Romina Verardo

Intramyocyte Detection of Epstein-Barr Virus Genome by Laser Capture Microdissection in Patients With Inflammatory Cardiomyopathy

Background—The causal role of Epstein-Barr virus (EBV) in inflammatory cardiomyopathy (IC) is still unclear, because this virus is present in latently infected circulating B lymphocytes in 90% of adults. Laser capture microdissection (LCM) has been applied on endomyocardial biopsy samples from patients with IC to assess the presence of EBV genome in separately dissected lymphocytes and myocytes. Methods and Results—Among 142 patients with cardiac dilation and dysfunction and a histological and immunohistochemical diagnosis of myocarditis, 44 had a myocardial viral infection detected by polymerase chain reaction on frozen endomyocardial biopsy samples. In 9 of them, the virus detected was EBV. LCM was performed on 5-&mgr;m-thick paraffin sections of EBV-infected hearts. Lymphocytes and myocytes were microdissected and analyzed separately by polymerase chain reaction analysis on DNA extracted from the collected cells. Blood and myocardial samples from patients with positive and negative serology for EBV were used as controls. EBV genome was detected in myocytes but not in infiltrating lymphocytes of patients, nor in myocardial samples from controls. Despite full conventional antifailure therapy, a progressive cardiac dilation and dysfunction was documented in patients with EBV-related IC at a mean of 31±14 months of follow-up. Conclusions—Intramyocyte detection of EBV can be obtained by LCM in up to 6.3% of patients with IC. This supports a cytopathic EBV role and suggests the opportunity for an antiviral/immunomodulatory therapy.

Increased oxidative stress contributes to cardiomyocyte dysfunction and death in patients with Fabry disease cardiomyopathy

Cardiac dysfunction of Fabry disease (FD) has been associated with myofilament damage and cell death as result of α-galactosidase A deficiency and globotriaosylceramide accumulation. We sought to evaluate the role of oxidative stress in FD cardiomyocyte dysfunction. Myocardial tissue from 18 patients with FD was investigated for the expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine by immunohistochemistry. Western blot analysis for nitrotyrosine was also performed. Oxidative damage to DNA was investigated by immunostaining for 8-hydroxydeoxyguanosine (8-OHdG), whereas apoptosis was evaluated by in situ ligation with hairpin probes. iNOS and nitrotyrosine expression was increased in FD hearts compared with hypertrophic cardiomyopathy and normal controls. Remarkably, immunostaining was homogeneously expressed in FD male cardiomyocytes, whereas it was only detected in the affected cardiomyocytes of FD females. Western blot analysis confirmed an increase in FD cardiomyocyte protein nitration compared with controls. 8-OHdG was expressed in 25% of cardiomyocyte nuclei from FD patients, whereas it was absent in controls. The intensity of immunostaining for iNOS/nitrotyrosine correlated with 8-OHdG expression in cardiomyocyte nuclei. Apoptosis of FD cardiomyocytes was 187-fold higher than in controls, and apoptotic nuclei were positive for 8-OHdG. Cardiac dysfunction of FD reflects increased myocardial nitric oxide production with oxidative damage of cardiomyocyte myofilaments and DNA, causing cell dysfunction and death.

Oxidative myocardial damage in human cocaine-related cardiomyopathy

The pathogenesis of cocaine‐related cardiomyopathy (CCM) is still unclear. Oxidative damage from cocaine‐generated reactive oxygen species (ROS) overcoming myocardial antioxidant reserve has been hypothesized by experimental studies.

High prevalence of intramural coronary infection in patients with drug-resistant cardiac syndrome X: comparison with chronic stable angina and normal controls

Background Coronary microvascular dysfunction has been reported along with myocardial viral infection. Whether intramural coronary vessels infection plays a role in patients with cardiac syndrome X (CSX) is unknown. Methods Thirteen consecutive patients (four men, nine women, mean age 51±10.5 years) with drug-resistant CSX underwent left ventricular endomyocardial biopsy. Myocardial tissue was examined for histology, immunohistochemistry and for the presence of cardiotropic viruses by PCR analysis. In the presence of a viral infection on the whole tissue, laser microdissection was performed to analyse the viral genome selectively in intramural vessels and cardiomyocytes. Controls were surgical cardiac biopsies from patients with chronic stable angina and from patients with mitral stenosis and normal cardiac function (normal controls). Results Histology showed hypertrophy and degeneration of cardiomyocytes with interstitial and replacement fibrosis in all CSX, while focal lymphocytic myocarditis was additionally recognised in three patients. No vasculitis was observed. Viral genomes were detected in nine of 13 CSX (Epstein–Barr virus in four, adenovirus in three, human herpes virus (HHV) 6 in one, Epstein–Barr adenovirus co-infection in one). Laser microdissection showed that Epstein–Barr and adenovirus localised both in cardiomyocytes and intramural vessels, while HHV-6 infection was confined to the vessel wall. Conclusions Viral genomes can be detected in intramural vessels of up to 69% of drug-resistant CSX. Coronary small vessels infection represents an alternative pathophysiological mechanism of this syndrome and can explain the poor response to anti-ischaemic drugs.

Myocardial and microvascular inflammation/infection in patients with HIV-associated pulmonary artery hypertension.

Background:Right ventricle compromise affects survival of patients with HIV-associated pulmonary artery hypertension (PAH). Design:Myocardial histology with viral assessment may clarify the mechanism of right ventricular deterioration and provide clues on PAH origin. Methods:Fifteen patients with HIV infection, PAH and right ventricular dysfunction underwent cardiac magnetic resonance, catheterization, coronary with ventricular angiography and biventricular endomyocardial biopsy. Endothelial expression of HLA-DR, ICAM-1, E-selectin and VCAM-1 was semi-quantitatively evaluated. PCR for HIV, hepatitis C virus, human herpes virus-6, human herpes virus-8, Epstein–Barr virus, adenovirus, cytomegalovirus, enterovirus, influenza A/B and parvovirus B19 was performed. In PCR-positive hearts, viral protein adenovirus-1 and TORDJI-22 were assessed by immunohistology. Results:New York Heart Association class was 2.4 ± 0.5, mean pulmonary artery pressure 49.93 ± 10.15 mmHg and wedge pressure 9.5 ± 2.19 mmHg. Coronaries were normal with slow flow. Left ventricular and/or right ventricular micro-aneurysms were seen in eight patients. Cardiac magnetic resonance documented increased right ventricular end-diastolic volume with reduced ejection fraction, normal left ventricular end-diastolic volume and left ventricular ejection fraction. Subepicardial/mesocardial oedema and delayed enhancement in the inter-ventricular junction and/or left ventricular inferolateral wall was detected in eight patients. Histology showed active lymphocytic myocarditis in 12 patients, with microvasculitis in three. Endothelial adhesion molecules were over-expressed in all patients. PCR was positive in four patients for hepatitis C virus and in two for adenovirus, and viruses localized both in cardiomyocytes and endothelial cells. Conclusions:Inflammation/infection of myocardium and intramural vessels is detectable in patients with HIV-associated PAH. It may adversely affect right ventricular function and have a role in the compromised pulmonary circulation.

Histological and proteomic profile of diabetic versus non-diabetic dilated cardiomyopathy

BACKGROUND Diabetic cardiomyopathy (DbCM) is indistinguishable from idiopathic dilated cardiomyopathy (IDCM) as specific histological and/or biochemical markers are unavailable. METHODS AND RESULTS Comparative histology, electron microscopy, morphometry for cell volume composition and myocardial fibrosis, reactive oxygen species (ROS), polymerase chain reaction for cardiotropic viruses, immunohistochemistry for nitrotyrosine, inducible nitric oxide synthase (iNOS), 8-hydroxydeoxyguanosine (8-OH-dG) and proteomics have been evaluated in endomyocardial biopsies from 9 patients (pts) (5 male and 4 female, mean age 61 ± 13 years) with DbCM (left ventricular end-diastolic diameter 65 ± 2.3mm; ejection fraction 27 ± 6) and type 2 diabetes mellitus and 9 pts with IDCM (mean age 60 ± 9 years) matched for sex, age and severity of left ventricular (LV) dysfunction. Controls were surgical biopsies from 9 pts with mitral stenosis and normal LV dimensions and function. No qualitative morphological changes were observed between DbCM and IDCM although mitochondrial damage and myofibrillolysis appeared more pronounced in DbCM. ROS were 5 times higher in DbCM than in IDCM and controls and were associated with higher expression of cytoplasm iNOS and nitrotyrosine and nuclear 8-OH-dG. Apoptosis was 14 times higher in DbCM than in IDCM and 41 times higher than in controls. Proteomic profile showed in DbCM a reduced expression of proteins related to beta-oxidation and detoxification pathway. CONCLUSIONS DbCM is a distinctive ROS-mediated disorder with oxidative damage of myocytes structural proteins and DNA causing cell dysfunction and death. Reduced expression of beta-oxidation proteins suggests a decline of energy production and of mitochondrial function.

Biopsy-proven autoimmune myocarditis in HIV-associated dilated cardiomyopathy

BackgroundDilated cardiomyopathy occurring in HIV-infected patients raises both diagnostic and therapeutic challenging problems. Indeed myocardial involvement in HIV infection has been variously attributed to several causes, including viral, toxic, nutritional and autoimmune, but no specific treatment capable to substantially improve patients’ prognosis has been recognized so far.Case PresentationHereby we describe the case of an autoimmune myocarditis manifesting with heart failure in a3 9-year-old man with HIV infection.Left ventricular endomyocardial biopsy showed a lymphocytic myocarditis characterized by over-expression of HLA-DR and negative polymerase chain reaction for cardiotropic viruses. Steroid treatment was followed by recovery of cardiac dimension and function.ConclusionPresence of auto-reactive myocarditis should be considered in patients with HIV-associated dilated cardiomyopathy. Its recognition by endomyocardial biopsy followed by steroid administration may result in a complete resolution of cardiac disease.

Cushing Syndrome Cardiomyopathy: Clinicopathologic Impact of Cortisol Normalization.

Endogenous Cushing syndrome (CS) is associated with systemic manifestations including abnormalities in glucose and lipid metabolism, alterations in coagulation factors, hypertension, cardiovascular disease, depression, and impaired health-related quality of life. Dilated cardiomyopathy is rarely associated with CS, with only few single cases previously reported in English literature.1 Histological changes and molecular pathways involved in CS-dilated cardiomyopathy are poorly understood, whereas structural outcome after cardiac recovery is unknown. We reported a research case of CS dilated cardiomyopathy in which noninvasive and invasive cardiac studies at baseline and follow-up were obtained after written patient consent. A 63-year-old woman had CS caused by adrenal adenoma. Echocardiographic left ventricular (LV) end-diastolic diameter, ejection fraction and maximal wall thickness, endomyocardial biopsy with assessment of cardiomyocyte diameter, % myofibrillolysis area, myocardial fibrosis and cell death, and myocardial atrogin-1 were evaluated at presentation and after 1-year cortisol normalization with adrenalectomy. At presentation, …

Myocardial expression of Toll-like receptor 4 predicts the response to immunosuppressive therapy in patients with virus-negative chronic inflammatory cardiomyopathy

We sought to determine whether myocardial expression of Toll‐like receptor 4 (TLR4) may predict the response to immunosuppression.

A-V block as presentation of cardiac amyloid: prominent infiltration of conduction tissue revealed by endomyocardial biopsy

Cardiac infiltration by amyloid generates a progressive restrictive cardiomyopathy culminating in diastolic heart failure. Conduction tissue can be affected as well by amyloidosis particularly in the advanced stage of the disease. Endomyocardial biopsy documentation of prominent infiltration of conduction tissue by cardiac amyloid causing A-V block and syncope, has never been provided before. A 68-year-old man with an untreated carpal tunnel syndrome was admitted because of recurrent syncopal episodes. The patient had normal blood pressure (130/80mmHg) and no peripheral oedema. ECG (Figure 1 panel A) documented a sinus rhythm with preserved QRS voltages and diffuse compromise of impulse conduction manifested by right bundle branch block, left anterior hemiblock, 1 and 2 degree A-V block with ventricular rate of 38 beats/min. At 2D-echo (Figure 1 panel B) a moderate diffuse thickening of left and right ventricular wall (maximal wall thickness 16 and 10mm, respectively) was recognized with left ventricular diastolic dysfunction and preserved systolic function (left ventricular ejection fraction 60%). The patient denied previous inflammations/infections and no monoclonal gammopathy was recognized at immunoelectrophoresis of serum and urines. He underwent a coronary angiography that showed a normal network and then a left ventricular endomyocardial biopsy. Histology of endomyocardial samples showed hypotrophic and degenerated cardiomyocytes surrounded by extensive areas of pale pink material at H&E (Figure 1, panel C) showing green birefringence at polarized light after Congo red staining (Figure 1, panel D) and suggesting cardiac amyloid. Electron microscopy confirmed the infiltrating material to consist of 100 Å wide amyloid fibrils (insert in Figure 1, panel D). In the tissue, samples were included sections of conduction tissue (CT) (Figure 1, panel E–F), appearing as small, loosely arranged myocytes positive to potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) immunostaining (Figure 1, panel G), supplied by a centrally placed arteriole and circumscribed by a fibrous membrane in a fascicle configuration (Monckeberg and Aschoff criteria). CT was nearly completely replaced by amyloid deposits positive to immunostaining for transthyretin (TTR) (insert in Figure 1, panel H). A genetic test revealed a mutation of TTR gene (TTRThr59Lys, p.Thr79Lys) in the affected patient and in some (a 30-year-old son and 9-yearold nephew) carrier family members still not manifesting the disease. The patient had a pacemaker implantation with functional recovery to NYHA class I. Substitution of Lys for the wild-type Thr residue at 59 position of TTR is known to be associated with autosomal dominant cardiac amyloidosis [1]. Early recognition of this entity may benefit from use of new drugs like tafamidis able to stabilize the mutant TTR, preventing the generation of amyloidogenic and toxic intermediates [2].