Filamin C is Essential for mammalian myocardial integrity

Abstract

FLNC, encoding filamin C, is one of the most mutated genes in dilated and hypertrophic cardiomyopathy. However, the precise role of filamin C in mammalian heart remains unclear. In this study, we demonstrated Flnc global (FlncgKO) and cardiomyocyte-specific knockout (FlnccKO) mice died in utero from severely ruptured ventricular myocardium, indicating filamin C is required to maintain the structural integrity of myocardium in the mammalian heart. Contrary to the common belief that filamin C acts as an integrin inactivator, we observed attenuated activation of β1 integrin specifically in the myocardium of FlncgKO mice. Although deleting β1 integrin from cardiomyocytes did not recapitulate the heart rupture phenotype in Flnc knockout mice, deleting both β1 integrin and filamin C from cardiomyocytes resulted in much more severe heart ruptures than deleting filamin C alone. Our results demonstrated that filamin C works in concert with β1 integrin to maintain the structural integrity of myocardium during mammalian heart development.

Fig 1. Filamin C is essential for mammalian heart development.

(A) Targeting strategy for generating Flnc knockout mice. Global Flnc knockout mice were generated by crossing Flnc floxed mice (Flnc^fl/fl) with Sox2^Cre mice, while cardiomyocyte-specific Flnc knockout mice were generated by crossing Flnc^fl/fl mice with Xmlc2^Cre mice or cTnT^Cre mice. After the deletion of exon 9–13 of the Flnc gene by Cre recombinase, a premature termination codon (PTC) will be introduced to exon 14 and trigger non-sense mediated mRNA decay (NMD) of Flnc mRNA. (B) Western blot confirms the complete depletion of filamin C protein in Flnc global knockout mice (Flnc^-/- or Flnc^gKO). GAPDH is used as a loading control. (C) Percentage of live wild-type (Flnc^+/+), heterozygous (Flnc^+/-) and knockout (Flnc^-/-) embryos from E8.5 to E12.5. Exact number of each genotype and each developmental stage: E8.5, 4:9:4 (Flnc^+/+: Flnc^+/-: Flnc^-/-); E9.5, 16:32:15; E10.5, 13:25:11; E11.5, 4:7:4*; E12.5, 4:6:0. *: dead/under resorption. (D) Wholemount images of control and Flnc global knockout (Flnc^gKO) embryos at E9.5, E10.5 and E11.5. Yellow arrows indicate pericardial effusion; Red arrow indicates heart rupture; Scale bar, 1 mm.

Fig 2. Filamin C maintains the integrity of myocardial wall.

(A) Wholemount brightfield/fluorescence merged images of control and Flnc^gKO embryos with red fluorescence myocardium indicator at E9.5. Yellow arrows indicate heart ruptures. Endo, endocardium; Myo, myocardium. Scale bar, 0.5 mm. (B and C) Representative immunofluorescence (IF) images of control and Flnc^gKO hearts at E9.5. Antibodies used for IF are indicated. Yellow arrows indicate CD31-positive thrombi. Scale bar, 0.1 mm. (D and E) Representative immunofluorescence (IF) images of control and Flnc^gKO hearts at E10.5. Antibodies used for IF are indicated. Yellow arrows indicate heart ruptures. CW, chest wall; Asterisk, chest wall overgrowth. Scale bar, 0.1 mm.

Fig 3. Wound healing and blood coagulation pathways were activated in Flnc^gKO.

(A) Heatmap showing gene expression changes in E9.5 control and Flnc^gKO hearts (n = 3 per group). (B) Volcano plot of differentially expressed genes (DEGs) [false discovery rate (FDR) < 0.05)] in E9.5 Flnc^gKO hearts compared with littermate controls. Notable DEGs are indicated. (C) Gene ontology analysis of differentially expressed genes of Flnc^gKO hearts compared with littermate controls at E9.5. BP, biological process; CC, cellular component; MF, molecular function; PW, KEGG pathway.

Fig 4. Extracellular matrix proteins are downregulated in the myocardium of Flnc^gKO.

(A-D) Representative immunofluorescence (IF) images of control and Flnc^gKO hearts at E9.5 using antibodies against α-actinin (actn2) or cardiac troponin T (cTnT), and pan-cadherin (A), desmoplakin (B), β-dystroglycan (C) or γ-sarcoglycan (D). Scale bar, 0.1 mm. (E) Representative collagen I and laminin immunofluorescence images of control and Flnc^gKO hearts at E9.5. yellow arrows: myocardium; magenta arrows: endocardium. Scale bar, 0.1 mm.

Fig 5. Both filamin C and β1 integrin is required to maintain the structural integrity of myocardium.

(A) Representative activated (9EG7) and total β1 integrin immunofluorescence images of control and Flnc^gKO hearts at E9.5. yellow arrows: myocardium; magenta arrows: endocardium. Scale bar, 0.1 mm. (B) The strategy for generating cardiomyocyte-specific Itgb1 knockout mice (Itgb1^cKO), cardiomyocyte-specific Flnc knockout mice (Flnc^cKO) and cardiomyocyte-specific Flnc/Itgb1 double knockout mice (Flnc/Itgb1^dcKO). (C) Wholemount images of control, Itgb1^cKO, Flnc^cKO and Flnc/Itgb1^dcKO embryos at E10.5. Yellow arrowheads indicate pericardial effusion; White arrows indicate heart ruptures. Scale bar, 0.5 mm.