马树红
中国医学科学院阜外医院
BACKGROUND:Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants.METHODS:We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant.RESULTS:Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts.CONCLUSIONS:These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.
Circulation 2024
BACKGROUND:Pseudoenzymes, catalytically deficient variants of active enzymes, have a wide range of regulatory functions. ADP-ribosylhydrolase-like 1 (ADPRHL1), a pseudoenzyme belonging to a small group of ADP-ribosylhydrolase enzymes that lacks the amino acid residues necessary for catalytic activity, may have a significant role in heart development based on accumulating evidence. However, the specific function of ADPRHL1 in this process has not been elucidated. To investigate the role of ADPRHL1 in the heart, we generated the first in vitro human embryonic stem cell model with an ADPRHL1 knockout.METHOD:Using the CRISPR/Cas9 system, we generated ADPRHL1 knockout in the human embryonic stem cell (hESC) H9 line. The cells were differentiated into cardiomyocytes using a chemically defined and xeno-free method. We employed confocal laser microscopy to detect calcium transients and microelectrode array (MEA) to assess the electrophysiological activity of ADPRHL1 deficiency cardiomyocytes. Additionally, we investigated the cellular mechanism of ADPRHL1 by Bulk RNA sequencing and western blot.RESULTS:The results indicate that the absence of ADPRHL1 in cardiomyocytes led to adhered abnormally, as well as perturbations in calcium transients and electrophysiological activity. We also revealed that disruption of focal adhesion formation in these cardiomyocytes was due to an excessive upregulation of the ROCK-myosin II pathway. Notably, inhibition of ROCK and myosin II effectively restores focal adhesions in ADPRHL1-deficient cardiomyocytes and improved electrical conduction and calcium activity.CONCLUSIONS:Our findings demonstrate that ADPRHL1 plays a critical role in maintaining the proper function of cardiomyocytes by regulating the ROCK-myosin II pathway, suggesting that it may serve as a potential drug target for the treatment of ADPRHL1-related diseases.
Stem cell research & therapy 2023
Alcoholic cardiomyopathy (ACM) is a myocardial injury caused by long-term heavy drinking. Existing evidence indicates that high levels of oxidative stress are the key to pathological cardiomyopathy caused by long-term exposure to high concentrations of alcohol, while angiotensin II (AngII) and its type 1 receptor (AT1R) play an important role in excessive drinking. Whether oxidative stress-induced damage in ACM is related to AngII and AT1R is unclear, and the effects of alcohol on the electrophysiology of myocardial cells have not been reported. Most existing studies have used animal models. This study established an in vitro model of ACM based on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The transcriptional profiling of alcohol treatment was performed by RNA-seq analysis. The role of oxidative stress, the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX), and the role of AngII and AT1R in the overactivation of oxidative stress were studied using fluorescent labeling, Western blotting, and high-content quantitative analysis. Real-time cell analysis(RTCA) and microelectrode array (MEA) were used to continuously monitor myocardial beating, observe the effects of alcohol on myocardial electrophysiological activity, and clarify the protective effects of the AT1R blocker losartan on ACM. We found that AngII and AT1R contribute to the effects of alcohol on the myocardium through oxidative stress damage, the mechanism of which may be achieved by regulating NOX.
Cell death discovery 2022
BACKGROUND:Existing methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably.METHODS:We induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/β-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs.RESULTS:WNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro.CONCLUSION:We provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction.
Stem cell research & therapy 2022
[Figure: see text].
Circulation research 2021
Stem cell-based therapy has been used to treat ischaemic heart diseases for two decades. However, optimal cell types and transplantation methods remain unclear. This study evaluated the therapeutic effects of human umbilical cord mesenchymal stem cell (hUCMSC) sheet on myocardial infarction (MI).METHODS:hUCMSCs expressing luciferase were generated by lentiviral transduction for in vivo bio-luminescent imaging tracking of cells. We applied a temperature-responsive cell culture surface-based method to form the hUCMSC sheet. Cell retention was evaluated using an in vivo bio-luminescent imaging tracking system. Unbiased transcriptional profiling of infarcted hearts and further immunohistochemical assessment of monocyte and macrophage subtypes were used to determine the mechanisms underlying the therapeutic effects of the hUCMSC sheet. Echocardiography and pathological analyses of heart sections were performed to evaluate cardiac function, angiogenesis and left ventricular remodelling.RESULTS:When transplanted to the infarcted mouse hearts, hUCMSC sheet significantly improved the retention and survival compared with cell suspension. At the early stage of MI, hUCMSC sheet modulated inflammation by decreasing Mcp1-positive monocytes and CD68-positive macrophages and increasing Cx3cr1-positive non-classical macrophages, preserving the cardiomyocytes from acute injury. Moreover, the extracellular matrix produced by hUCMSC sheet then served as bioactive scaffold for the host cells to graft and generate new epicardial tissue, providing mechanical support and routes for revascularsation. These effects of hUCMSC sheet treatment significantly improved the cardiac function at days 7 and 28 post-MI.CONCLUSIONS:hUCMSC sheet formation dramatically improved the biological functions of hUCMSCs, mitigating adverse post-MI remodelling by modulating the inflammatory response and providing bioactive scaffold upon transplantation into the heart.TRANSLATIONAL PERSPECTIVE:Due to its excellent availability as well as superior local cellular retention and survival, allogenic transplantation of hUCMSC sheets can more effectively acquire the biological functions of hUCMSCs, such as modulating inflammation and enhancing angiogenesis. Moreover, the hUCMSC sheet method allows the transfer of an intact extracellular matrix without introducing exogenous or synthetic biomaterial, further improving its clinical applicability.
Bioactive materials 2021
