Conservation of miR combo based direct cardiac reprogramming

Abstract

There is considerable interest in regenerating the injured heart by reprogramming resident fibroblasts into new functional cardiomyocytes. Cardiac reprogramming has been achieved via transcription factors or miRNAs. Transcription factor combinations appear to be species-specific as evidenced by the fact that combinations of transcription factors which are effective for the reprogramming of mouse fibroblasts are ineffective in pigs and humans. Whether miRNA based cardiac reprogramming suffers from the same limitation is unknown. We have previously demonstrated that mouse cardiac fibroblasts can be directly converted into cardiomyocytes both in vitro and in vivo via a combination of four microRNAs (miR-1, miR-133a, miR-208a and miR-499) termed "miR combo." To assess species-specificity, miR combo was transfected into cardiac fibroblasts isolated from the left ventricle of dogs, pigs and humans. QPCR analysis indicated that miR combo effectively reprogrammed fibroblasts from all of the tested mammalian species. Significant upregulation of cardiac developmental, sarcomere, and cardiac ion channel genes was observed. Through Actn2+ staining, we also found that miR combo transfection induced dog, pig and human cardiac fibroblasts to develop into cardiomyocyte-like cells. In conclusion, we have demonstrated that in contrast to transcription factor based approaches, miR combo effectively reprograms mammalian cardiac fibroblasts into cardiomyocyte-like cells.

Keywords: Cardiomyocytes; Fibroblasts; Reprogramming; miRNAs.

Fig. 1

miR combo induces cardiomyocyte gene expression in human cardiac fibroblasts. Fetal human cardiac fibroblasts isolated from the left ventricle were transfected with miR combo or the non-targeting control miRNA negmiR. After 14 days, RNA was extracted and pooled from three separate transfections. Pooled RNA was analyzed by high-throughput sequencing. Sequences were matched to the human genome and genes significantly up- and down-regulated by miR combo were determined. Up- and down-regulated genes were analyzed via gene ontology to determine their function. (A) Enrichment analysis displaying Gene Ontology (GO) terms which are over-represented in the set of genes up-regulated by miR combo. (B) Enrichment analysis displaying Gene Ontology (GO) terms which are under-represented in the set of genes up-regulated by miR combo. (C) Enrichment analysis displaying Gene Ontology (GO) terms which are over-represented in the set of genes down-regulated by miR combo.

Fig. 2

miR combo reprograms human cardiac fibroblasts into cardiomyocyte-like cells. Fetal human cardiac fibroblasts were transfected with the cardiac reprogramming cocktail miR combo. A non-targeting control miRNA (negmiR) were used as a control. (A) Four days post-transfection, RNA was extracted and analyzed for the expression of the cardiac commitment markers Gata4, Mef2C, Hand2, and Tbx5 by qPCR. Expression values are shown relative to the house keeping gene Gapdh. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Fourteen days post-transfection, RNA was extracted and analyzed for the expression of the indicated sarcomere genes by qPCR. Expression values are shown relative to the house keeping gene Gapdh. N = 4–7. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Cells were fixed fourteen days post-transfection and incubated with Actn2 binding antibodies to determine the number of cardiomyocyte-like cells. Representative images are shown (scale bar 100 μm) with quantification provided. N = 7. ***P < 0.001. (D) A representative image showing sarcomere formation in the cardiomyocyte-like cells. Scale bar 50 μm.

Fig. 3

miR combo reprograms pig and dog cardiac fibroblasts into cardiomyocyte-like cells. (A) Pig cardiac fibroblasts from the left ventricle were transfected with miR combo or the non-targeting miR negmiR. Fourteen days post-transfection, RNA was extracted and analyzed for the expression of the indicated cardiomyocyte-specific genes by qPCR. Expression values are shown relative to the house keeping gene Gapdh. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Pig cells were fixed fourteen days post-transfection and incubated with Actn2 binding antibodies to determine the number of cardiomyocyte-like cells. Representative images are shown (scale bar 100 μm) with quantification provided. Cardiomyocyte-like cell counts were determined from individual wells (N = 5). ***P < 0.001. (C) Dog cardiac fibroblasts from the left ventricle were transfected with miR combo or the non-targeting miR negmiR. Fourteen days post-transfection, RNA was extracted and analyzed for the expression of the indicated cardiomyocyte-specific genes by qPCR. Expression values are shown relative to the house keeping gene Gapdh. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Dog cells were fixed fourteen days post-transfection and incubated with Actn2 binding antibodies to determine the number of cardiomyocyte-like cells. Representative images are shown (scale bar 100 μm) with quantification provided. There were two populations of Actn2+ cells: low Actn2 expression and high Actn2 expression. Cell counts were determined for the high Actn2 expression population (N = 7). ***P < 0.001.