Coxsackievirus-induced myocarditis in mice: A model of autoimmune disease for studying immunotoxicity

Abstract

Excellent animal models are available for virus-induced and autoimmune heart disease that are remarkably similar to human disease. Developing good animal models for heart disease is crucial because cardiovascular disease is now the leading cause of death in the United States and is estimated to be the leading cause of death in the world by the year 2020. A significant proportion of heart disease in Western populations is associated with inflammation. Myocarditis, or inflammation of the heart muscle, is the major cause of sudden death in young adults. Although most individuals recover from acute myocarditis, genetically susceptible individuals may go on to develop chronic myocarditis and dilated cardiomyopathy (DCM) resulting in congestive heart failure. In this article, we describe a model of autoimmune myocarditis and DCM induced by inoculation with heart-passaged coxsackievirus B3 (CVB3). Intraperitoneal inoculation of susceptible mice with CVB3 induces acute cardiac inflammation from days 7 to 14 postinfection (pi) that progresses to chronic myocarditis and DCM from day 28 to at least 56 pi. The model of CVB3-induced myocarditis presented here allows dissection of the contribution of viral infection and xenobiotics on immune dysregulation and inflammation in the heart. An improved understanding of the interaction between environmental exposures and the development of heart disease represents a clear challenge for immunotoxicologists.

Introduction

Cardiovascular disease is the number one killer of men and women in the United States and the second most common cause of death worldwide [1], [2]. Although the true incidence of inflammation in the heart is unknown, it is estimated that 1 in 4 individuals in the United States have some form of inflammatory heart disease [1], [3]. Acute myocarditis is a principal cause of heart failure in young adults and often progresses to chronic myocarditis, DCM and congestive heart failure requiring heart transplantation [3]. CVB3 infection is believed to be a principle etiologic agent in human myocarditis, and the same virus strain that infects humans (ignoring the species barrier) induces biphasic myocarditis in genetically susceptible strains of mice [3], [4], [5], [6].

Currently, several animal models are available that closely resemble human myocarditis [7]. Murine models fall into two basic categories: acute cardiac inflammation and sudden death induced by direct viral damage (where nearly 70% of animals die at day 4–7 after infection) [8], [9], [10], [11], or inflammation triggered by adjuvant and cardiac myosin or virus and cardiac myosin (heart-passaged virus) resulting in acute myocarditis with no deaths that progresses to chronic heart disease and DCM [5], [6], [12], [13], [14], [15]. In this article, we describe a model of autoimmune myocarditis and DCM induced by inoculation with heart-passaged CVB3 [7], [15].

In the model of CVB3-induced myocarditis presented here, intraperitoneal (ip) inoculation of BALB/c mice with heart-passaged CVB3 (Nancy strain), which contains virus and cardiac myosin, induces inflammatory heart disease that is remarkably similar to disease induced by inoculation with adjuvant and cardiac myosin (experimental autoimmune myocarditis or EAM) [14], [15], [16], [17]. Acute CVB3-induced myocarditis develops in susceptible BALB/c mice from day 7 to 14 postinfection (pi) and progresses to chronic myocarditis and DCM from day 35 to at least 56 pi (Fig. 1A) [6], [15], [18], [19]. Resistant strains of mice (i.e. C57BL/6) do not develop the chronic phase of the disease (Fig. 1B). Furthermore, susceptible male mice develop significantly increased acute (Fig. 2A) and chronic (data not shown) myocarditis in response to CVB3 infection compared to female mice. Following infection with heart-passaged CVB3, mice quickly develop immunoglobulin (Ig)G autoantibodies specific for cardiac myosin, similar to those found in EAM and in patients with myocarditis and DCM [5], [6], [20], [21]. Acute CVB3-induced myocarditis is characterized by a focal cellular infiltrate with little necrosis or fibrosis, comprised primarily of macrophages, neutrophils, CD4⁺ T cells, and CD8⁺ T cells with lower numbers of B cells, mast cells, natural killer cells, and dendritic cells (Fig. 2B) [19]. In this model, virus replicates at relatively low levels in the heart during acute myocarditis (10²–10⁴ PFU) compared with other CVB3-induced models (10⁷–10⁹ PFU) [8], [9], [10], [11], [18], [19]. By day 14 pi, infectious virus is cleared from the heart and does not reactivate during chronic myocarditis [6], [15]. In genetically susceptible mice, a diffuse, lymphocytic infiltrate develops around day 35 pi with large areas of necrosis and fibrosis (Fig. 3A) as well as pericarditis (Fig. 3B) and DCM (Fig. 3D) [15]. Although viral genome can be detected in heart tissue during chronic myocarditis, persistent virus is found in both susceptible and resistant strains of mice [13], [22].

The development of autoimmune disease depends on a complex interaction between genetic, environmental, and endogenous factors [23], [24], [25]. Environmental agents, such as infections, diet, hormones and drugs, contribute significantly to the risk of developing and/or dying from heart disease [2], [14], [26], [27], [28]. The incidence and severity of heart disease is higher among men, but the incidence increases in women as they age [1], [29]. Young male BALB/c mice (6–8 weeks old) develop significantly greater acute and chronic myocarditis than female mice (Fig. 2), similar to humans [1], [30]. Public health measures have dramatically reduced exposure to many infections and toxic chemicals. However, the effect of xenobiotics on the immune system and the development of inflammatory heart disease following infection has not been fully evaluated. Recent studies suggest that low dose exposure to immunotoxicants, such as mercury, may exacerbate inflammatory heart disease induced by viral infection or adjuvant and cardiac myosin [31]. A clear challenge for immunotoxicologists is to better understand the mechanisms involved in the progression of inflammatory heart disease. Only then we can develop more effective prevention and intervention strategies for these diseases.