In Vitro Model Systems of Coxsackievirus B3-Induced Myocarditis: Comparison of Commonly Used Cell Lines and Characterization of CVB3-Infected iCell® Cardiomyocytes

Abstract

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.

Molecular Characteristics of Enterovirus B83 Strain Isolated from a Patient with Acute Viral Myocarditis and Global Transmission Dynamics

“…Enterovirus is one of the main pathogens that cause viral myocarditis by directly infecting myocardial cells, triggering inflammatory reaction, and leading to myocarditis [ 7 , 46 ]. Not all enterovirus infections can lead to viral myocarditis, but some enterovirus subtypes, especially coxsackievirus group B, especially CVB3, have a high risk of myocarditis [ 47 , 48 ]. Other enterovirus, such as echovirus and coxsackievirus group A, have also been reported to cause viral myocarditis [ 49 , 50 ].…”

Necroptosis and Viral Myocarditis: Tumor Necrosis Factor α as a Novel Biomarker for the Diagnosis of Viral Myocarditis

“…that have been used frequently to investigate the mechanism can only distantly represent human cardiomyocytes ( Claycomb et al, 1998 ). Not only the change of histopathology indicating the initiation of necroptosis was a key role in the pathology of VMC; the experiment implicates that the inhibition of RIPK1 and RIPK3 resulted in a reduction of viral replication by around half ( Kraft et al, 2021 ), suggesting that the activity of RIPK1/3 may also be crucial for CVB3 replication. However, how RIPK3 functions on CVB3 replication in the cardiomyocytes remains unknown, and the time element should be specially considered about the “paradox” until the exact mechanism is accurately defined.…”

“…The current research progress is the application of the human-induced pluripotent stem cells (hiPSCs) in establishing an in vitro model of CVB3-induced myocarditis, investigating the molecular mechanisms of viral myocarditis via iCell® Cardiomyocytes ( Kraft et al, 2021 ). It is said to be newly suitable for cardiomyocytes than other cardiac-like cells since cardiomyocytes represent the target cells of enteroviruses in the human heart, and those cardiac-like and non-cardiac cell lines (HeLa cells, HL-1 cells and H9c2 cells, etc.)…”

Alkaloids as potential antivirals. A comprehensive review

“…CVs are enteroviruses that cause ailments varying from gastrointestinal distress to pericarditis and myocarditis (CVB3-induced cardiomyopathy) [ 111 ], CVB3 thrives in the myocardium and produces severe coronary illnesses [ 112 ].…”