Downregulation of Survivin contributes to cell-cycle arrest during postnatal cardiac development in a severe spinal muscular atrophy mouse model

Abstract

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive degeneration of spinal-cord motor neurons, leading to atrophy of skeletal muscles. However, accumulating evidence indicates that it is a multi-system disorder, particularly in its severe forms. Several studies delineated structural and functional cardiac abnormalities in SMA patients and mouse models, yet the abnormalities have been primarily attributed to autonomic dysfunction. Here, we show in a severe mouse model that its cardiomyocytes undergo G0/G1 cell-cycle arrest and enhanced apoptosis during postnatal development. Microarray and real-time RT-PCR analyses revealed that a set of genes associated with cell cycle and apoptosis were dysregulated in newborn pups. Of particular interest, the Birc5 gene, which encodes Survivin, an essential protein for heart development, was down-regulated even on pre-symptomatic postnatal day 0. Interestingly, cultured cardiomyocytes depleted of SMN recapitulated the gene expression changes including downregulation of Survivin and abnormal cell-cycle progression; and overexpression of Survivin rescued the cell-cycle defect. Finally, increasing SMN in SMA mice with a therapeutic antisense oligonucleotide improved heart pathology and recovered expression of deregulated genes. Collectively, our data demonstrate that the cardiac malfunction of the severe SMA mouse model is mainly a cell-autonomous defect, caused by widespread gene deregulation in heart tissue, particularly of Birc5, resulting in developmental abnormalities through cell-cycle arrest and apoptosis.

Figure 1.

Heart weight and cardiomyocyte density of SMA mice are dramatically reduced, compared with heterozygotes. (A) Comparison of heart weight between SMA mice and their heterozygous littermates (Het) at five time points (P0, P2, P4, P6, and P8) during postnatal development (n = 8). (B) Representative pictures of hearts obtained from P4 SMA and heterozygous mice. (C) Comparison of the heart weight/body weight ratios from P0 to P8 between SMA and heterozygous mice (n = 8). (D) Hematoxylin and eosin (H&E) staining of sections from hearts of heterozygous (upper) and SMA (lower) mice on P0, P2, P4, and P6. The two enlargements below show irregular nuclei of cardiomyocytes (P4, SMA). Scale bar, 50 μm. (E) Quantitation of the number of cardiomyocytes per high-power field (HPF) (n = 4). **P < 0.01, ***P < 0.001.

Figure 2.

Cardiomyocyte proliferation and cell cycle are impaired in SMA mice. Heart sections from SMA (n = 3) and heterozygous (Het, n = 3) mice at four time points (P0, P2, P4, and P6) were stained with anti-Ki67 antibody (A) or anti-pH3 antibody (D) to label mitotic cells; nuclei were counterstained with DAPI. Total number of Ki67⁺ (B) or pH3⁺ cardiomyocytes (E) and percentage of Ki67⁺ (C) or pH3⁺ (F) cardiomyocytes per high-power field (HPF) was determined from three sections per heart, with three hearts per group (n = 9). **P < 0.01, ***P < 0.001.

Figure 3.

Cell cycle of SMA cardiomyocytes was analysed by flow cytometry. (A–D) DNA profiles of cardiomyocytes isolated from SMA mice aged P0, P2, P4, or P6. Heterozygous littermates (Het) were used as controls (n = 3). Cells were stained with propidium iodide. (E–H) Histograms of cell-cycle data from (A–D). *P < 0.05 **P < 0.01.

Figure 4.

Apoptosis is increased in cardiac tissues of SMA mice. (A) TUNEL staining of cardiac tissues from both heterozygous (Het) and SMA mice aged P0, P2, P4, and P6. Compared with heterozygous mice, the number of TUNEL-positive SMA cardiomyocytes is markedly increased on P4 and P6. Nuclei were counterstained with DAPI. Scale bar, 20 μm. (B) Quantification of apoptotic cardiomyocytes identified by TUNEL staining, as shown in (A) (3 mice per group and 3 counts per mouse). ***P < 0.001. (C) TEM images of heterozygous (upper) and SMA (lower) cardiac tissues. Mitochondria are normal in heterozygous mice, but swollen in SMA cardiomyocytes. The arrowheads point to degenerating mitochondria with cristae deformation.

Figure 5.

Expression of cell cycle-related genes during postnatal cardiac development of SMA mice. Heterozygous littermates (Het) were used as controls. (A) Microarray analysis of genes differentially expressed in the heart of SMA mice (n = 5). Tissue samples were collected on P5. Gene Ontology analysis shows that cell cycle-related genes are primarily affected. (B) Heat map showing log2 fold changes of expression levels for a subset of cell cycle-related genes (SMA over Het), analysed by real-time RT-PCR. Cardiac RNA samples were obtained from P0, P2, P4, and P6 mice (n = 3), respectively; gene-expression levels were normalized to Gapdh. (C–F) Histograms showing six mitosis-related genes that are downregulated in the heart of SMA mice on P0, P2, P4, and/or P6 (n = 4). (G) Western blotting analysis of Survivin, AURKB and SMN levels in hearts of mice aged P0, P2, P4, and P6. Protein levels were normalized to β-Tubulin. (H–J) Quantitation of the protein data from (G) and unpublished data (n = 3). For (C–F) and (H–J), *P< 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Expression of apoptosis-related genes during postnatal cardiac development of SMA mice. Heterozygous littermates (Het) were used as controls. (A) Heat map showing apoptosis-related genes that are upregulated. Heart tissues were collected on P0, P2, P4, and P6 (n = 3). Gene expression was analysed by real-time RT-PCR and presented as log2-transformed fold change. (B) Western blotting analysis of p53, p-p53 (Ser18), PUMA, NOXA and p21 levels. Protein levels were normalized to β-Tubulin. (C–G) Quantitation of the data in (A) plus unpublished data (n = 4). *P< 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

Effect of Survivin overexpression in SMN-depleted neonatal mouse cardiomyocytes. Cardiomyocytes isolated from P2 heterozygous mice were treated with two Smn1-specific siRNAs (siRNA-1 and siRNA-2), respectively, and a scrambled siRNA control. (A) Western blotting analysis showing that Survivin expression was markedly reduced in cardiomyocytes after knockdown of SMN with two independent siRNAs. Tubulin was used as a loading control. (B) Quantitation of SMN and Survivin protein levels after treatment with siRNA-1 or siRNA-2 in cardiomyocytes (n = 3). (C) Western blotting analysis showing proper expression of T7-tagged Survivin after transfection of the cDNA expression plasmid pCGT7-Survivin into SMN-depleted cardiomyocytes. Monoclonal anti-T7 antibody was used to detect T7-Survivin. (D) Quantitation of T7-Survivin levels after transfection of different amounts of pCGT7-Survivin. Tubulin was used as a loading control. (E–G) DNA profiles of cardiomyocytes transfected with control siRNA (E), or siRNA-2 with (G) or without (F) overexpression of Survivin (0.6 μg plasmid). Plasmid pCGT7-Survivin was transfected into cells 24 h post siRNA-2 transfection; 48 h later, cells were harvested for analysis (n = 3). (H) Histogram of cell-cycle data from (E–G). *P < 0.05 **P < 0.01.

Figure 8.

Subcutaneous delivery of ASO10–29 partially corrects the heart pathology of SMA mice. ASO in saline or saline alone was injected into mice at 90 mg/kg, twice between P0 and P1, and cardiac tissues were collected on P6 for various analyses. Heterozygous mice (Het) were used as controls. (A) Analysis of SMN2 exon 7 inclusion by radioactive RT–PCR after ASO treatment. FL, full-length transcript; Δ7, exon 7-skipped transcript; Incl %, 100 × FL/(FL + Δ7). (B) Western blotting analysis of SMN levels, normalized to β-tubulin. (C) Histogram of exon 7 inclusion data after ASO treatment as in (A) (n = 4). (D) Histogram of SMN levels from (B) (n = 3). (E) Heart weight after ASO treatment (n = 10). (F) Ratio of heart weight to body weight (mg/g, n = 10). (G) Quantitation of gene expression (Birc5, Aurkb, Mki67, cdkn1a/p21, Pmaip1/Noxa), normalized to Gapdh (n = 4). For (C–G), *P < 0.05, **P < 0.01, ***P < 0.001 versus Het; ^#P < 0.05, ^###P < 0.001 versus saline.