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. 2024 Apr 26;120(8):943–953. doi: 10.1093/cvr/cvae075

Serine protease inhibitor, SerpinA3n, regulates cardiac remodelling after myocardial infarction

Qihao Sun 1,2,3,4,5,6,#, Wei Chen 7,8,9,10,11,12,#, Rimao Wu 13,14,15,16,17,18, Bo Tao 19,20,21,22,23,24, Ping Wang 25,26,27,28,29,30, Baiming Sun 31,32,33,34,35,36, Juan F Alvarez 37,38,39,40,41,42, Feiyang Ma 43, David Ceja Galindo 44, Sean P Maroney 45, Anthony J Saviola 46, Kirk C Hansen 47, Shen Li 48,49,50,51,52,53,✉,3, Arjun Deb 54,55,56,57,58,59,✉
PMCID: PMC12098007  PMID: 38666458

Abstract

Aims

Following myocardial infarction (MI), the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix (ECM) by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure, and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors, and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human MI as well as non-ischaemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodelling is poorly understood. The aim of this study was to understand the role of Serpins in regulating scar formation after MI.

Methods and results

Using a SerpinA3n conditional knockout mice model, we observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homologue of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix, and significantly worse post-infarct cardiac function. Single-cell transcriptomics complemented with histology in SerpinA3n-deficient animals demonstrated increased inflammation, adverse myocyte hypertrophy, and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross-linking of ECM peptides consistent with increased proteolysis in SerpinA3n-deficient animals.

Conclusion

Our study demonstrates a hitherto unappreciated causal role of Serpins in regulating matrix function and post-infarct cardiac remodelling.

Keywords: Serpin, Serine proteases, Extracellular matrix, Remodelling, Myocardial infarction, Cardiac fibrosis

Time of primary review: 35 days

1. Introduction

The mammalian heart possesses a poor ability to regenerate after myocardial injury and heals via a fibrotic repair response.1 Following myocyte necrosis, inflammatory cells recruited to the site of injury release cytokines that activate fibroblasts.2–5 Activated fibroblasts or myofibroblasts deposit extracellular matrix (ECM) proteins that organize to form scar tissue in the infarcted region.6 Scar tissue is non-contractile, increases haemodynamic stress on the remaining viable myocardium, and leads to adverse ventricular remodelling characterized by the development of cardiac chamber dilatation, hypertrophy, and heart failure. The nascent scar deposited in the infarcted region is also susceptible to proteolytic degradation by a variety of proteases that are released primarily by inflammatory cells.7,8 Excessive proteolytic activity can lead to disruption of scar organization and worsen cardiac remodelling,9 and high collagen turnover has been described in human heart failure.10 The balance between matrix deposition and degradation in part determines the degree of ECM deposition as well as organization of scar tissue. Unchecked protease activity can also worsen inflammation by activation of protease-activated receptors as well as by releasing small peptide fragments of the matrix (matrikines) that can act as a ligand for further recruitment of inflammatory cells.

Excessive protease activity in the infarcted region is regulated by a system of protease inhibitors. Like proteases, protease inhibitors can be also classified according to the mechanism employed at the active site of protease inhibition and have been divided into five groups (serine, threonine, cysteine, aspartyl, and metalloproteinase inhibitors).11

Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of protease inhibitors and serve as critical regulatory molecules in regulating and attenuating excessive inflammatory and protease activity in tissue.12,13 Serpins are evolutionarily conserved and found in genomes of all kingdoms with many murine Serpin genes orthologous to human Serpin genes.13 The Serpin superfamily is divided into groups called clades (A–P) according to their sequence similarity, and the SerpinA group of Serpins is secreted or extracellular protease inhibitors resembling anti-trypsin like activity.12 Circulating SerpinA3 levels have been shown to improve prognostication in patients with non-ischaemic heart failure.14 Moreover, emerging clinical studies in individuals with acute myocardial infarction (MI) have shown that SerpinA3 levels significantly increased in the circulation after acute MI and higher SerpinA3 levels were associated with increased frequency of major adverse cardiovascular events (MACE) within the next 9 months of follow up.15 However little is known about the role of Serpins in regulating cardiac remodelling after acute cardiac injury. In this body of work, we demonstrate that Serpins are robustly expressed in the infarcted murine heart, and using conditional genetic loss-of-function approaches, complemented by single-cell transcriptomics and proteomics of scar tissue, we demonstrate the pivotal role of SerpinA3n (murine orthologue of human SerpinA3) in regulating post-infarct cardiac inflammation, fibrosis, and cardiac contractile function.

2. Methods

2.1. Animal care

All animal experiments were conducted in accordance with protocols approved by the Animal Research Committee, University of California, Los Angeles (UCLA). The animals were housed at the UCLA vivarium in accordance with the guidelines set by the American Association for Accreditation of Laboratory Animal Care. All animals belonged to the C57BL/6 strain, were healthy and immune-free, and had no prior exposure to drugs or tests. Furthermore, they had not been used in any other experimental procedures. Littermates were used as controls for all experiments.

Animal care and procedures in this manuscript conforms to the guidelines for the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Animals were euthanized before necropsy. The animals were exposed to 5% isoflurane for ∼3 min until they no longer responded to painful stimuli, after which they were euthanized by cervical dislocation.

Expanded materials and methods can be found within the supplementary material.

3. Results

3.1. Serpins are robustly up-regulated after acute MI

To determine the expression of Serpins after MI, we analysed transcriptomic data sets of hearts of adult male and female mice (C57Bl/6) subjected to acute MI 16 and observed robust expression of Serpins in the injured heart (compared with the uninjured heart) from post-injury Day 3 to Day 42. Out of the differentially up-regulated Serpins, we observed that SerpinA3n was up-regulated early after injury and its expression remained elevated at Day 42 after injury (Figure 1A). The average fold change in SerpinA3n expression was amongst the highest out of all the Serpins examined (see Supplementary material online, Table S1). The human orthologue of SerpinA3n (SerpinA3) has been associated in several clinical studies with outcomes after MI or human heart failure.14 Increased circulating SerpinA3 levels were associated with increased risk of adverse clinical events in a cohort of individuals with MI,15 coronary artery disease,17 and heart failure.14 Considering dramatic up-regulation of SerpinA3n in animal models of cardiac injury and clinical studies implicating a potential causal role of SerpinA3 in human MI or heart failure, we focused on the pathophysiological role of SerpinA3n in regulating post-infarct cardiac function. To confirm our bulk RNA-seq results, we first performed quantitative polymerase chain reaction (qPCR) on injured and uninjured hearts of wild-type mice subjected to MI via permanent ligation of the left anterior descending coronary artery and observed SerpinA3n expression to be increased in the infarcted heart by an order of magnitude with peak expression occurring at Day 14 after injury (Figure 1B). Western blotting confirmed increased SerpinA3n protein levels in the infarcted region at Day 14 after injury compared with uninjured regions of the heart (Figure 1C and D). We next analysed single-cell transcriptomics data sets of the mouse heart at 7 days after MI16 to determine the identity of population of cells that express SerpinA3n in the infarcted heart and observed that SerpinA3n was primarily expressed by the fibroblast population that co-expressed type I collagen genes (Figure 1E). Immunofluorescent staining demonstrated minimal expression of SerpinA3n in the uninjured heart (Figure 1F) but robust expression of SerpinA3n, primarily localized to the region of cardiac injury (Figure 1G). As SerpinA3n is a secreted protein, double immunofluorescent staining demonstrated the presence of abundant SerpinA3n around vimentin positive cardiac fibroblasts in the infarcted region (Figure 1H and I). These observations demonstrate that Serpins and in particular SerpinA3n is robustly expressed after cardiac injury by cardiac fibroblasts and is localized to the infarcted region.

Figure 1.

Figure 1

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SerpinA3n is expressed in the infarcted region of the heart by cardiac fibroblasts. (A) Heatmap of genes in Serpin family at different time points in the uninjured and injured regions of the heart after MI (n = 4 in each time point, P < 0.05 for all genes differentially expressed) (B) Relative expression levels of SerpinA3n in injured regions of the heart compared with uninjured at Day 3, 7, 14, and 21 days by qPCR (n = 6). (C) Representative immunoblots of SerpinA3n in uninjured and injured regions at 14 days post-MI (n = 4, uncropped immunoblots are available in Figure S13). (D) Densitometric quantification of SerpinA3n protein level normalized to GAPDH in uninjured and injured regions of the heart (n = 4). (E) Single-cell RNA-seq of non-myocytes at Day 7 after MI demonstrating cell phenotypes in clusters and the distribution of SerpinA3n and Col1a2 (n = 3). (F and G) H&E staining and immunostaining for SerpinA3n in the uninjured (F) and injured (G) regions at Day 7 after MI. Scale bar: 10 μm (high magnification). Low magnification: ×10. (H and I) Immunofluorescent staining for SerpinA3n, vimentin, and 4′,6-diamidino-2-phenylindole (DAPI) in sections from (H) uninjured and (I) injured region at 7 days after MI. Scale bar, 10 µm (representative images; n = 3, arrows point to SerpinA3n). Both male and female animals were used, and all data are shown as mean ± standard error of the mean (SEM); *P < 0.05, two-tailed Student’s t-test.

3.2. Genetic deletion of SerpinA3n leads to worsening post-infarct cardiac function

To determine the physiologic role of SerpinA3n in regulating cardiac repair after MI, we adopted a genetic loss-of-function approach. As SerpinA3n is primarily expressed by cardiac fibroblasts, we deleted SerpinA3n in cardiac fibroblasts by using Cre-lox approaches. For this purpose, we crossed animals with a fibroblast-driven Cre (Col1a2CreERT2) with animals that had both SerpinA3n alleles floxed.18 The Col1a2CreERT2 animal has a tamoxifen-inducible Cre recombinase upstream of a type I collagen enhancer element and has been used by us and others for genetic labelling of fibroblasts and fibroblast-specific deletion of genes.16,19–21 Progeny animals harbouring both alleles were administered tamoxifen for 12 days as described16 starting 5 days prior to injury and continuing for 7 days after injury to maximize capture of Col1a2-expressing cardiac fibroblasts and delete SerpinA3n (SerpinA3n conditional knockout or SerpinA3n CKO) (Figure 2A). To determine the efficiency of labelling with the Col1a2Cre driver, we crossed Cola2CreERT2 animals with the lineage reporter Rosa26tdtomato animals. Progeny animals were administered tamoxifen in an identical manner and subjected to MI. Hearts were harvested at 7 days after and enzymatically digested, and the non-myocyte layer was subjected to flow cytometry. We observed that the efficiency of fibroblast labelling was ∼71% (see Supplementary material online, Figure S1) and is consistent with previous observations made by us.20 Western blotting confirmed the deletion of SerpinA3n in the injured region (Figure 2B). Control animals (Cre-:SerpinA3nfl/fl) demonstrated robust up-regulation of SerpinA3n protein in the injured cardiac region, but no significant increase in SerpinA3n protein was observed in the SerpinA3n CKO animals at 7 days post-injury (Figure 2B and C). Quantitative densitometry revelated decrease of SerpinA3n protein in the SerpinA3n CKO animals by 74% compared with control littermates (Figure 2C). We next performed serial echocardiography to determine changes in cardiac function. Both B and M mode echocardiography demonstrated significantly greater dilatation of the cardiac chambers in the SerpinA3n CKO animals at 7 days post-injury with reduced cardiac contractile function (Figure 2D). Quantitative determination of cardiac contractile function demonstrated significant worsening of ejection fraction (EF) (control 41.66% ± 3.96% vs. SerpinA3n CKO 26.27% ± 3.50%) and fractional shortening (control 21.12% ± 2.22% vs. SerpinA3n CKO 12.55% ± 1.88%) in the SerpinA3n CKO animals as early as 7 days post-injury, and defects in cardiac contractile function persisted throughout the next 4 weeks (Figure 2E). The cardiac chambers in both systole (left ventricular internal dimension, LVIDs) and diastole (LVIDd) were also significantly more enlarged in SerpinA3n CKO animals compared with control animals (Figure 2E). In particular, the LVIDs in the SerpinA3n CKO animals was ∼36% greater than in the control animals at 4 weeks after cardiac injury (Figure 2E). We next examined the severity of worsening cardiac contractile function in the SerpinA3n CKO animals compared with controls and classified the severity of post-infarct heart failure as mild (EF > 40%), moderate (EF 20–40%), or severe (<20%), as described by us.19Both animals were subjected to similar degree of cardiac injury. Fifteen per cent of the control animals exhibited severe post-infarct heart failure, but almost 73% of the SerpinA3n CKO animals exhibited severe post-infarct heart failure (Figure 2F). As an additional control, we also examined Col1a2CreERT2:SerpinA3nwt/wt animals, administered them tamoxifen in an identical manner, and observed no difference in post-infarct function between Cre-:SerpinA3nfl/fl and Col1a2CreERT2:SerpinA3nwt/wt animals (see Supplementary material online, Figure S2). To eliminate potential effects of tamoxifen, we also examined another control for tamoxifen. We compared cardiac function of Col1a2CreERT2:SerpinA3nfl/fl animals subjected to MI but not treated with tamoxifen to the Cre-:SerpinA3nfl/fl treated with tamoxifen, and post-MI function was no different between these groups (see Supplementary material online, Figure S3). SerpinA3nCKO animals that were not subjected to cardiac injury did not demonstrate any cardiac phenotype compared with uninjured littermates (see Supplementary material online, Figure S4). We have previously demonstrated that the injured region of the heart becomes transcriptionally mature within 2 weeks after injury and transcriptional changes play a pivotal effect in regulating cardiac repair within the first 2 weeks after injury.16 To determine whether SerpinA3n contributes early to cardiac repair early after injury, we changed the timing of deletion of SerpinA3n by administering tamoxifen from Day 14 onwards in a subset of animals. Control SerpinA3nCKO animals received tamoxifen from Day −5 to Day 7 post-MI as described. We observed that early deletion of SerpinA3n dramatically worsened post-infarct cardiac function compared with deletion of SerpinA3n after the first 2 weeks, thus demonstrating the role of SerpinA3n early in cardiac remodelling (see Supplementary material online, Figure S5). Inflammatory cells that are recruited to the infarcted region within days of injury are the predominant source of proteases, and these results are consistent with the functional effects of SerpinA3n in inhibiting excessive protease activity arising from inflammatory activity early in cardiac repair. We also analysed the effects of SerpinA3n deletion in male vs. female animals and observed that both male and female animals exhibited a significant decline in post-infarct function (see Supplementary material online, Figure S6). Taken together, these observations demonstrate that SerpinA3n plays a pivotal role in cardiac remodelling and deficiency of SerpinA3n is associated with worsening cardiac repair and cardiac function. Post-infarct hypertrophy is known to be an adverse prognosticator of post-infarct outcomes, and we observed significantly higher heart weight/body weight ratios in the SerpinA3n CKO animals compared with the control animals with no change in body weight alone (Figure 2G). As post-infarct hypertrophy is known to vary between male and female mice, we analysed the heart weight/body weight ratios by sex and observed that male animals demonstrated a significant increase in HW/BW compared with female animals (see Supplementary material online, Figure S7). SerpinA3n is a serine protease inhibitor and binds to substrate proteases leading to irreversible inactivation of the enzyme complex. SerpinA3n is known to inhibit several serine proteases including granzyme B which is released by a variety of cytotoxic and non-cytotoxic cells.22 Granzyme B is known to be elevated after acute cardiac and neurological injury and contributes to matrix breakdown, apoptosis of target organs, worsening inflammation, and ECM remodelling.23 We thus measured granzyme B activity in the infarcted region at 7 days post-injury and observed that granzyme B activity was significantly increased by 26.5% ± 7.4% in the infarcted region of SerpinA3n CKO animals compared with control animals (Figure 2H). These observations suggest that decreased SerpinA3n in the infarcted heart is associated with adverse increase in granzyme B activity that is a known substrate/target of SerpinA3n.

Figure 2.

Figure 2

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SerpinA3n deletion in cardiac fibroblasts leads to worsening of post-infarct heart function. (A) Experimental outline for tamoxifen administration to generate SerpinA3n CKO animals. (B) Western blotting for SerpinA3n in infarcted hearts of control and SerpinA3 CKO animals and (C) quantitation for SerpinA3n expression in control and CKO mice at 7 days after MI (n = 5, uncropped immunoblots are available in Figure S13). (D) B and M mode echocardiography of littermate control and SerpinA3n CKO heart at 4 weeks after MI (red arrow, diastole diameter; yellow arrow, systolic diameter). (E) EF, fFS, as well as LV chamber size (LVID) in systole and diastole 4 weeks post-cardiac injury in control and SerpinA3n CKO animals (n = 25/control and 20/CKO at baseline and 1 week, n = 14/control and 10/CKO at 2 weeks, and n = 20/control and 15/CKO at 4 weeks). (F) Fraction of animals with mild, moderate, and severe reduction in EF. (G) HW, BW, and HW/BW ratios at 4 weeks post-MI (n = 19/control, n = 15/CKO). (H) Granzyme B activity in hearts of control and CKO mice at 7 days after MI (n = 4). Both male and female were used, and all data are shown as means ± SEM. **P < 0.01; *P < 0.05 by two-tailed Student's t-test (C, G, H), or by two-way ANOVA with Sidak's multiple comparisons test (E).

3.3. SerpinA3n CKO animals display disordered scar architecture, worsened post-infarct remodelling, and inflammation

We next examined the tissues of SerpinA3n CKO animals and observed significantly greater fibrosis both in the mid and apical myocardium (Figure 3A and B). Masson trichrome staining demonstrated loosely compacted scar tissue in the SerpinA3n CKO animals (Figure 3A). We next stratified the degree of fibrosis according to the ratio of fibrotic area to the left ventricular surface area, with <20% being mild fibrosis, 20–40% as moderate, and >40% being considered severe. In the control animals, we did not see severe degrees of fibrosis at 7 days after injury, but 25% of the SerpinA3n CKO animals exhibited severe fibrosis (Figure 3C). The degree of myocyte hypertrophy determined by assessment of myocyte surface area on histology was also significantly greater in the SerpinA3n CKO animals (Figure 3D). These observations taken together are consistent with worsening of cardiac function and chamber dilatation seen on echocardiography. As excessive protease and granzyme activity can worsen inflammation, we performed immunostaining for inflammatory cells at 3 days after cardiac injury and observed a significant increase in CD45- and CD68-expressing inflammatory cells (Figure 3E and F). We did not see any difference in the number of CD31 + endothelial cells at 4 weeks after cardiac injury (Figure 3E and F). We next determined whether the effects of increased fibrosis in the SerpinA3n CKO animals could be partly secondary to increased myocyte cell death. We harvested control and SerpinA3nCKO animals and performed TUNEL staining along with myocyte markers to detect myocyte apoptosis but did not detect increased myocyte cell death in the SerpinA3nCKO animals (see Supplementary material online, Figure S8). These findings suggest that the effects on fibrosis are likely secondary to the effects of SerpinA3n on cardiac remodelling.

Figure 3.

Figure 3

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Genetic deletion of SerpinA3n leads to increased fibrosis, hypertrophy, and inflammation after MI. (A) Histological sections of the apex and mid ventricle of control and SerpinA3n CKO animals stained with Masson trichrome. Scale bar: 200 μm. (B) Quantification of the fibrotic area in infarcted hearts (n = 16). (C) Fraction of animals with mild, moderate, and severe scarring at 4 weeks post-MI. (D) Representative image of cardiac troponin I and WGA and quantification of cardiomyocyte cross sectional area measured at 7 days post-MI (n = 7 in control and n = 6 in CKO). Scale bar: 10 μm. (E) Immunostaining of CD45 (nucleated haematopoietic cells, arrowheads), CD68 (macrophages, arrowheads), and CD31 (endothelial, arrowheads) in control and SerpinA3n CKO animals at 7 days post-MI. (F) Quantification of the percentage of CD45+ cells, CD68+ cells, and capillary density. Scale bar, 10 µm (n = 7 in control and n = 5 in CKO). Both male and female were used, and all data are shown as mean ± SEM; **P < 0.01; *P < 0.05, two-tailed Student’s t-test.

3.4. Single-cell transcriptomics demonstrates increased inflammatory gene expression in fibroblasts and myeloid cells in SerpinA3n CKO hearts after cardiac injury

Dysregulated or unchecked protease activity is known to induce apoptosis, worsen inflammation, and lead to degradation of ECM proteins that can further worsen inflammation or induce other remodelling changes.7 Such changes can lead to primary or secondary transcriptional changes in various cell populations in the injured heart. We subjected the control and SerpinA3n CKO animals to MI, harvested the hearts at Day 7 after injury, isolated the non-myocyte fraction, and performed single-cell transcriptomics by using the 10X Genomics platform. UMAP projections demonstrated the characteristic cell populations in the heart at 7 days after injury (Figure 4A, Supplementary material online, Figure S9). We examined the distribution of wild-type and SerpinA3n CKO genotypes and did not observe any significant differences in cell numbers between these two genotypes across all population of cells in the injured heart (Figure 4B and C). Violin plots of SerpinA3n demonstrated significantly lower expression of SerpinA3n in the fibroblast population (Figure 4D). In support of our findings, examination of human data sets also demonstrated the expression of SerpinA3 in cardiac fibroblasts in the uninjured heart as well as in infarcted human hearts (see Supplementary material online, Figure S10). As SerpinA3n was predominantly expressed by fibroblasts, we next performed subcluster analysis to determine the identity or subtype of fibroblasts expressing SerpinA3n. We stratified the fibroblasts into 7 subclusters (Figure 4E) based on distinct transcriptional signatures (see Supplementary material online, Figure  S11A). The genotypes (SerpinA3nCKO and littermates) were uniformly distributed across the fibroblast subclusters 0–5, and SerpinA3n exhibited lower expression in subcluster 6 (Figure 4F and G). SerpinA3n was expressed by fibroblast subclusters that were enriched in fibroblast marker vimentin (Figure 4H) and myofibroblast markers, alpha smooth muscle actin (Acta 2), and perisotin (Postn) (Figure 4I and J). Fibroblasts in cluster 6 that exhibited low expression of SerpinA3n also exhibited low expression of myofibroblast markers Acta2, transgelin (Tagln), and Myl9, a smooth muscle isoform enriched in smooth muscle cells (Figure 4I and J, Supplementary material online, Figure  S11A). We next examined the genes up-regulated in cardiac fibroblasts in SerpinA3n CKO hearts. Gene Ontology (GO) analysis demonstrated increased expression of ECM deposition pathways in cardiac fibroblasts consistent with increased fibrosis (Figure 4K). We also observed expression of inflammatory genes in serpinA3n CKO fibroblasts such as members of the CXCl family (Cxcl1, Cxcl5) which regulate recruitment of inflammatory cells, inflammatory cytokines (IL6), and the potent immunomodulatory transcription factor NFkb1 (see Supplementary material online, Figure  S11B). Analysis of differentially up-regulated genes in myeloid cells similarly demonstrated up-regulation of pro-inflammatory pathways with TNF signalling and Nf-kB pathway genes being the most significantly up-regulated (see Supplementary material online, Figure  S11C). Dot blot analysis of TNF signalling pathway genes demonstrated again members of the chemokine family (Cxcl1, Cxcl2), TNF, as well as IL1b and Nfkb1 to be up-regulated (see Supplementary material online, Figure  S11D). Taken together, these observations demonstrate and are consistent with the histological data demonstrating increased fibrosis and inflammation in the infarcted heart of SerpinA3n CKO animals.

Figure 4.

Figure 4

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Single-cell RNA sequencing of non-myocytes in control and SerpinA3n CKO animals at 7 days post-cardiac injury. (A) UMAP demonstrating different phenotypes of non-myocyte cell clusters in the injured heart and (B) distribution of control and SerpinA3n CKO genotypes across these clusters. (C) Quantitation from single-cell RNA-seq (% cells) of different types of non-myocyte in the SerpinA3n CKO and control animals at 7 days following injury (n = 3 animals/group; equal number of males and females were used between each group). (D) Violin plot demonstrating SerpinA3n expression (**adj P-value < 4e−280) significantly reduced in cardiac fibroblasts of SerpinA3n CKO vs. control animals. (E) UMAP demonstrating different phenotypes of fibroblast clusters in the injured heart and (F) distribution of control and SerpinA3n CKO cells across these clusters. (G) UMAP demonstrating the distribution of SerpinA3n, (H) fibroblast marker vimentin, and (I) myofibroblast marker Acta2. (J) Postn in the subclusters of fibroblast. (K) GO analysis of main pathways differentially up-regulated in cardiac fibroblasts in SerpinA3n CKO vs. control animals.

To determine transcriptional changes in myocytes, we harvested nuclei from the infarcted hearts (Day 7 post-injury) of control and SerpinA3n CKO animals and subjected the nuclei to single nuclear transcriptomics using the 10X Genomics platform. UMAP demonstrated the canonical cell population in the heart (see Supplementary material online, Figure  S12A), and there were no differences in cell numbers between control and SerpinA3n CKO genotypes across these cell population (see Supplementary material online, Figure  S12B). GO analysis of genes differentially up-regulated in myocytes demonstrated expression of pro-hypertrophic pathways and dilated cardiomyopathic pathways (Figure 5A). Post-infarct hypertrophy is an adverse prognostic indicator, and violin plots demonstrated increased expression of pro-hypertrophic sarcomeric genes such as Tnnc1, Tpm1 Ttn, and Myh6 in cardiomyocytes (Figure 5B). Genes down-regulated in cardiomyocytes included those primarily belonging to the focal adhesion pathway and actin cytoskeletal pathway (Figure 5C). Members of the integrin family (Itgb1, Itgb5, Itgb6, Itga7) or genes with adaptor function (Tln1) were down-regulated (Figure 5D). Integrins such as Itga7b1 act as a laminin receptor and bridges the ECM to sarcomeric proteins,24 and down-regulation of such proteins in the myocyte could potentially have deleterious consequences for force generation. Loss of integrin-linked kinase signalling as well as expression of various focal adhesion genes could lead to worsening myocyte contractile performance through disruption of attachment of sarcomeric proteins to the ECM (Figure 5D). Taken together, these observations are consistent with the principal phenotype of increased inflammation and decreased cardiac contraction seen in infarcted hearts of SerpinA3n CKO animals.

Figure 5.

Figure 5

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Single-nuclei RNA sequencing of cardiomyocytes in control and SerpinA3n CKO animals at 7 days post-cardiac injury. (A) GO analysis of main pathways up-regulated in cardiomyocytes in SerpinA3n CKO vs. control animals (n = 3 animals/group; equal number of males and females were used between each group). (B) Violin plot demonstrating increased expression of pro-hypertrophic sarcomeric genes in cardiomyocytes in SerpinA3n CKO animals (**adj P-value < 1e−14). (C) GO analysis of main pathways down-regulated in cardiomyocytes in SerpinA3n CKO vs. control animals. (D) Dot plot demonstrating focal adhesion genes significantly down-regulated in SerpinA3n CKO vs. control CFs 7 days after cardiac injury.

3.5. Proteomic characterization of scar tissue demonstrates distinct signatures in infarcted regions of control and SerpinA3n CKO animals

We have shown that genetic deletion of SerpinA3n worsens inflammation and post-infarct cardiac performance. Serine proteases degrade ECM proteins, and we performed proteomic analysis of the infarcted tissue of control and SerpinA3n CKO animals. The infarcted region was dissected, homogenized, and then subjected to protein extraction. Following enzymatic and chemical digestion, peptides were recovered and analysed by liquid chromatography–mass spectrometry (LC/MS). Principal component analysis demonstrated that proteins in the hearts of SerpinA3n CKO and control wild-type animals did not significantly differ in the uninjured heart but the proteomes of scar tissue of SerpinA3n CKO and control wild-type animals demonstrated a wide spatial separation (Figure 6A). Proteins that were observed to be differentially up-regulated in the infarcted hearts of SerpinA3n CKO animals related to pathways regulating collagenous ECM and inflammation (Figure 6B). Proteins down-regulated in SerpinA3n CKO infarcted tissue included those related to cross-linking consistent with unchecked protease activity (Figure 6C). A graphical representation of differentially expressed proteins in both the SerpinA3n CKO and wild-type control animal after injury demonstrated significant differences in key ECM proteins that are known to participate in wound healing such as Col XIV and Col X1 (Figure 6D). Col14a1 was down-regulated in injured hearts of SerpinA3n CKO compared with wild-type controls, and Col XIV is a fibril-associated collagen that is known to play critical roles for maintaining a collagen network under biomechanical stress.25 Col XI is similarly a fibril-forming collagen that regulates biomechanical properties of tissue, and SerpinA3n CKO animals demonstrated decreased expression of Col11a1. Loss of Col XI has been associated with disruption of normal mechanical properties of tissue.26 The mechanical properties of the nascent scar are important for optimal cardiac repair, and the proteomic analysis demonstrates altered expression of collagens and other matrix proteins that could be potentially deleterious to and underlie defective repair in SerpinA3n CKO animals. Taken together, these observations demonstrate proteins related to the matrix and cross-linking pathways are differentially expressed in the scar tissue of SerpinA3n CKO animals and could underlie defects in matrix architecture and cardiac dilatation, seen in these animals after cardiac injury.

Figure 6.

Figure 6

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Proteomic analysis of the infarcted regions of control and SerpinA3n CKO animals at 7 days post-cardiac injury. (A) Principal component analysis of proteome expression in infarcted and uninjured hearts of Ctrl and SerpinA3 CKO mice demonstrating differing proteomic expression profiles of injured hearts if SerpinA3CKO vs. Ctrl animals. Pathway analysis of proteins (B) that are significantly up-regulated and (C) down-regulated in SerpinA3n CKO infarcted scar tissue. (D) Graphical representation of genes up-regulated in SerpinA3n CKO animals and control animals after injury compared with respective uninjured hearts. Blue circles represent collagen proteins, and gray circles represent other matrix proteins. The red dotted line demonstrates proteins that exhibit equal changes in both SerpinA3n CKO and Ctrl animals after injury (n = 3 animals/group; both male and female were used, *P < 0.05).

4. Discussion

Serine protease inhibitors represent the largest family of protease inhibitors in mammals, are evolutionarily conserved, and subserve critical roles from regulation of coagulation to inflammation and matrix function. Although the role of Serpins is well described in the regulation of thrombosis, the effects of specific Serpins on cardiac disease are less clear. However, emerging biochemical and clinical evidence has implicated Serpins in key aspects of cardiovascular physiology. For instance, SerpinE1 (plasminogen activator inhibitor-1) is known to exert functions outside the coagulation cascade and promotes cardiac fibrosis by stimulating TGFbeta pathways, and animals deficient in PAI-1 are protected from angiotensin II-induced cardiac fibrosis.27In a recent study examining the prognostic significance of SerpinA3 levels in human individuals with heart failure, SerpinA3 transcripts were present at a higher level in the hearts of non-survivors vs. survivors and SerpinA3 levels were associated with worse cardiovascular mortality, demonstrating a strong association between SerpinA3 and human heart failure.14 Although it is difficult to distinguish causality vs. compensatory expression of candidate genes from such human studies, our animal studies demonstrate a definitive biological role of SerpinA3n, the murine homologue of human SerpinA3 in regulating cardiac remodelling and post-infarct heart failure. A recent proteomic study in humans demonstrated increased SerpinA3 levels in the circulation and in patients with ischaemic heart failure.28 In the infarcted heart, proteases are predominantly expressed by inflammatory cells and we observed that fibroblasts were the primary source of SerpinA3n expression. Inflammatory infiltrate decreases significantly in the infarcted heart after 2 weeks, and consistent with this, deletion of SerpinA3n after 2 weeks did not significantly worsen post-MI heart function. Deficiency of SerpinA3n led to increased activity of substrate protease granzyme B, and this was associated with poor matrix architecture, increased amount of matrix, and decreased cardiac function. Our study did not definitively identify the substrates of SerpinA3n that potentially exhibited increased proteolytic activity in the absence of SerpinA3n. However cathepsins, elastases, and chymotrypsin have been shown to be inactivated by SerpinA3n, and the large changes in the proteome observed by us likely reflect both primary and secondary changes affecting several proteolytic cascades in the absence of SerpinA3n. The increased amount of fibrosis is likely secondary to increased compensatory output of ECM proteins following increased proteolytic activity and not secondary to effects on myocyte cell death. Increased protease activity can worsen inflammation via release of ECM degradation products (matrikines) that are known to recruit inflammatory cells, in part through activation of chemokine receptors. Proteases can also worsen inflammation via activation of protease-activated receptors (PAR). Deficiency of SerpinA3n in our study resulted in increased inflammatory signatures with significantly greater accumulation of inflammatory cells. Myocyte cell death and apoptosis can worsen with inflammation, and our study suggests that increased inflammation along with the loss of focal adhesion and integrin proteins that are required for efficient cardiac force generation could underlie defects in cardiac contraction seen in SerpinA3n CKO animals. We have previously demonstrated that a defective matrix can lead to adverse cardiac remodelling and cardiac dilatation.16 Increased protease activity along with increased turnover of the matrix could have resulted in defective matrix function and cardiac dilatation in SerpinA3n CKO animals following MI. The myocyte hypertrophy seen in the SerpinA3n CKO animals may be compensatory to the decreased expression of focal adhesion and other key integrin proteins that are necessary for efficient myocyte force contraction.

In summary, our study highlights for the first time the role of Serpins and in particular SerpinA3n in regulating post-infarct cardiac remodelling. A large number of Serpins were differentially up-regulated in the heart after cardiac injury, and while we focus here on SerpinA3n, other Serpins not investigated by us could be playing important roles in remodelling. In summary, our study demonstrates the pleiotropic roles SerpinA3n plays in regulating transcriptional and cellular responses of both myocytes and non-myocytes in the infarcted heart and its critical necessity for optimal cardiac wound healing.

Supplementary Material

cvae075_Supplementary_Data
cvae075_supplementary_data.zip (5.3MB, zip)

Acknowledgements

We thank Dr Andrew Leask, University of Western Ontario, Canada, for providing the Col1a2CreERT2 animal.

Contributor Information

Qihao Sun, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Wei Chen, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Rimao Wu, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Bo Tao, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Ping Wang, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Baiming Sun, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Juan F Alvarez, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Feiyang Ma, Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.

David Ceja Galindo, Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

Sean P Maroney, Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

Anthony J Saviola, Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

Kirk C Hansen, Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.

Shen Li, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Arjun Deb, Division of Cardiology, Department of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Cardiovascular Theme, David Geffen School of Medicine, University of California, 675 Charles E Young Drive South, Los Angeles, California, 90095 CA, USA; Department of Molecular, Cell & Developmental Biology, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, 615 Charles E Young Drive S, Los Angeles, California, 90095 CA, USA; Molecular Biology Institute, University of California, 610 Charles E Young Dr S, Los Angeles, California, 90095 CA, USA; California NanoSystems Institute, University of California, 570 Westwood Plaza, Los Angeles, California, 90095 CA, USA.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Authors’ contributions

W.C. and Q.S. performed the majority of experiments and data analysis. R.W., S.L., and J.F.A. assisted in bench experiments. B.T., P.W., and B.S. performed animal surgeries. F.M. analysed scRNAseq and snRNA-seq. D.C.G., S.P.M., A.J.S., and K.C.H. performed proteomic experiments and data analysis. S.L. coordinated data analysis, experimental design, and supervision as well as manuscript preparation. A.d. conceptualized the project, designed all experiments, supervised all data collection, and wrote the manuscript.

Funding

This study was supported by grants from the National Institutes of Health, USA (HL149658, HL152176, HL149687, AR075867, and DK132735) and Department of Defense, USA (PR190268) to A.d.

Data availability

The data underlying this article are available in the NCBI's Gene Expression Omnibus database at https://www.ncbi.nlm.nih.gov/gds, and can be accessed with GSE236609.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

cvae075_Supplementary_Data
cvae075_supplementary_data.zip (5.3MB, zip)

Data Availability Statement

The data underlying this article are available in the NCBI's Gene Expression Omnibus database at https://www.ncbi.nlm.nih.gov/gds, and can be accessed with GSE236609.

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