Role of Stress Kinase JNK in Binge Alcohol-Evoked Atrial Arrhythmia.

BACKGROUND: Excessive binge alcohol drinking has acute cardiac arrhythmogenic effects, including promotion of atrial fibrillation (AF), which underlies "Holiday Heart Syndrome." The mechanism that couples binge alcohol abuse with AF susceptibility remains unclear. We previously reported stress-activated c-Jun N-terminal kinase (JNK) signaling contributes to AF development. This is interesting because JNK is implicated in alcohol-caused organ malfunction beyond the heart. OBJECTIVES: The purpose of this study was to detail how JNK promotes binge alcohol-evoked susceptibility to AF. METHODS: The authors found binge alcohol-exposure leads to activated JNK, specifically JNK2. Furthermore, binge alcohol induces AF (24- vs. 1.8-Hz burst pacing-induced episodes per attempt per animal), higher incidence of diastolic intracellular Ca2+ activity (Ca2+ waves, sarcoplasmic reticulum [SR] Ca2+ leakage), and membrane voltage (Vm) and systolic Ca2+ release spatiotemporal heterogeneity (ΔtVm-Ca). These changes were completely eliminated by JNK inhibition both in vivo and in vitro. calmodulin kinase II (CaMKII) is a proarrhythmic molecule known to drive SR Ca2+ mishandling. RESULTS: The authors report for the first time that binge alcohol activates JNK2, which subsequently phosphorylates the CaMKII protein, enhancing CaMKII-driven SR Ca2+ mishandling. CaMKII inhibition eliminates binge alcohol-evoked arrhythmic activities. CONCLUSIONS: Our studies demonstrate that binge alcohol exposure activates JNK2 in atria, which then drives CaMKII activation, prompting aberrant Ca2+ waves and, thus, enhanced susceptibility to atrial arrhythmia. Our results reveal a previously unrecognized form of alcohol-driven kinase-on-kinase proarrhythmic crosstalk. Atrial JNK2 function represents a potential novel therapeutic target to treat and/or prevent AF.

Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial Arrhythmogenesis.

RATIONALE: Atrial fibrillation (AF) is the most common arrhythmia, and advanced age is an inevitable and predominant AF risk factor. However, the mechanisms that couple aging and AF propensity remain unclear, making targeted therapeutic interventions unattainable. OBJECTIVE: To explore the functional role of an important stress response JNK (c-Jun N-terminal kinase) in sarcoplasmic reticulum Ca2+ handling and consequently Ca2+-mediated atrial arrhythmias. METHODS AND RESULTS: We used a series of cutting-edge electrophysiological and molecular techniques, exploited the power of transgenic mouse models to detail the molecular mechanism, and verified its clinical applicability in parallel studies on donor human hearts. We discovered that significantly increased activity of the stress response kinase JNK2 (JNK isoform 2) in the aged atria is involved in arrhythmic remodeling. The JNK-driven atrial proarrhythmic mechanism is supported by a pathway linking JNK, CaMKII (Ca2+/calmodulin-dependent kinase II), and sarcoplasmic reticulum Ca2+ release RyR2 (ryanodine receptor) channels. JNK2 activates CaMKII, a critical proarrhythmic molecule in cardiac muscle. In turn, activated CaMKII upregulates diastolic sarcoplasmic reticulum Ca2+ leak mediated by RyR2 channels. This leads to aberrant intracellular Ca2+ waves and enhanced AF propensity. In contrast, this mechanism is absent in young atria. In JNK challenged animal models, this is eliminated by JNK2 ablation or CaMKII inhibition. CONCLUSIONS: We have identified JNK2-driven CaMKII activation as a novel mode of kinase crosstalk and a causal factor in atrial arrhythmic remodeling, making JNK2 a compelling new therapeutic target for AF prevention and treatment.

The stress kinase JNK regulates gap junction Cx43 gene expression and promotes atrial fibrillation in the aged heart.

BACKGROUND: The stress kinase c-jun N-terminal kinase (JNK) is critical in the pathogenesis of cardiac diseases associated with an increased incidence of atrial fibrillation (AF), the most common arrhythmia in the elderly. We recently discovered that JNK activation is linked to the loss of gap junction connexin43 (Cx43) and enhanced atrial arrhythmogenicity. However, direct evidence for JNK-mediated impairment of intercellular coupling (cell-cell communication) in the intact aged atrium is lacking, as is evidence for whether and how JNK suppresses Cx43 in the aged human atrium. METHODS AND RESULTS: JNK activity in human atrial samples is correlated with both reduced Cx43 expression and increasing age. Using a unique technique of optical mapping space constant measurement, we found that impaired intercellular coupling and reduced Cx43 were linked to enhanced activation of JNK in intact aged rabbit atria. These JNK-associated alterations were further confirmed in naturally JNK activated aged mice and in cardiac-specific inducible MKK7D (JNK upstream activator) young mice. Moreover, JNK inhibition, using either JNK specific inhibitors in aged wild-type (WT) mice and JNK activator anisomycin-treated young WT mice or JNK1/2 dominant-negative mice with genetically inhibited cardiac JNK activity, completely eliminated these functional abnormalities. Furthermore, we discovered for the first time that long-term JNK activation downregulates Cx43 expression via c-jun suppressed transcriptional activity of the Cx43 gene promoter. CONCLUSION: Our results demonstrate that JNK is a critical regulator of Cx43 expression, and that augmented JNK activation in aged atria downregulates Cx43 to impair cell-cell communication and promote the development of AF. JNK inhibition may represent a promising therapeutic approach to prevent or treat AF in the elderly.

Voltage and calcium dual channel optical mapping of cultured HL-1 atrial myocyte monolayer.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

BACKGROUND: Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies. METHODS AND RESULTS: Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.