Figure 1. TLR3 inhibition inhibits maturation of reprogrammed fibroblasts into cardiomyocytes.

Neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with either vehicle or the TLR3 pharmacological inhibitor CU-CPT-4a (10uM) for a further 4 days. After incubation with the TLR3 pharmacological inhibitor, cells were cultured in normal growth media for a further 6 days. Quantitative PCR was used to analyze mRNA levels of 13 components of the cardiomyocyte sarcomere.

(A) Heat-map overview of the qPCR analysis. Expression values were normalized to the average expression of the negmiR vehicle samples and then averaged (N=3 technical replicates (fibroblasts were derived from single litter and seeded into 3 individual wells). Averages for each gene were then converted to Z-scores. Centroid linkage and Euclidean methods were employed for clustering and distance measurements respectively.

(B & C) Neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with either vehicle or the TLR3 pharmacological inhibitor CU-CPT-4a (10uM) for a further 4 days. After incubation with the TLR3 pharmacological inhibitor, cells were cultured in normal growth media for a further 10 days.

(B) RNA levels of the cardiomyocyte sarcomere components Myh6 (αmyosin heavy chain), Actn2 (αsarcomeric actinin) and Tnni3 (cardiac troponin-I) was determined by qPCR. N=4 independent experiments.

(C) Left: Cells were fixed and stained with anti-Actn2 antibodies (red). Nuclei were stained with DAPI (blue). Scale bar 50 microns. Right: Quantification of immunostaining shown in B. Cells expressing Actn2 were counted and expressed as a percentage of the total cell population. N=6 independent experiments.

(D & E) Neonatal cardiac fibroblasts were first transfected with either a control siRNA or a siRNA that targeted TLR3. Two days later, the cells were transfected again with either the negative control miR negmiR or miR combo. The day after transfection with miRNAs the media was replaced and the cells cultured in normal growth media for 14 days.

(D) Quantification of TLR3 knockdown by qPCR. N=3 independent experiments.

(E) RNA levels of the cardiomyocyte structural proteins Myh6 (αmyosin heavy chain), Actn2 (αsarcomeric actinin) and Tnni3 (cardiac troponin-I) was determined by qPCR. N=4 independent experiments. Data represented as Mean ± SEM. ***P<0.001, **P<0.01, *P<0.05, ns: not significant. For A, B and D comparisons are made between miR combo and negmiR for each group. For C, comparison is made between control siRNA and siRNA targeting TLR3.