Abstract Body: Background: The stress-responsive c-Jun N-terminal kinase isoform 2 (JNK2) pathway has been demonstrated by our group to causatively drive stress-evoked atrial fibrillation (AF) risk. Our pilot data suggest that JNK2 is present in blood, a finding not previously reported.
Objective: Test whether circulating JNK2 reflects cardiac JNK2 activation and serves as a biomarker of AF risk.
Methods: Human atrial tissue and plasma samples from organ donors and healthy subjects were analyzed along with samples from aged and holiday heart syndrome (HHS, binge alcohol exposure) mice with and without JNK2 inhibitor treatment (JNK2I, 5mg/kg, i.p. 4 doses). The heart-plasma JNK2 link was examined using cardiac-specific MKK7D (overexpression of active MKK7D, JNK upstream activator) and MKK7D-JNK2dn and MKK7D-JNK2KD models (expressing dominant-negative inactive JNK2 or 50% JNK2 knockdown). Plasma JNK levels and activity were assessed by immunoblotting and enzyme assays. Cardiac-derived EVs were tracked in CD63-Luc mice (overexpressing luciferase-tagged EV marker CD63) using luciferase assays.
Results: JNK2 was predominantly detected in human plasma among blood fractions using immunoblotting and kinase activity assays. Plasma and atrial JNK2 activation concordantly increased with aging across species (humans, mice and rabbits). In mice, in vivo JNK2 inhibition (JNK2I) normalized plasma JNK2 activation and AF inducibility in aged and HHS mice to young control levels, suggesting an atria-blood JNK2 link. Cardiac JNK activation in MKK7D mice increased plasma JNK levels and activity, whereas cardiac-specific JNK2 inhibition in MKK7D-JNK2dn mice restored them. Atrial-specific JNK2 inhibition via in vivo atrial painting gene transfer reduced AF inducibility and normalized plasma JNK2 in aged rabbits to young control levels, supporting a causal atria-blood JNK2 link. Circulating JNK2 was primarily carried by EVs as shown by immunoblotting and electron microscopy with JNK2 immunogold staining, with increased JNK activity in plasma EVs from aged rabbits and humans. EVs from MKK7D mice expressing cardiac-specific CD63-Luc showed higher luciferase activity than WT littermates, which was normalized by JNK2 knockdown in MKK7D-JNK2KD mice, alongside restoration of plasma EV JNK2 activity.
Conclusions: Elevated circulating JNK2, carried by heart-derived JNK2-containing EVs, reflects activated cardiac JNK2, identifies circulating JNK2 as a potential biomarker of cardiac stress and AF risk.