Inhibition of TGFβ signaling increases direct conversion of fibroblasts to induced cardiomyocytes

Abstract

Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.

Figure 1. Direct conversion of mouse embryonic fibroblasts to iCMs can be influenced by treatment with small molecules.

Schematic of direct reprogramming strategy of MEFs and timeline (A). Percentage of total cells with flashing Troponin T-GCaMP activity for control and small molecule treated MEFs (B). Representative immunocytochemistry images for HNGMT +DMSO (C), or +BIX (D), +CHIR+XAV (E), and +SB (F) treatments for cardiac Troponin T (green) and nuclei (red). * indicates p<0.05 and ** indicates p<0.01 compared to +DMSO control. Scale bar is 100 µM.

Figure 2. Inhibition of TGFβ early in the conversion process leads to the greatest increase in iCM yield.

Schematic depicting the addition of SB at different time points following induction of the transcription factors (A). Quantification at Day 14 of the number of cells with Troponin T-GCaMP activity upon addition of SB at Day -1 (D-1), 3 (D3), and 6 (D6) (B). * indicates p<0.05 and ** indicates p<0.01 compared to +DMSO control.

Figure 3. The increase in iCM number with SB treatment is not due to increased transgene expression or changes in cell proliferation.

Evaluation of gene expression of the transgenes at Day 2 via qPCR is not significantly different between +DMSO (black) control and +SB treatment groups (white, A). Evaluation of overall cell proliferation with Ki67 staining in MEFs with time for +DMSO (black) and +SB treatment groups (grey) (B). A time course evaluation of Troponin T (green) and Ki67 (red) did any dual labeled cells for either the +DMSO (top row) and +SB (bottom row) treated groups (C). Scale bar is 100 µM.

Figure 4. Direct conversion of adult mouse cardiac fibroblasts (CFs) to iCMs.

Conversion efficiency to iCMs increases in yield with increasing concentration of SB (A). Direct conversion of CFs to iCMs is reduced with addition of TGFβ1 (black, HNGMT+T1) and TGFβ2 (black, HNGMT+T2) to the culture medium, but not Activin A (black, HNGMT+AA). Addition of SB (grey) increases the yield of iCMs generated in the presence of TGFβ1 (grey, HNGMT+T1) and TGFβ2 (grey, HNGMT+T2 and Activin A (grey, HNGMT+AA) (B). Immunostaining for αActinin (red) and DAPI (blue) demonstrate increased sarcomeric structure with SB treatment for HNGMT+T1 (C). ** indicates p<0.01 compared to HNGMT+DMSO control.

Figure 5. Evaluation of gene expression changes in MEFs and CFs during conversion with and without SB.

Heatmap depicting the 49 and 38 genes up-regulated and down-regulated, respecively exclusively in MEFs for HNGMT + SB versus HNGMT+DMSO as well as the expression of the same genes in CFs (A). Venn diagrams demonstrating the overlap of 19 genes up-regulated HNGMT+SB for both MEFs and CFs as well as the 11 genes down-regulated in HNGMT+SB for both MEFs and CFs (B). The top five groups by p-value from as identified by functional analysis of the 19 commonly up-regulated and 11 commonly down-regulated genes in both MEFs and CFs for HNGMT+SB treatment (C). Quantitative RT-PCR results for genes identified to be involved in development of cardiac muscle and up-regulated by both MEFs and CFs (Bmp2, Pln, Ppargc1a, and Tgfbr3), or MEFs (Myh6 and Myocd), or down-regulated in both MEFs and CFs (Adamtsl2) (D). * indicates p<0.05 compared to the HNGMT+DMSO control for that gene and cell type by Students t-test.