Abstract Wed038: A Novel Role for AKAP1 in JNK2 Trafficking to Endoplasmic Reticulum and Ca ²⁺ Triggered Arrhythmic Activities

Evidence suggests that emotional stress is an independent risk factor for the onset of cardiac arrhythmias. Takotsubo Syndrome is a form of stress-induced cardiomyopathy with catecholamine surge-linked electrical and mechanical dysfunction in the heart, often associated with life-threatening ventricular arrhythmias. However, mechanisms of catecholamine-linked arrhythmias remain unclear. Recently, we revealed a previously unrecognized action of activated JNK2 in sarcoplasmic reticulum (SR) Ca 2+ mishandling and arrhythmogenesis under stress conditions. Catecholamines activate β-adrenergic signaling via PKA and A-Kinase Anchoring Protein-1 (AKAP1)-mediated complexes to effect subcellular trafficking. Here, we report a mechanistic link between AKAP1, PKA, and JNK2 in isoproterenol (ISO)-evoked ER Ca 2+ mishandling, using a heterologous HEK-RyR2 (expressing human RyR2 to mimic myocyte Ca 2+ cycling) cell model exposed to ISO (5 mM) for 24 h. Abnormal ER Ca 2+ leak was measured using a tetracaine-sensitive confocal Ca 2+ imaging protocol. We found enhanced JNK2 activation in ISO-treated HEK-RyR2 cells vs controls (n=3,3). Next, we fractionated FLAG-AKAP1/tGFP-JNK2 co-transfected HEK-RyR2 cells and saw that AKAP1 increased JNK2 localization to the membrane (p<0.01, n=7,6), implicating AKAP1 in subcellular trafficking of JNK2. Moreover, tGFP-JNK2 was found to co-immunoprecipitate (IP) with anti-FLAG antibody pulldown of FLAG-AKAP1 proteins. Functionally, ISO-evoked JNK2 activation increased ER Ca 2+ leak and AKAP1-mediated JNK2 membrane localization further enhanced this JNK2-driven ER Ca 2+ leak vs sham controls (p<0.01, n=34,45). AKAP1 alone showed no effect. Cells co-transfected with AKAP1 and inactive JNK2-APF (T/183/A&Y/185/F point mutations) had no effect either, suggesting a critical role for active JNK2 in ISO-evoked ER Ca 2+ leak, with AKAP1 augmenting this action. Disrupting AKAP1-PKA binding via AKAP1-A/328/P point mutation or PKA activity inhibition by PKI (1 μM, 24 h) normalized ISO-evoked ER Ca 2+ leak to sham control levels (p<0.05; n=34,24,14), implicating a key role for the AKAP1-PKA complex in augmented JNK2-driven ER Ca 2+ mishandling in response to ISO-stimulation. Overall, our findings reveal a novel role for AKAP1 in ISO-evoked membrane translocation of active-JNK2, and its critical contribution to ER Ca 2+ mishandling and arrhythmogenicity. The AKAP1-PKA-JNK2 pathway is a likely mechanism involved in catecholamine-evoked cardiac arrhythmogenesis.