Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

RATIONALE: Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. OBJECTIVES: We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. METHODS AND RESULTS: We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFß1 (transforming growth factor ß1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. CONCLUSIONS: Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.

Cardiovascular progenitor cells and tissue plasticity are reduced in a myocardium affected by Becker muscular dystrophy.

We describe the association of Becker muscular dystrophy (BMD) derived heart failure with the impairment of tissue homeostasis and remodeling capabilities of the affected heart tissue. We report that BMD heart failure is associated with a significantly decreased number of cardiovascular progenitor cells, reduced cardiac fibroblast migration, and ex vivo survival. BACKGROUND: Becker muscular dystrophy belongs to a class of genetically inherited dystrophin deficiencies. It affects male patients and results in progressive skeletal muscle degeneration and dilated cardiomyopathy leading to heart failure. It is a relatively mild form of dystrophin deficiency, which allows patients to be on a heart transplant list. In this unique situation, the explanted heart is a rare opportunity to study the degenerative process of dystrophin-deficient cardiac tissue. Heart tissue was excised, dissociated, and analyzed. The fractional content of c-kit+/CD45- cardiovascular progenitor cells (CVPCs) and cardiac fibroblast migration were compared to control samples of atrial tissue. Control tissue was obtained from the hearts of healthy organ donor's during heart transplantation procedures. RESULTS: We report significantly decreased CVPCs (c-kit+/CD45-) throughout the heart tissue of a BMD patient, and reduced numbers of phase-bright cells presenting c-kit positivity in the dystrophin-deficient cultured explants. In addition, ex vivo CVPCs survival and cardiac fibroblasts migration were significantly reduced, suggesting reduced homeostatic support and irreversible tissue remodeling. CONCLUSIONS: Our findings associate genetically derived heart failure in a dystrophin-deficient patient with decreased c-kit+/CD45- CVPCs and their resilience, possibly hinting at a lack of cardioprotective capability and/or reduced homeostatic support. This also correlates with reduced plasticity of the explanted cardiac tissue, related to the process of irreversible remodeling in the BMD patient's heart.

Identification of a Diagnostic Set of Endomyocardial Biopsy microRNAs for Acute Cellular Rejection Diagnostics in Patients after Heart Transplantation Using Next-Generation Sequencing.

INTRODUCTION: Acute cellular rejection (ACR) of heart allografts represents the most common reason for graft failure. Endomyocardial biopsies (EMB) are still subject to substantial interobserver variability. Novel biomarkers enabling precise ACR diagnostics may decrease interobserver variability. We aimed to identify a specific subset of microRNAs reflecting the presence of ACR. PATIENTS AND METHODS: Monocentric retrospective study. A total of 38 patients with the anamnesis of ACR were identified and for each patient three consecutive samples of EMB (with, prior and after ACR) were collected. Sixteen trios were used for next-generation sequencing (exploratory cohort); the resting 22 trios were used for validation with qRT-PCR (validation cohort). Statistical analysis was performed using R software. RESULTS: The analysis of the exploration cohort provided the total of 11 miRNAs that were altered during ACR, the three of which (miR-144, miR-589 and miR-182) were further validated in the validation cohort. Using the levels of all 11 miRNAs and principal component analysis, an ACR score was created with the specificity of 91% and sensitivity of 68% for detecting the presence of ACR in the EMB sample. CONCLUSION: We identified a set of microRNAs altered in endomyocardial biopsies during ACR and using their relative levels we created a diagnostic score that can be used for ACR diagnosis.

Viral genome changes and the impact of viral genome persistence in myocardium of patients with inflammatory cardiomyopathy.

INTRODUCTION: Viral infections are considered the most frequent cause of myocarditis and dilated cardiomyopathy (DCM). MATERIAL AND METHODS: We investigated the changes in viral presence and the impact of viral genome persistence in the myocardium on echocardiographic parameters, functional status and some laboratory parameters in a 6-month follow-up. Fifty-four patients with recent onset DCM, left ventricular ejection fraction < 40% and biopsy-proven myocarditis (> 14 mononuclear leukocytes/mm2 and/or > 7 T-lymphocytes/mm2) were enrolled. Polymerase chain reaction (PCR) was performed to detect pathogens in the myocardium. Patients were divided according to the administered therapy: standard heart failure medication (46 patients) and immunosuppressive therapy (8 patients). RESULTS: In the standard heart failure medication group viral clearance was observed in 13 patients and viral persistence in 24 patients in the follow-up period. Comparing both groups, there was no statistically significant difference - LVEF improvement of 12.0 ±11.4% vs. 18.3 ±12.6%, decrease in NYHA class of 0.7 ±0.7 vs. 1.0 ±0.7, decline in NT-proBNP of 1335 ±1933 ng/l vs. 1942 ±3242 ng/l and decrease in infiltrating leukocytes of 11.1 ±15.8 vs. 6.7 ±23.0 cells/mm2 and T-lymphocytes of 5.8 ±15.1 vs. 1.8 ±10.9 cells/mm2 (all p = NS). A decrease in PCR positive patients from 37 to 29 was observed. The number of PVB19 positive PCR findings decreased from 5 to 4 in patients with immunosuppressive therapy. CONCLUSIONS: A decrease in the number of positive PCR findings in control endomyocardial biopsy was observed. Viral genome persistence was not associated with worse outcome in short-term follow-up.

[Heart transplantation and follow-up treatment with AL-amyloidosis in 5 patients]. / Transplantace srdce a následující lécba AL-amyloidózy u 5 pacientu.

The prognosis for patients with cardiac impairment due to AL-amyloid deposition and severe cardiac insufficiency is poor, with a survival median in the order of months. The classical treatment of AL-amyloidosis in combination with cardiac insufficiency is very poorly tolerated and the treatment of such patients is associated with considerably higher mortality than among other patients with AL-amyloidosis. If, however, patients with an isolated or another dominating cardiac impairment, without severe damage to other organs and tissues, have a heart transplant performed, their cardiovascular condition will significantly improve as a result, along with their ability to tolerate any kind of treatment for AL-amyloidosis including that using high-dose chemotherapy with a transplant of autologous hematopoietic stem cells. The achievement of complete remission of AL-amyloidosis is a precondition for long-term survival, since when not achieved, amyloid deposition also arises in the transplanted heart. At the Centre for Cardiovascular and Transplantation Surgery, Brno, the first heart transplant due to its impairment by AL-amyloidosis was performed in 2010. By the year 2017 the number of patients with AL-amyloidosis, who had first undergone a heart transplant with subsequent treatment for AL-amyloidosis, increased to 5. The median age at which a heart transplant was performed is 60 (48-65) years. Four patients were men, one was a woman. The median monitoring equals 65 (88-15) months. Complete remission of AL-amyloidosis was achieved in all the patients. There were 5 lines of treatment needed for the first patient to attain it, of that twice high-dose melphalan with autologous stem cell transplantation, for the second patient a second-line treatment, high-dose melphalan and bortezomib-based therapy. No specific therapy was needed for the third patient, as immunosuppressive therapy following the heart transplant containing prednison led to complete remission of AL-amyloidosis. In the fourth case, sustainable complete remission was reached by high-dose melphalan and in the fifth case by one line of bortezomib-based therapy. The aforementioned data illustrate that a heart transplant is the first step which makes the patients with a severe heart failure, not tolerating any efficient therapy of AL-amyloidosis, capable of undergoing intense treatment of AL-amyloidosis. Sometimes one high-dose chemotherapy is sufficient, while at other times multiple treatment lines are needed to reach complete remission of AL-amyloidosis.Key words: AL-amyloidosis - autologous hematopoietic stem cells transplantation - bortezomib - cardiomyopathy - lenalidomide - thalidomide - heart transplantation.

First report of Sneathia sanguinegens together with Mycoplasma hominis in postpartum prosthetic valve infective endocarditis: a case report.

BACKGROUND: The presence of more than one bacterial agent is relatively rare in infective endocarditis, although more common in prosthetic cases. Molecular diagnosis from a removed heart tissue is considered a quick and effective way to diagnose fastidious or intracellular agents. CASE PRESENTATION: Here we describe the case of postpartum polymicrobial prosthetic valve endocarditis in a young woman. Sneathia sanguinegens and Mycoplasma hominis were simultaneously detected from the heart valve sample using broad range 16S rRNA polymerase chain reaction (PCR) followed by sequencing while culture remained negative. Results were confirmed by independent PCR combined with denaturing gradient gel electrophoresis. Before the final agent identification, the highly non-compliant patient left from the hospital against medical advice on empirical intravenous treatment with aminopenicillins, clavulanate and gentamicin switched to oral amoxycillin and clavulanate. Four months after surgery, no signs of inflammation were present despite new regurgitation and valve leaflet flail was detected. However, after another 5 months the patient died from sepsis and recurrent infective endocarditis of unclarified etiology. CONCLUSIONS: Mycoplasma hominis is a rare causative agent of infective endocarditis. To the best of our knowledge, presented case is the first report of Sneathia sanguinegens detected in this condition. Molecular techniques were shown to be useful even in polymicrobial infective endocarditis samples.

The First Manifestation of a Left Atrial Myxoma as a Consequence of Multiple Left Coronary Artery Embolisms.

A case of multiple embolisms in the left coronary artery as a rare first manifestation of left atrial myxoma is reported. A patient with embolic myocardial infarction and congestive heart failure was treated by percutaneous aspirations and balloon dilatations. Transesophageal echocardiography disclosed a villous myxoma with high embolic potential. Surgical resection of the tumour, suturing of a patent foramen ovale suture and an annuloplasty of the dilated tricuspid annulus was performed the third day after the admission. Recovery of the documented left ventricular systolic function can be explained by resorption of myxomatous material. The patient was discharged ten days after the surgery.

Improvement of left ventricular systolic function in inflammatory cardiomyopathy: What plays a role?

AIMS: To compare the differences between patients with inflammatory cardiomyopathy (ICM) with and without improvement in left ventricular (LV) systolic function and to identify the relevant predictors of LV improvement. PATIENTS AND METHODS: The study included 63 patients with biopsy-proven ICM and heart failure symptoms of at least NYHA II, symptom duration ≤ 6 months, LV ejection fraction (LVEF) ≤ 40% assessed by echocardiography and presence of >14 mononuclear leukocytes (LCA+ cells)/mm2 in biopsy samples. Patients were evaluated at baseline and after 6 months. RESULTS: In the group with LVEF improvement of ≥ 10% (I+ group, n = 41), LVEF increased from 24 ± 7% to 47 ± 8% (P < 0.001). In 22 patients (group I-), there was no or minimal LVEF increase (< 10%). In the I+ group, there were more LCA+ cells/mm2 at baseline (25.1 ± 16.5 vs. 18.5 ± 4.4 cells/mm2; P = 0.032) and a more significant decrease in LCA+ cells in the follow-up (reduction of 13.6 ± 14.3 cells/mm2 vs. 5.0 ± 7.7 cells/mm2 in the I- group; P = 0.009). The univariate logistic regression showed a possible association of number of LCA+ cells, LV end-diastolic diameter and N-terminal fragment of pro-brain natriuretic peptide (NTproBNP) value with LVEF improvement. In the multivariate analysis, only NTproBNP at diagnosis was confirmed as an independent predictor of LVEF improvement (OR=1.2; 1.003 to 1.394; P = 0.046). CONCLUSION: The LV systolic function improvement was observed in 65% of the patients. In these patients, the number of inflammatory cells at baseline was higher and decreased more but the higher baseline NTproBNP value was the only independent predictor of LVEF improvement.

Dirofilaria repens: emergence of autochthonous human infections in the Czech Republic (case reports).

BACKGROUND: Human dirofilariasis is a zoonotic infection that continues to spread to previously unaffected areas of Europe. In the South Moravian Region of the Czech Republic (CR), imported as well as autochthonous canine infections were recorded in the last decade, and parasite DNA was detected in mosquitoes of Aedes vexans. In the present paper, human Dirofilaria infections are reported from the country for the first time. CASE PRESENTATION: The samples from five patients with suspected tissue helminthiases were investigated. In particular cases, nematodes were isolated from various tissues including skin of lower leg, soft tissues of finger, subcutaneous tissue of hypogastrium, lymph node and peritoneum. The diagnosis was based on light microscopic morphology and/or DNA analysis of the worms. In addition, ELISA examination of patients' sera for anti-filaria IgG antibodies was performed. CONCLUSIONS: In the CR, five cases of human dirofilariasis caused by Dirofilaria repens were recorded during 2010-2014 (species determination for three of them was confirmed besides morphological also by DNA analysis). At least, three of the cases were of autochthonous origin (the patients are Czech citizens residing in South Moravian Region who have never travelled abroad). The findings confirm the natural setting of D. repens in South Moravian Region of the CR. Dirofilariasis should be therefore considered as endemic in this area where it may represent a significant risk factor for public health.

Correlations of the changes in bioptic findings with echocardiographic, clinical and laboratory parameters in patients with inflammatory cardiomyopathy.

Patients with myocarditis and left ventricular (LV) dysfunction may improve after standard heart failure therapy. This improvement seems to be related to retreat of myocardial inflammation. The aim of the present study was to assess changes in clinical, echocardiographic and some laboratory parameters and to correlate them with changes in the number of inflammatory infiltrating cells in endomyocardial biopsy (EMB) samples during the 6-month follow-up, and to define predictors of LV function improvement among baseline parameters. Forty patients with biopsy-proven myocarditis and impaired LV function (LV ejection fraction-LVEF <40 %) with heart failure symptoms ≤ 6 months were evaluated. Myocarditis was defined as the presence of >14 mononuclear leukocytes/mm(2) and/or >7 T-lymphocytes/mm(2) in the baseline EMB. The EMB, echocardiography and clinical evaluation were repeated after 6 months of standard heart failure therapy. LVEF improved on average from 25 ± 9 to 42 ± 12 % (p < 0.001); LV end-systolic volume and LV end-diastolic volume (LVEDV) decreased from 158 ± 61 to 111 ± 58 ml and from 211 ± 69 to 178 ± 63 ml (both p < 0.001). NYHA class decreased from 2.6 ± 0.5 to 1.6 ± 0.6 (p < 0.001) and NTproBNP from 2892 ± 3227 to 851 ± 1835 µg/ml (p < 0.001). A decrease in the number of infiltrating leukocytes (CD45+/LCA+) from 23 ± 15 to 13 ± 8 cells/mm(2) and in the number of infiltrating T lymphocytes (CD3+) from 7 ± 5 to 4 ± 3 cells/mm(2) (both p < 0.001) was observed. The decline in the number of infiltrating CD45+ cells significantly correlated with the change in LVEF (R = -0.43; p = 0.006), LVEDV (R = 0.39; p = 0.012), NYHA classification (R = 0.35; p = 0.025), and NTproBNP (R = 0.33; p = 0.045). The decrease in the number of CD3+ cells correlated with the change of systolic and diastolic diameters of the left ventricle (R = -0.33; p = 0.038 and R = -0.45; p = 0.003) and with the change in LVEDV (R = -0.43; p = 0.006). Tricuspid annular plane systolic excursion (TAPSE) (OR 0.61; p = 0.005) and early transmitral diastolic flow velocity (E wave) (OR 0.89; p = 0.002) were identified as predictors of LVEF improvement. Improvements in clinical status, LV function and NTproBNP levels correlated with decrease in the number of infiltrating inflammatory cells. TAPSE and E wave velocity were significant predictors of improvement in multivariate regression. Our observations suggest that contemporary guidelines-based therapy of heart failure is an effective treatment option in patients with recent onset biopsy-proven inflammatory cardiomyopathy.

[Recurrence of primary diseases after liver transplantation]. / Rekurence primárních chorob po transplantaci jater.

A majority of primary diseases for which orthotopic liver transplantation is carried out may recur in the liver allograft, mostly in adults. As the indication criteria, transplantation surgery and post-transplantation care improve, the patients survival lengthens as well, leading to concurrently increasing incidence as well as an increase in the relevance of recurrent diseases, which are the most significant cause of late liver graft dysfunction. The frequency, clinical consequences and therapeutic options of different disease recurrence vary considerably. Even recently the worst prognosis has been associated with hepatitis C for 100% reinfection, this situation is beginning to change with new oral antiviral drugs, as has already been successfully done with hepatitis B. Among immune-mediated disorders, primary biliary cirrhosis recurrence affects 30 - 50% of transplant patients, albeit with mild consequences. Graft loss and subsequent necessity of retransplantation are observed in almost 10% of patients with primary sclerosing cholangitis recurrence. 30% prevalence rates for autoimmune hepatitis recurrence are reported but the frequency of graft loss has declined considerably due to maintenance of corticosteroid therapy. Excessive relapse of alcohol consumption in patients with liver transplant for alcoholic liver disease leads most commonly to extrahepatic complications. Recurrent non-alcoholic steatohepatitis is rarely connected with graft loss in 5 - 10 years after transplantation. The diagnosis of a recurrent disease following liver transplantation is to a large extent based on histopathological features. In the differential diagnosis, other causes of graft dysfunction must be excluded.

Differential expression of anterior gradient protein 3 in intrahepatic cholangiocarcinoma and hepatocellular carcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer next to hepatocellular carcinoma (HCC). Despite the significant difference of the therapeutic strategy for both diseases, their histological appearance may be very similar. Thus the correct diagnosis is crucial for treatment choice but is often difficult to achieve. The aim of our study was to evaluate anterior gradient 3 (AGR3) as a new diagnostic marker helping to distinguish between ICC and HCC. AGR3 is a putative transmembrane protein implicated in breast, prostate and ovary tumorigenesis and belongs to the family of protein disulfide isomerases. Since there is little information on how AGR3 is expressed in normal and diseased tissues and what its exact function is, we analyzed its expression pattern in normal liver and tumor tissue of ICC and HCC. The immunohistochemical analysis in normal tissue revealed specific AGR3 expression in intrahepatic bile duct cholangiocytes which was not present in liver hepatocytes. Consequently we analyzed AGR3 expression in 74 representative samples of puncture biopsies, tissue excisions and resection specimens from which 48 samples were diagnosed as HCC and 26 as ICC. Our results showed AGR3 expression negative and weakly positive respectively in hepatocellular carcinomas compared to stronger AGR3 positivity in cholangiocellular carcinomas. AGR3 expression statistically significantly correlated to acid mucopolysaccharide expression and negatively correlated to glypican-3 expression. We conclude that according to receiver operating characteristics (ROC) analysis AGR3 expression is relatively specific for ICC and is potentially linked to mucosecretion, which may indicate potential implication in treatment resistance.

The variable clinical course of peripartum cardiomyopathy.

BACKGROUND: In Europe, peripartum cardiomyopathy (PPCM) is a rare disorder, often difficult to diagnose and it has a variable clinical course. The aim of this report was to describe and discuss the individual variability of this disorder and its management. PATIENTS AND METHODS: Three cases of PPCM manifesting as severe heart failure are compared. Common was the presence of myocardial inflammation detected by endomyocardial biopsy. Different were treatment methods and clinical course. Modern therapeutic concepts such as immunosuppressive therapy and bromocriptin administration are discussed, as well as non-pharmacological approaches. CONCLUSION: In the differential diagnostics of dyspnea associated with pregnancy and childbirth, PPCM should be considered. The potentially severe course of the disease requires hospitalization with the possibility of comprehensive heart failure treatment, including non-pharmacological approaches such as device therapy and heart transplantation.

[Pathology of urogenital schistosomiasis]. / Patologie urogenitální schistosomózy.

The authors submit the case history of their patient presenting with chronic cystitis, consequence of an infestation with the fluke Schistosoma haematobium. They also present the most frequent morphological forms of urogenital schistosomiasis, its complications and the possibilities of pathological differential diagnosis, based on literary data (including information from electronic data bases) on the pathomorphology and, more particularly, the histopathology of urogenital schistosomiasis. Among the general histopathological changes we see lesions that correspond to an active chronic infection with a chiefly granulomatous reaction. Calcification, pseudotumorous polypoid formations, ulcerations, obliterating fibrous lesions, epithelial transformations such as hyperplasia, metaplasia and dysplasia follow later. These changes are followed at the various sites by diverse clinical and morphological complications-posthaemorrhagic anaemia, adhesions, serious obstructions of urogenital openings and its consequences and, finally, an increased risk of pavement-cell carcinoma of the bladder. For all the above-mentioned reasons we should include the possibility of urogenital schistosomiasis into the large group of clinical and pathological aspects of differential diagnosis, when considering the etiology and treatment of inflammatory and tumorous lesions of the urogenital tract.