Cellular Biology

Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial ArrhythmogenesisNovelty and Significance

Jiajie Yan, Weiwei Zhao, Justin K. Thomson, Xianlong Gao, Dominic M. DeMarco, Elena Carrillo, Biyi Chen, Xiaomin Wu, Kenneth S. Ginsburg, Mamdouh Bakhos, Donald M. Bers, Mark E. Anderson, Long-Sheng Song, Michael Fill, Xun Ai
https://doi.org/10.1161/CIRCRESAHA.117.312536
Circulation Research. 2018;122:821-835
Originally published January 19, 2018

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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.

Introduction

Atrial fibrillation (AF) brings a high risk of mortality and associated morbidities, including stroke and heart failure (HF).1–3 Considering our growing elderly population, AF is becoming an enormous public health challenge and up to 10% to 15% of 70 to 80 year olds will develop AF.2–4 Current pharmacological AF treatment and prevention approaches are ineffective, and our understanding of the underlying mechanisms is incomplete, limiting the development of new therapeutic strategies.

Editorial, see p 799

In This Issue, see p 791

Meet the First Author, see p 792

JNK (c-Jun N-terminal kinase) is a well-characterized stress response kinase that is activated in response to various cellular stresses such as ultraviolet light, ischemia, inflammatory cytokines, and aging.5–9 Interestingly, many of these stresses are also well-established cardiovascular risk factors, and JNK activation has been observed in cardiovascular conditions like ischemia, hypertrophy, and HF. All these conditions are associated with increased AF risk.3,5–8,10–14 Not all hearts will experience a particular stress, but all hearts will inevitably age. And, the aged heart is more susceptible to some of the stress stimuli.15 Thus, age-associated JNK activation is a compelling experimental focus here. In the human atria, AF often involves complex pathological remodeling because of coexisting cardiovascular diseases with increasing age. Here, our focus is to explore the role of JNK activation in enhanced AF susceptibility in the aged heart lacking comorbid conditions (ie, with normal cardiac function, no AF history, no history of major cardiovascular diseases, and lacking structural remodeling). Once one specific pathogenic pathway is established, defining and understanding the contributions of all the other AF-associated circumstances, stresses, and factors become possible.

JNK2 (JNK isoform 2) is a major isoform in the heart.16 Our results reveal that JNK2 activation is markedly elevated in aged human, rabbit, and mouse atria. Further, this age-associated JNK2 activation causes abnormal intracellular calcium (Ca2+) waves and diastolic sarcoplasmic reticulum (SR) Ca2+ leak. It is well known that the action of CaMKII (Ca2+/calmodulin-dependent kinase II) on SR Ca2+ mishandling (ie, diastolic Ca2+ leak and waves) is proarrhythmic.17–22 Indeed, CaMKII inhibition has been considered as a potential antiarrhythmic intervention for HF.23 Here, we discovered the JNK2/CaMKII crosstalk as a previously unknown molecular mechanism of CaMKII activation. Activated JNK2 directly phosphorylates CaMKII proteins, driving CaMKII proarrhythmic effects on diastolic SR Ca2+ handling. In aged mice, JNK2 inhibition eliminated age-related CaMKII hyperactivation and the associated aberrant diastolic SR Ca2+ leak, Ca2+ waves, and AF susceptibility. These results identify JNK2 inhibition as a potential target in developing new therapeutic strategies to prevent or treat AF.

Methods

All data and supporting materials have been provided with the published article. An expanded Methods Section is available in the Online Data Supplement.

Animal and Cell Models

Wild-type (WT) C57B/6j male mice (Jackson Laboratory, ME) at 24 to 32 months (aged) and 2 to 2.5 months (young) were studied. Three mouse models were used to assess the contribution of JNK and CaMKII on Ca2+ dynamics and AF genesis. They were (1) a cardiac-specific inducible MKK7D (MAP kinase kinase 7) trangenic mouse strain (a generous gift from Dr Yibin Wang, University of California, Los Angeles [UCLA]) that can express cardiac MKK7D to robustly activate JNK with tamoxifen treatment,24 (2) homozygous JNK2KO (JNK2 knockout mice25 [Mapk9tm1Flv/J]; Jackson Laboratory), and (3) AC3-I mice,21 with cardiac-specific transgenic expression of a CaMKII peptide inhibitor. JNK2KO and AC3-I mice were treated with anisomycin as previously described.8,26 Young (6 months) and aged (60 months) New Zealand White male rabbits were also used. Four young rabbits were treated with a JNK activator anisomycin.27 Rabbit atrial myocytes were isolated as previously described with modification.17 All animal studies followed the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication, 8th Edition, 2011) and were approved by the Institutional Animal Care and Use Committees of Rush University Medical Center, Loyola University Chicago, University of Alabama at Birmingham, and University of Iowa.

A well-characterized cultured myocyte line (HL-1, from Dr William Claycomb, Louisiana State University) was used for our studies as previously described.7,8,26 Cells were treated with JNK activator anisomycin or infected with adenoviral JNK activator MKK7D to activate JNK in the presence or absence of JNK or CaMKII inhibition as described in the Online Data Supplement.

Human Atrial Specimens

Human right atrial tissues were obtained from donor hearts (Online Table I) provided by Illinois Gift of Hope Organ & Tissue Donor Network and Alabama Organ Center. The studies were approved by the Human Study Committees of Rush University Medical Center, Loyola University Chicago, University of Alabama at Birmingham, Alabama Organ Center, and Illinois Gift of Hope.

AF Induction in Mice In Vivo and in Human Atrial Preparations Ex Vivo

In vivo AF induction was conducted in sedated mice, and electrogram data were recorded using a 1.1F octapolar catheter inserted into the right atrium as previously described.19 Ex vivo AF induction in human donor atria was performed as described in the Online Data Supplement.

Confocal Ca2+ Imaging

Confocal line scanning Ca2+ images were obtained from intact atrium or atrial myocytes as previously described.28 Frequencies of Ca2+ waves/sparks and time constant (τ) of Ca2+ decay were analyzed from intrinsic sinus rhythm and recovered beats after the burst pacing (5–10 Hz) as previously described.28–30 Tetracaine-sensitive SR Ca2+ leak was measured using our well-established protocols as previously described.17,29,31

Intact Atrial Optical Mapping

Intact mouse hearts were preloaded with Rhod2-AM (5 µmol/L) followed by Rh237 (10 mmol/L; Invitrogen). Vm and Ca2+ signals were simultaneously recorded using a dual-channel optical imaging system as previously described.7,26 The SD of the difference between the activation time of action potential and calcium transient (ΔtVm−Ca) for a total of 400 channels within the mapping field was calculated to reflect the heterogeneity of the relationship between Vm and Ca2+ signals as previously described.32

Single RyR2 Recording

Single RyR channel function was measured by fusing isolated WT mouse SR vesicles into lipid bilayers as previously described.33 Anisomycin, alkaline phosphatase,17 the CaMKII inhibitors KN9317 and AIP, and the JNK2-specific inhibitor JNK2I-IX were applied to the cytosolic side of the reconstituted RyR2 (ryanodine receptor) channels.

CaMKII Activity Biosensor Imaging

The adenoviral mutant variant CaMKII sensor, Camui-vv (Förster resonance energy transfer [FRET]-based CaMKII sensor with mutated Met280Val & Met281Val; lacking the oxidation M280/M281 site but containing the intact autophosphorylation Thr286 site), was used to quantify the contribution of CaMKII phosphorylation and oxidation on CaMKII activation in anisomycin-treated isolated rabbit myocytes as previously described.34

Biochemical Assays

Immunoblotting was performed as previously described.8,17 Protein expressions were detected using specific antibodies, and JNK2 immunoprecipitation was also conducted using a JNK2-specific antibody as previously described.17 Human influenza hemagglutinin (HA)–tagged WT CaMKII-WT and mutant CaMKII-T286A vectors were constructed as previously described to determine the direct action of JNK2 on CaMKII phosphorylation, detected by immunoblotting and ADP-Glo Kinase assay (Promega), per manufacturer’s instructions.34

Statistical Analysis

All data are presented as mean±SEM. Differences between multiple groups or any 2 groups were evaluated using 1-way ANOVA with post hoc Tukey test or Student t test. When heterogeneity of variance was evidenced, a nonparametric Mann–Whitney test or nonparametric 1-way ANOVA was performed. A P value <0.05 was considered to be significant.

Results

JNK Activation and AF Susceptibility

As age increased, human atria showed markedly enhanced activation of JNK (JNK-P [phosphorylated JNK]), as assessed by immunoblotting (Figure 1A). This trend persisted when JNK-P was normalized to either unchanged total JNK2 proteins (Figure 1B; Online Figure IA and IB) or JNK1 (data not shown). Although we found unchanged total JNK proteins, JNK1 and JNK2 mRNA expression were also unchanged in human atria with increasing age (Online Figure IC and ID). The human atria were obtained from hearts of donors without a history of AF or major cardiovascular disease (Online Table I). We also measured AF inducibility in Langendorff-perfused human donor heart atrial preparations challenged with electric burst pacing. Three out of 4 aged donor hearts showed pacing-induced AF events after a train of electric stimulation. Figure 1C shows pacing-induced AF in 2 aged atrial preps subjected to 1 and 3 Hz pacing, respectively (30 s, 2× diastolic threshold; Figure 1C, long red arrows; n=4). In contrast, no pacing-induced AF events were found in any of the 4 young controls (even at a higher pacing frequency 4 Hz; Figure 1D; n=4).

Figure 1.
Figure 1.

Activated JNK (c-Jun N-terminal kinase) in human atria is associated with increasing age and arrhythmogenicity. A, Representative immunoblotting images and plot showing enhanced JNK-P (phosphorylated JNK) 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 atrial fibrillation (AF; after 1 or 3 Hz burst pacing) in 2 normal aged human hearts without a history of AF or coexisting cardiac diseases. D, Representative EG trace after 4 Hz burst pacing in a young healthy human donor heart. A.U. indicates arbitrary unit.

Like human atria, aged WT mouse atria also had elevated JNK-P (Figure 2A), and this was associated with a dramatically increased propensity for pacing-induced AF19 (Figure 2B through 2D) compared with that of young controls. These aged mice showed preserved cardiac function and an unchanged amount of atrial interstitial fibrosis (quantified using Trichrome staining as previously described30; Online Figure IIA and IIB) compared with young controls. A JNK2-specific inhibitor (JNK2I-IX with no action on JNK1 or other MAP kinases35) abolished pacing-induced AF when applied to WT aged mice (Figure 2B, right bar). When induced with tamoxifen, cardiac-specific MKK7D transgenic mice24 expressed constitutively active MKK7, an upstream JNK activator.5 In these mice, JNK activation was significantly increased by tamoxifen treatment (Figure 2E). The cardiac function of the MKK7D mice was the same before and 5 days after treatment (Online Figure IIC). In young MKK7D mice, induction of JNK activation resulted in an increased incidence of pacing-induced AF (n=4/5 versus 0/6 tamoxifen-treated WT littermates; Figure 2F and 2G). Thus, JNK activation promotes AF initiation in young mice similar to the action of age-associated JNK activation in aged WT mice.

Figure 2.
Figure 2.

Activated JNK (c-Jun N-terminal kinase) enhances atrial arrhythmogenicity in aged mice and cardiac-specific JNK activated young mice. A, Immunoblotting images and quantitative data showing increased JNK-P (phosphorylated JNK; activated JNK) in aged mouse atria. B, Summarized data of increased atrial fibrillation (AF) inducibility in aged wild-type (WT) mice compared with 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 intracardiac electrogram (EG) traces of burst pacing-induced AF in an aged mouse (C), whereas no arrhythmia was induced in a WT young mouse (D). E, Representative images and summarized quantitative immunoblotting results showing increased JNK-P in cardiac-specific tamoxifen-treated (Tamx; 1 dose per day for 5 days) MKK7D (MAP kinase kinase 7) mouse atria (MKK7D+) compared with 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, A trace of simultaneously recorded surface ECG. A.U. indicates arbitrary unit; and SRh, sinus rhythm.

JNK Activation and Abnormal Ca2+ Activities

Confocal Ca2+ imaging in Langendorff-perfused intact aged WT mouse atria revealed that there were frequent Ca2+ waves during the intrinsic sinus rhythm, but even more waves were evidenced after a bout of rapid electric pacing (Figure 3A and 3B). The Ca2+ transient decay time constant (τ) was also significantly prolonged in aged WT atria (Figure 3B, lower) compared with young controls. Simultaneous optical recordings of intracellular Ca2+ and Vm showed significantly increased spatiotemporal heterogeneity between the 2 signals (ΔtVm−Ca) in aged WT atria compared with young controls (Online Figure IIIA and IIIB). This increased heterogeneity of ΔtVm−Ca in the aged atrium is arrhythmogenic36 and aligned with the higher frequency of diastolic Ca2+ waves (Figure 3A and 3B).

Figure 3.
Figure 3.

Activated JNK (c-Jun N-terminal kinase) 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 wild-type (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 with that of WT young (Yg) controls. C, Representative electrogram (EG) traces of burst pacing-induced atrial fibrillation (AF) in anisomycin-treated (Aniso) WT mice and no arrhythmia induced in anisomycin-treated JNK2KO (JNK2 knockout) 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 and 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 SRh and in response to burst pacing in anisomycin (A)-treated WT young mouse atria compared with anisomycin-treated JNK2KO young mouse atria.

The contribution of JNK activation to the abnormal arrhythmogenic Ca2+ handling in the absence of aging was explored by treating young WT mice with a JNK activator, anisomycin.7,8 In young WT hearts, anisomycin treatment dramatically increased pacing-induced AF (Figure 3C and 3D). It also increased spontaneous and postpacing Ca2+ waves, although Ca2+ transient decay was prolonged (versus sham controls; Figure 3E and 3F). This is likely the same action of age-associated JNK activation in WT aged atria (Figures 2A through 2D, 3A, and 3B). To further assess JNK-specific action of anisomycin, we applied this agent to JNK2KO mice. We found that anisomycin-treated JNK2KO mice did not show aberrant Ca2+ handling or pacing-induced AF (Figure 3C through 3F). Note that aged and anisomycin-treated young mouse atria showed unchanged amounts of JNK2 and JNK1 proteins (Online Figure IIIC and IIID). The JNK2KO atria had the normal JNK1 but only trace amounts of JNK2 (Online Figure IIIC and IIID). These results indicate that there is a JNK2-specific action on aberrant Ca2+ activities and AF, suggesting that JNK2 is a critical determinant of abnormal events.

JNK Activation and Abnormal Diastolic Ca2+ Handling

We and others have previously shown that increased diastolic SR Ca2+ release causes abnormal ectopic activities, which can lead to arrhythmogenesis in diseased hearts.17,37,38 We monitored diastolic SR Ca2+ release using the tetracaine-sensitive SR Ca2+ leak protocol.17 SR Ca2+ leak was elevated in aged mouse atrial myocytes (Figure 4A) compared with young controls. The JNK2 inhibitor JNK2I-IX completely abolished this elevation. Confluent monolayers of HL-1 myocytes recapitulate in situ many genotypic and electric cardiac phenotypes, including the cell–cell interactions present in the heart.8,26 Intracellular Ca2+ handling in HL-1 myocytes was measured (Online Figure IVB and IVC). Anisomycin-treated HL-1 myocytes had significantly increased tetracaine-sensitive SR Ca2+ leak that was eliminated by JNK2 inhibition (Figure 4B and 4C). This is consistent with our results from freshly isolated aged mouse myocytes (Figure 4A). For confirmation in a larger animal model, JNK2 action on SR Ca2+ leak was also demonstrated in anisomycin-treated (24 hours) atrial myocytes isolated from young rabbits (Online Figure IVD). Together, our results indicate that JNK activation drives abnormal diastolic SR Ca2+ leak.

Figure 4.
Figure 4.

Activated JNK2 (c-Jun N-terminal kinase) causes markedly increased diastolic sarcoplasmic reticulum (SR) Ca2+ leak via increased probability of RyR (ryanodine receptor) single-channel opening (Po). 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 show that overexpression of inactivated JNK2dn (dominant-negative JNK2) proteins attenuates anisomycin (A)-induced SR diastolic Ca2+ leak (H) and overload (I), whereas inactivated JNK1dn (dominant-negative JNK1) has no such rescue effects. J, Ca2+ transient amplitude is unchanged in all groups. K, Sample single wild-type (WT) mouse RyR2 channel recordings before (control) and after cytosolic addition of 50 ng/mL anisomycin (without and with pretreatment of JNK2I-IX). The zero current levels are indicated by a dash. L, The mean single RyR2 Po 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).

In addition, JNK activation also increased SR Ca2+ content in both freshly isolated aged mouse atrial myocytes and anisomycin-treated cultured HL-1 myocytes compared with controls. This SR Ca2+ overload was eliminated by JNK2 inhibition using JNK2I-IX (Figure 4D and 4E). The systolic Ca2+ transient amplitude was not changed by JNK activation (Figure 4F). However, JNK activation prolonged the Ca2+ transient decay time constant, but not if JNK2 was inhibited (Figure 4G). We also assessed the contribution of NCX (Na+/Ca2+ exchanger) by measuring the Ca2+ decay rate of caffeine-induced Ca2+ transients. This decay rate was not altered by JNK activation (Online Figure IVE). To test JNK2 isoform specificity further, we applied an adenoviral dominant-negative approach to HL-1 myocytes (Online Figure VA through VD). JNK2dn (dominant-negative JNK2) abolished the anisomycin-evoked Ca2+ mishandling phenotype, whereas JNK1dn (dominant-negative JNK1) did not (Figure 4H through 4J). This confirms that the JNK action on diastolic SR Ca2+ leak and overload is indeed JNK2 specific.

Elevated SR Ca2+ diastolic leak implies dysfunction of the SR Ca2+ release channels (RyR2).39 Single RyR2 channel function was assessed by fusing heavy mouse SR microsomes into artificial lipid bilayers.40 Anisomycin application significantly increased RyR2 open probability (Po; RyR2 cytosolic Ca2+ sensitivity) but not when a JNK2 inhibitor was present (Figure 4K and 4L). The anisomycin action on Po was also prevented when either alkaline phosphatases or CaMKII inhibitor KN93 was present (Figure 4L; Online Figure VIA). The anisomycin action on RyR2 Po was also confirmed in human RyR channels (Online Figure VIB). The JNK-associated action on RyR2 Po explains the increased diastolic SR Ca2+ leak observed in atrial myocytes after JNK2 activation. The KN93 results imply that CaMKII has a role in linking JNK activation and RyR dysfunction. There was no additional JNK or CaMKII added to the SR vesicles, so the isolated RyR2s must have been associated with endogenous JNK and CaMKII. This was verified by measuring the component proteins of heavy SR microsomes (Online Figure VIC). An intriguing possibility is that JNK activation may promote CaMKII-dependent phosphorylation of RyR2.

JNK2 and CaMKII Crosstalk in Abnormal Ca2+ Activities and AF Susceptibility

CaMKII activation results in the phosphorylation of various SR Ca2+ handling proteins (eg, RyR2 and PLB [phospholamban]) and is proarrhythmic in diseased hearts.17 Blocking of CaMKII function seems to interrupt JNK-to-RyR2 signaling (Results). Immunoblotting revealed that activation of both CaMKII and JNK (ie, CaMKII-P and JNK-P) increased with age in human atria (Figure 5A). This trend persisted when CaMKII-P was normalized to total CaMKIIδ proteins (Online Figure IB). This JNK/CaMKII relationship was also present in aged rabbit atria and anisomycin-treated young rabbit atria (Figure 5B and 5C). The CaMKII-dependent phosphorylation of the RyR2 (RyR2815-P) and PLB (PLB17-P [phosphorylated PLB at Thr17]) proteins were assessed. Aged rabbit atria and young anisomycin-treated rabbit atria had elevated CaMKII-P and JNK-P levels and increased RyR2815-P (normalized to total RyR2) and PLB17-P levels (normalized to SERCA2 [sarco/endoplasmic reticulum Ca2+-ATPase 2]; Figure 5B and 5C). In contrast, NCX and SERCA were unaltered, and there was no change in PKA (protein kinase A)-dependent RyR2 and PLB phosphorylation (RyR2809-P and PLB16-P [phosphorylated PLB at Ser16]; Online Figure VIIB and VIIC). We did analogous testing in mouse models. A similar outcome was obtained in tamoxifen-induced MKK7D mouse atria with its constitutive JNK activation (Online Figure VIIA). In JNK2KO young atria, anisomycin treatment did not enhance CaMKII-P or increase the levels of RyR2815-P and PLB17-P (Figure 5D through 5F). Also, the PKA-mediated phosphorylation of RyR2809 and PLB16 remained at a level comparable to sham controls and anisomycin-treated WT mouse atria (Figure 5E and 5F). These control atria had similar JNK2 protein levels. JNK2KO atria had a normal JNK1 level but only trace amounts of JNK2 (Online Figure IIIC and IIID). In aged JNK2KO mouse atria with a long-term JNK2 ablation, there was dramatically reduced CaMKII activation (Figure 6A). Likewise, there was dramatically reduced CaMKII activation in aged WT atria that were treated with JNK2I-IX for 10 days (Figure 6B). These results indicate that atrial JNK2 activation results in phosphorylation of the CaMKII protein and in turn CaMKII-dependent phosphorylation of RyR2 and PLB.

Figure 5.
Figure 5.

Activated JNK2 (c-Jun N-terminal kinase isoform 2) leads to enhanced CaMKII (Ca2+/calmodulin-dependent kinase II) activation and promotes CaMKII-dependent phosphorylation of sarcoplasmic reticulum (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 (Yg) rabbits. And, this is linked to enhanced CaMKII-dependent phosphorylation of RyR2815 (RyR2815-P) and PLB17 (phospholamban; PLB17-P [phosphorylated PLB at Thr17]). D, Immunoblotting results suggest that enhanced JNK-P (phosphorylated JNK) is associated with enhanced CaMKII, but CaMKII activation in JNK2 knockout (KO) mice treated with anisomycin (Aniso) is significantly reduced compared with wild-type (WT) mice treated with Aniso. E, F, JNK2KO (JNK2 knockout) mouse atria attenuated CaMKII-dependent phosphorylation of RyR2815 and PLB17 (RyR2815-P, PLB17-P) compared with that of anisomycin-treated WT mice, although RyR2809 and PLB16 (phosphorylated PLB at Ser16) phosphorylation levels (RyR2809-P, PLB16-P) were unchanged. A.U. indicates arbitrary unit; and RyR, ryanodine receptor.

Figure 6.
Figure 6.

JNK2 (c-Jun N-terminal kinase isoform 2) enhances CaMKII (Ca2+/calmodulin-dependent kinase II) 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 [phosphorylated JNK]) in aged wild-type (WT) mouse atria. Dramatically reduced cardiac CaMKII activation in aged JNK2KO (JNK2 knockout) mice compared with that in WT aged mice. B, Summarized data showing JNK2 inhibition (in vivo treatment) reversed the CaMKII activation to the baseline of young hearts compared with markedly increased CaMKII-P in untreated aged mice. C, Representative electrograms (EGs) 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, whereas Aniso-induced JNK activation remains increased. F, Summarized data of diastolic sarcoplasmic reticulum (SR) Ca2+ leak in KN93+Aniso (KN93+A) treated and KN92+Aniso (KN92+A) treated HL-1 myocytes compared with sham controls (sham). SRh indicates sinus rhythm.

We also explored the JNK/CaMKII interaction in AC3-I mice, which have cardiac-specific expression of a CaMKII peptide inhibitor.21 In young AC3-I atria, anisomycin treatment did not enhance AF susceptibility or abnormal Ca2+ waves in AC3-I atria (Figure 6C and 6D). These AC3-I atria had substantially activated JNK but little (if any) CaMKII activation (Figure 6E). Together, these results imply that CaMKII activation may be required for JNK-driven arrhythmogenic activities. To test this, we pretreated HL-1 myocytes with the CaMKII inhibitor KN93 or its inactive congener (KN92). The CaMKII inhibition (KN93) eliminated the expected increase in anisomycin-evoked diastolic Ca2+ leak and Ca2+ transient decay time constant (Figure 6F and Online Figure VIIIA). In contrast, the KN92 pretreatment (leaving CaMKII function intact) did neither. In HL-1 myocytes, JNK2 inhibition, using either a dominant-negative approach or the JNK2 inhibitor JNKK2I-IX, substantially reduced anisomycin-driven CaMKII activation in HL-1 myocytes (Figure 7A through 7C). Moreover, coinfection of HL-1 myocytes with adenovirus JNK2 and constitutively activated adenovirus MKK7D to increase JNK2 activation levels resulted in significantly increased CaMKII-P (Online Figure VIIIB). In contrast, coinfected adenovirus JNK1 and adenovirus MKK7D did not alter CaMKII-P levels. Evidence of successful adenoviral infection is shown in Online Figure VE. The JNK2 isoform-specific action on CaMKII activation was also confirmed in isolated young rabbit atrial myocytes (Online Figure VIIIC).

Figure 7.
Figure 7.

JNK2 (c-Jun N-terminal kinase isoform 2) activates CaMKII (Ca2+/calmodulin-dependent kinase II) via direct phosphorylation of CaMKII proteins. A, B, Immunoblotting images of CaMKII-P and JNK-P (phosphorylated JNK) in Aniso-treated HL-1 myocytes with and without overexpressed inactivated JNK2dn (dominant-negative JNK2) or JNK2 inhibitor pretreatment. Bottom, Positive human influenza hemagglutinin (HA) signals in HA-tagged AdJNK2dn (adenovirus dominant-negative JNK2)-infected cells as evidence of successfully overexpressed JNK2dn proteins. C, Summarized data showing increased CaMKII activation in Aniso-treated HL-1 cells, whereas overexpressing AdJNK2dn or JNK2I treatment reversed CaMKII activation to the control level. D, Immunoblotting images of coimmunoprecipitated CaMKII with JNK2-specific antibody in human atrial homogenates. E, F, Blotting images of active hJNK2 (human JNK2) in phosphorylation of CaMKII in both human atrial tissue homogenates (E; in contrast to the even amount of α-actinin and GAPDH proteins) and pure hCaMKIIδ (human CaMKIIδ) proteins (F). Each assay was repeated at least 3×. G, Immunoblotting images showing increased phosphorylation of HA-immunoprecipitated (IPed) CaMKII-WT proteins but not HA-IPed CaMKII-T286A mutant (Mu) proteins compared with empty vector (V) controls (3 experimental repeats). Ponceau staining shows equal expression between CaMKII-wild-type (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 with CaMKII-WT sham controls without pure hJNK2 incubation. I, Summarized data showing increased ratio of CFP-camui-vv:FRET-camui-vv fluorescence signals in anisomycin-treated isolated rabbit atrial myocytes vs sham controls. CFP indicates cyan fluorescent protein; FRET, Förster resonance energy transfer; and IP, immunoprecipitation; and RLU, relative light units.

JNK2 Activates CaMKII

Next, we found that a JNK2-specific antibody pulled down the CaMKII protein from human atrial homogenates (Figure 7D), where the CaMKII was phosphorylated in a JNK2 dose-dependent manner (Figure 7E). The added JNK2 was the pure active hJNK (human full-length JNK2) protein. These hJNK2 proteins were incubated with pure hCaMKII (full-length human CaMKII proteins; ; without Ca2+/calmodulin present), and this also resulted in JNK2 dose-dependent phosphorylation of hCaMKII (Figure 7F). To further explore the JNK2/CaMKII interaction, we transfected HEK293 cells with CaMKII-WT or CaMKII-T286A HA-tagged vectors. The expressed CaMKII proteins were immunoprecipitated using an HA antibody and then incubated with hJNK2. The CaMKII-WT, not the CaMKII-T286A, protein was phosphorylated by the hJNK2 (Figure 7G). The ADP-Glo kinase phosphorylation assay was used to assess ATP consumption during the phosphorylation reaction. A significant level of hJNK2 phosphorylation of CaMKII-WT, not CaMKII-T286A, was detected (Figure 7H). Although we cannot exclude the possibility of JNK action on other phosphorylation sites of CaMKII, our results indicate that JNK2 directly activates the CaMKII protein.

Aging increases oxidative stress,11,41 and this can also activate CaMKII.42 To test the contribution of oxidation to CaMKII activation, we used a mutated CaMKII activity biosensor called camui-vv that lacks the oxidation-sensitive Met280/281 sites but still has the key Thr286 autophosphorylation site.34 We measured camui-vv FRET in anisomycin-treated (24 hours) rabbit atrial myocytes and found significantly increased CaMKII activity (Figure 7I). To corroborate this result, we pretreated HL-1 myocytes with anti-oxidant N-acetyl-L-cysteine and found that this did not alter anisomycin enhanced CaMKII-P status (Online Figure VIIID). These results suggest that the JNK2-driven increase in CaMKII activation can occur in the absence of CaMKII oxidation.

Discussion

We discovered that JNK signaling is associated with abnormal Ca2+ activities and enhanced AF propensity in the aged heart. Our finding reveals for the first time that the JNK2 isoform directly activates CaMKII. The activated CaMKII in turn enhances RyR2-mediated SR Ca2+ leak, enhancing AF propensity (Figure 8). This is very likely an important pathway linking cellular stresses (eg, aging) to abnormal diastolic Ca2+ activities.

Figure 8.
Figure 8.

Schematic diagram of proposed mechanism of JNK2 (c-Jun N-terminal kinase isoform 2)-driven CaMKII (Ca2+/calmodulin-dependent kinase II) activation that in turn promotes aberrant sarcoplasmic reticulum (SR) Ca2+ leak triggered arrhythmic activities.

JNK activation has been observed in various cardiovascular diseases associated with a dramatically increased AF propensity, including myocardial infarction and HF.5,8,10 AF, myocardial infarction, and HF frequently occur together in the aging population.2–4 However, the underlying mechanisms of AF arrhythmogenic substrate development in aged (but otherwise normal) hearts remain incompletely understood. Our current study revealed a link between JNK activation and enhanced arrhythmic susceptibility in human atria with increasing age but preserved cardiac function and no history of AF or any major cardiovascular diseases. Moreover, the striking antiarrhythmic effects of JNK inhibition in both aged animal and young animal JNK models strongly support the causal link between JNK and AF propensity.

One might argue that atrial structural remodeling could be another contributing proarrhythmic factor. Indeed, patchy fibrosis and generally more interstitial fibrosis is commonly associated with cardiovascular diseases like myocardial infarction, HF, and diabetic cardiomyopathy. However, we and others have reported that there is no evidence of age-associated structural remodeling in the aged rabbit and human atria without a history of coexisting cardiovascular disease or AF.8,30,43 Here, all aged animals and JNK challenged young animals showed preserved cardiac function and normal interstitial fibrosis level. Multiple types of cells can be involved in arrhythmogenic remodeling and AF development. Our studies include cellular Ca2+ imaging in isolated atrial myocytes and RyR2 single-channel recordings where cell identity is not an issue. And, our results in whole animals, isolated hearts, intact atria, atrial myocytes, and single RyR2 channels are consistent across this broad experimental spectrum. Thus, our conclusion that there is a critical proarrhythmogenic action of JNK-mediated CaMKII activation in atrial myocytes is well supported.

In addition to JNK, p38 is another major member of the stress response kinase MAPK (MAP kinase) family.44 In response to stress stimuli, the actions of JNK and p38 are dependent on cellular context. For example, JNK and p38 have opposite functions (activation or suppression) in cellular senescence.45,46 This is in agreement with our previous findings of unchanged p38 in both aged atria7,8 and in long-term anisomycin-treated atrial myocytes (data not shown). Although possible contributions of other MAPKs remain to be investigated, our results strongly point to JNK having a critical role in promoting atrial arrhythmias.

The JNK isoforms are generally targeted to distinct functions.47 In the heart, JNK1 is linked to helping preserve cardiac function and promoting apoptosis during ischemia–reperfusion in myocardial infarction hearts.47 However, to our knowledge, the JNK2 contribution to normal or pathological heart function is unknown to date. Our data show that JNK2 activation is involved in driving SR Ca2+ mishandling and thus enhancing AF propensity. This JNK2 action was not only present in aged hearts but also occurred in young animals challenged by JNK activation. Genetic JNK2 depletion or JNK2-specific inhibition reduced arrhythmogenicity in aged and young animal models. This implies that this pathway is not a specific ramification of age, but instead is ever-present and thus available to respond to cellular stresses. Specifically, activated JNK2 boosts SR Ca2+ handling, and this enhances AF propensity in the absence of coexisting cardiovascular disease.

Another novel finding here is our identification of a unique example of kinase pathway crosstalk as JNK2 directly activates CaMKII to drive the pathology. We and others have previously reported that CaMKII phosphorylation of Ca2+ handling proteins is a key proarrhythmic factor.17,22,38,39 Here, we show that JNK2-driven CaMKII activation results in CaMKII-dependent phosphorylation of RyR2815 and PLB17. The consequence is arrhythmogenic diastolic SR Ca2+ mishandling. Specifically, increased diastolic SR Ca2+ leak triggers aberrant Ca2+ activities. We present striking rescue results where either CaMKII or JNK2 inhibition eliminates this downstream diastolic Ca2+ handling dysfunction. Enhanced diastolic SR Ca2+ leak by itself will lower SR Ca2+ content. Interestingly, JNK activation increased both SR Ca2+ leak and content. This suggests that JNK activation enhances SR Ca2+ uptake sufficiently to overcome the larger RyR2-mediated SR Ca2+ leak. Consistent with Guo et al31 who explored CaMKII action in PLB KO mice, the enhanced SERCA function here did not accelerate the Ca2+ transient decay rate because SR Ca2+ leak was also enhanced. Previous results48 from cross-bred CaMKII-delta overexpression and PLB KO mice are also consistent with this outcome. We are aware that the CaMKII inhibitor KN93 used in the current studies may have off-target effects. However, we have also used the AC3-I mice (cardiac overexpression of CaMKII inhibitory peptide) and AIP in RyR channel measurement to confirm the action of CaMKII inhibition in JNK-driven abnormal Ca2+ activities in intact atria. The results were consistent with the rescue action of CaMKII inhibition using KN93 or JNK inhibition in diastolic SR Ca2+ leak in myocytes and RyR channel activities.

SR Ca2+ leak, uptake, and load are increased in paroxysmal AF, but CaMKII and RyR2 phosphorylation status have been reported to be unchanged.49 The role of CaMKII-dependent SR dysfunction in AF is clearly complex. Our target here was to define JNK contribution in arrhythmic Ca2+ handling in aged atrium in the absence of AF history. Age-associated atrial arrhythmogenicity may or may not be analogous to that in paroxysmal or chronic AF situations. However, Li et al50 have reported JNK activation in a tachypacing canine AF/HF model. Although the contribution of JNK in sustained AF clearly requires further investigation, we present compelling results that show age-associated JNK activation drives CaMKII-dependent atrial arrhythmogenicity via promotion of RyR2-mediated Ca2+ leak and this can be avoided by limiting JNK2 function. Suppression of CaMKII function is known to mitigate arrhythmias and various heart diseases in animal models provoking a great deal of interest in development of CaMKII inhibitors as possible antiarrhythmic therapeutic agents.23 We have revealed a novel example of a JNK2/CaMKII link where JNK2 directly activates CaMKII to drive arrhythmic remodeling.

Whether there are other mechanisms of JNK-driven Ca2+ mishandling and other ion channels that promote AF clearly warrants further investigation. For example, protein phosphatases are involved in regulating the CaMKII phosphorylation status. Although our results of cell free JNK2 and CaMKII protein binding studies suggest that JNK2 directly phosphorylates CaMKII without the presence of protein phosphatases, the role of protein phosphatases in this JNK/CaMKII relationship requires further investigation. However, the preponderance of our current results does suggest a central (albeit not necessarily an exclusive) role of JNK in AF.

It is well known that JNK activation is involved in the development of cancer, diabetes mellitus, and arthritis.5,6,10 To date, JNK inhibition has been explored as a possible anticancer and arthritis therapeutic target in clinical trials.10,47 Our results make exploring therapeutic strategies of JNK inhibition to address cardiac arrhythmias an attractive and still unexplored option. Last, aging is just one of the cellular stresses associated with JNK activation.8 Other stresses like inflammation, obesity, alcohol abuse, and HF are also linked with JNK activation, and some of these are associated with greater AF propensity. Therefore, the JNK2/CaMKII crosstalk discovered here might have broad potential therapeutic benefits in antiarrhythmic drug development. Further investigation is required to determine how JNK inhibition might influence other cellular properties to minimize/understand potential off-target effects of any JNK-targeted antiarrhythmic therapy.

Acknowledgments

We graciously thank the donor families at Gift of Hope who provided the gifts that made our research possible. We also sincerely thank Ms Alma Nani for her excellent technical assistance with cardiac sarcoplasmic reticulum (SR) vesicles isolation and RyR (ryanodine receptor) single-channel recording, Dr Yibin Wang (UCLA) for providing adenoviral MKK7D vector and MKK7D transgenic mice, Ms Jinying Yang for her assistance with AC3-I mouse breeding, Drs Xander Wehrens and Na Li (Baylor) for their suggestions, Dr Ryan Himes for their technical assistance with camui FRET imaging, Jollyn Tyryfter for her assistance with rabbit myocytes isolation, and Peter Caron for making accessories for our imaging equipment. J. Yan conducted the confocal Ca2+ imaging, optical mapping, and data analyses; assisted in writing and generating figures; W. Zhao conducted biochemical assays, animal preparations, and cell cultures; J.K. Thomson assisted with in vivo atrial fibrillation (AF) induction, analyzed AF data, and contributed to writing and generating figures; DM DeMarco assisted with preparing the article; E. Carrillo conducted camui FRET imaging and performed FRET analyses; B. Chen conducted some confocal Ca2+ imaging experiments and imaging data analyses; X. Gao constructed CaMKII-WT and CaMKII-T286A vectors, performed CaMKII activity assays, and analyzed data; X. Wu assisted with immunoblotting and FRET studies; K.S. Gingsburg assisted with cellular Ca2+ dynamic measurements and data analyses, as well as critical revision of the text; B. Mamdouh assisted with human tissue studies, procurement, and specimen collection; D.M. Bers provided Camui biosensors, assisted with cellular Ca2+ dynamic measurement/data analysis, and critically revised the text; M.E. Anderson provided AC3-I transgenic mice and critically revised the text; L.-S. Song conducted intact heart Ca2+ imaging and image analysis, and revised the text; M. Fill conducted single RyR channel recording/analyses and critically revised the text; X. Ai conceived and designed study, performed in vivo AF induction/biochemical studies, analyzed and interpreted data, and drafted/revised the text and figures. None of authors have any financial disclosures to report.

Sources of Funding

This research was supported by National Institutes of Health grants (HL080101 to D.M. Bers; HL079031, HL096652, HL070250, and HL071140 to M.E. Anderson; HL090905 and HL130346 to L.-S. Song; HL057832, AR054098, AA024769, and GM11397 to M. Fill; HL113640, AA024769, and HL062426 to X. Ai) and American Heart Association (10GRNT37700 to X. Ai).

Disclosures

None.

Footnotes

  • In December 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.60 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.312536/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    AF
    atrial fibrillation
    CaMKII
    Ca2+/calmodulin-dependent kinase II
    camui-vv
    FRET-based CaMKII sensor with mutated Met280Val & Met281Val
    hCaMKII
    human CaMKII
    HF
    heart failure
    hJNK
    human JNK
    JNK
    c-Jun N-terminal kinase
    JNK1dn
    dominant-negative JNK1
    JNK2
    JNK isoform 2
    JNK2dn
    dominant-negative JNK2
    JNK2KO
    JNK2 knockout
    JNK-P
    phosphorylated JNK
    NCX
    Na+/Ca2+ exchanger
    PKA
    protein kinase A
    PLB
    phospholamban
    PLB16-P
    phosphorylated PLB at Ser16
    PLB17-P
    phosphorylated PLB at Thr17
    RyR
    ryanodine receptor
    SR
    sarcoplasmic reticulum
    WT
    wild type

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Novelty and Significance

What Is Known?

  • Atrial fibrillation (AF) is the most common sustained arrhythmia and a major public health problem, which currently lacks effective pharmacological therapies.

  • Advanced age is a major risk factor for AF. Consequently, the burden of AF is growing exponentially as the mean age of many populations around the world grows.

What New Information Does This Article Contribute?

  • Kinase-on-kinase pathogenic crosstalk is critical in governing intercellular Ca2+ signaling and consequently Ca2+-mediated atrial arrhythmias.

  • Advancing age and other stresses (like alcohol, obesity, inflammation, etc.) drive JNK (c-Jun N-terminal kinase) activation and the JNK/CaMKII (Ca2+/calmodulin-dependent kinase II) crosstalk is likely a critical mechanism that couples arrhythmia and these stresses.

  • JNK2 inhibition may be a potential target in developing new therapeutic strategies to prevent or treat AF.

AF is associated with a high risk of mortality and associated morbidities, including stroke and heart failure. However, there is no clear molecular concept addressing the mechanism for enhanced atrial arrhythmogenicity in the aged heart. Here, we report that activation of the kinase JNK2 leads to arrhythmogenic diastolic Ca2+ mishandling. Further, we found that JNK2 is a critical activator of CaMKII, a highly validated proarrhythmic signal, through direct phosphorylation of CaMKII. The phosphorylated and hyperactivated CaMKII ultimately drives the diastolic Ca2+ dysfunction that triggers atrial arrhythmias. Our study reveals a previously unrecognized link between JNK2 activation and the age-related enhancement of AF propensity. This link involves a novel, previously unknown, form of pathogenic kinase-on-kinase crosstalk. Our studies reveal a new potential therapeutic target (JNK2) that can be leveraged to prevent the CaMKII hyperactivation and thus limit AF and potentially other cardiovascular diseases.

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  • Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial ArrhythmogenesisNovelty and Significance
    Jiajie Yan, Weiwei Zhao, Justin K. Thomson, Xianlong Gao, Dominic M. DeMarco, Elena Carrillo, Biyi Chen, Xiaomin Wu, Kenneth S. Ginsburg, Mamdouh Bakhos, Donald M. Bers, Mark E. Anderson, Long-Sheng Song, Michael Fill and Xun Ai
    Circulation Research. 2018;122:821-835, originally published January 19, 2018
    https://doi.org/10.1161/CIRCRESAHA.117.312536
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