Enhanced sensitivity of aged fibrotic hearts to angiotensin II- and hypokalemia-induced early afterdepolarization-mediated ventricular arrhythmias Aneesh Bapat, Thao P. Nguyen, Jong-Hwan Lee, Ali A. Sovari, Michael C. Fishbein, James N. Weiss and Hrayr S. Karagueuzian Am J Physiol Heart Circ Physiol 302:H2331-H2340, 2012. First published 30 March 2012; doi:10.1152/ajpheart.00094.2012 You might find this additional info useful... This article cites 39 articles, 20 of which can be accessed free at: http://ajpheart.physiology.org/content/302/11/H2331.full.html#ref-list-1 Updated information and services including high resolution figures, can be found at: http://ajpheart.physiology.org/content/302/11/H2331.full.html Additional material and information about AJP - Heart and Circulatory Physiology can be found at: http://www.the-aps.org/publications/ajpheart AJP - Heart and Circulatory Physiology publishes original investigations on the physiology of the heart, blood vessels, and lymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2012 by the American Physiological Society. ISSN: 0363-6135, ESSN: 1522-1539. Visit our website at http://www.the-aps.org/. Downloaded from ajpheart.physiology.org on June 4, 2012 This information is current as of June 4, 2012. Am J Physiol Heart Circ Physiol 302: H2331–H2340, 2012. First published March 30, 2012; doi:10.1152/ajpheart.00094.2012. TRANSLATIONAL PHYSIOLOGY Enhanced sensitivity of aged fibrotic hearts to angiotensin II- and hypokalemiainduced early afterdepolarization-mediated ventricular arrhythmias Aneesh Bapat,1* Thao P. Nguyen,1* Jong-Hwan Lee,3* Ali A. Sovari,1 Michael C. Fishbein,2 James N. Weiss,1 and Hrayr S. Karagueuzian1 1 Translational Arrhythmia Research Section, University of California-Los Angeles (UCLA) Cardiovascular Research Laboratory, and Division of Cardiology, Department of Medicine, UCLA, Los Angeles, California; 2Department Laboratory Medicine and Pathology, David Geffen School of Medicine, UCLA, Los Angeles, California; and 3Department of Anesthesiology and Pain Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Submitted 3 February 2012; accepted in final form 28 March 2012 ventricular fibrillation; triggered activity; optical mapping WE HAVE PREVIOUSLY SHOWN that ⬎90% of aged fibrotic rat ventricles develop ventricular cellular early afterdepolarizations (EADs), triggered activity, and ventricular fibrillation (VF) when exposed to oxidative stress (21) or glycolytic inhibition (20). The dramatic pro-VF effect of these two * A. Bapat, T. P. Nguyen, and J.-H. Lee contributed equally to this work. Address for reprint requests and other correspondence: H. S. Karagueuzian, Translational Arrhythmia Research Section, Cardiovascular Research Laboratory, David Geffen School of Medicine, Univ. of California-Los Angeles, 675 Charles E. Young Dr. S, MRL 1630, mail code: 176022, Los Angeles, CA 90095 (e-mail: hkaragueuzian@mednet.ucla.edu). http://www.ajpheart.org stressors in aged hearts is completely absent in young nonfibrotic hearts (20, 21). While fibrosis is known to facilitate the formation of EADs (23, 40), it is, however, also possible that aging-related cellular electrophysiological remodeling (1, 30) may directly increase the susceptibility of aged myocytes to EADs and triggered activity in response to clinically relevant stressors, such as an acute elevation of ANG II and hypokalemia. To investigate this issue, we directly compared the susceptibility of intact rat hearts and single rat ventricular myocytes isolated from both young and aged rats to two different clinically relevant stressors: 1) ANG II exposure, which is known to induce oxidative stress by activating endogenous ROS production via the activation of NADPH oxidase (41); and 2) hypokalemia (2.7 mM), an ionic stressor commonly encountered in clinical settings (24). METHODS This research protocol was approved by our Institutional Animal Care and Use Committee and followed the guidelines of the American Heart Association. Langendorff Preparation Male Fischer 344 young (3– 4 mo old, n ⫽ 23) and aged (24 –26 mo old, n ⫽ 30) rats were used in this study. Hearts of anesthetized rats were removed, and the ascending aorta was cannulated for retrograde perfusion through the coronary ostia, as we have previously described (20, 21). Optical Mapping Hearts were stained with RH-237 for voltage fluorescent optical imaging, as we have previously described (21). Cytochalasin D (5 ␮mol/l) was added to the perfusate to eliminate motion artifacts during optical recordings (20, 21). The stained heart was excited with green light (LED) at 532 nm, and the emitted fluorescence was collected using a CMOS camera (MiCAM Ultima, BrainVision, Tokyo, Japan) at 1 ms/frame and 100 ⫻ 100 pixels with a spatial resolution of 0.35 ⫻ 0.35 mm2/pixel covering the entire anterior left ventricular (LV) epicardial surface (18). Continuous single cell glass microelectrode recordings were made at the onset of VF from the LV epicardial surface (21). Pharmacological Interventions To determine if ANG II-induced VF was mediated through an alteration of the cellular redox state, the reducing agent N-acetylcysteine (NAC; 2 mmol/l) (13) was tested in four aged rats. The 0363-6135/12 Copyright © 2012 the American Physiological Society H2331 Downloaded from ajpheart.physiology.org on June 4, 2012 Bapat A, Nguyen TP, Lee JH, Sovari AA, Fishbein MC, Weiss JN, Karagueuzian HS. Enhanced sensitivity of aged fibrotic hearts to angiotensin II- and hypokalemia-induced early afterdepolarization-mediated ventricular arrhythmias. Am J Physiol Heart Circ Physiol 302: H2331–H2340, 2012. First published March 30, 2012; doi:10.1152/ajpheart.00094.2012.—Unlike young hearts, aged hearts are highly susceptible to early afterdepolarization (EAD)mediated ventricular fibrillation (VF). This differential may result from age-related structural remodeling (fibrosis) or electrical remodeling of ventricular myocytes or both. We used optical mapping and microelectrode recordings in Langendorff-perfused hearts and patchclamp recordings in isolated ventricular myocytes from aged (24 –26 mo) and young (3– 4 mo) rats to assess susceptibility to EADs and VF during either oxidative stress with ANG II (2 ␮M) or ionic stress with hypokalemia (2.7 mM). ANG II caused EAD-mediated VF in 16 of 19 aged hearts (83%) after 32 ⫾ 7 min but in 0 of 9 young hearts (0%). ANG II-mediated VF was suppressed with KN-93 (Ca2⫹/calmodulindependent kinase inhibitor) and the reducing agent N-acetylcysteine. Hypokalemia caused EAD-mediated VF in 11 of 11 aged hearts (100%) after 7.4 ⫾ 0.4 min. In 14 young hearts, however, VF did not occur in 6 hearts (43%) or was delayed in onset (31 ⫾ 22 min, P ⬍ 0.05) in 8 hearts (57%). In patch-clamped myocytes, ANG II and hypokalemia (n ⫽ 6) induced EADs and triggered activity in both age groups (P ⫽ not significant) at a cycle length of ⬎0.5 s. When myocytes of either age group were coupled to a virtual fibroblast using the dynamic patch-clamp technique, EADs arose in both groups at a cycle length of ⬍0.5 s. Aged ventricles had significantly greater fibrosis and reduced connexin43 gap junction density compared with young hearts. The lack of differential age-related sensitivity at the single cell level in EAD susceptibility indicates that increased ventricular fibrosis in the aged heart plays a key role in increasing vulnerability to VF induced by oxidative and ionic stress. H2332 FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS antioxidant therapy with NAC was initiated 15 min before and continued throughout the entire ANG II perfusion period (preventive therapy). Since an increased oxidant state activates Ca2⫹/calmodulindependent kinase II (CaMKII) (7, 41) and thus could potentially mediate ANG II effects, we also examined the preventive (n ⫽ 4) effect of the CaMKII inhibitor KN-93 (1 ␮mol/l) on ANG IImediated VF. Isolated Myocyte (Patch-Clamp) Experiments Suppression of ANG II-Induced VF in Aged Hearts With NAC and KN-93 Histological Analysis Percent tissue fibrosis and connexin (Cx)43 immunostain-positive spots were determined from 5-␮m-thick histological sections stained by Masson’s trichrome and Cx43 immunostaining methods as we have previously described (20). Statistical Analyses Significant differences in the incidence of VF (dichotomous comparisons) were determined using Fisher’s exact test. AP properties were determined using repeated-measures ANOVA. Differences among individual means were verified subsequently by xyz post hoc tests. Since normality of xyz distribution cannot be assumed to exist, we therefore used bootstrapping methods to detect significant differences (5, 17). The method randomly resampled 10,000 times with replacement. If the values of 10,000 random resampling were ⬎9,750 of the 10,000 values, we concluded that the F-value was statistically significant at the 0.025 level (two-sided test for ␣ ⫽ 0.05) (5, 17). We considered P values of ⱕ0.05 as significant and presented all data as means ⫾ SD. RESULTS Effects of ANG II on Cardiac Rhythm in Hearts Isolated From Aged Versus Young Rats All hearts in both age groups were in regular sinus rhythm when mounted in the tissue bath and perfused with normal To confirm that the arrhythmogenic effects of ANG II are mediated by oxidative activation of CaMKII (34, 37, 39, 41), we tested the effects of the reducing agent NAC (2 mM) and the CaKMII antagonist KN-93 (2 ␮M). Similar to H2O2induced VF (21), both agents prevented VF in three of four aged hearts each (P ⬍ 0.05; Fig. 2). Upon washout of NAC or KN-93, spontaneous VF reemerged within 30 min in all hearts. Effect of Hypokalemia on Cardiac Rhythm in Hearts Isolated From Young and Aged Rats When the K⫹ concentration in the Tyrode solution was decreased from 5.4 to 2.7 mM, VF spontaneously arose from sinus rhythm at a mean CL of 306 ⫾ 109 ms after an average of 7.4 ⫾ 0.4 min in all 11 aged hearts tested. VF was preceded by the emergence of EADs, followed by single, double, and short runs of triggered activity, which eventually progressed to VF at a CL of 43 ⫾ 16 (Fig. 3B). In young rat hearts, VF also arose from sinus rhythm at a mean CL of 357 ⫾ 172 in 8 of 14 hearts (P ⫽ NS compared with aged sinus CL). The mean CL during VF averaged 49 ⫾ 19 ms and was not significantly different from aged hearts. However, the time of onset of VF was significantly delayed (31 ⫾ 22 min) compared with aged AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org Downloaded from ajpheart.physiology.org on June 4, 2012 Solutions. The Tyrode perfusion solution was of the following composition (in mmol/l): 136 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with NaOH, and the Tyrode solution was also used for cell isolation and extracellular perfusion in patch-clamp experiments unless otherwise specified. Isolation of ventricular myocytes. The procedures used in this study conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996) and to University of California-Los Angeles Policy 990 on the Use of Laboratory Animal Subjects in Research (2001) (23). Patch clamp. Action potentials (APs) were recorded using borosilicate glass electrodes (tip resistance: 2–3 M⍀) and standard whole cell-patch clamp methods in the current-clamp mode. Corrections were made for liquid junction potentials. Data were acquired by an Axopatch 200B patch-clamp amplifier, a Digidata 1200 acquisition board, and Clampex 8.0 (Axon Instruments) and were filtered at 2 kHz. Experiments were performed at 34 –36°C. Dynamic clamp. A patch-clamped rat ventricular myocyte was bidirectionally coupled in real time to a virtual myofibroblast using the dynamic clamp technique (23). The dynamic clamp processed the myocyte membrane voltage signal from the patch-clamp amplifier and injected back into the myocyte a predicted virtual gap junction current proportional to the voltage difference and gap junction coupling conductance between the real myocyte membrane voltage and the virtual myofibroblast voltage using real-time Linux based software (23) (Real-Time eXperiment Interface, www.rtxi.org). The University of California-Los Angeles virtual myofibroblast model in the dynamic clamp experiments was modified from MacCannell’s “active” fibroblast model. Tyrode solution (Fig. 1, A and B). After 20 min of equilibration, 2 ␮M ANG II was added to the perfusate during sinus rhythm at a mean cycle length (CL) of 323 ⫾ 72 ms in nine young hearts and 310 ⫾ 109 ms in 19 aged hearts [P ⫽ not significant (NS)]. All nine young rat hearts exposed to ANG II remained in sinus rhythm during up to 90 min of ANG II perfusion (Fig. 1A). In contrast, 16 of 19 aged rat hearts developed VF (P ⬍ 0.01), which arose spontaneously from normal sinus rhythm without any preceding conduction block or rhythm irregularities. As shown in Fig. 1, B and C, the onset of VF was preceded by EADs, which developed 7.7 ⫾ 4.3 min after the onset of ANG II exposure, initially causing single and double triggered beats and then progressing to short runs of triggered activity causing nonsustained ventricular tachycardia (VT; mean CL of 93 ⫾ 11 ms), which after 32 ⫾ 7 min of exposure to ANG II degenerated to VF at a mean CL of 55 ⫾ 5 ms (Fig. 1C). VF required electrical shock for termination. The EADs in the microelectrode recordings shown in Fig. 1, B and C, occurred in late phase 3, and are therefore not likely to be at the site of origin of EAD-mediated triggered activity. However, our previous studies (20, 21) showed that endocardial cryoablation did not abolish EAD-mediated arrhythmias induced by oxidative stress, indicating that epicardial tissue is capable of EAD-mediated triggered activity in aged hearts. Optical mapping showed that EAD-mediated VF was associated with 5–10 focal activations near the epicardial base, which then converted to an irregular pattern of activation characterized by incomplete reentrant wavefronts mixed with focal activations. As shown in Fig. 1, D and E, after eight consecutive regularly propagating ectopic wavefronts (CL of ⬃100 ms), another focal activation arose midway between the base and apex of the heart that collided with the triggered focal activity arising from the base, leading to irregular activation wavefronts characteristic of VF (Fig. 1, D and E). VF required electrical shock for termination. Episodes of spontaneous VF were sometimes preceded by a sinus pause, as shown in Fig. 1, but this was not always the case. FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS H2333 Downloaded from ajpheart.physiology.org on June 4, 2012 Fig. 1. ANG II (ATII) induces early afterdepolarizations (EADs), ventricular tachycardia (VT), and ventricular fibrillation (VF) in aged but not young rat hearts. A–C: simultaneous microelectrode (ME) and pseudo-ECG recordings in a young heart (A) and an aged heart (B and C) exposed to ANG II (2 ␮mol/l). A: representative experiment in a young heart showing that sinus rhythm persists throughout 60 min of ANG II perfusion. B and C: recordings from an aged heart showing the emergence of EADs, triggered activity, and VF over the indicated time course. D: three isochronal activation maps, with the first being the last sinus beat (beat 1) before the onset of the VF shown at the bottom and indicated as 1 in the ECG (bottom). The sinus beat was followed by two consecutive focal activations that arose from the base of the heart (beats 2 and 3). The red arrows in D indicate the direction of propagation. The focal activation lasted for 8 –10 beats (the initial VT phase) and then degenerated to VF, as shown in E with four optical action potentials (APs; labeled 1– 4) shown in the adjacent schema. hearts (P ⬍ 0.05 by bootstrap; Fig. 3A). The remaining six young hearts maintained sinus rhythm and exhibited no arrhythmias during exposure to hypokalemia for up to 90 min. In both aged and young hearts, electrical shocks were ineffective in converting VF to sinus rhythm. However, return to normokalemic Tyrode solution restored sinus rhythm after 11 ⫾ 6 min in both age groups (Fig. 3, A and B, bottom recordings). Optical mapping at the onset of VF showed EAD-mediated triggered focal activation that arose near the base of the LV epicardium (Fig. 3C), similar to the pattern shown in Fig. 1D. We observed eight such episodes in three aged and three young hearts, in which epicardial EAD-mediated triggered activity degenerated to VF during hypokalemia. Arrhythmogenic Effects of ANG II and Hypokalemia on Isolated Young and Aged Isolated Ventricular Myocytes We next assessed the ability of ANG II (2 ␮M) and hypokalemia (2.7 mM) to evoke EADs and triggered activity in isolated ventricular myocytes from both age groups during pacing at CLs of 6 s, 1 s, 500 ms, and 300 ms. Under basal conditions, isolated myocytes from both aged and young hearts AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org H2334 FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS had similar resting membrane potential, AP duration at 90% repolarization, and maximum upstroke velocity (Table 1). Myocytes from both age groups developed low-amplitude (subthreshold) delayed afterdepolarizations (DADs), which did not produce triggered beats. However, the subsequent emergence of EADs was associated with larger DAD amplitudes, causing triggered activity at all CLs tested (Fig. 4). The summary data shown in Table 2 demonstrate that there was a trend for aged myocytes to exhibit a greater incidence of EADs and DADs than young myocytes. However, these differences did not reach statistical significance for the number of myocytes studied. Similar findings were obtained with hypokalemia, which promoted EADs and DADs in aged and young isolated myocytes at pacing CLs ⬎ 4 s (Fig. 5). As in the case of ANG II, there was a tendency for a higher incidence in aged myocytes, although the difference did not reach statistical significance. junction conductance of 3 nS (14). With the dynamic clamp turned off, the myocyte was then exposed to hypokalemia to induce EADs at a long pacing CL, which were suppressed by pacing at a short pacing CL of 0.5 s. Turning on the dynamic clamp to couple the myocyte to a virtual fibroblast caused EADs to appear at the shorter than 0.5 s pacing CL. Similar findings were obtained in six myocytes exposed to hypokalemia and six myocytes exposed to ANG II. Thus, myocyte-myofibroblast gap junction coupling promoted EAD formation at all pacing CLs tested up to 300 ms, which was the average sinus CL from which EADs emerged in isolated hearts exposed to hypokalemia or ANG II. These findings suggest that gap junction coupling between fibroblasts and myocytes, if it exists in aged rat ventricular tissue, could be an additional factor enhancing susceptibility to EADs. Dynamic Clamp Consistent with previous reports (20, 21), Masson’s trichrome stain revealed significantly greater fibrosis in aged ventricles than in young ventricles (38 ⫾ 10% vs. 2.8 ⫾ 1%, P ⬍ 0.001, n ⫽ 6) and reduced Cx43 immunostain-positive spots in aged ventricles compared with young ventricles (0.7 ⫾ 0.02% vs. 1.8 ⫾ 0.3%, P ⬍ 0.05). Since gap junction coupling between myocytes and myofibroblasts can promote arrhythmias (19, 23), we explored the theoretical possibility that the increased number of fibroblasts in aged hearts might directly contribute the increased susceptibility to EADs in tissue if they formed gap junctions with myocytes. Accordingly, we used the dynamic clamp technique to simulate the coupling of virtual fibroblasts to real myocytes in young adult and aged myocytes. Figure 6 shows the results from a patch-clamped isolated myocyte coupled to a virtual myofibroblast with a physiologically realistic capacitance of 50 pF, an uncoupled resting potential of ⫺50 mV, and a gap Ventricular Fibrosis and Cx43 DISCUSSION The major objective of this study was to investigate the relative importance of aging-related structural and cellular electrical remodeling in increasing the susceptibility of aged rat ventricles to EAD-mediated VF. To accomplish this objective, AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org Downloaded from ajpheart.physiology.org on June 4, 2012 Fig. 2. Effects of the Ca2⫹/calmodulin-dependent kinase II inhibitor KN-93 and N-acetylcysteine (NAC) on ANG II-induced EADs and VF in aged rat hearts. A and B: simultaneous pseudo-ECG and right ventricular (RV) bipolar recordings (BEG) in two aged rat hearts. A: recordings were made at baseline, for 1 h in the presence of combined ANG II ⫹ KN-93, and then in the presence of only ANG II. Notice the emergence of VF after 35 min of KN-93 washout. B: recordings at baseline and then for 1 h with combined ANG II ⫹ NAC for 1 h. Upon NAC washout, VF emerged after 30 min of continued perfusion of ANG II. FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS H2335 Downloaded from ajpheart.physiology.org on June 4, 2012 Fig. 3. Effects of increasing the duration of hypokalemia in young and aged rat hearts. A–D: simultaneous ME (top) and pseudo-ECG (bottom) recordings in a young heart (A) and in an aged heart (B) with optical recordings shown in C and D. Hypokalemia (2.7 mmol/l) initiated EADs and VF in the young heart after 27 min (A) and in the aged heart after 6 min (B). A switch to normal Tyrode solution perfusion terminated the VF and restored normal sinus rhythm within 14 and 11 min in the young and aged heart, respectively. C and D: optical mapping snapshots at the onset of VF in the aged heart showing the last sinus beat (beat 1) followed by five EAD-mediated triggered beats leading to VF (ECG, bottom). In these snapshots, the red indicates the onset of activation, i.e., the ectopic source. Notice that after the last sinus beat, the three beats (beats 2– 4) were caused by a single focal activation that arose near the base of the heart; however, the fifth and sixth beats were associated with two simultaneous foci, with one focus arising from near the base and the second from midway between the base and the apex (white arrows in beats 5 and 6). D: EAD-mediated five propagating triggered APs at the onset of the VF episode shown in C. The optical APs were recorded from sites 1–4, as shown in the diagram to the left. we subjected isolated ventricular myocytes and whole hearts from young and aged rats to two physiologically relevant EAD-promoting stressors: ANG II and hypokalemia. Our results show that isolated myocytes from both aged and young rat hearts develop EADs and DADs when exposed to ANG II or hypokalemia. In contrast, however, EADs and triggered VF developed only in aged whole hearts. This discrepancy suggests that the aging-related cellular electrical remodeling does AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org H2336 FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS Table 1. Baseline AP properties of isolated ventricular myocytes Resting membrane potential, mV AP duration at 90% repolarization, ms Maximum upstroke velocity, V/s Young Myocytes Aged Myocytes P Value ⫺78 ⫾ 4 119 ⫾ 34 214 ⫾ 36 ⫺80 ⫾ 3 129 ⫾ 42 194 ⫾ 45 NS NS NS Values are means ⫾ SD. AP, action potential; NS, not significant. Mechanisms of ANG II- and Hypokalemia-Induced Arrhythmias ANG II signaling intersects with many cellular pathways, but one of its major G protein-mediated actions is to stimulate ROS generation via NADPH oxidase. Our findings that the arrhythmogenic effects of ANG II in intact aged rat hearts were blocked by the antioxidant NAC and the CaMKII inhibitor KN-93 suggest that ANG II induces these arrhythmias by a similar mechanism to H2O2, which has previously been shown to involve the oxidative activation of CaMKII (28, 35, 39). CaMKII activation promotes EADs and DADs by inducing late Na⫹ current and modifying L-type Ca2⫹ current, which both reduces repolarization reserve and promotes intracellular Ca2⫹ loading, as observed in transgenic mice overexpressing cardiac CaMKII (6, 34, 37). It should be noted that in addition to acute oxidative stress induced by ANG II in promoting cardiac arrhythmias, chronic ANG II could also promote atrial and ventricular arrhythmias by other mechanisms (9, 12, 16). In contrast, hypokalemia promotes afterdepolarizations by altogether different mechanisms. The initial effect of hypokalemia is to reduce repolarization reserve by decreasing the conductances of outward K⫹ currents important for repolarization, particularly the fast component of the delayed rectifier K⫹ current and the inward rectifier K⫹ current (24). These K⫹ channel conductances exhibit a strong dependence on extracellular K⫹ concentration, such that even though hypokalemia increases the driving force for K⫹ efflux, the current amplitude (the product of driving force and conductance) is significantly reduced. The second major effect of hypokalemia is suppression of the Na⫹-K⫹ pump, which also occurs immediately but whose consequences take longer period of time to develop (24). As intracellular Na⫹ gradually rises, inhibition of Na⫹/Ca2⫹ exchange leads to intracellular Ca2⫹ overload, afterdepolarizations, and triggered activity by a mechanism similar to digitalis (24). Thus, early arrhythmias induced by hypokalemia are attributable to reduced repolarization reserve caused by a suppression of repolarizing K⫹ currents, whereas later arrhythmias are due to the combined effects of K⫹ current suppression and Ca2⫹ overload from Na⫹-K⫹ pump inhibition. This may explain why 58% of young hearts eventually developed afterdepolarizations, triggered activity, and VF after prolonged (31 ⫾ 22 min) exposure to hypokalemia, whereas ANG II, H2O2, or glycolytic inhibition (20, 21) failed to induce arrhythmias in young hearts even for exposure periods of ⬎60 min. In AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org Downloaded from ajpheart.physiology.org on June 4, 2012 not explain the increased susceptibility of aged rat hearts to VF and that additional tissue-related factors are important. A striking difference between aged versus young hearts is the degree of ventricular fibrosis and reduction in Cx43, manifestations of structural remodeling due to aging (10, 20, 21, 29). By interposing collagen bundles between myocardial tissues, fibrosis creates regions of slow conduction, which are well known to predispose to reentrant arrhythmias. However, slow conduction does not directly account for the increased susceptibility to EADs. More relevant to EAD formation (40), fibrosis also alters the local source-to-sink relationship in cardiac tissue. Since ventricular myocytes are normally coupled via gap junctions to an average of 11 nearest neighbors (11), the source-to-sink relationship exerts a powerful influence in suppressing EADs and DADs. That is, as soon as the voltage of a myocyte primed to have an afterdepolarization begins to deviate from that of its neighbors, electrotonic current flows to minimize the voltage difference, forcing the errant myocyte to behave like its normal neighbors. From simulations, it has been estimated that in normal well-coupled ventricular muscle, literally hundreds of thousands of adjacent myocytes all have to simultaneously be primed to develop an EAD or DAD to generate a propagating AP (40). Since EADs and DADs tend to occur irregularly (26, 36), the source-to-sink mismatch is a powerful factor protecting well-coupled myocardium from afterdepolarization-induced premature ventricular contractions (PVCs) and triggered activity as it occurs in young nonfibrotic hearts. The presence of fibrosis in aged hearts, however, greatly weakens this protective effect by interposing collagen bundles between myocytes, preventing myocytes from maintaining normal gap junction coupling and greatly reducing the number of myocytes required to generate afterdepolarizations, which can propagate as premature ventricular depolarizations (PVDs; triggered activity in the tissue) (40), causing VF in the whole heart. Moreover, at the same time that fibrosis alters local source-tosink relationships to promote the emergence of EADs, the slow conduction induced by fibrosis predisposes to localized conduction block, increasing the likelihood that the PVDs and triggered beats will induce reentry and VF. This scenario is consistent with the optical and microelectrode mapping results in aged hearts exposed to either ANG II or hypokalemia, with EADs promoting focal PVCs and triggered activity causing focal VT, followed by mixed focal-reentrant VF (Figs. 1 and 3). In addition to the structural effects of fibrosis, fibroblasts can potentially directly contribute to arrhythmogenesis by paracrine actions (33) or by forming gap junctions with cardiac myocytes, which affects their electrophysiology, including inducing automaticity and promoting afterdepolarizations (19, 23). In the present study, we used the dynamic clamp technique to confirm that coupling a myocyte to a virtual fibroblast facilitated EAD formation during ANG II or hypokalemia (Fig. 6). Whether significant myocyte-myofibroblast gap junction coupling exists in intact native cardiac tissue remains highly controversial (4, 25). Nevertheless, our findings raise the theoretical possibility that if myocyte-fibroblast gap junction coupling occurs in aged fibrotic rat ventricles, it may potentiate the emergence of ANG II- and hypokalemia-induced EADs and VF. For example, myocyte-myofibroblast coupling might explain the finding that EAD and arrhythmia emergence in the intact fibrotic heart is not bradycardia dependent and can also occur during regular sinus rhythm at CLs of ⬍500 ms. Reduction of aging-related gap junction distribution also could play an important role in decreasing myocyte coupling and compromising the protective source-to-sink mismatch effect. Consistent with our previous studies (20, 21), we found a significant decrease in Cx43 density in aged compared with young ventricular tissue. FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS H2337 Downloaded from ajpheart.physiology.org on June 4, 2012 Fig. 4. ANG II induces EADs in myocytes isolated from young and aged rat hearts. A and B: effects of progressively shorter pacing cycle length (PCL) on ANG II (2 ␮M)-induced EADs and triggered activity in isolated single young (A) and aged (B) myocytes. Both young and aged myocytes manifested EADs and triggered activity. There was, however, a greater preponderance of EAD-mediated triggered beats at a PCL of 300 ms in the aged myocyte compared with the young myocyte. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org H2338 FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS Table 2. EADs and triggered activity initiated by ANG II and hypokalemia in young and aged isolated single rat ventricular myocytes Pacing Cycle Length 6s % 1s n % 0.5 s 0.25 s n % n % n 15 13 36 67 11 9 30 57 10 7 ANG II (2 ␮mol/l) Incidence of EADs and repolarization failure Young myocytes Aged myocytes P value Incidence of triggered activities Young myocytes Aged myocytes P value 82 100 17 14 67 85 NS 63 100 NS 16 14 44 92 ⬍0.01 NS 16 13 33 88 ⬍0.05 NS 12 8 55 80 ⬍0.05 11 5 NS Hypokalemia (2.7 mmol/l) 89 88 27 16 43 70 NS 93 81 7 10 7 40 NS 27 16 NS 43 50 14 5 0 0 NS 7 10 7 40 NS NS 14 5 NS 5 5 0 0 5 5 NS Incidence or fraction of myocytes in which an arrhythmia triggered early afterdepolarization (EAD; i.e., low-amplitude depolarization with no regenerative upstroke), repolarization failure, or triggered activity (spontaneous AP without pacing) induced by stress. n, number of ventricular myocytes tested. contrast, all of the aged rat hearts studied developed EADmediated VF after a much shorter exposure to hypokalemia, averaging 7.4 ⫾ 0.4 min, suggesting that their threshold for hypokalemia-induced VF was significantly lower than young hearts. That is, reduced repolarization reserve, without secondary intracellular Ca2⫹ overload from Na⫹-K⫹ pump inhibition, was perhaps sufficient to induce VF in aged rat hearts, whereas young rat hearts required both factors. The scenario leading to EADs in the whole heart, however, requires fibrosis (first hit) followed by reduced repolarization reserve (second hit) to promote EADs and triggered VF with ease. Fig. 5. A and B: hypokalemia-induced EADs (*) and triggered beats (arrows) in single myocytes isolated from young (A) and aged (B) rat hearts. The effects of progressively shorter PCLs of hypokalemia (2.7 mmol/l) are shown in myocytes isolated from both age groups. Cells in both age groups manifested EADs and repolarization failure at PCL ⬎ 1 s. EADs were suppressed at a PCL of shorter than 1 s in the young myocyte and at a PCL of 250 ms in the aged myocyte. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org Downloaded from ajpheart.physiology.org on June 4, 2012 Incidence of EADs and repolarization failure Young myocytes Aged myocytes P value Incidence of triggered activities Young myocytes Aged myocytes P value H2339 FIBROSIS IN AGING AND VENTRICULAR ARRHYTHMIAS Fig. 6. A and B: effects of coupling of a young rat ventricular myocyte (A) and an aged rat ventricular myocyte (B) to a virtual myofibroblast and the induction of EADs during hypokalemia at short PCLs. During the uncoupled state, hypokalemia failed to promote EADs during pacing at a cycle length of 0.5 s in both young (A) and aged (B) rats. However, coupling to a virtual myofibroblast, with a gap junction coupling conductance of 3 nS, a capacitance of 50 pF, and a resting membrane potential of ⫺50 mV, promoted EADs in both young (C) and aged (D) ventricular myocytes. Clinical Implications VF is the most common cause of sudden cardiac death and prematurely claims the lives of ⬃300,000 people every year in the United States. Animal models of spontaneous VF occurring from an otherwise regular cardiac rhythm are rare, so ANG II- and hypokalemia-induced arrhythmias in aged and young rat hearts may be a valuable model for the study of EAD-mediated VF, particularly since these two stressors are clinically relevant. A number of experimental and clinical studies have demonstrated therapeutic benefits of angiotensin-converting enzyme inhibitors (2) and ANG II receptor blockers in reducing arrhythmia risk in clinical settings (8, 16, 22). While the antiarrhythmic effects of chronic therapy with angiotensin-converting enzyme inhibitors and ANG II receptor blockers are usually attributed to long-term effects such as inhibition of fibrosis and hypertrophy, the present study and a prior study (41) suggest that blocking sudden elevation in ANG II may also contribute to reduced VF risk by suppressing ROS-mediated arrhythmias. Hypokalemia, at the level used in this study, occurs in a variety of clinical conditions, often as a side effect of diuretic therapy, and is known to promote arrhythmias and sudden cardiac death (3, 15, 27). Our findings emphasize the importance of maintaining normokalemia in patients with heart disease. In patients without heart disease, aging does not greatly increase VF risk (2), and ventricular fibrosis in otherwise normal aged hearts is less extensive than in rats (31). The aged rats used in this study also had normal lifespans, despite the dramatic cardiac fibrosis, suggesting that fibrosis alone is relatively benign unless coupled to a second hit, such as an ionic, oxidative, or metabolic stress consistent with the multihit hypothesis of VF (32). Limitations It could be argued that LV epicardial surface mapping may miss endocardial Purkinje cells in generating focal activity, and, as a result, the epicardial focal sites may reflect breakthrough activation. We cannot exclude a possible role of Purkinje fibers in the genesis of EADs since we did not perform endocardial cryoablation to eliminate this possibility. However, we did confirm in our previous studies (20, 21) that endocardial cryoablation did not abolish EAD-mediated arrhythmias induced by oxidative stress or glycolytic inhibition in aged rat ventricles, indicating that the epicardium is fully capable of generating EADs under these conditions. Moreover, the ability of isolated ventricular myocytes to generate EADs, DADs, and triggered activity indicates that ventricular myocytes have an intrinsic ability to generate EADs. Finally, the observation that young rat hearts did not exhibit EAD-mediated arrhythmias in response to ANG II, H2O2, or glycolytic inhibition (20, 21) implies that aging would have to significantly remodel His-Purkinje system properties to account for the increased susceptibility of aged rat hearts to EAD-mediated arrhythmias. GRANTS This work was supported by National Heart, Lung, and Blood Institute Grants P01-HL-78931 and R01-HL-103662 (to J. N. Weiss), the Laubisch and Kawata Endowments (to J. N. Weiss), a Sarnoff Cardiovascular Research Fellowship (to A. Bapat), and an American Heart Association-Western States Affiliate Postdoctoral Fellowship (to T. P. Nguyen). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). None AUTHOR CONTRIBUTIONS Author contributions: A.B., T.P.N., J.-H.L., and A.A.S. performed experiments; A.B., T.P.N., J.-H.L., A.A.S., M.C.F., and H.S.K. analyzed data; A.B., T.P.N., J.-H.L., M.C.F., J.N.W., and H.S.K. interpreted results of experiments; A.B., T.P.N., J.-H.L., A.A.S., M.C.F., and H.S.K. prepared figures; A.B. and H.S.K. drafted manuscript; A.B., T.P.N., J.-H.L., A.A.S., M.C.F., J.N.W., and AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00094.2012 • www.ajpheart.org Downloaded from ajpheart.physiology.org on June 4, 2012 The lack of fibrosis prevents the promotion of hypokalemiamediated EADs and triggered VF in young hearts (43%) despite reduced repolarization reserve (single hit). However, as the duration of hypokalemia increases, an additional mechanism may emerge (i.e., Ca2⫹ overload secondary to Na⫹-K⫹ pump inhibition), which could act as the second hit, promoting EADs and triggered VF in 57% of the young hearts. 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