Replacement Fibrosis Research Articles

Hypertrophy and inflammation: too much for one heart

Hypertrophic cardiomyopathy (HCM) is a genetically determined disease characterized by heterogeneous genetic, morphologic, and clinical patterns. Although it is half a century since Teare1 first described the disease, interest in HCM is growing as we move from understanding disease pathology towards prognostic assessment and risk stratification. Microscopic findings of HCM are distinctive and include myocardial hypertrophy and gross disorganization of cardiac fibres. Cellular disarray and disorganization of the myofibrillar architecture within a given cell are standard.2 Patients with HCM often exhibit myocyte necrosis and replacement fibrosis in the left ventricle. A spectrum of severity and distribution is observed, ranging from isolated small scars to extensive transmural fibrosis. These pathological features are considered the consequence of myocardial ischaemia due to coronary microvascular dysfunction.3 Myocardial fibrosis may contribute to the increased ventricular chamber stiffness and impaired relaxation identifiable in most patients with HCM and is the substrate for the genesis of ventricular arrhythmias and sudden death. The clinical course of HCM varies. Some patients' symptoms are absent or mild and remain stable. However, most patients develop symptoms of variable degree that progress and worsen over time. A general relationship exists between the extent of hypertrophy and the severity of symptoms, but this relationship is not absolute, and some patients have severe symptoms with only mild and apparently localized hypertrophy. The transition from a hypertrophied and non-dilated state with intact contractility to one of systolic dysfunction may evolve gradually or have a more abrupt presentation and occurs in … *Corresponding author. Tel: +34 93 2919258; fax: +34 93 5565603. E-mail address : abayesgenis{at}santpau.es

Left ventricular aneurysm in a patient with mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome): clinical and pathological correlation

A 22-year-old man was diagnosed with mucopolysaccharidosis type VI, also known as Maroteaux-Lamy syndrome, which is a known cause of cardiac valvular disease. He presented with exercise intolerance and was diagnosed with a large, apical, left ventricular aneurysm (LV aneurysm) and subsequently underwent left ventricular aneurysmectomy with improvement in clinical status. Previous echocardiograms revealed that the LV aneurysm was new and is, therefore, likely acquired rather than congenital. Pathology confirmed a true aneurysm, replacement fibrosis, and PAS-positive material in cardiomyocytes. Subsequent echocardiography revealed progression of valvular heart disease with moderate stenosis of the aortic and mitral valves. We propose that altered metabolism of glycosaminoglycans in the extracellular matrix may have contributed to the development of the LV aneurysm in this patient.

THE ROLE OF CARDIOMYOCYTE APOPTOSIS IN THE DEVELOPMENT OF CARDIAC FIBROSIS.

Objective Cardiomyocyte (CM) apoptosis is elevated in end-stage heart failure. Cardiac fibrosis is a common phenotype of heart failure. In previous studies we have shown that in SR-uPA transgenic (SR-uPA+/o) mice there is an increase in plasmin-dependent cardiac fibrosis. We hypothesize that in these mice, plasmin causes the pericellular proteolysis of laminin, an extracellular adhesive glycoprotein that acts as a bridge between proteins attached to the cell membrane and the basal lamina. Consequently, due to the loss of cell-matrix contact, apoptosis is induced. Apoptosis of CM then leads to replacement fibrosis. To test this hypothesis we measured apoptosis in the hearts of SR-uPA+/o and nontransgenic (SR-uPAo/o) littermates. Methods We measured apoptosis in the hearts of SR-uPA+/o and SR-uPAo/o mice using TUNEL (Roche) staining of apoptotic nuclei in situ with a hematoxylin counterstain. In addition, since caspases are known mediators in the apoptotic pathway, we performed immunostaining and immunoblotting for cleaved caspase 3 (Cell Signaling Technology), a downstream effector caspase, in SR-uPA+/o and SR-uPAo/o heart sections. Mouse thymus served as a positive control for both experiments. Additionally, lysates of camptothecin-treated and nontreated 293 cells were positive and negative controls, respectively, for the immunoblotting. Results Increased numbers of TUNEL-positive cells were seen in 6-week-old SR-uPA+/o hearts compared with SR-uPAo/o littermates (51.5 [33-67] vs 17.5 [12-28] TUNEL positive cells per 400× field, n = 8, p = .003). Preliminary analysis of counterstained sections reveals TUNEL-positive cells are not CM. In our cleaved caspase-3 studies two SR-uPA+/o hearts had increased numbers of staining cells compared with SR-uPAo/o littermate controls. Furthermore, the immunoblotting was suggestive of greater cleaved caspase-3 activity in 6-week-old SR-uPA+/o versus SR-uPAo/o mice. Conclusions In SR-uPA+/o mice we found an increase in TUNEL-positive cells and increased cleaved caspase-3 activity. These data indicate that apoptosis may be an important mediator of cardiac fibrosis. However, contrary to our hypothesis, the cells undergoing apoptosis do not appear to be CM and, instead, could be macrophages. Further immunostaining will be needed to conclusively identify the cell type. Additionally, we will increase our sample size in our cleaved caspase-3 immunostaining experiment.

Cardiac matrix metalloproteinase-2 expression independently induces marked ventricular remodeling and systolic dysfunction

Although enhanced cardiac matrix metalloproteinase (MMP)-2 synthesis has been associated with ventricular remodeling and failure, whether MMP-2 expression is a direct mediator of this process is unknown. We generated transgenic mice expressing active MMP-2 driven by the alpha-myosin heavy chain promoter. At 4 mo MMP-2 transgenic hearts demonstrated expression of the MMP-2 transgene, myocyte hypertrophy, breakdown of Z-band registration, lysis of myofilaments, disruption of sarcomere and mitochondrial architecture, and cardiac fibroblast proliferation. Hearts from 8-mo-old transgenic mice displayed extensive myocyte disorganization and dropout with replacement fibrosis and perivascular fibrosis. Older transgenic mice also exhibited a massive increase in cardiac MMP-2 expression, representing recruitment of endogenous MMP-2 synthesis, with associated expression of MMP-9 and membrane type 1 MMP. Increases in diastolic [control (C) 33 +/- 3 vs. MMP 51 +/- 12 microl; P = 0.003] and systolic (C 7 +/- 2 vs. MMP 28 +/- 14 microl; P = 0.003) left ventricular (LV) volumes and relatively preserved stroke volume (C 26 +/- 4 vs. MMP 23 +/- 3 microl; P = 0.16) resulted in markedly decreased LV ejection fraction (C 78 +/- 7% vs. MMP 48 +/- 16%; P = 0.0006). Markedly impaired systolic function in the MMP transgenic mice was demonstrated in the reduced preload-adjusted maximal power (C 240 +/- 84 vs. MMP 78 +/- 49 mW/microl(2); P = 0.0003) and decreased end-systolic pressure-volume relation (C 7.5 +/- 1.5 vs. MMP 4.7 +/- 2.0; P = 0.016). Expression of active MMP-2 is sufficient to induce severe ventricular remodeling and systolic dysfunction in the absence of superimposed injury.

Increased cardiac mRNA expression of matrix metalloproteinase-1 (MMP–1) and its inhibitor (TIMP–1) in DCM patients

Left ventricular dilation and myocardial remodeling are hallmarks of dilated cardiomyopathy (DCM). It is assumed that left ventricular dilation is caused by the disintegration of the collagenous network by increased collagenolytic activity of matrix metalloproteinases (MMPs) and their adequate tissue inhibitors (TIMPs). In this study the myocardial MMP-1 and TIMP-1 mRNA expressions were investigated by using real-time quantitative PCR analysis from right septal endomyocardial biopsies of patients with dilated cardiomyopathy (n = 46) and control subjects (n = 11). The volume density (Vv%) of collagen was measured morphometrically. Classification was done according to LV diameters [left ventricular enddiastolic diameter (LVEDD, cm) calculated to body surface area (BSA, m(2))] into three DCM groups: group I (LVEDD-BSA > 2.7-3.0 cm/m(2)), group II ( > 3.0-3.6 cm/m(2)), group III ( > 3.6 cm/m(2)), controls (< 2.7 cm/m(2)). Compared with controls, the MMP-1 expression in patients with DCM was significantly increased (119.2 +/- 45.2 vs. 1.3 +/- 0.4; p < 0.001) as was TIMP-1 expression (9.6 +/- 1.2 vs. 1.3 +/- 0.4; p < 0.01). Moreover the MMP-1 and TIMP-1 expression varied according to LV diameter: group I (MMP-1: 8.7 +/- 3.5; p = 0.33; TIMP- 1: 4.5 +/- 1.2; p < 0.01); group II (MMP-1: 211.4 +/- 86.0; p < 0.001; TIMP-1: 12.5 +/- 1.9 ; p < 0.001); group III (MMP-1: 38.8 +/- 22.6; p < 0.01; TIMP-1: 8.1 +/- 1.7; p < 0.001). Compared with controls, the collagen level in DCMPt. was significantly increased: 5.0 +/- 0.6 vol% vs 1.2 +/- 0.2 vol% p < 0.001 and correlates with LV diameter. This study reveals that the overexpression of MMP-1, which is associated with an increased ratio of MMP-1/TIMP-1 in DCM, indicates an activated collagenolytic system while replacement fibrosis is accumulating. The MMP-1 overexpression is mainly found in moderately dilated DCM hearts (group II) indicating the dynamic process of LV dilation and the importance of collagenases in the early phase of LV remodeling.

Selective matrix metalloproteinase inhibition attenuates progression of left ventricular dysfunction and remodeling in dogs with chronic heart failure

Matrix metalloproteinases (MMPs) contribute to the progression of left ventricular (LV) dysfunction and remodeling associated with heart failure (HF). The present study examined the long-term effects of a selective MMP inhibitor PG-530742 (PG) on the progression of LV dysfunction and remodeling in dogs with HF. Chronic HF [LV ejection fraction (LVEF), </=36%] was produced by multiple sequential intracoronary microembolizations in 24 dogs. Two weeks after the last embolization, dogs were randomized to 3 mo of therapy with either high-dose (HD) PG (3.5 mg/kg, n = 8), low-dose (LD) PG (0.2 mg/kg, n = 8), or to a matched placebo (PL, n = 8). PG has been shown to produce complete inhibition of MMP-2, -3, -9, and -13, while sparing MMPs-1 and -7. Hemodynamic and echocardiographic measurements were made before and 3 mo after initiating therapy. In PL and LD dogs, LVEF decreased significantly, and LV end-systolic volume (ESV) and LV end-diastolic volume (EDV) increased significantly during the 3-mo follow-up period. Whereas in HD dogs ejection fraction increased from 36 +/- 1 to 40 +/- 1% (P = 0.003), EDV and ESV decreased (59 +/- 4 vs. 57 +/- 4 ml, P = 0.02; and 38 +/- 2 vs. 34 +/- 2 ml, P = 0.00001, respectively). When compared with controls, HD-treated dogs showed 30% reduction in replacement fibrosis, 29% reduction in interstitial fibrosis, and 28% reduction in myocyte cross-sectional area. mRNA expression of selective MMPs was also reduced in LV tissue in HD- but not LD-treated dogs. In conclusion, in dogs with moderate HF, long-term monotherapy with HD selective MMP inhibitor PG prevents LV remodeling and the progression of global LV dysfunction.

Wasting and mortality in beef cattle parasitized by Eurytrema coelomaticum in the State of Paraná, southern Brazil

A sporadic wasting syndrome affecting beef cattle herds parasitized by Eurytrema coelomaticum is described in the State of Paraná, southern Brazil. The disease was characterized by progressive weight loss, poor body condition despite plenty of good quality forage available, and death. Annual losses ranged from 1 to 3%. The clinical course of the disease varied from 2 to 10 months after the onset of the first clinical signs. At necropsy, one of the three distinct following patterns of lesions were observed: the pancreas was of normal size and color; small, shrunken, white and markedly and diffusely firm (fibrosis); or slightly enlarged and dark with a shriveled capsular surface. Myriads of leaf-shaped trematodes of the genus Eurytrema were packed inside multiple dilated ducts with thickened, whitish fibrous walls, or the flukes were embedded in the remaining pancreatic parenchyma. Microscopic findings included extensive loss of the pancreatic parenchyma with replacement fibrosis, intralesional flukes and eggs, and ductal hyperplasia. Inflammatory reaction varied from absent to severe with the presence of a granulomatous reaction around the trematode eggs. One affected animal had high plasma amylase concentration (1580 U/L) suggesting exocrine pancreatic insufficiency. Glucose blood levels were not significantly increased. We suggest that diffuse and marked fibrosis of the pancreas induced by the presence of large amounts of pancreatic flukes is the cause of the sporadic cases of chronic wasting and death of cattle in this geographic area.

Left Ventricular Histomorphometric Findings in Dogs with Heart Failure Treated with the Acorn Cardiac Support Device

Progressive left ventricular (LV) dilation in the setting of heart failure is associated with increased mortality and morbidity. The Acorn Cardiac Support Device (CSD, Acorn Cardiovascular, Inc., St. Paul, MN) is a preformed polyester device that is surgically placed over the cardiac ventricles, anchored to the AV-groove and tailored anteriorly to fit snugly over the epicardial surface of the heart. The CSD was shown to prevent progressive LV enlargement and, indeed, reduce LV size and attenuate global LV remodeling in both animal models of experimentally-induced heart failure as well as in patients with advanced heart failure. This review will examine the CSD from two histologic perspectives namely, (1) the interaction of the CSD with the epicardial surface of the heart and (2) the effects of long-term therapy with the CSD on cellular remodeling. The review will be based on available pre-clinical data generated in dogs with coronary microembolization-induced heart failure that underwent long-term (3 and 6 months) monotherapy with the CSD. The data will show that long-term implantation leads to encapsulation of the CSD by connective tissue that matures with time and that does not invade the underlying myocardium. Furthermore that implantation of the CSD has no adverse impact on epicardial coronary vessel. At the cellular level, existing data will show that long-term monotherapy with the CSD is associated with reduced cardiomyocyte hypertrophy, reduced volume fraction of replacement and interstitial fibrosis, normalization of capillary density and oxygen diffusion distance and attenuation of cardiomyocyte apoptosis. The outcomes strongly argue in favor of a structural modification of the failing myocardium by CSD therapy that is consistent with "reverse cellular remodeling".

Circulating Levels of Heart-Type Fatty Acid-Binding Protein and Its Relation to Thallium-201 Perfusion Defects in Patients With Hypertrophic Cardiomyopathy

Myocyte loss and replacement fibrosis have been observed in patients with hypertrophic cardiomyopathy (HC) with heart failure. This study was designed to elucidate whether heart-type fatty acid-binding protein (H-FABP), a sensitive biochemical marker for myocardial damage, indicates ongoing myocardial damage in patients with HC. We studied 48 patients with HC and 17 control subjects. Patients with HC were divided into 2 groups according to the New York Heart Association (NYHA) functional class: NYHA I + II (n = 40) and NYHA III + IV (n = 8). Serum H-FABP and myoglobin levels were measured, and extent score was used to assess the extent of thallium-201 perfusion defect. Serum H-FABP levels were significantly higher in patients with HC than in control subjects (3.8 +/- 1.6 vs 2.6 +/- 0.7 ng/ml, p = 0.0032). Furthermore, serum H-FABP levels were significantly higher in NYHA III + IV than in NYHA I + II (5.2 +/- 1.3 vs 3.5 +/- 1.5 ng/ml, p = 0.0043). Serum myoglobin levels showed no significant difference among the 3 groups (control, 46.6 +/- 15.0 ng/ml; NYHA I + II, 55.5 +/- 26.4 ng/ml; NYHA III + IV, 65.1 +/- 33.6 ng/ml, p = 0.2115). Extent score correlated positively with serum H-FABP levels (r = 0.420, p = 0.0026) and negatively with fractional shortening (r = -0.542, p <0.0001). Increased H-FABP levels indicate ongoing myocardial damage, which could result in clinical deterioration in patients with HC.

Yin and Yang of Myocardial Transforming Growth Factor-β 1

Ischemic heart disease is a leading cause of chronic myocardial failure and mortality in the United States.1 After myocardial infarction (MI), the heart undergoes a well-characterized process of deleterious structural and molecular remodeling, marked by ventricular dilation, infarct wall thinning, and replacement fibrosis, with a corresponding progressive impairment of contractile function.2 Recent work has suggested that supplementation of myocardial cells with exogenous bone marrow–derived stem cell populations may have the potential to regenerate lost cardiomyocytes and slow, or even reverse, the remodeling process.3 Although the importance of both postinjury myocardial remodeling and regeneration are well accepted, the critical mechanisms that underlie these processes remain unclear. As insight into the biology of myocardial injury and dysfunction increases, the role of stress-activated cytokines has achieved particular prominence.4 Two articles in this issue of Circulation highlight the importance of the stress-activated cytokine, transforming growth factor (TGF)-β1, in mediating the complex processes of cardiac remodeling and regeneration. See pp 2430 and 2438 First isolated >20 years ago, TGF-β was identified from sarcoma cells based on its ability to potentiate the transforming and proliferative actions of epidermal growth factor on non-neoplastic fibroblasts.5,6 Since then, the TGF-β family has grown rapidly and at present consists of >30 structurally related members, including TGF-β1. This diverse cytokine superfamily has been subgrouped according to sequence similarity and specific downstream signaling pathways into the TGF-β/activin/nodal family and the bone morphogenic protein/growth and differentiation factor/Muellerian inhibiting …

The interaction of coronary tone and cardiac fibrosis

Regulation of coronary vascular tone is critical for proper perfusion and function of the myocardium. Many disease processes result in compromised regulation of coronary vascular tone and impaired myocardial perfusion. A common result of coronary vascular dysfunction is the development of areas of replacement fibrosis within the myocardium and surrounding the vasculature. Both intravascular processes, such as coronary atherosclerosis and endothelial dysfunction, and extravascular processes, including compromised myocardial metabolism, hormone excesses, and altered local signaling, may result in coronary vascular dysregulation. Coronary occlusion events, in turn, lead to myocardial damage and the activation of inflammatory cells and fibroblasts. The role of fibroblasts in regulating myocardial fibrosis and the contribution of myofibroblasts, cells that have limited contractile potential while retaining many of the extracellular matrix regulating processes of the fibroblast, may also contribute to the development of myocardial disease. In this review we examine the recent literature on myocardial fibrosis and myofibroblast activity, highlighting the effects of several classes of cardiovascular agents on the remodeling process.

Gene Therapy for Repair of Cardiac Fibrosis

Because of the increase in life expectancy and a high survival rate after myocardial infarction, cardiac fibrosis is becoming one of the most important problems in cardiology. It is characterized by excessive accumulation of fibrillar collagen in the extracellular space, either because of a loss of cardiomyocytes (replacement fibrosis) and/or as an interstitial response to various chronic cardiovascular diseases such as hypertension, myocarditis, and congestive heart failure (reactive fibrosis).1 In humans, cardiac fibrosis is universal in the aging heart. Activated fibroblasts play a pivotal role in the formation and maintenance of fibrous tissue by the production of various extracellular-matrix proteins, including collagen and fibronectin. It is now recognized that even in areas with long-standing fibrosis, such as postinfarction scars, fibroblasts remain metabolically active.2 Their activity is regulated by various autocrine and paracrine factors, such as angiotensin II, aldosterone, endothelins, cytokines, and growth factors.3 The renin-angiotensin-aldosterone system is considered to be of major importance for the regulation of cardiac fibrosis. Increased tissue levels of angiotensin II have shown to promote, and ACE inhibitors are able to delay the development of, cardiac fibrosis.4,5 See p 394 Interstitial fibrosis reduces the electrical coupling between cardiac myocytes because fibroblasts produce smaller or larger collagenous septa, which electrically insulate cardiac cells or muscle bundles. As a result, the normal myocardial architecture becomes disrupted and is transformed into a pathological substrate characterized by the presence of multiple insulating barriers, which force the depolarization wave to spread nonuniformly. As predicted by computer simulations and demonstrated in cell cultures, in such a medium, long local conduction delays occur that may slow the effective conduction velocity to very low values. Unlike a decrease of the rapid sodium current, which will result in conduction failure when conduction velocity is depressed to approximately 30% of control, slow …

Histopathological Study of Tissue Reaction to Pacemaker Electrodes Implanted in the Endocardium

Limited information is available about histopathological reactions to the implanted endocardial electrodes of pacemakers (PM). Gross anatomic and histologic studies of tissue reactions to PM electrodes were made in thirteen autopsy cases (nine men and four women, ages 25 approximately 89 years, mean age 71.8) who died two months to twenty-one years after PM implantation. Nine of them had complete atrioventricular (AV) block, three had sick sinus syndrome, and one had bradycardia-tachycardia syndrome. The direct causes of death were not related to their PM. The tip with projecting tines was implanted in the right ventricle in all patients. At the contact area between the electrode and the endocardium, no tissue reaction was observed in one patient with a history of over sixteen years of PM implantation. However, cardiomyocytes under the tip had been replaced by fibrotic tissue in many other patients. In two patients in particular where the electrode had been implanted at the apex of each right ventricle, all cardiomyocytes had disappeared and only fibrotic tissue and adipose tissue were observed under the tip. These findings suggest that mechanical stress caused by attaching the tip tightly damages cardiomyocytes and brings about changes in the pacing thresholds. In three patients, a space was seen between the tip and the endocardium. A fibrous sheath covering the electrode extended to the tip and formed a thick fibrous cap. This non-excitable fibrous cap acted as a virtual electrode and possibly affected the elevation of the threshold in these patients. In four patients, extensive myocardial fibrosis due to disease, e. g. previous myocardial infarction, dilated cardiomyopathy, amyloidosis, or sarcoidosis, was found in the area surrounding the tip and also might affect the elevation of the threshold. We concluded that elevation of pacing thresholds after PM implantation is not due to reactive endocardial thickening. The space between the tip and the endocardium is occupied by a fibrous sheath, and an overly tight attachment damages cardiomyocytes causing replacement fibrosis. Thus, it is not desirable in some patients to insert the electrodes into the apex, where the myocardium is thin. To avoid the elevation of thresholds, development of further devices is necessary to allow electrode fixation to the endocardium with a more suitable pressure level.

Circulating tenascin-C levels in patients with idiopathic dilated cardiomyopathy

Circulating serum tenascin-C (an extracellular matrix glycoprotein) levels in patients with idiopathic dilated cardiomyopathy (IDC) were measured. Serum tenascin-C levels were increased in proportion to the severity of left ventricular dysfunction in patients with IDC. The associations of serum tenascin-C levels with serum troponin T and procollagen type III aminoterminal peptide levels suggest that increased levels of serum tenascin-C indicate ongoing replacement fibrosis after myocardial damage in IDC.

How structurally normal are human atria in patients with atrial fibrillation?

Atrial myocardium in humans shows structural changes that increase with aging. The most conspicuous are fibro-fatty replacement and patchy replacement fibrosis. The present study reveals that these structural changes are more extensive in hearts of patients with paroxysmal atrial fibrillation (AF) than in "no AF" hearts. The changes involve the myocardial sleeves on pulmonary veins and sites of rapid conduction, such as the terminal crest and Bachmann's bundle. These structural changes should be taken into consideration as potential substrates for initiation and maintenance of atrial arrhythmias.

Scar formation after ischemic myocardial injury in MRL mice

Introduction MRL is a strain of mice with the unusual ability to recover from injuries without forming scar tissue. It was recently reported that the MRL mice can recover with little or no scar formation following a cryogenically induced myocardial infarction of the right ventricle. These findings suggest that scarless recovery from myocardial injury is possible in mammalian ventricles. If applicable to humans, these results could have significant clinical implications in the management of patients with myocardial infarction. However, myocardial infarction in human patients usually results from ischemic injury instead of cryoinjury. Myocardial infarction usually occurs in the left ventricle. Methods We performed a prospective study creating left ventricular myocardial infarction in 32 MRL mice using coronary ligation. Results Among them, 21 mice survived surgery and had myocardial infarction documented by delayed contrast-enhanced magnetic resonance imaging (MRI) 35±10 days after surgery. All 21 mice had evidence of myocardial infarction in follow-up MRI performed 116±21 days after surgery. The infarct size was 27±12% of the area of visualized myocardium during the first MRI study and was 29±11% during the second MRI study ( P=NS). Large myocardial infarction with aneurysm formation was documented by histological examinations in all 21 mice. The ventricular aneurysm showed transmural infarction with replacement fibrosis and mummified necrotic myocardium surrounded by collagenous scar tissue. Occasional surviving myocytes are present within the aneurysm. Conclusion Ischemic myocardial injury in MRL mice is not followed by scarless recovery.

Human hibernating myocardium is jeopardized by apoptotic and autophagic cell death

ObjectivesThe aim of the present study was to objectify the loss of myocytes and the mechanism by which myocytes die in human hibernating myocardium (HHM). BackgroundIntracellular degeneration, reduced cellular protein synthesis, and the replacement fibrosis contribute to structural disintegration of HHM. MethodsIn 14 patients, HHM was diagnosed by dobutamine echocardiography, radionuclide ventriculography, and thallium-201 scintigraphy. Functional recovery was documented by repeating the preoperative clinical investigations three months after successful coronary artery bypass graft surgery (CABG). During CABG, transmural biopsies were taken from the center of HHM regions and studied by electron microscopy, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) method, reverse transcription-polymerase chain reaction, and Western blotting. Control samples were taken from nondiseased human myocardium. ResultsAll patients showed significant improvement or normalization of the regional function of HHM. Ubiquitin-related autophagic cell death was evident ultrastructurally by the occurrence of autophagic vacuoles, cellular degeneration, and nuclear disassembly. Ubiquitin-protein complexes were found in 0.03 ± 0.008% (control: 0%, p < 0.005) of all myocytes. The proteasome 20S subunit/total myocytes were reduced from 63.3 ± 9.6% in control myocardium to 36.9 ± 8.4% in HHM. Complement-9, indicating oncosis, was found in only one of 14 biopsies. TUNEL-positive myocytes were 0.002 ± 0.0003%. Electron microscopy showed apoptotic cells in 3 of 14 samples. However, the bcl-2/baxratio was significantly reduced. Moreover, caspase-3 messenger ribonucleic acid was 8.5 times upregulated, and caspase-3 was activated. Cell death was absent in controls. ConclusionsIn HHM, ubiquitin-related autophagic cell death and apoptosis cause a loss of myocytes. This plays an important role in progressive tissue damage and causes a reduction of the extent of functional recovery of HHM.

108th ENMC International Workshop, 3rd Workshop of the MYO-CLUSTER project: EUROMEN, 7th International Emery-Dreifuss Muscular Dystrophy (EDMD) Workshop, 13–15 September 2002, Naarden, The Netherlands

Inserm UR582 (ex 523), Institut de Myologie, Bâtiment Babinski, G.H. Pitie-Salpetriere, 47, boulevard de l’Hopital, 75 651 Paris Cedex 13, France Laboratoire de Genetique Moleculaire et Chromosomique, IURC, Montpellier, France Institute of Cardiology, University of Bologna, Bologna, Italy Department of Paediatrics and Neonatal Medicine, Imperial College School of Medicine, Hammersmith Campus, London, UK Department of Neurology, Academic Medical Centre, Amsterdam, The Netherlands Service de Cardiologie, GH Cochin, Paris, France Randall Center, Kings College, London, UK Neuromuscular Unit, M.R.C. Polish Academy of Sciences, Warsaw, Poland ITOI, Unit of Bologna, c/o IOR, Bologna, Italy Istituto Ortopedico Rizzoli, Neuromuscular Unit, Bologna, Italy MRIC, North East Wales Institute, Wrexham, UK Department of Internal Medicine and Cardiology Medical University of Warsaw, Warsaw, Poland Department of Cardiology, University Hospital Maastricht, Maastricht, The Netherlands Dipartimento di Biopatologia e Diagnostica per Immagini, Rome, Italy Istituto di Genetica Biochimica ed Evoluzionistica, CNR (IGBE-CNR), Pavia, Italy Division of Medical Genetics, University of Leicester, Leicester, UK Institute of Human Genetics, Greifswald, Germany

Fatal Kawasaki Disease (KD) due to Early Fibrous Obliterative Coronary Artery Disease

The purpose of the study is to describe 2 fatal cases of KD due to nonthrombotic obstructive coronary artery disease. Patient 1. 4yr Caucasian male, URTI, low grade fever, eventually had 4 of 5 KD criteria. Given IVIG x 2 about day 15 with no response: persistent fevers and hemolytic anaemia 1 month, small joint arthritis & arthralgias 2nd month, abdominal pains 3rd month. Initial coronary artery (CA) dilatation progressed with the development of unstable angina and low cardiac output 4 months after presentation. Died after anaesthetic induction for cardiac catheterization. Post mortem histology showed thick walled obstructed triple vessel CA disease. Histology showed marked fibrocellular intimal proliferation. There was recent myocardial and duodenal infarction. Patient 2. 6 month Caucasian male developed fever and 4 criteria for KD but no peripheral oedema or redness. Given IVIG day 8 and day 20 due to irritability without fever. Echo day 8 and day 50 showed mild CA dilatation. He represented day 95 with congestive heart failure, cardiogenic shock, poor LV function and died. Post mortem showed minor CA aneurysmal changes only. There was marked luminal obstruction caused by replacement fibrosis of the intimal and media. There was patchy chronic inflammatory infiltration.In summary, these 2 patients showed atypical KD histology with early intimal fibrosis and luminal obstruction. Conclusions:1. KD may be fatal due to early (within 3-4 months from onset) fibrotic obliterative CA disease rather than due to giant CA aneurysms and complications.2. New treatment strategies for KD are required for late presenters or non-responders to IVIG when there is evidence of continued disease process.

Cardiac Troponin I in Feline Hypertrophic Cardiomyopathy

Measurement of plasma cardiac troponin I concentration ([cTnI]) is a sensitive and specific means for detecting myocardial damage in many mammalian species. Studies have shown that [cTnI] increases rapidly after cardiomyocyte injury. The molecular structure of cTnl is highly conserved across species, and current assays developed for its detection in humans have been validated in many species. In this study, [cTnI] was quantified using a 2‐site sandwich assay in plasma of healthy control cats (n = 33) and cats with moderate to severe hypertrophic cardiomyopathy (HCM) (n = 20). [cTnI] was significantly higher in cats with HCM (median, 0.66 ng/mL; range, 0.05–10.93 ng/mL) as compared with normal cats (median, &lt;0.03 ng/mL; range, &lt;0.03‐0.16 ng/mL) (P &lt; .0001). An increase in [cTnI] was also highly sensitive (sensitivity = 85%) and specific (specificity = 97%) for differentiating cats with moderate to severe HCM from normal cats. [cTnI] was weakly correlated with diastolic thickness of the left ventricular free wall (r2= .354; P= .009) but not with the diastolic thickness of the interventricular septum (P= .8467) or the left atrium: aorta ratio (P= .0652). Furthermore, cats with congestive heart failure at the time of cTnl analysis had a significantly higher [cTnI] than did cats that had never had heart failure and those whose heart failure was controlled at the time of analysis (P= .0095 and P= .0201, respectively). These data indicate that cats with HCM have ongoing myocardial damage. Although the origin of this damage is unknown, it most likely explains the replacement fibrosis that is consistently identified in cats with moderate to severe HCM.