Abstract
Aims
Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated ...markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection.
Methods and results
Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-CoV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with coronavirus disease 2019. Infection of induced pluripotent stem cell-derived cardiomyocytes was dependent on cathepsins and angiotensin-converting enzyme 2, and was blocked by remdesivir.
Conclusion
This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an angiotensin-converting enzyme 2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir.
Graphical Abstract
Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered ...a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.
Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial ...dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.
Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges ...identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay.
The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of ...preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.
Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated ...decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.
There is an outmost need for the identification of specific antiviral compounds. Current antivirals lack specificity, making them susceptible to off-target effects, and highlighting importance of ...development of assays to discover antivirals targeting viral specific proteins. Previous studies for identification of inhibitors of RNA-dependent RNA polymerase (RdRp) mostly relied on radioactive methods. This study describes a fluorometric approach to assess in vitro activity of viral RdRp for drug screening. Using readily available DNA- and RNA-specific fluorophores, we determined an optimum fluorometric approach that could be used in antiviral discovery specifically for RNA viruses by targeting RdRp. Here, we show that double-stranded RNA could be successfully distinguished from single-stranded RNA. In addition, we provide a strategy based on self-priming RNA to assess RdRp activity.
Graphical abstract
IntroductionThe adult mammalian heart has a limited capacity for regeneration and repair. Instead, fibrotic tissue replaces scarred myocardium. However, there is a short time during postnatal ...development, between postnatal day 1 (P1) and day 7 (P7), in which the mouse heart possesses the potential to fully regenerate. Yet, this regenerative potential is lost after P7. Long non-coding RNAs play a crucial role in various aspects of cardiovascular biology. Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a well conserved and highly expressed lncRNA which regulates endothelial cell proliferation and is required for vessel growth. However, its role in cardiac regeneration is unclear.HypothesisSince MALAT1 is upregulated after myocardial infarction (MI) at P1, we assessed its role in postnatal cardiac and vascular regeneration.ResultsPreliminary data indicate that induction of ischemic myocardial infarction by ligation of the left anterior descending (LAD) coronary artery at postnatal day 3 (P3) results in impaired regeneration in MALAT1-/- neonatal pups in comparison to wild type MALAT1+/+ litter mates. Furthermore, the fibrotic scar formation in MALAT1+/+ wild neonate pups was significantly reduced in comparison to the MALAT1-/- knockout pups (p<0.05, n=7-8). Whereas MALAT1+/+ mice showed a robust cardiac regeneration mediated by proliferation of cardiomyocytes, this proliferative response was impaired in MALAT1-/- mice (p<0.05, n=5-8). Additionally, vessel area and endothelial cell proliferation was higher in in the border zone of MALAT1+/+ pups compared to the knockout litter mates (p<0.05, n=5-8). To determine whether cardiomyocytes proliferation might be directly affected, we silenced MALAT1 expression by Gapmer in murine cardiomyocytes. Indeed, MALAT1 silencing also decreased cell proliferation in vitro (p<0.01, n=4).ConclusionsTaken together, our findings suggest that genetic deletion or pharmacological inhibition blocked cardiomyocyte proliferation indicating that MALAT1 is required for neonatal cardiac regeneration.
BackgroundRNA sequencing revealed that the majority of the human genome is transcribed as non-coding transcripts, which mostly consist of long non-coding RNAs (lncRNAs). Although some lncRNAs were ...shown to control biological processes, their function in cardiovascular diseases is still largely unexplored. Here we characterized lncRNA expression in cardiac tissue of patients with heart failure and explored the function of the regulated candidates.ResultsRNA sequencing identified differentially regulated IncRNAs in heart failure samples in comparison to healthy controls (n=10 per group, p<0.01). Among the significantly regulated transcripts, Snhg12, which was profoundly down-regulated (4.3 fold) showed the highest expression in iPS-derived human cardiomyocytes (hiPSC-CM) (N=3). Since Snhg12 was also highly expressed in endothelial cells and fibroblasts, we explored its function in all cell types by using Gapmer-mediated silencing. Silencing of Snhg12 (60 % inhibition) lead to increased tube formation (n=9; p<0.05) and angiogenic sprouting of HUVECs (n=8; p<0.01) and reduced hypertrophy of hiPSC-CM (N≥3, p<0.05). Fibroblast proliferation and collagen production was not affected. Since the Snhg12 locus hosts four different non-coding snoRNAs, which are fundamental for ribosomal functions, we determined the regulation of these RNAs. Indeed, silencing of Snhg12 lead to an upregulation of snoRNA-61 in HUVEC, but the other three hosted snoRNAs remain unchanged. Since snoRNA functions in angiogenesis are unexplored, we determined the effect of snoRNA-61. Indeed, Gapmer-mediated silencing of snoRNA-61, decreased cell viability, angiogenic sprouting and tube formation of HUVECs (n=4; p<0.05). In conclusion, Snhg12 is downregulated in heart failure patients. Silencing of Snhg12 increased angiogenic functions of HUVECs and prevented cardiomyocyte hypertrophy. The pro-angiogenic effect of Snhg12 silencing might be mediated by the up-regulated of snoRNA-61, which promoted endothelial cell sprouting. The down-regulation of Snhg12 during heart failure may present a compensatory mechanism.
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