beneficial_effect_of_calcium_treatment_for359.pdf

LABORATORY INVESTIGATION Beneficial Effect of Calcium Treatment for...

LABORATORY INVESTIGATION Beneficial Effect of Calcium Treatment for Hyperkalemia Is Not Due to “Membrane Stabilization” Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 Joseph S. Piktel, MD1 OBJECTIVES: Hyperkalemia is a common life-threatening condition causing se- Xiaoping Wan, MD, PhD2 vere electrophysiologic derangements and arrhythmias. The beneficial effects of Shalen Kouk, MD3 calcium (Ca2+) treatment for hyperkalemia have been attributed to “membrane sta- bilization,” by restoration of resting membrane potential (RMP). However, the un- Kenneth R. Laurita, PhD4 derlying mechanisms remain poorly understood. Our objective was to investigate Lance D. Wilson, MD1 the mechanisms underlying adverse electrophysiologic effects of hyperkalemia and the therapeutic effects of Ca2+ treatment. DESIGN: Controlled experimental trial. SETTING: Laboratory investigation. SUBJECTS: Canine myocytes and tissue preparations. INTERVENTIONS AND MEASUREMENTS: Optical action potentials and volume averaged electrocardiograms were recorded from the transmural wall of ventricular wedge preparations (n = 7) at baseline (4 mM potassium), hyperka- lemia (8–12 mM), and hyperkalemia + Ca2+ (3.6 mM). Isolated myocytes were studied during hyperkalemia (8 mM) and after Ca2+ treatment (6 mM) to determine cellular RMP. MAIN RESULTS: Hyperkalemia markedly slowed conduction velocity (CV, by 67% ± 7%; p < 0.001) and homogeneously shortened action potential duration (APD, by 20% ± 10%; p < 0.002). In all preparations, this resulted in QRS widen- ing and the “sine wave” pattern observed in severe hyperkalemia. Ca2+ treatment restored CV (increase by 44% ± 18%; p < 0.02), resulting in narrowing of the QRS and normalization of the electrocardiogram, but did not restore APD. RMP was significantly elevated by hyperkalemia; however, it was not restored with Ca2+ treatment suggesting a mechanism unrelated to “membrane stabilization.” In addition, the effect of Ca2+ was attenuated during L-type Ca2+ channel blockade, suggesting a mechanism related to Ca2+-dependent (rather than normally sodium- dependent) conduction. CONCLUSIONS: These data suggest that Ca2+ treatment for hyperkalemia restores conduction through Ca2+-dependent propagation, rather than restoration of membrane potential or “membrane stabilization.” Our findings provide a mech- anistic rationale for Ca2+ treatment when hyperkalemia produces abnormalities of conduction (i.e., QRS prolongation). KEYWORDS: calcium; cardiac conduction; electrocardiography; hyperkalemia; resting membrane potential H yperkalemia is a common electrolyte abnormality seen in critical care Copyright © 2024 by the Society of settings and is potentially life threatening (1, 2). Elevated extracellular Critical Care Medicine and Wolters potassium concentration can result in well-described electrocardiogram Kluwer Health, Inc. All Rights (ECG) changes. Generally, during mild to moderate hyperkalemia, shortened, Reserved. peaked T waves are observed, indicating an effect on ventricular repolarization. DOI: 10.1097/CCM.0000000000006376 Critical Care Medicine www.ccmjournal.org     1 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Piktel et al treating hyperkalemia with Ca2+ (12). Whether IV Ca2+ is protective by improving hyperkalemic abnormalities KEY POINTS in only conduction, repolarization, or both is poorly understood. Furthermore, improved understanding of Question: To determine the mechanisms under- these effects would help guide clinicians in appropriate lying adverse electrophysiologic effects of hyper- use of Ca2+ treatment in hyperkalemia. Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy kalemia and the therapeutic effects of calcium In this study we use ex vivo tissue and in vitro cel- (Ca2+) to better inform treatment of hyperkalemia. lular approaches to determine the electrophysiologic wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 Findings: These data demonstrate that Ca2+ basis for the beneficial effect of Ca2+ on the ECG and treatment for hyperkalemia restores conduction arrhythmia substrates. Given that Ca2+ improves con- by promoting calcium inward current-dependent duction in conditions when cardiomyocyte RMP is propagation, rather than restoration of resting membrane potential or “membrane stabilization.” markedly elevated, such as in acute ischemia (7), we hypothesized that the beneficial effects of Ca2+ treat- Meaning: This study provides a mechanistic ra- ment during hyperkalemia are based on direct pres- tionale for Ca2+ treatment when hyperkalemia ervation of cardiac conduction, but not effects on produces abnormalities of conduction (i.e., QRS prolongation). membrane stabilization or cardiac repolarization. MATERIALS AND METHODS During more severe hyperkalemia, conduction slow- All experiments were carried out in accordance with ing occurs, producing a widened QRS interval on the Public Health Service guidelines for the care and use of ECG, which when severe, eventually merges with the T laboratory animals and approved by our institutional wave, creating a sine wave pattern. Arrhythmias attrib- animal care and use committee (Board name: CWRU utable to hyperkalemia include bradycardias, conduc- IACUC, Study Number: Protocol 070161, Study title: tion block, and ventricular tachycardia and fibrillation, Cell Repolarization, Alternans, and Arrhythmogenesis, resulting in cardiovascular dysfunction and death (1, Approval Date: January 18, 2008, Animal Welfare 3, 4). However, the effects of elevated potassium on Assurance No. A3145-01). Eight adult, male, random arrhythmia pathophysiology (i.e., substrates) are not source, mongrel dogs were used in this study. To min- fully understood (1). Previously it has been shown that imize animal pain and suffering, these experiments hyperkalemia heterogeneously alters the action poten- were conducted on tissue collected by organ harvest tial (AP) duration (APD) in epicardial and endocardial only after deep surgical anesthesia was established with myocytes (5). Heterogeneities in APD increase spatial pentobarbital. The canine species was chosen because dispersion of repolarization (DOR), which can lead of the well-known electrophysiologic similarities to to the development of reentrant arrhythmias, ventric- humans and the canine wedge preparation is an estab- ular tachycardia/fibrillation, and sudden cardiac death. lished model for the human ECG (13). Male dogs were Hyperkalemia also elevates cellular resting membrane specifically used to eliminate variability induced by sex- potential (RMP), which decreases cellular fast sodium related heterogeneities in repolarization and sensitivity channel function and excitability, resulting in slowed to potassium (14). We used data from our prior work conduction and block, a prerequisite for the develop- in the canine wedge preparation, which examined con- ment of reentrant arrhythmias (1, 6, 7). duction and repolarization heterogeneities underlying IV calcium (Ca2+) is a common treatment for the ad- arrhythmias, as the basis for a sample size calculation verse cardiac effects of hyperkalemia, which have been used to estimate the number experiments and dogs re- attributed to its “membrane stabilizing effects,” a vague quired (please see Animal Research: Reporting of In term frequently used, and typically attributed to nor- Vivo Experiments guidelines for further detail) (15). malization of RMP (8–11). However, despite decades of We used two complimentary approaches: 1) the canine everyday use, the mechanism by which Ca2+ improves wedge preparation which allowed examination of the cardiac conduction and function in hyperkalemia re- effects of potassium and calcium on conduction, repo- mains incompletely understood (1). Consequently, there larization, and the ECG in a well-established tissue is significant heterogeneity in the clinical threshold for model and 2) isolated canine myocytes, which allows 2     www.ccmjournal.org XXX 2024 Volume 52 Number 00 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Laboratory Investigation for precise determination of cellular RMP under a va- and RG vectors between nearby sites of the 256 site riety of conditions but does not allow determination of array were calculated and averaged across the max- cell-to-cell conduction. imal direction of depolarization (CV) and repolariza- tion (RG) to obtain the average CV and RG per wedge. Optical Mapping in the Canine Wedge Measurements were always made during endocardial Preparation pacing (to reproduce normal endocardial to epicardial Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy activation in the heart). We have previously described our methods for optical wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 mapping in the canine wedge preparation (15–18). Isolated Cardiomyocyte Studies Briefly, the intact heart was rapidly excised by right lateral thoracotomy performed under pentobarbital Patch-Clamp Recordings. The amphotericin perforated (50 mg/mL IV) anesthesia. Transmural wedges of left patch technique was used to obtain whole-cell recordings ventricular myocardium were isolated and the branch of of membrane voltage under current-clamp conditions as the corresponding coronary artery was cannulated and described previously (23). Briefly, the cells were bathed perfused with Tyrode’s solution (140 mM sodium chlo- in a chamber continuously perfused with Tyrode’s solu- ride [NaCl], 4.0 mM potassium chloride [KCl], 1.8 mM tion composed of (mmol/L) NaCl 137, KCl 5.4, calcium Ca2+ chloride, 5.5 mM dextrose, 0.5 mM magnesium sul- chloride (CaCl2) 2.0, MgSO4 1.0, glucose 10, HEPES 10, fate [MgSo4], 0.9 mM monosodium phosphate, 10 mM and pH to 7.35 with NaOH. Patch pipettes were pulled 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid from borosilicate capillary glass and lightly fire-polished [HEPES], sodium hydroxide [NaOH] titrated to a pH to resistance 0.9–1.5 mol/LΩ when filled with electrode 7.41, and oxygenated with 100% oxygen). The wedge solution composed of (mmol/L) aspartic acid 120, KCl was then placed in the chamber with the transmural sur- 20, NaCl 10, magnesium chloride, HEPES 5, 240 μg/mL face against the glass imaging plate and perfused with a of amphotericin B (Sigma, St Louis, Mo), and pH 7.3. A voltage sensitive dye di-4-aminonaphthenyl-pyridinum- gigaseal was rapidly formed. Typically, 10 minutes later, propylsulfonate (8 µM). APs were recorded from 256 sites amphotericin pores lowered the resistance sufficiently simultaneously with high spatial (0.89–1.1 mm), tem- to current-clamp the cells. Myocytes were paced using poral (1 ms), and voltage (0.5 mv) resolution. Blebbistatin a 1.5–2 diastolic threshold and a 5-ms current pulse. (6 µM) was used to eliminate motion artifact. The im- Experiments were performed at 30°C. Command and aging chamber was insulated and temperature closely data acquisition were operated with an Axopatch 200B regulated by an insulated water circuit and measured patch-clamp amplifier controlled by a personal com- using a digital temperature probe (Omega Engineering puter using a Digidata 1200 acquisition board driven by Inc., Norwalk, CT) in the water bath, allowing for tem- pCLAMP 7.0 software (Axon Instruments, Foster City, perature precision of ±0.1°C (15). Measurements were CA). Cells were superfused with different [K+]o concen- made at baseline (4 mM) potassium concentration [K+]o tration at 4 and 8 mM KCl with and without additional in the Tyrode’s solution, and then increased to 8 mM, CaCl to increase Ca2+ concentration to 6 mM, with and and 12 mM [K+]o. Ca2+ was added to the 12 mM [K+]o without addition of verapamil (10 uM) or tetrodotoxin solution to create a Ca2+ concentration that was 2× the (120 uM). Myocytes were stimulated at a baseline stim- normal (3.6 mM). This is consistent with expected rise of ulation rate of 150 beats/min. After a period of stimula- Ca2+ observed after 20 mg/kg of IV Ca2+ (19). tion to establish steady state, measurements were made Groups and Methods of Measurement. APD was for the subsequent 20 beats/min. APD was measured at measured in any one transmural layer by averaging 90% repolarization. 5 epicardial, mid-myocardial, and endocardial cell APDs, respectively. Transmural cell types were defined Statistical Analysis by previously validated anatomic and functional cri- terion (18, 20). DOR was defined as the difference be- Statistical analysis was performed using IBM SPSS tween the APD of the longest and shortest cell type. Statistics 24 (IBM Corp, Armonk, NY) and Excel Conduction velocity (CV) and maximal repolarization (Microsoft, Redmond, WA). One-way analysis of gradients (RGs) were determined by a previously vali- variance was used to compare differences mean dated vector analysis technique (21, 22). Briefly, CV APD, CV, and DOR across wedge experiments Critical Care Medicine www.ccmjournal.org     3 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Piktel et al under different potassium concentrations and least square differences post hoc test was applied to test specific means. Student t tests (two-way, unpaired) were used to analyze the differences in RMP and APD under hyperkalemic conditions in isolated myocytes. All mean data are represented with the Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy value and sd. Previous power analyses suggested n = 5 per group to detect a significant difference in DOR wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 in the canine wedge with a power of 0.8 and an error of 0.05 (15). RESULTS The Effect of Hyperkalemia on Cardiac Figure 1. Hyperkalemia slows conduction, which is rescued by Conduction and Repolarization calcium treatment. Top row shows the effect of hyperkalemia and calcium (Ca2+) treatment on the electrocardiogram (ECG). Figure 1 shows the effect of hyperkalemia on the During hyperkalemia, a wide QRS showing a sine wave pattern is ECG, myocardial conduction, and repolarization. observed, which is normalized by Ca2+ treatment. Second row Hyperkalemia produced marked conduction slowing, shows endocardial (ENDO) and epicardial (EPI) action potentials (APs) at baseline, during hyperkalemia, and Ca2+ treatment. as evidenced by: 1) QRS widening of the ECG (upper At baseline, transmural conduction time (difference between row, ECG 4 vs. 12 mM [K+]o), 2) increase in time be- activation time of early activated ENDO and late activated tween depolarization of the early activated endocar- EPI APs is relatively short [red hatched lines]). Difference in dial AP and late activated epicardial AP (middle rows, repolarization times (a marker of dispersion of repolarization 4 vs. 12 mM [K+]o, red hatched boxes), and 3) marked [DOR]) is also shown (orange hatched lines). During hyperkalemia, transmural conduction slowing and block, in which marked conduction slowing occurs, with small prolongations in DOR. During Ca2+ treatment, conduction time normalizes. hyperkalemia (12 mM) shows significant crowding of Representative isochronal contour maps of conduction time activation time isochrones compared with baseline (third row) are also shown under the three conditions. Earliest (4 mM, lower row). Of note, there is also conduction conduction is in white, while later conduction or repolarization block observed in the subepicardium (solid line) dur- is shown by darker colors. Note that with 12 mM [K+], there ing hyperkalemia. Summary data of CV over all exper- is marked conduction slowing, as indicated by crowding of iments is shown in Figure 2. Hyperkalemia clearly isochrones and conduction block occurs in the subepicardium (dark line). With Ca2+ treatment, conduction velocity normalizes, slowed transmural conduction (by 67% ± 7%; p < 0.002 and conduction block is prevented. Summary data for mean action at 12 mM [K+]o). potential duration (APD) under each condition is also shown at As expected, hyperkalemia also shortened APD. the bottom row. Shown in Figure 1, middle rows, are endocardial and epicardial APs, where there is moderate shortening of DOR. Taken together, these data show that although endocardial and epicardial APs, with an average 20% APD is shortened during hyperkalemia, APD hetero- ± 10% decrease (p < 0.002). This observation was con- geneity does not increase and therefore would not nec- firmed in isolated myocytes, where APD shortening essarily contribute to increased arrhythmia risk. was observed at 8 mM [K+]o (average 47% decrease; p < 0.002; Fig. 3A, control vs. high K tracings). As AP The Effect of Ca2+ on Hyperkalemia-Induced heterogeneity and increased DOR can be a potent Conduction Slowing and Action Potential substrate for arrhythmias, we next examined whether Duration hyperkalemia increased heterogeneity of APD in the wedge preparation. Figure 4 shows average APD from Figure 1, far right column, shows normalization of endocardial and epicardial cell types across the trans- the ECG (upper row), when hyperkalemia is treated mural wall. Similar APD shortening during 12 mM with Ca2+ when compared with hyperkalemia alone [K+]o was observed between the different cell types, (center column). This is associated with normaliza- demonstrating there was no change in transmural tion of CV, demonstrated by decreased time between 4     www.ccmjournal.org XXX 2024 Volume 52 Number 00 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Laboratory Investigation endocardial and epicardial activation (middle rows), as well as normalization of CV (lower row), as com- pared with hyperkalemia alone (center column). Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy Note that although con- duction block is observed wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 at 12 mM, this is no longer evident in the presence of Ca2+ treatment. Summary data over all experiments (Fig. 2) demonstrates at- tenuation of hyperkalemia- induced CV slowing by Ca2+ and this effect was dose dependent (Supplemental Fig. 1, http://links.lww. com/CCM/H567). In the Figure 2. Summary data of conduction velocity (CV) over all experiments is shown. The greatest wedge preparation, we did CV slowing from baseline (4 mM) was observed between 8 and 12 mM [K+]. Calcium (Ca2+) not observe any signifi- improved conduction, which is expected to be antiarrhythmic (n = 7 preparations under all cant changes in APD and conditions except 8 mM, n = 5, *p < 0.02, **p < 0.001). DOR with Ca2+ treatment during hyperkalemia (Fig. 4). In contrast to what was observed in tissue preparations, in isolated myocytes, the addition of Ca2+ partially mitigated hyperkalemia-induced APD shortening (high K vs. high K + Ca2+ and sum- mary data in Fig. 3B). Mechanisms Underlying Restoration of CV by Ca2+ During Hyperkalemia We next examined the effect of hyperkalemia and Ca2+ on RMP. As this can- not be evaluated using optical mapping in tis- Figure 3. Effect of hyperkalemia and experimental conditions on the action potential (AP). sue, this was tested in iso- A, Shown are tracings of cellular APs recorded at baseline (black), hyperkalemia (8 mM, gray), lated myocytes. As shown and after calcium (Ca2+) treatment (stippled) from isolated cardiomyocytes. Marked AP shortening occurs with hyperkalemia, which is improved with Ca2+ treatment (*p < 0.05, ** p < 0.01, n = 7). in Figure 5A, as expected, B, Summary data for action potential duration (APD) under the three conditions is shown. hyperkalemia (8 mM) sig- C, Summary data for high K + Ca condition, with and without verapamil, are shown. nificantly increased RMP. Critical Care Medicine www.ccmjournal.org     5 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Piktel et al Ca2+ treatment is involved in the mechanism of increased CV. To ensure verapamil had no effects on RMP, this was investigated in single cell studies, which showed Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy no effect of verapamil on hyperkalemia-induced wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 RMP elevation during Ca2+ treatment (Fig. 5, A and C). In addition, the pres- ence of verapamil mitigated the partial restoration of hyperkalemia-induced APD shortening by Ca2+ (Fig. 3A, high K + Ca2+ vs. high K + Ca2+ plus verapamil tracings and Fig. 3C). This suggests Figure 4. Effect of hyperkalemia and Ca+ on transmural action potential duration (APD) and that increased L-type Ca2+ dispersion of repolarization (DOR). Summary data of APD as determined in the canine wedge current secondary to Ca2+ preparation is shown. Compared with baseline (4 mM [K+]) at high potassium, APD is shortened, administration is also re- but DOR is not significantly (NS) affected (difference between epicardial and endocardial APD) even in presence of calcium treatment (*p < 0.05). sponsible for the APD pro- longation we observed in isolated myocytes. Finally, to Interestingly, treatment with Ca had no effect on investigate whether the effect of Ca2+ treatment during 2+ RMP, which was reproducible over all experiments hyperkalemia was mediated by an effect on the sodium (summary data in Fig. 5B). This strongly suggests that channel, in isolated myocytes, we conducted studies in the mitigation of CV we observed was not secondary the presence of the sodium channel blocker tetrodotoxin to an effect on RMP or “membrane stabilization.” (120 mM). No attenuation of the effect of Ca2+ on RMP We next investigated a potential alternative mech- or APD was seen in isolated myocytes in the presence of anism for improvement in CV secondary to Ca2+ treat- tetrodotoxin, n = 7 (data shown in Supplemental Fig. ment, based on calcium inward current-dependent 2, http://links.lww.com/CCM/H567), further suggest- propagation (7, 24) using a pharmacological approach. ing that the mechanism by which Ca2+ restores CV dur- In Figure 6A are representative isochrone maps from a ing hyperkalemia was not related to enhanced sodium wedge preparation at baseline (4 mM [K+]o, upper row, channel excitability. We observed differences between far left), after induction of hyperkalemia (12 mM [K+]o, tetrodotoxin and verapamil on APD under conditions no treatment), treatment with Ca2+ and then Ca2+ treat- of hyperkalemia and Ca2+ treatment, where verapamil, ment in the presence of the L-type Ca2+ channel blocker but not tetrodotoxin, shortened APD (Supplemental verapamil (20 uM). As expected, during hyperkalemia, Fig. 2, A and C, http://links.lww.com/CCM/H567), sug- marked conduction slowing, as noted by crowding of gesting a more preferential effect on L-type Ca2+ by ve- isochrones, and conduction block (solid line), were rapamil in our experiments. observed. In the presence of Ca2+, conduction slowing is mitigated, and block is prevented. However, in the DISCUSSION presence of verapamil, conduction slowing and block returns. Summary data shows that verapamil prevents High potassium significantly slows conduction and CV improvements by Ca2+ treatment during hyperka- shortens APD, creating arrhythmogenic substrates. lemia (Fig. 6B). Taken together, this suggests that dur- Ca2+ treatment results in improved conduction, nor- ing hyperkalemia, L-type Ca2+ current augmentation by malizing the ECG. Our data are consistent with known 6     www.ccmjournal.org XXX 2024 Volume 52 Number 00 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Laboratory Investigation on cardiac excitability and “membrane stabilization” is based on early observations by Winkler et al (25) in 1939 where Ca2+ prevented car- diac standstill during hyper- Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy kalemia and by additional experimental observations wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 that at markedly high Ca2+ concentrations, Ca2+ itself elevates RMP (6, 8, 9, 26). However, in the current study, at physiologically relevant calcium concen- trations in isolated myo- cytes, we did not observe a change in RMP by increas- ing Ca2+ (19). Nonetheless, we observed that Ca2+ ad- ministration restored CV Figure 5. Effect of hyperkalemia and Ca+ resting membrane potential (RMP). A, Shown are RMP and normalized the ECG. and phase 0 depolarization of representative action potentials in control (black), hyperkalemia (8 mM potassium, black stippled) and hyperkalemia with calcium (Ca2+) treatment (gray). Note that Furthermore, our data although RMP rises during hyperkalemia, it is not affected by Ca2+ treatment. B, Summary data for demonstrating that L-type RMP is shown (*p < 0.01, n = 7). C, Summary data for RMP during hyperkalemia and Ca2+ channel blockade pre- Ca2+ treatment, with and without verapamil (Verap), is shown. NS = not significant. vents improvements in CV by Ca2+ treatment during effects of hyperkalemia on RMP, whereby as hyperka- hyperkalemia (without a change in RMP), suggests a lemia progresses, RMP increases. Also consistent with mechanism related to Ca2+ mediated conduction, rather clinical and experimental observations, we observed than an effect on RMP. Ca2+ mediated conduction has improved CV with Ca2+ treatment (1, 7, 9). However, been described in ischemia and other conditions with we observed no improvement in RMP with Ca2+ treat- elevated RMP (7, 24, 27) and involves maintenance of ment, strongly suggesting that the effect we observed is impulse propagation by cellular excitability through not related to “membrane stabilization.” Furthermore, L-type Ca2+ current. An additional, potentially com- these data provide a mechanistic rationale for clinical plimentary, mechanism is that changes in extracellular use of Ca2+ treatment for hyperkalemia when the ECG Ca2+ concentrations may promote ephaptic coupling, reveals slow conduction (i.e., QRS prolongation). thereby improving CV. Ephaptic coupling, refers to the activation of voltage gated ion channels and impulse Effect of Hyperkalemia and Calcium on propagation by extracellular potential gradients in the Conduction Velocity and “Membrane perinexal space between adjacent myocytes. Specifically, Stabilization” regarding hyperkalemia, increased Ca2+ concentrations As hyperkalemia progresses, the initial rise in RMP have been shown to decrease the width of the perinexus, decreases the threshold for sodium current activation which is expected to enhance ephaptic coupling and im- thereby increasing conduction. However, a further rise in prove conduction (28, 29). RMP will decrease sodium channel availability, thereby Effects of Hyperkalemia and Calcium on decreasing conduction. This balance is likely why an Repolarization and the T Wave increase in CV is observed at lower ranges of hyperka- lemia, then slowed conduction at more severe hyperka- As expected, hyperkalemia shortened APD, which has lemia (1, 6). The evidence of a protective effect of Ca2+ been attributed to the changes in peaked and narrowed Critical Care Medicine www.ccmjournal.org     7 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Piktel et al of hyperkalemia and Ca2+ treatment were more appar- ent in single-cell prepa- rations. Importantly, our examination of effects in both isolated myocytes and Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy intact tissue, therefore, pro- vides additional insight into wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 mechanisms relevant to the whole heart. Limitations To achieve ECG changes consistent with severe hyperkalemia, including a sine wave pattern ECG, we used 12 mM [K+]o to signif- icantly slow CV, which may be less clinically relevant than the 6 mM [K+]o used in cellular studies. However, Figure 6. Effect of verapamil and calcium (Ca ) on transmural conduction. A, Upper row shows 2+ differences in susceptibility (in order left to right) transmural conduction maps at baseline, hyperkalemia, hyperkalemia + Ca2+ to hyperkalemia in experi- treatment, and hyperkalemia + Ca2+ treatment in the presence of L-type Ca2+ channel blockade mental mammalian prepa- with verapamil. Conduction slows during hyperkalemia and subsequent subepicardial conduction rations and man have been block is observed. Ca2+ treatment improves conduction and ameliorates conduction block. The addition of verapamil again slows conduction and conduction block returns. B, Summary data for noted before (1, 5). In addi- conduction velocity (CV) during hyperkalemia and Ca2+ treatment, with and without verapamil, is tion, a biphasic response to shown (*p = 0.02, **p < 0.001, n = 3). ENDO = endocardial, EPI = epicardial. hyperkalemia on cardiac conduction in multicellular T waves of the ECG in moderate elevations of potas- preparations is well described, where significant conduc- sium (1, 3). However, we did not observe that hyper- tion slowing is only observed at concentrations greater kalemia promoted APD heterogeneities that would than 8 mM [K+]o in guinea pig and human (30–32). potentially promote reentrant arrhythmias, nor did There are several limitations of the wedge preparation. Ca2+ treatment affect APD heterogeneities. This sug- Whole-heart Purkinje and bundle branch conduction gests that Ca2+ treatment would likely have minimal are not taken into account, and potassium derange- effect on RGs, other than those produced by conduc- ments have known preferential effects on the conduc- tion slowing. tion system and Purkinje fibers (1, 4, 33), particularly We observed differences in the effect of Ca2+ treat- at the Purkinje-myocyte junction (34). Nevertheless, the ment on APD in isolated myocytes vs. the wedge prep- wedge preparation is an established model used to ex- aration and less sensitivity to hyperkalemia in the amine the electrophysiological heterogeneities and basis wedge preparation. Effects observed in isolated cells of the T wave under normal and pathologic conditions are known to be different from what is observed in (13, 20, 35). Additionally, using the wedge preparation, multicellular preparations due to, in part, the effects of we cannot comment on the effects of hyperkalemia on cell-to-cell coupling by gap junctions, which decreases the SA or AV node and associated abnormal automa- intrinsic electrophysiologic heterogeneities in tissue. ticity and bradycardia that occurs with hyperkalemia Therefore, known heterogeneities in myocyte sensi- (1, 4, 7). Finally, we could not reliably induce arrhyth- tivity to hyperkalemia (5) may explain why the effects mias or observe spontaneous arrhythmias in the wedge 8     www.ccmjournal.org XXX 2024 Volume 52 Number 00 Copyright © 2024 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.  Laboratory Investigation preparation because of known size limitations and be- inform its use during bradycardia, or other arrhythmias, cause of the inability to capture the preparation at short as is currently clinically indicated. cycle lengths necessary to perform programmed elec- trical stimulation. A lack of specificity of tetrodotoxin 1 Department of Emergency Medicine, Emergency Care for sodium currents and potential effect on L-type Ca2+ and Research and Innovation, MetroHealth Campus, Case current has been suggested, but this is controversial (32, Western Reserve University, Cleveland, OH. Downloaded from http://journals.lww.com/ccmjournal by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCy 36–39). We did not perform studies with verapamil or 2 Department of Physiology & Cell Biology, The Ohio State University, College of Medicine, Columbus, OH. tetrodotoxin in the absence of hyperkalemia or in dose- wCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 08/03/2024 3 Orthopedic Surgery and Sports Medicine, Mercy Clinic, St. response studies with Ca2+ treatment. We also did not Louis, MO. test Ca2+ treatment at 8 mM [K+]o. 4 The Heart and Vascular Research Center, MetroHealth Campus, Case Western Reserve University, Cleveland, OH. Supplemental digital content is available for this article. Direct Clinical Implications URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website Although these data provide a mechanistic rationale for (http://journals.lww.com/ccmjournal). Ca2+ treatment when there is evidence of hyperkalemia- This study was supported by Departmental MetroHealth induced conduction slowing, it should be noted that Foundation Grant (to Drs. Piktel and Wilson), an Emergency additional considerations are important when con- Medicine Foundation Career Development Grant (to Dr. Piktel), sidering Ca2+ treatment. For example, there are well a grant from the ZOLL Foundation (to Dr. Piktel), and National Institutes of Health/National Heart, Lung, and Blood Institute described side effects of Ca2+ administration, in- (HL142754; to Drs. Piktel, Laurita, and Wilson). cluding skin injury if infiltrated and unpleasant hot Presented, in part, at the American College of Emergency Physicians flushes or chalky taste, but severe complications, such Scientific Sessions, Las Vegas, NV, September 28, 2010. as hypotension, bradycardia, and arrhythmias are un- Drs. Piktel, Laurita, and Wilson received support for article re- common (10, 40). An additional consideration is that search from the National Institutes of Health. The remaining authors have disclosed that they do not have any potential con- ECG changes in these patients may evolve quickly and flicts of interest. patients may develop life-threatening complications, For information regarding this article, E-mail: lwilson@metro- and the typical treatment error is failure to treat with health.org Ca2+, rather than overtreatment. However, there are data to suggest that in patients with adverse even