Differentiation of cardiomyocytes requires functional serine residues within the amino-terminal domain of desmin

Abstract

Desmin contributes to the stability of the myocardium and its amino-terminal domain influences intermediate filament formation and interacts with a variety of proteins and DNAs. Specific serine residues located in this domain are reversibly phosphorylated in a cell cycle and developmental stage-dependent manner as has been demonstrated also for other cytoplasmic type III intermediate filament proteins. Although absence of desmin apparently does not affect cardiomyogenesis, homozygous deletion of the amino-terminal domain of desmin severely inhibited in vitro cardiomyogenesis. To demonstrate the significance of phosphorylation of this domain in cardiomyogenic commitment and differentiation, we inhibited phosphorylation of serine residues 6, 7, and 8 by mutation to alanine, and investigated early cardiomyogenesis in heterozygous embryoid bodies. As control, serine residues 31 and 32, which are not phosphorylated by kinases mutating serine residues 6, 7, and 8, were mutated to alanine in a second set. Desmin(S6,7,8A) interfered with cardiomyogenesis and myofibrillogenesis in a dominant negative fashion, whereas desmin(S31,32A) produced only a mild phenotype. Desmin(S6,7,8A) led to the down-regulation of the transcription factor genes brachyury, goosecoid, nkx2.5, and mef2C and increased apoptosis of presumptive mesoderm and differentiating cardiomyocytes. Surviving cardiomyocytes which were few in number had no myofibrils. Demonstration that some but not any mutant desmin interfered with the very beginning of cardiomyogenesis suggests an important function of temporarily phosphorylated serine residues 6, 7, and 8 in the amino-terminal domain of desmin in cardiomyogenic commitment and differentiation.

Fig. 1. Desmin^S6,7,8A and desmin^S31,32A affect cardiomyogenesis in embryoid bodies (EBs) in a dominant negative fashion.

(A) Development of beating cardiomyocytes in EBs generated from embryonic stem cells (ESCs) of genotypes as indicated. K1 and K2 are two different ESC clones. (B) Initial rate of differentiation of cardioblasts to beating cardiomyocytes. Starting 1 day after the first beating cardiomyocytes were observed, increase of beating, cardiomyocytes per day were measured for 3 consecutive days. (C) Influence of mutant desmin on the extent of cardiomyogenesis in EBs. Means were calculated from the day of maximum beating activity ± 2 days. (A–C) Data are means of at least four independent experiments, each performed in triplicate. Number of EBs analyzed in each case, N = 311; except for des^+/+, N = 916. Error bars, standard deviation σ_{x(n – 1)}. p-values relate to control (des^+/+). (D) Western blot analysis of intermediate filament preparations from EBs at day 16. Immuno-detection of desmin, vimentin, and connexin 43. Ponceau S-stained gel as loading control.

Fig. 2. Desmin^S6,7,8A destroys the morphology of myofibrils in cardiomyocytes.

(A) Merged confocal double immunofluorescence micrographs of typical cardiomyocyte clusters in embryoid bodies (EBs) with desmin alleles as indicated. EBs were double stained with a polyclonal antibody to desmin and a secondary TRITC-conjugated antibody (red), a monoclonal antibody to cTnT and a secondary FITC-conjugated antibody (green) at day 16 after embryonic stem cells aggregation. Areas of colocalization are yellow. Scale bar: 25 µm. (B) High-resolution confocal double immunofluorescence micrographs of cardiomyocytes stained as in (A). Arrow-heads, irregularly assembled sarcomeres. Arrows, cytoplasmic aggregates of desmin. Right column, merged images. Scale bar, 10 µm.

Fig. 3. Desmin^S6,7,8A negatively affects commitment and early pro-liferation of cardiomyocytes and hampers rhythmic contraction.

(A) Number of beating cardiomyocyte clusters per embryoid body (EB). Number of EBs analyzed in each case, N = 311; except for des^+/+, N = 916. (B) Size distribution of cardiomyocyte clusters presented as the relative proportion of small clusters with less than 10 (dark gray bars) versus large clusters with up to several hundred cardiomyocytes (light gray bars). Number of EBs analyzed in each case: N = 386. (C) Beating rates of cardiomyocytes collected at day 16 ± 4 days. Number of cardiomyocyte clusters analyzed: des^+/+, N = 85; all others N = 52. Error bars indicate standard deviation σ_{x(n − 1).} p-values relate to control (des^+/+).

Fig. 4

Desmin^S6,7,8A causes increased cell death in differentiating cardiomyocytes. Confocal micrographs of areas of cardiomyogenesis in embryoid bodies triple stained for nuclear DNA with DAPI (blue, left column), for damaged DNA by transferase-mediated dNTP-fluorescein nick end labeling assay (green, middle column), and with cTnT antibodies (red) at day 8 (A) and at day 12 (B) after embryonic stem cells aggregation. Genotypes as indicated in the left column. Scale bar, 25 µm.

Fig. 5

Desmin^S6,7,8A negatively influences the onset of mesodermal and myocardial transcription factor expression. mRNA was isolated and reverse transcribed from embryonic stem cells (day 0) and embryoid bodies (EBs) of genotypes as indicated at days 4, 6, and 8. Semi-quantitative RT-PCR was performed with primer pairs as indicated. (A) Typical data from three independent experiments with each two clones are shown. GAPDH RT-PCR, loading control. (B) Time course of the expression of brachyury, goosecoid, nkx2.5, and mef2c in EBs between days 4 and 8. Statistical analysis of six independent experiments. Expression levels normalized to expression in wild-type EBs for each day. *, p-values were all smaller than 0.05 except for brachyury and goosecoid expression on day 8; p-values relate to control (des^+/+) with an arbitrary expression level of 1.0.