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

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: Cardiac fibrosis; Extracellular matrix; Myocardial infarction; Remodelling; Serine proteases; Serpin.

Figure 1

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.

Figure 2

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).

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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).