Mycobacterium tuberculosis suppresses host antimicrobial peptides by dehydrogenating L-alanine.

Antimicrobial peptides (AMPs), ancient scavengers of bacteria, are very poorly induced in macrophages infected by Mycobacterium tuberculosis (M. tuberculosis), but the underlying mechanism remains unknown. Here, we report that L-alanine interacts with PRSS1 and unfreezes the inhibitory effect of PRSS1 on the activation of NF-κB pathway to induce the expression of AMPs, but mycobacterial alanine dehydrogenase (Ald) Rv2780 hydrolyzes L-alanine and reduces the level of L-alanine in macrophages, thereby suppressing the expression of AMPs to facilitate survival of mycobacteria. Mechanistically, PRSS1 associates with TAK1 and disruptes the formation of TAK1/TAB1 complex to inhibit TAK1-mediated activation of NF-κB pathway, but interaction of L-alanine with PRSS1, disables PRSS1-mediated impairment on TAK1/TAB1 complex formation, thereby triggering the activation of NF-κB pathway to induce expression of AMPs. Moreover, deletion of antimicrobial peptide gene ß-defensin 4 (Defb4) impairs the virulence by Rv2780 during infection in mice. Both L-alanine and the Rv2780 inhibitor, GWP-042, exhibits excellent inhibitory activity against M. tuberculosis infection in vivo. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses its own alanine dehydrogenase to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.

Mycobacterium tuberculosis produces D-serine under hypoxia to limit CD8+ T cell-dependent immunity in mice.

Adaptation to hypoxia is a major challenge for the survival of Mycobacterium tuberculosis (Mtb) in vivo. Interferon (IFN)-γ-producing CD8+ T cells contribute to control of Mtb infection, in part by promoting antimicrobial activities of macrophages. Whether Mtb counters these responses, particularly during hypoxic conditions, remains unknown. Using metabolomic, proteomic and genetic approaches, here we show that Mtb induced Rv0884c (SerC), an Mtb phosphoserine aminotransferase, to produce D-serine. This activity increased Mtb pathogenesis in mice but did not directly affect intramacrophage Mtb survival. Instead, D-serine inhibited IFN-γ production by CD8+ T cells, which indirectly reduced the ability of macrophages to restrict Mtb upon co-culture. Mechanistically, D-serine interacted with WDR24 and inhibited mTORC1 activation in CD8+ T cells. This decreased T-bet expression and reduced IFN-γ production by CD8+ T cells. Our findings suggest an Mtb evasion mechanism where pathogen metabolic adaptation to hypoxia leads to amino acid-dependent suppression of adaptive anti-TB immunity.

Mycobacterium tuberculosis inhibits METTL14-mediated m6A methylation of Nox2 mRNA and suppresses anti-TB immunity.

Internal N6-methyladenosine (m6A) modifications are among the most abundant modifications of messenger RNA, playing a critical role in diverse biological and pathological processes. However, the functional role and regulatory mechanism of m6A modifications in the immune response to Mycobacterium tuberculosis infection remains unknown. Here, we report that methyltransferase-like 14 (METTL14)-dependent m6A methylation of NAPDH oxidase 2 (Nox2) mRNA was crucial for the host immune defense against M. tuberculosis infection and that M. tuberculosis-secreted antigen EsxB (Rv3874) inhibited METTL14-dependent m6A methylation of Nox2 mRNA. Mechanistically, EsxB interacted with p38 MAP kinase and disrupted the association of TAB1 with p38, thus inhibiting the TAB1-mediated autophosphorylation of p38. Interaction of EsxB with p38 also impeded the binding of p38 with METTL14, thereby inhibiting the p38-mediated phosphorylation of METTL14 at Thr72. Inhibition of p38 by EsxB restrained liquid-liquid phase separation (LLPS) of METTL14 and its subsequent interaction with METTL3, preventing the m6A modification of Nox2 mRNA and its association with the m6A-binding protein IGF2BP1 to destabilize Nox2 mRNA, reduce ROS levels, and increase intracellular survival of M. tuberculosis. Moreover, deletion or mutation of the phosphorylation site on METTL14 impaired the inhibition of ROS level by EsxB and increased bacterial burden or histological damage in the lungs during infection in mice. These findings identify a previously unknown mechanism that M. tuberculosis employs to suppress host immunity, providing insights that may empower the development of effective immunomodulators that target M. tuberculosis.

Mycobacterium tuberculosis suppresses host DNA repair to boost its intracellular survival.

Mycobacterium tuberculosis (Mtb) triggers distinct changes in macrophages, resulting in the formation of lipid droplets that serve as a nutrient source. We discover that Mtb promotes lipid droplets by inhibiting DNA repair responses, resulting in the activation of the type-I IFN pathway and scavenger receptor-A1 (SR-A1)-mediated lipid droplet formation. Bacterial urease C (UreC, Rv1850) inhibits host DNA repair by interacting with RuvB-like protein 2 (RUVBL2) and impeding the formation of the RUVBL1-RUVBL2-RAD51 DNA repair complex. The suppression of this repair pathway increases the abundance of micronuclei that trigger the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway and subsequent interferon-ß (IFN-ß) production. UreC-mediated activation of the IFN-ß pathway upregulates the expression of SR-A1 to form lipid droplets that facilitate Mtb replication. UreC inhibition via a urease inhibitor impaired Mtb growth within macrophages and in vivo. Thus, our findings identify mechanisms by which Mtb triggers a cascade of cellular events that establish a nutrient-rich replicative niche.

Electroporation induced by internal defibrillation shock with and without recovery in intact rabbit hearts.

Defibrillation shocks from implantable cardioverter defibrillators can be lifesaving but can also damage cardiac tissues via electroporation. This study characterizes the spatial distribution and extent of defibrillation shock-induced electroporation with and without a 45-min postshock period for cell membranes to recover. Langendorff-perfused rabbit hearts (n = 31) with and without a chronic left ventricular (LV) myocardial infarction (MI) were studied. Mean defibrillation threshold (DFT) was determined to be 161.4 ± 17.1 V and 1.65 ± 0.44 J in MI hearts for internally delivered 8-ms monophasic truncated exponential (MTE) shocks during sustained ventricular fibrillation (>20 s, SVF). A single 300-V MTE shock (twice determined DFT voltage) was used to terminate SVF. Shock-induced electroporation was assessed by propidium iodide (PI) uptake. Ventricular PI staining was quantified by fluorescent imaging. Histological analysis was performed using Masson's Trichrome staining. Results showed PI staining concentrated near the shock electrode in all hearts. Without recovery, PI staining was similar between normal and MI groups around the shock electrode and over the whole ventricles. However, MI hearts had greater total PI uptake in anterior (P < 0.01) and posterior (P < 0.01) LV epicardial regions. Postrecovery, PI staining was reduced substantially, but residual staining remained significant with similar spacial distributions. PI staining under SVF was similar to previously studied paced hearts. In conclusion, electroporation was spatially correlated with the active region of the shock electrode. Additional electroporation occurred in the LV epicardium of MI hearts, in the infarct border zone. Recovery of membrane integrity postelectroporation is likely a prolonged process. Short periods of SVF did not affect electroporation injury.

CGAS is a micronucleophagy receptor for the clearance of micronuclei.

Micronuclei are constantly considered as a marker of genome instability and very recently found to be a trigger of innate immune responses. An increased frequency of micronuclei is associated with many diseases, but the mechanism underlying the regulation of micronuclei homeostasis remains largely unknown. Here, we report that CGAS (cyclic GMP-AMP synthase), a known regulator of DNA sensing and DNA repair, reduces the abundance of micronuclei under genotoxic stress in an autophagy-dependent manner. CGAS accumulates in the autophagic machinery and directly interacts with MAP1LC3B/LC3B in a manner dependent upon its MAP1LC3-interacting region (LIR). Importantly, the interaction is essential for MAP1LC3 recruitment to micronuclei and subsequent clearance of micronuclei via autophagy (micronucleophagy) in response to genotoxic stress. Moreover, in contrast to its DNA sensing function to activate micronuclei-driven inflammation, CGAS-mediated micronucleophagy blunts the production of cyclic GMP-AMP (cGAMP) induced by genotoxic stress. We therefore conclude that CGAS is a receptor for the selective autophagic clearance of micronuclei and uncovered an unprecedented role of CGAS in micronuclei homeostasis to dampen innate immune surveillance.Abbreviations: ATG: autophagy-related; CGAS: cyclic GMP-AMP synthase; CQ: chloroquine; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; LIR, MAP1LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NDZ: nocodazole; STING1: stimulator of interferon response cGAMP interactor 1.

Membrane time constant during internal defibrillation strength shocks in intact heart: effects of Na+ and Ca2+ channel blockers.

INTRODUCTION: We assessed defibrillation strength shock-induced changes of the membrane time constant (tau) and membrane potential (DeltaVm) in intact rabbit hearts after administration of lidocaine, a sodium (Na(+)) channel blocker, or nifedipine, a L-type calcium (Ca(2+)) channel blocker. METHODS AND RESULTS: We optically mapped anterior, epicardial, electrical activity during monophasic shocks (+/-100, +/-130, +/-160, +/-190, and +/-220 V; 150 microF; 8 ms) applied at 25%, 50%, and 75% of the action potential duration via a shock lead system in Langendorff-perfused hearts. The protocol was run twice for each heart under control and after lidocaine (15 microM, n = 6) or nifedipine (2 microM, n = 6) addition. tau in the virtual electrode area away from the shock lead was approximated with single-exponential fits from a total of 121,125 recordings. The same data set was used to calculate DeltaVm. We found (1) Under all conditions, there is inverse relationship between tau and DeltaVm with respect to changes of shock strength, regardless of shock polarity and phase of application: a stronger shock resulted in a larger DeltaVm, which corresponded to a smaller tau (faster cellular response); (2) Lidocaine did not cause appreciable changes in either tau or DeltaVm versus control, and (3) Nifedipine significantly increased both tau and DeltaVm in the virtual cathode area; in contrast, in the virtual anode area, this effect depended on the phase of shock application. CONCLUSION: tau and DeltaVm are inversely related. Na(+) channel blocker has minimal impact on either tau or DeltaVm. Ca(2+) blocker caused polarity and phase-dependent significant changes in tau and DeltaVm.

Spatial distribution and extent of electroporation by strong internal shock in intact structurally normal and chronically infarcted rabbit hearts.

INTRODUCTION: Although life-saving, a strong internal defibrillation shock may temporarily or permanently damage the heart via disruption of cell membranes (electroporation). Spatial extent of electroporation in intact, normal, or infarcted hearts has not been investigated. In this study, shock-induced electroporation in intact rabbit hearts with and without chronic (>4 weeks) left ventricular myocardial infarction (MI) was characterized. METHODS AND RESULTS: A coil shock electrode was inserted in the right ventricle of Langendorff-perfused hearts. One truncated exponential monophasic shock (+300 V, 8 ms) was delivered by a 150 microF capacitor clinical defibrillator while the heart was perfused with membrane-impermeant dye propidium iodide (PI). The heart was sectioned transversely, and uptake of PI into ventricular myocardium through electropores was quantified. Histological evaluation was performed via Masson's trichrome staining. PI accumulation was minimal in the control (n = 3) and MI (n = 3) hearts without shock. Following shock delivery, (1) in control (n = 5) and MI (n = 5) hearts, electroporation mostly occurred near the shock electrode and was longitudinally distributed along the active region of the shock electrode; (2) in MI group, electroporation was significantly increased (P < 0.05) in the surviving anterior epicardial layers of the infarcted region; and (3) between the control and MI groups, the overall extent of electroporation was similar. CONCLUSION: Shock-induced electroporation was spatially dependent on the location and dimension of the active region of the shock electrode. The overall extent of electroporation in the MI heart was comparable with the control heart, but the surviving anterior epicardial layers in the infarcted region were more susceptible to electroporation.

Optical mapping of shock-induced arrhythmogenesis in the rabbit heart with healed myocardial infarction: fluorescent imaging with a photodiode array.

Optical mapping of electrical activity in the heart employs digital imaging and voltage-sensitive dyes. These methods have become an increasingly common research tools in basic cardiac electrophysiology. Significant advantages of this approach include simultaneous noncontact recording of entire action potentials free of electrical stimulus-induced artifacts from multiple closely adjacent sites, and adjustable spatial and temporal resolutions. In this way, the activation pattern as well as the repolarization pattern can be monitored by dynamic registration of transmembrane potential changes. As a result, the success of these techniques is most evident in the investigation of the mechanisms of pacing, vulnerability, and defibrillation, in which conventional electrical recordings are hampered by stimulus-induced artifacts. Using optical mapping technology and instrumentation driven by LabVIEW software, we mapped changes in transmembrane voltage during defibrillation shocks and identified the mechanisms of vulnerability and defibrillation in rabbit hearts with healed myocardial infarction (>or=4 wk postinfarction).

An experimental rabbit model for off-pump left ventricular reconstruction following left ventricular aneurysm.

BACKGROUND: Cardiac electromechanical remodeling following left ventricular reconstruction (LVR) surgery is not fully understood. Further development of an animal model will facilitate investigations in this area. In the present study, we aimed to establish a novel LVR procedure without the use of cardiopulmonary bypass in a rabbit left ventricular (LV) aneurysm model. METHODS: LV aneurysm was created in 6 rabbits by ligation of the distal left coronary artery. More than a month later, LVR aneurysm surgery was performed off-pump using a purse-string suture around the aneurysm. Cardiac dimensions and function were evaluated using echocardiographic techniques perioperatively and 4 weeks after LVR surgery. Six structurally normal hearts were used as controls. RESULTS: LVR surgery was successfully performed in all 6 rabbits. Both LV end-diastolic volume (LVEDV, 4.6 +/- 0.9 to 3.3 +/- 0.6 mL; P < .01) and LV end-systolic volume (LVESV, 2.5 +/- 0.6 to 1.5 +/- 0.2 mL, P < .01) were decreased immediately postsurgery versus presurgery, and LV ejection fraction (LVEF) was increased (44.5 +/- 5.3 to 55.6 +/- 4.8%, P < .001). For comparison, in normal rabbits (n = 6), LVEDV, LVESV and LVEF were 3.1 +/- 0.7 mL, 1.2 +/- 0.5 mL, and 64.5 +/- 8.8%, respectively. During follow-up, one rabbit died 3 weeks after surgery from an unknown cause. In the remaining 5 animals, improvements of LVEDV (3.7+/- 0.4 mL, P < .05), LVESV (1.7 +/- 0. 3 mL, P < .01), and LVEF (53.1 +/- 2.8%, P < .01) were maintained versus presurgery values for more than 4 weeks after LVR. CONCLUSIONS: Off-pump LVR of rabbit LV aneurysm is an effective and less invasive surgery that resulted in sustained improvement in cardiac function with no gross intraoperative or postoperative mortality. This may be a useful model for investigations of electromechanical remodeling following LVR.

Longitudinal study of cardiac remodelling in rabbits following infarction.

BACKGROUND: Cardiac remodelling following myocardial infarction (MI) is a complex, dynamic process. There have been few longitudinal studies of these changes. METHODS: A 2-dimensional transthoracic echocardiography was performed on 20 rabbits, before and 1, 2, 4, 8, and 12 weeks after MI (n = 14) and twice for controls (n = 6). Chronic left ventricular (LV) infarct size was histologically characterized and correlated with mechanical function. A linear mixed model was used to analyze longitudinal and infarct size-related changes in LV end-systolic volume (ESV), end-diastolic volume (EDV), ejection fraction (EF), sphericity, circumferential strain, and wall motion score index. RESULTS: Mean LV infarct size was 28.9% ± 9.3%. After MI, rapid remodelling occurred in LVESV, LVEF, and sphericity for 2 weeks and LVEDV for 4 weeks, with slower changes afterwards. LV infarct size correlated with LVESV (r = 0.76), LVEDV (r = 0.71), and LVEF (r = 0.69). Larger infarcts resulted in greater LVESV dilation (P = 0.04) and faster LVEDV (P < 0.01), LVEF (P < 0.01), and sphericity (P < 0.01) remodelling. Apical global circumferential strain and wall motion score index increased for 1 week, then stabilized, regardless of infarct size, and apical global circumferential strain was correlated with apical infarction (r = 0.58). Additionally, regional circumferential strain decreased in segments with severe (> 80%) infarction more quickly (P < 0.01) and by a greater degree (P = 0.04) compared with segments with minor (< 20%) infarction. CONCLUSIONS: The most dynamic remodelling of cardiac function in this model occurred during the first 4 weeks, stabilizing thereafter, with changes maintained up to 12 weeks. Infarct size affected both the early rate and long-term extent of mechanical remodelling.

How similar are the mice to men? Between-species comparison of left ventricular mechanics using strain imaging.

BACKGROUND: While mammalian heart size maintains constant proportion to whole body size, scaling of left ventricular (LV) function parameters shows a more complex scaling pattern. We used 2-D speckle tracking strain imaging to determine whether LV myocardial strains and strain rates scale to heart size. METHODS: We studied 18 mice, 15 rats, 6 rabbits, 12 dogs and 20 human volunteers by 2-D echocardiography. Relationship between longitudinal or circumferential strains/strain rates (S(Long)/SR(Long), S(Circ)/SR(Circ)), and LV end-diastolic volume (EDV) or mass were assessed by the allometric (power-law) equation Y = kM(ß). RESULTS: Mean LV mass in individual species varied from 0.038 to 134 g, LV EDV varied from 0.015 to 102 ml, while RR interval varied from 81 to 1090 ms. While S(Long) increased with increasing LV EDV or mass (ß values 0.047±0.006 and 0.051±0.005, p<0.0001 vs. 0 for both) S(Circ) was unchanged (p = NS for both LV EDV or mass). Systolic and diastolic SR(Long) and SR(Circ) showed inverse correlations to LV EDV or mass (p<0.0001 vs. 0 for all comparisons). The ratio between S(Long) and S(Circ) increased with increasing values of LV EDV or mass (ß values 0.039±0.010 and 0.040±0.011, p>0.0003 for both). CONCLUSIONS: While S(Circ) is unchanged, S(Long) increases with increasing heart size, indicating that large mammals rely more on long axis contribution to systolic function. SR(Long) and SR(Circ), both diastolic and systolic, show an expected decrease with increasing heart size.

Optical mapping of ventricular arrhythmias in LQTS mice with SCN5A mutation N1325S.

Transgenic expression of SCN5A mutation N1325S creates a mouse model for type-3 long QT syndrome (LQT3), TG-NS/LQT3. Optical mapping is a high temporal and spatial resolution fluorescence mapping system that records 256 action potentials simultaneously in a Langendorff-perfused heart. Here for the first-time, we provide a spatial view of VT in a genetic LQT3 model using optical mapping. Spontaneous VT was detected in TG-NS/LQT3 hearts, but not in littermate control hearts. VT was initiated primarily by activation of a new firing focus as well as functional conduction block of new activation waves. New firing was initiated at many different Loci in the heart, suggesting that "increased automaticity" is a key mechanism for initiation of VT. The sustained VT was maintained by a reentry mechanism. Nifedipine, an L-type calcium channel blocker, decreased the frequency of VT, indicating the involvement of abnormalities of the calcium homeostasis in the genesis of VT in TG-NS/LQT3 mice.

Scaling of diastolic intraventricular pressure gradients is related to filling time duration.

In early diastole, pressure is lower in the apex than in the base of the left ventricle (LV). This early intraventricular pressure difference (IVPD) facilitates LV filling. We assessed how LV diastolic IVPD and intraventricular pressure gradient (IVPG), defined as IVPD divided by length, scale to the heart size and other physiological variables. We studied 10 mice, 10 rats, 5 rabbits, 12 dogs, and 21 humans by echocardiography. Color Doppler M-mode data were postprocessed to reconstruct IVPD and IVPG. Normalized LV filling time was calculated by dividing filling time by RR interval. The relationship between IVPD, IVPG, normalized LV filling time, and LV end-diastolic volume (or mass) as fit to the general scaling equation Y = kM beta, where M is LV heart size parameter, Y is a dependent variable, k is a constant, and beta is the power of the scaling exponent. LV mass varied from 0.049 to 194 g, whereas end-diastolic volume varied from 0.011 to 149 ml. The beta values relating normalized LV filling time with LV mass and end-diastolic volume were 0.091 (SD 0.011) and 0.083 (SD 0.009), respectively (P < 0.0001 vs. 0 for both). The beta values relating IVPD with LV mass and end-diastolic volume were similarly significant at 0.271 (SD 0.039) and 0.243 (SD 0.0361), respectively (P < 0.0001 vs. 0 for both). Finally, beta values relating IVPG with LV mass and end-diastolic volume were -0.118 (SD 0.013) and -0.104 (SD 0.011), respectively (P < 0.0001 vs. 0 for both). As a result, there was an inverse relationship between IVPG and normalized LV filling time (r = -0.65, P < 0.001). We conclude that IVPD decrease, while IVPG increase with decreasing animal size. High IVPG in small mammals may be an adaptive mechanism to short filling times.

Mechanisms of enhanced shock-induced arrhythmogenesis in the rabbit heart with healed myocardial infarction.

Shock-induced vulnerability and defibrillation have been mostly studied in structurally normal hearts. However, defibrillation therapy is normally applied to patients with diseased hearts, frequently those with prior myocardial infarction (MI). Shock-induced vulnerability and defibrillation have not been well studied under this condition. We sought to examine the mechanisms of shock-induced arrhythmogenesis and arrhythmia maintenance in a rabbit model of healed MI (4 wk or more postinfarction). Ligation of the lateral division or posterolateral division of the left coronary artery at a level of 40-70% from the apex was performed 53 +/- 21 days before acute experiments. Shock-induced vulnerability was assessed in infarcted (n = 8) and structurally normal (n = 8) hearts by delivering internal monophasic shocks at different shock strengths and delivery phases. Electrical activities from the anterior epicardium during shock application and during shock-induced arrhythmias were optically recorded and quantitatively analyzed. Ligation resulted in a transmural left ventricular free wall infarction mainly located at the apical region with a consistent endocardial border zone (BZ) as confirmed by histological studies. There were significant increases in the incidence, severity, and duration of shock-induced arrhythmias in the infarcted hearts versus controls due to 1) postshock break-excitation wavefronts that frequently originated near the infarction BZ and 2) the existence of an infarction BZ that created an anatomic reentry pathway and facilitated arrhythmia maintenance. In conclusion, the infarction BZ contributes to both increased shock-induced arrhythmogenesis and arrhythmia maintenance in the rabbit model of healed MI.

Differences in left ventricular long-axis function from mice to humans follow allometric scaling to ventricular size.

While the heart size maintains a constant proportion to body size, heart function parameters, such as heart rate and cardiac output, show a more complex scaling pattern. How these phenomena affect the long-axis left ventricular (LV) function is unknown. We studied 10 mice, 15 rats, 6 rabbits, 8 mongrel dogs and 38 human volunteers. Doppler tissue echocardiography data were postprocessed to reconstruct mitral annulus (MA) peak systolic velocity and displacement. The relationship between MA peak velocity, MA displacement and LV ejection time, and LV end-diastolic volume (and mass) were fit to an allometric (power-law) equation Y=kMbeta. LV mass varied from 0.062 to 255 g, while end-diastolic volume varied from 0.014 to 205 ml. beta values of the relation between LV ejection time and LV end-diastolic volume and mass were 0.247+/-0.017 and 0.267+/-0.018, respectively. beta values of the relationship between MA displacement and LV end-diastolic volume and mass were 0.358+/-0.047 and 0.390+/-0.051 (P<0.023 versus beta of LV ejection time). beta values of the relationship between MA peak systolic velocity and LV end-diastolic volume and mass were 0.096+/-0.012 and 0.100+/-0.013, respectively (P<0.0001 versus 0). Finally, beta values of the relationship between the long-to-short axis displacement ratio and LV end-diastolic volume and mass were 0.077+/-0.017 and 0.086+/-0.019 (P<0.0001 versus 0). We conclude that MA velocity, displacement, and long-to-short axis displacement ratio scale allometrically to heart size. This reduces the relative long-axis contribution to heart function in small mammals.

Mechanisms of shock-induced arrhythmogenesis during acute global ischemia.

Little is known about the mechanisms of vulnerability and defibrillation under ischemic conditions. We investigated these mechanisms in 18 Langendorff-perfused rabbit hearts during 75% reduced-flow ischemia. Electrical activity was optically mapped from the anterior epicardium during right ventricular shocks applied at various phases of the cardiac cycle while the excitation-contraction decoupler 2,3-butanedione monoxime (BDM; 15 mM) was used to suppress motion artifacts caused by contraction of the heart. During ischemia, vulnerable window width increased [from 30-90% of the action potential duration (APD) in the control to -10 to 100% of the APD in ischemia]. Moreover, arrhythmia severity increased along with the reduction of APD (176 +/- 9 ms in control and 129 +/- 26 ms in ischemia, P < 0.01) and increased dispersion of repolarization (45 +/- 17 ms in control and 73 +/- 28 ms in ischemia, P < 0.01). Shock-induced virtual electrode polarization was preserved. Depolarizing (contrary to hyperpolarizing) response time constants increased. Virtual electrode-induced wavefronts of excitation had much more tortuous pathways leading to wavefront fractionation. Defibrillation failure at all shock strengths was observed in four hearts. Optical mapping revealed that the shock extinguished the arrhythmia; however, the arrhythmia self-originated after an isoelectric window of 339 +/- 189 ms. In conclusion, in most cases, virtual electrode-induced phase singularity (VEIPS) was responsible for shock-induced arrhythmogenesis during acute global ischemia. Enhancement of arrhythmogenesis was associated with an increased dispersion of repolarization and altered deexcitation. In four hearts, arrhythmogenesis could not be explained by VEIPS.

Shock-induced arrhythmogenesis is enhanced by 2,3-butanedione monoxime compared with cytochalasin D.

Investigation of the mechanisms of arrhythmia genesis and maintenance has benefited from the use of optical mapping techniques that employ excitation-contraction uncouplers. We investigated the effects of the excitation-contraction uncouplers 2,3-butanedione monoxime (BDM) and cytochalasin D (Cyto D) on the induction and maintenance of arrhythmia by electric shocks. Electrical activity was optically mapped from anterior epicardium of rabbit hearts (n = 9) during shocks (-100 V, 8 ms) applied from a ventricular lead at various phases of action potential duration (APD). Restitution curves were obtained using S1-S2 protocol and measurement of APD values at 70% of repolarization. Compared with Cyto D, BDM significantly shortened APD at 90% of repolarization, although no significant difference in dispersion of repolarization was observed. Wavelength was also shortened with BDM. In general, shock-induced arrhythmias with BDM and Cyto D were ventricular tachycardic in nature. With respect to shock-induced sustained arrhythmias, the vulnerable window was wider and the incidence was higher with BDM than with Cyto D. There was also a difference in the morphology of ventricular tachycardia (VT) between the two agents. The arrhythmias with BDM usually resembled monomorphic VT, especially those that lasted >30 s. In contrast, arrhythmias with Cyto D more resembled polymorphic VT. However, the average number of phase singularities increased under Cyto D vs. BDM, whereas no significant difference in the dominant frequency of shock-induced sustained arrhythmia was observed. BDM reduced the slope of the restitution curve compared with Cyto D, but duration of arrhythmia under BDM was significantly increased compared with Cyto D. In conclusion, BDM increased arrhythmia genesis and maintenance relative to Cyto D.