Animals and animal model

All animal handling procedures in this study were in accordance with the Declaration of the National Institutes of Health guide for the care and use of Laboratory animals in Ophthalmic and Vision Research and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University under approval number20220301A.

Cardiac-conditional USP38 knockout (USP38-CKO) and USP38 cardiac-specific transgene (USP38-TG) mice were purchased from Cyagen and constructed in a C57BL6/J background by Cre-loxp system technology. The PCR primer for the Cre promoter was: F: 5’-ATGACAGACAGATCCCTCCTATCTCC-3’, R: 5’-CTCATCACTCGTTGCATCATCGAC-3’. The gene mice construction process and schematic diagrams are shown in Supplemental Data S1 and S2. The mice were conditioned in-house and provided with tap water and a commercial diet. Mice were housed at a temperature of 22 ± 2 ◦C and a 12:12-h light/dark cycle with free access to food and water.

MI models were established in adult male mice at 6–8 weeks by ligating the left descending coronary artery (LAD) as previously described (Snider et al. 2021). In brief, mice were anaesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital. After successful anesthesia, artificial respiration was performed to maintain normal pH, PO2, and PCO2, followed by incision of the pericardium at the third or fourth intercostal space and permanently ligation of the LAD with 7 − 0 silk sutures approximately 2 mm from its origin. The muscle and skin were sutured after the ligation was completed. Electrocardiography (ECG) was recorded synchronously, and changes in the ST-segment were detected. The success of MI construction was judged according to the changes in the ST segment of the ECG and the color of the apex. The sham procedure consisted of a similar operation, except that no ligation was performed. Mice were processed on days 3 and 7 after MI, respectively.

Echocardiography analysis

Cardiac function was assessed by echocardiography at 7 days after MI. Echocardiography was carried out using the VINNO 6VET ultrasound system (Vinno Technology, Suzhou, Jiangsu, China) which equipped with a 23-MHz linear array transducer (Vinno Technology, Suzhou, Jiangsu, China).

Cytokine measurement

The blood samples from mice were collected and centrifuged at 3400 rpm (4 °C) for 30 min to obtain serum. Protein concentrations of IL-1β (GEM0002, Servicebio, Wuhan, China), IL-6 (GEM0001, Servicebio, Wuhan, China), and IL-10 (GEM0003, Servicebio, Wuhan, China) in serum were determined using standard ELISA kits according to the instructions.

Histological analysis

The left atrial (LA) tissue section from day 7 post-MI was embedded in paraffin and cut into 5-µm-thick sections. The degree of LA fibrosis was evaluated using Masson Trichrome staining. Sections were viewed under a microscope and the data were measured with Image Pro-Plus software. Immunofluorescence and histochemical staining analysis was performed to identify macrophages (CD68, iNOS, and CD163), which were immunolabeled with CD68, iNOS, and CD163 antibodies. All images were analyzed by Image-Pro Plus software.

Electrocardiograph analysis

The ECG telemetry transmitter (China YiSense Biomedical Technology Co., Ltd.) was fixed to the back of the mice, and the leads were placed subcutaneously on the upper right and lower left chest and the ECG was recorded continuously for 24 h. The data were analyzed with LabChart 7 software.

Electrophysiological studies

Langendorff-perfused hearts were prepared according to our previously published methods (Shuai et al. 2019). In brief, electrophysiological studies in isolated perfused hearts were conducted using the Langendorff apparatus with HEPES-buffered Tyrode’s solution (130 mM NaCl; 5.4 mM KCl; 1.8 mM CaCl2; 1mM MgCl2; 0.3 mM Na2HPO4; 10 mM HEPES; 10 mM glucose; pH adjusted to 7.4 with NaOH), bubbled with 95% O2–5% CO2 at 37 °C and at a constant pressure of 60 mmHg to evaluate the induction of AF, interatrial conduction time (IACT), and atrial effective refractory periods (AERP). Langendorff-perfused hearts were stimulated with a pair of electrodes placed on the right atrial (RA). All isolated hearts were stabilized for 20 min by perfusion at a constant flow before programmed electric stimulation. The hearts that did not recover to a regular spontaneous rhythm or had irreversible myocardial ischemia were discarded.

Teflon-coated (except at the tips) silver bipolar electrodes were placed on the appendages of the RA, LA, and left ventricle. The interelectrode distance between the RA and LA was set at 5 mm to measure the IACT. The ERPs of the left and right atria were measured by the S2 extra-stimulus method using eight regularly paced beats with cycle lengths of 100, 80, 60, and 40 ms. IACT was measured during RA pacing. AF inducibility was tested by burst pacing methods. The induction of AF was tested by applying three chains of a 2-second burst pacing using the electronic stimulator. Specifically, the first 2-second burst had a cycle length (CL) of 40 ms (pulse duration = 5 ms). Following 3 min of stabilization, the second 2-second burst was applied with a CL of 20 ms (pulse duration = 5 ms). After 3 min of stabilization, the last 2-second burst with a CL of 20 ms was applied with a 10 ms pulse duration. AF was defined as a rapid and irregular atrial rhythm with irregular RR intervals lasting at least 1 s. The duration of AF was measured from the end of burst pacing to the first P wave detected after the rapid irregular atrial rhythm.

Western blot analysis and quantitative real-time PCR

The total proteins were extracted from the frozen LA tissues. Protein concentrations were determined and normalized using the Bicinchoninic Acid (BCA) Protein Assay Kit (G2026, Servicebio, Wuhan, China). Following that, proteins were separated by sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), then transferred onto a polyvinylidene difluoride (PVDF) membrane and incubated with primary antibodies (Supplemental Table S1) overnight at 4°C. Finally, secondary antibodies were incubated with the membranes for 60 min at room temperature. Intensive chemiluminescence (BL523B, Biosharp, Anhui, China) was used to visualize the signals. The protein expression levels of target genes were normalized to the internal reference gene GAPDH.

Total RNA was purified from the atrial samples using RNAiso Plus reagent (9109, Takara, Japan). RNA was transcribed into complementary DNA with the PrimeScript RT reagent Kit (#RR047A, TaKaRa). Then, qRT-PCR was conducted in a 20 µl reaction system containing cDNA, forward primers, reverse primers, and SYBR Premix Ex Taq (#RR420A, TaKaRa). The sequences of the primers used for qRT-PCR are described in the Supplemental Table S2.

Statistical analysis

All the data were expressed as mean ± standard error (SEM) or percentages, and the data were analyzed by GraphPad Prism software (GraphPad Software, San Diego). All data were analyzed by the Shapiro-Wilk normality test for normal distribution. For comparisons between the two groups, a two-tailed Student t-test was applied for normal data, and the Mann-Whitney test was applied for nonnormal data. For comparisons between multiple groups, one-way ANOVA with Bonferroni post hoc analysis was used for normal data, and the Kruskal-Wallis test was used for nonnormal data. Categorical data were analyzed using the Fisher exact test. Values of P < 0.05 were considered to be statistically significant.