Intron polymorphism in Camk2d is associated with ventricular arrhythmias in normal adult Sprague-Dawley rats

Abstract

Calcium/calmodulin-dependent protein kinase II (CAMKII) is a critical regulator of cardiac electrophysiology. However, the role of the four bases deletion polymorphism in Camk2d which codes delta subunit of CAMKII, particularly those involving intron sequences, remains poorly understood. This study aimed to investigate the impact of Camk2d c.1044+125_128delGTTT missing polymorphism on cardiac morphology and arrhythmogenesis in normal adult Sprague-Dawley (SD) rats. A total of 85 SD rats were genotyped by Sanger sequencing, revealing a distribution of 25.9% wild-type (WT), 48.2% heterozygous, and 25.9% homozygous variants. Echocardiography, Hematoxylin-Eosin staining, Masson’s trichrome staining and transmission electron microscopy indicated no significant differences in cardiac structure or baseline function among the three groups. In freely moving rats, premature atrial arrhythmias were detected in 2 of 9 WT rats, 1 of 9 heterozygous rats, and 1 of 9 homozygous rats. Premature ventricular contractions (PVCs) were observed in none of 9 WT or homozygous rats, 3 of 9 heterozygous rats, with one heterozygous rat exhibiting frequent PVCs. Electrical programmed stimulation revealed a higher incidence of inducible atrial fibrillation in homozygous rats compared to WT rats and a higher incidence of inducible ventricular tachycardia in heterozygous rats compared to WT rats. These findings suggest that deletion polymorphism in the intron sequences of Camk2d are unexpectedly common in normal SD rat populations and that such polymorphism predispose to ventricular arrhythmias without overt structural heart disease. Our study highlights the potential arrhythmogenic risk associated with non-coding DNA sequence alterations in Camk2d and underscores the importance of genetic screening in experimental animal models.

Introduction

Ventricular arrhythmias, including premature ventricular contractions (PVCs) and ventricular tachycardia (VT), are major contributors of morbidity and sudden cardiac death. They have traditionally been associated with structural heart diseases such as ischemic or hypertrophic cardiomyopathy [1]. However, growing evidence suggests that ventricular arrhythmias can also occur in healthy adults with structurally normal hearts [2]. These idiopathic arrhythmias remain difficult to predict and manage in clinical practice.

Therapeutic strategies such as antiarrhythmic drug therapy and catheter ablation are well-established and effective for many patients with structural heart disease [3]. These approaches have also shown benefit in patients without structural abnormalities but with a high symptom burden. Conversely, in patients with non-structural arrhythmias and minimal symptoms, a conservative approach is generally preferred. This typically involves observation, lifestyle modification, and avoidance of unnecessary pharmacologic intervention, unless symptoms progress or arrhythmia frequency increases. A deeper understanding of the molecular mechanisms underlying ventricular arrhythmias in structurally normal hearts may pave the way for novel preventive and therapeutic strategies.

In recent years, non-coding genomic regions, including intron sequences, have attracted attention as critical regulators of gene expression and cellular function. Introns are no longer regarded as mere passive genetic elements; rather, they possess independent regulatory roles, particularly under stress conditions [4]. Serval studies have shown that intron DNAs can actively modulate cellular responses to nutrient deprivation, enhancing organismal survival under stress [5, 6]. This emerging evidence suggests that introns may serve as dynamic mediators of gene-environment interactions. Furthermore, deletion mutations within intron DNA sequences may alter RNA splicing, stability, and the regulation of protein translation, leading to pathological changes without modifying the coding sequence itself [7, 8]. While the role of coding mutations in inherited arrhythmia syndromes is well recognized, the effects of deletion mutations or polymorphisms in intron regions on cardiac electrophysiology remain poorly characterized. Some studies have suggested that aberrations in intron-mediated RNA regulation can contribute to electrical instability, thereby promoting the development of ventricular arrhythmias even in the absence of overt structural abnormalities [9]. Therefore, understanding the impact of deletion polymorphisms in non-coding sequences represents a promising new frontier in arrhythmia research.

Calcium/calmodulin-dependent protein kinase II (CAMKII) is a well-established regulator of cardiac calcium handling, membrane excitability, and arrhythmogenesis [10]. Dysregulation of Camk2d, which codes the delta subunit of CAMKII, has been implicated in both atrial fibrillation and ventricular arrhythmias [11]. Although amounting evidence has focused on mutations of Camk2d coding sequences, little is known about the role of deletion polymorphism in its intron sequences. Given the functional importance of intronic regions under stress and their ability to modulate RNA splicing, stability, and the regulation of protein translation [5, 6], it is important that intronic mutations/polymorphisms in Camk2d could have arrhythmogenic consequences independent of structural remodeling.

In the previous study, we found a c.1044+125_128delGTTT in Sprague-Dawley (SD) rats by accident. Therefore, we investigated the occurrence of Camk2d intronic deletion polymorphisms and the impact of Camk2d intronic deletion polymorphisms on cardiac electrophysiology in SD rats. This well-established animal model offers a stable genetic background and is widely used in cardiovascular research, making it ideal for studying subtle genetic influences on cardiac electrophysiology [12, 13]. Our findings revealed a surprisingly high prevalence of 4 bases deletion polymorphism in Camk2d introns among normal SD rats, with associated increases in susceptibility to ventricular arrhythmias despite normal cardiac structure.

Studying deletion polymorphism in intron DNA sequences of key regulatory genes such as Camk2d holds great potential for elucidating the mechanisms underlying ventricular arrhythmias in healthy adults. By focusing on non-coding regions, this research expands current paradigms that primarily emphasize structural defects or coding variants. Moreover, identifying the arrhythmogenic potential of such intronic alterations may facilitate the development of RNA-targeted diagnostic tools and therapies. In this context, investigating intronic deletions polymorphism provides a valuable opportunity to bridge the gap between genetic variation and arrhythmic risk in populations traditionally considered low-risk.

Materials and Methods

The SD rat (Rattus norvegicus) model was purchased from Dossy Experimental Animals Co., Ltd. (Chengdu, China). The genotype of each animal was confirmed via Sanger sequencing. Heterozygotic, homozygotic and WT littermates weighing 400–500 g were used for the experiments. The rats were housed at a constant temperature (24 ± 1°C) under a 12/12 h light-dark cycle, with free access to food and water. All procedures were approved by the Ethics Committee for Animal Care and Use in Chengdu Medical College (2024-62), China.

Sanger sequencing

The DNA was extracted as the following method. Briefly, 0.1 g of fingertip tissue was cut under isoflurane anesthesia and washed twice with pure water, then incubated overnight at 56°C in the following mixed liquor: 100 ml 5% chelex-100, 20 µl 1% protease K, 5 ml 15.43% dithiothreitol. After that, the liquid was bathed at 100°C for 8 min. After shaked and mixed well, the liquid was centrifuged at 13,000 RPM for 5 min. The liquid is DNA. Then, we measured the DNA concentration and Polymerase Chain Reaction (PCR) was performed. The PCR primer sequence is as follows:

Forward: CCTTCAGTGACTAAACATGCTC.

Reverse: GAGAAACCATTGCACGATCAGT.

The PCR products were sent to Sangon Biotech Ltd., for sequencing with the same primer. If the target sequence is “GTTTTGTTTGTTTGTTTGTTTGTTTGTTTT” (c.1044+104_133), it was regarded as wildtype. If the sequence is “GTTTTGTTTGTTTGTTTGTTTGTTTT”, which missed four bases “GTTT”, the genotype is homozygote. If two sequences were presented, the heterozygote was determined.

Western blotting

Protein samples were extracted from the apex of right ventricle in cold RIPA lysis buffer (BL-504A, Biosharp Biotechnology, Hefei, China) containing a complete protease inhibitor cocktail (HY-K0010, MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (P1082, Beyotime Biotechnology, Shanghai, China). Protein samples were separated by SDS–polyacrylamide gel electrophoresis and transferred to a 0.45 µm PVDF membrane (IPVH00010, Millipore, Billerica, MA, USA), which was detected using specific primary antibodies (anti-p-CAMKII, 1:1,000, ab182647, Abcam, Cambridge, MA, USA; CAMKII, 1:1,000, 4436S, Cell Signaling Technology, Danvers, MA, USA). Briefly, the membrane was first probed with an antibody against phosphorylated p-CaMKII, followed by incubation with a rabbit peroxidase-conjugated secondary antibody (ab6721, Abcam) and visualization by enhanced chemiluminescence in a chemiluminescence imaging system (ChemiDoc XRS+, BIO-RAD, Hercules, CA, USA). After imaging, the membrane was stripped using a stripping buffer composed of 15 g glycine, 1 g SDS, and 10 ml Tween-20 dissolved in 800 ml distilled water, adjusted to pH 2.2 with HCl, and brought to a final volume of 1 l. Following stripping and thorough washing, the membrane was re-blocked and re-probed with an antibody against total CaMKII. Signal detection was performed using the same procedure. The band intensity was quantified by using Image J (National Institutes of Health, Bethesda, MD, USA).

Echocardiography

Rats were anesthetized with 5% isoflurane (induction) and maintained with 1.5% isoflurane for transthoracic echocardiography (Vevo 3100 imaging digital ultrasound system, FUJIFILM VisualSonics, Inc., Toronto, Canada). The heart was visualized from the parasternal long-axis view. The left ventricle ejection fraction (EF) and fractional shortening (FS) were calculated from the measurements of wall thickness and chamber diameters. Left ventricle posterior wall diastolic thickness (LVPWd) and Left ventricle anterior wall diastolic thickness (LVAWd) were measured using M-mode.

Hematoxylin-eosin (HE) staining

Cardiac tissues were harvested in four-chamber view and immersion-fixed in 10% neutral buffered formalin for 8 h. Then the tissues were embedded in paraffin and cut at 4 µm thickness. After dewaxing in xylene and graded ethanol rehydration, slides were rinsed in distilled water. Excess moisture was removed prior to hematoxylin immersion (5 min). Acid-alcohol was used for differentiation, followed by ammonia solution treatment. Sections were water-rinsed, ethanol-rehydrated, and counterstained with eosin (2 min). Graded ethanol dehydration preceded xylene clearing. Coverslips were mounted with Permount TM Mounting Medium. Images were captured using a brightfield microscope (HS6 series, Yuyao, China) with integrated analysis software.

Masson’s trichrome protocol

Tissue specimens are fixed in 10% neutral buffered formalin. Then paraffin embedding is performed, and sections are cut at 4 µm thickness. Following xylene dewaxing and ethanol gradient rehydration, slides were rinsed. Iron hematoxylin staining (4 min) and acid-alcohol differentiation preceded rinsing. Sections were treated with Ponceau acid fuchsin (4 min), followed by 1% acetic acid for 1 min, phosphomolybdic acid (1 min), and aniline blue (1 min), with intermittent water rinses. Glacial acetic acid differentiation (1%) and ethanol dehydration were conducted. After xylene clearing, Permount-mounted slides were imaged under identical microscopy. Fibrosis quantification used Image-Pro Plus 6.0.

Transmission electron microscopy

Rats were anesthetized and sacrificed with 200 mg/Kg sodium pentobarbital. The free wall tissues of the myocardium from the papillary muscle section were taken, with a volume no larger than 1mm*1mm*1mm. they were rapidly immersed in 2% paraformaldehyde and 2.5% glutaraldehyde solution and fixed for 4 h, then in 1% osmium acid in 0.1M phosphate buffer for 2 h, followed by gradient alcohol dehydration and osmosis in acetone. After that, the samples were embedded with Epon 812 epoxy resin and sliced into ultra-thin 60 nm sections, and stained with 2% uranium acetate in saturated alcohol solution for 15 min and followed by stained in lead citrate for 15 min. Then the samples were dried at room temperature overnight. The ultrastructures were photographed by transmission electron microscopy (Hitachi HT7800, Hitachi, Tokyo, Japan).

Electrocardiogram measurements with biotelemetry

Surgery was performed aseptically on wildtype, heterozygotic and homozygotic rats anesthetized with pentobarbital sodium (40 mg/kg). A catheter attached to a transmitter (Data Sciences International, St. Paul, MN, USA) was inserted into the abdominal wall for ECG monitoring. The electrode line runs under the skin (outside the abdominal wall muscles), and the negative electrode is sutured to the muscle tissue 0.5 cm below the clavicle in the right midclavicular line. The positive electrode is placed in the muscle tissue about 1 cm above the costal arch in the left anterior axillary line, and the body of the transmitter was implanted in the abdominal cavity. The rats were housed individually and inspected daily for motor activity, signs of infection, and food and water intake. ECGs of freely moving rats after two weeks of recovery were measured with a data acquisition system (Data Sciences International, St. Paul, MN, USA). 15 min of ECGs were was analyzed at about 16 o’clock with noise using LabChart 8.2.3 (AD Instruments, Bella Vista, NSW, Australia). We selected 16:00 as the ECG analysis time point for the following reasons. Sixteen o’clock in rats corresponds to the pre-awakening phase in humans, during which a higher incidence of cardiac events has been reported [14, 15]. Specifically, previous clinical studies demonstrated an increased onset of sudden cardiac death and sustained ventricular tachycardia during the early hours before awakening [14, 15]. In our preliminary analysis, we compared ECG data obtained at both 4:00 (data not shown) and 16:00, and found that arrhythmic events were more frequently detected at 16:00. In addition, beginning observations at 16:00, prior to their active period, helped maintain consistent baseline conditions across all animals.

Electrical programmed stimulation

Briefly, the rats were anesthetized with ethyl carbamate at 1g/kg (KELUN, Industry Group, Chengdu, China). A tracheal cannula was inserted. Isoflurane of 1.5% vaporized with room air was inhaled with a volume-limited ventilator (ALC-V8; Shanghai Alcott Biotech Co., Ltd., Shanghai, China.). Tidal volume and respiratory rate were set at 8 ml/kg and 80 breaths/min, respectively. Lead II ECG was recorded with subdermal electrodes placed in the limbs. The right femoral vein was cannulated for saline infusion and the right femoral artery was punctured to monitor the blood pressure. The right jugular vein was cannulated to position an electrode catheter (3F, inter-electrode distance of 4 mm, SMC-304; Physio-Tech, Tokyo, Japan) into the right atrium. A stimulator (SEN-8203; Jinjiang Ltd., Chengdu, China) with rectangular pulses (with 2 times the diastolic threshold voltage and 3-ms width) was used for programmed stimulation. The atrial/ventricular effective refractory period (ERP) was measured with a train of eight beats (S1) of basic CLs at 150 (ERP [CL_{150 ms}]), 120 (ERP [CL_{120 ms}]) and 100 (ERP [CL_{100 ms}]) followed by an extra stimulus (S2). The ERP was defined as the shortest S1S2 interval that captured the atrium/ventricles. An episode of more than 3 continuous premature atrial/ventricular contractions was regarded as an incidence of atrial fibrillation/ventricular tachycardia (VT) [16]. Before programmed electrical stimulation (PES), twelve recordings of consecutive complexes were used to calculate the mean for the electrophysiological variables. In addition, three recording of consecutive wave of blood pressure was evaluated for the mean of blood pressure.

Statistical analysis

All data are presented as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Dunnett’s multiple test using GraphPad Prism 6.0 (version 6.07, GraphPad Software, Inc., San Diego, CA, USA). The chi-square (χ²) independence test was used to test whether or not there is a statistical difference for VT and AF. A value of P<0.05 was considered as statistically significant.

Results

A high incidence of c.1044+125_128delGTTT polymorphism in Camk2d intronic DNA sequences was detected in SD rat

Sanger sequencing showed that 22 out of 85 rats (25.9%) had no c.1044+125_128delGTTT mutations (WT, Fig. 1A), 41 rats (48.2%) were heterozygous for the deletion polymorphism (Heter, Fig. 1B), and 22 rats (25.9%) were homozygous (Homo, Fig. 1C, Table 1).

Fig. 1.

Gene sequence of wild type rat (WT) (A), heterozygous rat (Heter) (B), and homozygous rat (Homo) (C).

Table 1. Proportions of wild-type, heterozygous, and homozygous rats with different phenotypes in 85 SD rats.

	WT	Heter	Homo
n	22	41	22
%	25.90%	48.20%	25.90%

wild type rat: WT; heterozygous rat: Heter; homozygous rat: Homo.

c.1044+125_128delGTTT polymorphism did not alter the protein level of CAMKII

Western blot (WB) showed that p- CAMKII and CAMKII were similar in WT, heterozygous, and homozygous rats. These results suggested that c.1044+125_128delGTTT polymorphism did not affect the protein level of CAMKII (Fig. 2).

Fig. 2.

Western blot analysis and quantification of phosphonate calcium/calmodulin-dependent protein kinase II delta (p-CAMKII) and CAMKII protein levels. Each value represents mean ± SD (n=3 per group, one-way analysis of variance + Dunnett’s multiple test). (wild type rat: WT; heterozygous rat: Heter; homozygous rat: Homo)

Cardiac structure and function were not affected by c.1044+125_128delGTTT polymorphism

Representative echocardiogram traces from WT, heterozygous, and homozygous rats are shown in Fig. 3A-C and supplementary file 1. Heart rate (Fig. 3D), EF (Fig. 3E) and FS (Fig. 3F) did not differ significantly among the three group. Additionally, no significant difference was observed in the LVPWd and LVAWd (Fig. 3G–H). Histological analysis using HE (Fig. 3I–K) and Masson’s trichrome staining (Fig. 3L–O) demonstrated no significant difference in structure and fibrosis among the three group. Further analysis of transmission electron microscopy (Fig.3 P-R) showed no abnormal ultrastructure in these three groups.

Fig. 3.

The cardiac structure and function was similar among WT, heterozygous, and homozygous rats. A–C: Representative echocardiography from parasternal long axis images in a WT (A), heterozygous (B), and homozygous rat (C). D–H: A summary of echocardiographic parameters is listed below. Each value represents mean ± SD (n=6 for each group, one-way analysis of variance + Dunnett’s multiple test). Heart rate (Heart rate, D), Ejection fraction (EF, E), fractional shortening (FS, F), Left ventricular posterior wall in diastole (LVPWd, G), left ventricular anterior wall in diastole (LVAWd, H), wild type rat: WT; heterozygous rat: Heter; homozygous rat: Homo; Hematoxylin and eosin: HE. Data are presented as the mean ± SD. I–O: Representative traces from four chamber images by HE and Masson’s trichrome in a WT, heterozygous, and homozygous rat. No significant difference in fibrosis was detected among three groups (n=3 for each group, one-way analysis of variance + Dunnett’s multiple test). P–R: Representative images of ultrastructure from electron microscopy indicated no abnormal ultrastructure in these three groups.

c.1044+125_128delGTTT polymorphism in Camk2d increased susceptibility to PVC in freely moving rats

Representative electrocardiographic traces from WT, heterozygous, and homozygous freely moving rats are shown in Figs. 4A–C. It demonstrated that spontaneous premature atrial contractions (PACs) occurred in 22.2% of WT, 11.1% of heterozygous, and 11.1% of homozygous rats (Figs. 4D–G). Spontaneous PVCs were observed only in heterozygous rats (33%, Fig. 4H). Notably, in one heterozygous rat, 1,080 PVCs were recorded within a 15-min period (Figs. 4B and E).

Fig. 4.

Spontaneous arrythmia in the WT, heterozygous, and homozygous rats with DSI instrument. A–C: Representative electrocardiogram recordings in a WT rat (A), heterozygous (B) and homozygous rat (C). Note that typical traces of frequent premature ventricular complex in a heterozygous rat (red arrows). D–F: Summary of spontaneous arrhythmia in the WT, heterozygous, and homozygous rats. Each column indicates the onset of spontaneous arrhythmia in each rat. The columns are marked according to the type of arrhythmias developed each 30 s. Yellow column: premature atrial complex, red column: premature ventricular complex. G–H: The incidence of premature atrial complex and premature ventricular complex in the freely moving WT, heterozygous, and homozygous rats. (n=9 for each group, one-way analysis of variance + Dunnett’s multiple test). (wild type rat: WT; heterozygous rat: Heter; homozygous rat: Homo; Data Sciences International: DSI)

c.1044+125_128delGTTT polymorphism in Camk2d have no effect on blood pressure and cardiac electrophysiology in anesthesia rats

Representative blood pressure traces from WT, heterozygous, and homozygous in anesthesia rats are shown in Figs. 5A–C. There is no significant difference with heart rate, P duration, PR interval, QRS width, QT interval and mean blood pressure (Figs. 5D–I).

Fig. 5.

Electrophysiology and hemodynamic parameters in the WT, heterozygous, and homozygous rats under anesthesia. A–C: Representative blood pressure recordings in a WT rat (A), heterozygous (B) and homozygous rat (C). D–I: Summary of Electrophysiology and hemodynamic parameters, including Heart rate (D), P wave duration (E), PR interval (F), QRS width (G), QT interval (H), and mean blood pressure (MBP, I) in the WT, heterozygous, and homozygous rats. Each value represents mean ± SD (n=8 for each group, one-way analysis of variance + Dunnett’s multiple test). (wild type rat: WT; heterozygous rat: Heter; homozygous rat: Homo)

c.1044+125_128delGTTT polymorphism in Camk2d increased susceptibility to PES-induced VT

During PES, representative intracardiac and surface ECG traces from wild-type, heterozygous, and homozygous rats are shown in Figs. 6A–C. The incidence of VT increased significantly in heterozygous rats compared to wild-type. At a cycle length of 150 ms, VT was observed in 50% of heterozygous rats, 12.5% of homozygous rats, and 0% of wild-type rats (Fig. 6D). At 120 ms, the incidence of VT rose to 75% in heterozygous rats and 25% in homozygous rats, while still absent in wild-type rats (Fig. 6E). At 100 ms, VT was detected in 37.5% of heterozygous rats but not in wild-type or homozygous rats (Fig. 6F). Statistical differences in VT incidence were found between the wild-type and heterozygous groups at pacing intervals of 150 ms and 120 ms. During PES, representative intracardiac and surface electrocardiographic traces from wild-type, heterozygous and homozygous rats are shown in Figs. 6G–I. At a pacing cycle length of 150 ms, AF was induced in 37.5% of heterozygotes, 50% of homozygotes and 12.5% of wild-type animals (Fig. 6J). At 120 ms, AF occurred in 50% of homozygotes but in 0% of heterozygotes and wild-type rats (Fig. 6K). At 100 ms, AF was not observed in any genotype (Fig. 6L).

Fig. 6.

Electrophysiology by programmed stimulation in anaesthetized WT, heterozygotic and homozygotic rats. A–C: Typical trace of ventricular tachycardia induced by electrophysiological programmed stimulation from WT (A), heterozygous (B), and homozygous rats (C). D–F: The number of rats showing ventricular tachycardia (VT, black bar) at pacing cycle lengths of 150 ms, 120 ms and 100 ms in the WT, heterozygotic and homozygotic rats. Data are presented as the mean ± SD. (n=8 for each group, chi-square (χ²) independence test). * P<0.05 vs. WT. G–I: Typical trace of atrial arrhythmias induced by electrophysiological programmed stimulation from WT (G), heterozygous (H), and homozygous rats (I). J–L: The number of rats showing atrial arrhythmias (AF, black bar) at pacing cycle lengths of 150 ms, 120 ms and 100 ms in the WT, heterozygotic and homozygotic rats. Data are presented as the mean ± SD. (n=8 for each group, chi-square (χ²) independence test). *P<0.05 vs. WT. (wild type rat: WT; heterozygous rat: Heter; homozygous rat: Homo)

Discussion

This study provides novel evidence that deletion polymorphism within the intron sequences of Camk2d significantly increase susceptibility to ventricular arrhythmias without accompanying structural heart disease in normal SD rats. Our findings demonstrate that such non-coding polymorphism traditionally considered less impactful than coding sequence alterations, can profoundly influence cardiac electrophysiological stability, emphasizing the importance of comprehensive genetic screening in arrhythmia research.

CAMKII is a ubiquitous mediator, belonging to the serine/threonine calcium/calmodulin-dependent protein kinase superfamily [17]. CaMKII consists of four subunits called CaMKIIα, β, γ, and δ that are encoded by four different genes [18, 19]. The CaMKIIδ was encoded by gene of Camk2d, located in chromosome 2 and included 20 exons. The coding DNA sequence (CDS) is 1602 bases and code 533 amino acids. The c.1044+125_128delGTTT polymorphism was located in the intron which is between the 14th and 15th exon. However, this intron sequence is not found in human. Consistent with previous research underscoring CAMK II’s critical role in cardiac excitation-contraction coupling and arrhythmogenesis [20], our data reveal that Camk2d intronic deletion polymorphism may disrupt normal regulatory processes even in the absence of overt myocardial remodeling. Interestingly, echocardiographic assessments confirmed that cardiac structure and baseline function remained similar across all genotypes, reinforcing the concept that arrhythmias can arise from molecular dysfunction alone, independent of structural abnormalities [21]. The absence of significant differences in structural parameters between WT, heterozygous, and homozygous rats strengthens the argument that the observed arrhythmogenic phenotype is primarily electrophysiological in nature.

The elevated incidence of inducible VT in heterozygous rats during PES suggests a heightened arrhythmogenic substrate linked specifically to intronic deletion polymorphism. This finding aligns with prior studies indicating that subtle alterations in ion channel regulation, calcium handling, or intracellular signaling pathways all known to be modulated by CAMKII can predispose to arrhythmias even in the absence of overt cardiomyopathy [22, 23]. Moreover, the PVCs observed in freely moving heterozygous animals suggest an increased burden of ectopic activity, which may act as triggers for more sustained arrhythmic events.

An unexpected finding of this study was the relatively high prevalence of Camk2d intron deletion polymorphism in the normal SD rat population. This observation raises important questions regarding the baseline genetic variability within commonly used laboratory animal models and highlights the need for careful genetic characterization when interpreting experimental cardiac electrophysiology results. Given that genetic background can significantly influence susceptibility to arrhythmias [24, 25], our results suggest the potential confounding effects of unrecognized genetic polymorphism in experimental settings.

Both heterozygous and homozygous animals exhibited a higher overall arrhythmia incidence compared with wild-type controls, suggesting that intronic polymorphisms in Camk2d predispose to cardiac electrical instability. Interestingly, heterozygous carriers of the Camk2d deletion polymorphism exhibited a higher incidence of ventricular arrhythmias, whereas homozygous animals displayed a higher susceptibility to atrial arrhythmias. Intronic variants can perturb splicing and broader transcriptional regulation, and have been increasingly implicated in the molecular etiology of inherited cardiac disease [6, 26, 27]. These observations may reflect a dosage-dependent effect of Camk2d signaling on calcium handling and electrophysiological stability: partial loss of function in heterozygotes may be sufficient to destabilize ventricular repolarization and promote triggered activity or re-entrant circuits, whereas complete loss in homozygotes may elicit developmental or molecular compensatory pathways that attenuate ventricular excitability but incompletely protect the atria, which possess distinct action potential dynamics and ion-channel expression.

The c.1044+125_128delGTTT polymorphism identified in this study is located within an intronic region between exons 14 and 15 of the rat Camk2d gene. Although this specific sequence is not conserved in the human orthologue, its position within a regulatory non-coding region adjacent to essential exons underscores its potential functional importance. Intronic variants are increasingly recognized for their ability to modulate splicing, transcriptional regulation, and chromatin architecture [28, 29]. Our data further suggest that both heterozygous and homozygous carriers of this polymorphism may display an intrinsic susceptibility to arrhythmogenesis. Consequently, genetic background at this locus could represent a previously underappreciated confounder in experimental studies of cardiac electrophysiology, potentially influencing outcomes and interpretation. For this reason, future investigations should preferentially employ wild-type animals at the Camk2d locus to avoid genotype-related bias.

It should be noted that the present findings are based on a 15-min ECG detection period determined by system constraints. Therefore, the results reflect arrhythmic events within this limited time window, and longer continuous analysis may further improve the accuracy of arrhythmia assessment in future studies. Future research should focus on elucidating the specific molecular pathways by which these intronic alterations disrupt cardiac electrophysiology. One limitation is the modest sample size in each genotype group, particularly for detecting subtle differences in arrhythmogenic susceptibility between homozygous and heterozygous animals. Although the genotype distribution was consistent with Mendelian expectations and allowed for preliminary analysis, the statistical power may be insufficient to fully resolve genotype-dependent effects. Ongoing efforts to expand the cohort will help address this limitation in future studies. The other limitation is insufficient to fully dissect these chamber-specific mechanisms. Future studies will incorporate chamber-targeted electrophysiological recordings, calcium transient imaging, and transcriptomic profiling to characterize potential compensatory pathways and clarify their differential effects on atrial versus ventricular substrates.

In conclusion, our study identified deletion polymorphism within Camk2d intron sequences as novel arrhythmogenic substrates in structurally normal SD rat hearts. Intron RNA sequences protect rats from ventricular arrhytnmia during mechanical stimulation. These findings expanded the current understanding of the genetic basis of ventricular arrhythmias, emphasize the critical role of non-coding genomic regions, and call for greater attention to genetic screening in experimental electrophysiology. Moreover, the rat model described here provides a valuable experimental platform to dissect the electrophysiological and molecular consequences of intronic mutations, thereby offering translational insights into the functional interpretation of non-coding variants frequently uncovered by human genome-wide association studies. Further investigation into the precise molecular mechanisms involved may pave the way for innovative therapies aimed at preventing sudden cardiac death in at risk but previously unrecognized populations.

Author Contributions

Rong Luo and Xin Cao: conceptualization, data curation, formal analysis, investigation, methodology, visualization, and writing-original draft preparation. Chunyun Zhao: data curation, formal analysis, methodology, and resources. Yi Wang and Yilin He: data curation and methodology. Chang Liu: methodology. Xiaoping Li: conceptualization, visualization and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82274414, 32171182), National Natural Science Foundation of Sichuan (2024NSFSC2118), Chengdu University of TCM (QJRC2022037) for cultivation of young sci-tech talents.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by grants from the National Natural Science Foundation of China (82274414, 32171182), National Natural Science Foundation of Sichuan (2024NSFSC2118), Chengdu University of TCM (QJRC2022037) for cultivation of young sci-tech talents.

Data Availability Statement

The data presented in this study are available from the corresponding author upon reasonable request.