心筋への直接転換における細胞周期の機能解析

筑波大学 (University of Tsukuba)201

心筋への直接転換における細胞周期の機能解析

筑波大学 (University of Tsukuba)201

Ameliorating the Fibrotic Remodeling of the Heart through Direct Cardiac Reprogramming

Coronary artery disease is the most common form of cardiovascular diseases, resulting in the loss of cardiomyocytes (CM) at the site of ischemic injury. To compensate for the loss of CMs, cardiac fibroblasts quickly respond to injury and initiate cardiac remodeling in an injured heart. In the remodeling process, cardiac fibroblasts proliferate and differentiate into myofibroblasts, which secrete extracellular matrix to support the intact structure of the heart, and eventually differentiate into matrifibrocytes to form chronic scar tissue. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate this pathologic remodeling and replace the cardiac fibrotic scar with myocardium in situ. Since the first discovery in 2010, many progresses have been made to improve the efficiency and efficacy of reprogramming by understanding the mechanisms and signaling pathways that are activated during direct cardiac reprogramming. Here, we overview the development and recent progresses of direct cardiac reprogramming and discuss future directions in order to translate this promising technology into an effective therapeutic paradigm to reverse cardiac pathological remodeling in an injured heart

Long Non-Coding RNAs in Atrial Fibrillation: Pluripotent Stem Cell-Derived Cardiomyocytes as a Model System

Atrial fibrillation (AF) is a type of sustained arrhythmia in humans often characterized by devastating alterations to the cardiac conduction system as well as the structure of the atria. AF can lead to decreased cardiac function, heart failure, and other complications. Long non-coding RNAs (lncRNAs) have been shown to play important roles in the cardiovascular system, including AF; however, a large group of lncRNAs is not conserved between mouse and human. Furthermore, AF has complex networks showing variations in mechanisms in different species, making it challenging to utilize conventional animal models to investigate the functional roles and potential therapeutic benefits of lncRNAs for AF. Fortunately, pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) offer a reliable platform to study lncRNA functions in AF because of certain electrophysiological and molecular similarities with native human CMs. In this review, we first summarize the broad aspects of lncRNAs in various heart disease settings, then focus on their potential roles in AF development and pathophysiology. We also discuss current uses of PSCs in AF research and describe how these studies could be developed into novel therapeutics for AF and other cardiovascular diseases

Additional HAND2 or microRNA-1 could facilitate the progress of human iCM-reprogramming by GMTEMMZ 7 factors (7Fs).

<p>A) A t-SNE embedding was utilized to visualize the overall reprogramming degree in individual iCMs reprogrammed by 7Fs plus one extra factor, including microRNA-1 (n = 39), <i>HAND2</i> (n = 61), <i>RXRG</i> (n = 18), <i>SMYD1</i> (n = 74), and <i>TBX20</i> (n = 74). Two dash lines were inserted to assort iCMs into three populations: fibroblast-like, intermediate-, and CM-like reprogrammed iCMs. The data of H9CMs, H9Fs, 4 weeks-reprogrammed 7Fs-iCMs (4W-7Fs-iCMs) and 12W-7Fs-iCMs were included as control groups. B) The quantification of three subpopulations in panel A showed that additional HAND2 or microRNA-1 significantly decreased the subpopulation of fibroblast-like reprogrammed iCMs, while the CM-like subpopulation was significantly enhanced only in 12W-7Fs-iCMs. *p<0.05 vs. 4W-7Fs-iCMs. C) The expression of HAND2 in individual H9Fs and iCMs reprogrammed by 7Fs or 7Fs+HAND2. 7Fs+HAND2-reprogrammed iCMs were classified into three sub-populations with low-, medium- and high-expression of HAND2. D) Comparison of cardiac (upper panel) and fibroblast-enriched (lower panel) genes among low-HAND2 (n = 5), medium-HAND2 (n = 14) and high-HAND2 (n = 42) subpopulations of 7Fs+HAND2-reprogrammed iCMs. *p<0.05, ** p<0.01, *** p<0.001.</p

Single cell qPCR reveals that additional <i>HAND2</i> and microRNA-1 facilitate the early reprogramming progress of seven-factor-induced human myocytes

<div><p>The direct reprogramming of cardiac fibroblasts into induced cardiomyocyte (CM)-like cells (iCMs) holds great promise in restoring heart function. We previously found that human fibroblasts could be reprogrammed toward CM-like cells by 7 reprogramming factors; however, iCM reprogramming in human fibroblasts is both more difficult and more time-intensive than that in mouse cells. In this study, we investigated if additional reprogramming factors could quantitatively and/or qualitatively improve 7-factor-mediated human iCM reprogramming by single-cell quantitative PCR. We first validated 46 pairs of TaqMan^® primers/probes that had sufficient efficiency and sensitivity to detect the significant difference of gene expression between individual H9 human embryonic stem cell (ESC)-differentiated CMs (H9CMs) and human fibroblasts. The expression profile of these 46 genes revealed an improved reprogramming in 12-week iCMs compared to 4-week iCMs reprogrammed by 7 factors, indicating a prolonged stochastic phase during human iCM reprogramming. Although none of additional one reprogramming factor yielded a greater number of iCMs, our single-cell qPCR revealed that additional <i>HAND2</i> or microRNA-1 could facilitate the silencing of fibroblast genes and yield a better degree of reprogramming in more reprogrammed iCMs. Noticeably, the more <i>HAND2</i> expressed, the higher-level were cardiac genes activated in 7Fs+HAND2-reprogrammed iCMs. In conclusion, <i>HAND2</i> and microRNA-1 could help 7 factors to facilitate the early progress of iCM-reprogramming from human fibroblasts. Our study provides valuable information to further optimize a method of direct iCM-reprogramming in human cells.</p></div

Additional reprogramming factors didn’t increase the yield of αMHC-mCherry⁺ iCMs.

<p>A) Heatmap of gene expression profiles for a panel of transcription factors that were expressed at a significantly higher level in pooled CMs (H9CMs and fetal human CMs [fHCMs]) than that in H9Fs and HDFs. B) Fold changes (normalized to H9Fs) of cardiac transcription factors activated in pooled human iCMs reprogrammed by 7 factors (7Fs) of GMTEMMZ for 4 weeks (H9FiCMs-4W) and 12 weeks (H9FiCMs-12W). C-D) The effect of adding one extra transcription factor (C) or microRNA-1 (D) on the induction of αMHC-mCherry⁺ (upper panel, n = 6) or cardiac troponin T (cTnT⁺, lower panel, n = 4) iCMs reprogrammed by 7Fs. *p<0.05, **p<0.01 compared to the control group of 7Fs+GFP.</p

Validation of 96 TaqMan^® primers/probes for single-cell qPCR in individual H9CMs (n = 56), H9Fs (n = 45), and HDFs (n = 41).

<p>Pooled samples of H9CMs and human fibroblasts (P-CMs and P-HFs) were used as positive controls. A) Heatmap of gene expression profiles in our microarray assay of H9Fs and H9CMs. B) A panel of 46 primers/probes had sufficient efficiency and sensitivity for single-cell qPCR. C) A principal component analysis (PCA) displayed a global view of the expression profile of those 46 genes in individual H9CMs, H9Fs, and HDFs.</p

The expression profile of the identified 46 genes is able to estimate the quality of human iCM reprogramming.

<p>A) A hierarchical clustering analysis showed that most human iCMs reprogrammed from H9Fs by 7 factors of GMTEMMZ for 12 weeks (H9FiCM-12W, n = 39) were clustered close to H9CMs, while many iCMs reprogrammed from H9Fs (H9FiCM-4W, n = 36) and HDFs (HDFiCM-4W, n = 37) for 4 weeks were clustered close to fibroblasts. B) Principal component (PC) projections of individual cells. The dash-line circle surrounds a population of iCMs that had a good quality of reprogramming; the arrow indicates another population of iCMs that were poorly reprogrammed.</p