Single-cell chromatin profiling reveals genetic programs activating proregenerative states in nonmyocyte cells.

Cardiac regeneration requires coordinated participation of multiple cell types whereby their communications result in transient activation of proregenerative cell states. Although the molecular characteristics and lineage origins of these activated cell states and their contribution to cardiac regeneration have been studied, the extracellular signaling and the intrinsic genetic program underlying the activation of the transient functional cell states remain largely unexplored. In this study, we delineated the chromatin landscapes of the noncardiomyocytes (nonCMs) of the regenerating heart at the single-cell level and inferred the cis-regulatory architectures and trans-acting factors that control cell type-specific gene expression programs. Moreover, further motif analysis and cell-specific genetic manipulations suggest that the macrophage-derived inflammatory signal tumor necrosis factor-α, acting via its downstream transcription factor complex activator protein-1, functions cooperatively with discrete transcription regulators to activate respective nonCM cell types critical for cardiac regeneration. Thus, our study defines the regulatory architectures and intercellular communication principles in zebrafish heart regeneration.

Fibroblast Reprogramming in Cardiac Repair.

Cardiovascular disease is one of the major causes of death worldwide. Limited proliferative capacity of adult mammalian cardiomyocytes has prompted researchers to exploit regenerative therapy after myocardial injury, such as myocardial infarction, to attenuate heart dysfunction caused by such injury. Direct cardiac reprogramming is a recently emerged promising approach to repair damaged myocardium by directly converting resident cardiac fibroblasts into cardiomyocyte-like cells. The achievement of in vivo direct reprogramming of fibroblasts has been shown, by multiple laboratories independently, to improve cardiac function and mitigate fibrosis post-myocardial infarction, which holds great potential for clinical application. There have been numerous pieces of valuable work in both basic and translational research to enhance our understanding and continued refinement of direct cardiac reprogramming in recent years. However, there remain many challenges to overcome before we can truly take advantage of this technique to treat patients with ischemic cardiac diseases. Here, we review recent progress of fibroblast reprogramming in cardiac repair, including the optimization of several reprogramming strategies, mechanistic exploration, and translational efforts, and we make recommendations for future research to further understand and translate direct cardiac reprogramming from bench to bedside. Challenges relating to these efforts will also be discussed.

Can we stop one heart from breaking: triumphs and challenges in cardiac reprogramming.

Ischemic cardiac injury causes irreversible muscle loss and scarring, but recent years have seen dramatic advances in cardiac reprogramming, the field focused on regenerating cardiac muscle. With SARS-CoV2 increasing the age-adjusted cardiovascular disease mortality rate, it is worth evaluating the state of this field. Here, we summarize novel innovations in reprogramming strategies, insights into their mechanisms, and technologies for factor delivery. We also propose a broad model of reprogramming to suggest directions for future research. Poet Emily Dickinson wrote, "If I can stop one heart from breaking, I shall not live in vain." Today, researchers studying cardiac reprogramming view this line as a call to action to translate this revolutionary approach into life-saving treatments for patients with cardiovascular diseases.

Tracing the history of a heart.

Newly developed tools will help researchers understand how the human heart develops and build better models to study and treat congenital heart disease.

Fine-tuned repression of Drp1-driven mitochondrial fission primes a 'stem/progenitor-like state' to support neoplastic transformation.

Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model, we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem/progenitor cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks' level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.

Strategy of Isolating 'Primed' Tumor Initiating Cells Based on Mitochondrial Transmembrane Potential.

Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are "primed" by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these "primed" cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol can be directly used or modified to be used in various cell types. Thus, this protocol is anticipated to be invaluable for the basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.

New quantitative approach reveals heterogeneity in mitochondrial structure-function relations in tumor-initiating cells.

Steady-state mitochondrial structure or morphology is primarily maintained by a balance of opposing fission and fusion events between individual mitochondria, which is collectively referred to as mitochondrial dynamics. The details of the bidirectional relationship between the status of mitochondrial dynamics (structure) and energetics (function) require methods to integrate these mitochondrial aspects. To study the quantitative relationship between the status of mitochondrial dynamics (fission, fusion, matrix continuity and diameter) and energetics (ATP and redox), we have developed an analytical approach called mito-SinCe2 After validating and providing proof of principle, we applied mito-SinCe2 on ovarian tumor-initiating cells (ovTICs). Mito-SinCe2 analyses led to the hypothesis that mitochondria-dependent ovTICs interconvert between three states, that have distinct relationships between mitochondrial energetics and dynamics. Interestingly, fusion and ATP increase linearly with each other only once a certain level of fusion is attained. Moreover, mitochondrial dynamics status changes linearly with ATP or with redox, but not simultaneously with both. Furthermore, mito-SinCe2 analyses can potentially predict new quantitative features of the opposing fission versus fusion relationship and classify cells into functional classes based on their mito-SinCe2 states.This article has an associated First Person interview with the first author of the paper.

Crosstalk between the mitochondrial fission protein, Drp1, and the cell cycle is identified across various cancer types and can impact survival of epithelial ovarian cancer patients.

Mitochondrial metabolic reprogramming is a hallmark of tumorigenesis. Although mitochondrial function can impact cell cycle regulation it has been an understudied area in cancer research. Our study highlights a specific involvement of mitochondria in cell cycle regulation across cancer types. The mitochondrial fission process, which is regulated at the core by Drp1, impacts various cellular functions. Drp1 has been implicated in various cancer types with no common mechanism reported. Our Drp1-directed large-scale analyses of the publically available cancer genomes reveal a robust correlation of Drp1 with cell-cycle genes in 29 of the 31 cancer types examined. Hypothesis driven investigation on epithelial ovarian cancer (EOC) revealed that Drp1 co-expresses specifically with the cell-cycle module responsible for mitotic transition. Repression of Drp1 in EOC cells can specifically attenuate mitotic transition, establishing a potential casual role of Drp1 in mitotic transition. Interestingly, Drp1-Cell-Cycle co-expression module is specifically detected in primary epithelial ovarian tumors that robustly responded to chemotherapy, suggesting that Drp1 driven mitosis may underlie chemo-sensitivity of the primary tumors. Analyses of matched primary and relapsed EOC samples revealed a Drp1-based-gene-expression-signature that could identify patients with poor survival probabilities from their primary tumors. Our results imply that around 60% of platinum-sensitive EOC patients undergoing relapse show poor survival, potentially due to further activation of a mitochondria driven cell-cycle regime in their recurrent disease. We speculate that this patient group could possibly benefit from mitochondria directed therapies that are being currently evaluated at various levels, thus enabling targeted or personalized therapy based cancer management.