Mutations in Alström protein impair terminal differentiation of cardiomyocytes

Abstract

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Figure 1. Increased cardiomyocyte proliferation in people with ALMS1 mutations

(a, b) Representative confocal images of the proband heart vs. age-matched control; PH3 (green), troponin T (red), DAPI (blue). (c) Higher magnification confocal images of PH3-positive cardiomyocytes in the proband; PH3 (green), troponin T (red), DAPI (blue). (d.) Additional high-magnification confocal image of a PH3-positive cardiomyocyte in the proband; PH3 (green), troponin T (red), DAPI (blue). (e.) Phospho-aurora kinase (PAK) staining (green) is present in a dividing cardiomyocyte nucleus in the proband; PAK (green), α-sarcomeric actinin (red), DAPI (blue (f) The number of PAK-positive cardiomyocytes in the heart of the proband was compared to three age-matched controls with failing ventricles. (g) The number of PH3-positive cardiomyocytes in affected individuals was compared to three age-matched controls with failing ventricles. Error bars represent standard error of mean (SEM). All scale bars represent 10μm.

Figure 2. PH3-positive cardiomyocytes in the proband and another affected individual

Additional representative confocal microscopic images from the heart of the proband (a) and another individual with mitogenic cardiomyopathy (b) have high levels of phosphohistone-H3 (Ph3)-positivity. Images were obtained with confocal microscopy using immunostaining for Ph3 (green), α-sarcomeric actinin or troponin T (red), wheat germ agglutinin (white) to outline cell boundaries, and DAPI (blue) to highlight nuclei. Panels a and b – DAPI; panels a* and b* – Ph3; panels a** and b** – cTnT; panels a*** and b*** – merged images with DAPI, Ph3, cTnT, and WGA. Scale bars all indicate 10 μM.

Figure 3. Human cells analyzed for ploidy status

(a.) The DNA content in dividing cardiomyocyte nuclei was compared to non-dividing nuclei (both cardiomyocytes and non-cardiomyocytes) in the proband using quantitative confocal laser cytometry with DAPI nuclear staining. The ratio of DNA in nuclei from proliferating cardiomyocytes compared to non-proliferating (cardiomyocyte and lymphocyte) nuclei in the proband is 1.9, indicating predominantly 4N chromosomal content in these cells. (b.) Summary of the ploidy analysis using centromeric probes for 4 affected individuals and 2 unaffected controls. Error bars indicate S.E.M.. (c.) An example of ploidy analysis using one of the centromere FISH probes for chromosome 8 (CEP8) in an affected individual. Color staining is shown in the bottom left. Scale bars indicate 10 μM; These five panels are the same field with: C) DAPI; C*) CEP8; C**) DAPI and CEP8; C***) DAPI, CEP8 and sarcomeric actinin; and C****) DAPI, CEP8, sarcomeric actinin, and WGA.

Figure 4. Knockdown of Alms1 increases the number of cells in G2/M phases in cardiomyocyte enriched cultures

Comparison of the % of cells in different phases of cell cycle between control transfection and Alms1 knockdown (KD) using siRNA; red = G0 or G1 phase, white = S phase, blue = G2 or M phase. (*) refers to P< 0.05 using unpaired Student’s t-test.

Figure 5. Increased cardiomyocyte proliferation in cultured cells after Alms1 knockdown

(a.) Comparison of α-MHC-GFP positive cardiomyocytes in different phases of cell cycle after control siRNA transfection compared with Alms1 knockdown (KD) by siRNA; red = G0 or G1 phase, white = S phase, blue = G2 or M phase; N=3. Alms1 KD increases the proportion of cells in G2/M phase (blue). (b., c., d., e.) PAK-positive cardiomyocytes in different phases of mitosis; cTnT (red), PAK (green), and DAPI (blue); b is prophase, c is metaphase, d is anaphase, e is telophase. (f.) Flow cytometry of puromycin-selected cardiomyocytes (cTnT⁺ cells) shows that after Alms1 knockdown (KD), there is increased PAK expression compared to controls; N=3. (g.) Scatter plot showing increased incorporation of EdU (Y-axis) and increased DNA content by DAPI (X-axis) in puromycin-selected (cTnT⁺) cardiomyocytes with ALMS1 KD; N=15. (h.) Total number of puromycin-selected cardiomyocytes after Alms1 knockdown (KD) is increased compared to controls; N=4. In all Figures, (*) refers to P<0.05 using Student’s t-test. Error bars represent standard error of mean (SEM), and all scale bars represent 5 μM.

Figure 6. Increased cardiomyocyte proliferation in homozygous Alms1^Gt/Gt mutant mice

(a, b.) Representative confocal images demonstrating EdU (green) incorporation in Alms1^Gt/Gt vs. wild-type littermate control mouse cardiac myocytes; α-sarcomeric actinin (red), wheat germ agglutinin (WGA; white), and DAPI (blue). (c.) Bar graph comparing cardiomyocyte EdU incorporation in Alms1^Gt/Gt mutant (KO) vs. wild-type littermate control mice (N=4 each). (d, e.) Representative confocal images demonstrating PH3 (green) in a cardiomyocyte nucleus in a Alms1^Gt/Gt mutant vs. wild-type littermate control mouse, with cTnT (red), wheat germ agglutinin (white), and DAPI (blue). (f.) Comparison of the number of PH3-positive cardiomyocytes in Alms1^Gt/Gt (KO) vs. wild-type littermate control mice (N=3 each). (g, h.) Representative confocal images demonstrating PAK staining (green) is present in a dividing cardiomyocyte nucleus in an Alms1^Gt/Gt mouse (KO); DAPI (blue) highlights the nucleus, which is surrounded by α-sarcomeric actinin (red) to highlight cardiomyocytes and WGA (white) to identify cell boundaries. (i.) Comparison of the number of PAK-positive cardiomyocytes in Alms1^Gt/Gt (KO) vs. wild-type controls. All scale bars represent 10μm. Error bars represent S.E.M.. N=3 each.

Figure 7. Increased cardiomyocyte density and normalized heart size in homozygous Alms1^Gt/Gt mutant mice

Representative images of (A.) wild-type and (B.) Alms1^Gt/Gt hearts stained with wheat germ agglutinin (white) and cTnT (red); scale bars = 50 uM. (C) Measured cardiomyocyte cross sectional area, N=12 each. (D) Heart/body weight ratios, N=16 for WT and N=7 for Alms1−/−; error bars represent S.E.M., (*) indicates P<0.05 using Student’s t-test.

Figure 8. Perinatal expression of Alms1 in murine cardiomyocytes

Murine GFP-positive cardiomyocytes were isolated from α-MHC-GFP transgenic mice by FACS. Alms1 mRNA levels were normalized to Gapdh. Relative Alms1 mRNA levels at embryonic day 15.5 (ED15.5; N=4) and postnatal day 0 (P0; N=4) were compared by student’s T-Test to postnatal days 5.5/6.5 (P5/6; N=10), p=0.02 and p=0.013, respectively. (*) denotes P<0.05. There was no significant difference in Alms1 expression between days E15.5 and P0; error bars indicate S.E.M.