Cell‐cycle regulation of sarcomere integrity—Role for Actn2 phosphorylation

Figures

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  FIGURE 1. Actn2 expression during muscle differentiation. C2C12 cells were differentiated to myotubes according to standard protocols. At the indicated times, protein extracts were analyzed for Actn2 expression. For comparison, β‐Actin levels are shown. The graph shows the relative expression of Actn2 for three independent experiments. Statistics: One‐way ANOVA with Bonferroni post hoc tests. *p < 0.05, **p <0.01.

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  FIGURE 2. Actn2 is phosphorylated by CDK1 at amino acid T308. (a) iPSCs were differentiated under standard conditions to generate cardiomyocytes. Protein extracts were isolated at the indicated times in the differentiation process and analyzed with an antibody detecting CDK1 phosphorylated proteins. GAPDH was used as a loading control. (b) CDK1 phosphorylates substrates at a S/TPx(x)R/K consensus motif. Only one sequence in Actn2 matches this consensus motif: TPEK. TPEK is conserved in all species with a three‐ and four‐chambered heart. (c) CDK1 kinase assay: CDK1 enzyme was incubated with an Actn2 peptide (21‐mer) containing the TPEK consensus motif. A positive control and negative control peptide were used. The negative control was a scrambled Actn2 peptide, whereby TPEK was converted to APEK. Peptide phosphorylation was determined by measuring the incorporation of a fluorescently tagged ATP. Standard curves were used to convert absorbance to molar amount. N = 5. (d) Actn2 was immunoprecipitated from iPSC‐derived cardiac progenitors or iPSC‐derived cardiomyocytes. Immunoprecipitates were probed with an antibody which detects CDK1 phosphorylated proteins. Representative immunoblots from…

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  FIGURE 3. Preventing Actn2 T308 phosphorylation by gene editing promotes C2C12 myotube differentiation and sarcomere formation. (a) Overview of the CRISPR‐Cas9 homology‐directed repair (HDR) approach. (b–d) C2C12 cells were transfected with Cas9 and the gRNAs needed to convert the Actn2 T308 residue to A308 (prevents phosphorylation) or D308 (mimics phosphorylation). After 7 days, cells were analyzed for gene editing or seeded for differentiation. Analysis of gene‐editing efficiency (panel b) was determined by qPCR with primers designed for the introduced SNP. Differentiation was assessed 6 days after seeding. In the first instance (panel c), myotube number was calculated by staining for Actn2. Results of five independent experiments are shown. For each experiment, four images per well were taken and the average myotube number reported. A further analysis of differentiation was carried out by calculating the percentage of myotubes displaying striated sarcomeres (panel d). N = 5. Statistics: One‐way ANOVA with Bonferroni post hoc testing, **p < 0.01, ***p < 0.001.

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  FIGURE 4. Actn2 T308 phosphorylation correlates with increased proliferative capacity. C2C12 cells were transfected with the Actn2 T308A or Actn2 T308D expression plasmids. Cell number was determined by MTS assay at the indicated times post‐transfection. N = 3. ***p < 0.001.

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  FIGURE 5. Actn2 T308 phosphorylation and sarcomere assembly. The data provided in this study suggests a mechanism whereby Actn2 T308 phosphorylation regulates sarcomere assembly. In the presence of the cell‐cycle kinase CDK1, Actn2 T308 is phosphorylated and sarcomeres become unstable. When cells exit the cell‐cycle and CDK1 activity declines, Actn2 T308 is dephosphorylated. In the dephosphorylated state, Actn2 promotes sarcomere formation.