Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice

Abstract

Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we test a prevailing hypothesis that NE ruptures trigger the pathological cGAS-STING cytosolic DNA-sensing pathway using a mouse model of Lamin cardiomyopathy. The reduction of Lamin A/C in cardio-myocyte of adult mice causes pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures are followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remains inactive. Deleting cGas or Sting does not rescue cardiomyopathy in the mouse model. The lack of cGAS-STING activation is likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling is activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin cardiomyopathy.

Keywords: CP: Cell biology; ECM; Lamin A/C; Lmna; cGAS STING; dilated cardiomyopathy; extracellular matrix; laminopathy; nuclear envelope rupture; nuclear lamina.

Figure 1.. Lamin A/C reduction in cardiomyocytes causes dilated cardiomyopathy in adult mice

(A) Tamoxifen (Tam) induces Lmna deletion in cardiomyocytes in adult mice. See Figure S1 for related analyses. (B) Lamin A/C immunoblot in isolated cardiomyocytes (top) with signal quantification (bottom). n = 2–3 mice/genotype. (C) Lamin A/C immunohistochemistry (brown) with hematoxylin counterstaining (blue) in mouse heart tissues. Scale bar: 20 μm. (D) Hematoxylin and eosin staining of hearts. Scale bar: 1 mm. (E) Left ventricular ejection fraction with interquartile range (box) measured by echocardiography. p value, Wilcoxon test. n = 5–10 mice/genotype. (F) Kaplan-Meier survival analysis. Wild type (WT) n = 48, Lmna^CKO n = 42. p value, log-rank test. (G) Volcano plot comparing RNA-seq read counts in Lmna^CKO versus WT hearts. Number, upregulated or downregulated gene count. n = 3–7 mice/genotype. (H) Top 10 Gene Ontology (GO) terms overrepresented among differentially expressed genes. p value is computed by Metascape. (I) Transcript abundance in hearts quantified by RNA-seq. TPM, normalized transcripts per million. Bar, mean. n = 5–7 mice/genotype. P, false discovery rate (FDR)-adjusted p value computed by a generalized linear model in DESeq2. (J) Immunofluorescence of heart tissue sections for CD45 (pan-leukocyte, red) and CD68 (macrophage, green). Scale bar: 20 μm. (K) Masson’s trichrome staining of heart sections. Arrow, collagen deposition. Scale bar: 20 mm. (L) Summary.

Figure 2.. Lamin A/C reduction causes localized nuclear envelope rupture in cardiomyocytes

(A) Lamin A/C immunohistochemistry in heart tissue. Arrow, DNA protruded from nuclei. Scale bar: 20 μm. See Figure S2 for related analyses. (B) Top: transmission electron micrograph of heart sections focusing on cardiomyocytes. Bottom: close-up image of the area indicated by rectangle in the top image. Arrow, protruded chromatin. Scale bar: 1 μm. (C) Immunofluorescence for Lamin A/C in isolated cardiomyocytes. Phalloidin stains F-actin. Arrowhead, local loss of Lamin A/C with protruded DNA. Scale bar: 20 μm. (D) Same as (C) but for PCM1 immunofluorescence. (E) Native NLS-tdTomato signals in isolated cardiomyocytes with PCM-1 immunofluorescence. Scale bar: 20 μm. (F) NLS-tdTomato signal intensity in nucleus and cytoplasm of cardiomyocytes (box, interquartile range). Cardiomyocytes are stratified by the presence or absence of local PCM-1 loss at the nuclear envelope. WT n = 51, Lmna^CKO n = 53 (PCM1-loss 34, intact 19) cardiomyocytes. p value, Wilcoxon test. (G) MyoAAV-mediated GFP-icGAS expression in cardiomyocytes in vivo. (H) Native GFP-icGAS signals in isolated cardiomyocytes with PCM1 immunofluorescence. Scale bar: 20 μm. (I) Percentage of cardiomyocytes with GFP-icGAS punctum at nuclear tip (bar, mean). n = 3 mice/genotype. p value, unpaired one-tailed Welch’s t test. (J) Percentage of cardiomyocytes with DNA protrusion from PCM1-lost nuclear tip (bar, mean). n = 3 mice/genotype. p value, same as (I). (K) Percentage of gamma-H2AX-positive nuclei in heart section (bar, mean). n = 4–5 mice/genotype. p value, same as (F). (L) Number of TUNEL-positive cells in heart section (bar, mean). p value, same as (F). 3 mice/genotype. (M) Summary.

Figure 3.. cGAS and STING are not required for inflammation-related gene expression and DCM in Lmna^CKO mice

(A) Normalized cGAS and STING abundance quantified by immunoblot shown in Figures S3A and S3B (bar, mean). n = 2 mice/genotype. (B) Left: classification of upregulated genes in Lmna^CKO hearts at 2 weeks post-tamoxifen into cardiomyocyte-derived or non-cardiomyocyte-derived genes based on 4sU labeling in SLAM-IT-seq (n = 3–8 mice/genotype). Right: top 10 GO terms overrepresented in each class of upregulated genes. p value is computed by Metascape. (C) MyoAAV-mediated cGAS overexpression in cardiomyocytes in vivo. (D) Top: anti-cGAS and Lamin A/C immunofluorescence in isolated cardiomyocytes. Bottom: close-up image of the area indicated by rectangle in the top image. Arrowhead, cGAS at nuclear tip. Scale bar: 20 μm. (E) Transcript abundance in isolated cardiomyocytes normalized by Actb abundance quantified by quantitative PCR. Bar, mean. n = 3–5 mice/genotype. p value, Wilcoxon test. (F) Volcano plot comparing RNA-seq read counts. Left: Cgas^+/+;Lmna^CKO (n = 6) versus Cgas^+/+;Lmna^Het (n = 6). Right: Cgas^−/−;Lmna^CKO (n = 4) versus Cgas^+/+;Lmna^CKO (n = 6). (G) Transcript abundance quantified by RNA-seq. Bar, mean. P, FDR-adjusted p value computed by a generalized linear model in DESeq2. (H) Left ventricular ejection fraction measured by echocardiography (box, interquartile range). n = 6–12 mice/genotype. p value, same as (E). (I) Masson’s trichrome staining of heart sections. Scale bar: 20 μm. (J) Kaplan-Meier survival analysis. n = 5–11 mice/genotype. p value, log-rank test. (K) Summary.

Figure 4.. ECM-mediated signaling from Lmna^CKO cardiomyocytes is predicted to activate fibroblasts

(A) snRNA-seq uniform manifold approximation and projection (UMAP) plot for WT and Lmna^CKO heart nuclei. CM, cardiomyocyte; Fib, fibroblast; Mac, macrophage; EC, endothelial cell; CEC, coronary EC; LEC, lymphatic EC; PC, pericyte. See Figure S4 for additional analyses. (B) WT and Lmna^CKO nucleus count within each cell type cluster. Cell types combined in later analyses are indicated below the graph. (C) snRNA-seq pseudo-bulk volcano plot comparing Lmna^CKO versus WT heart nuclei. Red, upregulated genes. Blue, downregulated genes. The number of differentially expressed genes is indicated above the plot. (D) GO terms enriched among upregulated genes within indicated cell type. p value is computed by Metascape. (E) Pseudo-bulk transcript abundance of ECM-related genes. Point, mean of normalized read count per replicate. Bar, mean across replicates. P, FDR-adjusted p value computed by a generalized linear model in DESeq2. (F) Same as (E) but for immune-related genes. (G) Predicted signaling from sender cells (x axis) to receiver cells (y axis). Color: signaling gains in Lmna^CKO hearts relative to WT hearts. (H) Predicted signaling mediators for the signaling indicated by box in (G) with alphabetical labels. (I) Predicted intercellular signaling indicated in (G) by alphabetical labels in Lmna^CKO hearts. (J) Model.