Cardiomyocyte Proliferative Capacity Is Restricted in Mice With Lmna Mutation

Abstract

LMNA is one of the leading causative genes of genetically inherited dilated cardiomyopathy (DCM). Unlike most DCM-causative genes, which encode sarcomeric or sarcomere-related proteins, LMNA encodes nuclear envelope proteins, lamin A and C, and does not directly associate with contractile function. However, a mutation in this gene could lead to the development of DCM. The molecular mechanism of how LMNA mutation contributes to DCM development remains largely unclear and yet to be elucidated. The objective of this study was to clarify the mechanism of developing DCM caused by LMNA mutation. Methods and Results: We assessed cardiomyocyte phenotypes and characteristics focusing on cell cycle activity in mice with Lmna mutation. Both cell number and cell size were reduced, cardiomyocytes were immature, and cell cycle activity was retarded in Lmna mutant mice at both 5 weeks and 2 years of age. RNA-sequencing and pathway analysis revealed "proliferation of cells" had the most substantial impact on Lmna mutant mice. Cdkn1a, which encodes the cell cycle regulating protein p21, was strongly upregulated in Lmna mutants, and upregulation of p21 was confirmed by Western blot and immunostaining. DNA damage, which is known to upregulate Cdkn1a, was more abundantly detected in Lmna mutant mice. To assess the proliferative capacity of cardiomyocytes, the apex of the neonate mouse heart was resected, and recovery from the insult was observed. A restricted cardiomyocyte proliferating capacity after resecting the apex of the heart was observed in Lmna mutant mice. Conclusions: Our results strongly suggest that loss of lamin function contributes to impaired cell proliferation through cell cycle defects. The inadequate inborn or responsive cell proliferation capacity plays an essential role in developing DCM with LMNA mutation.

Keywords: cell cycle; dilated cardiomyopathy; lamin A/C; p21; repressed proliferating capacity.

Figure 1

Cardiomyocyte cell number and size in wild type (wt) and Lmna^−/− mice. Both the cell number and size of cardiomyocytes were significantly reduced in Lmna^−/− mice (3 weeks old). (A) Total counts of cardiomyocytes in 10 tissue layers of the heart (n = 5 in each group). (B) Cross-section area of cardiomyocytes measured in a tissue section (n = 5 in each group). (C,D) Tissue sections stained with wheat germ agglutinin in wt (C) and Lmna^−/− (D) mice. The open circle represents wt, and the solid circle represents Lmna^−/− mice. The significance of differences between two groups was determined using the Student's 2 tailed T-test. Scale bar: 1 mm in lower magnification and 50 μm in higher magnification in inset.

Figure 2

Nucleation and cell cycle markers of cardiomyocytes in wild type (wt) and Lmna^−/− mice. (A) Nucleation of cardiomyocytes in 8 days old (n = 7 in each group) and 3 weeks old (n = 12 in wt and 11 in Lmna^−/−) showed more mononuclear and less bi- or poly-nuclear cardiomyocytes in Lmna^−/− mice. (B) Percentage of phospho-histone H₃ positive cardiomyocytes in 8 days old (n = 7 in each group) and of EdU positive cells in 3 weeks old (n = 5 in wt and 6 in Lmna^−/−) showed retarded cell cycle in Lmna^−/− mice. Dots represent individual mouse data. The significance of differences between two groups was determined using the Student's 2 tailed T-test.

Figure 3

Nucleation of cardiomyocytes in young and old wild type (wt) and Lmna^+/− mice. (A) Nucleation of cardiomyocytes in 3 weeks old (n = 12 in wt and 5 in Lmna^+/−) was similar between wt and Lmna^+/− mice. (B) Nucleation of cardiomyocytes in 2 years old (n = 4 in each group) showed more mononuclear and less binuclear cardiomyocytes in Lmna^+/− mice. Dots represent individual mouse data. The significance of differences between two groups was determined using the Student's 2 tailed T-test.

Figure 4

Protein expression of p21 and p53, and DNA damage of cardiomyocyte in 5 weeks. (A) Immunostaining against anti-p21 antibody in heart tissue section of wt, Lmna^+/−, and Lmna^−/−. The top panel shows p21 staining, and the bottom panel shows merged figures of anti-p21 antibody (green), WGA (red), and DAPI (blue). Scale bar: 10 μm. (B) Western blot analysis of LV tissue hybridized with p53, p21, and β-actin Abs. The data shown are representative of three independent experiments. (C) Protein expression level of p53 and p21 normalized by β-actin. *P < 0.05 vs. respective wt. (D) Percentage of phospho histone H₂ AX staining counted in cardiomyocytes (Supplementary Figure 2) (n = 3 in each group). Dots represent individual mouse data. The significance of differences between 3 groups was determined with 1-way ANOVA. Post-hoc pairwise comparisons were performed with the Tukey–Kramer test.

Figure 5

EdU incorporation and the percentage of EdU positive myocyte nuclei after apical resection. (A) Representative immunostaining for EdU in cardiac tissue sections of wt, Lmna^+/−, and Lmna^−/−. The top panel shows EdU staining, and the bottom panel shows merged figures of immunostaining for EdU (red), WGA, and troponin I (green) and DAPI (blue). Yellow arrows indicate EdU positive nucleus only in cardiomyocytes. Scale bar: 10 μm. (B) Percentage of EdU incorporated myocyte nuclei (n = 7 to 11 in each group). A circle indicates data from mice with the sham operation, square with apical resection. Dots represent individual mouse data. The significance of differences between 3 groups was determined with 1-way ANOVA. Post-hoc pairwise comparisons were performed with Tukey–Kramer test.

Figure 6

Our model depicting a possible mechanism in the development of DCM with a Lmna mutation. Black boxes denote findings from this study.