Regulation and functions of NLRP3 inflammasome in cardiac fibrosis: Current knowledge and clinical significance.

Cardiac fibrosis can lead to heart failure, arrhythmia, and sudden cardiac death, representing one of the leading causes of death due to cardiovascular diseases. Cardiac fibrosis involves several multifactorial processes that cannot be effectively controlled by the available therapies. Therefore, current research has focused on the development of novel drugs that can be used to prevent cardiac fibrosis. Recent studies on the functions of inflammasome have provided an in-depth understanding of the regulatory functions of inflammasome in cardiac fibrosis. This review summarizes the latest research on the functions of the NLRP3 inflammasome in various cardiovascular diseases. The latest findings indicate that the NLRP3 inflammasome mediates several inflammatory responses and is associated with pyroptosis, mitochondrial regulation, and myofibroblast differentiation in cardiac fibrosis. These novel findings provide insight into the vital role of the NLRP3 inflammasome in the pathogenesis of cardiac fibrosis, which can be used to identify new targets for its prevention and treatment.

ALDH2 Overexpression Alleviates High Glucose-Induced Cardiotoxicity by Inhibiting NLRP3 Inflammasome Activation.

Although the underlying mechanisms of diabetes-induced myocardial injury have not been fully illuminated, the inflammation reaction has been reported intently linked with diabetes. The nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, the key component of pyroptosis, is involved in inflammation reaction, which may be one of the important mechanisms in diabetes-induced myocardial injury. The purpose of this study was to investigate the changes of NLRP3 inflammasome and pyroptosis in high glucose-induced H9C2 cardiac cell injury and investigate whether overexpression of mitochondrial aldehyde dehydrogenase 2 (ALDH2) can reduce the occurrence of pyroptosis. The H9C2 cardiac cells were exposed to 35 mM glucose for 24 h to induce cytotoxicity. Mitochondrial ALDH2 overexpression cardiac cell line was constructed. The results showed in high glucose condition that ALDH2 overexpression significantly increased H9C2 cardiac cell viability, increased mitochondrial ALDH2 activity and protein expression, and reduced mitochondrial reactive oxygen species (ROS) production, 4-hydroxynonenal (4-HNE), and lactate dehydrogenase (LDH) levels; meanwhile, the pyroptosis key components-NLRP3 inflammasome-related proteins, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteine-containing aspartate specific protease 1 (Caspase-1), and interleukin-18 (IL-18) protein expressions-were significantly decreased, and IL-18 and interleukin-1ß (IL-1ß) levels were also decreased. In high glucose-induced cardiac cell injury, ALDH2 overexpression may reduce ROS production, thereby inhibiting the activation of NLRP3 inflammation and cell pyroptosis. ALDH2 gene might play the potential role in the treatment of high glucose-induced H9C2 cardiac cell injury.

Variable Cardiac Responses to Immunosuppressive Therapy in Anti-Mitochondrial Antibody-Positive Myositis.

We describe a case of anti-mitochondrial antibody-positive myositis associated with cardiovascular involvement. An electrophysiological study (EPS) showed binodal dysfunction, and cardiac magnetic resonance (CMR) imaging revealed left ventricular dysfunction with diffuse, patchy T2 high-intensity areas and late gadolinium enhancement indicative of inflammation and fibrosis. The left ventricular dysfunction was successfully treated with immunosuppressive therapy as documented by CMR. Persistence of conduction system dysfunction was confirmed by EPS, and a pacemaker was implanted. CMR and EPS concisely documented the variable cardiac response to treatment in anti-mitochondrial antibody-positive myositis. We demonstrate the utility of cardiac investigations in this rare disorder.

Fundamental Mechanisms of Regulated Cell Death and Implications for Heart Disease.

Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.

Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria.

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.

Preventive effect of apigenin against isoproterenol-induced apoptosis in cardiomyoblasts.

We investigated the effect of apigenin, a dietary flavonoid, on isoproterenol hydrochloride (ISO)-induced apoptotic signaling in cardiomyoblast H9C2 cells. The results showed that apigenin treatment (10 µM) prevented ISO (31.25 µM)-induced lipid peroxidative levels and antioxidants status in H9C2 cells. Furthermore, apigenin inhibited expression of inflammatory markers in ISO-treated cells. In addition, apigenin prevented ISO-induced DNA damage and apoptotic signaling through modulating the expression of Bax, caspase-3, -8 and -9, cytochrome c, and Fas proteins in H9C2 cells. It is concluded that apigenin prevents ISO-induced antioxidants depletion, oxidative DNA damage, inflammatory, and apoptotic signaling in H9C2 cells. Thus, the present results demonstrated that apigenin has a cardioprotective effect on cardiomyoblasts cells.

Inhibition of programmed necrosis limits infarct size through altered mitochondrial and immune responses in the aged female rat heart.

Both advancing age and estrogen loss exacerbate acute myocardial infarction in the female heart. However, the mechanistic underpinnings of age-related differences in cell death after ischemia-reperfusion (I/R) injury in female subjects and reductions in cardioprotective reserve capacity remain largely unexplored. The aim of the present study was to determine the efficacy of programmed necrosis inhibition on infarct size reduction and preservation of left ventricular (LV) function after I/R injury with female aging. Fischer 344 rats were ovariectomized (OVX) at 15 mo and studied at 24 mo (MO OVX) versus adult rats with intact ovaries (6 mo). After in vivo coronary artery ligation (55-min ischemia and 2- or 6-h reperfusion), necrostatin-1 (Nec-1; 3.5 or 5.7 mg/kg) delivered upon reperfusion significantly reduced infarct size by 37% and improved LV function in the MO OVX group ( P < 0.01). Although age-associated elevations in cyclophilin D and mitochondrial acetylation ( P < 0.001) were unaffected by Nec-1, profound reductions in IL-1, IL-6, and TNF-α ( P < 0.05) as well as cardiac immune cell infiltration were observed in MO OVX but not adult rats. We conclude that chronic inflammation and postmenopausal estrogen deficiency conspire to exacerbate acute infarction through a mechanism involving exaggerated mitochondria-mediated programmed necrosis through receptor-interacting protein 1 signaling. Modulatory effects of programmed necrosis inhibition on proinflammatory cytokine production after I/R reveal a potentially important mechanistic target to restore and preserve cardiac function in the OVX aged female heart. NEW & NOTEWORTHY Myocardial infarct size reduction by inhibition of programmed necrosis in aged female subjects suggests a dominant cell death pathway. Alterations in mitochondrial protein levels and acetylation underscore a mitochondria-dependent mechanism, whereas the profound cytokine reduction in aged subjects alone points to a divergent role for immune modulation of programmed necrosis and viable therapeutic target.

TNF-α induces Drp1-mediated mitochondrial fragmentation during inflammatory cardiomyocyte injury.

Dynamin-related peptide 1 (Drpl)-mediated mitochondrial fission is an important process associated with cardiac dysfunction under different pathological conditions. The aim of the present study was to investigate the expression of Drpl during inflammatory myocardial injury. Sprague­Dawley rats were treated intraperitoneally with lipopolysaccharides (LPS). Furthermore, cultured H9C2 cardiomyocytes were treated with LPS, interleukin­6 (IL­6) and tumor necrosis factor­α (TNF­α). Total and mitochondrial proteins were isolated from the heart tissue of rats and from the H9C2 cardiomyocytes. Expression levels of Drp1 and RhoA were analyzed by western blotting. Mitochondrial morphology was determined using confocal laser microscopy. The levels of mitochondrial Drp1 and phosphorylated­Drp1 (p­Drp1) Ser616 were revealed to be increased in rats 6 h after injection with LPS (5, 10 or 20 mg/kg). Furthermore, treatment with LPS and IL­6 did not demonstrate a significant effect on the expression of total and mitochondrial Drp1 in H9C2 cardiomyocytes in vitro; however, treatment with TNF­α (20 ng/ml) significantly enhanced the levels of mitochondrial Drp1 and p­Drp1 Ser616. Following TNF­α treatment, the expression of Ras homolog gene family member A (RhoA) was also revealed to increase. Treatment with both Y­27632 and fasudil, [Rho kinase (ROCK) inhibitors], was demonstrated to attenuate the otherwise TNF­α­induced increase in p­Drp1 Ser616 and mitochondrial Drp1. In addition, it was revealed that Y­27632 and fasudil may also attenuate the TNF­α­induced increase in mitochondrial fragmentation and cell viability. Therefore, the findings of the present study suggest that TNF­α is the predominant inducer of Drp1 S616 phosphorylation during sepsis. The results of the present study also suggest that the RhoA/ROCK pathway may be involved in the phosphorylation and mitochondrial translocation of Drp1, which leads to mitochondrial fragmentation.

Antiapoptotic effect of exercise training on ovariectomized rat hearts.

The purpose of this study was to evaluate the effects of exercise training on cardiac Fas receptor-dependent and mitochondria-dependent apoptotic pathways in ovariectomized rats. Histopathological analysis, TUNEL assay, and Western blotting were performed on the excised hearts from three groups of Sprague-Dawley rats, which were divided into a sham-operated group, a bilaterally ovariectomized group (OVX), and a bilaterally ovariectomized group that underwent treadmill running exercise for 60 min/day, 5 sessions/wk, for 10 wk (OVX-EX). The abnormal myocardial architecture, cardiac trichome-stained fibrosis and cardiac TUNEL-positive apoptotic cells in ovariectomized rats improved after exercise training. The protein levels of tumor necrosis factor-α, tumor necrosis factor receptor 1, Fas ligand, Fas receptors, Fas-associated death domain, activated caspase-8 and activated caspase-3 (Fas receptor-dependent apoptotic pathways), as well as t-Bid, Bad, Bak, Bax, cytosolic cytochrome c, activated caspase-9, and activated caspase-3 (mitochondria-dependent apoptotic pathways) were decreased in the OVX-EX group compared with the OVX group. Exercise training suppressed ovariectomy-induced cardiac Fas receptor-dependent and mitochondria-dependent apoptotic pathways in ovariectomized rat models. These findings might indicate a new therapeutic effect for exercise training to prevent cardiac apoptosis in menopausal or bilaterally oophorectomized women.

Nur77 is involved in graft infiltrating T lymphocyte apoptosis in rat cardiac transplantation model.

Acute allograft rejection is initiated by a large number of recipient T cells that recognize donor alloantigens. Apoptotic signals trigger Nur77 production. Nur77 translocates from the nucleus to the mitochondria to induce a loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators, including HtrA2/Omi. In this study, we investigated the relationship between Nur77, HtrA2/Omi, and T lymphocyte apoptosis during acute allograft rejection in a rat cardiac transplantation model. The median survival time of the isograft group was longer than that of the allograft group. The cardiac grafts in isogenic (Lewis to Lewis) and allogenic (Wistar to Lewis) models were subjected to HE stain, showing that no rejection occurred in the isografts and that the rejection level was increased in allografts. Compared with the rare expression in syngeneic Lewis rat hearts by western blot analysis, Nur77 protein level in allograft increased from day 1, peaked at day 5 after transplantation, and maintained the highest level until day 7. Double immunofluorescence staining on allograft tissues at day 5 showed Nur77 immunocompetence in most CD3(+) cells, and Nur77 positive T cells also showed HtrA2/Omi-positive signal. Meanwhile, active caspase-3 was apparent in these HtrA2/Omi-positive T cells. Immunohistochemical results suggested that both Nur77 and active caspase-3 were expressed in increasing infiltrating lymphocytes. Our results demonstrated that upregulated Nur77 may promote graft-infiltrating T lymphocyte apoptosis by translocating and inducing HtrA2/Omi release from mitochondria in acute rejection after cardiac transplantation.

Downregulation of adenine nucleotide translocator 1 exacerbates tumor necrosis factor-α-mediated cardiac inflammatory responses.

Inflammation contributes significantly to cardiac dysfunction. Although the initial phase of inflammation is essential for repair and healing, excessive proinflammatory cytokines are detrimental to the heart. We found that adenine nucleotide translocator isoform-1 (ANT1) protein levels were significantly decreased in the inflamed heart of C57BL/6 mice following cecal ligation and puncture. To understand the molecular mechanisms involved, we performed small-interfering RNA-mediated knockdown of ANT1 and studied tumor necrosis factor-α (TNFα)-induced inflammatory responses in myocardium-derived H9c2 cells and cardiomyocytes. ANT1 knockdown significantly increased swollen mitochondria and mitochondrial reactive oxygen species, concomitant with increased TNFα-induced NF-κB reporter gene activity and interleukin-6 and TNFα expression. A mitochondrial-targeted antioxidant mito-TEMPO attenuated TNFα-induced mitochondrial reactive oxygen species, NF-κB reporter gene activity, and cytokine expression in ANT1 knockdown cells. Interestingly, TNFα or lipopolysaccharide (LPS) treatment significantly decreased ANT1 protein levels, suggesting a feed-forward regulation of proinflammatory cytokine expression activated by ANT1 downregulation. These data suggest that ANT1 downregulation contributes to cardiac inflammation post-cecal ligation and puncture. Preventing ANT1 downregulation could provide a novel molecular target to temper cardiac inflammation.

Design and use of peptide-based antibodies decreasing superoxide production by mitochondrial complex I and complex II.

Mitochondria are the major source of reactive oxygen species. Both complex I and complex II mediate O2*- production in mitochondria and host reactive protein thiols. To explore the functions of the specific domains involved in the redox modifications of complexes I and II, various peptide-based antibodies were generated against these complexes, and their inhibitory effects were subsequently measured. The redox domains involved in S-glutathionylation and nitration, as well as the binding 2011. motif of the iron-sulfur cluster (N1a) of the complexes I and II were utilized to design B-cell epitopes for generating antibodies. The effect of antibody binding on enzyme-mediated O2*- generation was measured by EPR spin trapping. Binding of either antibody AbGSCA206 or AbGSCB367 against glutathione (GS)-binding domain to complex I inhibit its O2*- generation, but does not affect electron transfer efficiency. Binding of antibody (Ab24N1a) against the binding motif of N1a to complex I modestly suppresses both O2*- generation and electron transfer efficiency. Binding of either antibody Ab75 or Ab24 against nonredox domain decreases electron leakage production. In complex II, binding of antibody AbGSC90 against GS-binding domain to complex II marginally decreases both O2*- generation and electron transfer activity. Binding of antibody AbY142 to complex II against the nitrated domain modestly inhibits electron leakage, but does not affect the electron transfer activity of complex II. In conclusion, mediation of O2*- generation by complexes I and II can be regulated by specific redox and nonredox domains.

Sepsis-induced cardiomyopathy: a review of pathophysiologic mechanisms.

Cardiac dysfunction is a well-recognized complication of severe sepsis and septic shock. Cardiac dysfunction in sepsis is characterized by ventricular dilatation, reduction in ejection fraction and reduced contractility. Initially, cardiac dysfunction was considered to occur only during the "hypodynamic" phase of shock. But we now know that it occurs very early in sepsis even during the "hyperdynamic" phase of septic shock. Circulating blood-borne factors were suspected to be involved in the evolution of sepsis induced cardiomyopathy, but it is not until recently that the cellular and molecular events are being targeted by researchers in a quest to understand this enigmatic process. Septic cardiomyopathy has been the subject of investigation for nearly half a century now and yet controversies exist in understanding it's pathophysiology. Here, we discuss our understanding of the pathogenesis of septic cardiomyopathy and the complex roles played by nitric oxide, mitochondrial dysfunction, complements and cytokines.

Presence of antimitochondrial autoantibodies in patients with autoimmune hepatitis.

BACKGROUND AND AIM: Antimitochondrial autoantibodies (AMA) are known to be a hallmark of primary biliary cirrhosis, and it has been suggested that AMA play a crucial role in generating biliary changes. Biliary tract lesions are not uncommon in patients with autoimmune hepatitis (AIH) and previous works have demonstrated that AMA are occasionally detectable in sera of patients with AIH. Therefore, the role of AMA as a cause of bile duct lesions in AIH livers should be addressed. The aim of the present study was to determine whether the presence of AMA is associated with clinical features, especially the occurrence of bile duct lesions, in patients with AIH. METHODS: Forty-one patients diagnosed as having AIH according to the revised scoring system of the International Autoimmune Hepatitis Group were enrolled in this study. Clinical data were retrospectively reviewed, and histological findings of the liver were investigated. AMA reactivity was determined by immunoblotting using beef heart mitochondria as antigens. RESULTS: Although not found in any enrolled patient by conventional indirect immunofluorescence, AMA were detectable in 14 out of 41 patients (34%). Clinical parameters including biochemistry, autoantibody profile, and responses to treatment were similar irrespective of AMA status. Bile duct lesions were noted in 14/14 (100%) and 23/27 (85%) of AMA-positive and -negative patients with AIH, respectively (P = 0.134). There was no statistically significant difference in the grade of inflammation or stage of fibrosis between the two groups. CONCLUSION: Antimitochondrial autoantibodies were found to be present in sera of patients with AIH more frequently than expected, even at very low titer. However, clinical features and histological findings of AIH were not influenced by the AMA status.

[Ultrastructural peculiarities of heart of rats of various ages at different levels of antigen load].

The aim of the research was to investigate electron-microscopic peculiarities of heart at different levels of antigen loading using adult and old aged gnotobiont rats, conventional rats and rats with experimental autoimmune cardiomyopathy. The results of investigations showed that the ultrasrtuctural characteristics of heart greatly depend on microbiological status and age of experimental animals. The ultrasrtucture of heart of adult rats mostly depend on the level of intensity of antigen loading. In all experimental groups of old rats the subcellular differences had the same directions. Although, it must be mentioned that there was not significant correlation between the level of heart damage and intensity of antigen loading. According to this, age-related changes depend not only on microbiological status of organism, but on genetically determined peculiarities of internal regulative processes taking place during the ontogenesis. Thus, the received results may be used to identify age-related changes, which give us the possibility for the differentiation between age-related and pathological peculiarities.

Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten.

The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-gamma]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten, which may be liberated following cardiomyocyte destruction during the inflammation of the heart.

Assessment of membrane-bound mammal mitochondrial adenine nucleotide translocase topography by experimental antibodies.

To gain insight into the immunogenicity of mitochondrial adenine nucleotide translocase (ANT), we raised antibodies against purified bovine heart ANT by induction of ascitic fluid in male Balb/c mice. We identified the antigenic determinants detected by these antibodies by (1) immunodetection of GST-ANT fusion proteins and selected partial constructs of ANT, (2) immunodetection of chemically synthesized overlapping peptides on solid support, and (3) back-titration ELISA. Results revealed a short epitope spreading of the antibodies, resulting in a small number of antigenic determinants. Thus, each antibody detects one or two major epitopes located in the putative hydrophilic loops M2 and M3. No evidence for the antigenicity of the first 133 amino acids of ANT was obtained. These well-characterized antibodies were used to study the topography of the membrane-bound ANT by back-titration ELISA with mitochondrial membranes. We demonstrated that amino acids 145-150 and 230-237 are fully accessible to the antibodies in native ANT, whereas regions 133-140 and 244-251 are not. Furthermore, we used mitochondria devoid of the outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) to establish the matrix or cytosolic orientation of loops M2 and M3. The results clearly show that these loops have a matrix orientation and thus support the six transmembrane segment model of ANT topography in the inner mitochondrial membrane.

Possible association of HLA-DRB1 gene with the autoantibody against myocardial mitochondria ADP/ATP carrier in dilated cardiomyopathy.

To probe the genetic background and immunopathogenesis of dilated cardiomyopathy (DCM) 77 patients with DCM, HLA-DRB1 gene polymorphism were analyzed by using the polymerase chain reaction/sequence specific primer (PCR/SSP) technique and autoantibody against myocardial mitochondria ADP/ATP carrier were examined by using the Immunoblot analysis. The frequency of HLA-DRB1*0901 allele was significantly higher in DCM patients in which autoantibody against ADP/ATP carrier of myocardial mitochondria is positive in contrast with those in which the autoantibody is negative (25.46% vs 3.45%, P < 0.05), the relative risk (RR) being 9.56. The other frequencies of HLA-DRB1 alleles have no significant difference in the antibody positive group and negative group. It is possible that a subset of DCM patients may exist in which autoimmunity is associated with genetic factors.

Autoantibodies in the sera of patients with rheumatic heart disease: characterization of myocardial antigens by two-dimensional immunoblotting and N-terminal sequence analysis.

The concept of antigenic mimicry in autoimmune diseases such as rheumatic fever has been under investigation for decades and the range of cross-reactive tissue antigens for streptococcal-induced antibodies identified in rheumatic heart disease is still expanding. To identify heart tissue-reactive antigens which may be implicated in the secondary immunopathogenesis of rheumatic fever, sera from 56 patients with acute rheumatic heart disease were probed in two-dimensional Western blots for reactivity against heart tissue antigens. After two-dimensional immunoblot analysis, proteins were submitted to N-terminal amino acid sequence analysis. This analysis identified creatine kinase, two mitochondrial proteins and, at a low level, various stress proteins as cross-reactive myocardial antigens. Therefore, in addition to myosin, creatine kinase may represent another major antigen for autoreactive antibodies in rheumatic heart disease. Mitochondrial proteins have been implicated in the pathogenesis of inflammatory heart disease for some years, and in this study we have identified two mitochondrial proteins as relevant antigens in rheumatic heart disease.

Natural and disease associated autoantibodies to the autoantigen, dihydrolipoamide acetyltransferase, recognise different epitopes.

Naturally occurring autoantibodies are ubiquitous and may serve physiological functions. We examined the relationship of natural and disease-associated autoantibodies in the context of autoantibodies to dihydrolipoamide acetyltransferase, the 74 kDa E2 sub-unit of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), characteristic of primary biliary cirrhosis (PBC). We tested for natural autoantibodies to PDC-E2 in normal sera, and compared epitopes recognised by natural and disease-associated autoantibodies. Methods included affinity purification of anti-PDC-E2 from normal and PBC sera, ELISA and immunoblotting, capacity of antibodies to inhibit the enzyme function of the pyruvate dehydrogenase complex (PDC), use of F(ab)2 fragments of anti-PDC-E2 in inhibition assays, and testing affinity purified anti-PDC-E2 on peptide fragments of PDC-E2. We found that natural auto-antibodies to PDC-E2 of IgG class were demonstrable in all healthy human sera (10/10). However, their reactivity differed from that of disease-associated autoantibodies, in that anti-PDC-E2 from normal serum failed to inhibit the catalytic activity of PDC; and F(ab)2 fragments from PBC sera potently blocked the binding of anti-PDC-E2 from PBC sera to PDC-E2, but not the binding of natural anti-PDC-E2 to PDC-E2. Immunoblotting on fragments of PDC-E2 using affinity-purified preparations from PBC sera and normal sera failed to provide evidence for gross differences in epitope reactivity. We conclude that normal human sera contain natural IgG autoantibodies to the immunodominant inner lipoyl domain of PDC-E2, as seen characteristically in PBC. However, there is evidence for differences in fine epitope recognition.