Let-7 family of microRNA is required for maturation and adult-like metabolism in stem cell-derived cardiomyocytes

Abstract

In metazoans, transition from fetal to adult heart is accompanied by a switch in energy metabolism-glycolysis to fatty acid oxidation. The molecular factors regulating this metabolic switch remain largely unexplored. We first demonstrate that the molecular signatures in 1-year (y) matured human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are similar to those seen in in vivo-derived mature cardiac tissues, thus making them an excellent model to study human cardiac maturation. We further show that let-7 is the most highly up-regulated microRNA (miRNA) family during in vitro human cardiac maturation. Gain- and loss-of-function analyses of let-7g in hESC-CMs demonstrate it is both required and sufficient for maturation, but not for early differentiation of CMs. Overexpression of let-7 family members in hESC-CMs enhances cell size, sarcomere length, force of contraction, and respiratory capacity. Interestingly, large-scale expression data, target analysis, and metabolic flux assays suggest this let-7-driven CM maturation could be a result of down-regulation of the phosphoinositide 3 kinase (PI3K)/AKT protein kinase/insulin pathway and an up-regulation of fatty acid metabolism. These results indicate let-7 is an important mediator in augmenting metabolic energetics in maturing CMs. Promoting maturation of hESC-CMs with let-7 overexpression will be highly significant for basic and applied research.

Keywords: cardiac maturation; hESC-cardiomyocyte; let-7; metabolism; microRNA.

Fig. 1.

The molecular signatures of in vitro cardiac maturation reflect in vivo cardiac maturation. (A) Schematic representation of large-scale mRNA and miRNA sequencing using Illumina platform from day 20-CMs and in vitro-matured CMs derived from hESC (H7). (B and C) qPCR analysis of maturation markers in day 20-CMs and in vitro-matured CMs. Means ± SEM are shown. **P ≤ 0.05 (Student's t test). (D) 2D principal component analysis using genomewide expression data for day 20-CMs, 1y-CMs, HAH, HFA, and HFV samples attained using R. (E) Heat map depicting changes in gene expression of 12 different pathways between day 20-CMs, 1y-CMs, HAH, HFA, and HFV samples attained using R. The rows reflect read counts and are standardized individually and colored according to the Z score. Yellow and blue represent up- and down-regulation, respectively. (F–I) Density plots using R generated with fold change expression of genes from four representative categories for HAH (F), 1y-CM (G), HFA (H), and HFV (I) relative to gene expression of day 20-CMs. X axis indicates log₂ fold change in gene expression. Black line indicates expression of all genes. Colored lines toward the left and right side of the black line indicate down-regulation and up-regulation of pathways, respectively. All experiments were repeated at least three times.

Fig. 2.

Genome-wide sequencing of in vitro-matured CMs reveals let-7 as the most highly expressed miRNA family. (A) Plot depicting expression of all miRNAs with deducible read counts. The x axis indicates ranks of miRNAs based on relative fold change expression (y axis). Colored points highlight members of various miRNA families, including let-7d, let-7g, let-7f, let-7b, and let-7i; mir-378f, mir-378g, mir-378e, mir-378b, mir-378a, mir-378i, and mir-378c; mir-30b; mir-129–5p; and mir-502–5p. (B) Heat map generated using multiexpression viewer (mev.tm4.org) includes fold changes of all significantly regulated miRNAs (FC ≥ 2 and P ≤ 0.001) in common between 1y-CMs and cEHTs relative to day 20-CMs. Yellow and blue indicate up- and down-regulation, respectively. Numbers: 1 and 2 indicate significantly up- or down-regulated miRNAs, respectively. (C) miRNA-mRNA target analysis using IPA with 1y-CM expression datasets: three miRNAs with the highest number of targets in 1y-CMs. P values reflect a one-sided Fisher’s exact test calculated using the total number of targets for each miRNA and the number of targets present in the dataset.

Fig. 3.

Let-7 is required for hESC-CM maturation. (A–K) All analyses done in EV control, Lin28a OE, and Lin28a OE+ let-7g OE CMs. (A–C) qPCR analysis to (A) examine Lin28a expression, (B) demonstrate that let-7g is down-regulated in Lin28a OE CMs but its expression is rescued in response to let-7g OE using let-7g mimics, and (C) evaluate the expression of maturation markers. (D) α-Actinin (green) and DAPI (blue) staining of representative CMs from the three treatments. (Scale bar = 25 µm.) (E–H) Compared with EV control, Lin28a OE CMs showed significant decrease in (E) cell perimeter, (F) cell area, and (G) sarcomere length and (H) an increase in circularity index. The phenotype was partially rescued in Lin28a OE CMs+let-7g OE. (I–N) All analyses done in SCRAMBLE (SCM) control and let-7g antagomir-treated CMs. (I) qPCR analysis to examine let-7g expression. (J) α-Actinin (green) and DAPI (blue) staining of representative CMs from the two treatments. (Scale bar = 25 µm.) (K–N) Compared with SCM control, let-7g KD CMs showed significant decrease in (K) cell perimeter, (L) cell area, and (M) sarcomere length and an increase in (N) circularity index. n = 50 cells per condition, three biological replicates. Means ± SEM are shown. **P ≤ 0.05 (Student’s t test). All experiments were repeated at least three times, and representative results are shown for D and J.

Fig. 4.

Let-7 is sufficient for hESC-CM maturation. (A–C) qPCR analysis to (A) validate let-7i and let-7g expression derived from miRNA sequencing analysis from day 20-CM, in vitro-matured CMs, and HAH, and (B) demonstrate that let-7i and let-7g OE in RUES2-CMs results in increased expression of the two members. EV indicates empty vector control in RUES2-CMs (three biological replicates were analyzed for let-7 OE and EV samples). (C) Examine the expression of maturation markers in H7 day 20-CMs, EV, and RUES2-CMs. Gene expression is shown normalized first to GAPDH and then normalized to EV control. (D) α-Actinin (green), α-actin (red), and DAPI (blue) staining of representative EV control, let-7i OE, and let-7g OE CMs. (Scale bar = 50 µm.) Compared with EV control, let-7 OE CMs showed significant changes in (E) cell perimeter, (F) cell area, (G) circularity index, and (H) sarcomere length. n = 50 cells per condition, three biological replicates. (I) Representative force traces in EV control and let-7 OE CMs. (J) Significant increase in twitch force in let-7 OE CMs. n = 25 for EV control, n = 32 for let-7i OE, and n = 29 for let-7g OE from a total of three biological replicates. (K) Frequency of beating CMs. Compared with EV control, let-7 OE CMs show an increase in (L–N) APD, APD90, and (O) APD50/APD90. EV, let-7i OE, and let-7g OE CMs are collected at day 30 and hence are 10 d older than day 20 samples. Means ± SEM are shown. **P ≤ 0.05 and ***P ≤ 0.001 (Student’s t test). All experiments were repeated at least three times and representative images are shown for D.

Fig. 5.

Let-7 is critical for cardiac maturation (A, B, E, and F are analyses done with gene expression analyses and C and D are analyses based on splice variant signatures). (A) Scatter plot of let-7g OE (y axis) vs. EV control (x axis) from the mRNA sequencing dataset. Red dots indicate maturation marker genes in the dataset. A few are labeled in the plot: troponin I type 3 (TNNI3); gap junction protein alpha 1 (GJA1); actin alpha cardiac muscle 1 (ACTC1); myosin heavy chain 7 (MYH7); ryanodine receptor 2 (RYR2); potassium channel, subfamily J2 (KCNJ2); sodium channel protein 5 alpha (SCN5A); sarco endoplasmic reticulum Ca²⁺ATPase 2 (SERCA2); troponin T type 2 (TNNT2); calcium channel, voltage dependent, alpha 1C (CACNA1C). (B) 2D-PCA using mRNA signatures from 12 pathways (indicated in Fig. 1E) across the analyzed samples, as indicated in the figure. (C) Heat map showing the proportion of each of the 80 isoforms identified as differentially spliced across each condition. Each value is the estimated proportion of that isoform among all expressed isoforms of the same gene in that condition. (D) 2D-PCA based on the proportions of the 80 identified differentially spliced transcripts, applied to all replicates from these eight conditions. (E) Heat map demonstrating changes in gene expression of 12 different pathways between EV control and let-7g OE CMs. Left to right, columns 1–2 and 3–5 represent biological replicates of EV and let-7g OE CMs, respectively. The rows reflect read counts of various genes in the different categories. Rows are standardized individually and colored according to the Z score. Yellow and blue represent up- and down-regulation, respectively. (F) Density plots using R generated with fold change expression (x axis indicates log₂-fold change) of genes from four categories, indicative of cardiac function for let-7 OE/EV CMs. Black curve indicates expression of all genes. Curves toward the left and right side of the black curve indicate down-regulation and up-regulation of pathways, respectively.

Fig. 6.

Let-7 OE accelerates CM maturation. (A–F) Let-7 OE results in down-regulation of the PI3/AKT/Insulin pathway and up-regulation of fatty acid metabolism. Comparisons were done between let-7 OE and EV control for all assays. (A) Density plots using R generated with fold change expression (let-7g OE/EV) of genes for fatty acid metabolism and PI3/AKT/insulin signaling. (B) qPCR analysis of candidate let-7 targets and genes from the fatty acid metabolism. (C) Representative OCR profile in response to ATP synthase inhibitor oligomycin, uncoupler of electron transport and oxidative phosphorylation, FCCP, and electron transport chain blockers rotenone and antimycin during mito-stress assay. (D) Quantification of maximal respiration capacity; that is, changes in response to FCCP treatment after inhibition of ATP synthase by oligomycin. n = 24 from three biological replicates. (E) Representative OCR trace of let-7g OE CMs for fatty acid stress measuring Etomoxir (ETO)-responsive OCR changes after the second dose of palmitate addition. (F) Quantification of changes in OCR in response to ETO. n = 32 from three biological replicates. Means ± SEM are shown. **P ≤ 0.05 (Student’s t test). (G–K) Knock-down of IRS2 and EZH2 results in up-regulation of fatty acid metabolism and improved expression of cardiac maturation markers. (G and H) Representative OCR profile and quantification of maximal respiratory capacity in siIRS2-CM and siEZH2-CM. (I and J) Representative OCR trace for fatty acid stress using palmitate and quantification of OCR change (E). (K) qPCR analysis of cardiac maturation markers, fatty acid metabolism genes.