RESUMEN
Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.
Asunto(s)
Angiotensina II , COVID-19 , Células Madre Pluripotentes Inducidas , Dispositivos Laboratorio en un Chip , Miocitos Cardíacos , Humanos , Angiotensina II/farmacología , Apoptosis/efectos de los fármacos , COVID-19/virología , COVID-19/metabolismo , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , SARS-CoV-2/fisiologíaRESUMEN
Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-ß (IFN-ß) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-ß production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-ß production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-ß production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.
Asunto(s)
Infecciones por Coxsackievirus , Enterovirus Humano B , Interferón Tipo I , Miocarditis , Miocitos Cardíacos , ARN Viral , Miocarditis/virología , Miocarditis/inmunología , Miocarditis/genética , Animales , Miocitos Cardíacos/virología , Miocitos Cardíacos/metabolismo , Ratones , Enterovirus Humano B/inmunología , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/genética , Interferón Tipo I/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Humanos , Inmunidad Innata , Transducción de Señal , Interferón beta/metabolismo , Interferón beta/genética , Interferón beta/inmunología , Masculino , Regiones no Traducidas 5'RESUMEN
Viral myocarditis, an inflammatory disease of the myocardium, is a significant cause of sudden death in children and young adults. The current coronavirus disease 19 pandemic emphasizes the need to understand the pathogenesis mechanisms and potential treatment strategies for viral myocarditis. Here, we found that TRIM29 was highly induced by cardiotropic viruses and promoted protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses that promote viral replication in cardiomyocytes in vitro. TRIM29 deficiency protected mice from viral myocarditis by promoting cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive monocytic myeloid-derived suppressor cells (mMDSC) in vivo. Mechanistically, TRIM29 interacted with PERK to promote SUMOylation of PERK to maintain its stability, thereby promoting PERK-mediated signaling pathways. Finally, we demonstrated that the PERK inhibitor GSK2656157 mitigated viral myocarditis by disrupting the TRIM29-PERK connection, thereby bolstering cardiac function, enhancing cardiac antiviral responses, and curbing inflammation and immunosuppressive mMDSC in vivo. Our findings offer insight into how cardiotropic viruses exploit TRIM29-regulated PERK signaling pathways to instigate viral myocarditis, suggesting that targeting the TRIM29-PERK axis could mitigate disease severity.
Asunto(s)
Adenina , Estrés del Retículo Endoplásmico , Indoles , Miocarditis , Miocitos Cardíacos , eIF-2 Quinasa , Animales , Humanos , Masculino , Ratones , Adenina/análogos & derivados , Apoptosis , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Miocarditis/virología , Miocarditis/metabolismo , Miocarditis/patología , Miocardio/patología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Miocitos Cardíacos/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Replicación ViralRESUMEN
AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15â kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.
Asunto(s)
Infecciones por Coxsackievirus , Citocinas , Metabolismo Energético , Glucólisis , Mitocondrias Cardíacas , Miocitos Cardíacos , Ubiquitinas , Animales , Humanos , Masculino , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Enterovirus Humano B/patogenicidad , Enterovirus Humano B/metabolismo , Interacciones Huésped-Patógeno , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Miocitos Cardíacos/patología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Ubiquitinas/metabolismo , Ubiquitinas/genéticaRESUMEN
Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.
Asunto(s)
Apoptosis , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Infecciones por Coxsackievirus , Modelos Animales de Enfermedad , Miocarditis , Miocitos Cardíacos , FN-kappa B , Proteínas de Unión al ARN , Receptores de Interleucina-1 , Transducción de Señal , Animales , Masculino , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proliferación Celular , Células Cultivadas , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/enzimología , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/patología , Enterovirus Humano B/patogenicidad , Mediadores de Inflamación/metabolismo , Ratones Endogámicos BALB C , Miocarditis/metabolismo , Miocarditis/genética , Miocarditis/patología , Miocarditis/virología , Miocarditis/enzimología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , FN-kappa B/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Función Ventricular IzquierdaRESUMEN
Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis.
Asunto(s)
Apoptosis , Proteína 11 Similar a Bcl2 , Infecciones por Coxsackievirus , Enterovirus Humano B , Proteína Forkhead Box O1 , Miocarditis , Miocitos Cardíacos , Proteína 11 Similar a Bcl2/metabolismo , Proteína 11 Similar a Bcl2/genética , Apoptosis/genética , Miocitos Cardíacos/virología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Animales , Miocarditis/virología , Miocarditis/metabolismo , Miocarditis/genética , Miocarditis/patología , Enterovirus Humano B/fisiología , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/patología , Acetilación , Humanos , Masculino , Ratones , Transducción de Señal , RatasRESUMEN
Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5' terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5' terminal deletions in CVB3 genomic RNAs decreased the levels of 2A proteinase activity but could not abrogate it. Using newly generated viral reporters encoding nano-luciferase, we found that 5' terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were cotransfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5' terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans-dominant inhibitors. IMPORTANCE Enteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5' terminally deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full-length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.
Asunto(s)
Cardiomiopatía Dilatada , Infecciones por Coxsackievirus , Enterovirus Humano B , Humanos , Antígenos Virales , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/virología , Infecciones por Coxsackievirus/complicaciones , Enterovirus Humano B/metabolismo , Genómica , Miocitos Cardíacos/virología , Péptido Hidrolasas , Infección Persistente , ARN Viral/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Sars-Cov-2 may trigger molecular and functional alterations of cardiomyocytes (CMs) of the heart due to the presence of receptor angiotensin-converting enzyme 2 (ACE2) of the host cells. While the endocytic itinerary of the virus via cleavage of the spike protein of Sars-Cov-2 is well understood, the role of the remaining part of the spike protein subunit and ACE2 complex is still elusive. Herein, the possible effects of this complex are investigated by using synthetic spike proteins of Sars-Cov-2, human-induced pluripotent stem cells (hiPSC), and a culture device made of an arrayed monolayer of cross-linked nanofibers. hiPSCs are first differentiated into CMs that form cardiac tissue-like constructs with regular beating and expression of both ACE2 and gap junction protein Connexin 43. When incubated with the spike proteins, the hiPSC-CMs undergo a rhythmic fluctuation with overstretched sarcomere structures and dispersed gap junction proteins. When incubated with the spike proteins and supplementary angiotensin II, the damage of the spike protein on hiPSC-CMs is enhanced due to downregulated ACE2, chromatin margination, altered Connexin 43 expression, sarcomere disruption, and beating break. This discovery may imply latent effects of the spike proteins on the heart.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Miocitos Cardíacos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Angiotensina II/farmacología , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/patología , COVID-19/fisiopatología , Conexina 43/metabolismo , Técnicas de Cultivo , Humanos , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miocitos Cardíacos/virología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Cardiomyocyte injury and troponin T elevation has been reported within COVID-19 patients and are associated with a worse prognosis. Limited data report this association among COVID-19 pregnant patients. OBJECTIVE: We aimed to analyze the association between troponin T levels in severe COVID-19 pregnant women and risk of viral sepsis, intensive care unit (ICU) admission, or maternal death. METHODS: We performed a prospective cohort of all obstetrics emergency admissions from a Mexican National Institute. All pregnant women diagnosed by reverse transcription-polymerase chain reaction (RT-qPCR) for SARS-CoV-2 infection between October 2020 and May 2021 were included. Clinical data were collected, and routine blood samples were obtained at hospital admission. Seric troponin T was measured at admission. RESULTS: From 87 included patients, 31 (35.63%) had severe COVID-19 pneumonia, and 6 (6.89%) maternal deaths. ROC showed a significant relationship between troponin T and maternal death (AUC 0.979, CI 0.500-1.000). At a cutoff point of 7 ng/mL the detection rate for severe pneumonia was 83.3% (95%CI: 0.500-0.100) at 10% false-positive rate. CONCLUSION: COVID-19 pregnant women with elevated levels of troponin T present a higher risk of death and severe pneumonia.
Asunto(s)
COVID-19/complicaciones , COVID-19/mortalidad , Mortalidad Materna , Neumonía/mortalidad , Complicaciones Infecciosas del Embarazo/mortalidad , Complicaciones Infecciosas del Embarazo/virología , Troponina T/sangre , Adulto , COVID-19/epidemiología , Femenino , Hospitalización , Humanos , México/epidemiología , Miocitos Cardíacos/patología , Miocitos Cardíacos/virología , Neumonía/epidemiología , Neumonía/virología , Embarazo , Complicaciones Infecciosas del Embarazo/epidemiología , Estudios Prospectivos , Factores de Riesgo , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: To investigate the effect of A20 and how A20 is regulated in viral myocarditis (VMC). METHODS: BABL/C mice, primary neonatal rat cardiomyocytes and H9c2 cells were infected with Coxsackie virus B3 (CVB3) to establish animal and cellular models of VMC. H&E staining revealed the pathologic condition of myocardium. ELISA measured the serum levels of creatine kinase, creatine kinase isoenzyme and cardiac troponin I. The effects of A20, miR-1a-3p and ADAR1 were investigated using gain and loss of function approaches. ELISA measured the levels of IL-6, IL-18 and TNF-α in serum or cell culture supernatant. TUNEL staining and flow cytometry assessed the apoptosis of myocardium and cardiomyocytes, respectively. RNA-binding protein immunoprecipitation and dual-luciferase reporter assays verified the binding between A20 and miR-1a-3p. Co-immunoprecipitation assay verified the binding between ADAR1 and Dicer. RESULTS: A20 was underexpressed and miR-1a-3p was overexpressed in the myocardium of VMC mice as well as in CVB3-infected cardiomyocytes. Overexpression of A20 suppressed cardiomyocyte inflammation and apoptosis in vivo and in vitro. miR-1a-3p promoted CVB3-induced inflammation and apoptosis in cardiomyocytes by binding to A20. The expression of miR-1a-3p was regulated by ADAR1. ADAR1 promoted the slicing of miR-1a-3p precursor by binding to Dicer. CONCLUSION: A20, regulated by ADAR1/miR-1a-3p, suppresses inflammation and cardiomyocyte apoptosis in VMC.
Asunto(s)
Adenosina Desaminasa/genética , Regulación de la Expresión Génica , Miocarditis/genética , Miocitos Cardíacos/metabolismo , ARN/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Adenosina Desaminasa/biosíntesis , Animales , Animales Recién Nacidos , Apoptosis , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Miocarditis/patología , Miocarditis/virología , Miocitos Cardíacos/patología , Miocitos Cardíacos/virología , Proteínas Nucleares , Ratas , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.
Asunto(s)
COVID-19/inmunología , Células Madre Pluripotentes Inducidas , Interleucina-10/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Miocitos Cardíacos , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/virología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/patología , Miocitos Cardíacos/virologíaRESUMEN
Efficient translation of human induced pluripotent stem cells (hiPSCs) requires scalable cell manufacturing strategies for optimal self-renewal and functional differentiation. Traditional manual cell culture is variable and labor intensive, posing challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient- and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation and generated functional neurons, cardiomyocytes, and hepatocytes. To validate our approach, we compared robotic and manual cell culture operations and performed comprehensive molecular and cellular characterizations (e.g., single-cell transcriptomics, mass cytometry, metabolism, electrophysiology) to benchmark industrial-scale cell culture operations toward building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Robótica , Automatización , Linaje de la Célula , Células Cultivadas , Cuerpos Embrioides/citología , Hepatocitos/citología , Hepatocitos/virología , Células Madre Embrionarias Humanas/citología , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/virología , Neuronas/citología , RNA-Seq , Estándares de Referencia , Análisis de la Célula Individual , Infección por el Virus Zika/patologíaRESUMEN
Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses.
Asunto(s)
Infecciones por Coxsackievirus/inmunología , Interacciones Huésped-Patógeno/inmunología , Miocarditis , Infecciones por Parvoviridae/inmunología , Parvovirus B19 Humano/fisiología , Replicación Viral , Infecciones por Coxsackievirus/patología , Humanos , Miocarditis/inmunología , Miocarditis/patología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/patología , Miocitos Cardíacos/virología , Infecciones por Parvoviridae/patología , Receptores Virales/inmunologíaRESUMEN
Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.
Asunto(s)
COVID-19/virología , Miocitos Cardíacos/virología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Catepsina B/metabolismo , Fusión Celular , Chlorocebus aethiops , Células Madre Embrionarias/metabolismo , Exocitosis , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Confocal , Serina Endopeptidasas/metabolismo , Células Vero , Proteínas Virales/metabolismo , Internalización del Virus , Replicación ViralRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.
Asunto(s)
COVID-19/complicaciones , Enfermedades Cardiovasculares/inmunología , Síndrome de Liberación de Citoquinas/inmunología , SARS-CoV-2/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Enfermedades Cardiovasculares/virología , Diferenciación Celular , Línea Celular , Biología Computacional , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Síndrome de Liberación de Citoquinas/patología , Síndrome de Liberación de Citoquinas/virología , Humanos , Células Madre Pluripotentes Inducidas , Miocardio/citología , Miocardio/inmunología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Fosfoproteínas/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Regulación hacia Arriba/inmunología , Internalización del Virus/efectos de los fármacosRESUMEN
Coxsackievirus B3 (CVB3) belongs to the enteroviruses, which are a well-known cause of acute and chronic myocarditis, primarily infecting cardiac myocytes. As primary human cardiomyocytes are difficult to obtain, viral myocarditis is quite frequently studied in vitro in different non-cardiac and cardiac-like cell lines. Recently, cardiomyocytes that have been differentiated from human-induced pluripotent stem cells have been described as a new model system to study CVB3 infection. Here, we compared iCell® Cardiomyocytes with other cell lines that are commonly used to study CVB3 infection regarding their susceptibility and patterns of infection and the mode of cell death. iCell® Cardiomyocytes, HeLa cells, HL-1 cells and H9c2 cells were infected with CVB3 (Nancy strain). The viral load, CVB3 RNA genome localization, VP1 expression (including the intracellular localization), cellular morphology and the expression of cell death markers were compared. The various cell lines clearly differed in their permissiveness to CVB3 infection, patterns of infection, viral load, and mode of cell death. When studying the mode of cell death of CVB3-infected iCell® Cardiomyocytes in more detail, especially regarding the necroptosis key players RIPK1 and RIPK3, we found that RIPK1 is cleaved during CVB3 infection. iCell® Cardiomyocytes represent well the natural host of CVB3 in the heart and are thus the most appropriate model system to study molecular mechanisms of CVB3-induced myocarditis in vitro. Doubts are raised about the suitability of commonly used cell lines such as HeLa cells, HL-1 cells and H9c2 cells to evaluate molecular pathways and processes occurring in vivo in enteroviral myocarditis.
Asunto(s)
Línea Celular/virología , Enterovirus Humano B/fisiología , Modelos Biológicos , Miocarditis/virología , Miocitos Cardíacos/virología , Animales , Proteínas de la Cápside/biosíntesis , Muerte Celular , Chlorocebus aethiops , Enterovirus Humano B/ultraestructura , Células HeLa , Humanos , Técnicas In Vitro , Ratones , Péptido Hidrolasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células Vero , Carga Viral , Replicación ViralRESUMEN
Heart injury has been reported in up to 20% of COVID-19 patients, yet the cause of myocardial histopathology remains unknown. Here, using an established in vivo hamster model, we demonstrate that SARS-CoV-2 can be detected in cardiomyocytes of infected animals. Furthermore, we found damaged cardiomyocytes in hamsters and COVID-19 autopsy samples. To explore the mechanism, we show that both human pluripotent stem cell-derived cardiomyocytes (hPSC-derived CMs) and adult cardiomyocytes (CMs) can be productively infected by SARS-CoV-2, leading to secretion of the monocyte chemoattractant cytokine CCL2 and subsequent monocyte recruitment. Increased CCL2 expression and monocyte infiltration was also observed in the hearts of infected hamsters. Although infected CMs suffer damage, we find that the presence of macrophages significantly reduces SARS-CoV-2-infected CMs. Overall, our study provides direct evidence that SARS-CoV-2 infects CMs in vivo and suggests a mechanism of immune cell infiltration and histopathology in heart tissues of COVID-19 patients.
Asunto(s)
COVID-19/patología , Quimiocina CCL2/metabolismo , Lesiones Cardíacas/virología , Monocitos/inmunología , Miocitos Cardíacos/metabolismo , Animales , Comunicación Celular/fisiología , Línea Celular , Chlorocebus aethiops , Cricetinae , Modelos Animales de Enfermedad , Humanos , Macrófagos/inmunología , Masculino , Miocitos Cardíacos/virología , Células Madre Pluripotentes/citología , Células VeroRESUMEN
OBJECTIVE: Despite growing evidence about myocardial injury in hospitalized COronaVIrus Disease 2019 (COVID-19) patients, the mechanism behind this injury is only poorly understood and little is known about its association with SARS-CoV-2-mediated myocarditis. Furthermore, definite evidence of the presence and role of SARS-CoV-2 in cardiomyocytes in the clinical scenario is still lacking. METHODS: We histologically characterized myocardial tissue of 40 patients deceased with severe SARS-CoV-2 infection during the first wave of the pandemic. Clinical data were also recorded and analyzed. In case of findings supportive of myocardial inflammation, histological analysis was complemented by RT-PCR and immunohistochemistry for SARS-CoV-2 viral antigens and in situ RNA hybridization for the detection of viral genomes. RESULTS: Both chronic and acute myocardial damage was invariably present, correlating with the age and comorbidities of our population. Myocarditis of overt entity was found in one case (2.5%). SARS-CoV-2 genome was not found in the cardiomyocytes of the patient with myocarditis, while it was focally and negligibly present in cardiomyocytes of patients with known viral persistence in the lungs and no signs of myocardial inflammation. The presence of myocardial injury was not associated with myocardial inflammatory infiltrates. CONCLUSIONS: In this autopsy cohort of COVID-19 patients, myocarditis is rarely found and not associated with SARS-CoV-2 presence in cardiomyocytes. Chronic and acute forms of myocardial damage are constantly found and correlate with the severity of COVID-19 disease and pre-existing comorbidities.
Asunto(s)
COVID-19/complicaciones , Inflamación/virología , Miocarditis/virología , Miocardio/patología , Anciano , Anciano de 80 o más Años , Autopsia , Estudios de Cohortes , Femenino , Humanos , Inflamación/epidemiología , Masculino , Miocarditis/epidemiología , Miocitos Cardíacos/virología , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la EnfermedadRESUMEN
Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Células Madre Embrionarias Humanas/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/virología , SARS-CoV-2/fisiología , Benzotropina/farmacología , Humanos , Miocitos Cardíacos/citología , Péptidos/farmacologíaRESUMEN
We review some of the important discoveries and advances made in basic and translational cardiac research in 2020. For example, in the field of myocardial infarction (MI), new aspects of autophagy and the importance of eosinophils were described. Novel approaches, such as a glycocalyx mimetic, were used to improve cardiac recovery following MI. The strategy of 3D bio-printing was shown to allow the fabrication of a chambered cardiac organoid. The benefit of combining tissue engineering with paracrine therapy to heal injured myocardium is discussed. We highlight the importance of cell-to-cell communication, in particular, the relevance of extracellular vesicles, such as exosomes, which transport proteins, lipids, non-coding RNAs, and mRNAs and actively contribute to angiogenesis and myocardial regeneration. In this rapidly growing field, new strategies were developed to stimulate the release of reparative exosomes in ischaemic myocardium. Single-cell sequencing technology is causing a revolution in the study of transcriptional expression at cellular resolution, revealing unanticipated heterogeneity within cardiomyocytes, pericytes and fibroblasts, and revealing a unique subpopulation of cardiac fibroblasts. Several studies demonstrated that exosome- and non-coding RNA-mediated approaches can enhance human induced pluripotent stem cell (iPSC) viability and differentiation into mature cardiomyocytes. Important details of the mitochondrial Ca2+ uniporter and its relevance were elucidated. Novel aspects of cancer therapeutic-induced cardiotoxicity were described, such as the novel circular RNA circITCH, which may lead to novel treatments. Finally, we provide some insights into the effects of SARS-CoV-2 on the heart.