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

FIGURE 1. Binge Alcohol Increases the Inducibility of Atrial Arrhythmia in the Human Donor Heart and Rabbit Heart (A) Representative electrogram trace showing burst pacing (3 Hz)-induced AF in a binge alcohol-exposed human donor heart (with normal cardiac function). (B) Representative EG recording from a paced age-matched control human donor heart and there was no AF upon cessation of pacing. (C) Representative electrogram trace showing burst pacing (for 30 s at 6× diastolic threshold; cycle length = 100 ms)-induced AF immediately after cessation of the pacing in open-chest rabbits with binge alcohol pre-exposure, whereas the same burst pacing protocol did not induce any incidence of atrial arrhythmia in sham control rabbit. (D) Summarized data showing enhanced AF inducibility and increased average duration of pacing-induced AF in binge alcohol-exposed rabbits compared to that of sham controls. AF = atrial fibrillation; Alc = alcohol; EG = electrogram; NSR = normal sinus rhythm.

FIGURE 2. JNK Activation in Binge Alcohol-Exposed Atria (A) Summarized quantitative immunoblotting data and representative immunoblotting images show increased levels of JNK-P and unchanged expression of total JNK1 and JNK2 proteins in the atrial tissue from binge alcohol-exposed human donor hearts compared to non–alcohol-exposed controls. (B, C) Summarized data and representative immunoblotting images show enhanced level of JNK-P in alcohol-exposed rabbit and mouse atria. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; JNK = c-Jun N-terminal kinase; JNK-P = phosphorylated JNK; WT = wild type; other abbreviation as in Figure 1.

FIGURE 3. JNK Inhibition Precludes Binge Alcohol-Evoked Atrial Arrhythmogenicity in Intact Mouse Atria (A) Representative electrogram traces showing burst-pacing induced atrial arrhythmia in a Langendorff-perfused WT mouse heart with binge alcohol pre-exposure (1.), and self-restored NSR after burst pacing in sham control mouse hearts (2.) as well as binge alcohol-exposed JNK1/2dn mouse hearts with overexpression of cardiac-specific inactivated dominant negative JNK (3.). (B, C) Summarized data of enhanced AF inducibility and increased average duration of pacing-induced AF in binge alcohol-exposed WT mouse hearts compared to that of sham controls. JNK1/2dn = inactivated dominant negative JNK1 and JNK2; other abbreviations as in Figures 1 and 2.

FIGURE 4. JNK Inhibition Abolished Binge Alcohol-Evoked Abnormal Atrial Ca2+ Waves in Intact Mouse Atria (A) Representative confocal images show increased frequency of Ca2+ sparks (yellow arrows) and Ca2+ waves (blue arrows) in binge alcohol-exposed WT mouse atria after 10-Hz burst pacing (1.) and reversion of NSR in either control WT mice (2.) or JNK1/2dn mice with overexpression of cardiac-specific JNK1/2dn (3.). (B) Summarized data show increased frequency of Ca2+ waves in binge alcohol-exposed mouse atria, while dominant negative overexpression of JNK1/2 precluded binge alcohol-prompted Ca2+waves. (C) Atrial Ca2+decay constant τ was increased in binge alcohol-exposed WT mice, but binge alcohol-exposed JNK1/2dn mice exhibited unchanged τ of Ca2+ decay compared to that of sham controls. Ca2+ = calcium; other abbreviations as in Figures 1 to 3.

FIGURE 5. JNK2 Inhibition (In Vivo Treatment) Eliminates Binge Alcohol-Evoked Abnormal Ca2+ Handling in Intact Atria and Atrial Myocytes (A) Summarized data show enhanced JNK2 kinase activity (measured by ADP production) in JNK2 antibody-specific pulldown JNK2 proteins but unchanged JNK1 activity. (B) Summarized data show enhanced heterogeneity of ΔtVm-Ca in alcohol-exposed mouse left atria, whereas JNK2 inhibitor in vivo treatment abolished this arrhythmogenic abnormality. (C) Representative optical isochronal maps show heterogeneous wavefront propagation of the Vm and Ca2+ signals in an alcohol-exposed atrium compared to a WT sham control. JNK2 inhibition effectively reversed this alcohol-evoked heterogeneity. (D, E) Summarized data show increased tetracaine-sensitive diastolic SR Ca2+ leakage in alcohol-exposed rabbit atrial myocytes (D) and HL-1 myocytes (E), while JNK2 inhibition abolished this alcohol-induced SR Ca2+ leak. ADP = adenosine diphosphate; JNK2I = JNK2 inhibitor; NS = no statistical significance; RLU = relative light units; Vm = membrane potential; other abbreviations as in Figures 1 to 3.

FIGURE 6. Like the Effect of JNK2 Inhibition, CaMKII Inhibition Also Abolishes This Binge Alcohol-Evoked Abnormal Ca2+ Handling in Atrial Myocytes and Intact Atria (A) Summarized data showing CaMKII inhibition with the inhibitor KN93 (but not its inactive analog KN92) also prevented alcohol-evoked tetracaine-sensitive diastolic SR Ca2+ leakage in HL-1 myocytes. (B) (C) In vivo treatment with a single dose of KN93 eliminated alcohol-evoked Ca2+ waves and shortened [Ca2+]i decay constant τ compared to KN92-treated alcohol hearts. Abbreviations as in Figure 1.

FIGURE 7. Alcohol-Evoked JNK2 Regulates CaMKII Activity (A) Summarized data show increased ADP production (reflecting an increased ATP consumption in the CaMKII reaction with its substrate) in HA-pulldown CaMKII-WT proteins from alcohol-exposed cells, while JNK2I suppressed alcohol-evoked CaMKII activity. (B) Pooled ADP production assay data and representative immunoblotting images show that active pure JNK2 proteins increase the phosphorylation of CaMKII-WT, whereas autophosphorylation site mutation prevents this JNK2 action on HA-pulldown CaMKII-T286A proteins. (C) Summarized data show alcohol-enhanced CaMKII activity is not affected by the mutation of CaMKII oxidative sites Met280/281. (D) Summarized quantitative immunoblotting data and representative images show JNK2 inhibition prevented alcohol-enhanced phosphorylation of CaMKII (activated) in cultured HL-1 myocytes. CaMKII = calmodulin kinase; CaMKII-WT = wildtype CaMKII; CaMKII-T286A = CaMKII with Thr287Ala mutation; CaMKII-VV = CaMKII with Met280/281Val mutation; JNK2I = JNK2 inhibitor. Other abbreviations as in Figures 1 and 2.

CENTRAL ILLUSTRATION. Alcohol-Evoked JNK Activates CaMKII to Promote Atrial Fibrillation Proposed mechanism showing that binge alcohol-evoked JNK2 activates CaMKII, which in turn, promotes aberrant SR Ca2+ handling and atrial arrhythmias. Ca2+ = calcium; CaMKII = calmodulin kinase II; JNK2 = c-Jun N-terminal kinase 2; SR = sarcoplasmic reticulum.