Downregulation of EB1 impedes Cx43 localization and cardiac conduction after hypothermic ischemia-reperfusion in rats

Animals

Specific pathogen free (SPF) grade male SD rats were purchased from Changsha (SYXK (Xiang) 2023-0002; Tianqin Biotechnology Co., Ltd., Tianjin, China), including 18 rats aged 8–10 weeks and 40 rats aged 4–6 weeks, and housed them at the Translational Medicine Center of Guizhou Medical University. The rats were housed individually and fed a standard rodent diet, maintained under controlled conditions of temperature (22–24 °C) and humidity (45–50%), with a 12-h light/dark cycle. One week of acclimatization feeding was completed prior to experiments. The rats involved in the experiment were confirmed dead after their hearts were removed under anesthesia and the remaining ones were euthanized before the end of experiment program. The rats were euthanized by inhalation of carbon dioxide gas (flow rate: 50–60% of cage volume/min, no prefilled chambers), ensuring that no other animals were present at the execution site. The bodies were properly handled only after the rats were confirmed dead. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. The Animal Care and Use Committee of Guizhou Medical University (NO. 2303271) reviewed and approved each experiment.

lsolated heart ischemia-reperfusion model

Rats aged 8–10 weeks were randomly grouped into the continuous perfusion (CP group) or ischemia-reperfusion (IR group) (six rats in each group). The rats were anesthetized with sodium pentobarbital (60 mg/kg) and anticoagulated with sodium heparin (2000 U/100 kg). When the rats reached deep narcosis, a thoracotomy was conducted, and the hearts were rapidly removed and placed in a 4 °C Krebs-Henseleit (K-H) solution. Then the Langendorff heart perfusion technique was used for retrograde perfusion of the heart via the aorta. The non-cyclic Langendorff device provided a constant pressure of 70 mmHg at (37 ± 0.5) °C from an oxygen-containing glass reservoir. Rat heart returned to normal sinus rhythm within 3 min of balanced perfusion and maintained a heart rate ≥180 beats per minute were included in this study. After 30 min equilibration perfusion with 37 °C K-H solution, cardiac arrest was induced by 4 °C Thomas solution infusion and maintained for 60 min. During arrest, myocardial protection was achieved by continuous immersion in 4 °C K-H solution, with half-volume replenishment of 4 °C Thomas solution at 30 min arrest interval. Post-arrest reperfusion was performed with 37 °C K-H solution for 30 min. The CP group hearts were continuously perfused with K-H solution for 120 min. The K-H solution (NaCl 120 mM, KCl 4.5 mM, CaCl₂ 1.25 mM, MgCl₂·6H₂O 1.2 mM, KH₂PO₄ 1.2 mM, NaHCO₃ 20 mM, C₆H₁₂O₆ 10 mM) was oxygenated with 95% O₂/5% CO₂ and maintained at pH 7.4.

Animal experimental design

A total of 4–6 week old male SD rats were randomly divided into treatment groups and a non-treatment group (Group C). In the treatment groups, the rats received an injection of a negative control adenovirus (AAV9-CON group) or an adenoviral vector containing Mapre1 (AAV9-EB1 group) (1 × 10¹² v.g/rat; Shanghai Gene Chem Co., Ltd., Shanghai, China) or an equal volume of saline (IR group) via the tail vein (eight rats in each group). Four weeks later, the treatment group underwent ex vivo myocardial ischemia-reperfusion, while the non-treatment group was subjected to only 5 min of continuous perfusion (to expel residual blood from the myocardium). Throughout the study, no rats exhibited infections, rapid weight loss, or sudden death. However, if such conditions arose, euthanasia would be promptly administered to minimize animal distress.

Ex vivo electrophysiological recordings

Once the isolated heart is placed in the perfusion device, electrodes are fixed to the surface of the left ventricle (determined by the anatomical landmarks of the left anterior descending branch, aorta, and atrium). The 64-channel MAP system (EMS64-USB-1003, Oxford, UK) is used to collect electrical conduction at time points T₀, T₁, and T₂ (T₀, balanced perfusion for 30 min; T₁, reperfusion for 15 min; T₂, reperfusion for 30 min). The heart’s electrical conduction data is calculated by EMapRecord 5.0 software (MappingLab Ltd., Oxford, UK), including absolute inhomogeneity (P_5–95) and the inhomogeneity index (P_5–95/P₅₀).

Hematoxylin-Eosin staining

Rats cardiac tissues were fixed with 4% paraformaldehyde (BL539A; Biosharp, Hefei City, China) for 36 h, followed by dehydration in a series of alcohol gradients (70%, 80%, 90%, 95%, 100%) for 5 min each. The conventional method of staining involved hematoxylin-eosin (HE) staining. Finally, the sections were observed under an optical microscope (Olympus, Tokyo, Japan).

Immunohistochemical staining

Myocardial tissues were immersed in a 4% paraformaldehyde solution for 36 h, embedded in paraffin, and cut into 5 μm thick sections. After de-paraffinization and rehydration, sections underwent heat-induced epitope retrieval using an antigen retrieval solution (Servicebio, Hubei, China). The sections were then incubated overnight with primary antibody at 4 °C (Cx43, 1:1500, Sigma-Aldrich, St. Louis, MO, USA, C6219). Subsequently, the sections were incubated at 37 °C for 30 min with biotinylated goat anti-rabbit IgG antibody, followed by a 30-min reaction with Strept Avidin–Biotin Complex at 37 °C. Color development was performed with DAB substrate with hematoxylin counterstaining.

Immunofluorescence staining

After the completion of ex vivo perfusion, fresh rat myocardium was collected and frozen for sectioning, followed by immunofluorescence staining. Sections were washed three times on a shaker with PBS for 10 min each time. To enhance cell membrane permeability and porosity, the sections were incubated in 0.5% Triton X at room temperature for 30 min. Subsequently, the sections were washed again with PBS three times for 10 min each time. The sections were then incubated in 5% bovine serum albumin (BSA) at room temperature for 1 h. Next, the sections were incubated overnight at 4 °C with primary antibodies (Cx43, 1:500, C6219; N-cadherin, 1:1,000, Cell Signaling Technology, Danvers, MA, USA, #14215; MAPRE1, 1:200, Abmart, Shanghai, China, TD13549). The following day, the sections were rinsed in PBS three times for 10 minutes each time, followed by the addition of a diluted mixture of fluorescent secondary antibodies(FITC-Goat Anti-Rabbit IgG (H+L),1:400, proteintech, Rosemont, IL, USA, SA00003-2; CY3-Goat Anti-Mouse lgG(H+L), 1:400, biopm, PMK-014-095M/S). The sections were then kept in a dark room at room temperature for 1.5 h. Myocardial sections were washed on a shaker with PBS three times for 5 min each time. DAPI (Servicebio, Hubei, China) was applied to the sections and incubated for 5 min, followed by three washes for 5 min each. Finally, the sections were sealed with an anti-fade mounting medium. Imaging was performed using a confocal microscope (SpinSR10; Olympus, Toyo, Japan).

Extraction of tubulin fractions

To separate the tubulin protein components into free and aggregated forms. We homogenized fresh heart tissue in 1 ml of microtube stabilizing buffer and 0.1% Triton X, where the buffer components included 2 mM ethylenediaminetetraacetic acid, 2 mM ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid, 0.5 mM MgCl₂, 20% glycerol, and 0.1 M piperazine-N,N′-bis (2-ethanesulfonic acid, pH 6.8). The homogenate was centrifuged at 100,000 g for 15 min at 4 °C. The supernatant was collected as the free tubulin protein fraction, and the remaining portion was resuspended in lysis buffer and incubated on ice. After 1 h of incubation, it was centrifuged at 100,000 g for 15 min at 4 °C. The supernatant post-centrifugation was kept as the aggregated tubulin protein component.

RT-qPCR

Total RNA was extracted from 20 mg fresh heart tissues using TRIzol (Invitrogen, Carlsbad, CA, USA). The quality and quantity of the RNA were assessed through agarose gel electrophoresis (1%, 135 V, 25 min) and NanoDrop One^c (701-058108108; Thermo Scientific, Waltham, MA, USA). A total of 1,000 ng of RNA in a volume of 20 µl volume was reverse transcribed into cDNA according to the manufacturer’s instructions (Yeasen, Shanghai, China). The quantitative Real-Time PCR analysis was performed on a CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA) using SYBR Green Master Mix kit (Yeasen, Shanghai, China). The following primers were used: Mapre1 forward: 5′-TGCTTGGGTGAAGAGAGG-3′; Mapre1 reverse: 5′-CAGAGATGTGGGCAGGTC-3′; β-actin forward: 5′-TGTCACCAACTGGGACGATA-3′; β-actin reverse: 5′-GGGGTGTTGAAGGTCTCAAA-3′. The PCR reaction was run at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 20 s, and 72 °C for 20 s. β-actin was used as an internal reference and the expression was calculated by ∆∆Ct method.

Western blotting

Total protein was extracted using RIPA buffer (Solarbio, Beijing, China) and quantified using the BCA Protein Assay kit (Solarbio, Beijing, China). The membrane and cytoplasmic protein extraction kit (KeyGEN BioTECH, Nanjing, China) was utilized to prepare cell membrane and cytoplasmic protein extracts. After denaturation, the proteins were separated with SDS-page (PG112; Shanghai Yaenzyme Biotechnology Co., Ltd, Shanghai, China) and transferred to PVDF membranes(IPVH00010; Merck Millipore, Billerica, MA, USA). Membrane blockade was conducted with the Protein Free Rapid Blocking Buffer (PS108P; Shanghai Yaenzyme Biotechnology Co., Ltd., Shanghai, China) for 10 min at ambient temperature. The primary antibodies were diluted by Antibody diluent (G2025; Servicebio, Hubei, China) and incubated with the membrane at 4 °C overnight. The primary antibodies included Anti-MAPRE1/EB1 antibody (1:1,000; ab308076; Abcam, Cambridge, UK), Anti-Connexin 43 antibody (1:8,000; C6219; Sigma-Aldrich, St. Louis, MO, USA), N-cadherin Mouse mAb (1:1,000; #14215; Cell Signaling Technology, Danvers, MA, USA), GAPDH antibody (1:10,000; 1E6D9; Proteintech, Rosemont, IL, USA), VDAC1/2 antibody (1:1,000; 10866-1-AP; Proteintech, Rosemont, IL, USA), α-Tubulin (DM1A) Mouse mAb (1:1,000; #3873; Cell Signaling Technology, Danvers, MA, USA) and Anti-Sodium Potassium ATPase antibody (1:20,000; ab76020; Abcam, Cambridge, UK). After washing with TBST, the membrane was incubated with HRP-conjugated Affinipure Goat Anti-Mouse lgG (H+L) (1:1,000, China) or HRP-conjugated Affinipure Goat Anti-Rabbit lgG (H+L) (1:1,000, China) at room temperature for 1 h. Subsequently, the membrane was visualized using the electrochemiluminescence (ECL) reagent (Bio-Rad Laboratories, Hercules, CA, USA) and bands were detected using the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). The relative protein levels were represented as the ratio of the grayscale values of the target protein to α-Tubulin, ATPase, voltage-dependent anion channels (VDAC) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Statistical analysis

The GraphPad Prism 9.5.2 was used to analyze the data. Statistical analyses were performed by one-way ANOVA between groups and two-way ANOVA at different time points within groups. One-way ANOVA with Benjamini-Hochberg correction was employed to evaluate the differences among multiple groups for the absolute inhomogeneity and inhomogeneity index at T₀, T₁, and T₂. For other one-way ANOVA, Dunnett’s multiple comparison test was used. Additionally, two-way ANOVA followed by Tukey’s multiple comparison test was utilized to assess the differences among multiple groups. Comparisons between the two groups were performed by an unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 were considered as statistically significant with mean ± SD.

EB1 downregulation and reduced Cx43 in gap junctions in ischemia-reperfusion model

Figure 1A shows the Langendorff ex vivo perfusion experimental procedure. To ensure the successful construction of the hypothermic ischemia-reperfusion model, we used HE staining to detect morphological changes in myocardial tissue. As shown in Fig. 1B, the myocardial tissue exhibited varying degrees of cell swelling and nuclear enlargement after hypothermic ischemia-reperfusion. Previous studies have shown that EB1 reduction in myocardial IDs of heart failure patients is accompanied by a decrease in Cx43 in gap junctions (Smyth et al., 2010), suggesting that EB1 may be a biological target for improving Cx43 distribution and predicting arrhythmia. Similarly, this study observed a downregulation of EB1 in myocardial tissue following hypothermic ischaemia-reperfusion (Fig. 1C), accompanied by a reduction in membrane Cx43 (intercellular gap junction Cx43) (Fig. 1D).

Overexpression of EB1 reduces the incidence and duration of hypothermic reperfusion arrhythmia

To further understand the role of EB1 in hypothermic ischemia-reperfusion arrhythmia, we overexpressed EB1 in rat myocardium (Fig. 2A). We detected the expression of the EB1 gene through RT-qPCR, confirming successful virus transfection (Fig. 2B). We used 64-channel MEA mapping technology and ECG to detect changes in cardiac conduction and arrhythmia after hypothermic ischemia-reperfusion. The study found that hypothermic ischemia-reperfusion led to arrhythmia (Figs. 2C–2F). Figure 2C shows typical reperfusion arrhythmia graphs, primarily ventricular premature beats, with atrioventricular conduction block, ventricular tachycardia, and ventricular fibrillation (Fig. 2D). In our study, the frequency and duration of reperfusion arrhythmia were reduced after EB1 overexpression (Figs. 2E and 2F). These results indicate that overexpression of EB1 can reduce reperfusion arrhythmias.

EB1 improves reperfusion arrhythmia by correcting myocardial conduction after hypothermic ischemia-reperfusion

To further explore the protective mechanism of EB1 in reperfused myocardium, we monitored the electrophysiological changes in hypothermic ischemia-reperfused myocardium in each group. This study used a multi-electrode array mapping technique to further investigate the following abnormalities of myocardial electrical conduction: (1) The increased temporal and spatial dispersion of conduction; (2) inhomogeneity indices.

The study found that hypothermic ischemia-reperfusion caused changes in the location and time of electrical activation. In the IR and AAV9-CON groups, compared with T₀, we observed changes in activation locations and prolonged activation times at T₁ and T₂ after reperfusion from the conduction activation maps, whereas the AAV9-EB1 group remained stable (Fig. 3A). These findings suggest that post-reperfusion electrophysiological remodeling, characterized by altered conduction velocity and/or pathological propagation pathways, may underlie the development of spatiotemporal activation heterogeneity across myocardial regions. Statistical analysis of heart rate parameters revealed no significant differences across the observed time points (P > 0.05) (Fig. 3B). We postulate that there are two possible reasons for the absence of statistically significant differences in heart rate changes. First, arrhythmias do not necessarily occur precisely at the acquisition time points of T₀, T₁, and T₂. Second, as we observed, arrhythmias mainly occur within 15 min after ischemia-reperfusion, and they become less common at the T₁ time point.

Myocardial electrical conduction abnormalities serve as pathophysiological biomarkers for reperfusion arrhythmogenesis, where increased conduction heterogeneity establishes the electrophysiological substrate for re-entrant circuits. Notably, conduction heterogeneity metrics, specifically absolute conduction heterogeneity and heterogeneity index, provide quantitative assessment of regional tissue susceptibility to arrhythmic re-entry (Lammers et al., 1990). Figure 3A is the activation map, while Fig. 4A is the dispersion map at that time point, with higher dispersion indicating greater cardiac electrical conduction inhomogeneity. In the IR and AAV9-CON groups, compared with T₀, we observed increased dispersion at T₁ and T₂ after reperfusion from the dispersion maps, while the AAV9-EB1 group remained stable (Fig. 4A), which was consistent with the changes in conduction activation time in Fig. 3A. Additionally, we recorded and statistically analyzed the absolute inhomogeneity (P_5–95) and inhomogeneity index (P_5–95/P₅₀) of conduction. In the IR and AAV9-CON groups, the inhomogeneity of rat myocardium increased after hypothermic ischemia-reperfusion (Figs. 4H and 4I). However, compared with the IR group, the inhomogeneity index and absolute inhomogeneity of myocardial conduction after reperfusion (T₁ and T₂) were significantly reduced in the AAV9-EB1 group (Figs. 4C, 4D, 4F, 4G). These results suggest that overexpression of EB1 improved cardiac conduction.

Overexpression of EB1 rescues Cx43 after hypothermic ischemia-reperfusion

Our previous research found that after ischemia-reperfusion, both EB1 and gap junction protein Cx43 were reduced. Next, we investigated the reasons why EB1 improved cardiac conduction after ischemia-reperfusion. Therefore, we used different experimental methods to investigate the relationship between EB1 and Cx43 in myocardial ischemia-reperfusion. In the fluorescence images (Fig. 5A), we observed that EB1 was distributed vertically towards N-cadherin and tended to localize at N-cadherin, which was consistent with the distribution of EB1 along microtubules. We hypothesize that EB1 concentrates towards N-cadherin to deliver Cx43 to the IDs, promoting the distribution of Cx43 at the IDs. EB1 was significantly expressed in the AAV9-EB1 group (Figs. 5A and 5B), while N-cadherin was not affected by EB1 overexpression. We also observed that the reduced Cx43 in cardiomyocytes after ischemia-reperfusion was prevented by the overexpression of EB1, rescuing the loss of Cx43 after ischemia-reperfusion (Fig. 5B).

Our previous sequencing analysis results indicated the involvement of microtubules in ischemia-reperfusion (Huang et al., 2024). Microtubules can assist in the transport of Cx43, as well as a multitude of other proteins including Na_V1.5. Therefore, we also assessed the relationship between EB1 and microtubule stability. Western blot analysis showed that, compared to the control group, the polymerization of microtubules increased and depolymerization decreased in the EB1 overexpression group, indicating that EB1 overexpression can enhance the stability of microtubules after ischemia-reperfusion (Figs. 5C and 5D).

EB1 promotes Cx43 localization to IDs and reduces Cx43 lateralization

To further elucidate whether EB1 overexpression improves reperfusion arrhythmia by rescuing the loss of gap junction Cx43 at IDs, we used IHC and IF to detect the distribution of Cx43 to evaluate the role of EB1. In both IHC and IF, we observed that, compared to the IR and AAV9-CON groups, Cx43 is more concentrated at the GJs in the C group and AAV9-EB1 group, with intact morphology and orderly arrangement (Figs. 6A and 6B). In the immunohistochemical images, we noted the lateralization of Cx43 (Fig. 6A, indicated by arrows). Given that N-cadherin is present at GJs and exhibits high stability (Kostetskii et al., 2005), we used N-cadherin as a control in the IF detection experiments. In the fluorescence images, we also observed prominent lateralization of Cx43 in the IR and AAV9-CON groups. In the right-sided insets, the upper image shows the co-localization of Cx43 with N-cadherin, representing the GJs, while the lower image, indicated by arrows, shows the lateralization of Cx43 (Fig. 6B), consistent with the immunohistochemical (IHC) findings. In addition, we measured myocardial membrane Cx43 levels by immunoblotting. Compared with the IR group, membrane Cx43(gap junction Cx43 in cardiomyocytes) was significantly increased in rat cardiomyocytes overexpressing EB1 (Fig. 6C). These results indicate that EB1 promotes the localization of Cx43 at gap junctions following hypothermic ischemia-reperfusion and reduces the lateralization of Cx43.