Environmental factors influence somatic cell reprogramming to cardiomyocyte-like cells.

Direct cardiac reprogramming, which refers to somatic cell (i.e. fibroblast) fate conversion to cardiomyocyte-like cell without transitioning through an intermediate pluripotent state, provides a novel therapeutic strategy for heart regeneration by converting resident cardiac fibroblasts to cardiomyocytes in situ. However, several limitations need to be addressed prior to clinical translation of this technology. They include low efficiency of reprogramming, heterogeneity of starting fibroblasts, functional immaturity of induced cardiomyocytes (iCMs), virus immunogenicity and toxicity, incomplete understanding of changes in the epigenetic landscape as fibroblasts undergo reprogramming, and the environmental factors that influence fate conversion. Several studies have demonstrated that a combination of enforced expression of cardiac transcription factors along with certain cytokines and growth factors in the presence of favorable environmental cues (including extracellular matrix, topography, and mechanical properties) enhance the efficiency and quality of direct reprogramming. This paper reviews the literature on the influence of the microenvironment on direct cardiac reprogramming in vitro and in vivo.

Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies.

To date, no viable therapeutic options exist for the effective and sustained reversal of cardiac failure, other than heart transplantation and mechanical circulatory assist devices. Therefore, divergent strategies aiming at the de novo formation of contractile tissue, as a prerequisite for the restoration of cardiac pump function, are currently being pursued. Clinical trials involving the transplantation of somatic progenitor cells failed. The search for alternative cell-based strategies to combat the consequences of ischemic injury has sparked widespread interest in the genetic and pharmacologic reprogramming of fibroblasts into cardiomyocytes, harnessing the abundant in vivo pool of cardiac fibroblasts. Here, we provide a comprehensive overview of in vitro and in vivo cardiac reprogramming studies identified in an extensive literature search. We systematically review and evaluate feasibility, efficiency, and reproducibility of the different technologies currently being explored. Finally, we discuss potential safety issues deduced from preclinical studies and identify obstacles that must be overcome before clinical translation.-Klose, K., Gossen, M., Stamm, C. Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies.

S-phase Synchronization Facilitates the Early Progression of Induced-Cardiomyocyte Reprogramming through Enhanced Cell-Cycle Exit.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFPhigh iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFPlow cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.

PON2 ameliorates Ang II-induced cardiomyocyte injury by targeting the CANX/NOX4 signaling pathway.

BACKGROUND: The incidence of heart failure (HF) presents an escalating trend annually, second only to cancer. Few literatures are available regarding on the role of paraoxonase 2 (PON2) in HF so far despite the protective role of PON2 in cardiovascular diseases. METHODS: PON2 expression in AC16 cells was examined with reverse transcriptase-quantitative polymerase chain reaction and western blot following angiotensin II (Ang II) challenging. After PON2 elevation, 2, 7-dichlorofluorescein diacetate assay estimated reactive oxygen species content, related kits appraised oxidative stress, enzyme-linked immunosorbent assay evaluated inflammatory levels, and Western blot was applied to the analysis of apoptosis levels. Research on cytoskeleton was conducted by immunofluorescence (IF), and Western blot analysis of the expressions of hypertrophy-related proteins was performed. BioGRID and GeneMania databases were used to analyze the relationship between PON2 and Calnexin (CANX), which was corroborated by co-immunoprecipitation experiment. Subsequently, PON2 and CANX were simultaneously overexpressed in AC16 cells induced by Ang II to further figure out the mechanism. RESULTS: PON2 expression was depleted in Ang II-induced AC16 cells. PON2 might mediate CANX/NOX4 signaling to inhibit oxidation, inflammatory, hypertrophy, and damage in Ang II-induced AC16 cells. CONCLUSION: PON2 can ease Ang II-induced cardiomyocyte injury via targeting CANX/NOX4 signaling.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction.

BACKGROUND: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. METHODS: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. RESULTS: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA-expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. CONCLUSIONS: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.

AIFM1, negatively regulated by miR-145-5p, aggravates hypoxia-induced cardiomyocyte injury.

BACKGROUND: Hypoxia-induced apoptosis is linked to the pathogenesis of myocardial infarction. The role of apoptosis-inducing factor mitochondria associated 1 (AIFM1) in cardiomyocyte injury remains unclear. This study was aimed at probing into the role and the underlying regulatory mechanism of AIFM1 in myocardial injury. METHODS: H9c2 cardiomyocytes and C57BL/6 mice were used for myocardial hypoxic/ischemic injury and myocardial infarction animal models. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the expression levels of AIFM1 mRNA and miR-145-5p. Western blot was used for examining the expression levels of AIFM1, caspase-3, cleaved caspase-3, p-53, and γ-H2AX. Cell viability was examined by cell counting kit-8 (CCK-8) assay and BrdU assay. Interaction between AIFM1 and miR-145-5p was determined by bioinformatics analysis, qRT-PCR, Western blot, and dual-luciferase reporter assay. RESULTS: AIFM1 expression was markedly highly elevated, while miR-145-5p expression was significantly down-regulated in the myocardial infarction animal model and H9c2 cells under hypoxia. Augmentation of AIFM1 led to a dramatic decrease of cell viability, accompanied by an increase of the secretion of the inflammatory cytokines IL-1ß, TNF-α, IL-6, and the expression of cleaved caspase-3. Furthermore, AIFM1 was identified as a target of miR-145-5p. In addition, miR-145-5p/AIFM1 axis regulated the expression of p53. CONCLUSION: AIFM1 may exacerbate myocardial ischemic injury by promoting inflammation and the injury of cardiomyocytes, and its up-regulation may be partly due to the down-regulation of miR-145-5p.

Silibinin eliminates mitochondrial ROS and restores autophagy through IL6ST/JAK2/STAT3 signaling pathway to protect cardiomyocytes from doxorubicin-induced injury.

Growing evidence indicates that silibinin (SLB), a main component extracted from Chinese herb Silybum marianum, can effectively antagonize doxorubicin (DOX) induced myocardial injury (DIMI), but the specific molecular mechanism is still unelucidated. Herein, DOX induced human AC16 cardiomyocyte injury model and Network Pharmacology are used to predict and verify the potential mechanism. The analysis results of the core PPI network of SLB against DIMI show that JAK/STAT signaling pathway and autophagy are significantly enriched. Molecular docking results indicate that SLB has stronger binding ability to signaling key proteins IL6ST, JAK2 and STAT3 (affinity ≤ -7.0 kcal/mol). The detection results of pathway activation and autophagy level demonstrate that SLB significantly alleviates DOX induced IL6ST/JAK2/STAT3 signaling pathway inhibition and autophagy inhibition, reduces the death rate of cardiomyocytes. This protective effect of SLB is eliminated when key pathway proteins (IL6ST, JAK2, STAT3) are knocked down or autophagy is inhibited (3-MA or Beclin1 knockdown). These results suggest that the regulation of IL6ST/JAK2/STAT3 signaling pathway and autophagy may be important mechanism for SLB's protective effect on DOX injured cardiomyocytes. Further experimental results prove that knockdown of IL6ST, JAK2 and STAT3 eliminate the mitochondrial ROS scavenging effect and autophagy promoting effect of SLB. In sum, SLB can decrease the mitochondrial ROS and restore autophagy to antagonize DOX-induced cardiomyocyte injury by activating IL6ST/JAK2/STAT3 signaling pathway.

In Vitro Conversion of Murine Fibroblasts into Cardiomyocyte-Like Cells.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise as a potential treatment for cardiovascular disease, many of which are associated with tremendous loss of functional cardiomyocytes and simultaneous formation of scar tissue. Burgeoning studies have shown that the introduction of three minimal transcriptional factors, Gata4, Mef2c, and Tbx5 (G/M/T), could convert murine fibroblasts into iCMs that closely resemble endogenous CMs both in vitro and in vivo. Recent studies on iCM cell fate determination have demonstrated that the removal of genetic and epigenetic barriers could facilitate iCM reprogramming. However, varied reprogramming efficiency among research groups hinders its further study and potential applicability. Here, we provide a newly updated and detailed protocol for in vitro generation and evaluation of functional iCMs from mouse embryonic fibroblasts and neonatal cardiac fibroblasts using retroviral polycistronic construct encoding optimal expression of G/M/T factors. We hope that this optimized protocol will lay the foundation for future mechanistic studies of murine iCMs and further improvement of iCM generation.

Inhibition of CREB-CBP Signaling Improves Fibroblast Plasticity for Direct Cardiac Reprogramming.

Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a promising approach but remains a challenge in heart regeneration. Efforts have focused on improving the efficiency by understanding fundamental mechanisms. One major challenge is that the plasticity of cultured fibroblast varies batch to batch with unknown mechanisms. Here, we noticed a portion of in vitro cultured fibroblasts have been activated to differentiate into myofibroblasts, marked by the expression of αSMA, even in primary cell cultures. Both forskolin, which increases cAMP levels, and TGFß inhibitor SB431542 can efficiently suppress myofibroblast differentiation of cultured fibroblasts. However, SB431542 improved but forskolin blocked iCM reprogramming of fibroblasts that were infected with retroviruses of Gata4, Mef2c, and Tbx5 (GMT). Moreover, inhibitors of cAMP downstream signaling pathways, PKA or CREB-CBP, significantly improved the efficiency of reprogramming. Consistently, inhibition of p38/MAPK, another upstream regulator of CREB-CBP, also improved reprogramming efficiency. We then investigated if inhibition of these signaling pathways in primary cultured fibroblasts could improve their plasticity for reprogramming and found that preconditioning of cultured fibroblasts with CREB-CBP inhibitor significantly improved the cellular plasticity of fibroblasts to be reprogrammed, yielding ~2-fold more iCMs than untreated control cells. In conclusion, suppression of CREB-CBP signaling improves fibroblast plasticity for direct cardiac reprogramming.

DDX3X deficiency alleviates LPS-induced H9c2 cardiomyocytes pyroptosis by suppressing activation of NLRP3 inflammasome.

Increasing evidence suggest that NOD-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis may be the underlying pathological mechanism of sepsis-induced cardiomyopathy. DDX3X, an ATP-dependent RNA helicase, plays a vital role in the formation of the NLRP3 inflammasome by directly interacting with cytoplasmic NLRP3. However, whether DDX3X has a direct impact on lipopolysaccharide (LPS)-induced cardiomyocyte injury by regulating NLRP3 inflammasome assembly remains unclear. The present study aimed to investigate the role of DDX3X in the activation of the NLRP3 inflammasome and determine the molecular mechanism of DDX3X action in LPS-induced pyroptosis in H9c2 cardiomyocytes. H9c2 cardiomyocytes were treated with LPS to simulate sepsis in vitro. The results demonstrated that LPS stimulation upregulated DDX3X expression in H9c2 cardiomyocytes. Furthermore, Ddx3x knockdown significantly attenuated pyroptosis and cell injury in LPS-treated H9c2 cells by suppressing NLRP3 inflammasome activation. Taken together, these results suggest that DDX3X is involved in LPS-induced cardiomyocyte pyroptosis, and DDX3X deficiency mitigates cardiomyocyte damage induced by LPS treatment.

Mitochondria-Rich Extracellular Vesicles Rescue Patient-Specific Cardiomyocytes From Doxorubicin Injury: Insights Into the SENECA Trial.

BACKGROUND: Anthracycline-induced cardiomyopathy (AIC) is a significant source of morbidity and mortality in cancer survivors. The role of mesenchymal stem cells (MSCs) in treating AIC was evaluated in the SENECA trial, a Phase 1 National Heart, Lung, and Blood Institute-sponsored study, but the mechanisms underpinning efficacy in human tissue need clarification. OBJECTIVES: The purpose of this study was to perform an in vitro clinical trial evaluating the efficacy and putative mechanisms of SENECA trial-specific MSCs in treating doxorubicin (DOX) injury, using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iCMs) generated from SENECA patients. METHODS: Patient-specific iCMs were injured with 1 µmol/L DOX for 24 hours, treated with extracellular vesicles (EVs) from MSCs by either coculture or direct incubation and then assessed for viability and markers of improved cellular physiology. MSC-derived EVs were separated into large extracellular vesicles (L-EVs) (>200 nm) and small EVs (<220nm) using a novel filtration system. RESULTS: iCMs cocultured with MSCs in a transwell system demonstrated improved iCM viability and attenuated apoptosis. L-EVs but not small EVs recapitulated this therapeutic effect. L-EVs were found to be enriched in mitochondria, which were shown to be taken up by iCMs. iCMs treated with L-EVs demonstrated improved contractility, reactive oxygen species production, ATP production, and mitochondrial biogenesis. Inhibiting L-EV mitochondrial function with 1-methyl-4-phenylpyridinium attenuated efficacy. CONCLUSIONS: L-EV-mediated mitochondrial transfer mitigates DOX injury in patient-specific iCMs. Although SENECA was not designed to test MSC efficacy, consistent tendencies toward a positive effect were observed across endpoints. Our results suggest a mechanism by which MSCs may improve cardiovascular performance in AIC independent of regeneration, which could inform future trial design evaluating the therapeutic potential of MSCs.

Cardiac regeneration with pluripotent stem cell-derived cardiomyocytes and direct cardiac reprogramming.

Cardiovascular disease is the leading cause of death globally. Cardiomyocytes (CMs) have poor regenerative capacity, and pharmacological therapies have limited efficacy in severe heart failure. Currently, there are several promising strategies for cardiac regeneration. The most promising approach to remuscularize failing hearts is cell transplantation therapy using newly generated CMs from exogenous sources, such as pluripotent stem cells. Alternatively, approaches to generate new CMs from endogenous cell sources in situ may also repair the injured heart and improve cardiac function. Direct cardiac reprogramming has emerged as a novel therapeutic approach to regenerate injured hearts by directly converting endogenous cardiac fibroblasts into CM-like cells. Through cell transplantation and direct cardiac reprogramming, new CMs can be generated and scar tissue reduced to improve cardiac function; therefore, cardiac regeneration may serve as a powerful strategy for treatment of severe heart failure. While substantial progress has been made in these two strategies for cardiac regeneration over the past several years, challenges remain for clinical translation. This review provide an overview of previous reports and current challenges in this field.

Less may be more: Using small molecules to reprogram human cells into functional cardiomyocytes.

The prospect of genetically reprogramming cardiac fibroblasts into induced cardiomyocytes by using cardio-differentiating transcription factors represents a significant advantage over previous strategies involving stem cell implantation or the delivery of angiogenic factors. Remarkably, intramyocardial administration of cardio-differentiating factors consistently results in 20% to 30% improvements in postinfarct ejection fraction and nearly a 50% reduction in myocardial fibrosis in murine models. Despite these encouraging observations, few breakthroughs have been made in the reprogramming of human cells, which have more rigorous epigenetic constraints and gene regulatory networks that oppose reprogramming. As a potential solution to this challenge, Cao and colleagues used a cocktail of 9 chemicals capable of reprogramming human fibroblasts into contractile cardiomyocyte-like cells, albeit at a low efficiency. This strategy would obviate the concerns with viral vectors and appears to partially overcome the epigenetic constraints in human cells. Nevertheless, significant challenges, including drug-drug interactions, low reprogramming efficiency, and lack of in vivo data must be overcome before future clinical application.

Direct Cardiac Reprogramming as a Novel Therapeutic Strategy for Treatment of Myocardial Infarction.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds great promise as a novel therapy for the treatment of heart failure, a common and morbid disease that is usually caused by irreversible loss of functional cardiomyocytes (CMs). Recently, we and others showed that in a murine model of acute myocardial infarction, delivery of three transcription factors, Gata4, Mef2c, and Tbx5 converted endogenous cardiac fibroblasts into functional iCMs. These iCMs integrated electrically and mechanically with surrounding myocardium, resulting in a reduction in scar size and an improvement in heart function. Our findings suggest that iCM reprogramming may be a means of regenerating functional CMs in vivo for patients with heart disease. However, because relatively little is known about the factors that regulate iCM reprogramming, the applicability of iCM reprogramming is currently limited to the experimental settings in which it has been attempted. Specific hurdles include the relatively low conversion rate of iCMs and the need for reprogramming to occur in the context of acute injury. Therefore, before this treatment can become a viable therapy for human heart disease, the optimal condition for efficient iCM generation must be determined. Here, we provide a detailed protocol for both in vitro and in vivo iCM generation that has been optimized so far in our lab. We hope that this protocol will lay a foundation for future further improvement of iCM generation and provide a platform for mechanistic studies.

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and quantitatively efficient direct cardiac reprogramming. Taken together, this study provides new insights into the complexity of cell fate conversion and better understanding of the roles of transcriptional activators, signaling pathways and protein kinases in increasing the efficiency of direct cardiac reprogramming and maturity of iCMs.

Effect of biophysical cues on reprogramming to cardiomyocytes.

Reprogramming of fibroblasts to cardiomyocytes offers exciting potential in cell therapy and regenerative medicine, but has low efficiency. We hypothesize that physical cues may positively affect the reprogramming process, and studied the effects of periodic mechanical stretch, substrate stiffness and microgrooved substrate on reprogramming yield. Subjecting reprogramming fibroblasts to periodic mechanical stretch and different substrate stiffness did not improve reprogramming yield. On the other hand, culturing the cells on microgrooved substrate enhanced the expression of cardiomyocyte genes by day 2 and improved the yield of partially reprogrammed cells at day 10. By combining microgrooved substrate with an existing optimized culture protocol, yield of reprogrammed cardiomyocytes with striated cardiac troponin T staining and spontaneous contractile activity was increased. We identified the regulation of Mkl1 activity as a new mechanism by which microgroove can affect reprogramming. Biochemical approach could only partially recapitulate the effect of microgroove. Microgroove demonstrated an additional effect of enhancing organization of sarcomeric structure, which could not be recapitulated by biochemical approach. This study provides insights into new mechanisms by which topographical cues can affect cellular reprogramming.

Conversion of mouse fibroblasts into cardiomyocyte-like cells using small molecule treatments.

The possibility of controlling cell fates by overexpressing specific transcription factors has led to numerous studies in stem cell research. Small molecules can be used, instead of transcription factors, to induce the de-differentiation of somatic cells or to induce pluripotent cells (iPSCs). Here we reported that combinations of small molecules could convert mouse fibroblasts into cardiomyocyte-like cell without requiring transcription factor expression. Treatment with specific combinations of small molecules that are enhancer for iPSC induction converted mouse fibroblasts into spontaneously contracting, cardiac troponin T-positive, cardiomyocyte-like cells. We specifically identified five small molecules that can induce mouse fibroblasts to form these cardiomyocyte-like cells. These cells are similar to primary cardiomyocytes in terms of marker gene expression, epigenetic status of cardiac-specific genes, and subcellular structure. Our findings indicate that lineage conversion can be induced not only by transcription factors, but also by small molecules.