Small-molecule ISRIB reduces susceptibility to postinfarct atrial fibrillation in rats via the inhibition of integrated stress responses

Phosphorylation of the eukaryotic translation initiation factor 2 α-subunit (eIF2ɑ),which subsequently upregulates activating transcription factor 4 (ATF4), is the core event in the integrated stress response (ISR) pathway. Previous studies indicate phosphorylation of eIF2ɑ in atrial tissue in response to atrial fibrillation (AF). This study investigated the role of ISR pathway in experimental AF by using a small-molecule ISR inhibitor (ISRIB). Accordingly, rats were subjected to coronary artery occlusion to induce myocardial infarction (MI), or sham operation, and received either trans-ISRIB (2 mg/kg/day, i.p.) or vehicle for seven days. Thereafter, animals were subjected to the AF inducibility test by transesophageal rapid burst pacing followed by procurement of left atrium (LA) for assessment of atrial fibrosis, inflammatory indices, autophagy-related proteins, ISR activation, ion channel, and connexin43 expression. Results showed a significant increase in the AF vulnerability and the activation of ISR in LA as evidenced by enhanced eIF2α phosphorylation. ISRIB treatment suppressed upregulation of ATF4, fibrosis as indexed by determination of α-smooth muscle actin and collagen levels, inflammatory macrophage infiltration (i.e., CD68 and iNOS/CD68positive macrophage) and autophagy as determined by expression of LC3. Further, ISRIB treatment reversed the expression of relevant ion channel (i.e., Nav1.5, Cav1.2, and Kv4.3) and Cx43 remodeling. Collectively, the results suggest that the ISR is a key pathway in pathogenesis of AF, post-MI, and represents a novel target for treatment of AF.


Introduction
Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical practice, and associated with stroke, congestive heart failure (CHF), and mortality (Guo et al., 2015;Lippi et al., 2020). With the aging of the population, AF has become a remarkable public health problem (Krijthe et al., 2013) associated with high costs of medical care of affected individuals (Stewart et al., 2002). Nowadays, available therapies for AF include Antiarrhythmic drug, catheter ablation and surgical ablation, each with limitations like adverse effects, inconsistent efficacy and high recurrence rate (Heijman et al., 2016). Recent studies identified new pathophysiological mechanisms of AF including cardiac fibrosis (Nattel, 2017), oxidative stress (Antonopoulos et al., 2019), apoptosis, autophagy (Chen et al., 2011;Wiersma et al., 2017), inflammation (Zhou and Dudley, 2020) , and atrial ion-channel dysfunction, Ca 2+ -handling abnormalities, autonomic neural dysregulation, and atrial remodeling (Andrade et al., 2014).
AF mechanism might be associated with the integrated stress response (ISR), which is always activated in chronic and persistent stress (Pakos-Zebrucka et al., 2016;Santos-Ribeiro et al., 2018). The ISR pathway is upregulated in atria of AF animal models indicating by enhanced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequently upregulation of activating transcription factor 4 (ATF4) (Pakos-Zebrucka et al., 2016). Previous studies have demonstrated eIF2α phosphorylation and ATF4 upregulation in the atria of experimental and human AF (Meyer-Roxlau et al., 2017;Wiersma et al., 2017;Freundt et al., 2018). ISR is also involved in atrial inflammation during chronic and persistent stress because it triggers artery inflammasome, interleukin-1beta secretion and 7 atherosclerosis (Onat et al., 2019).
Therefore, we hypothesize that ISRIB may inhibit atrial fibrosis, inflammation, autophagy and dysfunction of ion channel, and AF susceptibility because chronic stress plays important role of in atrial remodeling and AF (Zhang et al., 2014;Wiersma et al., 2017).

Myocardial Infarction
MI was induced by coronary artery ligation at the site of the LAD as we have previously described (Wang et al., 2011;Zhu et al., 2015;Wang et al., 2017;Wu et al., 2020). The success of the MI protocol was judged by the ST-segment elevation on ECG (RM6240, Chengdu, China) and pallor transition in heart immediately after ligation. Rats in sham groups underwent the same procedures without LAD occlusion (Wu et al., 2020).

Ultrasound Cardiogram (UCG)
Seven days after MI, cardiac function was assessed blindly by a skilled echocardiologist (Zhu et al., 2015) using a HP SONOS 5500 (Philips Medical System, Ultrasound Inc., USA) which was equipped with a 21 MHz ultrasound transducer. Parasternal long-axis and shortaxis views were acquired to measure the internal diameters of the left ventricles (LV) at enddiastole (LVIDd) and end-systole (LVIDs). The ejection fraction (EF) and the fractional shortening (FS) were calculated (Wang et al., 2011).

Electrocardiogram (ECG) and AF inducibility
This article has not been copyedited and formatted. The final version may differ from this version. The ECG trace was continuously recorded by a biological signal recording and analysis system (RM6240, Chengdu, China) which filtered out the signals below 10 Hz and above 100 Hz. The basic ECG was recorded consciously for 10 min for calculating ECG parameters such as P wave, QRS complex, PR interval, and QT intervals (Wang et al., 2011;Wu et al., 2020

Masson staining
Six left atrial (LA) tissue samples were dissected from every group and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into sections (5 µm thick). Sections of LA were stained by Masson's staining to detect collagen as previously reported . The level of cardiac fibrosis was determined by the percentage of the fibrosis area to the total area (% cardiac fibrosis) using the ImageJ software (Media Cybernetics, Inc.) (Wu et al., 2020).

Immunohistochemistry and immunofluorescence labeling
Paraffin-embedded tissue sections of LA were processed and incubated with different primary antibodies (Supplementary Table 1) at 4 °C overnight (Wang et al., 2011). For This article has not been copyedited and formatted. The final version may differ from this version. immunohistochemistry, six sections from every group were stained with the Envision system (Dako) and counterstained with hematoxylin. For immunofluorescence, secondary antibodies conjugated with fluorescein (FITC/CY3) were incubated for 1 h at room temperature. Then, 4,6-diamino-2-phenylindole (DAPI) was used to counterstain nuclei for further evaluation.
The negative control staining was performed using the same protocol without adding any primary antibody. Images were recorded using a microscope (Nikon Eclipse C1, Tokyo, Japan) and quantified using the ImageJ software through calculating as the percentage of positively stained area to total area at high magnification (×400). For quantification of CD68 + macrophages, the number of cells was counted in high magnification fields.

Western Blot
Protein expression was assayed using Western blot as described previously . In brief, six LA tissues, from each group, were processed and protein concentrations measured by the bicinchoninic acid assay. Equal amounts of proteins (50μg) were separated using 5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membrane was then blocked with 5% non-fat dry milk in blocking buffer (10 mM Tris-HCl, 150 mM NaCl, and 0.2% Tween-20, pH 7.6) for 2 h at 37 °C and incubated with primary antibodies (Seen in supplementary Table 1) at certain dilutions at 4 °C overnight. The antibody binding was detected using a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Sigma) and visualized using an ECL kit for quantification for blots image (a Quantity-One software, Bio-Rad).

Statistical analysis
This article has not been copyedited and formatted. The final version may differ from this version. Values were expressed as means ± s.e.m. Significant differences between means were determined using the one-way ANOVA coupled with the Bonferroni post hoc test for multiple comparisons. The Mann-Whitney U test was used to test the difference of AF duration between the two MI groups. The Chi-square test was used to compare the inducibility of AF.
A value of P < 0.05 was considered significant difference.

ISRIB improves cardiac function in post-MI rats
Within the first 24 hours，2 rats and 4 rats died in sham and MI group, respectively (13.3% vs 26.7%, P = 0.3894). From the second day, remaining rats received ISRIB or vehicle injection. On the second and third days, 2 rats died in MI group and one rat died in each other three groups (supplementary Fig. S1). No significant differences in animal survival rate were noted between sham and MI groups with or without ISRIB intervention (P =0.8571).
On the seventh day, two rats in the Sham group one rat in the Sham+ ISRIB group died while under anesthesia and, thus, did not complete UCG and ECG assessment. (Supplementary Fig.   S1). To assess whether ISRIB administration affects cardiac function post-MI, we investigated the echocardiographic parameters after ISRIB treatment in post-MI rats ( Table 1). As shown in Table 1, rats in the MI group exhibited significantly reduced EF(%) and FS(%), expanded cardiac systolic volume and LVIDs compared with sham group. ISRIB treatment significantly ameliorated the decline of EF, FS, and ventricular enlargement in MI rats but the treatment did not affect the aforementioned parameters in sham-operated animals.

ISRIB inhibits AF inducibility in post-MI rats
This article has not been copyedited and formatted. The final version may differ from this version. To determine whether ISRIB may be beneficial in AF, we tested the AF inducibility and ECG parameters in post-MI rats. Figure 1A shows that atrial burst rapid pacing could induce AF. Compared with the sham group, the MI group exhibited significantly vulnerability to AF (81.82% vs 0%, P < 0.05). ISRIB significantly reduced the AF inducibility in MI rats (33.3% vs 81.82%, P < 0.05) (Fig. 1B). The longest AF duration was not different between the two MI groups (Fig. 1C). We also observed increases in P wave duration, QRS wave complexes, PR interval and QT interval compared with the sham group (P < 0.01). ISRIB significantly reduced these changes in MI rats (P < 0.01, Table 2). Figure 2A shows representative illustrations for atrial fibrosis. ISRIB treatment significantly reduced fibrotic areas of the LA in MI rats but not in normal rats (Fig. 2D). As shown in Figure 2B, the expression levels of the alpha-smooth muscle actin (α-SMA, a marker for the proliferation of myofibroblasts) in MI rats were significantly higher than those in normal rats. ISRIB significantly ameliorated the α-SMA expression in LA from the MI group but not from the normal group (Fig. 2E). As shown in Figure 3C, a remarkable deposition of collagen I in the LA of the MI group was observed compared with that of the sham group. ISRIB treatment significantly reduced collagen I productions in MI rats but not in normal rats (Fig. 2F). These results indicated that ISR had a role in atrial fibrosis by promoting myofibroblast transition and collagen deposition after MI.

ISRIB restores ATF4 expression without affecting p-eIF2α in the LA in post-MI rats
ISR activation leads to global reduction in protein synthesis but preferentially enhances  3A and B). ISRIB treatment significantly reversed ATF4 level in post-MI rats ( Fig. 3A and B).
Western blot further confirmed that ISRIB treatment significantly inhibited the ATF4 protein expression in the LA from MI rats but not from normal rats ( Fig. 3C and E). The key regulation step in the ISR is the phosphorylation of eIF2α. We found that ISRIB treatment did not alter the expression levels of eIF2α and p-eIF2α in both sham and MI rats ( Fig. 3C and D).
These results indicate that ISRIB restores ATF4 protein expression in the nuclei of myocytes from LA through suppressing the downstream of the eIF2α pathway.

ISRIB regulates atrial macrophage infiltration in post-MI rats
Previous studies have reported inflammatory response and macrophage infiltration in atrial tissue including M1 (iNOS + /CD68 + ) and M2 subtypes (CD163 + /CD68 + ) Liu et al., 2019). As shown in Figures 4A and C, a remarkable increase of total CD68+ macrophages was observed in LA from MI rats, which could be reduced by ISRIB treatment.
Moreover, the distributions of iNOS-positive and CD163-positive immunofluorescence signals are not limited to macrophages and increase significantly in atrial tissue from MI rats ( Fig. 4A and B). ISRIB treatment reduced iNOS + /CD68 + cells (Fig. 4D) without changing CD163 + /CD68 + cells (Fig. 4E). These results indicate that ISR may play an important role in macrophages infiltration into atrial tissue from post-MI rats. We also determined the expression of autophagy markers, LC3, which is activated and incorporated into autophagosomes (Wiersma et al., 2017;Yuan et al., 2018). Using fluorescent immunostaining, we found a significant increase in the expression of the autophagy marker LC3 in LA in MI rats which was completely reversed by ISRIB ( Fig. 5A and C). Western blot analysis also confirmed an increase of LC3-II and LC3-II/LC3-I ratio in LA, and ISRIB treatment significantly reduced the upregulation of LC3 in MI rats ( Fig. 5B and D). These results suggest a biologically contribution of ISR to endoplasmic reticulum stress and autophagy.

ISRIB stabilizes the connexin43 (Cx43) expression in the LA in post-MI rats
To determine whether the reduction in ISR activation affects the Cx43 expression and distribution in atria, we tested the Cx43 level using fluorescent immunostaining. The Cx43 was distributed in the intercalated disk (end of myocardial cell) in the sham-operated group but redistributed to the lateral cell membrane in the MI group (supplementary Fig. S2A and   B). As shown in Figure 6A, Cx43 immunostaining was markedly reduced in LA in MI rats.
Accordingly, the relative value of the Cx43-positive area decreased significantly in the MI group which was reversed by ISRIB treatment (Fig. 6B). These results suggest that the ISR activation may be associated with the gap junction remodeling.

ISRIB keeps ion channel expression in the LA in post-MI rat
The abnormal ion channel is closely related to the action potentials of atrial myocytes, which facilitates atrial re-entry via the atrial heterogeneous conduction and focal trigger activity (Nattel et al., 2007). The protein expression of some ion channel-related proteins were assessed to learn the role of ISR in the generation of AF (Nattel et al., 2007). We found significant reduction in protein expression for Nav1.5 ( Fig. 7A and B), Cav1.2 ( Fig. 7A and C), and Kv4.3 ( Fig. 7A and C) in LA in MI rats (Fig 7). Moreover, ISRIB treatment significantly restored the protein expression levels of all three ion channels (Fig. 7). These results support the notion that ISR results in the down regulation of the protein expression of the above ion channels in LA.

Discussion
This study demonstrates that ISR activation in the LA is associated with post-MI AF. We have also observed that MI promoted atrial fibrosis, inflammation, autophagy, and intact, which is helpful for neurological diseases (Rabouw et al., 2019). Thus, ISRIB may protect against chronic ISR-related diseases such as atherosclerosis (Onat et al., 2019) and type 2 diabetes mellitus (Pandey et al., 2019). Recent studies indicate the involvement of endoplasmic reticulum stress which appeared as upregulation of eIF2α and ATF4, in atria of experimental and human AF (Wiersma et al., 2017;Freundt et al., 2018). Therefore, ISRIB may inhibit AF by suppressing ISR in atria. Indeed, the present study showed that ISRIB significantly reduced the AF inducibility to rapid atrial stimulation. We deduce that ISR may be involved in the atrial proarrhythmic substrates of AF in post-MI rats.
The atrial fibrosis is associated with low-conduction velocity and long activation time (Krul et al., 2015). Suppression of atrial fibrosis prevent AF through "upstream therapy", including renin-angiotensin-aldosterone system, inflammatory mediators, and other signaling targets (Li et al., 2001;Nattel et al., 2020). Under stress conditions, the transforming growth factor beta1 (TGFβ1) promotes fibroblasts to proliferate and differentiate into profibrotic collagen-secreting myofibroblasts, leading to atrial collagen deposition, extracellular matrix remodeling, and fibrosis (Nattel et al., 2020). In the present study, we observed that the ISRIB reduced more than 50% of the fibrotic areas and inhibited the collagen type I deposition and α-SMA expression in the LA from MI rats thereby suggesting that ISR is involved in atrial fibrosis.
The atrial inflammation mediates electrical and structural remodeling, and modulates calcium homeostasis and gap junction Packer, 2020 Zhou and Dudley, 2020). In this study, ISRIB reduces total CD68 + macrophage infiltration in LA, suggesting involvement of ISR in atrial inflammation. We also observed that some infiltrated CD68 + macrophages in LA from MI rats were iNOS-positive but CD163-negative. ISRIB treatment reduced the level of iNOS but not CD163 in atria.
CD163-positive macrophages have been known as a marker of M2 subtype (Liu et al., 2019), also have potential pro-fibrotic role in atrium from AF (Watson et al., 2020). Thus, the ISR plays an important role in regulating atrial proinflammatory response. Further study is needed to clarify the detailed mechanism of ISR in atrial inflammation and the occurrence of AF.
Previous studies have showed that the autophagy-related protein LC3 increased in the left atrium of human chronic AF (Shingu et al., 2020) and AF animals (Wiersma et al., 2017;Yuan et al., 2018). Our results showed that MI promoted LC3 expression in LA which was partly reversed by ISRIB treatment. Autophagy may be an important mechanism of AF (Kroemer et al., 2010) because previous studies have confirmed that autophagy could induce atrial electrical remodeling including L-type Ca 2+ -current reduction and selective degradation of Cav1.2 (Wiersma et al., 2017;Yuan et al., 2018). Autophagy also leads to eIF2α phosphorylation and ATF4 upregulation in experimental and human AF (Wiersma et al., 2017).
Thus, ISR activation might be associated with autophagy and play an important role of AF post-MI.
In the present study, we found that ISRIB ameliorates the reduction in the protein expression levels of Nav1.5, Cav1.2, and Kv4.2 in LA from post-MI. We also found that ISRIB improves the Cx43 expression and distribution. Cx43 is a major component of gap junction between myocytes and its abnormality may cause atrial spatial heterogeneity of conduction (Nattel et al., 2007). In a rat model of spontaneous hypertension, the AF vulnerability is associated with slow conduction velocity and low Cx43 expression, phosphorylation and redistribution (Parikh et al., 2013). Thus, our results suggest that ISRIB may attenuate the atrial electrical remodeling by preventing the downregulation of functional transmembrane proteins including ion channel and Cx43 in LA.
As key mechanisms of initial and maintenance of AF, ion channels and gap junction may be altered by ischemia through atrial fibrosis (Nattel, 2017), inflammation (Zhou and Dudley, 2020), and autophagy in LA (Wiersma et al., 2017;Packer, 2020). In the present study, ISRIB inhibited atrial fibrosis, inflammation and autophagy in LA. Therefore, ISR-pathway might contribute to the proarrhythmic mechanism of AF. Overall, ISRIB blocks the ATF4 protein expression without affecting p-eIF2α in LA post-MI, which results in the restoration of normal functional protein synthesis (including ion channels and gap junction) by suppressing This article has not been copyedited and formatted. The final version may differ from this version. This study has several limitations. First, we did not determine spontaneous AF in nonanesthesia rats due to lack of relevant physiological monitoring system for small animal.
Second, we did not evaluate the electrophysiological functions of ion channel (Na + , K + , and Ca 2+ ) from atrial myocytes although we have determined the protein expression of Nav1.5, Cav1.2, and Kv4.2. Third, other signaling pathways relevant to ISR may also play roles in AF.
Fourth, further studies are needed to establish the long-term therapeutic effects of ISRIB on AF in the context of assessment of its potential toxic effects.

Conclusions
Despite the existence of several limitations, this study has revealed that the trans-ISRIB could suppress ISR in ischemia atria which are at risk for AF. The possible mechanisms may be related to cardiac fibrosis, inflammatory cell infiltration, autophagy-related protein LC3 expression, and the remodeling of ion channel and Cx43. Considering the limitations of current therapies for AF, this study suggests that the ISR is a key pathway in pathogenesis of AF, and represents a novel target for treatment of AF.

Performed data analysis: Deguo Wang and Wen Xing.
Wrote or contributed to the writing of the manuscript: Nengwei Hu and Deguo Wang.

Competing financial interests
We have no conflict of interest to disclose.
The data used to support the findings of this study are included within the article and supplementary materials.
This article has not been copyedited and formatted. The final version may differ from this version.     atria. D. Quantitative analysis of the expression of phosphorylated eIF2α relative to total eIF2α. E.
Quantitative analysis of the expression of ATF4 relative to GAPDH. Data are shown as means ± s.e.m. Significant differences between means were determined by one-way ANOVA with the Bonferroni post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01 vs. Sham group; # P < 0.05, ## P < 0.01 vs. MI group. and CD68-positive (CD68 + ，iNOS + /CD68 + , and CD163 + /CD68 + ) area of cells. Data are shown as means ±s.e.m. Significant differences between means were determined by one-way ANOVA with the Bonferroni post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01 vs.
Sham group; # P < 0.05, ## P < 0.01 vs. MI group.  Data are shown as means ±s.e.m. Significant differences between means were determined by one-way ANOVA with Bonferroni post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01 vs.