Up‐regulated Cx43 phosphorylation at Ser368 prolongs QRS duration in myocarditis

Abstract Prolongation of QRS duration in electrocardiogram is one of the risk factors for morbidity and mortality in many kinds of cardiac diseases. However, its molecular mechanism is unknown. In this study, utilizing experimental autoimmune myocarditis (EAM) as a disease model, we show that the prolongation of QRS duration is accompanied by elevated phosphorylation of connexin 43 (Cx43) at Ser368 (pS 368Cx43). In cultured cells, inflammatory cytokine IL‐1β activates p38 MAPK to up‐regulate pS 368Cx43 and impairs cell‐to‐cell communication. In isolated hearts of normal rats, perfusion of IL‐1β not only increases pS 368Cx43 but also impairs cell‐to‐cell communication and prolongs QRS duration. Furthermore, blockade of p38 MAPK down‐regulates pS 368Cx43, improves cell‐to‐cell communication and reduces QRS duration in EAM. These findings suggest that up‐regulation of pS 368Cx43 by IL‐1β via p38 MAPK contributes to the prolongation of QRS duration and could be a therapeutic target for myocarditis‐induced prolongation of QRS duration.

coronary artery disease, 6,7 ischemic cardiomyopathy, 8 myocardial infarction 9 and heart failure. 10 Furthermore, the prolongation of QRS duration has been shown to be associated with death risk in right bundle branch block, 11 worsen left ventricular function, 12 atrial fibrillation, 13 ventricular tachyarrhythmias, 14 etc. For example, comparing the groups with duration of QRS ≥ 120 ms vs QRS < 120 ms (median follow-up, 45 months), the mortality in patients with heart failure is 51% vs 34% and the sudden death rate is 25% vs 17% respectively. 8 The relative risk of recurrent ventricular arrhythmia is nearly fourfold higher in patients who had the prolongation of QRS duration (≥120 ms) than in those with a normal QRS duration. 15 A prolonged QRS duration in patients with heart failure has been shown to be associated with more advanced myocardial disease, worse left ventricular function, poorer prognosis and a higher allcause mortality rate compared with patients with a narrow QRS complex. 12 The risk of inducible sustained monomorphic ventricular tachycardia increases by 2.4% for each 1 ms prolongation in QRS duration. 16 For every 10 ms prolongation in QRS duration, mortality rate increases 10% for ventricular arrhythmias, 17 18%-26% for bundle branch block 11,18 and 6% for myocardial infarction 9 respectively.
Thus, understanding the molecular mechanism of the prolongation of QRS duration is of clinical significance.
The duration of the QRS complex is determined by the ventricular depolarization and the propagation of the excitatory cardiac impulse throughout the ventricle. The prolongation of the QRS complex reflects ventricular conduction delay, a substrate for arrhythmogenicity. 19 Gap junction channels form an intercellular pathway for electrical cell-to-cell coupling and are essential for normal cardiac impulse propagation. It has been shown that alterations in electrical coupling via gap junction channels contribute to abnormal conduction and arrhythmogenesis in the heart. 20 Pathological alterations in connexin abundance or function can lead to slowing of conduction. 21,22 In mammalian ventricular muscle, connexin 43 (Cx43) is the predominant gap junction channel. 20,23 Impaired propagation, reflected in the prolongation of QRS duration, reduces coordinated ventricular contraction and forms a substrate for cardiac arrhythmias, 22 which has been observed in cardiac-restricted Cx43 "knockout" mice. 21 Homozygous ablation of Cx43 in cardiomyocytes leads to low voltage QRS and significant prolongation of QRS duration. 24 QRS duration was significantly prolonged in Cx43(+/À) mice than in wild type, but P-wave duration and amplitude did not differ. 25 Genetic knockout of Cx43 in mice is associated with conduction slowing, prolongation of QRS duration and increased susceptibility to ventricular arrhythmias. 21,25,26 Replacement of Cx43 by Cx31 in the heart leads to significant prolongation of QRS duration. 27 In addition, some preclinical and marketed drugs have been shown to cause QRS prolongation via Cx43 uncoupling. 28 Therefore, as a principal conductor of intercellular current in the ventricle, 25 Cx43 is one of the molecular determinants for the prolongation of QRS duration.
A model of rat experimental autoimmune myocarditis (EAM) resembles human giant cell myocarditis, and the recurrent form of EAM leads to dilated cardiomyopathy. In this study, utilizing EAM as a disease model, we show that the elevation of p S368 Cx43 by IL-1b via the p38 MAPK signalling pathway contributes to the prolongation of QRS duration in myocarditis.

| Induction of EAM model
Male Lewis rats (180-200 g), aged 6-8 weeks, purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China) were maintained in Xiamen University animal experimental centre. All animal care and experiments were performed in accordance with procedures approved by the Animal Care and Use Committee of Xiamen University. Porcine cardiac myosin was prepared from the ventricular muscle of porcine hearts as previously described. 29 To produce EAM, each rat was immunized on day 0 with 0.2 mL emulsion containing 1 mg porcine cardiac myosin with an equal volume of complete Freund's adjuvant supplemented with mycobacterium tuberculosis H37RA at a concentration of 10 mg/mL by a single subcutaneous injection in both footpads. The rats in the control group were only immunized with complete Freund's adjuvant.
2.2 | In vivo administration of SB203580 to EAM rats SB203580 was dissolved in DMSO, then intraperitoneally injected every 2 days at a concentration of 20 mg/kg to the rats from day 14 to day 20. The rats in the control group were intraperitoneally injected with same volume of DMSO. Relative mRNA expression and protein levels were measured by quantitative real-time RT-PCR and Western blot analysis.

| Electrocardiography (ECG) recording
The ECG of all rats was recorded under light isoflurane anaesthesia and analysed off-line using BL-420S bio-experiment system (Chengdu Techman Software Co. LTD, China). Heart rate (HR), P wave, QRS complex and PR interval were evaluated. QRS duration was measured in lead II from the earliest to the latest deflection of the QRS complex, while its amplitude was measured from the nadir to the top of each QRS complex.

| Immunohistochemical analysis
Hearts were fixed in 10% formalin, embedded and cut into 5 lm thick sections. After deparaffinized and rehydrated, endogenous peroxidase was blocked by incubation in 3% H 2 0 2 in methanol for 10 minutes. For antigen retrieval, the sections were heated at 120°C in citric acid buffer for 15 minutes and then cooled for 30 minutes at room temperature. After washed 3 times in PBS, the sections were blocked by 10% bovine serum albumin for 30 minutes and incubated with anti-connexin43 antibody (Sigma, 1:2000), anti-phospho-Cx43 antibody (Ser368) (Cell Signalling, 1:100) overnight at 4°C.
Afterwards, the sections were washed 3 times in PBS, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson, 1: 500) for 1 hour at room temperature. The immune reaction was performed using a DAB Kit (MXB, China) under microscope. The sections were counterstained with hematoxylin, gradient alcohol dehydration and mounted. Afterwards, the membranes were washed in PBS containing 0.05% Tween 20, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson, 1:10000) for 1 hour at room temperature. The immunoreactivity was visualized by an enhanced chemiluminescence (ECL) advanced kit (Millipore). Signal intensities were analysed using Image J software.  absolute level of fluorescence, were counted using a fluorescence microscope to judge activity of the Cx43 gap junction channels as previously described. 30

| Preparation of isolated perfused heart
Rats were anesthetized with isoflurane after treatment with heparin (10 mg/kg) for 30 minutes. A thoracotomy was performed to excise the heart. During preparation, the heart was washed with cold Krebs-Henseleit solution to decrease its contractility. Then, it was connected to Langendorff's apparatus and perfused with modified

| Scrape-loading dye transfer
The isolated heart was perfused for 10 minutes to rinse the blood and immersed in cold Krebs-Henseleit solution for 10 minutes at 4°C to decrease its contractility. Then, the apex of heart was sectioned and incubated for 1 minutes in Lucifer yellow at 4°C according to a previously described method. 31 Afterwards, the heart was immediately fixed in liquid nitrogen and embedded in OCT compound. The heart was sliced (50 lm) and imaged with fluorescence microscope.

| Immunofluorescence analysis
Cryostat sections (5 lm) of hearts were fixed in 4% paraformaldehyde for 10 minutes, and processed for immunostaining as previously described. 32 The H9c2 cells cultured on six-well glass chamber slides were fixed in 4% paraformaldehyde, blocked by 10% bovine serum albumin for 30 minutes and stained with antibodies overnight at 4°C. Afterwards, the sections were washed 3 times in PBS and incubated with Alexa 549-conjugated goat anti-rabbit antibodies (Rockland) for 1 hour at room temperature. Nuclei were stained with DAPI. Slices were mounted with antifade mounting medium (Applygen, China) and analysed using Olympus microscope (IX51).

| Statistical analysis
All data are shown as mean AE SEM. Data were analysed statistically by one-way analysis of variance (ANOVA) followed by the Bonferroni test for multiple comparisons. Origin7.0 (Microcal Software, Inc.) or Prism 5.0 (GraphPad Software, Inc) software was used for statistical analyses. Values of P < .05 were considered to be statistically significant.

| Experimental autoimmune myocarditis induces prolongation of QRS duration in ECG
To investigate whether EAM could result in the prolongation of QRS duration, we carried out ECG recordings on the rats. Representative lead II ECG recordings on day 0 and during the progression of EAM is shown in Figure 1A. On day 0, the QRS duration was with that of the prolongation of QRS duration. 21,24 In contrast, the QRS duration and its amplitude were not changed on the corresponding day of the control rats ( Figure 1B). Similar result has been reported in DBA/2 mice with experimentally induced myocarditis. 2

| Experimental autoimmune myocarditis induces up-regulation of p S368 Cx43
Next, we investigated whether EAM affected Cx43, since it has been reported that the prolongation of QRS duration is closely associated with the dysfunction of Cx43. 21,24 Figure 2A showed that the ratio between phosphorylated and non-phosphorylated Cx43 in the cardiac ventricles was significantly increased on day 14 and peaked on day 21, which was reduced on day 28 but was still higher than that of day 0. The relative level of the p S368 Cx43 was significantly increased from 0.21 AE 0.01 on day 0 to 0.59 AE 0.05 on day 14, 0.87 AE 0.11 on day 21 and reduced to 0.42 AE 0.02 on day 28 (Figure 2B). In agreement with this result, immunohistochemical staining of the p S368 Cx43 showed that the p S368 Cx43 was obviously increased on day 14 and 21 of EAM (coloured brown in Figure 2C).
As we have previously shown that the p S368 Cx43 affects the distribution of Cx43 in H9c2 cells, 30 (Figure 3E), even though the EAM serum could phosphorylate Erk to make it activation ( Figure S1). These results suggest that both PKC and p38 MAPK signalling pathways contribute to the elevation of the p S368 Cx43 level induced by the myocarditis serum. In addition, we found that PKC inhibitor (Ro-32-0432) could partly suppress phosphorylation of p38 (p-p38) induced by the EAM serum ( Figure 3G). Similarly, p38 inhibitor (SB203580) could also partly suppress the EAM serum up-regulated phosphorylation of MARCKS (p-MARCKS, Figure 3H), which is a marker for PKC activation. These results imply that there might be interaction between p38 MAPK and PKC signalling pathways. As effect of PKC on the p S368 Cx43 has been intensively studied, 30,32,33 we focus on the effect of p38 MAPK on the p S368 Cx43 in the following experiments.
Increase in the p S368 Cx43 has been shown to decrease gap junction mediated cell-to-cell communication. 30,32,33 To test whether the elevation of the p S368 Cx43 level by the EAM serum could affect   [34][35][36][37] Besides, levels of inflammatory cytokines, such as TNF-a and IL-1b were raised in patients with acute myocarditis. 37 We therefore examined whether cytokines in the EAM serum contribute to the EAM induced elevation of p S368 Cx43. We found that treatment of the cells with IL-1b increased the phosphorylated (p-p38) and thus, activated form of p38 MAPK ( Figure 5A). The activated p38 MAPK promoted the elevation of the p S368 Cx43 with a maximal induction at 15 minutes ( Figure 5B), the time course of which coincided with that of the EAM serum induced elevation of p S368 Cx43 and p-38 ( Figure 3B and F). Furthermore, the effect of IL-1b on the p S368 Cx43 was inhibited by p38 inhibitors (SB203580 and SB202190) ( Figure 5C Figure 5E). In contrast, treatment of the cells with TNF-a only slightly increased the p S368 Cx43 and the phosphorylated form of p38 ( Figure S2 A and C), which was inhibited by SB203580 as well (Figure S2 B). These results suggest that the effect of the EAM serum on the p S368 Cx43 is mainly due to the ability of IL-1b to activate p38 MAPK to phosphorylate Cx43 at Ser368. In agreement with this result, it has been reported in astrocytes that IL-1b induces p38 activation and inhibits dye coupling of Cx43-mediated gap junction. 38 3.5 | IL-1b up-regulates p S368 Cx43, impairs cell-tocell communication and prolongs QRS duration in isolated rat heart In order to investigate whether the impairment of cell-to-cell communication at the cellular level could also apply to the heart level, we perfused isolated heart of normal rats with IL-1b. Representative ECG recordings ( Figure 6A) and data of relative QRS duration ( Figure 6D). In addition, immunohistochemical staining of p S368 Cx43 ( Figure 6E) and Cx43 ( Figure 6F) showed that IL-1b caused distribution of both p S368 Cx43 and Cx43 from a dense manner to a disperse manner at the intercalated discs of the ventricular myocardium. Furthermore, IL-1b reduced diffusion of the fluorescent dye (Lucifer yellow) from the cut edge into the centre of the isolated heart within 1 minutes (middle panel, Figure 6G). In contrast, the dye diffused toward the heart centre deeply within 1 minutes in the control condition (left panel, Figure 6G). It is worthy to note that the dye could diffuse deeply if it was allowed to diffuse 5 minutes (right panel, Figure 6G), suggesting that the function of Cx43 is maintained somehow. These results strongly suggest that elevation of p S368 Cx43 by IL-1b directly impairs cell-to-cell communication and thus prolongs the QRS duration.
3.6 | Blockade of p38 MAPK suppresses p S368 Cx43, improves cell-to-cell communication and reduces QRS duration in EAM Based on the above results that the elevation of p S368 Cx43 via p38 MAPK could cause the prolongation of QRS duration, it can be inferred that inhibition of the p38 MAPK should prevent the EAM-induced prolongation of QRS duration. Inflammation is a complex process involving numerous mediators of cellular and plasma origin with elaborate, interrelated biological effects. Therefore, to avoid action of inflammation as many as possible, we applied p38 MAPK inhibitor SB203580 every 2 days after the most severe inflammation period (day 14) to day 20 of EAM, during which the QRS prolongation has been established. Figure 7A shows representative time courses of ECG recorded from the same rat of control, EAM or SB203580 treated EAM rats. In the control rats, the QRS duration (~18 ms) and QRS amplitude (~800 lV) were almost not altered from day 14 to day 21 ( Figure 7B Figure 7F). Furthermore, SB203580 partially restored the dye transfer ability of the heart, which was significantly impaired in the heart of EAM ( Figure 7G-I). These results suggest that inhibition of p38 MAPK activity could prevent not only the myocarditis induced elevation of p S368 Cx43 but also the impairment of cell-to-cell communication and the prolongation of the QRS duration.

| DISCUSSION
In this study, we provided several lines of evidence to support that the elevation of p S368 Cx43 contributes to the prolongation of QRS duration in EAM. (i) The time course of changes in the QRS duration was coincided with that of the p S368 Cx43 in EAM rats ( Figures 1C   and 2B); (ii) When the elevation of p S368 Cx43 was suppressed by p38 MAPK blocker, the prolongation of QRS duration was prevented in EAM as well ( Figure 7); (iii) When the level of p S368 Cx43 was upregulated by IL-1b, the QRS duration was prolonged even for the isolated normal heart (Figure 6).
In EAM rats, the QRS duration prolonged from day 10 to day 14, then reduced. Meanwhile, the p S368 Cx43 level was also significantly increased, then decreased. What is the relationship between the prolongation of QRS duration and the elevation of the p S368 Cx43 level?
It has been demonstrated that mutation of Ser368 to Ala to prevent its phosphorylation makes the Cx43 form constitutively open channels, 33 while mutation of Ser368 to Asp to mimic p S368 Cx43 results in a closed state of the gap junctional channel, 39 suggesting that upregulation of the p S368 Cx43 could lead to impairment of cell-to-cell communication. As a matter of fact, many studies have shown that up-regulation of p S368 Cx43 reduces cell-to-cell communication. 30,32,40 In this study, we demonstrated that the elevation of p S368 Cx43 by the EAM serum or IL-1b caused impairment of the cell-to-cell communication in the cultured cells. Extent of cell-to-cell communication through gap junction channels is one of the major factors to affect propagation of the electrical impulse through the heart. 19 And impaired propagation of electrical impulse has been shown to be able to cause the prolongation of QRS duration. 21,22 Our study showed that blockade of p38 MAPK in EAM rats not only prevented the elevation of p S368 Cx43 but also prevented the prolongation of QRS duration. Most importantly, in isolated hearts, which F I G U R E 7 Blockade of p38 MAPK prevents the prolongation of QRS duration by suppressing the elevation of p S368 Cx43 in EAM. A, Representative electrocardiogram (ECG) of control, EAM and SB203580 (20 mg/kg) treated EAM rats. SB203580 was intraperitoneally injected every two days to the rats from day 14 to day 20. Each set of ECG was recorded from the same rat on day 14, 17 and 21. The solid and doted lines indicate the period of QRS duration. B and C, Statistical summary on QRS duration (B) and QRS amplitude (C) from control, EAM and SB203580-treated EAM rats (n = 6-8). D, Western blot analysis of relative p S368 Cx43 in the cardiac ventricles of control, EAM and SB203580-treated EAM rats (n = 6-8). Upper panel: Western blot; Lower panel: Statistical summary. **P < .01 vs control; ***P < .001 vs control; # P < .05 vs EAM; ## P < .01 vs EAM. E and F, Immunohistochemical staining of p S368 Cx43 (E, brown) and Cx43 (F, brown) in the ventricular sections of the control, EAM and SB203580-treated EAM rats on day 21 of EAM (n = 6). Bar = 20 lm. G, Representative snapshots of dye (LY, Lucifer yellow) diffusion for 1 minutes in the slice of control, EAM or SB203580-treated EAM heart. H and I, Quantification of dye diffusion measured as the fluorescence intensity vs distance from the edge (H) and as the area under the curve (I). Bar = 100 lm. Dotted line: the edge of dye transferred. White arrows: cutting edge at the apex of the heart eliminates most of the factors that could influence ECG, perfusion of IL-1b could still lead to the elevation of p S368 Cx43, the impairment of cell-to-cell communication and thus the prolongation of QRS duration. Therefore, these results suggest that EAM induced elevation of p S368 Cx43 might be responsible for the prolongation of QRS duration in EAM rats.
Many studies have shown that p S368 Cx43 is up-regulated through the PKC signalling pathway. 30,32,33 In this study, we showed that p38 MAPK is involved in the up-regulation of p S368 Cx43 as well. Furthermore, we found that IL-1b, one of the major inflammatory cytokines released in the early stage of EAM, could activate p38 MAPK, up-regulate the p S368 Cx43 level and suppress cell-to-cell communication. However, this does not exclude the contribution of PKC in the process. In fact, we found that blockade of either PKC or p38 MAPK could suppress the elevation of p S368 Cx43. In good agreement with this result, it has been demonstrated that Cx43 forms a complex with protein kinases Src, p38 MAPK and PKC. 41,42 Therefore, it is highly likely that both PKC and p38 MAPK signalling pathways contribute to the elevation of the p S368 Cx43 level in EAM.
Taken together, in this study, we identified a novel regulatory mechanism that up-regulation of p S368 Cx43 by IL-1b via p38 MAPK signalling contributes to the prolongation of QRS duration in myocarditis. Thus, blockade of p38 MAPK in EAM rats prevents both the EAM-induced elevation of p S368 Cx43 level and the EAM-induced prolongation of QRS duration, suggesting that p38 MAPK should be a novel therapeutic target for myocarditis.