International Journal of Cardiology 194 (2015) 21–22
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Clinical severity of viral myocarditis is not associated with a mutation of dystrophin gene cleavage sites Mirae Lee a, Jin-Oh Choi b, Soo-Hyun Yun b, Eun-Seon Ju b, Yoo-Jung Lee b, Jong-Won Kim c, Byung-Kwan Lim d, Eun-Seok Jeon b,⁎ a
Division of Cardiology, Department of Medicine, Samsung Changwon Hospital, Sungkyunkwan University, School of Medicine, Changwon-Si, Gyeongsangnam-Do, Republic of Korea Division of Cardiology, Cardiac and Vascular Center, Department of Medicine, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea d Department of Biomedical Science, Jungwon University, Goesan-gun, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 18 April 2015 Accepted 7 May 2015 Available online 9 May 2015 Keywords: Myocarditis Coxackievirus Dystrophin gene
Myocarditis, an inflammatory disorder of the heart, most often results from a common viral infection. Particularly, coxsackievirus B3 (CVB3) might explain 20–30% of all myocarditis cases, and the pathogenesis of viral myocarditis has been mostly studied in enteroviral myocarditis [1]. A previous study showed that CVB3 has direct cytopathic effects on cardiomyocytes [2]. Later studies demonstrated that enteroviral protease 2A cleaves dystrophin at two dystrophin gene cleavage sites resulting in dilated cardiomyopathy [3–5]. However, the varying severity and penetrance of viral myocarditis has not been fully explained, although many studies suggest genetic factors are associated with viral myocarditis [6–8]. We hypothesized that some mutation(s) at the dystrophin cleavage sites facilitates dystrophin cleavage and leads to severe left ventricular (LV) dysfunction. To test this hypothesis, we performed a genetic analysis of the dystrophin gene cleavage sites in patients suspected of viral myocarditis. Consecutive patients with acute myocarditis between 2003 and 2013 were prospectively enrolled in our hospital's myocarditis registry. In the patients who gave us informed consent, patients' blood sample were collected and stored for study purpose. Patients were included in this study if they met all of the following criteria: (1) distinct onset of
⁎ Corresponding author at: Division of Cardiology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University, School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-710, Korea. E-mail address: [email protected] (E.-S. Jeon).
http://dx.doi.org/10.1016/j.ijcard.2015.05.037 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
symptoms such as dyspnea, chest pain, or palpitations with a viral prodrome; (2) evidence of LV systolic dysfunction on echocardiography or increased cardiac enzyme levels without significant stenosis on coronary angiography; and (3) presence of inflammatory cell infiltration in a myocardial biopsy specimen. Fulminant myocarditis (FM) was defined as cases requiring mechanical circulatory support and/or highdose inotropic support. Otherwise, patients were classified into the non-fulminant myocarditis (NFM) group. Special tests for enteroviral infection were performed in some patients using the CVB antibody neutralization test in sera more than 2 weeks apart, immunohistochemical staining for the enteroviral VP1 capsid protein in biopsied heart tissue, reverse transcription-polymerase chain reaction or the Quantigene multiplex assay for the CVB3 VP1 capsid RNA detection in biopsied heart tissue. Additional informed consent was obtained to analyze genetic sequences before the present study. Genomic DNA was isolated from peripheral blood leukocytes using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). Exons 15 and 50 of the dystrophin gene, which include residues 588 and 2434, known as the enteroviral protease 2A target cleavage sites, were amplified in an ABI 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) [3]. Conventional Sanger sequencing was performed using the BigDye Terminator Cycle Sequencing Kit 3.1 (Applied Biosystems) on an ABI prism 3730 Genetic analyzer (Applied Biosystems). This research was approved by the Ethics Committee of the Institutional Review Board.
Table 1 Study patients with fulminant myocarditis (FM) and non-fulminant myocarditis (NFM) showing their enteroviral infection status and pathologic diagnoses (n = 80).
Age, year Male gender, n (%) Enterovirus Positive, n (%) Negative, n (%) Not tested, n (%) Pathologic diagnosis Lymphocytic myocarditis, n (%) Non-lymphocytic myocarditis, n (%)
FM (N = 45)
NFM (N = 35)
45 ± 15 19 (42)
38 ± 14 24 (69)
22 (49) 4 (9) 19 (42)
5 (14) 5 (14) 25 (71)
19 (42) 26 (58)
12 (34) 23 (66)
22
M. Lee et al. / International Journal of Cardiology 194 (2015) 21–22
Fig. 1. Direct DNA sequencing of exons 15 and 50. Arrow indicates the target sequence at nucleotide 588 in exon 15 (A) and nucleotide 2434 in exon 50 (B).
Eighty patients were classified into the FM (n = 45) and NFM (n = 35) groups. Among them, 36 (45%) patients were tested for enteroviral infection. Twenty-two (49%) patients and five (14%) patients showed evidence of enteroviral infection in the FM and NFM groups, respectively (Table 1). Nineteen (42%) and 12 (34%) patients had lymphocytic myocarditis in the FM and NFM groups, respectively, on the pathologic examination. Other patients had eosinophilic myocarditis, giant cell myocarditis, or mixed cell infiltration. A genetic analysis was performed in all patients. All amino acid sequences in exons 15 and 50, including residues 588 and 2434, respectively, were identical with the reference sequence in all patients regardless of myocarditis severity or pathological diagnosis (Fig. 1). We investigated any mutation at the dystrophin gene cleavage sites by enteroviral protease 2A in patients with FM or NFM and confirmed no mutation at the sites in the study patients. This is the first report to analyze the genetic sequence of the known dystrophin gene cleavage sites in human cases with acute myocarditis. The results show that the genetic predisposition for viral myocarditis is not explained by a simple mutation at the dystrophin gene cleavage sites. Recently several studies suggested multiple host factors associated with viral myocarditis demonstrating multiple chromosome loci, other target molecules such as serum response factor, and immunologic role in inflammatory response of the disease [8–10]. Considering the studies along with our finding, the genetic predisposition in viral myocarditis seems not to be caused by a single factor. Further studies to understand the host factors susceptible to viral myocarditis are needed.
Conflict of interest None.
Acknowledgments Byung-Kwan Lim and Mirae Lee contributed equally to this study. This work was supported by grants from the National Research Foundation (NRF) of Korea provided by the Korean Government (No. NRF2012R1A1A2008640), and Samsung Biomedical Research Institute (SBRI GL1-B2-2711). References [1] C. Gauntt, S. Huber, Coxsackievirus experimental heart diseases, Front. Biosci. 8 (2003) e23–e35. [2] R. Wessely, A. Henke, R. Zell, R. Kandolf, K.U. Knowlton, Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: an approach to the study of enteroviral persistence in cardiac myocytes, Circulation 98 (1998) 450–457. [3] C. Badorff, G.H. Lee, B.J. Lamphear, et al., Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy, Nat. Med. 5 (1999) 320–326. [4] D. Xiong, T. Yajima, B.K. Lim, et al., Inducible cardiac-restricted expression of enteroviral protease 2A is sufficient to induce dilated cardiomyopathy, Circulation 115 (2007) 94–102. [5] C. Badorff, K.U. Knowlton, Dystrophin disruption in enterovirus-induced myocarditis and dilated cardiomyopathy: from bench to bedside, Med. Microbiol. Immunol. 193 (2004) 121–126. [6] D. Xiong, G.H. Lee, C. Badorff, et al., Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: a genetic predisposition to viral heart disease, Nat. Med. 8 (2002) 872–877. [7] W. Poller, U. Kuhl, C. Tschoepe, M. Pauschinger, H. Fechner, H.P. Schultheiss, Genome-environment interactions in the molecular pathogenesis of dilated cardiomyopathy, J. Mol. Med. (Berl.) 83 (2005) 579–586. [8] M. Aly, S. Wiltshire, G. Chahrour, J.C. Osti, S.M. Vidal, Complex genetic control of host susceptibility to coxsackievirus B3-induced myocarditis, Genes Immun. 8 (2007) 193–204. [9] J. Wong, J. Zhang, B. Yanagawa, et al., Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function, Cell Res. 22 (2012) 360–371. [10] R. Deonarain, D. Cerullo, K. Fuse, P.P. Liu, E.N. Fish, Protective role for interferon-beta in coxsackievirus B3 infection, Circulation 110 (2004) 3540–3543.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Clinical severity of viral myocarditis is not associated with a mutation of dystrophin gene cleavage sites Mirae Lee a, Jin-Oh Choi b, Soo-Hyun Yun b, Eun-Seon Ju b, Yoo-Jung Lee b, Jong-Won Kim c, Byung-Kwan Lim d, Eun-Seok Jeon b,⁎ a
Division of Cardiology, Department of Medicine, Samsung Changwon Hospital, Sungkyunkwan University, School of Medicine, Changwon-Si, Gyeongsangnam-Do, Republic of Korea Division of Cardiology, Cardiac and Vascular Center, Department of Medicine, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul, Republic of Korea d Department of Biomedical Science, Jungwon University, Goesan-gun, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 18 April 2015 Accepted 7 May 2015 Available online 9 May 2015 Keywords: Myocarditis Coxackievirus Dystrophin gene
Myocarditis, an inflammatory disorder of the heart, most often results from a common viral infection. Particularly, coxsackievirus B3 (CVB3) might explain 20–30% of all myocarditis cases, and the pathogenesis of viral myocarditis has been mostly studied in enteroviral myocarditis [1]. A previous study showed that CVB3 has direct cytopathic effects on cardiomyocytes [2]. Later studies demonstrated that enteroviral protease 2A cleaves dystrophin at two dystrophin gene cleavage sites resulting in dilated cardiomyopathy [3–5]. However, the varying severity and penetrance of viral myocarditis has not been fully explained, although many studies suggest genetic factors are associated with viral myocarditis [6–8]. We hypothesized that some mutation(s) at the dystrophin cleavage sites facilitates dystrophin cleavage and leads to severe left ventricular (LV) dysfunction. To test this hypothesis, we performed a genetic analysis of the dystrophin gene cleavage sites in patients suspected of viral myocarditis. Consecutive patients with acute myocarditis between 2003 and 2013 were prospectively enrolled in our hospital's myocarditis registry. In the patients who gave us informed consent, patients' blood sample were collected and stored for study purpose. Patients were included in this study if they met all of the following criteria: (1) distinct onset of
⁎ Corresponding author at: Division of Cardiology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University, School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 135-710, Korea. E-mail address: [email protected] (E.-S. Jeon).
http://dx.doi.org/10.1016/j.ijcard.2015.05.037 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
symptoms such as dyspnea, chest pain, or palpitations with a viral prodrome; (2) evidence of LV systolic dysfunction on echocardiography or increased cardiac enzyme levels without significant stenosis on coronary angiography; and (3) presence of inflammatory cell infiltration in a myocardial biopsy specimen. Fulminant myocarditis (FM) was defined as cases requiring mechanical circulatory support and/or highdose inotropic support. Otherwise, patients were classified into the non-fulminant myocarditis (NFM) group. Special tests for enteroviral infection were performed in some patients using the CVB antibody neutralization test in sera more than 2 weeks apart, immunohistochemical staining for the enteroviral VP1 capsid protein in biopsied heart tissue, reverse transcription-polymerase chain reaction or the Quantigene multiplex assay for the CVB3 VP1 capsid RNA detection in biopsied heart tissue. Additional informed consent was obtained to analyze genetic sequences before the present study. Genomic DNA was isolated from peripheral blood leukocytes using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). Exons 15 and 50 of the dystrophin gene, which include residues 588 and 2434, known as the enteroviral protease 2A target cleavage sites, were amplified in an ABI 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) [3]. Conventional Sanger sequencing was performed using the BigDye Terminator Cycle Sequencing Kit 3.1 (Applied Biosystems) on an ABI prism 3730 Genetic analyzer (Applied Biosystems). This research was approved by the Ethics Committee of the Institutional Review Board.
Table 1 Study patients with fulminant myocarditis (FM) and non-fulminant myocarditis (NFM) showing their enteroviral infection status and pathologic diagnoses (n = 80).
Age, year Male gender, n (%) Enterovirus Positive, n (%) Negative, n (%) Not tested, n (%) Pathologic diagnosis Lymphocytic myocarditis, n (%) Non-lymphocytic myocarditis, n (%)
FM (N = 45)
NFM (N = 35)
45 ± 15 19 (42)
38 ± 14 24 (69)
22 (49) 4 (9) 19 (42)
5 (14) 5 (14) 25 (71)
19 (42) 26 (58)
12 (34) 23 (66)
22
M. Lee et al. / International Journal of Cardiology 194 (2015) 21–22
Fig. 1. Direct DNA sequencing of exons 15 and 50. Arrow indicates the target sequence at nucleotide 588 in exon 15 (A) and nucleotide 2434 in exon 50 (B).
Eighty patients were classified into the FM (n = 45) and NFM (n = 35) groups. Among them, 36 (45%) patients were tested for enteroviral infection. Twenty-two (49%) patients and five (14%) patients showed evidence of enteroviral infection in the FM and NFM groups, respectively (Table 1). Nineteen (42%) and 12 (34%) patients had lymphocytic myocarditis in the FM and NFM groups, respectively, on the pathologic examination. Other patients had eosinophilic myocarditis, giant cell myocarditis, or mixed cell infiltration. A genetic analysis was performed in all patients. All amino acid sequences in exons 15 and 50, including residues 588 and 2434, respectively, were identical with the reference sequence in all patients regardless of myocarditis severity or pathological diagnosis (Fig. 1). We investigated any mutation at the dystrophin gene cleavage sites by enteroviral protease 2A in patients with FM or NFM and confirmed no mutation at the sites in the study patients. This is the first report to analyze the genetic sequence of the known dystrophin gene cleavage sites in human cases with acute myocarditis. The results show that the genetic predisposition for viral myocarditis is not explained by a simple mutation at the dystrophin gene cleavage sites. Recently several studies suggested multiple host factors associated with viral myocarditis demonstrating multiple chromosome loci, other target molecules such as serum response factor, and immunologic role in inflammatory response of the disease [8–10]. Considering the studies along with our finding, the genetic predisposition in viral myocarditis seems not to be caused by a single factor. Further studies to understand the host factors susceptible to viral myocarditis are needed.
Conflict of interest None.
Acknowledgments Byung-Kwan Lim and Mirae Lee contributed equally to this study. This work was supported by grants from the National Research Foundation (NRF) of Korea provided by the Korean Government (No. NRF2012R1A1A2008640), and Samsung Biomedical Research Institute (SBRI GL1-B2-2711). References [1] C. Gauntt, S. Huber, Coxsackievirus experimental heart diseases, Front. Biosci. 8 (2003) e23–e35. [2] R. Wessely, A. Henke, R. Zell, R. Kandolf, K.U. Knowlton, Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: an approach to the study of enteroviral persistence in cardiac myocytes, Circulation 98 (1998) 450–457. [3] C. Badorff, G.H. Lee, B.J. Lamphear, et al., Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy, Nat. Med. 5 (1999) 320–326. [4] D. Xiong, T. Yajima, B.K. Lim, et al., Inducible cardiac-restricted expression of enteroviral protease 2A is sufficient to induce dilated cardiomyopathy, Circulation 115 (2007) 94–102. [5] C. Badorff, K.U. Knowlton, Dystrophin disruption in enterovirus-induced myocarditis and dilated cardiomyopathy: from bench to bedside, Med. Microbiol. Immunol. 193 (2004) 121–126. [6] D. Xiong, G.H. Lee, C. Badorff, et al., Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: a genetic predisposition to viral heart disease, Nat. Med. 8 (2002) 872–877. [7] W. Poller, U. Kuhl, C. Tschoepe, M. Pauschinger, H. Fechner, H.P. Schultheiss, Genome-environment interactions in the molecular pathogenesis of dilated cardiomyopathy, J. Mol. Med. (Berl.) 83 (2005) 579–586. [8] M. Aly, S. Wiltshire, G. Chahrour, J.C. Osti, S.M. Vidal, Complex genetic control of host susceptibility to coxsackievirus B3-induced myocarditis, Genes Immun. 8 (2007) 193–204. [9] J. Wong, J. Zhang, B. Yanagawa, et al., Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function, Cell Res. 22 (2012) 360–371. [10] R. Deonarain, D. Cerullo, K. Fuse, P.P. Liu, E.N. Fish, Protective role for interferon-beta in coxsackievirus B3 infection, Circulation 110 (2004) 3540–3543.
