Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial Arrhythmogenesis

Abstract

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.

Keywords: aged; atrial fibrillation; calcium-calmodulin-dependent protein kinase type 2; phosphorylation; ryanodine receptor 2; sarcoplasmic reticulum; stress-activated protein kinase JNK2.

Figure 1. Activated JNK in human atria is associated with increasing age and arrhythmogenicity

A ) Representative immunoblotting images and plot showing enhanced phosphorylated JNK (JNK-P) in aged human atria. B) Similar increase in the ratio of JNK-P to total JNK2 proteins. C) Representative electrogram (EG) traces of burst pacing induced AF (after 1Hz or 3 Hz burst pacing) in two normal aged human hearts without a history of AF or coexisting cardiac diseases. D) Representative EG trace after 4Hz burst pacing in a young healthy human donor heart.

Figure 2. Activated JNK enhances atrial arrhythmogenicity in aged mice and cardiac-specific JNK activated young mice

A ) Immunoblotting images and quantitative data showing increased phosphorylated JNK (JNK-P; activated JNK) in aged mouse atria. B) Summarized data of increased AF inducibility in aged WT mice compared to that of young (Yg) controls. Summarized data showing JNK2 specific inhibitor (JNK2I-XI) in vivo treatment in aged WT mice strikingly attenuated pacing induced AF events (far right bar) as seen in untreated aged WT mice. C-D) Representative intra-cardiac electrogram (EG) traces of burst pacing induced AF in an aged mouse (C), while no arrhythmia was induced in a wild-type (WT) young mouse (D). E) Representative images and summarized quantitative immunoblotting results showing increased JNK-P in cardiac specific tamoxifen-treated (Tamx; one dose per day for 5 days) MKK7D mouse atria (MKK7D⁺) compared to tamoxifen-treated WT littermates (MKK7D^-). F-G) Summarized AF inducibility and representative EG trace of burst pacing induced AF in a tamoxifen-treated MKK7D mouse. Lower panel shows a trace of simultaneously recorded surface ECG. SRh=sinus rhythm.

Figure 3. Activated JNK enhances abnormal Ca²⁺ activities in intact aged mouse atria and cardiac-specific JNK activated young mouse atria

A ) Representative confocal images of Ca²⁺ waves and Ca²⁺ transients after burst pacing in Langendorff-perfused aged intact mouse atria and restored sinus Ca²⁺ transients after burst-pacing in sham WT intact mouse atria. B) Summarized bar graphs showing significantly increased frequency of spontaneous (sinus rhythm (SRh) before bust pacing) and pacing-induced Ca²⁺ waves along with prolonged relaxation time of Ca²⁺ transients during the recovery period (when burst pacing was stopped) in aged mouse atria compared to that of WT young controls. C) Representative electrogram traces of burst pacing induced AF in anisomycin-treated (Aniso) WT mice and no arrhythmia induced in anisomycin-treated JNK2 knockout (JNK2KO) mice. D) Summarized data of AF inducibility in anisomycin-treated WT and JNK2KO mice vs sham controls. E) Example confocal images of increased Ca²⁺ sparks & waves in anisomycin(Aniso)-treated WT mouse atria after burst pacing (upper), and restored sinus Ca²⁺ transients after burst pacing in anisomycin-treated JNK2KO mouse atria (lower). F) Summarized data showing significantly increased Ca²⁺ waves and prolonged τ of Ca²⁺ decay during sinus rhythm (SRh) and in response to burst-pacing in anisomycin(A)-treated WT young mouse atria compared to anisomycin-treated JNK2KO young mouse atria.

Figure 4. Activated JNK2 causes markedly increased diastolic SR Ca²⁺ leak via increased probability of RyR single channel opening (P_o)

A ) Summarized data showing increased diastolic SR Ca²⁺ leak in freshly isolated aged mouse myocytes; JNK2 specific inhibitor JNK2I-IX (JNK2I) treatment completely reversed the Ca²⁺ leak. B) anisomycin-treated (Aniso or A) HL-1 myocytes also showed dramatically increased diastolic SR Ca²⁺ leak; JNK2 specific inhibition completely prevented these anisomycin actions. C) Example traces of Aniso-treated vs sham control (Ctl) HL-1 myocytes in the tetracaine-sensitive leak confocal measurement protocol. D-E) Summarized data showing increased SR Ca²⁺ content (examined with caffeine-induced Ca²⁺-released) in aged mouse myocytes and anisomycin-treated HL-1 myocytes, which is reversed by JNK2I treatment. F-G) Unchanged Ca²⁺ transient amplitude (F) but prolonged τ of Ca²⁺ decay (G; which is also reversed by JNK2I treatment) in Aniso (A)-treated HL-1 myocytes. H-I) Summarized data shows that overexpression of inactivated JNK2dn proteins attenuates anisomycin (A)-induced SR diastolic Ca²⁺ leak (H) and overload (I), while inactivated JNK1dn has no such rescue effects. J) Ca²⁺ transient amplitude is unchanged in all groups. K) Sample single WT mouse RyR2 channel recordings before (control) and after cytosolic addition of 50ng/ml anisomycin (without and with pre-treatment of JNK2I-IX). The zero current levels are indicated by a dash. L) The mean single RyR2 P_o after anisomycin treatment alone (open square), and with KN93 present (filled triangle) or with alkaline phosphatase present (Alk.Ph., filled square) or with JNK2I-IX present (open triangle) are shown in the inset. Filled circle is WT RyR2 data. Anisomycin action on RyR2 cytosolic Ca²⁺ sensitivity is also illustrated (inset)

Figure 5. Activated JNK2 leads to enhanced CaMKII activation and promotes CaMKII-dependent phosphorylation of SR Ca²⁺ handling proteins

A ) Example images and pooled immunoblotting data showing that significantly increased CaMKII-P is positively correlated with enhanced JNK activation in human atria with increasing age. B-C) Representative images and summarized data showing that markedly increased CaMKII-P in JNK activated atria from aged rabbits and anisomycin-treated young rabbits. And, this is linked to enhanced CaMKII-dependent phosphorylation of RyR2815 (RyR2815-P) and PLB17 (PLB17-P). D) Immunoblotting results suggest that enhanced JNK-P is associated with enhanced CaMKII, but CaMKII activation in JNK2KO mice treated with anisomycin (Aniso) is significantly reduced compared to WT mice treated with Aniso. E-F) JNK2KO mouse atria attenuated CaMKII-dependent phosphorylation of RyR2815 and PLB17 (RyR2815-P, PLB17-P) compared to that of anisomycin-treated WT mice, while RyR2809 and PLB16 phosphorylation levels (RyR2809-P, PLB16-P) were unchanged.

Figure 6. JNK2 enhances CaMKII activation and CaMKII inhibition prevented JNK-induced arrhythmic activities

A ) Representative images and summarized immunoblotting data showing increased CaMKII-P along with enhanced activation of JNK (JNK-P) in aged WT mouse atria. Dramatically reduced cardiac CaMKII activation in aged JNK2KO mice compared to that of WT aged mice. B) Summarized data showing JNK2 inhibition (in vivo treatment) reversed the CaMKII activation to the baseline of young hearts compared to markedly increased CaMKII-P in untreated aged mice. C) Representative electrograms of burst-pacing followed by self-reversion to sinus rhythm (no arrhythmia induced) in anisomycin-treated (Aniso) AC3-I mice and AC3-I-sham control mice (n=0/6, 0/6). This suggests that CaMKII inhibition in AC3-I mice prevents Aniso-induced atrial arrhythmias. D) Summarized data suggest that CaMKII inhibition in AC3-I mice completely abolished Aniso-induced aberrant atrial Ca²⁺ waves and prolonged τ of Ca²⁺ decay. E) Immunoblotting images of attenuated activated CaMKII in AC3-I mice, while Aniso-induced JNK activation remains increased. F) Summarized data of diastolic SR Ca²⁺ leak in KN93+Aniso (KN93+A) treated as well as KN92+Aniso (KN92+A) treated HL-1 myocytes compared to sham controls (sham).

Figure 7. JNK2 activates CaMKII via direct phosphorylation of CaMKII proteins

A-B) Immunoblotting images of CaMKII-P and JNK-P in Aniso-treated HL-1 myocytes with and without overexpressed inactivated JNK2dn or JNK2 inhibitor pretreatment. Bottom panel shows positive HA signals in HA-tagged AdJNK2dn-infected cells as evidence of successfully overexpressed JNK2dn proteins. C) Summarized data showing increased CaMKII activation in Aniso-treated HL-1 cells while overexpressing AdJNK2dn or JNK2I-treatment reversed CaMKII activation to the control level. D) Immunoblotting images of co-immunoprecipitated CaMKII with JNK2 specific antibody in human atrial homogenates. E-F) Blotting images of active human JNK2 (hJNK2) in phosphorylation of CaMKII in both human atrial tissue homogenates (E; in contrast to the even amount of alpha-actinin and GAPDH proteins) and pure human CaMKIIδ (hCaMKIIδ) proteins (F). Each assay was repeated at least three times. G) Immunoblotting images showing increased phosphorylation of HA-IPed CaMKII-WT proteins but not HA-IPed CaMKII-T286A mutant (Mu) proteins compared to empty vector (V) controls (three experimental repeats). Ponceau staining shows equal expression between CaMKII-WT and mutant CaMKII-T286A samples. H) Summarized data of increased ADP production from CaMKII phosphorylation by pure active hJNK2 proteins in HA-IPed CaMKII-WT samples but not HA-IPed mutant CaMKII-T286A compared to CaMKII-WT sham-controls without pure hJNK2 incubation. I) Summarized data showing increased ratio of CFP-camui-vv to FRET-camui-vv fluorescence signals in anisomycin-treated isolated rabbit atrial myocytes vs sham-controls.

Figure 8. Schematic diagram of proposed mechanism of JNK2-driven CaMKII activation that in turn promotes aberrant SR Ca²⁺ leak triggered arrhythmic activities