The influence of conditional Ski knockdown in primary cardiac myofibroblasts: fibrosis markers, Taz mRNA abundance and alternative Ski exon splicing.
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Date
2022-06-22
Authors
Fehr, Rebeca
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Abstract
Background: SKI is a protein that functions as a transcriptional cofactor and is expressed in cardiac muscle. It is encoded by 7 exons in mouse and its primary function is that of a negative regulator for TGF-β1 signaling. While TGFβ1 stimulates cardiac scar formation and fibrosis, SKI is known to negatively regulate TGFβ1 and deactivate cardiac myofibroblasts, the main cells responsible for cardiac fibrosis. Our lab has recently shown that SKI may influence the Hippo signaling pathway however Ski downregulation has not been studied in heart. Hypotheses: In vitro Ski knockdown in primary cardiac myofibroblasts modifies gene transcription of Hippo pathway component proteins and marker genes for cardiac fibrosis. Further, SKI may exist in two different exon splice variants in mouse heart. Methods: In all experiments, P0 primary cardiac myofibroblasts from Ski-floxed (transgenic) and wild-type (non-transgenic) mice were cultured. After optimization, these cells were infected with adenoviral-GFP as control or adenoviral-Cre (Ad-Cre) to specifically excise Ski exons 2 and 3; and harvested 72h hours after infection. Samples were prepared for qPCR, cytotoxicity (live-dead) assay, and DNA sequencing. Results: Two different transcriptional variants (Tv) of mouse Ski were confirmed in Ski-floxed and control cells, with and without exon 2. The presence of Ad-Cre did not influence cell death in treated control or Ski-floxed cells. Excision of exons 2 and 3 (as well as significant knockdown of exons 1, and 4 - 7) was confirmed in Ad-Cre-treated Ski-floxed cells in the presence of Cre, and exon frameshift was noted following this excision. Ad-Cre was shown to influence mRNA expression of both control and Ski-floxed treated cells – Cre treated myofibroblasts were compared to GFP infected controls. We observed that Taz mRNA expression is significantly elevated in basal conditions with Ad-Cre treated Ski knockdown P0 myofibroblasts vs Ad-GFP treated controls. On the other hand, the mRNA expression of fibrillar collagens I α 1, collagen III α 1, Eda- fibronectin, Limd1, Lats2, α-Sma and Pdgfr-α genes were not altered in Ski-floxed Ad-Cre treated cells vs controls. Conclusion: In vitro Ski downregulation in P0 myofibroblasts (in the absence of any other stimulus and in serum-starved conditions) does not directly influence fibrillar collagen gene transcription. This notwithstanding the steady state mRNA abundance of the Hippo effector protein Taz is significantly upregulated by Ski knockdown vs Ad-GFP treated controls. The discovery of novel differentially spliced Ski transcripts may impact future investigation of SKI function in heart. We suggest that Ski knockdown will provide a useful model to investigate the role of Ski in activation of various cytokines, including TGFβ1.
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Keywords
SKI, Cardiac Fibrosis, Knockdown, Cardiac Myofibroblast, Cre-LoxP