Cardiac Myosin Heavy Chain Alpha Research Articles

Cardiac myosin-specific TCR transgenic mouse models for studying dilated cardiomyopathy

Abstract We previously reported that the cardiac myosin heavy chain-alpha (Myhc) 334–352 encompasses epitopes for both CD4 and CD8 T cells that can induce autoimmune myocarditis/dilated cardiomyopathy (DCM) in A/J mice expressing the IAk and IEk molecules. More recently, we made an unexpected observation that, Myhc 334–352 although by being a poor binder of IAb molecule in C57Bl/6 mice, could induce strong T cell responses, but myocarditogenicity was severely dented. In our efforts to delineate the roles of Myhc-specific, CD4 and CD8 T cells in the DCM pathogenesis, we have now created the T cell receptor (TCR) transgenic (tg) mice on C57Bl/6 background for Myhc 334–352, and demonstrate that the Myhc 334–352-specific TCR was found common to both CD4 and CD8 T cells. While, the naïve tg T cells do not respond, antigen-primed CD4 and CD8 T cells from immunized mice respond to Myhc 334–352 that produce predominantly, interferon-γ. Furthermore, although mild, the immunized tg mice developed heart lesions containing the mononuclear cells, and such a phenotype was more apparent in females than males. As the tg mice become available on both resistant (C57Bl/6) and susceptible (A/J) backgrounds, our TCR tg system can be used as a helpful tool to dissect the complexities of antigen-specific CD4 and CD8 T cells in the causation of DCM in various experimental settings.

Gender differences in the development of autoimmune myocarditis induced with cardiac myosin heavy chain-alpha, 334-352 in C57Bl/6 mice (BA15P.222)

Abstract Various animal models of experimental autoimmune myocarditis (EAM) have been developed to understand the immune pathogenesis of myocardial injuries. Although, genetic susceptibility is one major predisposing factor in the causation of myocarditis, other factors like gender-bias can also influence the disease outcome. For example, A/J mice carrying IAk allele on ‘A’ background are highly susceptible to EAM induced with cardiac myosin heavy chain-alpha (Myhc); whereas C57Bl/6 mice on ‘B’ background possessing IAb allele are resistant. Whether these phenotypic differences are due to inherent variations in the presentation of immunodominant epitopes of cardiac antigens are not known. We addressed this question using Myhc 334-352 as a putative cardiac antigen that induces EAM in A/J mice. Unexpectedly, we discovered that C57Bl/6 mice immunized with Myhc 334-352 showed antigen-specific proliferative responses regardless of gender as determined by T cell proliferation assay and major histocompatibility complex (MHC) class II/IAb dextramer staining. However, in contrast to females, only male mice developed myocarditis which showed higher frequencies of CD4 T cells producing T helper 17 family cytokines namely, interleukin (IL)-17A and IL-22 but not IL-17F. The observation that both IAb and IAk alleles can present single peptide but with varied disease outcomes provides opportunities to dissect the influence of non-MHC genes and also sexual dimorphism on myocarditis-susceptibility.

Delineating the role of cardiac myosin-specific CD8 T cells in autoimmune myocarditis (BA15P.220)

Abstract Enteroviruses like coxsackievirus B3 are common suspects in patients with myocarditis/dilated cardiomyopathy. Previously, it was shown that autoreactive cytotoxic T lymphocytes, also called CD8 T cells, have a role in the mediation of myocarditis induced by coxsackievirus B3. But identification of their target antigens in cardiac tissues continued to be a challenge. We have discovered that CD8 T cells have the ability to induce myocarditis in A/J mice by recognizing cardiac myosin heavy chain-alpha (Myhc) as the target antigen. We have localized two peptides designated, trunk 4 and trunk 5 within Myhc that can stimulate CD8 T cells and CD4 T cells. Furthermore, expectedly, CD8 T cells recognizing the Myhc peptides produced predominantly, interferon-gamma and tumor necrosis factor-alpha, but not interleukin-17, and A/J mice immunized with the CD8 T cell epitopes, develop myocarditis by active immunization. It has been widely believed that Myhc-induced autoimmune myocarditis is a CD4 T cell-mediated disease. Our discovery creates a paradigm shift in the understanding of how cardiac damage can occur by multiple immune cells that recognize the same target antigens but through different mechanisms.

Bacillus infantis NRRL B-14911-induced myocarditis: a novel disease model to study the autoimmune mechanisms of myocardial injuries in the myocarditis-susceptible, A/J mice (BA15P.221)

Abstract We recently discovered that Bacillus spp., NRRL B-14911, a marine microbe, has the ability to induce myocarditis through molecular mimicry. The bacterium contains a mimicry epitope designated, BAC 25-40, and it induces autoimmune myocarditis by generating cross-reactive T cells for cardiac myosin heavy chain-alpha 334-352 (Myhc). We have analysed the complete genome sequence leading us to identify this microorganism as Bacillus infantis NRRL B-14911, a close relative of the strain that has been isolated in sepsis of newborn infants. Conditions have been optimized for the bacterium to grow in a Zobell marine medium at 37oC in an aerobic environment. Our preliminary studies indicate that A/J mice infected with Bacillus infantis NRRL B-14911 show the generation of CD4 T cells that can react with both BAC 25-40 and Myhc, as determined by major histocompatibility complex class II/IAk tetramer staining. We propose that Bacillus infantis NRRL B-14911-induced myocarditis can serve as a useful disease model to study the immune-mediated mechanisms of bacterial myocarditis as they relate to molecular mimicry and/or epitope spreading.

Evaluation of Cardioprotective Effect of 3,5,3′-Tri-iodo-L-thyronine in Isoproterenol-Induced Cardiotoxicity

T3 (3,5,3′-triiodothyronine) has drawn relatively little attention in relation to cardiovascular (CVS) diseases. The present study was designed to evaluate the cardioprotective action of T3 in isoproterenol-(ISO-) induced cardiac toxicity. Female Wistar rats were exposed with ISO (100 mg/kg, body weight, subcutaneously) for 2 days at the interval of 24 h followed by T3 (3 μg/kg, body weight, orally) treatment for 3 days. Positive control rats received only ISO (100 mg/kg, body weight, subcutaneously) for 2 days at the interval of 24 hrs. Control group animals received normal saline as a vehicle. As expected, ISO-induced significant changes were observed in low-density lipoprotein, total cholesterol, ALT, CK-MB to TCK ratio, and prolongation of QT interval in electrocardiogram, which is toward normalization after T3 treatment. Lower heart weight, upregulation of cardiac myosin heavy chain alpha (MHC-α), and reduced inflammatory cell infiltration, myonecrosis, vacuolar changes, and a trend toward normal cardiac muscle fiber architecture in microscopic examination of cardiac tissue further support the cardioprotective effect of T3.

Detection of cardiac myosin-specific CD4 T cells by using MHC II / IAk tetramers in A/J mice (93.12)

Abstract Myocarditis is one of the predominant causes of heart failure in young adolescents, and Coxsackievirus B strains (CVB1-6) are common suspects. CVB3 isolates from humans induce myocarditis in A/J mice, but the infectious virus cannot be isolated 18-21 days postinoculation. However viral nucleic acids showing deletions in the 5’ non-translated regions persist. The extent to which continuing low level replication of CVB causes inflammation is not known and an autoimmune response is suspected in late disease progression, and cardiac myosin heavy chain-alpha (MYHC-α) has been identified as a dominant autoantigen. To delineate the contribution of autoimmune response to postinfectious myocarditis, it is critical to demonstrate the generation of pathogenic autoreactive T cells as they expand to a significant proportion to cause disease. To this end, we have created major histocompatibility complex class II / IAk tetramers for MYHC-α, 334-352. By using IAk / RNase 43-56 tetramers as controls, we have optimized the conditions to detect MYHC-α 334-352-specific CD4 T cells in lymph node cell cultures derived from A/J mice immunized with MYHC-α 334-352. Preliminary studies suggest that the reagents can also be used to enumerate the frequency of myosin-specific T cells ex vivo in immunized mice. These reagents will now permit us to determine the generation and functions of myosin-reactive T cells as an indicator of autoimmune response to cardiac myosin in mice infected with CVB3.

Electrophoretic variants of cardiac myosin heavy chain-alpha in Sprague Dawley rats.

Analysis of cardiac myosin revealed differences in gel electrophoretic migration patterns of the alpha-isoform of myosin heavy chain, but not the beta-isoform, in Sprague Dawley rats. No differences in the migration patterns of the alpha-or beta-isoforms were observed in other rat strains. Three electrophoretic migration patterns of the alpha-isoforms were observed in individual rats: a slower migrating isoform alone (4% of all rats tested), a faster migrating isoform alone (55%), and both isoforms (41%). The isoform expression pattern was identical in all myocardial regions in each rat. Frequency of expression patterns suggests multiple gene sequences for alpha-cardiac myosin heavy chain in Sprague Dawley rats. Sequence analysis of amplified regions of the Sprague Dawley and Brown Norway rat alpha-myosin genes, specifically the 5'-untranslated region, exons 1-3, and associated introns, showed numerous single nucleotide polymorphisms in coding and noncoding regions, including putative regulatory sites in Sprague Dawley rats, but not in Brown Norway rats. All Sprague Dawley rats varied from Brown Norway rats and no heterogeneity was observed in Brown Norway rats. Several deletions and dimorphic positions were also observed. Dimorphic positions were evident on automated sequencing comparisons. The data indicate that at least two alpha-myosin heavy chain isoforms exist in Sprague Dawley rats and these rats exhibit sequence diversity within that portion of the alpha-myosin heavy chain gene reported in this study.

Characterization of the TBX5 binding site and analysis of mutations that cause Holt-Oram syndrome.

Holt-Oram syndrome is caused by mutations in TBX5, a member of the T-box gene family. In order to identify DNA sequences to which the TBX5 protein binds, we have performed an in vitro binding site selection assay. We have identified an 8 bp core sequence that is part of the Brachyury consensus-binding site. We show that TBX5 binds to the full palindromic Brachyury binding site and to the half-palindrome, whereas Brachyury does not bind to the TBX5 site. Amino acids 1-237 of TBX5 are required for DNA binding. Analysis of the effects of specific substitution mutations that arise in Holt-Oram patients indicates that G80R and R237Q eliminate binding to the target site. DNA database analysis reveals that target sites are present in the upstream regions of several cardiac-expressed genes including cardiac alpha actin, atrial natriuretic factor, cardiac myosin heavy chain alpha, cardiac myosin heavy chain beta, myosin light chain 1A, myosin light chain 1V and Nkx2.5. Cell transfection studies demonstrate that TBX5 activates the transcription of an atrial natriuretic factor reporter construct and this effect is significantly reduced by deletion of the TBX5 binding site.

Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.

Immune interactions in the heart were studied using a murine model of myosin-induced autoimmune myocarditis. A T cell hybridoma specific for mouse cardiac myosin was generated from A/J mice and used to demonstrate that endogenous myosin/I-Ak complexes are constitutively expressed on antigen-presenting cells in the heart. This T cell hybridoma, Seu.5, was used as a functional probe to identify a myocarditis-inducing epitope of cardiac myosin. Overlapping peptides based on the cardiac myosin heavy chain alpha (myhc alpha) sequences were synthesized and tested for their ability to stimulate Seu.5 T cells. One peptide, myhc alpha (325-357) strongly stimulated the Seu.5 T cells, localizing the epitope to this region of the myhc alpha molecule. Using truncated peptides, the epitope was further localized to residues 334-352. The myhc alpha (334-352) peptide strongly induced myocarditis when administered to A/J mice, which was histologically indistinguishable from that induced by myosin. The myhc alpha (334-352) epitope was present in cardiac myosin and not skeletal muscle myosins, providing a biochemical basis for the cardiac specificity of this autoimmune disease. Induction of myocarditis by this epitope was restricted to the myhc alpha isoform and not the myhc beta isoform, suggesting there may be a difference in the efficiency of generating tolerance to these isoforms of cardiac myosin, which are differentially developmentally regulated. The myhc alpha (334-352) epitope bound to purified I-Ak molecules in a similar manner to other I-Ak-restricted immunogenic epitopes, HEL(48-61) and RNase(43-56). Importantly, the myhc alpha (334-352) epitope was able to bind to I-Ak molecules on the surface of antigen-presenting cells in a stable manner. These findings demonstrate that autoantigenic epitopes can behave in a dominant manner and constitutively bind to class II molecules in the target organ in a similar manner to foreign immunogenic epitopes.

Insulin and substrate consumption induced changes in cardiac myosin heavy chain alpha mRNA and beta mRNA levels in the diabetic rat heart

D by streptozotocin show decreased velocity of cardiac contraction. We have shown that these changes are accompanied by increased myosin (M) V3 and decreased VI predominance in D hearts [normal ( ) heart Vl-72%, V3-15%; D Vl-15% V3-68%]1. Administration of 3 U protamin zinc insulin (PZI) for 3 weeks completely normalizes M isoforms (Miso) levels. MV1 is composed of 2 myosin heavy chains (MHC) α and MV3 of 2 MHC β. We wanted to determine whether changes in MHCa and MHCβ mRNA accompany Miso alterations, examine the insulin (I) responsiveness, and determine whether lowering of increased fatty acid oxidation which occurs in D hearts leads to changes in MHC mRNA.