Ann Thorac Surg 2004;77:1684-1689
© 2004 The Society of Thoracic Surgeons
Original article: cardiovascular
Myocyte contractility with caspase inhibition and simulated hyperkalemic cardioplegic arrest
Rupak Mukherjee, PhDa*,
William M. Yarbrough, MDa,
Emily S. Reesea,
Jennifer S. Leisera,
Jeffrey A. Samplea,
Joseph T. Mingoiaa,
Amy E. Hardina,
Robert E. Strouda,
Julie E. McLeana,
Jennifer W. Hendricka,
Francis G. Spinale, MD, PhDa
a Division of Cardiothoracic Surgery Research, Medical University of South Carolina, Charleston, South Carolina, USA
Accepted for publication October 8, 2003.
* Address reprint requests to Dr Mukherjee, Cardiothoracic Surgery, Strom Thurmond Research Bldg, 770 MUSC Complex, Suite 625, Medical University of South Carolina, Charleston, SC 29425, USA
e-mail: mukherr{at}musc.edu
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Abstract
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BACKGROUND: Exposure of left ventricular (LV) myocytes to simulated hyperkalemic cardioplegic arrest (HCA) has been demonstrated to perturb ionic homeostasis and adversely affect myocyte contractility on rewarming. Altered ionic homeostasis can cause cytosolic activation of the caspases. While caspases participate in apoptosis, these proteases can degrade myocyte contractile proteins, and thereby alter myocyte contractility. Accordingly, this study tested the hypothesis that caspase inhibition during HCA would attenuate the degree of myocyte contractile dysfunction upon rewarming, independent of a loss in myocyte viability.
METHODS: Porcine (n = 8) LV myocytes were isolated and assigned to the following treatment groups: normothermic control: incubation in cell culture media for 2 hours at 37°C; HCA only: incubation for 2 hours in hypothermic HCA solution (4°C, 24 mEq K+); or incubation in hypothermic HCA solution supplemented with 10 µM of the caspase inhibitor, z-VAD (z-Val-Ala-Asp-fluoromethyl-ketone, HCA+zVAD). Myocyte viability, assayed as a function of mitochondrial function, was determined to be similar in the normothermic and both HCA groups.
RESULTS: The HCA caused a significant reduction in myocyte shortening velocity compared with normothermic control values (41 ± 6 versus 86 ± 8 µm/s, p < 0.05). The HCA+zVAD group had significantly improved myocyte shortening velocity compared with the HCA only group (63 ± 7 µm/s, p < 0.05).
CONCLUSIONS: Independent of changes in viability, caspase inhibition attenuated myocyte contractile dysfunction after HCA and rewarming. Thus, caspase activation during HCA contributes, at least in part, to impaired myocyte contractility with rewarming. Supplementation of HCA with caspase inhibitors may provide a means to preserve myocyte contractile function after cardioplegic arrest.
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Introduction
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A continued strategy for achieving myocardial quiescence during cardiac surgery is cardioplegic arrest, most commonly achieved through the delivery of a hyperkalemic solution [1–4]. However, transient left ventricular (LV) dysfunction can occur after cardioplegic arrest and reperfusion [2, 4–7]. Numerous ex vivo and in vitro studies have demonstrated that prolonged exposure to a normoxic, hyperkalemic environment perturbed intracellular ionic homeostatic processes [5, 8–11]. Abnormalities in intracellular ionic homeostasis can interfere with the excitation-contraction coupling process and adversely affect myocyte contractile function upon reperfusion and rewarming [5, 12–14]. Importantly, another potential outcome of altered ionic homeostasis is cytosolic activation of the caspases [15, 16]. The caspases are a family of cysteine proteases and a number of these proteins have been identified to be present in cardiac myocytes [15, 17, 18]. While historically thought to solely participate in programmed cell death, ie, apoptosis, recent studies have demonstrated that the caspases can proteolyze contractile proteins [19, 20]. Therefore, caspase activation within LV myocytes after exposure to a hyperkalemic environment may cause derangements of the contractile apparatus, before committing the myocyte into the apoptotic pathway. However, it remains unclear whether caspase activation may cause, at least in part, the impairment in myocyte contractile function after simulated cardioplegic arrest and rewarming. Accordingly, the present project tested the central hypothesis that caspase inhibition during simulated hyperkalemic cardioplegia would attenuate the degree of myocyte contractile dysfunction upon rewarming, independent of a loss in cell viability.
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Material and methods
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In the present study, an isolated myocyte system was used to examine the effects of hypothermic, hyperkalemic cardioplegic arrest (HCA) without and with caspase inhibitor supplementation on myocyte contractile function. Myocytes were isolated from the left ventricular free wall of pigs (Yorkshire, n = 8, 30 kg). All animals were treated and cared for in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (National Research Council, Washington, 1996).
Myocyte isolation and contraction analysis
Myocyte isolation and determination of myocyte contractile function were performed as previously described [13, 14, 21]. A 2-mL aliquot of the isolated myocyte suspension was plated onto coverslips previously coated with a basement membrane substrate (Matrigel, Collaborative Research Inc, Bedford, MA), stabilized at 37°C in oxygenated media for 60 minutes, and then randomly assigned to treatment. Viable myocytes included those that retained a rod shape, excluded trypan blue, and remained quiescent in culture. Yield of viable myocytes from all isolations was greater than 60%.
Isolated myocyte contractile measurements were performed using computer-assisted videomicroscopy as described previously [13, 14]. Myocytes were thermostatically maintained at 37°C and electrically field stimulated at a frequency of 1 Hz. The measurements computed from the digitized contraction profiles included percent shortening (%), shortening and relengthening velocities (µm/s), total contraction duration (ms), time to peak contraction (ms), and time to 50% relaxation (ms). All measurements were calculated for each contraction and the results averaged for 20 consecutive contractions.
Experimental design and rationale
The objective of this study was to define the interactive effects of HCA and concomitant caspase inhibition on myocyte contractile function. Accordingly, myocytes were randomly assigned to one of three treatment protocols: (1) normothermic control group: incubation in a physiologic solution (Medium 199, Gibco, Carlsbad, CA) at 37°C and in a 95% oxygen environment for 2 hours; (2) HCA only group: incubation in Ringer's solution at 4°C containing 24 mEq/L K+ and 30 mEq/L HCO3–, and stored at 4°C for 2 hours (O2 saturation: 120 to 150 mm Hg); and (3) HCA with concomitant caspase inhibition group (HCA+zVAD): incubation at 4°C for 2 hours in HCA solution supplemented with 10 µmol/L of the caspase inhibitor, zVAD (z-Val-Ala-Asp-fluoromethyl-ketone, Sigma, St. Louis, MO). The zVAD concentration was chosen based on the findings of a past study that documented effective inhibition of apoptosis in cell culture preparations [22].
After completion of the respective incubation periods, myocytes were resuspended with either cell culture media (normothermic and HCA only groups) or zVAD supplemented cell culture media at 37°C (10 µM zVAD, HCA+zVAD group) and myocyte contractile function determined. After measurement of basal contractile function, myocytes were exposed to 25 nmol/L isoproterenol and contractile function measurements were repeated. This concentration of isoproterenol has been previously shown to produce a maximal response in porcine myocytes [21].
Myocyte viability assay
Reduction of the tetrazolium salt 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) by mitochodrial dehydrogenases to an insoluble chromagen was used to assay myocyte viability [23]. Myocytes from each of the incubation protocols listed above were incubated with MTT (0.1 mg/mL) for 1 hour at 37°C in a 96-well plate (104 cells/well). The wells were washed with phosphate buffered saline and 200 µL of 0.5N HCl added to each well. The resultant absorbance was read at 540 nm (Labsystems 355, Helsinki, Finland) to determine myocyte viability. Quadruplicate samples from each of the incubation protocols were assayed and the absorbance readings averaged.
Immunohistochemistry
After incubation for 2 hours in either normothermic cell culture media, HCA, or HCA supplemented with zVAD, a subset of the LV myocytes were fixed and immunohistochemically stained for 
-actinin using methods described previously [24]. Briefly, LV myocytes that were previously fixed in 70% ethanol and stored in an EDTA containing buffer were plated on microscope slides coated with poly-L-lysine. Immediately before being immunostained, the myocytes were permeabilized with 1% Triton-X-100 and washed with phosphate-buffered saline. Myocyte preparations were incubated with 15% goat serum for 2 hours at 4°C, washed with phosphate-buffered saline, flooded with primary antiserum for -actinin (1:500), and incubated for 2 hours at 4°C. After incubation and a stringent wash, myocytes were incubated with a 1:1000 dilution of mouse antigoat secondary antibody conjugated with Texas Red. The immunostained LV myocytes were then examined and imaged by fluorescence microscopy (Zeiss Axioskop2, München-Hallbergmoos, Germany). Negative controls for the immunostaining procedure included substitution of the primary antiserum with phosphate-buffered saline.
Data analysis
Indices of myocyte function were compared between the normothermic and HCA groups were using analysis of variance (ANOVA). Each pig was considered a complete block. Thus, the number of myocytes studied from each animal was considered as repeated observations within each block. If the ANOVA revealed significant differences, pairwise tests of individual group means were compared with the use of Bonferroni's probabilities. All statistical analysis was performed with standard statistical software programs (BMDP Statistical Software, University of California Press). Results are presented as mean ± SEM. Values of p less than 0.05 were considered to be statistically significant.
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Results
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Myocyte viability
Myocyte viability was assayed after 2 hours of incubation in normothermic cell culture medium, simulated HCA, or in HCA augmented with the caspase inhibitor, zVAD (HCA+zVAD). In the normothermic group, the absorbance of the mitochondrial oxidative endproduct was 0.028 ± 0.005 and was not different from the HCA only (0.0273 ± 0.007) or the HCA+zVAD groups (0.0252 ± 0.011). Nevertheless, clearly defined alterations with respect to sarcomere registry could be visualized in those myocytes in the HCA only group. Specifically, immunohistochemical staining for -actinin (Fig 1)
revealed that staining for this protein was diffuse (Fig 1, B) when compared with myocytes incubated in normothermic cell culture medium (Fig 1, A). For myocytes in the HCA+zVAD group (Fig 1, C), the staining pattern for -actinin revealed a punctuate sarcomeric registration.
Myocyte contractile function
Contractility was measured in more than 2,500 individual myocytes (minimum of 208 myocytes per pig). However, contractile function determined from all myocytes of each pig was averaged and the final sample size reported is the number of pigs. Representative steady-state contraction profiles for myocytes from the normothermic, HCA only, and HCA+zVAD groups are shown in Figure 2.
Resting myocyte length and contractile function for the normothermic, HCA only, and HCA+zVAD groups are summarized in Table 1.
Myocyte resting length was significantly lower both HCA groups compared with normothermic controls. A significant reduction in contractile function was observed for myocytes in the HCA only group. For example, myocyte percent shortening was reduced by greater than 40% in myocytes in the HCA only group relative to the normothermic values. In both HCA groups, basal myocyte shortening velocity was lower than the normothermic group. However, shortening velocity in the HCA+zVAD group was higher than HCA only values. Similarly, relengthening velocity was higher in the HCA+zVAD group than HCA only values. An index of active relaxation, the time to 50% relaxation, tended to be greater in the HCA only group than normothermic values (p = 0.12).
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Fig 2. Representative myocyte contraction profiles after 2 hours of incubation in normothermic cell culture medium (solid circles), simulated hyperkalemic cardioplegic arrest (HCA [squares]), or in HCA augmented with the caspase inhibitor, zVAD (triangles). Myocytes were electrically stimulated at 1 Hz from a control myocyte and changes in myocyte length were digitized. Myocyte resting lengths and contraction amplitudes were lower in both HCA groups compared with normothermic values.
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Table 1. Isolated Myocyte Contractile Function After Simulated Hyperkalemic Cardioplegic Arrest (HCA) and Rewarming: Effects of Broad-Spectrum Caspase Inhibition With zVAD-fmk (HCA + zVAD)
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In light of the fact that basal myocyte shortening velocity was lower in the HCA groups, myocyte inotropic responsiveness was determined as the change in myocyte shortening velocity after ß-adrenergic stimulation and the results are summarized in Figure 3.
While myocyte shortening velocity with ß-adrenergic stimulation increased from baseline values in all groups, the change in myocyte shortening velocity was lower in both HCA groups compared with normothermic values. In the HCA+zVAD group, however, myocyte shortening velocity after ß-adrenergic stimulation was higher than HCA only values.
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Fig 3. Myocyte ß-adrenergic response in normothermic myocytes, myocytes exposed to simulated hyperkalemic cardioplegic arrest (HCA), and in myocytes incubated in HCA augmented with the caspase inhibitor, zVAD (HCA+zVAD). Myocyte ß-adrenergic responsiveness was reduced from normothermic values in both HCA groups. Compared with the HCA-only group, myocyte ß-adrenergic responsiveness was increased in the HCA+zVAD group. *p less than 0.05 versus normothermic; +p less than 0.05 versus HCA.
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Comment
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Hyperkalemic cardioplegic arrest has remained the mainstay for achieving myocardial quiescence during cardiac surgery [1, 3]. However, transient left ventricular (LV) pump dysfunction can occur after HCA and rewarming [2, 4]. Past studies from this laboratory have demonstrated that a cellular mechanism that can contribute to LV pump dysfunction after HCA and rewarming is a reduction in isolated myocyte contractility [12–14]. The caspases, which are intracellular proteases, can degrade components of the myofibrillar apparatus [19, 20], and a past study has demonstrated that caspase inhibition attenuated the degree of contractile dysfunction in ex vivo myocardial preparations [19]. The present study examined whether caspase inhibition during simulated HCA would improve myocyte contractile function upon rewarming. The significant finding of the present study was that supplementation of HCA solution with a broad-spectrum caspase inhibitor attenuated the decline in myocyte contractility upon rewarming, independent of changes in myocyte viability.
Hyperkalemic exposure of myocytes is associated with increased intracellular Ca2+ and H+ levels upon reperfusion [5, 8, 9, 11]. Alterations in ionic homeostasis can induce a number of changes in the myocyte structure and function including cellular edema and shrinkage, perturbation of membrane excitation, and disturbances in the functioning of intracellular and sarcolemmal ionic processes [9, 12, 25–28]. Specifically, increased intracellular Ca2+ levels can alter myofilament sensitivity to Ca2+ and disrupt crossbridge formation and dissociation [25, 26]. In addition, increases in intracellular Ca2+ levels have been demonstrated to activate intracellular proteases that can degrade components of the myofilament apparatus [19, 20]. Therefore, hyperkalemia induced changes in intracellular Ca2+ levels may have contributed, through direct or indirect actions on the myofilament apparatus, to myocyte contractile dysfunction that was observed in the present study.
The caspases are intracellular cysteine proteases that have been described to mediate the process of programed cell death, or apoptosis [17–19]. To date, at least 14 caspases have been characterized in mammalian tissues, with several being identified to be present in cardiac myocytes [15, 17]. Although the precise signaling pathways are not fully understood, an important intracellular stimulus of caspase activation is elevation of cytosolic Ca2+ levels [16]. Substrates that have been identified to be activated or degraded by activated caspases include DNases, ribose polymerase, kinases, and structural proteins such as fodrin and vimentin [17]. In the present study, myocytes in the HCA only group demonstrated a loss of sarcomeric registration with cardioplegic arrest, suggesting alterations in the contractile apparatus. Caspase inhibition appeared to attenuate this change in sarcomeric structure and was concordant with an improvement in contractile function. These results suggest that caspase inhibition improved myofilament alignment within the myocyte and thereby provides a subcellular mechanism for the improvement in contractile function with caspase inhibition.
These observations are consistent with recent studies that have suggested that caspase activation can cause contractile protein degradation within viable myocytes and contribute to changes in contractile performance. Specifically, Communal and associates [19] demonstrated that the myofibrillar proteins such as -actin, -actinin, and the troponin complex could be proteolyzed by caspases. In addition, these investigators demonstrated that exposure of muscle fibers to caspases reduced contraction force, and that this effect could be ameliorated with the addition of a caspase inhibitor [19]. In the present study, supplementation of the HCA solution with a membrane-permeable caspase inhibitor attenuated the degree of myocyte contractile dysfunction with rewarming. Therefore, these results suggest that hyperkalemic exposure caused intracellular caspase activation and that myocyte contractile dysfunction after HCA and rewarming was due, at least in part, to caspase-mediated proteolysis of the myofibrillar apparatus. It is important to note that in the present study, myocyte viability was not altered by simulated HCA and rewarming. This finding suggests that the effects of caspase inhibition on myocyte contractility after HCA and rewarming occurred independently of a commitment of the myocytes to the apoptotic cascade. Nevertheless, future studies that more carefully examine the effects of caspase inhibition on components of the apoptotic cascade in the setting of simulated hyperkalemic arrest are warranted.
Stimulation of the ß-adrenergic receptors results in the activation of an intracellular signaling cascade that modulates Ca2+ dynamics, and causes an increase in intracellular Ca2+ content [21, 29]. Past studies have demonstrated that independent of other known stimuli, ß-adrenergic receptor stimulation can cause caspase activation in ventricular myocytes [19, 30, 31]. Therefore, the combination of hyperkalemic exposure and ß-adrenergic receptor stimulation, as was employed in the present study, may have provided an additive or synergistic effects on myocyte intracellular Ca2+ levels, and thereby, an increase in Ca2+-mediated caspase activation. While this issue remains speculative, the present study demonstrated that myocyte contractile response to ß-adrenergic stimulation after HCA and rewarming was improved with caspase inhibition. In light of the fact that ß-adrenergic receptor agonists are administered to patients in the postcardiopulmonary bypass setting, this observation may hold particular clinical relevance.
Study limitations, future directions, and conclusions
Through the use of a caspase inhibitor, the role of caspase activation in myocyte contractile dysfunction after HCA and rewarming was indirectly assessed. Moreover, whether and to what degree caspase-mediated proteolysis of the contractile proteins occurred with simulated HCA and rewarming was not determined. In light of the findings of the present study, careful examination of these intracellular processes is warranted. In addition, the present study was not designed to separate effects of hyperkalemia from those of hypothermia on myocyte contractile function when the HCA solution was supplemented with a caspase inhibitor. A past study has demonstrated that hypothermic incubation followed by rewarming did not cause a decline in myocyte contractile function when compared with myocytes incubated in normothermic cell culture medium [13]. Thus, it is likely that the effects of caspase inhibition with HCA occurred due to modulation of intracellular processes induced by hyperkalemic exposure. Finally, it must be recognized that beneficial effects of caspase inhibitor supplementation of the HCA solution occurred in an isolated myocyte model and may not reflect conditions encountered in vivo. Therefore, extrapolation of the findings of the present study to the clinical situation must be undertaken with caution. Nevertheless, the findings from the present study suggest that supplementation of hyperkalemia solutions with caspase inhibitors may provide a novel and useful therapeutic modality.
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Acknowledgments
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-66029, HL-45024, HL-97012, and PO1–48788.
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Abstract
Introduction
