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

Lisa Kraft,Guiscard Seebohm,Karin Klingel,Martina Sauter

doi:10.3390/v13091835

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.

Highlights

Coxsackievirus B3 (CVB3) is a non-enveloped, positive-sense single-stranded RNA virus belonging to the enteroviruses
Sharma et al demonstrated that cardiomyocytes that have been differentiated from human-induced pluripotent stem cells (hiPSCs) express the coxsackievirus and adenovirus receptor (CAR), which is an important prerequisite for CVB3 entry. They demonstrated that these cardiomyocytes are susceptible to infection with a luciferase-expressing CVB3 strain (CVB3-Luc), establishing this cardiomyocyte/CVB3-Luc system as a platform to quantitatively assess the efficacy of antiviral compounds in reducing coxsackievirus proliferation [19]. As these findings suggest that cardiomyocytes being differentiated from hiPSCs represent a suitable model system to investigate molecular mechanisms in CVB3-induced myocarditis, we aimed to compare those cells with different cell lines that are commonly used to study CVB3-induced myocarditis
To assess the infection efficacy, the viral load in CVB3-infected iCell® Cardiomyocytes was compared with three cell lines that are commonly used to study the mechanisms of CVB3 infection

Summary

Introduction

Coxsackievirus B3 (CVB3) is a non-enveloped, positive-sense single-stranded RNA virus belonging to the enteroviruses. The molecular mechanisms of viral myocarditis are studied in vitro in human cardiomyocytes. As primary human heart tissue is difficult to obtain, different cell lines are used to study CVB3-induced myocarditis. HL-1 cells are cardiac muscle cells that have been derived from the AT-1 mouse atrial cardiomyocyte tumor lineage They are supposed to show characteristics typical of embryonic atrial cardiac muscle cells but are not representative for adult human cardiomyocytes [4]. H9c2 cells have been derived from embryonic BDIX rat heart tissue and are supposed to exhibit many of the properties of skeletal muscle [6]. It is common knowledge that these cell lines represent human cardiomyocytes only distantly, they are used quite frequently to study cellular processes in CVB3-induced myocarditis

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