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Ann Thorac Surg 2007;84:857-862
© 2007 The Society of Thoracic Surgeons
Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri
Accepted for publication April 24, 2007.
* Address correspondence to Dr Lawton, Washington University School of Medicine, 660 S Euclid Ave, Campus Box 8234, St. Louis, MO 63110 (Email: lawtonj{at}wustl.edu).
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Abstract |
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Methods: Isolated rabbit myocytes were perfused with Tyrode’s control solution for 20 minutes, followed by test solution for 20 minutes. Test solutions included (1) Tyrode’s control, (2) a metabolic inhibition solution containing sodium cyanide and 2-deoxyglucose, (3) metabolic inhibition plus diazoxide, (4) metabolic inhibition plus diazoxide plus HMR1098 (a sarcolemmal KATP-channel blocker), or (5) metabolic inhibition plus diazoxide plus 5-hydroxydeconoate (a mitochondrial KATP-channel blocker). Myocytes were then reexposed to Tyrode’s solution for 20 minutes. Volume measurements were taken every 5 minutes. Contractility was recorded using edge-detection software at baseline and at 10 and 20 minutes of reexposure to Tyrode’s solution.
Results: Simulated ischemia (metabolic inhibition) caused significant myocyte swelling and associated reduced contractility. The addition of diazoxide abolished myocyte swelling and attenuated the associated reduced contractility. Observations with diazoxide were unchanged by the addition of HMR 1098 or 5-hydroxydeconoate.
Conclusions: Diazoxide, with or without either KATP-channel blocker, attenuated the significant myocyte swelling and reduced contractility secondary to metabolic inhibition. These data suggest a role for diazoxide, independent of the KATP channel, in myocyte volume homeostasis. In addition, the prevention of myocyte swelling resulted in improved contractility, consistent with previous data and the hypothesis that myocyte swelling may participate in the phenomenon of myocardial stunning.
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Introduction |
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The KATP channel is felt to be cardioprotective, is intrinsically linked to cellular metabolism and ATP levels, and has the highest density of all channels in the heart (approximately 2000 to 3000 channels per cell) [4]. Of interest is that other investigators have demonstrated the prevention of myocardial edema in an isolated heart model by the opening of the KATP channel [5]. In addition, ischemic preconditioning has been found to provide resistance to osmotic cell swelling and to ameliorate ischemic cell swelling at the cellular and mitochondrial levels, and the KATP channel is known to be an important component of the phenomenon of ischemic preconditioning [6, 7]. We have also noted that the addition of a KATP-channel opener to normal physiologic solution is associated with a reduction in myocyte volume at normothermic temperatures [2]. These findings suggest a potential role of the KATP channel—or the channel opener—in myocyte volume regulation.
Ischemia is also known to independently result in significant myocyte swelling [8]. The investigation of myocyte structural derangement secondary to ischemia, its relationship to myocyte function, and the effect of the addition of a KATP-channel opener will enhance the knowledge of the role of the KATP channel in myocyte volume regulation. This study was performed to determine if simulated myocyte ischemia (metabolic inhibition) results in significant myocyte swelling and associated contractile dysfunction and if these detrimental consequences may be prevented by the addition of diazoxide, a KATP-channel opener.
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Material and Methods |
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Isolation of Rabbit Ventricular Myocytes
New Zealand White rabbits (either sex, 3 to 4 kg) were anesthetized intramuscularly with xylazine (14.0 mg/kg), acepromazine (1.3 mg/kg), and ketamine (83.0 mg/kg), and received 3000 units of intravenous heparin. A maximum of 2 myocytes were used from each rabbit for analysis. Each animal underwent sternotomy and rapid cardiectomy.
Each heart was attached to a Langendorff column for retrograde perfusion (37°C) for 5 minutes with a solution containing 1.8 mM of CaCl2 in Tyrode’s physiologic solution consisting of (in mmol/L) 130 NaCl, 5 KCl, 0.4 KHPO4, 3 MgCl2, 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 15 taurine, 10 glucose, and 5.7 creatine (pH adjusted to 7.25 by 20% NaOH titration). Extracellular Ca2+ was washed out by perfusion of a solution containing 0.1 mM of sodium ethylene glycol tetraacetic acid [EGTA] in Tyrode’s physiologic solution for 5 minutes. Solutions were equilibrated with 95% O2 and 5% CO2.
The hearts were then perfused with 1500 mg/L bovine serum albumin (Sigma, St. Louis, MO), 400 mg/L collagenase type II (Worthington Biomedical, Freehold, NJ), and 50 mg/L of protease (Sigma) in Tyrode’s physiologic solution for 18 minutes to provide enzymatic digestion.
Ventricles were removed and minced over nylon mesh to remove debris, placed into Kraftbruhe (KB) solution containing (in mM/L) 120 potassium glutamate, 10 KCl, 10 KH2PO4, 1.8 MgSO4, 0.5 K2EGTA, 10 taurine, 10 HEPES, and 20 glucose, and stored at room temperature for 30 minutes to allow settling.
Myocyte Imaging and Volume Measurement
Myocytes were visualized on a slide on a glass-bottomed chamber using an inverted microscope stage (Leitz, Wetzlar, Germany) equipped with Hoffman modulation optics (Modulation Optics, Greenvale, NY). After a 5-minute stabilization period, the chamber was perfused at a rate of 3 mL/min with an altered control solution of Tyrode’s consisting of (in mM/L) 130 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 24 NaHCO3, 1.75 Na2HPO4, and 10 glucose buffered to a pH of 7.4 using 95% O2 and 5% CO2. Chamber temperature was maintained throughout the experiments at 37°C by a water bath system (Thermo Haake, Karlsruhe, Germany).
Cell images were displayed on a video monitor using a charge-coupled device camera (KPM1U, Hitachi Denshi, Tokyo, Japan). The cell images were captured using a video-frame grabber (Scion Corporation, Frederick, MD). Cell borders were manually traced and length, width, and area were measured using Scion Image software (Scion Corporation, Frederick, MD).
Assuming that the changes in cell width and thickness were proportional, relative cell volume was determined by the following formula volume t / volume c = (area t x width t)/(area c x width c) [9] where t and c refer to test and control, respectively. On the basis of repeated measurements of single images and measurements of multiple images of a cell, this methodology for estimating cell volume has been shown to be reproducible to less than 1% [9].
Myocyte Contractility
Myocyte contractility was measured using a video-based edge detection system (IonOptix, Milton, MA). Cells were paced using a field stimulator (MyoPacer, IonOptix, Milton, MA) at a voltage 10% above threshold at a frequency of 1 Hz with a 5-millisecond duration to avoid the occurrence of fusion beats. Polarity of the stimulator was altered at every other stimulation to avoid possible build-up of electrolyte by-products at one electrode. After a 5-minute stimulation period, data were obtained from 25 to 30 consecutive beats and averaged.
The variables measured were percentage of cell shortening, maximal velocity of shortening, and maximal velocity of relengthening. Percentage of shortening is the amount of contraction along the longitudinal plane of the cell compared with the resting length of the cell. Velocity of shortening is the briskness of contraction, defined as the derivative of the change in length from resting length until maximal shortening over time. Velocity of relengthening is the cell’s ability to quickly recover from contraction, defined as the derivative of the change in length from maximal shortening until return to resting length over time. Cells that showed less than 7% cell shortening at baseline period were excluded. These variables were measured at baseline and after 10 and 20 minutes of reexposure to control Tyrode’s solution.
Experimental Protocol
Cells were perfused for 20 minutes in control solution at 37°C to obtain baseline volume and contractility measurements. Cells (n = 8 myocytes in each group, up to 2 myocytes per rabbit) were then perfused for 20 minutes with test solution at 37°C, followed by a 20-minute reexposure period to the control solution at 37°C.
Test solutions included: (1) control, (2) metabolic inhibition solution, (3) metabolic inhibition with diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine-1,1-dioxide 100 µM/L, Sigma), a KATP-channel opener, (4) metabolic inhibition with diazoxide and HMR 1098 (40 µM/L, a sarcolemmal KATP-channel blocker, gift from Aventis Pharma Deutschland Gmbh, Frankfurt, Germany), and (5) metabolic inhibition with diazoxide and 100 µM/L 5-hyrdroxydecanoate (5HD), a mitochondrial KATP-channel blocker (Sigma). The metabolic inhibition solution consisted of (in mM/L) 130 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 24 NaHCO3, 1.75 Na2HPO4, 5 2-deoxyglucose (2DG), and 5 NaCN (sodium cyanide); pH 7.4 and 310 mOsm. The diazoxide dose of 100 µM/L was effective in ameliorating cell swelling secondary to hyperkalemic cardioplegia in previous studies and has been used by other investigators [10]. Ischemia simulated by the use of 2DG and sodium cyanide results in complete metabolic inhibition [11–14].
Stock solutions of diazoxide and HMR 1098 were made by dissolving each reagent in 0.1% dimethyl sulfoxide, which has no effect on cell volume [9]. A stock solution of 5HD was made by dissolving it in deionized water.
Statistical Analysis
Data were analyzed using SYSTAT 11 (SYSTAT Software Inc, Point Richmond, CA). All data are presented as mean value ± standard error of the mean. A repeated-measures analysis of variance was used for sequential time-based measurements for each test solution against its own baseline value. The Fisher least significant difference test was used to make separate post hoc multiple comparisons between different test groups during the test solution and reexposure periods. Values of p < 0.05 were considered significant.
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Results |
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Comment |
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This and previous studies have documented that three independent stresses each individually result in significant myocyte swelling: hyperkalemic cardioplegia (9°C) [2], hyposmotic stress [3], and metabolic inhibition (present study). Exposure to hyperkalemic cardioplegia results in a hyposmotic extracellular environment, leading to cell swelling [9]. Hyposmotic stress results in cell swelling due to exposure to a hyposmotic extracellular environment. Metabolic inhibition results in myocyte swelling due to buildup of intracellular metabolites and a resultant hyperosmotic intracellular milieu [8]. Drewnowska and colleagues [9] have also suggested that hyperkalemic cardioplegia specifically results in cellular swelling due to K+ and Cl– influx because swelling was partially blocked by replacing Cl– with an impermeant anion. It is therefore intriguing that diazoxide, a KATP-channel opener, was found to prevent myocyte swelling due to all three stresses.
Diazoxide Mechanism of Action
The KATP channel provides endogenous protection by means of its unique property of coupling cell membrane potential to myocardial metabolism; however, the exact mechanism of its cardioprotection is unknown [16, 17]. Opening the sarcolemmal KATP channel results in K+ efflux from the cell, whereas opening of the mitochondrial KATP channel is associated with K+ influx from the cytosol into the mitochondria. In this study, the addition of diazoxide, a KATP-channel opener, prevented the detrimental increase in myocyte volume and ameliorated the associated decline in myocyte function after metabolic inhibition. This would support a hypothesis that the KATP channel—or the channel opener—plays some cardioprotective role during stress, perhaps through the maintenance of volume homeostasis related to K+ flux (accompanied by anion and obligated water movement).
Potential alternate mechanisms involving adenosine, protein kinase C, or chloride (Cl–)-channel opening are suggested by the work of Diaz and colleagues [7], who documented that ischemic preconditioning by brief ischemic episodes or pharmacologic methods limits myocyte swelling during subsequent myocardial ischemia. Inhibition of adenosine receptors, protein kinase C, or Cl– channels blocked the beneficial effect on cell volume maintenance during subsequent ischemia [7]. They postulated that a cell volume regulatory mechanism was placed in motion by ischemic preconditioning, was still functional, and limited subsequent ischemic cell swelling [7]. The work of Diaz and colleagues is applicable to the results of this study because the KATP channel is known to be an important component in the phenomenon of ischemic preconditioning; however, this protocol was not designed to mimic conditions of ischemic preconditioning [6].
In the present study, the addition of 5HD (mitochondrial KATP-channel blocker) or HMR 1098 (sarcolemmal KATP-channel blocker) did not reverse the beneficial effect observed by diazoxide after metabolic inhibition. These findings are consistent with our previous work in which HMR 1098 did not inhibit the beneficial effects of diazoxide or pinacidil in animal and human myocytes [2; unpublished data] and the work of others who have found that 5HD does not inhibit the protective effects of diazoxide [18]. However, these results are not consistent with other reports documenting a reversal or a partial reversal of diazoxide’s cardioprotection with the use of 5HD [14, 17, 19, 20]. Some of these differences may be explained by the diversity in the models and drug dosages used. These conflicting results may also suggest the nonspecificity of channel openers or blockers or a KATP channel–independent site of action for diazoxide.
The specificity of KATP-channel openers and blockers has been challenged by other investigators. Diazoxide (claimed mitochondrial specific KATP-channel opener) has been demonstrated to open sarcolemmal KATP channels in the presence of adenosine diphosphate, and HMR 1098 (claimed specific sarcolemmal KATP-channel inhibitor) may not be entirely specific to the sarcolemmal channel [16, 21]. Other investigators have criticized the use of 5HD because of its potential lack of specificity to the mitochondrial KATP channel and its channel-independent actions [22]. KATP channel–independent targets of diazoxide have been described; most frequently, a decrease in succinate oxidation by the inhibition of the enzyme succinate dehydrogenase, which is also vital, by generation of flavin adenine dinucleotide (FADH2), to the respiratory chain [23, 24].
Investigators have proposed that some other effect on mitochondrial metabolism is responsible for cardioprotection, such as a link between partial inhibition of electron transport and pharmacologic preconditioning, perhaps by way of the generation of reactive oxygen species [24, 25]. In addition, Halestrap and colleagues [16] suggest that other pharmacologic effects of "specific" mitochondrial KATP-channel openers or inhibitors are responsible for the cardioprotective findings, such as the activation of protein kinase pathways. Rodrigo and colleagues [25] proposed that diazoxide works by an indirect mechanism at the level of the mitochondria rather than by opening the sarcolemmal KATP channel.
Other researchers have attributed the beneficial effect of diazoxide to its activation of a mitochondrial KATP channel [14], and the opening of the mitochondrial KATP channel has been suggested to be cardioprotective in multiple animal models [26]. However, the mere existence of the mitochondrial KATP channel has been questioned because its genetic composition is unknown [16, 22, 23, 27, 28]. Much of the data available claiming to support the existence of a mitochondrial KATP channel have relied on its pharmacologic "opening" or "inhibition" by drugs that are proposed to be specific to the mitochondrial channel [22–24]. Direct mitochondrial KATP channel activity has been suggested by various methods, including measurement of mitochondrial flavoprotein oxidation by assay or patch clamping of the inner mitochondrial membrane; however, some investigators have criticized these methods [16, 23, 29].
Some investigators suggest that succinate dehydrogenase forms a structural and a functional component of a supercomplex that forms the mitochondrial KATP channel and that succinate dehydrogenase regulates mitochondrial KATP by means of its physical interaction and not by means of its action on oxidative phosphorylation [28]. Others reiterate that any definitive role of the mitochondrial KATP channel awaits discovery of the molecular structure of this channel and stress that there are no sequences in the human genomic database that are likely candidates for a mitochondrial sulfonylurea receptor [23, 25]. The pharmacologic manipulation of the channel alone may therefore only suggest or presume a role of the channel itself; thus, the protective mechanism of diazoxide remains unclear and further work is needed to confirm its role in myocyte volume homeostasis.
Relationship Between Myocyte Volume and Function
These data confirm that exposure of isolated myocytes to simulated ischemia (metabolic inhibition) results in a significant increase in myocyte volume that is associated with a decline in myocyte function. It has been our hypothesis that myocyte swelling due to ischemia or hyperkalemic cardioplegia, or both, is one mechanism of myocardial stunning. In previous work, exposure to hyperkalemic cardioplegia alone resulted in an approximate 6% increase in myocyte volume that was associated with a 10% to 20% reduction in contractility [2]. Similar results of about a 6% increase in volume and a 10% reduction in contractility were noted after mild (0.9 times control) hyposmotic stress [3]. In the present study, an approximate 10% increase in myocyte volume was associated with an approximate 20% reduction in myocyte contractility after metabolic inhibition. Thus, an inverse relationship between myocyte volume derangement and myocyte contractility derangement may be established.
Previous work using hyperosmotic stress is also consistent with an inverse relationship between myocyte volume and contractility: an approximate 30% reduction in cellular volume secondary to hyperosmotic stress was associated with an increase of about 10% in myocyte contractility [3]. Of interest was that the amelioration of myocyte swelling of up to 10% of baseline volume secondary to each stress (hyperkalemic cardioplegia, hyposmotic stress, or metabolic inhibition) also resulted in elimination of the associated contractility reduction [1–3]. Thus, the volume derangement and the functional derangement appear to be related.
Mechanisms Underlying Myocyte Functional Derangement
The mechanisms underlying a derangement in excitation–contraction coupling after myocyte swelling are largely unknown [8]. A mechanical distortion of the plasma membrane may alter the structure of ion channels, such as the inward currents of Na+ or Ca2+, or both. These alterations would lead to significant changes in action potential duration and would limit the subsequent triggering of Ca2+ release by the sarcoplasmic reticulum, thus limiting calcium availability for binding to troponin C and the contractile proteins [30]. Li and colleagues [8] documented a biphasic (an increase followed by a decrease) alteration in cell shortening during 20 minutes of exposure to hyposmotic stress in isolated myocytes. The late (after 8 minutes) decrease in cell shortening was paralleled by a decrease in action potential duration, decrease in intracellular Ca2+, and a decrease in Ca2+ current, suggesting a relationship to the functional change [8]. Cell shortening also continued to be reduced upon reexposure to an isotonic solution, which is consistent with the results of this study.
Clearly, the limitation of availability of calcium to the contractile apparatus will significantly alter the force of subsequent contraction [30]. In addition, the opening of swell-activated channels may play a role in the derangement of contractility at the myocyte level. These observations provide an opportunity for future work to investigate the mechanisms involved after volume alteration at the cellular level.
Because of its relationship to myocyte function, myocyte swelling after stress may provide mechanistic insight into the phenomenon of myocardial stunning. Diazoxide has a beneficial effect on myocyte volume homeostasis during stress and provides improved myocyte functional recovery after simulated ischemia. Similar results were noted after isolated myocyte exposure to hypothermic hyperkalemic cardioplegia and mild hyposmotic stress. The mechanisms remain largely unknown. After the elucidation of its mechanism of action, diazoxide may prove to be clinically useful by its ability to limit functional derangement (stunning) after myocardial ischemia.
Study Limitations
Isolated myocytes were used because they allow for the repeated measurements of cell volume and function. Myocytes were exposed to crystalloid solutions in the absence of blood. Caution must be taken in applying the findings in this study to the whole organ or organism level because they do not reflect the complexity of the extracellular space and vascular, hormonal, and neural elements.
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Acknowledgments |
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