studied; there was no variation in the voltage for myocyte stimulation. Polarity of the stimulating electrodes was alternated at every pulse to prevent the build up of electrochemical by-products. Myocyte contractions were imaged using a charge-coupled device with a non-interlaced scan rate of 240 Hz (GPCD60, Panasonic, Secaucus, NJ). Myocyte motion signals were captured with the cell parallel to the video raster lines, and this video signal input through an edge detector system (Crescent Electronics, Sandy, Utah), and displayed on a video monitor, as well as recorded on video tape using a standard VHS recorder (HSU 32; Mitsubishi Electronics, Cypress, CA). To incorporate the higher scanning frequency, the CCD camera divided the standard video (RS170) vertical scanning field of 16.7 ms into four sub-fields. After the standard 1.3 ms blanking pulse, each sub-field was scanned in 3.3 ms and followed by a 0.7 ms blanking pulse. The edge detection system used the changes in light intensity at the edges of the myocytes to track myocyte motion and was calibrated by recording the image of a stage micrometer (Ziess, Oberkochen, Germany) with grating spaced 10 micro meter apart using the two objectives. [19,20]During playback, the voltage output of the edge detector was recorded for a known distance between the left and right edges. This process was repeated for a minimum of five different distances. Regression analysis was then performed to determine the relationship between the edge detector output voltage and the distance between the left and right edges. Regression analysis revealed a strong linear relationship between the edge detector output voltage and the distance between the edges (r = 0.998; P < 0.001). The distance between the left and right myocyte edges was converted into a voltage signal, digitized at 240 Hz and input into an 80286 computer (ZBV2526; Zenith Data Systems, St. Joseph, MI) for subsequent analysis. For the reconstruction of a digitized signal, the Nyquist criterion states that the sampling frequency must be at least twice the maximum band-limited frequency contained in that signal. Specifically, the highest frequency component that could be reconstructed in the current series of experiments was 120 Hz. Further, Sato et al. reported that a temporal resolution of 16.7 ms adequately captured changes in sarcomere length. In the current study, the on-camera magnification of the myocyte images was 9.25 pixels/micro meter. This pixel resolution provided an adequate spatial resolution for the determination of myocyte motion by video-based edge detection. In a previous study, Fourier analysis of myocyte contractile data revealed that 99% of the frequency power spectrum was below 4 Hz when the myocytes were stimulated at 0.5 Hz, implying that all the contractile information was contained within the first 8 harmonics of the principal frequency. The current study obtained myocyte contractile motion signals that far exceeded these computed minimum requirements. The velocity of myocyte motion was computed from the contraction profile using the central-difference algorithm. [19,23]Noise spikes were eliminated by examining the velocity profile for transient double-zero crossings. A minimum of 20 consecutive contraction profiles were digitized from each cardiocyte for analysis.
Stimulated myocytes were allowed a 5 min stabilization period after electrical stimulation, and contraction data for each myocyte was recorded from a minimum of 20 consecutive contractions. Parameters computed from the digitized contraction profiles included: percent shortening (%), velocity of shortening (micro meter/s), velocity of relengthening (micro meter/s), total contraction duration (ms), and time to peak contraction (ms). Myocyte percent shortening (%) was determined as the percent difference between maximum and minimum cell length for each contraction. Myocyte velocity computations were obtained by differentiating the digitized contraction profiles. The time to peak contraction was computed by calculating the time required for the differentiated velocity profile to reach zero velocity after the start of contraction. All parameters were calculated for each contraction and the results averaged for the 20 contractions observed.
Cardioplegia and Drug Administration Protocol
After collection of baseline myocyte contractile performance, myocytes were randomly assigned to one of the following two treatment protocols: normothermic, control group: incubation with 37 degrees Celsius Ringer's solution containing 30 mEq/I HCO3(pH 7.8, 280 mOSM) and then stored for 2 h at 37 degrees Celsius in a 95% O2environment; cardioplegia group: incubation with 4 degrees Celsius Ringer's solution containing 24 mEq/I potassium and 30 mEq/I HCO3, then stored at 4 degrees Celsius for 2 h with subsequent rewarming. Steady-state myocyte contractile function in the control group, obtained in the current study, was similar to values reported previously by this laboratory for myocytes without prior incubation. Thus, incubation of myocytes at 37 degrees Celsius for 2 h had no effect on myocyte contractile function and was, therefore, used as the control for comparison in the current study. After the respective treatment protocol, the myocytes were transferred to the stimulation chamber and infused with warm, oxygenated media at 37 degrees Celsius for exactly 10 min. After approximately 5 min of rewarming, myocytes had recovered to steady-state contractile function. Once the myocytes had been rewarmed, electrical stimulation was initiated and steady-state contractile measurements were obtained as described in the previous section. After measurement of baseline function, myocytes were exposed to increasing concentrations of extracellular Calcium2+ (3-10 mM) or isoproterenol (2-100 nM) for 5 min and measurements of myocyte function were repeated.
Myocyte incubation media from both experimental groups was sampled in triplicate at the beginning of each study, before initiation of treatment protocols, and after 2 h at the completion of each experimental protocol for determination of pH, pCO2, POsub 2, HCO3- and measurement of sodium and potassium concentrations (1312 Blood Gas Manager, Instrumentation Laboratory, Lexington, Mass). To examine the effects of various periods of cardioplegic arrest on myocyte contractile function, myocytes were incubated at 4 degrees in cardioplegic solution for 30, 60, 90 and 120 min. After rewarming, steady-state contractile measurements were obtained.
Effect of Hypoxia on Myocyte Contractile Function
To determine whether hypoxia with subsequent reoxygenation had a direct effect on myocyte contractile function, or played a mechanistic role in the change in contractile function after HHCA, an additional set of experiments were performed. In this portion of the study, isolated myocytes were randomly assigned to one of four treatment protocols after isolation and plating onto coverslips. 1) Normothermia and Oxygenation: myocytes were maintained under normothermic, oxygenated conditions for 2 h as described in the previous section; 2) Cardioplegia and Oxygenation: myocytes were exposed to 2 h of crystalloid cardioplegic arrest and subsequent rewarming; 3) Normothermia and Hypoxia: normothermic hypoxia for 2 h followed by reoxygenation with standard cell culture media; 4) Cardioplegia and Hypoxia: cardioplegic arrest with hypoxic cardioplegic solution for 2 h, with subsequent rewarming and reoxygenation in cell culture media. For the hypoxia experiments, myocytes were placed in either normothermic cell culture media or crystalloid cardioplegic solution that had been continuously gassed with 100% N2O for 30 min. For the 2 h of normothermic hypoxia or cardioplegic arrest, the solutions were continuously gassed with 100% N2O to maintain the percent oxygen content below 5%. Percent oxygen of the solutions was continuously monitored using an oxygen selective microelectrode system (OM-4, Microelectrodes, Londonderry, NH). The hypoxic conditions uniformly caused a 20% loss of myocytes in all treatment groups. After the treatment protocol for each of the four groups, myocyte contractile function was analyzed as previously described.
Resting myocyte length was measured at baseline (normothermia) and after HHCA and rewarming. Furthermore, to ensure that changes in initial length did not affect indexes of myocyte contractile function, myocyte velocity of shortening and lengthening was expressed as a function of resting length, as described previously by this laboratory and others. [12,18]Myocytes returned to 99% of the resting length for each contraction or the myocyte was not used for computations. To more carefully examine the issue of myocyte swelling after HHCA, myocytes under normothermic conditions and after HHCA were immediately placed in a buffered sodium cacodylate solution containing 2% paraformaldehyde and 2% glutaraldehyde (pH 7.4, 325 mOsm). The isolated myocytes were then imaged using a 10x phase contrast objective. The image was input into an image analysis system (IBAS 2000 Image Analysis System, Ziess/Kontron, Oberkochen, Germany), and the images were digitized at 512 x 512 line resolution and 256 gray levels. Individual cell profiles were automatically discriminated by gray level to determine myocyte profile surface area. .
Changes in indexes of myocyte function between the control and cardioplegia groups were examined using multiway analysis of variance. If the analysis of variance revealed significant differences, pairwise tests of individual group means were compared using Bonferroni's probabilities. To determine whether relative hypoxia and cardioplegic arrest with rewarming had independent, additive effects on myocyte contractile processes, a two-way analysis of variance with interactions was performed. All statistical analysis was performed using standard statistical software programs (BMDP Statistical Software, University of California, Los Angeles, CA). Results are presented as mean plus/minus SEM. Values of P < 0.05 were considered to be statistically significant.
Myocytes were successfully harvested from all pigs used in this study and were either exposed to 2 h of hypothermic hyperkalemic cardioplegic arrest and rewarming (cardioplegia group) or incubated for 2 h at 37 degrees Celsius in standard culture medium (control group). The pH, PCO2, and PO2of the incubation media were not significantly different between study groups at the beginning or end of the experimental protocol. After 2 h of incubation in either normothermic culture medium or after HHCA, the POranged from 34-36 Torr and the pH was 7.3 to 7.5. Repetitive stimulation of myocytes for 20 min after the 2-h incubation had no effect on these values.
Representative contraction profiles at baseline for control and cardioplegia myocytes are shown in Figure 1and steady-state myocyte contractile function for these two treatment groups is summarized in Table 1and Table 2. Myocytes exposed to HHCA and rewarming displayed a significant reduction in baseline myocyte contractile function. Specifically, isolated myocyte percent shortening was 40% less and myocyte velocity of shortening was 43% less in the cardioplegia group compared to the control group. There were no significant baseline differences in the time to peak myocyte contraction and duration of myocyte contraction and duration of myocyte contraction for the two groups. The initial baseline, resting myocyte length was, however, significantly reduced for myocytes after HHCA relative to control, normothermic myocytes.
Myocytes exposed to less than 2 h of HHCA did not display any reduction in contractile function (Figure 2). Specifically, cardioplegic arrest for 30, 60, or 90 min did not result in any decrease in the percent or velocity of myocyte shortening. Accordingly, myocyte contractile function in the current study was examined after 2 h of HHCA. This time interval was also chosen because it reflects a common duration of cardioplegic arrest encountered clinically.
To determine the effects of extracellular Calcium2+ on myocyte contractile function, extracellular Calcium2+ was added in a step-wise fashion to the media of both the control and cardioplegia myocytes. The results from this portion of the study are summarized in Table 1. In control myocytes, contractile function increased in a dose-dependent manner with increased concentrations of extracellular Calcium2+. Specifically, a significant increase in myocyte contractile function from baseline was observed in the presence of 4 mM extracellular Calcium2+ or greater for control myocytes. A maximum contractile response for control myocytes was observed at 8 mM extracellular Calcium2+, where the velocity of shortening increased by more than 100% from baseline values. In contrast, increased extracellular Calcium2+ only minimally improved myocyte contractile function in the cardioplegia group. Myocyte percent and velocity of shortening were significantly lower in the cardioplegia group compared to control at all extracellular Calcium2+ concentrations. A significant increase in myocyte percent shortening occurred in the cardioplegia group but only in the presence of 10 mM Calcium2+, the highest extracellular Calcium2+ concentration studied. The velocity of shortening did not significantly improve in the cardioplegia group at any increased Calcium2+ concentration. When the velocity of shortening was normalized to end diastolic length, mild, yet significant, increases in percent velocity of shortening occurred in myocytes in the cardioplegic group at 8 mM extracellular Calcium2+. However, the percent velocity of shortening at 8 mM extracellular Calcium2+ was increased by only 69% for cardioplegic myocytes, whereas control myocytes displayed an increase of 117% in percent velocity of shortening. Although contractile response to extracellular Calcium2+ in myocytes exposed to HHCA appeared to be shifted toward higher Calcium2+ concentrations, it is noteworthy that a significant reduction in myocyte viability occurred when myocytes were exposed to higher than 10 mM extracellular Calcium2+. Resting end-diastolic myocyte length was not affected by increased extracellular Calcium2+ concentrations in cardioplegic myocytes. End-diastolic length was similar between control and cardioplegic myocytes at all concentrations of extracellular Calcium2+ with the exception of a significant reduction in cardioplegic myocytes in the presence of 10 mM extracellular Calcium2+. A significantly prolonged duration of myocyte contraction was observed in the cardioplegia group when extracellular Calcium2+ concentration was increased, without any significant change in the time to myocyte peak contraction. The velocity of relengthening was depressed in the cardioplegia group compared to control at all Calcium2+ concentrations.
To determine the effects of beta AR stimulation on myocyte contractile function after HHCA and rewarming, isoproterenol was added in a step-wise fashion to the medium of both control and cardioplegia myocytes. The results for this portion of the study are summarized in Table 2. Isoproterenol produced a significant, dose-dependent increase in contractile function for both control and cardioplegia myocytes when compared to baseline. In contrast to the results obtained with extracellular Calcium2+, the lowest concentration of isoproterenol used in the current study, 2 nM, caused a significant increase in myocyte contractile function from baseline values for both groups. However, contractile function was significantly decreased in cardioplegia myocytes compared to control myocytes at every concentration of isoproterenol. At the highest dose of isoproterenol, 100 nM, the increase in myocyte contractile function was blunted relative to 10 and 50 nM and viability was reduced. Resting myocyte length was similar between control and cardioplegic myocytes at all concentrations of isoproterenol with the exception of a significant reduction in end-diastolic length in cardioplegic myocytes at 2 nM isoproterenol.
The disparity between myocyte contractile responsiveness to extracellular Calcium2+ and beta-adrenergic stimulation after HHCA and rewarming is exemplified in Figure 3. The maximum increase in myocyte contractile function occurred between 10-50 nM isoproterenol in the control group, and at 10 nM isoproterenol in the cardioplegia group. The extracellular Calcium2+ concentration that produced the most pronounced increase in myocyte percent and velocity of shortening in the control group, 8 mM, only minimally increased myocyte contractile function in the cardioplegia group.
To determine the effect of hypoxia with subsequent reoxygenation on myocyte contractile function, myocytes were exposed to relative hypoxia under normothermic conditions and during HHCA. Myocytes subjected to 2 h of normothermic hypoxia displayed significant reductions in contractile performance relative to normothermic oxygenated myocytes (Table 3). Myocyte velocity of shortening decreased by more than 41% with 2 h of hypoxia and reoxygenation when compared to normothermic, oxygenated control values. When myocytes were exposed to both hypoxia and cardioplegic arrest for 2 h with subsequent rewarming and reoxygenation, myocyte contractile performance was significantly reduced from both normothermic oxygenated control values and oxygenated cardioplegia values (Table 3). Two hours of hypoxic cardioplegic arrest resulted in more than a 44% decline in myocyte velocity of shortening from normothermic oxygenated control values and a 24% decline compared to oxygenated cardioplegic arrest values. Two-way analysis of variance revealed that the effects of hypoxia and cardioplegic arrest on myocyte percent shortening and velocity of shortening had independent, additive effects, with no significant interaction. As illustrated in Table 3, myocyte active relaxation properties were also significantly affected by hypoxia with subsequent reoxygenation. Two hours of hypoxia and cardioplegic arrest caused a 59% decline in myocyte velocity of relengthening when compared to normothermic oxygenated control values, and a 33% decline relative to oxygenated cardioplegic arrest values (Table 3). In contrast to the findings for myocyte shortening characteristics, analysis of variance revealed that hypoxia and cardioplegic arrest induced significant interactive effects on myocyte velocity of relengthening (F = 7.85, P = 0.0056). Thus, combined hypoxia and cardioplegic arrest with subsequent rewarming and reoxygenation had significant interactive effects on myocyte contractile function.
To further examine the effects of hypoxia alone and in combination with cardioplegic arrest and rewarming on myocyte contractile function, myocyte beta-adrenergic responsiveness was examined. As shown in a previous section, beta-adrenergic responsiveness of myocytes was significantly impaired under conditions of oxygenated cardioplegic arrest relative to oxygenated normothermia. Myocyte beta-adrenergic responsiveness was further reduced by hypoxia under normothermic conditions. Myocyte velocity of shortening in the presence of 25 nM isoproterenol declined by 43% after 2 h of hypoxia with subsequent reoxygenation when compared to normothermic oxygenated control values. Interestingly, myocyte velocity of shortening in the presence of 25 nM isoproterenol for myocytes after hypoxia and cardioplegic arrest was not significantly reduced when compared to myocytes exposed to cardioplegia alone (Table 3). These results suggest that cardioplegic arrest provided protective effects on myocyte beta-adrenergic responsiveness under conditions of relative hypoxia.
To determine any alteration in myocyte dimension after HHCA, isolated myocyte profile surface area was measured using computer digitization methods for both normothermic myocytes and myocytes exposed to HHCA. As illustrated in Figure 4A, myocyte profile surface area formed a Gaussian distribution in control, normothermic myocytes, with a mean value of 3,438 plus/minus 56 micro meter2. HHCA resulted in a significant increase in myocyte profile surface area (4,439 plus/minus 60 micro meter2, P < 0.05) relative to control values (Figure 4(B)).
Despite significant improvements in myocardial preservation techniques during CPB, [26,27]depressed LV function occurs after hypothermic cardioplegic arrest and rewarming. [28,29]The cellular basis and the optimal treatment for such ventricular dysfunction is unclear. In the current study, myocyte contractile function was examined after HHCA, in control normothermic conditions at baseline, after exposure to either Calcium2+ or a beta AR agonist, and under hypoxic conditions to gain a better understanding of the mechanism of myocardial dysfunction and the agents best suited to improve contractile function after cardioplegic arrest.
Administration of Calcium2+ is common after HHCA and rewarming in an attempt to improve ventricular performance. [6,8]Increasing extracellular Calcium2+ increases the Calcium2+ gradient, and thereby results in a higher concentration of intracellular Calcium2+ after myocyte depolarization. This increased intracellular Calcium2+ should enhance myofilament cross-bridging and thereby improve myocyte contractile performance. In the current study, increased extracellular Calcium2+ produced the expected dose-dependent increases in percent and velocity of myocyte shortening in control, normothermic myocytes. However, increased extracellular Calcium2+ only minimally improved indexes of myocyte contractile function after hypothermic cardioplegic arrest and rewarming. This study, therefore, provides direct evidence that increased extracellular Calcium2+ has a reduced effect on myocyte contractile function after HHCA, and this limited myocyte contractile function enhancement helps explain the failure of Calcium2+ infusion in prior studies to improve indexes of LV pump function in patients after HHCA. [9-11]The disparity in myocyte contractile function in the current study may be directly related to a reduced sensitivity of myofilaments to Calcium sup 2+, which has been observed after ischemia and hypothermic cardioplegic arrest. [30-32]Such reduced myofilament sensitivity to Calcium2+ may be the result of alterations in excitation-contraction coupling and may be mediated by a number of intracellular factors, including acidosis and alterations in myofilament cytoarchitecture, both commonly observed in the ischemic myocytes. Furthermore, changes in sodium potassium ATPase activity, sodium-calcium exchange, or abnormalities in calcium homeostasis probably are important contributory mechanisms for the changes in myocyte contractile function and inotropic responsiveness observed after HHCA and rewarming.
Myocyte contraction duration was significantly increased in cardioplegia myocytes compared to control at higher Calcium2+ concentrations, without a change in time to peak contraction. The increased contraction duration appears to be the result of a significantly reduced velocity of relengthening in cardioplegia myocytes exposed to calcium compared to controls. These findings suggest that Calcium2+ may impair myocyte relaxation properties after HHCA. Increased intracellular Calcium2+ concentrations in diastole have been shown to be associated with deterioration of ventricular compliance. Such alterations in Calcium2+ homeostasis occur after cardioplegic arrest and could play a role in the abnormal myocyte relaxation. [35,36]The ability of the sarcoplasmic reticulum of a postischemic myocyte to take up Calcium2+ may be impaired, [37,38]which would result in an increased intracellular Calcium2+ concentration in diastole. Increased extracellular Calcium2+ concentration after cardioplegic arrest also could contribute to intracellular Calcium2+ overload by enhanced Calcium2+ influx through passive diffusion or increased Sodium2+ /Calcium2+ exchange in reperfused tissue. [39,40].
Myocytes exposed to HHCA displayed a significant increase in percent shortening between 8 and 10 mM extracellular Calcium2+, whereas there was no change in the velocity of shortening. The apparent disparity between the changes in myocyte percent and velocity of shortening in the presence of 10 mM Calcium2+ can be attributed to prolongation in the total duration of contraction. As discussed previously, alterations in Calcium2+ homeostasis with a subsequent prolongation of active relaxation processes may have occurred in the presence of high extracellular Calcium2+. Increased contraction duration results in an increase in cytosolic Calcium2+ availability because of enhanced Calcium2+ release and reduced uptake. The increased Calcium2+ presence results in more cross-bridging of actin and myosin, with a greater degree of sarcomere shortening.
Beta AR agonists commonly are used to improve depressed LV pump function after HHCA and rewarming. beta AR stimulation results in the downstream production of cyclic AMP (cAMP). Increased cAMP causes a number of intracellular events, including (1) phosphorylation of sarcolemmal Calcium2+ channels, thereby increasing Calcium2+ entry into the myocyte; (2) enhanced Calcium2+ sequestration into the sarcoplasmic reticulum; (3) increased rate of actomyosin complex cross-bridge cycling; and (4) decreased sensitivity of the contractile protein troponin to Calcium2+, which also enhances cross-bridge cycling. [41-43]In the current study, beta AR stimulation produced a dose-dependent increase in contractile function in normothermic, control myocytes. In contrast to extracellular Calcium2+, beta AR stimulation markedly increased myocyte contractile function after HHCA. The disparity of these results suggests that the mechanism of beta AR stimulation after HHCA and rewarming is not simply increased Calcium2+ availability but probably alternative mechanisms, which may include changes in myofilament sensitivity to Calcium2+. Although beta AR stimulation increased myocyte contractile function, it failed to normalize myocyte contractile function up to control levels after cardioplegic arrest. These results suggest that abnormalities in myocyte contractile function persist after hypothermic cardioplegic arrest and rewarming, which cannot be overcome by beta AR stimulation.
The dose-dependent increase in myocyte contractile function with increased concentrations of isoproterenol is consistent with previous reports from this laboratory. In control, normothermic myocytes, high concentrations of isoproterenol caused a reduction in myocyte contractile function. This is consistent with dose-response curves in which high concentrations of isoproterenol have been shown to cause a decline in contractile performance. Potential mechanisms for this effect include increased activation of beta2receptors, internalization of beta1receptors, and a generalized desensitization of beta-receptor transduction due to elevated intracellular cyclic AMP concentrations. At high concentrations of the beta-agonist isoproterenol (100 nM), myocyte contractile function and viability was significantly reduced after HHCA and rewarming. The mechanisms for these apparent toxic effects of high concentrations of isoproterenol are probably alterations in ionic homeostasis. Specifically, beta AR stimulation with subsequent cAMP production has been shown to directly increase L-type calcium channel current. In addition, isoproterenol at high concentrations has been associated with stimulation of the sodium-potassium ATPase system. HHCA and rewarming has been shown to cause changes in ionic homeostasis. Thus, high concentrations of isoproterenol after HHCA may further exacerbate alterations in ionic homeostasis, which in turn compromise myocyte contractile function and viability.
Myocyte beta-adrenergic responsiveness was reduced from normothermic values after hypothermic cardioplegic arrest. In a prior report, after CPB and hypothermic cardioplegic arrest in dogs, beta-receptor density decreased and cyclic AMP production was reduced. Thus, contributory mechanisms for the reduced myocyte beta-adrenergic responsiveness that occurred in the current study after HHCA and rewarming includes alterations in beta-receptor transduction. It is well established that plasma catecholamine levels significantly increase after hypothermic arrest and CPB. The current study demonstrates for the first time that reduced myocyte beta-adrenergic responsiveness occurs after HHCA and is independent of neurohormonal influences.
Current cardioplegic techniques employ electromechanical arrest and interruption of myocardial blood flow. Results from the current study demonstrated that electromechanical arrest of isolated myocytes using an oxygenated hyperkalemic cardioplegic solution caused abnormalities in steady-state contractile performance with subsequent rewarming. However, it remained unclear how relative hypoxia, which may occur clinically with cardioplegic arrest, would affect myocyte contractile processes with subsequent rewarming and reoxygenation. To address this issue, myocytes were subjected to relative hypoxia with and without cardioplegic arrest. Results from this series of experiments demonstrated that hypoxia alone under normothermic conditions significantly reduced steady-state myocyte contractile function and beta-adrenergic responsiveness. Furthermore, hypoxia in combination with cardioplegic arrest compounded the negative effects on myocyte contractile processes. The significance of these findings are twofold. First, contributory mechanisms for the reduction in myocyte contractile performance after cardioplegic arrest and rewarming are independent of relative hypoxic conditions. Second, hypoxia with subsequent reoxygenation has a direct and negative effect on myocyte contractile function. Taken together, these findings suggest that the abnormalities in LV function that occur clinically after cardioplegic arrest and rewarming result from the direct effects of electromechanical uncoupling due to hyperkalemia and can potentially result from hypoxic conditions, if present.
In the current study, 2 h of normothermic hypoxia with subsequent reoxygenation caused a significant decline in myocyte beta-adrenergic responsiveness. In contrast, when hypoxia was coupled with hypothermic cardioplegic arrest in the presence of beta-adrenergic stimulation, myocyte velocity of shortening and relengthening was similar to oxygenated cardioplegic arrest values. These unique findings suggest that HHCA under hypoxic conditions has a relative protective effect on myocyte beta AR transduction but not on other indexes of myocyte contractile function. In contrast, combined hypoxia and cardioplegic arrest appeared to affect myocyte velocity of relengthening to a greater degree. Specifically, analysis of variance revealed that the effects of hypoxia and cardioplegic arrest caused more than an independent, additive effect on myocyte relaxation. Myocyte shortening is a relatively energy-independent process in that the release of Calcium2+ from the cytosol and cross-bridge formation do not require hydrolysis of significant amounts of ATP. In contrast, myocyte relaxation is an energy-dependent process by which ATP is hydrolyzed to resequester Calcium2+, release actin-myosin cross-bridges, and restore myocyte membrane potentials. Results from the current study demonstrate that hypoxia in combination with cardioplegic arrest and rewarming has a selective and significant interactive effect on myocyte velocity of relengthening (myocyte active relaxation). These findings suggest that a contributory mechanism for the reduction in myocyte contractile function with hypoxia is a direct effect on myocyte active relaxation processes. Future studies that specifically address the intracellular mechanisms for the effects of hypoxia and reoxygenation in this isolated myocyte system would be appropriate.
A common sequelae of HHCA is myocardial edema. With subsequent reperfusion after HHCA, myocardial water content has been shown to significantly increase and be strongly associated with abnormalities in LV compliance. However, it is unclear whether the increased myocardial edema is primarily in the extracellular compartment, the cellular compartment, or both. To address this issue, the current project examined isolated myocyte size. In the current study, HHCA with subsequent reperfusion and rewarming resulted in significant increases in myocyte profile surface area. Because myocyte profile surface area is directly proportional to myocyte volume, a marked cellular swelling appears to occur in myocytes after HHCA. These findings provide a cellular basis for past studies that have reported increased myocardial edema in vivo. More importantly, these results suggest that fundamental abnormalities in cell volume regulation occur with HHCA and subsequent rewarming. Extracellular hyperkalemia results in sodium influx, which in turn results in an influx of calcium via the sodium-calcium exchanger. [51-53]Furthermore, the membrane depolarization induced by hyperkalemia causes a leakage of calcium from the sarcoplasmic reticulum and an influx of calcium by way of the "widow current." Thus, with prolonged depolarization in a hyperkalemic environment, alterations in ionic homeostasis can occur and cause fundamental abnormalities in volume regulation with subsequent reperfusion and rewarming. In the current study, hyperkalemic cardioplegic arrest with subsequent rewarming caused myocyte swelling and contractile dysfunction. Furthermore, myocyte responsiveness to extracellular calcium and beta AR stimulation were reduced. A probable contributory mechanism for these alterations in myocyte volume regulation and contractile function is an alteration in intracellular calcium homeostasis.
Future studies using this isolated myocyte model may be advantageous in determining specific mechanisms responsible for the depressive effects of HHCA and rewarming on contractile performance. Although the isolated myocyte model has the advantage of performing direct measurements of myocyte contractile function, it has several limitations. First, the buffering and osmotic influences of the extracellular environment, which may play an important role in vivo, have been removed. Thus, the hyperkalemic solution used in the current study was not buffered by extracellular proteins, which may exist in vivo. Second, this isolated system provides maximal solute diffusion capacity between the cytosol and the extracellular space. In vivo, the coronary vasculature and capillary diffusion distances are affected by coronary artery disease, hypertrophy, and other pathologic states. However, these limitations also define the strength of this model, as questions can be asked concerning the direct effects of HHCA and rewarming on the basic functional unit of the heart, the cardiac myocyte.
In summary, in the current study, exposure of isolated myocytes to HHCA resulted in depressed contractile function after rewarming. This study provides a cellular basis for the depressed LV function observed after HHCA. Increased extracellular Calcium2+ only minimally improved myocyte contractile function after HHCA and rewarming. In contrast, beta AR stimulation significantly improved myocyte contractile function after HHCA and rewarming. These observations suggest that beta AR stimulation improved myocyte contractile function in this setting by alternative mechanisms other than augmenting intracellular Calcium2+. These results also suggest that myocyte contractile dysfunction after HHCA and rewarming is not due to simply myocyte Calcium2+ depletion. Direct measurements of intracellular Calcium2+ depletion. Direct measurements of intracellular Calcium2+ in myocytes in future studies would be valuable in determining the influence of intracellular Calcium2+ levels on contractile function after HHCA.
*Institute of Laboratory Animal Resources, Committee on Care and Use of Laboratory Animals, National Research Council: Guide for the Care and Use of Laboratory Animals, National Institutes of Health publication 86-123, 1985.