Desmin stimulates differentiation of cardiomyocytes and up-regulation of brachyury and nkx2.5

Abstract

Desmin contributes to structural integrity and function of the myocardium but its function seems to be redundant in early cardiomyogenesis in the desmin null mouse model. To test the hypothesis that desmin also plays a supportive role in cardiomyogenic commitment and early differentiation of cardiomyocytes we investigated cardiomyogenesis in embryoid bodies expressing different desmin alleles. Constitutive expression of desmin and increased synthesis during mesoderm formation led to the up-regulation of brachyury and nkx2.5 genes, accelerated early cardiomyogenesis and resulted in the development of large, proliferating, highly interconnected, and synchronously beating cardiomyocyte clusters, whereas desmin null cardiomyocytes featured an opposite phenotype. In contrast, constitutive expression of amino-terminally truncated desmin(Delta1-48) interfered with the beginning of cardiomyogenesis, caused down-regulation of mesodermal and myocardial transcription factors, and hampered myofibrillogenesis and survival of cardiomyocytes. These results provide first evidence that a type III intermediate filament protein takes part in regulating the differentiation of mesoderm to cardiomyocytes at the very beginning of cardiomyogenesis.

Fig. 1. Expression and synthesis of desmin and desmin^Δ1−48 in embryonic stem cells and embryoid bodies (EBs).

(A) Western blot analysis of intermediate filament preparations from embryonic stem cells with an additional ectopic allele constitutively expressing desmin (des^+/+ des^ect), two wild-type desmin alleles (des^+/+), two null alleles (des^−/−), and two constitutively expressed mutant alleles lacking codons 1−48 (des^{Δ1−48/Δ1−48}), with antibodies against desmin, vimentin, and connexin 43. Heart: positive control. Connexin 43 immuno-blot and CBB, Coomassie brilliant blue stained gel, loading controls. *Note, smaller size of the constitutively synthesized desmin lacking amino acids 1−48. (B) Semi-quantitative RT-PCR of des^+/+ and des^+/+ des^ect EBs with desmin and GAPDH primer pairs at different stages of EB development. Rows 1−4 represent steps of tenfold dilution of the cDNA for each day. (C) Expression of wild type, ectopic and mutant desmin proteins in EBs. Western blot analysis of intermediate filament preparations from EBs at day 16. Antibodies and genotypes as indicated.

Fig. 2. Premature synthesis of desmin causes early onset of cardiomyogenesis in embryoid bodies (EBs), but lack of its amino-terminal domain severely hampers cardiomyogenesis.

(A) Development of beating cardiomyocytes in EBs generated from embryonic stem cells of genotypes as indicated. Note, that only des^+/+des^ect cardiomyocytes started to beat on day 6 (arrow) and onset of cardiomyogenesis in des^{Δ1−48/Δ1−48} EBs was delayed by 2 days. (B) Acceleration of development to beating cardiomyocytes by desmin. Starting 1 day after the first beating cardiomyocytes were observed, increase of beating cardiomyocytes per day was measured for 3 consecutive days. (C) Influence of desmin on the extent of cardiomyogenesis in EBs. Means were calculated from the day of maximum beating activity ± 2 days. (A−C) Data from experiments with two des^+/+des^ect clones were indistinguishable and thus combined. Data are means of six independent experiments. Number of EBs analyzed in each case: N = 386. Error bars, standard deviation σ_{x(n − 1)}. Denoted significant p-values relate to control (des^+/+).

Fig. 3. Desmin increases size and interconnections of cardiomyocyte clusters but desmin^Δ1−48 causes disorganization of contractile apparatus.

(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) and a monoclonal antibody to cTnT and a secondary FITC-conjugated antibody (green) at day 16 after embryonic stem cells aggregation. Note, ectopic synthesis of desmin in all cells of des^+/+des^ect and des^{Δ1−48/Δ1−48} EBs. Scale bar, 25 μm. (B) High-resolution confocal double immunofluorescence micrographs of cardiomyocytes stained as in (A). Right column, merged images. Scale bar, 10 μm.

Fig. 4. Desmin promotes commitment and proliferation of cardiomyocytes in embryoid bodies (EBs).

(A) Number of beating cardiomyocyte clusters per EB indicating commitment of cardiomyocytes. Number of EBs analyzed in each case, N = 311; except for des^+/+ and des^+/+des^ect, N = 916. (B) Size distribution of cardiomyocyte clusters which is directly proportional to the proliferation of differentiating cardiomyocytes, presented as the relative proportion of small clusters with <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) Rhythmic contraction of cardiomyocytes was measured on day 16 ± 4. Number of cardiomyocyte clusters analyzed: des^+/+, N = 85; des^+/+des^ect, N =109; all others N = 52. Data are means of six independent experiments. Error bars, standard deviation σ_{x(n − 1)}. Denoted significant p-values relate to control (des^+/+).

Fig. 5

Apoptosis is absent in early differentiating cardiomyocytes but increases with age in des^−/− and desmin^Δ1−48 expressing cardiomyocytes. Embryoid bodies (EBs) fixed on days 8 and 16 after embryonic stem cells aggregation, respectively, and indirectly stained with DAPI for nuclear DNA (blue), for single-strand brakes of DNA by TUNEL assay with fluorescein-labelled dNTPs (green), and for cTnT with anti-cTnT and secondary TRITC-conjugated antibodies (red). Genotypes as indicated. (A) Merged confocal triple immunofluorescence micrographs of typical cardiomyogenic areas in EBs at day 8. In the case of des^{Δ1−48/Δ1−48} EBs images from different EBs were combined in order to show a larger number of the widely dispersed and rarely found cardiomyocytes. Arrow, two of 1,083 cardiomyocytes positive for TUNEL staining. Insets, examples of rarely found typical apoptotic cells with phosphatidylserine at the outer side of the plasma membranes. FITC-conjugated annexin V (green). (B) Cardiomyocytes in EBs at day 16. Note, that cTnT positive des^−/− and des^{Δ1−48/Δ1−48} cardiomyocytes are surrounded by TUNEL positive cells but very rarely are positive for TUNEL assay and cTnT (inset lower right panel). Scale bar, 25 μm.

Fig. 6

Premature synthesis of desmin causes increased expression of mesodermal and myocardial transcription factor genes in embryoid bodies (EBs). mRNA was isolated and reverse transcribed from embryonic stem cells (day 0) and EBs of genotypes as indicated in the lower left corner at days 4, 6, and 8, respectively. (A) Semiquantitative RT-PCR was performed in at least three independent experiments (one typical shown) with primer pairs as indicated. cDNA was normalized by levelling the GAPDH RT-PCR product. Statistical analysis of (B) brachyury and (C) nkx2.5 expression in EBs between days 4 and 6 of six independent experiments. Expression levels normalized to expression in wild-type EBs. *p-values smaller than 0.05 relate to control (des^+/+).