**2.2 Propranolol ameliorates and epinephrine exacerbates progression of acute and chronic viral myocarditis**

Recent studies point to important interactions between proinflammatory cytokines and neurohumoral mediators in heart failure. We investigated the influence of the β-adrenergic system on cytokines and neurohumoral factors and the sequelae of viral myocarditis (Wang et al., 2005). In an experimental model with virus-infected BALB/c mice, we studied the acute and chronic effects of epinephrine and propranolol on myocardial morphology, cytokine gene expression, and survival. BALB/c mice were inoculated with the encephalomyocarditis virus (EMCV) or sham inoculated with saline and followed for 30 days. Epinephrine increased the severity of inflammatory cell infiltration and myocardial necrosis induced by EMCV-inoculated mice. Survival rate after 30 days was reduced to 40% in epinephrine-treated EMCV-inoculated mice compared with 70% in untreated EMCVinoculated mice (P<0.05). Treatment with the β-blocker propranolol significantly decreased

Exacerbation of Viral Myocarditis by

myocarditis (Ensing, 1985).

clozapine-induced myocarditis.

**3. Tobacco smoke** 

Tobacco Smoke: The Catecholamine Hypothesis 175

cellular pH, shift in osmolarity, or likely depletion of high-energy stores necessary for protective proteolytic enzyme activity. Of course, it is likely that the effects of cocaine on the animal or a patient with myocarditis are complex and involve several mechanisms or conditioning factors, including drug diluents with intrinsic pharmacological activity or sensitizing effects (Ensing, 1985; Wolf and Blum, 1983). However, the observation that exacerbation of myocarditis seems to occur with cocaine but not other commonly abused drugs without prominent cardiac effects, including heroin and phencyclidine, suggests that cocaine may have a unique combination of properties that make its use in patients exposed or infected with viral pathogens likely to enhance development of myocarditis and its sequelae. Our data indicate that the unique ability of cocaine to increase local release and circulating levels of catecholamines is the primary factor responsible for exacerbation of

**2.4 Clozapine-induced myocarditis: role of catecholamines in a murine model** 

Clozapine, an atypical neuroleptic agent, is very effective in the treatment of resistant schizophrenia. However, cardiotoxicity of clozapine, particularly in young patients, has raised concerns about its its safety and smoking may have a significant effect on serum concentrations of clozapine (Seppala et al., 1999). A particularly high incidence of myocarditis has been reported among patients treated with atypical neuroleptic drugs, including clozapine and risperidone. Clozapine has been shown to markedly increase circulating levels of catecholamines (Coulter et al., 2001; Elman et al., 2002). Increased catecholamines have been postulated to trigger an inflammatory response resulting in myocarditis, dilated cardiomyopathy, and death, although this has not yet been thoroughly studied. Here (Wang et al., 2008), we used the mouse to study whether clozapine administration could caused adverse myocarditis associated with an increase in catecholamines. Male BALB/c mice, age ~6 weeks, were administered 5, 10 or 25 mg/kg clozapine daily for 7 and 14 days; one group was administered 25 mg/kg clozapine plus 2 mg/kg propranolol for 14 days. Saline-treated mice served as controls. Heart sections were stained with hematoxylin and eosin for histopathological examination. Plasma catecholamines were measured with HPLC. Myocardial TNF-α concentrations were determined by ELISA. Histopathologic examination of clozapine-treated mice showed a significant dose-related increase in myocardial inflammation that correlated with plasma catecholamine levels and release of TNF-α. Propranolol significantly attenuated these effects. A hypercatecholaminergic state induced by clozapine could explain the occurrence of myocarditis in some patients. Our data suggest that βadrenergic blocking agents may be effective in reducing the incidence and severity of

**3.1 Exacerbation of viral myocarditis by tobacco smoke as a cause of heart failure**  In this study (Bae et al. 2010), we determined whether exposure to tobacco smoke will exacerbate the severity of viral myocarditis in mice. Viral myocarditis was generated n 4 week old male BALB/c mice by ί.ρ. injection of encephalomyocarditis virus (EMCV). Mice were exposed to cigarette smoke for 90 minutes/day 5x/week. Four groups were studied: 1) Control (C, no smoke and no virus), 2) Smoke (S) only, and 3) Virus only (V), and 4) Preexposure to smoke for l week prior to virus injection (S+V). We observed an over 2-fold

mortality, myocardial necrosis and infiltration of inflammatory cells in EMCV-inoculated mice. Propranolol also suppressed gene expression of the cytokines TNF-α, IL-6 and IL-10 involved in inflammation. A single dose of epinephrine 120 days after EMCV inoculation caused death in 70% of infected mice; propranolol significantly reduced the incidence of death to 33%. These results indicate that acute and chronic stages of viral myocarditis are modulated by the β-adrenergic system and its interactions with proinflammatory factors.

#### **2.3 Cocaine enhances myocarditis induced by encephalomyocarditis virus in murine models**

This study (Wang et al., 2002a) was designed to investigate whether cocaine can exacerbate viral myocarditis and increase its incidence. Clinical evidence suggests that cocaine abuse increases the incidence of myocarditis. However, it had not been directly demonstrated that cocaine exposure enhances murine myocarditis. BALB/c mice were divided into eight groups saline control, encephalomyocarditis virus (EMCV), 10 mg/kg cocaine (Coc-10), 30 mg/kg cocaine (Coc-30), 50 mg/kg cocaine (Coc-30), EMCV+Coc-50. After inoculation with EMCV, the mice were daily treated with either cocaine or saline for 90 days. Mice were euthanized at different days after EMCV inoculation. Mortality was recorded and myocarditis severity was evaluated. The mortality of the myocarditis mice treated with cocaine increased significantly from 22% (EMCV) to 25.7% (Coc-10+ EMCV), 41.4% 9 (Coc-30+EMCV) and 51.4% (Coc-50+EMCV) (P<0.05) respectively. The incidence and severity of inflammatory cell infiltration and myocardial lesions was higher in infected mice exposed to cocaine. Cocaine administered only before infection did not exacerbate myocarditis. Norepinephrine assay showed that cocaine exposure significantly increased myocardial norepinephrine concentration, but this increase was partially inhibited in infected animals. Adrenalectomy abolished the effect of cocaine on mortality. Furthermore, propranolol, a βblocker, significantly decreased the enhancing effects of cocaine on myocarditis mice. In conclusion, cocaine increases the severity and mortality of viral myocarditis in mice. Increased catecholamines may be a major factor in this effect.

A variety of viruses have been reported as causative agents of myocarditis in man, including Coxsackie, echo and influenza viruses, cytomegalovirus, poliomyelitis virus, Epstein Barr virus, herpes simplex virus, adenovirus and several others (Rodeheffer & Gersh, 2001; Ensley et al., 1995; Abelmann, 1966). Our laboratory has demonstrated that cocaine can exacerbate viral myocarditis in a murine model (Wang et al., 2002a). Possible mechanisms of such an interaction include, (a) cocaine-induced damage to the endothelial/endocardial cells in the extracellular matrix or to the myocytes themselves, thereby reducing structural and immunological barriers to cellular penetration of the virus and increasing the myocardial and vascular permeability and infectivity of viral particles. Evidence supporting "possibility (a)" includes reports suggesting that cocaine can damage the endothelial lining of cells after even a single exposure, thereby accelerating atherosclerosis in animal models (Egashira et al., 1991; Bacharach et al., 1992). Cocaine has also been reported to increase natural killer cell activity (Van Dyke et al., 1986). Both lymphocytic and eosinophilic myocarditis have been reported in cocaine abusers (Virmani et al., 1988; Tazelaar et al., 1987) and heart failure is a common finding (Weiner et al., 1986; Duell, 1987). Alternatively, "possibility (b)", cocaine may exacerbate viral myocarditis by permitting enhanced viral replication of the viral agent once it has penetrated the cell membrane. Such an effect may occur through a direct or catecholamine-mediated alteration in the cellular milieu that in turn could alter viral transcription and replication. Enhanced viral replication could occur through a change in

mortality, myocardial necrosis and infiltration of inflammatory cells in EMCV-inoculated mice. Propranolol also suppressed gene expression of the cytokines TNF-α, IL-6 and IL-10 involved in inflammation. A single dose of epinephrine 120 days after EMCV inoculation caused death in 70% of infected mice; propranolol significantly reduced the incidence of death to 33%. These results indicate that acute and chronic stages of viral myocarditis are modulated by the β-adrenergic system and its interactions with proinflammatory factors.

**2.3 Cocaine enhances myocarditis induced by encephalomyocarditis virus in murine** 

This study (Wang et al., 2002a) was designed to investigate whether cocaine can exacerbate viral myocarditis and increase its incidence. Clinical evidence suggests that cocaine abuse increases the incidence of myocarditis. However, it had not been directly demonstrated that cocaine exposure enhances murine myocarditis. BALB/c mice were divided into eight groups saline control, encephalomyocarditis virus (EMCV), 10 mg/kg cocaine (Coc-10), 30 mg/kg cocaine (Coc-30), 50 mg/kg cocaine (Coc-30), EMCV+Coc-50. After inoculation with EMCV, the mice were daily treated with either cocaine or saline for 90 days. Mice were euthanized at different days after EMCV inoculation. Mortality was recorded and myocarditis severity was evaluated. The mortality of the myocarditis mice treated with cocaine increased significantly from 22% (EMCV) to 25.7% (Coc-10+ EMCV), 41.4% 9 (Coc-30+EMCV) and 51.4% (Coc-50+EMCV) (P<0.05) respectively. The incidence and severity of inflammatory cell infiltration and myocardial lesions was higher in infected mice exposed to cocaine. Cocaine administered only before infection did not exacerbate myocarditis. Norepinephrine assay showed that cocaine exposure significantly increased myocardial norepinephrine concentration, but this increase was partially inhibited in infected animals. Adrenalectomy abolished the effect of cocaine on mortality. Furthermore, propranolol, a βblocker, significantly decreased the enhancing effects of cocaine on myocarditis mice. In conclusion, cocaine increases the severity and mortality of viral myocarditis in mice.

A variety of viruses have been reported as causative agents of myocarditis in man, including Coxsackie, echo and influenza viruses, cytomegalovirus, poliomyelitis virus, Epstein Barr virus, herpes simplex virus, adenovirus and several others (Rodeheffer & Gersh, 2001; Ensley et al., 1995; Abelmann, 1966). Our laboratory has demonstrated that cocaine can exacerbate viral myocarditis in a murine model (Wang et al., 2002a). Possible mechanisms of such an interaction include, (a) cocaine-induced damage to the endothelial/endocardial cells in the extracellular matrix or to the myocytes themselves, thereby reducing structural and immunological barriers to cellular penetration of the virus and increasing the myocardial and vascular permeability and infectivity of viral particles. Evidence supporting "possibility (a)" includes reports suggesting that cocaine can damage the endothelial lining of cells after even a single exposure, thereby accelerating atherosclerosis in animal models (Egashira et al., 1991; Bacharach et al., 1992). Cocaine has also been reported to increase natural killer cell activity (Van Dyke et al., 1986). Both lymphocytic and eosinophilic myocarditis have been reported in cocaine abusers (Virmani et al., 1988; Tazelaar et al., 1987) and heart failure is a common finding (Weiner et al., 1986; Duell, 1987). Alternatively, "possibility (b)", cocaine may exacerbate viral myocarditis by permitting enhanced viral replication of the viral agent once it has penetrated the cell membrane. Such an effect may occur through a direct or catecholamine-mediated alteration in the cellular milieu that in turn could alter viral transcription and replication. Enhanced viral replication could occur through a change in

Increased catecholamines may be a major factor in this effect.

**models** 

cellular pH, shift in osmolarity, or likely depletion of high-energy stores necessary for protective proteolytic enzyme activity. Of course, it is likely that the effects of cocaine on the animal or a patient with myocarditis are complex and involve several mechanisms or conditioning factors, including drug diluents with intrinsic pharmacological activity or sensitizing effects (Ensing, 1985; Wolf and Blum, 1983). However, the observation that exacerbation of myocarditis seems to occur with cocaine but not other commonly abused drugs without prominent cardiac effects, including heroin and phencyclidine, suggests that cocaine may have a unique combination of properties that make its use in patients exposed or infected with viral pathogens likely to enhance development of myocarditis and its sequelae. Our data indicate that the unique ability of cocaine to increase local release and circulating levels of catecholamines is the primary factor responsible for exacerbation of myocarditis (Ensing, 1985).
