In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and ...further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prompt coronary catheterization and revascularization have markedly improved the outcomes of myocardial infarction, but have also resulted in a growing number of surviving patients with permanent ...structural damage of the heart, which frequently leads to heart failure. There is an unmet clinical need for treatments for this condition
, particularly given the inability of cardiomyocytes to replicate and thereby regenerate the lost contractile tissue
. Here we show that expression of human microRNA-199a in infarcted pig hearts can stimulate cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. However, subsequent persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however dosage of this therapy needs to be tightly controlled.
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IJS, KISLJ, NUK, UL, UM, UPUK
COVID-19 is a deadly pulmonary disease with peculiar characteristics, which include variable clinical course and thrombophilia. A thorough understanding of the pathological correlates of the disease ...is still missing.
Here we report the systematic analysis of 41 consecutive post-mortem samples from individuals who died of COVID-19. Histological analysis is complemented by immunohistochemistry for cellular and viral antigens and the detection of viral genomes by in situ RNA hybridization.
COVID-19 is characterized by extensive alveolar damage (41/41 of patients) and thrombosis of the lung micro- and macro-vasculature (29/41, 71%). Thrombi were in different stages of organization, consistent with their local origin. Pneumocytes and endothelial cells contained viral RNA even at the later stages of the disease. An additional feature was the common presence of a large number of dysmorphic pneumocytes, often forming syncytial elements (36/41, 87%). Despite occasional detection of virus-positive cells, no overt signs of viral infection were detected in other organs, which showed non-specific alterations.
COVID-19 is a unique disease characterized by extensive lung thrombosis, long-term persistence of viral RNA in pneumocytes and endothelial cells, along with the presence of infected cell syncytia. Several of COVID-19 features might be consequent to the persistence of virus-infected cells for the duration of the disease.
This work was supported by a King's Together Rapid COVID-19 Call grant from King's College London. MG is supported by the European Research Council (ERC) Advanced Grant 787971 “CuRE” and by Programme Grant RG/19/11/34633 from the British Heart Foundation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cardiomyocyte proliferation stops at birth when the heart is no longer exposed to maternal blood and, likewise, to regulatory T cells (Tregs) that are expanded to promote maternal tolerance towards ...the fetus. Here, we report a role of Tregs in promoting cardiomyocyte proliferation. Treg-conditioned medium promotes cardiomyocyte proliferation, similar to the serum from pregnant animals. Proliferative cardiomyocytes are detected in the heart of pregnant mothers, and Treg depletion during pregnancy decreases both maternal and fetal cardiomyocyte proliferation. Treg depletion after myocardial infarction results in depressed cardiac function, massive inflammation, and scarce collagen deposition. In contrast, Treg injection reduces infarct size, preserves contractility, and increases the number of proliferating cardiomyocytes. The overexpression of six factors secreted by Tregs (Cst7, Tnfsf11, Il33, Fgl2, Matn2, and Igf2) reproduces the therapeutic effect. In conclusion, Tregs promote fetal and maternal cardiomyocyte proliferation in a paracrine manner and improve the outcome of myocardial infarction.
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in ...virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The identification of the molecular mechanisms controlling cardiomyocyte proliferation during the embryonic, fetal, and early neonatal life appears of paramount interest in regard to exploiting this ...information to promote cardiac regeneration. Here, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the Notch pathway. We found that Notch1 is expressed in proliferating ventricular immature cardiac myocytes (ICMs) both in vitro and in vivo, and that the number of Notch1-positive cells in the heart declines with age. Notch1 expression in ICMs paralleled the expression of its Jagged1 ligand on non-myocyte supporting cells. The inhibition of Notch signaling in ICMs blocked their proliferation and induced apoptosis; in contrast, its activation by Jagged1 or by the constitutive expression of its activated form using an adeno-associated virus markedly stimulated proliferative signaling and promoted ICM expansion. Maintenance or reactivation of Notch signaling in cardiac myocytes might represent an interesting target for innovative regenerative therapy.
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BFBNIB, NUK, PNG, UL, UM, UPUK
BACKGROUND—Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed ...in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth.
METHODS AND RESULTS—Here, we report tight control of cardiomyocyte hypertrophy through miR-378. In isolated primary cardiomyocytes, miR-378 was found to be both necessary and sufficient to repress cardiomyocyte hypertrophy. Bioinformatic prediction suggested that factors of the mitogen-activated protein kinase (MAPK) pathway are enriched among miR-378 targets. Using mRNA and protein expression analysis along with luciferase assays, we validated 4 key components of the MAPK pathway as targets of miR-378MAPK1 itself, insulin-like growth factor receptor 1, growth factor receptor-bound protein 2, and kinase suppressor of ras 1. RNA interference with these targets prevented the prohypertrophic effect of antimiR-378, suggesting their functional relation with miR-378. Because miR-378 significantly decreases in cardiac disease, we sought to compensate for its loss through adeno-associated virus–mediated, cardiomyocyte-targeted expression of miR-378 in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction). Restoration of miR-378 levels significantly attenuated thoracic aortic constriction–induced cardiac hypertrophy and improved cardiac function.
CONCLUSIONS—Our data identify miR-378 as a regulator of cardiomyocyte hypertrophy, which exerts its activity by suppressing the MAPK signaling pathway on several distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted adeno-associated virus–miR-378 may prove to be an effective therapeutic strategy in myocardial disease.
Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer ...cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.
Nanoscale manipulations of the extracellular microenvironment are increasingly attracting attention in tissue engineering. Here, combining microscopy, biological, and single-cell electrophysiological ...methodologies, we demonstrate that neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes interact with carbon nanotubes by forming tight contacts and show increased viability and proliferation. Furthermore, we observed changes in the electrophysiological properties of cardiomyocytes, suggesting that carbon nanotubes are able to promote cardiomyocyte maturation.
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IJS, KILJ, NUK, PNG, UL, UM
Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington's disease. One possible mechanism is a ...diminished nuclear translocation of the transcription factor sterol regulatory element-binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP2-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2 (hSREBP2). Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington's disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed dopamine receptor D2 (Drd2) transcript levels decline, cleared mutant huntingtin aggregates and attenuated behavioural deficits. We conclude that glial SREBP2 participates in Huntington's disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.
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