Liu Liu

Reprogramming fibroblasts into induced cardiomyocytes (iCMs) holds great potential for cardiac regeneration. However, low conversion rate both in vitro and in vivo remains a significant challenge. To address this challenge, we focused on potential epigenetic barriers and screened 33 small-molecule inhibitors/degraders targeting histone acetyl post-translational modifications and related epigenetic factors. BET degraders were found to enhance cardiac reprogramming efficiency by degrading BRD4 and repressing genes involved in immune response, particularly those in the JAK/STAT pathway. We further identified that macrophage/oncostatin M activated the JAK/STAT pathway to repress cardiac reprogramming. BRD4 degrader treatment improved iCMs formation by inhibiting macrophage/oncostatin M-induced activation of the JAK/STAT pathway. Moreover, BRD4 degrader treatment enhanced MGT-mediated cardiac regeneration in vivo and improved myocardial performance post-myocardial infarction. These findings provide new insights into BRD4, macrophage/oncostatin M and JAK/STAT pathway in fibroblast to cardiomyocyte-like cell conversion and offer promising targets and small molecules to improve iCM reprogramming for clinical applications. Highlights Screen of histone acetylation modifiers identified BRD4, a histone acetylation reader, as an important regulator in cardiac reprogramming. BRD4 degradation promoted cardiac reprogramming by repressing inflammation and the JAK-STAT pathway. BRD4 degradation inhibited macrophage/oncostatin M-induced activation of the JAK/STAT pathway. BRD4 degradation enhanced MGT-mediated cardiac regeneration in vivo and improved myocardial performance post-myocardial infarction.

The phosphorylation of 14-3-3 binding motif is involved in many cellular processes. A strategy that enables targeted degradation of phospho-14-3-3-binding-motif embedded (P-14-3-3-BME) proteins for studying their functions is highly desirable for basic research. Here, we report a phosphorylation-induced, ubiquitin-proteasome-system-mediated targeted protein degradation (TPD) strategy that allows specific degradation of P-14-3-3-BME proteins. Specifically, by ligating a modified von Hippel-Lindau E3-ligase with an engineered 14-3-3 bait, we generated a protein chimera referred to as Targeted Degradation of P-14-3-3-BME Protein (TDPP). TDPP can serve as a universal degrader for P-14-3-3-BME proteins based on the specific recognition of the phosphorylation in 14-3-3 binding motifs. TDPP shows high efficiency and specificity to a difopein-EGFP reporter, general and specific P-14-3-3-BME proteins. TDPP can also be applied for the validation of P-14-3-3-BME proteins. These results strongly support TDPP as a powerful tool for 14-3-3 related research.

Direct reprogramming of fibroblasts into CM-like cells has emerged as an attractive strategy to generate induced CMs (iCMs) in heart regeneration. However, low conversion rate, poor purity, and the lack of precise conversion of iCMs are still present as significant challenges. In this review, we summarize the recent development in understanding the molecular mechanisms of cardiac reprogramming with various strategies to achieve more efficient iCMs. reprogramming. Specifically, we focus on the identified critical roles of transcriptional regulation, epigenetic modification, signaling pathways from the cellular microenvironment, and cell cycling regulation in cardiac reprogramming. We also discuss the progress in delivery system optimization and cardiac reprogramming in human cells related to preclinical applications. We anticipate that this will translate cardiac reprogramming-based heart therapy into clinical applications. In addition to optimizing the cardiogenesis related transcriptional regulation and signaling pathways, an important strategy is to modulate the pathological microenvironment associated with heart injury, including inflammation, pro-fibrotic signaling pathways, and the mechanical properties of the damaged myocardium. We are optimistic that cardiac reprogramming will provide a powerful therapy in heart regenerative medicine.

Myocardial infarction (MI) is accompanied by severe energy deprivation and extensive epigenetic changes. However, how energy metabolism and chromatin modifications are interlinked during MI and heart repair has been poorly explored. Here, we examined the effect of different carbon sources that involved in the major metabolic pathways of acetyl-CoA synthesis on myocardial infarction and found that elevation of acetyl-CoA significantly improved heart function in I/R rats by administration of sodium octanoate (8C). Mechanistically, 8C prevented I/R injury by promoting histone acetylation which in turn activated the expression of antioxidant genes HO1, NQO1 and SOD2 and inhibited cardiomyocyte apoptosis. Furthermore, we identified that 8C-promoted histone acetylation and heart repair were carried out by metabolic enzyme medium-chain acyl-CoA dehydrogenase (MCAD) and histone acetyltransferase Kat2a. Therefore, our results demonstrate that 8C dramatically improves cardiac function through metabolic acetyl-CoA-mediated histone acetylation. This study uncovers an interlinked metabolic/epigenetic network comprising 8C, acetyl-CoA, MCAD, and Kat2a in stimulating histone acetylation and anti-oxidative stress gene expression to combat heart injury.

Reprogramming fibroblasts into induced cardiomyocytes (iCMs) is a potentially promising strategy for heart regeneration. Yet a major challenge is the low conversion rate. To address this challenge, we screened and identified four chemicals, insulin-like growth factor-1, Mll1 inhibitor MM589, transforming growth factor-β inhibitor A83-01, and Bmi1 inhibitor PTC-209, termed as IMAP, that coordinately enhanced reprogramming efficiency. Using α-muscle heavy chain -green fluorescent protein mouse embryo fibroblasts as the staring cell type, we observed a six-fold increase of iCM formation with IMAP treatment. IMAP stimulated higher cardiac troponin T and α-actinin expression and more sarcomere formation with up-regulation of many cardiac genes and down-regulation of fibroblast genes. Furthermore, IMAP promoted higher spontaneous beating and calcium transient activities of iCMs derived from neonatal cardiac fibroblasts. Intriguingly, we identified that many genes involved in immune responses, particularly those in specific C-C chemokine signaling pathways, were repressed with IMAP treatment. We next tested C-C chemokine ligands Ccl3, Ccl6, and Ccl17 in cardiac reprogramming and observed inhibitory effect on iCM formation, while corresponding inhibitors of Ccr1, Ccr4, and Ccr5 had the opposite effect. These results indicated that suppression of specific C-C chemokine signaling pathways was a direct down-stream event of IMAP treatment that enhanced cardiac reprogramming. In conclusion, we identified a combination of four chemicals IMAP in suppressing specific C-C chemokine signaling pathways and facilitating MGT-induced cardiac reprogramming. Our studies revealed the role of these specific C-C chemokine signaling pathways in cardiac reprogramming and provide potential targets in iCM formation and its clinical applications.