Signal Recognition Granule Receptor Beta Subunit Promotes Arrhythmogenic Remodeling in the Heart Failure Mice

Abstract

Background

Heart failure is a major cause of global morbidity and mortality, often complicated by ventricular arrhythmias (VAs) that worsen prognosis. The Srprb (signal recognition particle receptor beta subunit) is an endoplasmic reticulum membrane‐anchored protein involved in protein processing.

Methods

Male C57BL/6 mice received tail vein injections of adeno‐associated virus to cardiac‐specifically overexpress or knock down Srprb. A pressure overload‐induced heart failure model was established via aortic banding 3 weeks later. Cardiac function, structural/electrical remodeling, and VA susceptibility were evaluated 4 weeks post aortic banding using echocardiography, ECG, in vivo electrophysiology, and molecular/pathological analyses. In vitro, primary neonatal mouse cardiomyocytes and fibroblasts were transfected with Srprb‐modulating adenoviruses to investigate its role in hypertrophy and fibrosis. Pathway inhibitors were used to confirm mechanisms.

Results

Srprb knockdown improved pressure overload‐induced cardiac structural and electrical remodeling, reducing VAs. Conversely, Srprb overexpression exacerbated these abnormalities and increased VA incidence. In vitro, Srprb knockdown alleviated angiotensin II‐induced cardiomyocyte hypertrophy and TGF‐β (transforming growth factor‐β)‐induced fibroblast fibrosis, whereas its overexpression aggravated them. Mechanistically, Srprb promoted VA vulnerability by activating endoplasmic reticulum stress and the TLR4/CaMKII/NF‐κB (Toll‐like receptor 4/Ca2+/calmodulin‐dependent protein kinase II/nuclear factor kappa B) signaling pathway.

Conclusions

Srprb knockdown improved ventricular remodeling and suppressed VAs in mice with heart failure, whereas its overexpression has opposite effects. These actions were mediated through regulation of endoplasmic reticulum stress and the TLR4/CaMKII/NF‐κB pathway.

Nonstandard Abbreviations and Acronyms

AAV

adeno‐associated virus

AB

aortic banding

ALT

the threshold of alternans MAPs

Ang II

angiotensin II

APD

action potential duration

α‐SMA

α‐smooth muscle actin

β‐MHC

β‐myosin heavy chain

CaMKII

Ca2+/calmodulin‐dependent protein kinase II

Chop

C/EBP homologous protein

Cx43

connexin 43

ERS

endoplasmic reticulum stress

GRP78

glucose regulated protein 78kD

IκB

inhibitor of nuclear factor kappa B

PERK

protein kinase RNA‐like endoplasmic reticulum kinase

PLB

phospholamban

SCD

sudden cardiac death

SERCA 2a

sarcoendoplasmic reticulum calcium ATPase 2a

SRP

signal recognition particle

SRPR

Signal recognition particle receptor

Srprb

Signal recognition granule receptor beta subunit

TGF‐β

transforming growth factor‐β

TLR4

Toll‐like receptor 4

VAs

Ventricular arrhythmias

Research Perspective.

What Is New?

• This study pioneeringly demonstrates that Srprb (signal recognition particle receptor beta subunit) governs postheart failure ventricular arrhythmias by modulating endoplasmic reticulum stress and the TLR4/CaMKII/NF‐κB (Toll‐like receptor 4/Ca2+/calmodulin‐dependent protein kinase II/nuclear factor kappa B) pathway, providing novel insights into the upstream regulatory mechanisms and clinical relevance.

• Our findings have important clinical implications for preventing sudden cardiac death under pressure overload and guide the development of new therapeutic approaches for heart failure.

What Question Should Be Addressed Next?

• The precise mechanism by which Srprb regulates endoplasmic reticulum stress to promote ventricular arrhythmias remains elusive, necessitating further in‐depth investigation.

Heart failure (HF) remains a major global cause of mortality and morbidity. ¹ Among patients with HF, arrhythmias and sudden cardiac death (SCD) are frequent, with roughly half of all HF‐related deaths attributable to ventricular arrhythmias (VAs). ² , ³ , ⁴ The clinical spectrum of VAs ranges from asymptomatic presentations to life‐threatening events such as sustained ventricular tachycardia, ventricular fibrillation, and SCD. Therefore, elucidating the mechanisms underlying VAs in the setting of HF and developing novel preventive and therapeutic strategies are essential to improving clinical outcomes and reducing mortality in this population.

Ventricular remodeling is a key mechanism in the development of VAs post HF, involving both structural and electrical remodeling. Structural remodeling includes myocardial hypertrophy, inflammatory infiltration, and fibrosis, leading to disrupted myocardial excitation‐contraction coupling and impaired myocardial systolic and diastolic functions, ultimately resulting in arrhythmias. ⁵ Electrical remodeling can be divided into primary and secondary remodeling: primary remodeling occurs in response to functional damage, such as changes in electrical activation sequences, and secondary remodeling is a consequence of structural changes, characterized by abnormal ventricular repolarization, particularly changes in action potential duration (APD). The molecular basis of electrical remodeling includes abnormal expression and function of ion channels, intracellular calcium dysregulation, and sodium and potassium channel remodeling, which can lead to afterdepolarizations and triggered activity, causing arrhythmias. ⁶ Therefore, targeting cardiac remodeling has become an important strategy for preventing and treating VAs post HF.

The endoplasmic reticulum (ER) is a membrane‐bound organelle involved in the synthesis, folding, maturation, and posttranslational modification of secretory and transmembrane proteins. The ER also plays a crucial role in calcium storage, release, and signaling, making it essential for maintaining cellular homeostasis and the balance between health and disease. ⁷ Endoplasmic reticulum stress (ERS) is a stress response triggered by the accumulation of misfolded or unfolded proteins when the ER environment is disrupted. ⁸ Pathophysiological factors in HF, such as metabolic disturbances, hypoxia, and inflammation, place high demands on the protein‐folding capacity of the ER. When the ER cannot meet these demands, ERS is induced. ⁹ In turn, ERS triggers inflammation and oxidative stress, and the maladaptive unfolded protein response induces apoptosis, exacerbating HF progression. ¹⁰

ERS also plays a significant role in the development of VAs, primarily through 2 mechanisms: intracellular calcium homeostasis and ion channel protein synthesis homeostasis. Under stress conditions such as HF, the expression of ryanodine receptor and SERCA 2a (sarcoendoplasmic reticulum calcium ATPase 2a) in the ER is suppressed, leading to impaired calcium transport and release, which reduces cardiac contractile and diastolic functions. ¹¹ Additionally, ERS affects the expression of calcium regulatory proteins such as CaMKII (Ca²⁺/calmodulin‐dependent protein kinase II), which is activated by increased intracellular calcium levels, further exacerbating arrhythmias. ¹²

TLR4 (Toll‐like receptor 4), a pattern recognition receptor expressed on the cell membranes of various cell types, including cardiomyocytes, plays a significant role in the development of cardiovascular diseases. ¹³ Several studies have reported that TLR4, as an upstream signal of cardiac CaMKII, mediates CaMKII activation. ¹⁴ , ¹⁵ Under stress conditions such as HF and myocardial ischemia, CaMKII activation mediates ion channel damage and calcium protein homeostasis disruption, driving the expression of inflammatory genes in arrhythmias. ¹⁶ , ¹⁷ TLR4 also activates NF‐κB (nuclear factor kappa B), regulating macrophage polarization and the expression of various proinflammatory mediators such as IL‐1β (interleukin‐1β), IL‐6, iNOS (inducible nitric oxide synthase), and TNF‐α (tumor necrosis factor‐α), further stimulating inflammatory responses. ¹⁸

Protein synthesis in cells begins with free ribosomes, where polypeptide chains are initiated and then translocated to the ER with the help of signal peptides, signal recognition particles (SRPs), signal recognition particle receptors (SRPRs), and translocons, where they are extended until the peptide chain is complete. ¹⁹ SRP is a complex consisting of a 7S small RNA and 6 polypeptide subunits, and SRPR is a membrane‐integrated protein located on the ER. ²⁰ Mammalian SRPR is an α/β heterodimer. Srpra (signal recognition granule receptor α subunit) is a subunit of the SRPR, a peripheral membrane GTPase containing an N‐terminal X domain and a C‐terminal NG domain. ²¹ Srprb is another subunit of SRPR, a membrane‐anchored protein with an N‐terminal transmembrane domain and a G domain that binds GTP and forms a heterodimer with the X domain of Srpra (SRX/β heterodimer). Additionally, Srprb anchors the α subunit to the ER membrane. ²² Loffredo et al. demonstrated through parabiosis experiments that heart‐derived secreted proteins are necessary for age‐related cardiac dysfunction and structural remodeling. ²³ More recently, secreted proteins have been established as critical players either in the development of congestive HF or as immediate phenotypic manifestations of congestive HF. ²⁴ Previous large‐scale proteomic studies have also identified several secreted biomarkers associated with congestive HF. ²⁵ , ²⁶ , ²⁷ , ²⁸ Advances in high‐throughput technologies have now enabled the creation of large‐scale screening platforms to identify novel secretory mediators involved in HF. Nevertheless, the upstream triggers that initiate the synthesis of these secretory proteins remain poorly understood. The SRP and its receptor (SRPR) constitute a central cellular machinery responsible for the cotranslational translocation of approximately one third of the proteome to membrane compartments. SRP, along with the SRPR (composed of alpha and beta subunits), directs the nascent polypeptide‐ribosome complex to the ER, thereby facilitating entry into the secretory pathway. Based on integrative omics data mining and subsequent experimental validation, we have selected Srprb as a candidate for further investigation. We hypothesize that Srprb, an ER‐anchored subunit of the SRP receptor, serves as an early initiator of ERS induced by congestive HF.

METHODS

The authors declare that all supporting data are available within the article and its online supplementary files.

A more detailed description of the methods can be found in Data S1.

Animal and Animal Model

All animal experiments were conducted in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85‐23, revised 1996) and authorized by the Animal Care and Use Committee of Renmin Hospital of Wuhan University under the approval number: 20221207B. Male C57BL/6J wild‐type mice aged approximately 6 weeks (weighing 18–20 g) were used for this study. C57 wild‐type mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Upon arrival, mice were acclimatized in a quarantine room for 1 week. All animals were housed in a specific pathogen‐free barrier environment (12‐hour light/dark cycle, temperature 22±2 °C, humidity 50±15%) with free access to food and water. Drinking water was autoclaved and provided in the cages, and feed was sterilized by 60 °C irradiation before being placed in the feed boxes. To maintain a clean environment, professional caretakers regularly changed bedding, and all cages and bedding were sterilized before being placed in the barrier environment. All animal experiments were conducted after approval by the Animal Ethics Committee of Renmin Hospital of Wuhan University and in strict compliance with the Regulations on the Management of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals. For cell experiments, primary neonatal mouse cardiomyocytes and fibroblasts were used.

Experimental Grouping

1. Animal experiment grouping: After 1 week of acclimatization, 6‐week‐old mice were injected with adeno‐associated virus (AAV) via the tail vein. In the study, 4 types of viruses were injected: (1) Srprb‐overexpressing AAV (AAV‐Srprb) group: mice received tail vein injection of AAV9‐cTnT (cardiac troponin T)‐Srprb; (2) Srprb‐overexpressing control AAV (AAV‐NC) group: mice received tail vein injection of AAV9‐cTnT‐NC; (3) Srprb‐knockdown AAV (AAV‐Sh‐Srprb): mice received tail vein injection of AAV9‐cTNT‐Sh‐Srprb; and (4) Srprb‐knockdown control AAV (AAV‐Sh‐NC): mice received tail vein injection of AAV9‐cTNT‐short hairpin RNA. Each mouse received a tail vein injection at a dose of 3×10¹⁰vg. These vectors contained the cTnT cardiac promoter, which targeted specific Srprb genes in cardiac tissue. There are 40 mice in each group. Three weeks post injection, 21 mice from each virus‐injected group underwent aortic banding (AB) surgery, while the remaining 19 mice underwent sham surgery as controls (Sham). Thus, the animal experiment consisted of 8 groups: AAV‐NC+Sham, AAV‐Srprb+Sham, AAV‐NC+AB, AAV‐Srprb+AB, AAV‐Sh‐NC+Sham, AAV‐Sh‐Srprb+Sham, AAV‐Sh‐NC+AB, and AAV‐Sh‐Srprb+AB.

2. Cell experiment grouping: To further validate the effects of Srprb on mouse myocardium, primary neonatal mouse cardiomyocytes and fibroblasts were isolated, cultured, and transfected with Srprb‐overexpressing adenovirus (Ad‐Srprb), Srprb‐overexpressing control adenovirus (Ad‐NC), Srprb‐knockdown adenovirus (Ad‐Sh‐Srprb), or Srprb‐knockdown control adenovirus (Ad‐Sh‐NC). Cells were then stimulated with angiotensin II (Ang II)/TGF‐β (transforming growth factor‐β) or PBS for 24 hours. Thus, the primary neonatal mouse ventricular cardiomyocyte experiment was divided into 8 groups: Ad‐NC+PBS, Ad‐Srprb+PBS, Ad‐NC+Ang II, Ad‐Srprb+Ang II, Ad‐Sh‐NC+PBS, Ad‐Sh‐Srprb+PBS, Ad‐Sh‐NC+Ang II, and Ad‐Sh‐Srprb+Ang II. Similarly, the primary neonatal mouse fibroblast experiment was also divided into 8 groups: Ad‐NC+PBS, Ad‐Srprb+PBS, Ad‐NC+TGF‐β, Ad‐Srprb+TGF‐β, Ad‐Sh‐NC+PBS, Ad‐Sh‐Srprb+PBS, Ad‐Sh‐NC+TGF‐β, and Ad‐Sh‐Srprb+TGF‐β.

Statistical Analysis

GraphPad Prism 8.0 (Inc., La Jolla, CA, USA) was used to perform statistical analyses. All data were represented as mean±SEM. For comparisons across multiple groups, a 1‐way ANOVA was employed. When the overall ANOVA test showed significance (P<0.05), Tukey’s honestly significant difference test was used for post hoc pairwise comparisons. In these post hoc analyses, a Tukey’s honestly significant difference‐adjusted P alue of <0.05 was considered statistically significant for specific between‐group differences. For direct comparisons between 2 groups, an unpaired Student’s t test was applied. When P<0.05, values were considered statistically significant.

RESULTS

The Expression of Srprb Is Upregulated in Human Failing Hearts, Murine Hypertrophic Hearts, and Ang II Exposed Cardiomyocytes

First, through preliminary bioinformatics data mining and analysis, we found that the Srprb gene was significantly upregulated in human HF tissues (Figure 1A). Subsequently, we established a pressure overload‐induced HF model in C57 mice using AB surgery and detected Srprb expression in the heart of mice 4 weeks post surgery. Immunofluorescence staining (Figure 1H,D) and Western blot (Figure 1B,E) showed that Srprb expression was significantly higher in the hearts of AB‐induced mice compared with sham mice (P<0.01). In vitro, we isolated and cultured primary ventricular cardiomyocytes from 1‐ to 3‐day‐old neonatal mice and stimulated them with Ang II for 24 hours to observe changes in Srprb expression. As shown in Figure 1I,F and Figure 1C,G, immunofluorescence (Figure 1I,F) and Western blot (Figure 1C,G) results indicated that Srprb expression in cardiomyocytes significantly increased after Ang II stimulation compared with the PBS group (P<0.01). Moreover, AB‐induced HF mice exhibited deterioration in cardiac function and increased vulnerability and duration of VAs (Figure S1). These results suggest that Srprb may participate in the progression of AB‐induced HF mice ventricle remodeling.

Figure 1. Srprb is upregulated in human failing hearts, murine hypertrophic hearts, and Ang II‐exposed cardiomyocytes.

A, Bioinformatics analysis results of Srprb expression in patients with HF vs patients without HF; H, D, Representative immunofluorescence images and statistical analysis of Srprb expression in Sham mice vs AB mice (n=6/group). B, E, Representative Western blot images and statistical analysis of Srprb expression in Sham mice vs AB mice (n=6/group). I, F, Representative immunofluorescence images and statistical analysis of Srprb expression in Ang II‐stimulated primary neonatal mouse ventricular cardiomyocytes vs PBS‐treated primary neonatal mouse ventricular cardiomyocytes (n=6/group). C, G, Representative Western blot images and statistical analysis of Srprb expression in Ang II‐stimulated primary neonatal mouse ventricular cardiomyocytes vs PBS‐treated primary neonatal mouse ventricular cardiomyocytes (n=6/group). Data were presented as mean±SEM. AB indicates aortic banding; Ang II, angiotensin II; cTnt, cardiac troponin T; HF, heart failure; and Srprb, signal recognition particle receptor beta subunit.

To study the impact of Srprb on VAs in HF mice and its mechanism, we used AAV to overexpress or knock down Srprb in the mouse heart and adenovirus to overexpress or knock down Srprb in primary neonatal mouse ventricular cardiomyocytes/fibroblasts. As shown in Figure S2, Western blot results indicated that AAV‐Srprb significantly increased Srprb expression in the mouse heart (P<0.01) (Figure S2A,B), whereas AAV‐Sh‐Srprb significantly reduced Srprb expression in the mouse heart (P<0.01) (Figure S2C,D). Similarly, Ad‐Srprb significantly increased Srprb expression in primary neonatal mouse ventricular cardiomyocytes (P<0.01) (Figure S2E,F), and Ad‐Sh‐Srprb significantly reduced Srprb expression in primary neonatal mouse ventricular cardiomyocytes (P<0.01) (Figure S2G,H). Ad‐Srprb also significantly increased Srprb expression in primary neonatal mouse fibroblasts (P<0.01) (Figure S2I,J), whereas Ad‐Sh‐Srprb significantly reduced Srprb expression in primary neonatal mouse fibroblasts (P<0.01) (Figure S2K,L).

The Effect of Srprb Knockdown on Cardiac Function in Pressure Overload‐Induced HF Mice

To investigate the effect of Srprb knockdown on cardiac function in HF mice, we used echocardiography and surface ECG to analyze cardiac function and in vivo electrophysiology in different groups of mice. As shown in Figure 2B–F, compared with the Sham group, mice 4 weeks post AB surgery showed thickened ventricular walls, enlarged ventricular cavities, and reduced left ventricular (LV) systolic function, as indicated by significantly increased LV end‐systolic dimension (P<0.01) and LV end‐diastolic dimension (P<0.01) and significantly decreased fractional shortening (P<0.01) and LV ejection fraction (P<0.01). Under physiological conditions, Srprb knockdown did not affect cardiac function or morphology. However, 4 weeks post AB surgery, Srprb‐knockdown mice showed alleviated changes in LV end‐diastolic dimension (P<0.01), LV end‐systolic dimension (P<0.01), LV ejection fraction (P<0.01), and fractional shortening (P<0.05) compared with AAV‐Sh‐NC mice. As shown in Figure 2G,H, the QRS (P<0.01) and QTc (P<0.01) intervals were significantly longer in the AB group compared with the Sham group, and the QRS (P<0.05) and QTc (P<0.01) intervals were significantly shorter in the AAV‐Sh‐Srprb+AB group compared with the AAV‐Sh‐NC+AB group, whereas there was no significant difference between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P>0.05). Additionally, there were no significant differences in RR and PR intervals among the 4 groups (P>0.05).

Figure 2. The effect of Srprb knockdown on cardiac function in mice.

A, The schematic illustration of the animal experimental workflow; B, Representative echocardiographic images of the 4 groups; C–F, Statistical analysis of echocardiographic parameters in the 4 groups (n=6/group); G, Representative surface ECG images of the 4 groups; H, Statistical analysis of ECG parameters in the 4 groups (n=6/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; cTnt, cardiac troponin T; FS, fractional shortening; LVEDd, left ventricular end‐diastolic diameter; LVEF, left ventricular ejection fraction; LVESd, left ventricular end‐systolic diameter; NC, control; and Srprb, signal recognition particle receptor beta subunit.

The Effect of Srprb Overexpression on Cardiac Function in Pressure Overload‐Induced HF Mice

As shown in Figure 3A–E, compared with the Sham group, the AB group showed significantly increased LV end‐systolic dimension (P<0.01) and LV end‐diastolic dimension (P<0.01) and significantly decreased fractional shortening (P<0.01) and LV ejection fraction (P<0.01). Compared with the AAV‐NC+Sham group, the AAV‐Srprb+Sham group showed no significant changes in cardiac function (P>0.05). However, compared with the AAV‐NC+AB group, the AAV‐Srprb+AB group showed further deterioration in cardiac function, with further reductions in LV ejection fraction (P<0.05) and fractional shortening (P<0.01) and further increases in LV end‐systolic dimension (P<0.05) and LV end‐diastolic dimension (P<0.01). As shown in Figure 3F,G, the QRS (P<0.01) and QTc (P<0.01) intervals were significantly longer in the AB group compared with the Sham group, and the QRS (P<0.05) and QTc (P<0.01) intervals were significantly longer in the AAV‐Srprb+AB group compared with the AAV‐NC+AB group, whereas there was no significant difference between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05). Additionally, there were no significant differences in RR and PR intervals among the 4 groups (P>0.05).

Figure 3. The effect of Srprb overexpression on cardiac function in mice.

A, Representative echocardiographic images of the 4 groups; B–E, Statistical analysis of echocardiographic parameters in the 4 groups (n=6/group); F, Representative surface ECG images of the 4 groups; G, Statistical analysis of ECG parameters in the 4 groups (n=6/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; FS, fractional shortening; LVEDd, left ventricular end‐diastolic diameter; LVEF, left ventricular ejection fraction; LVESd, left ventricular end‐systolic diameter; NC, control; and Srprb, signal recognition particle receptor beta subunit.

Combined, these results indicate that Srprb knockdown alleviated AB‐induced cardiac dysfunction in HF mice, whereas Srprb overexpression exacerbated AB‐induced cardiac dysfunction in HF mice.

The Effect of Srprb on APD, APD Alternate Threshold, ERP, and VAs Susceptibility in Pressure Overload‐Induced HF Mice

The results showed that Srprb knockdown prevented the prolongation of QRS and QTc intervals in AB mice, whereas Srprb overexpression exacerbated the prolongation of QRS and QTc intervals in AB mice. Therefore, we used in vivo electrophysiology to further explore the impact of Srprb on VAs in pressure overload‐induced HF mice. APD90 was defined as the average repolarization time of 90% of 6 to 8 consecutive monophasic action potential when the pacing cycle length was 150 ms. In order to induce APD alternate threshold (ALT), pacing cycle length was decreased started at 150 ms and gradually reduced by 10 ms and then by 5 ms from 100 to 50 ms until APD alternans. The S1–S2 stimulation protocol (consisting of 8 consecutive S1 stimuli at 150 ms intervals followed by an additional S2 stimulus) is used to determine the ventricular effective refractory period (ERP).

As shown in Figure 4, compared with the Sham group, APD90 (P<0.01), ALT (P<0.01) were longer, and ERP (P<0.01) was shorter significantly in the AB group. In AB mice, APD90 (P<0.01), ALT (P<0.01) were shorter and ERP (P<0.05) was longer obviously in the AAV‐Sh‐Srprb group compared with the AAV‐Sh‐NC group, whereas there was no significant difference between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P<0.05). Finally, burst stimulation showed that no VAs were induced in the AAV‐Sh‐NC+Sham group, and there was 1 mouse induced VA in the AAV‐Sh‐Srprb+Sham group, whereas the probability of inducing VAs was significantly increased in AB group. However, the VAs induction rate was significantly lower and the duration of VAs was markedly decreased in the AAV‐Sh‐Srprb+AB group compared with the AAV‐Sh‐NC+AB group (P<0.05).

Figure 4. The effect of Srprb knockdown on action potential duration, alternans threshold, effective refractory period, and ventricular arrhythmia induction rate in mice.

A, B, Representative monophasic action potential waveforms and statistical analysis of APD in the 4 groups (n=6/group); C, D, Representative alternans waveforms and statistical analysis of ALT in the 4 groups (n=6/group); E, Statistical analysis of ERP in the 4 groups (n=6/group); F–H, Representative VAs waveforms induced by burst stimulation and statistical analysis of VAs induction rate and duration of VAs in the 4 groups (n=11–13/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; ALT, alternans threshold; APD, action potential duration; ERP, effective refractory period; NC, control; NSR, normal sinus rhythm; PCL, pacing cycle length; Srprb, signal recognition particle receptor beta subunit; VAs, ventricular arrhythmias; and VT, ventricular tachycardia.

As shown in Figure 5, APD90 (P<0.01), ALT (P<0.01) were prolonged and ERP (P<0.01) was shorter significantly in the AB group compared with the Sham group. Compared with the AAV‐NC+AB group, APD90 (P<0.05) and ALT (P<0.01) were significantly longer and ERP (P<0.05) was shorter significantly in the AAV‐Srprb+AB group, whereas there was no significant difference between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05). Burst stimulation showed that there was 1 mouse induced VA in both the AAV‐NC+Sham and AAV‐Srprb+Sham groups, and the probability of inducing VAs was increased and the duration of VAs (P<0.01) was prolonged significantly in AB mice. Although the VAs induction rate was significantly higher and the duration of VAs was markedly increased in the AAV‐Srprb+AB group compared with the AAV‐NC+AB group (P<0.05).

Figure 5. The effect of Srprb overexpression on action potential duration, alternans threshold, effective refractory period, and ventricular arrhythmia induction rate in mice.

A, B, Representative monophasic action potential waveforms and statistical analysis of APD in the 4 groups (n=6/group); C, D, Representative alternans waveforms and statistical analysis of ALT in the 4 groups (n=6/group); E, Statistical analysis of ERP in the 4 groups (n=6/group); F–H, Representative VAs waveforms induced by burst stimulation and statistical analysis of VAs induction rate and duration of VAs in the 4 groups (n=11–13/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; ALT, alternans threshold; APD, action potential duration; ERP, effective refractory period; NC, control; NSR, normal sinus rhythm; PCL, pacing cycle length; Srprb, signal recognition particle receptor beta subunit; VAs, ventricular arrhythmias; VF, ventricular fibrillation; and VT, ventricular tachycardia.

In summary, Srprb knockdown significantly improved VAs in AB‐induced HF mice, whereas Srprb overexpression exacerbated VAs in AB‐induced HF mice.

The Effect of Srprb on Pathological Hypertrophy and Fibrosis in Pressure Overload‐Induced HF Mice

The formation of reentry circuits can lead to VAs, and pathological myocardial hypertrophy and fibrosis can promote the formation of reentry circuits. Therefore, in this study, we investigated the effect of Srprb on AB‐induced ventricular hypertrophy and fibrosis.

First, through measurements of basic cardiac parameters (Figure 6B), hematoxylin and eosin staining (Figure 6A), wheat germ agglutinin staining (Figure 6A), and Western blot (Figure 6F), we found that compared with the Sham group, heart weight/body weight, heart weight/tibia length (Figure 6B), cardiomyocyte cross‐sectional area (Figure 6C), and the expression levels of hypertrophy markers BNP (brain natriuretic peptide) and β‐MHC (β‐myosin heavy chain) (Figure 6G) were significantly increased in the AB group (P<0.01). In AB mice, these parameters were significantly lower in the AAV‐Sh‐Srprb group compared with the AAV‐Sh‐NC group (P<0.05), whereas there was no significant difference between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P>0.05). Additionally, we assessed the degree of myocardial fibrosis, another major manifestation of myocardial structural remodeling. Masson and PSR staining showed that fibrosis was significantly increased in the AB group compared with the Sham group (P<0.01). Fibrosis area (Figure 6D,E), and the protein expression levels of fibrosis markers collagen I (P<0.01) and collagen III (P<0.01) were significantly lower in the AAV‐Sh‐Srprb+AB group compared with the AAV‐Sh‐NC+AB group (Figure 6F,G). There were no significant differences in fibrosis‐related parameters between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P>0.05).

Figure 6. The effect of Srprb knockdown on cardiac hypertrophy and fibrosis in mice.

A, C, Representative HE and WGA staining images of the ventricles and statistical analysis of cardiomyocyte CSA in the 4 groups (n=6/group); B, Statistical analysis of HW/BW, LW/BW, and HW/TL in the 4 groups (n=6/group); D–E, Representative Masson and PSR staining images of the ventricles and statistical analysis of ventricular fibrosis area in the 4 groups (n=6/group); F–G, Representative Western blot images and statistical analysis of hypertrophy and fibrosis markers BNP, β‐MHC, collagen I, and collagen III in the 4 groups (n=6/group) (the quantitative data were derived from Sirius Red staining). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; β‐MHC, β‐myosin heavy chain; BNP, brain natriuretic peptide; CSA, cross‐sectional area; HE, hematoxylin and eosin; HW/BW, heart weight/body weight; HW/TL, heart weight/tibia length; LW/BW, lung weight/body weight; NC, control; Srprb, signal recognition particle receptor beta subunit; and WGA, wheat germ agglutinin.

As shown in Figure 7, compared with the AAV‐NC+AB group, heart weight/body weight (P<0.01), heart weight/tibia length (P<0.01) (Figure 7B), cardiomyocyte cross‐sectional area (P<0.05) (Figure 7A,C), and the expression levels of hypertrophy markers BNP (P<0.01) and β‐MHC (P<0.01) (Figure 7F,G) were significantly increased in the AAV‐Srprb+AB group. There were no significant differences in these parameters between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05). Masson and PSR staining showed that fibrosis area was significantly higher in the AAV‐Srprb+AB group compared with the AAV‐NC+AB group (P<0.01) (Figure 7D,E), and the protein expression levels of collagen I (P<0.01) and collagen III (P<0.01) were significantly higher in the AAV‐Srprb+AB group compared with the AAV‐NC+AB group (Figure 7F,G). There were no significant differences in fibrosis‐related parameters between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05).

Figure 7. The effect of Srprb overexpression on cardiac hypertrophy and fibrosis in mice.

A, C, Representative HE and WGA staining images of the ventricles and statistical analysis of cardiomyocyte CSA in the 4 groups (n=6/group); B, Statistical analysis of HW/BW, LW/BW, and HW/TL in the 4 groups (n=6/group); D–E, Representative Masson and PSR staining images of the ventricles and statistical analysis of ventricular fibrosis area in the 4 groups (n=6/group); F–G, Representative Western blot images and statistical analysis of hypertrophy and fibrosis markers BNP, β‐MHC, collagen I, and collagen III in the 4 groups (n=6/group) (the quantitative data were derived from Sirius Red staining). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; β‐MHC, β‐myosin heavy chain; BNP, brain natriuretic peptide; CSA, cross‐sectional area; HE, hematoxylin and eosin; HW/BW, heart weight/body weight; HW/TL, heart weight/tibia length; LW/BW, lung weight/body weight; NC, control; Srprb, signal recognition particle receptor beta subunit; and WGA, wheat germ agglutinin.

To further validate the effect of Srprb on cardiac structural remodeling, we isolated ventricular myocytes and fibroblasts from 1‐ to 3‐day‐old neonatal mice and transfected them with Ad‐Sh‐Srprb or Ad‐Srprb for 24 hours, followed by stimulation with Ang II or TGF‐β. Hematoxylin and eosin staining, α‐actin immunofluorescence, α‐SMA (α‐smooth muscle actin) immunohistochemistry, and Western blot were performed. As shown in Figure S3B–F, compared with the PBS group, ventricular myocytes surface area and the expression levels of BNP and β‐MHC were significantly increased in the Ang II group (P<0.01). Compared with the Ad‐Sh‐NC+Ang II group, ventricular myocytes surface area (P<0.05) and the expression levels of BNP (P<0.01) and β‐MHC (P<0.05) were significantly reduced in the Ad‐Sh‐Srprb+Ang II group. There were no significant differences in these parameters between the Ad‐Sh‐NC+PBS and Ad‐Sh‐Srprb+PBS groups (P>0.05). As shown in Figure S2G–J, compared with the PBS group, the expression levels of α‐SMA, collagen I, and collagen III were significantly increased in the TGF‐β group (P<0.01). Compared with the Ad‐Sh‐NC+TGF‐β group, the expression levels of α‐SMA (P<0.05), collagen I (P<0.01), and collagen III (P<0.05) were significantly reduced in the Ad‐Sh‐Srprb+TGF‐β group. There were no significant differences in these parameters between the Ad‐Sh‐NC+PBS and Ad‐Sh‐Srprb+PBS groups (P>0.05). As shown in Figure S4A–E, compared with the Ad‐NC+Ang II group, ventricular myocytes surface area and the expression levels of BNP and β‐MHC were significantly increased in the Ad‐Srprb+Ang II group (P<0.01). There were no significant differences in these parameters between the Ad‐NC+PBS and Ad‐Srprb+PBS groups (P>0.05). As shown in Figure S4F–I, compared with the Ad‐NC+TGF‐β group, the expression levels of α‐SMA (P<0.01), collagen I (P<0.01), and collagen III (P<0.05) were significantly increased in the Ad‐Srprb+TGF‐β group. There were no significant differences in these parameters between the Ad‐NC+PBS and Ad‐Srprb+PBS groups (P>0.05).

These results indicate that Srprb knockdown significantly improved pathological cardiac structural remodeling in AB‐induced HF mice, including hypertrophy and fibrosis, whereas Srprb overexpression exacerbated pathological cardiac hypertrophy and fibrosis in AB‐induced HF mice.

The Effect of Srprb on Ion Channels and Gap Junction Proteins Related to Normal Cardiac Rhythm in Pressure Overload‐Induced HF Mice

Another important mechanism of arrhythmias is cardiac electrical remodeling. Several studies have shown that abnormal expression of cardiac ion channels and gap junction proteins is a prerequisite for VAs. Therefore, we used Western blot and polymerase chain reaction to detect the mRNA and protein expression levels of major ion channel subunits related to normal cardiac rhythm, including the main sodium channel Nav1.5, potassium channel subunits Kv1.5, Kir2.1, Kv4.3, and the L‐type calcium channel subunit Cav1.2. Additionally, we used Western blot and immunohistochemistry to detect the expression of Cx43 (connexin 43), a gap junction protein.

As shown in Figure 8A–C, compared with the Sham group, the mRNA and protein expression levels of Nav1.5, Kv1.5, Kir2.1, Kv4.3, and Cav1.2 were significantly reduced in the AB group (P<0.01). However, these downregulated ion channels were partially restored in the AAV‐Sh‐Srprb group (P<0.05). There were no significant differences in these parameters between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P>0.05). As shown in Figure 8D–G, compared with the Sham group, Cx43 expression was significantly reduced in the AB group (P<0.01). There was no significant difference in Cx43 expression between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P>0.05). However, compared with the AAV‐Sh‐NC+AB group, Cx43 expression was significantly increased in the AAV‐Sh‐Srprb+AB group (P<0.01).

Figure 8. The effect of Srprb knockdown on ion channels and gap junction proteins related to normal cardiac rhythm in mice.

A, B, Representative Western blot images and statistical analysis of Kv1.5, Kir2.1, Kv4.3, Nav1.5, and Cav1.2 in the ventricles of the 4 groups (n=6/group); C, Statistical analysis of Kv1.5, Kir2.1, Kv4.3, Nav1.5, and Cav1.2 mRNA expression in the ventricles of the 4 groups (n=6/group); D, E, Representative Western blot images and statistical analysis of Cx43 in the ventricles of the 4 groups (n=6/group); F, G, Representative immunohistochemistry images and statistical analysis of Cx43 in the ventricles of the 4 groups (n=6/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; Cx43, connexin 43; NC, control; and Srprb, signal recognition particle receptor beta subunit.

As shown in Figure 9A–C, compared with the Sham group, the mRNA and protein expression levels of Nav1.5, Kv1.5, Kir2.1, Kv4.3, and Cav1.2 were significantly reduced in the AB group (P<0.01). Compared with the AAV‐NC+AB group, these downregulated ion channels were further exacerbated in the AAV‐Srprb+AB group (P<0.05). There were no significant differences in these parameters between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05). As shown in Figure 9D–G, compared with the Sham group, Cx43 expression was significantly reduced in the AB group (P<0.01). There was no significant difference in Cx43 expression between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05). However, compared with the AAV‐NC+AB group, Cx43 expression was significantly reduced in the AAV‐Srprb+AB group (P<0.01).

Figure 9. The effect of Srprb overexpression on ion channels and gap junction proteins related to normal cardiac rhythm in mice.

A, B, Representative Western blot images and statistical analysis of Kv1.5, Kir2.1, Kv4.3, Nav1.5, and Cav1.2 in the ventricles of the 4 groups (n=6/group); C, Statistical analysis of Kv1.5, Kir2.1, Kv4.3, Nav1.5, and Cav1.2 mRNA expression in the ventricles of the 4 groups (n=6/group); D, E, Representative Western blot images and statistical analysis of Cx43 in the ventricles of the 4 groups (n=6/group); F–G, Representative immunohistochemistry images and statistical analysis of Cx43 in the ventricles of the four groups (n=6/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; Cx43, connexin 43; NC, control; and Srprb, signal recognition particle receptor beta subunit.

To further validate the effect of Srprb on cardiac electrical remodeling, we conducted cell experiment. Western blot and polymerase chain reaction were used to detect the expression of ion channels related to normal cardiac rhythm. As shown in Figure S5, compared with the PBS group, the mRNA and protein expression levels of Nav1.5, Kv1.5, Kir2.1, Kv4.3, and Cav1.2 were significantly reduced in the Ang II group (P<0.01). Compared with the Ad‐Sh‐NC+Ang II group, the mRNA and protein expression levels of these ion channels were significantly increased in the Ad‐Sh‐Srprb+Ang II group (P<0.05). There were no significant differences in these parameters between the Ad‐Sh‐NC+PBS and Ad‐Sh‐Srprb+PBS groups (P>0.05). As shown in Figure S6, compared with the Ad‐NC+Ang II group, the mRNA and protein expression levels of these ion channels were significantly increased in the Ad‐Srprb+Ang II group (P<0.05). There were no significant differences in these parameters between the Ad‐NC+PBS and Ad‐Srprb+PBS groups (P>0.05).

These results indicate that Srprb knockdown significantly improved the abnormal expression and distribution of ion channels and gap junction proteins related to normal cardiac rhythm in AB‐induced HF mice, whereas Srprb overexpression exacerbated these abnormalities.

Srprb May Influence VAs in Pressure Overload‐Induced HF Mice by Regulating Endoplasmic Reticulum Stress and the TLR4 Signaling Pathway

To clarify the mechanism by which Srprb influences VAs in pressure overload‐induced HF mice, we performed transcriptomic sequencing to analyze differentially expressed genes and affected pathways in the ventricular tissues of AAV‐NC+AB and AAV‐Srprb+AB mice. As shown in Figure 10A, Kyoto Encyclopedia of Genes and Genomes analysis revealed that the downstream signaling pathways were enriched in protein processing in ER and TLR signaling pathway. Subsequently, through immunofluorescence colocalization of Srprb and the ER‐specific marker Calnexin in ventricular myocytes, we found that Srprb highly colocalized with Calnexin (Figure 10B,C).

Figure 10. Srprb may influence VAs in HF mice by regulating endoplasmic reticulum stress and the TLR4 signaling pathway.

A, KEGG enrichment analysis in the ventricular tissues between AAV‐NC+AB and AAV‐Srprb+AB mice (|log2FC|>1.2, P value<0.05); B, Representative immunofluorescence images of Srprb and Calnexin colocalization in Sham and AB mouse heart tissues; C, Representative immunofluorescence images of Srprb and Calnexin colocalization between PBS and Ang II‐stimulated primary neonatal mouse ventricular cardiomyocytes. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; Ang II, angiotensin II; HF, heart failure; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, control; NF‐kappa B, nuclear factor kappa B; Srprb, signal recognition particle receptor beta subunit; TLR4, Toll‐like receptor 4; and VA, ventricular arrhythmia.

The Effect of Srprb on Endoplasmic Reticulum Stress in Pressure Overload‐Induced HF Mice

The Kyoto Encyclopedia of Genes and Genomes analysis of transcriptomic sequencing revealed enrichment in the Protein processing in ER pathway, which is closely related to ERS. Combined with the immunofluorescence colocalization of Srprb and Calnexin, we hypothesized that Srprb may influence VAs in pressure overload‐induced HF mice by regulating ERS. Therefore, we conducted experiments to validate this hypothesis. First, we used Western blot to detect the expression of ERS‐related proteins. As shown in Figure 11A,C, compared with the Sham group, the expression levels of GRP78 (glucose regulated protein 78kD), p‐PERK (phosphorylated protein kinase RNA‐like endoplasmic reticulum kinase), p‐IRE1α (phosphorylated inositol requiring enzyme 1α), and CHOP (C/EBP homologous protein) were significantly increased in the AB group (P<0.01). Compared with the AAV‐Sh‐NC+AB group, the expression levels of GRP78 (P<0.05), p‐PERK (P<0.05), p‐IRE1α (P<0.01), and CHOP (P<0.01) were significantly reduced in the AAV‐Sh‐Srprb+AB group. There were no significant differences in the expression of these ERS‐related proteins between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P>0.05). There were no significant differences in the protein expression levels of PERK, IRE1α, and ATF6 (activating transcription factor 6) among the 4 groups (Figure S8B,F) (P>0.05). As shown in Figure 11D,F, compared with the AAV‐NC+AB group, the expression levels of GRP78 (P<0.05), p‐PERK (P<0.05), p‐IRE1α (P<0.01), and CHOP (P<0.01) were significantly increased in the AAV‐Srprb+AB group (P<0.05). There were no significant differences in the expression of these ERS‐related proteins between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05). The protein expression levels of PERK, IRE1α, and ATF6 showed no significant differences among the 4 groups (Figure S8A,E) (P>0.05).

Figure 11. The effect of Srprb on endoplasmic reticulum stress in mice.

A, C, Representative Western blot images and statistical analysis of ERS markers GRP78, p‐PERK, p‐IRE1α, and CHOP in the ventricles of the 4 groups (n=6/group); B, Transmission electron microscopy images of the ER and mitochondria in the ventricles of the 4 groups (n=6/group); D, F, Representative Western blot images and statistical analysis of ERS markers GRP78, p‐PERK, p‐IRE1α, and CHOP in the ventricles of the 4 groups (n=6/group); E, Transmission electron microscopy images of the ER and mitochondria in the ventricles of the 4 groups (n=6/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; CHOP, C/EBP homologous protein; ERS, endoplasmic reticulum stress; GRP78, glucose regulated protein 78kD; NC, control; p‐IRE1α, phosphorylated inositol requiring enzyme 1α; p‐PERK, phosphorylated protein kinase RNA‐like endoplasmic reticulum kinase; and Srprb, signal recognition particle receptor beta subunit.

Because ERS is closely related to mitochondrial damage, we used transmission electron microscopy to examine the ultrastructure of the ER and mitochondria in cardiomyocytes from HF mice. As shown in Figure 11B, compared with the Sham group, the ultrastructure of the ER and mitochondria in cardiomyocytes from the AB group showed significant abnormalities (loss of granules on the ER membrane, fragmentation of the ER into sheets or vesicles, significant swelling of mitochondria and disorganization of mitochondrial cristae). Compared with the AAV‐Sh‐NC+AB group, the damage to the ER and mitochondria was significantly reduced in the AAV‐Sh‐Srprb+AB group. There were no significant differences in the ultrastructure of the ER and mitochondria between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups. As shown in Figure 11E, compared with the AAV‐NC+AB group, the damage to the ER and mitochondria was significantly worsened in the AAV‐Srprb+AB group. There were no significant differences in the ultrastructure of the ER and mitochondria between the AAV‐NC+Sham and AAV‐Srprb+Sham groups.

In vitro cell experiments also confirmed these findings. As shown in Figure S7A,B, compared with the PBS group, the expression levels of GRP78, p‐PERK, p‐IRE1α, and CHOP were significantly increased in Ang II‐stimulated primary neonatal mouse ventricular cardiomyocytes (P<0.01). Compared with the Ad‐Sh‐NC+Ang II group, the expression levels of GRP78, p‐PERK, p‐IRE1α, and CHOP were significantly reduced in the Ad‐Sh‐Srprb+Ang II group (P<0.05). There were no significant differences in the expression of these ERS‐related proteins between the Ad‐Sh‐NC+PBS and Ad‐Sh‐Srprb+PBS groups (P>0.05). No significant differences were observed in the protein expression levels of PERK, IRE1α, and ATF6 across the 4 groups (Figure S8D,H) (P>0.05). As shown in Figure S7C,D, compared with the Ad‐NC+Ang II group, the expression levels of GRP78 (P<0.01), p‐PERK (P<0.05), p‐IRE1α (P<0.01), and CHOP (P<0.01) were significantly increased in the Ad‐Srprb+Ang II group. There were no significant differences in the expression of these ERS‐related proteins between the Ad‐NC+PBS and Ad‐Srprb+PBS groups (P>0.05). No significant differences in the protein expression of PERK, IRE1α, and ATF6 were detected among the 4 groups (Figure S8C,G) (P>0.05).

These results indicated that Srprb knockdown significantly improved ERS in cardiomyocytes from HF mice, whereas Srprb overexpression significantly exacerbated ERS in cardiomyocytes in HF mice.

The Effect of Srprb on the TLR4/CaMKII/NF‐κB Signaling Pathway in Pressure Overload‐Induced HF Mice

The Kyoto Encyclopedia of Genes and Genomes analysis of transcriptomic sequencing revealed enrichment in the TLR signaling pathway. We then used Western blot and immunohistochemistry to validate this finding. As shown in Figure 12A,D, Western blot results showed that compared with the Sham group, the expression levels of TLR4 (P<0.01), p‐CaMKII/CaMKII (P<0.01), pp65/p65 (P<0.01), (Phospho‐Phospholamban) PPLB/PLB (phospholamban) (P<0.01), and p‐IκBα/IκBα (inhibitor of nuclear factor kappa) (P<0.05) were significantly increased, while SERCA 2a (P<0.01) expression was significantly reduced in the AB group. Compared with the AAV‐Sh‐NC+AB group, the expression levels of TLR4 (P<0.01), p‐CaMKII/CaMKII (P<0.01), pp65/p65 (P<0.05), PPLB/PLB (P<0.05), and p‐IκBα/IκBα (P<0.05) were significantly reduced, whereas SERCA 2a (P<0.01) expression was significantly increased in the AAV‐Sh‐Srprb+AB group. There were no significant differences in the expression of these proteins between the AAV‐Sh‐NC+Sham and AAV‐Sh‐Srprb+Sham groups (P>0.05). As shown in Figure 12B,C,E,F, immunohistochemistry results showed that compared with the AAV‐Sh‐NC+AB group, the expression levels of p‐CaMKII and pp65 were significantly reduced in the AAV‐Sh‐Srprb+AB group (P<0.05).

Figure 12. The effect of Srprb knockdown on the TLR4/CaMKII/NF‐κB signaling pathway in mice.

A, D, Representative Western blot images and statistical analysis of TLR4, p‐CaMKII, CaMKII, SERCA 2a, pp65, p65, PPLB, PLB, p‐IκBα, and IκBα in the ventricles of the 4 groups (n=6/group); B, E, Representative immunohistochemistry images and statistical analysis of p‐CaMKII in the ventricles of the 4 groups (n=6/group); C, F, Representative immunohistochemistry images and statistical analysis of pp65 in the ventricles of the 4 groups (n=6/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; CaMKII, Ca2+/calmodulin‐dependent protein kinase II; IκBα, inhibitor of nuclear factor kappa; NC, control; NF‐κB, nuclear factor kappa B; PLB, phospholamban; PPLB, Phospho‐Phospholamban; SERCA 2a, sarcoendoplasmic reticulum calcium ATPase 2a; Srprb, signal recognition particle receptor beta subunit; and TLR4, Toll‐like receptor 4.

As shown in Figure 13A,D, Western Blot results showed that compared with the AAV‐NC+AB group, the expression levels of TLR4 (P<0.01), p‐CaMKII/CaMKII (P<0.05), pp65/p65 (P<0.05), PPLB/PLB (P<0.01), and p‐IκBα/IκBα (P<0.05) were significantly increased, and SERCA 2a (P<0.01) expression was significantly reduced in the AAV‐Srprb+AB group. There were no significant differences in the expression of these proteins between the AAV‐NC+Sham and AAV‐Srprb+Sham groups (P>0.05). As shown in Figure 13B,C,E,F, immunohistochemistry results showed that compared with the AAV‐NC+AB group, the expression levels of p‐CaMKII and pp65 were significantly increased in the AAV‐Srprb+AB group (P<0.05).

Figure 13. The effect of Srprb overexpression on the TLR4/CaMKII/NF‐κB signaling pathway in mice.

A, D, Representative Western blot images and statistical analysis of TLR4, p‐CaMKII, CaMKII, SERCA 2a, pp65, p65, PPLB, PLB, p‐IκBα, and IκBα in the ventricles of the 4 groups (n=6/group); B, E, Representative immunohistochemistry images and statistical analysis of p‐CaMKII in the ventricles of the 4 groups (n=6/group); C, F, Representative immunohistochemistry images and statistical analysis of pp65 in the ventricles of the 4 groups (n=6/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; CaMKII, Ca2+/calmodulin‐dependent protein kinase II; IκBα, inhibitor of nuclear factor kappa; NC, control; NF‐κB, nuclear factor kappa B; PLB, phospholamban; PPLB, Phospho‐Phospholamban; SERCA 2a, sarcoendoplasmic reticulum calcium ATPase 2a; Srprb, signal recognition particle receptor beta subunit; and TLR4, Toll‐like receptor 4.

As shown in Figure S9 A‐B, compared with the PBS group, the expression levels of TLR4, p‐CaMKII/CaMKII, pp65/p65, PPLB/PLB, and p‐IκBα/IκBα were significantly increased, whereas SERCA 2a expression was significantly reduced in Ang II‐stimulated primary neonatal mouse ventricular cardiomyocytes (P<0.01). Compared with the Ad‐Sh‐NC+Ang II group, the expression levels of TLR4 (P<0.01), p‐CaMKII/CaMKII (P<0.01), pp65/p65 (P<0.01), PPLB/PLB (P<0.05), and p‐IκBα/IκBα (P<0.01) were significantly reduced, whereas SERCA 2a (P<0.01) expression was significantly increased in the Ad‐Sh‐Srprb+Ang II group. There were no significant differences in the expression of these proteins between the Ad‐Sh‐NC+PBS and Ad‐Sh‐Srprb+PBS groups (P>0.05). As shown in Figure S9C,D, compared with the Ad‐NC+Ang II group, the expression levels of TLR4 (P<0.01), p‐CaMKII/CaMKII (P<0.05), pp65/p65 (P<0.01), PPLB/PLB (P<0.01), and p‐IκBα/IκBα (P<0.01) were significantly increased, whereas SERCA 2a (P<0.05) expression was significantly reduced in the Ad‐Srprb+Ang II group. There were no significant differences in the expression of these proteins between the Ad‐NC+PBS and Ad‐Srprb+PBS groups (P>0.05).

These results indicated that Srprb knockdown significantly reduced the activity of the TLR4/CaMKII/NF‐κB signaling pathway in pressure overload‐induced HF mice, whereas Srprb overexpression significantly increased the activity of the pathway.

Inhibition of TLR4 or ERS Reduces Srprb Overexpression‐Mediated Aggravation of Aortic Banding‐Induced VAs

As shown in Figure 14, compared with the AAV‐Srprb+AB+DMSO group, APD90 (P<0.01), ALT (P<0.01) were shorter and ERP (P<0.05) was longer significantly in the AAV‐Srprb+AB+TAK‐242 group or AAV‐Srprb+AB+4‐phenylbutyric acid (4‐PBA) group. Burst stimulation showed that the VAs induction rate was lower and the duration of VAs was decreased distinctly in the AAV‐Srprb+AB+TAK‐242 group or AAV‐Srprb+AB+4‐PBA group compared with the AAV‐Srprb+AB+DMSO group (P<0.05). Furthermore, as shown in Figure S10, cardiac dysfunction and ventricular remodeling, aggravated by Srprb overexpression, can be alleviated by inhibiting TLR4 or ERS.

Figure 14. Inhibition of TLR4 or ERS reduces Srprb overexpression‐mediated aggravation of aortic banding‐induced VAs.

A, The schematic illustration of the animal experimental workflow; B, C, Representative monophasic action potential waveforms and statistical analysis of APD in the 3 groups (n=6/group); D, E, Representative alternans waveforms and statistical analysis of ALT in the 3 groups (n=6/group); F, Statistical analysis of ERP in the 3 groups (n=6/group); G–I, Representative VA waveforms induced by burst stimulation and statistical analysis of VA induction rate and duration of VAs in the 3 groups (n=11–13/group). Data were presented as mean±SEM. AAV9 indicates adeno‐associated virus serotype 9; AB, aortic banding; ALT, alternans threshold; APD, action potential duration; cTnT, cardiac troponin T; ERP, effective refractory period; NSR, normal sinus rhythm; PCL, pacing cycle length; Srprb, signal recognition particle receptor beta subunit; TLR4, Toll‐like receptor 4; VAs, ventricular arrhythmias; VF, ventricular fibrillation; and VT, ventricular tachycardia.

As shown in Figure S11 B‐C, compared with the Ad‐Srprb+Ang II group, the expression levels of p‐CaMKII/CaMKII (P<0.01), PPLB/PLB (P<0.01), pp65/p65 (P<0.01), and p‐IκBα/IκBα (P<0.01) were significantly reduced, whereas SERCA 2a (P<0.05) expression was significantly increased in the Ad‐Srprb+Ang II+TAK‐242 group. Similarly, compared with the Ad‐Srprb+Ang II group, the expression levels of p‐CaMKII/CaMKII (P<0.01), PPLB/PLB (P<0.01), pp65/p65 (P<0.01), and p‐IκBα/IκBα (P<0.01) were significantly reduced, whereas SERCA 2a (P<0.05) expression was significantly increased in the Ad‐Srprb+Ang II+4‐PBA group.

As shown in Figure S11 D‐H, compared with the Ad‐Srprb+Ang II group, cardiomyocyte surface area (P<0.05) and the expression levels of BNP (P<0.01) and β‐MHC (P<0.01) were significantly reduced in the Ad‐Srprb+Ang II+TAK‐242 group. Similarly, compared with the Ad‐Srprb+Ang II group, cardiomyocyte surface area (P<0.05) and the expression levels of BNP (P<0.01) and β‐MHC (P<0.01) were significantly reduced in the Ad‐Srprb+Ang II+4‐PBA group. As shown in Figure S8 I‐L, compared with the Ad‐Srprb+TGF‐β group, the expression levels of α‐SMA (P<0.01), collagen I (P<0.01), and collagen III (P<0.01) were significantly reduced in the Ad‐Srprb+TGF‐β+TAK‐242 group. Similarly, compared with the Ad‐Srprb+TGF‐β group, the expression levels of α‐SMA (P<0.01), collagen I (P<0.01), and collagen III (P<0.01) were significantly reduced in the Ad‐Srprb+TGF‐β+4‐PBA group.

As shown in Figure S11 M‐O, compared with the Ad‐Srprb+Ang II group, the expression levels of Nav1.5, Kv1.5, Kir2.1, Kv4.3, and Cav1.2 were significantly increased in the Ad‐Srprb+Ang II+TAK‐242 group (P<0.05). Similarly, compared with the Ad‐Srprb+Ang II group, the expression levels of Nav1.5, Kv1.5, Kir2.1, Kv4.3, and Cav1.2 were significantly increased in the Ad‐Srprb+Ang II+4‐PBA group (P<0.05).

Therefore, this study demonstrated that the TLR4 inhibitor TAK‐242 and the ERS inhibitor 4‐PBA both inhibited Srprb overexpression‐mediated aggravation of AB‐induced VAs. Meanwhile, in vitro cell experiment showed that TLR4 inhibitor TAK‐242 and the ERS inhibitor 4‐PBA both suppressed Srprb overexpression‐induced activation of the CaMKII/NF‐κB signaling pathway, upregulated the expression of ion channels in cardiomyocytes, alleviated cardiomyocyte hypertrophy, and reduced fibroblast fibrosis, further confirming that Srprb regulates VAs post HF by modulating ERS and the TLR4/CaMKII/NF‐κB signaling pathway.

DISCUSSION

Cardiac remodeling is a significant pathophysiological manifestation of various cardiovascular diseases progressing to HF, characterized by myocardial hypertrophy and fibrosis, which have been identified as determinants of the clinical course of heart failure. ²⁹ Heart failure is often complicated by VAs, which frequently lead to poor prognosis in patients. ³⁰ Another critical pathological basis for VAs is ventricular electrical remodeling, including the downregulation of numerous cardiac ion channels and transporters, leading to prolonged QT interval on the surface ECG, prolonged APD, and increased risk of arrhythmias. ³¹ Understanding the molecular mechanisms underlying ventricular arrhythmias in HF is pivotal for developing novel therapeutic strategies to reduce the incidence of VAs caused by HF. The study first presents the evidence that Srprb, which is upregulated in human failing hearts, murine hypertrophic hearts, and Ang II‐exposed cardiomyocytes, plays a crucial part in the occurrence of VA after HF. Knockdown of Srprb improves ventricular structural and electrical remodeling in HF mice, alleviating the occurrence of VAs, whereas overexpression of Srprb exacerbates ventricular structural and electrical remodeling and promotes VAs. In addition, through comprehensive mechanistic research, we have demonstrated that overexpression of Srprb promotes the occurrence of VAs after HF by activating ERS and the TLR4/CaMKII/NF‐κB signaling pathway.

The SRP is a key molecule in the transport of secretory and membrane proteins to the cell membrane, ¹⁹ and the SRPR is a membrane‐integrated protein located on the ER. ²⁰ The mammalian SRPR, which determines the activity of SRP, typically consists of 2 subunits: α (Srpra) and β (Srprb). Srprb has a single transmembrane domain that anchors it to the ER membrane, influencing protein processing within the ER. Through transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes analysis of the ventricles between AAV‐NC+AB and AAV‐Srprb+AB mice, we found that the downstream signaling pathways were enriched in Protein processing in ER and TLR signaling pathways. Subsequently, immunofluorescence experiments revealed a high degree of colocalization between Srprb and the ER‐specific marker Calnexin. Combined with the structural sites of Srprb, we hypothesized that Srprb is closely related to endoplasmic reticulum function. Through a series of mechanistic explorations, we further identified the mechanism by which Srprb affects ventricular arrhythmias after heart failure.

In cardiovascular diseases, cardiac ion channels are often downregulated. ³² Patients with HF exhibit reduced expression of the Na⁺ channel (Nav1.5) protein and decreased current (I_Na), leading to a slower rate of action potential upstroke (dV/dtmax), which further slows conduction—a prerequisite for reentrant arrhythmias—and impairs impulse propagation in cardiac tissue. ³³ Human and animal studies have shown that multiple K⁺ channel currents are downregulated in patients with cardiomyopathy, associated with reduced transcriptional and translational levels of the corresponding channel subunits. The reduction in K⁺ channel repolarizing currents leads to prolonged APD and corresponding QT interval prolongation. ³⁴ QT interval prolongation is also associated with another arrhythmia mechanism, triggered activity, which can lead to polymorphic ventricular tachycardia, such as torsades de pointes. ³⁵ Nonuniform prolongation of ventricular action potentials leads to enhanced dispersion of repolarization, a hallmark of electrical remodeling in HF, which can trigger ventricular tachycardia. The prolongation APD in HF may be partly due to the downregulation of inward rectifier potassium currents and calcium‐independent transient outward potassium currents, leading to early depolarization and triggering ventricular tachycardia. ³⁶ Gap junctions, specialized membrane channels composed of protein subunits called connexins, provide the basis for electrical, physical, and chemical communication between adjacent cardiomyocytes, with Cx43 being the primary ventricular connexin. ³⁷ In failing hearts, heterogeneous changes in the expression, distribution, and density of these gap junction proteins play an important role in the pathogenesis of abnormal impulse propagation and VAs. In HF, Cx43 expression is reduced by up to 50%, with partial protein dephosphorylation leading to gap junction uncoupling. ³⁸ Consistent with previous studies, we found that HF mice with Srprb knockdown exhibited shortened QRS and QTc intervals, reduced APD, decreased ALT, and lower VAs inducibility, along with upregulation of cardiac ion channels and gap junction protein Cx43. Conversely, HF mice overexpressing Srprb exhibited prolonged QRS and QTc intervals, increased APD, elevated ALT, and higher VAs inducibility, along with downregulation of cardiac ion channels and Cx43.

ERS plays an important role in the development of cardiac hypertrophy and HF. Significant ERS has been observed in failing human hearts and hypertrophic or failing mouse hearts, with elevated expression levels of Xbp1 (X‐box binding protein 1), GRP78, ATF4 (activating transcription factor 4), and CHOP in cardiomyocytes, along with significant mitochondrial swelling and loss of cristae. ⁹ , ³⁹ Similarly, we confirmed that compared with the Sham group, HF mice induced by AB surgery exhibited significantly increased expression levels of GRP78, p‐PERK, p‐IRE1α, and CHOP proteins in the myocardium. Under electron microscopy, granules on the ER membrane of cardiomyocytes showed varying degrees of loss, the ER was fragmented into sheets or vesicles of varying sizes, and mitochondria exhibited significant swelling with shortened and disorganized cristae. HF mice with Srprb knockdown showed significantly reduced expression levels of GRP78, p‐PERK, p‐IRE1α, and CHOP proteins in the myocardium, and under electron microscopy, the damage to the ER and mitochondria was significantly alleviated. Conversely, HF mice overexpressing Srprb exhibited significantly increased expression levels of GRP78, p‐PERK, p‐IRE1α, and CHOP proteins in the myocardium, and under electron microscopy, the damage to the ER and mitochondria was significantly worsened. Under mild ERS, the unfolded protein response acts as an adaptive mechanism, promoting the refolding of ER proteins, degrading misfolded proteins, and restoring ER homeostasis. However, under severe ERS, activation of the UPR, particularly the PERK and IRE1α pathways, can shut down the synthesis of many important proteins, including multiple cardiac ion channel proteins. The PERK pathway primarily downregulates multiple cardiac ion channels by increasing mRNA degradation, playing a detrimental role in HF and myocardial infarction, ⁴⁰ , ⁴¹ leading to increased risk of arrhythmias due to electrical remodeling. For example, activation of PERK in HF and myocardial infarction inhibits Nav1.5 expression ⁴⁰ , ⁴¹ and downregulates multiple K⁺ channels, ⁴⁰ , ⁴¹ , ⁴² whereas inhibition of PERK has a protective effect on cardiovascular diseases and reduces the risk of VAs. ⁴⁰ Under physiological conditions, inhibition of IRE1α downregulates certain channels and prolongs APD, ⁴² indicating that the IRE1α branch helps maintain the expression levels of channel proteins. However, when cardiomyocytes are under ERS, APD is prolonged, and inhibition of the IRE1α branch increases the expression of certain channel proteins, shortening APD. ⁴² This suggests that under pathological conditions, activation of ERS downregulates certain channel proteins, promoting the occurrence and progression of arrhythmias. Our study also confirmed that treatment with the ERS inhibitor 4‐PBA upregulated the expression of multiple ion channels critical for maintaining normal myocardial rhythm. This was accompanied by attenuated histopathological remodeling, improved cardiac function, and inhibition of the CaMKII/NF‐κB pathway activation, collectively leading to a reduction in VAs after HF. Overall, this evidence solidifies that Srprb overexpression promotes VAs by activating ERS.

The TLR4 signaling pathway is considered one of the primary triggers of inflammation induced by pressure overload. Previous studies have confirmed that TLR4, as an upstream signal of cardiac CaMKII, plays a crucial role in HF‐related cardiac remodeling. ⁴³ Myocardial Ca²⁺ homeostasis is regulated by the CaMKII‐PLB‐SERCA 2a axis, and abnormal activation of CaMKII promotes the phosphorylation of PLB and inhibits the expression of SERCA 2a, leading to imbalance in excitation‐contraction coupling and the occurrence of life‐threatening VAs. ¹⁷ , ⁴⁴ Under HF stress conditions, activation of CaMKII mediates damage to ion channels and disruption of Ca²⁺ homeostasis proteins, driving the expression of inflammation‐related factors (TNF‐α, IL‐6, IL‐1β) in the occurrence of arrhythmias. ⁴⁴ , ⁴⁵ Therefore, we believe that TLR4 plays an important role in arrhythmias. Further experimental validation revealed that the TLR4 inhibitor TAK‐242 restored the expression and localization of key ion channels, ameliorated histopathological remodeling, and improved cardiac function, thereby alleviating VAs following HF. It is well established that TLR4 acts as a upstream regulator of both CaMKII and NF‐κB signaling pathways, playing a critical role in HF‐associated cardiac remodeling. ⁴⁶ Both CaMKII and NF‐κB are also recognized as key regulators of VAs. ⁴⁷ Consistent with this established role, we found that TAK‐242 significantly suppressed the activation of the CaMKII/NF‐κB signaling pathway. This inhibition subsequently mitigated both myocardial structural and electrical remodeling, leading to a reduced incidence of VAs.

Our data position Srprb as an upstream ER‐anchored checkpoint that integrates 2 major arrhythmogenic axes—endoplasmic‐reticulum stress and TLR4/CaMKII/NF‐κB signaling—thereby enabling concurrent suppression of ERS and inflammatory–calcium‐handling pathways. Consistent with this nodal role, Srprb knockdown (1) regressed adverse structural remodeling (hypertrophy/fibrosis), (2) normalized electrical remodeling (restored Nav1.5, Kv subunits, Cav1.2, and Cx43), and (3) improved in vivo electrophysiology (shorter APD/ALT, longer ERP) with reduced VA inducibility, while exerting minimal effects under physiological conditions. By comparison, single‐axis interventions (eg, TLR4 or ERS inhibition alone) partially phenocopied the benefits but did not recapitulate the breadth of substrate modification seen with Srprb modulation, and conventional antiarrhythmic or device‐based strategies may lessen events without reversing the proarrhythmic substrate. Together, these findings suggest that Srprb represents a mechanistically convergent and disease‐selective target with potential for combination therapies (eg, alongside TLR4 or ERS inhibitors) to achieve deeper substrate control in HF‐associated VAs. Therefore, the research clarifies the impact and mechanism of Srprb on VAs in HF, providing a new theoretical basis for the clinical prevention and treatment of sudden death after HF, and has certain guiding value for the development of new prevention and treatment strategies.

CONCLUSIONS

In conclusion, our findings highlight the crucial role of Srprb in exacerbating susceptibility to VAs in HF mice. Knockdown of Srprb ameliorated pressure overload‐induced ventricular structural and electrical remodeling in HF mice, inhibiting the occurrence of VAs. Overexpression of Srprb deteriorated pressure overload‐induced ventricular structural and electrical remodeling, facilitating the occurrence of VAs. These effects are related to the regulation of ERS and the TLR4/CaMKII/NF‐κB signaling pathway. Therefore, targeting the Srprb may offer a potential therapeutic strategy for VAs in HF.

Sources of Funding

This work was supported by grants from the Natural Science Foundation of Hubei Province of China (No. 2022CFB708).

Disclosures

None.

Supporting information

Data S1. Supplemental Methods

Figures S1–S11

Unedited gels