Titin in muscular dystrophy and cardiomyopathy: Urinary titin as a novel marker.

Titin, encoded by the gene TTN, is the largest human protein, and plays central roles in sarcomeric structures and functions in skeletal and cardiac muscles. Mutations of TTN are causally related to specific types of muscular dystrophies and cardiomyopathies. A developed methodology of next generation sequencing has recently led to the identification of novel TTN mutations in such diseases. The clinical significance of titin is now emerging as a target for genetic strategies. Titin-related muscular dystrophies include tibial muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, hereditary myopathy with early respiratory failure, central core myopathy, centronuclear myopathies, and Salih myopathy. Truncation mutations of TTN have been identified as the most frequent genetic cause of dilated cardiomyopathy. In this review article, we highlight the role of titin and impact of TTN mutations in the pathogenesis of muscular dystrophies and cardiomyopathies. Recently, a novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of the urinary titin N-terminal fragments (U-TN) has been established. We discuss the clinical significance of U-TN in the diagnosis of muscular dystrophies and differential diagnosis of cardiomyopathies, as well as risk stratification in dilated cardiomyopathy.

Urinary 8-Hydroxy-2'-Deoxyguanosine as a Myocardial Oxidative Stress Marker Is Associated With Ventricular Tachycardia in Patients With Active Cardiac Sarcoidosis.

BACKGROUND: Recently, we reported that urinary 8-hydroxy-2'-deoxyguanosine (U-8-OHdG), an oxidative stress marker, reflected inflammatory activity in cardiac sarcoidosis (CS). Here, we investigated whether U-8-OHdG levels were associated with ventricular tachycardia (VT) in patients with CS. METHODS AND RESULTS: This prospective cohort study enrolled 62 consecutive patients with CS, of whom 36 were diagnosed as having active CS based on abnormal 18F-flurodeoxyglucose accumulation in the heart on positron-emission tomography/computed tomography. The 36 patients with active CS were subdivided as having CS with sustained VT (CS-VT group; n=18) or CS without sustained VT (CS-nVT group; n=18). Twenty-seven patients diagnosed with idiopathic dilated cardiomyopathy served as heart failure controls. U-8-OHdG, brain natriuretic peptide, cardiac function indices, and immunohistological data from subendomyocardial biopsy samples were compared across groups. Immunohistochemical examination of ventricle biopsy samples revealed that the anti-8-OHdG antibody-positive area of cardiac tissue was significantly greater in CS-VT than in CS-nVT or dilated cardiomyopathy and significantly correlated with U-8-OHdG levels (n=58; R=0.61; P<0.00001), which were significantly higher in CS-VT than in CS-nVT (24.6±7.1 versus 15.2±3.8 ng/mg·Cr; P<0.0001). Other baseline characteristics did not differ between the groups. Multivariate analysis indicated that U-8-OHdG was an independent determinant factor for VT. Receiver operating characteristic curve analysis to identify patients with VT resulted in a U-8-OHdG cutoff value of 17.5 ng/mg·Cr (sensitivity, 89%; specificity, 83%; area under the curve, 0.90). CONCLUSIONS: U-8-OHdG levels are associated with VT in patients with active CS diagnosed by 18F-flurodeoxyglucose positron-emission tomography, providing additive and relevant information about the arrhythmia substrate.

Urinary Proteomics in Predicting Heart Transplantation Outcomes (uPROPHET)-Rationale and database description.

OBJECTIVES: Urinary Proteomics in Predicting Heart Transplantation Outcomes (uPROPHET; NCT03152422) aims: (i) to construct new multidimensional urinary proteomic (UP) classifiers that after heart transplantation (HTx) help in detecting graft vasculopathy, monitoring immune system activity and graft performance, and in adjusting immunosuppression; (ii) to sequence UP peptide fragments and to identify key proteins mediating HTx-related complications; (iii) to validate UP classifiers by demonstrating analogy between UP profiles and tissue proteomic signatures (TP) in diseased explanted hearts, to be compared with normal donor hearts; (iv) and to identify new drug targets. This article describes the uPROPHET database construction, follow-up strategies and baseline characteristics of the HTx patients. METHODS: HTx patients enrolled at the University Hospital Gasthuisberg (Leuven) collected mid-morning urine samples. Cardiac biopsies were obtained at HTx. UP and TP methods and the statistical work flow in pursuit of the research objectives are described in detail in the Data supplement. RESULTS: Of 352 participants in the UP study (24.4% women), 38.9%, 40.3%, 5.7% and 15.1% had ischemic, dilated, hypertrophic or other cardiomyopathy. The median interval between HTx and first UP assessment (baseline) was 7.8 years. At baseline, mean values were 56.5 years for age, 25.2 kg/m2 for body mass index, 142.3/84.8 mm Hg and 124.2/79.8 mm Hg for office and 24-h ambulatory systolic/diastolic pressure, and 58.6 mL/min/1.73 m2 for the estimated glomerular filtration rate. Of all patients, 37.2% and 6.5% had a history of mild (grade = 1B) or severe (grade ≥ 2) cellular rejection. Anti-body mediated rejection had occurred in 6.2% patients. The number of follow-up urine samples available for future analyses totals over 950. The TP study currently includes biopsies from 7 healthy donors and 15, 14, and 3 patients with ischemic, dilated, and hypertrophic cardiomyopathy. CONCLUSIONS: uPROPHET constitutes a solid resources for UP and TP research in the field of HTx and has the ambition to lay the foundation for the clinical application of UP in risk stratification in HTx patients.

An oxidative stress biomarker, urinary 8-hydroxy-2'-deoxyguanosine, predicts cardiovascular-related death after steroid therapy for patients with active cardiac sarcoidosis.

BACKGROUND: We investigated whether urinary 8-hydroxy-2'-deoxyguanosine (U-8-OHdG), a marker of oxidative DNA damage, is a prognosticator of cardiovascular-related death in patients with cardiac sarcoidosis (CS). METHODS AND RESULTS: In this prospective study, 30 consecutive patients were divided into the active CS (n=20) and non-active CS (n=10) groups, based on abnormal isotope accumulation in the heart on (18)F-fluorodeoxyglucose positron-emission tomography/computed tomography ((18)F-FDG PET/CT) imaging. Nineteen patients in the active CS group underwent corticosteroid therapy. Before corticosteroid therapy initiation, U-8-OHdG, brain natriuretic peptide (BNP), other biomarkers, and indices of cardiac function were measured. Patients were followed-up for a median of 48months. The primary endpoint was the incidence of cardiovascular-related death. During the follow-up period, in the corticosteroid-treated active CS group, 7 of 19 patients experienced cardiovascular-related death. By contrast, in the non-active CS group, 1 of 10 patients died from cardiovascular-related causes. Univariate and multivariate analyses showed that U-8-OHdG and BNP were independent predictors for cardiovascular-related death. The cut-off values for predicting cardiovascular death in corticosteroid-treated patients with active CS were 19.1ng/mg·Cr and 209pg/mL for U-8-OHdG and BNP, respectively. Patients with a U-8-OHdG concentration ≥19.1ng/mg·Cr or a BNP concentration ≥209pg/mL had a significantly higher cardiovascular-related death risk, but U-8-OHdG had better predictive value compared with BNP. CONCLUSION: These findings suggested that U-8-OHdG was a powerful predictor of cardiovascular-related death in patients with CS, suggesting that active CS patients with elevated U-8-OHdG levels might be resistant to corticosteroid therapy.

Relationship between Urinary Level of Phytate and Valvular Calcification in an Elderly Population: A Cross-Sectional Study.

Pathological calcification generally consists of the formation of solid deposits of hydroxyapatite (calcium phosphate) in soft tissues. Supersaturation is the thermodynamic driving force for crystallization, so it is believed that higher blood levels of calcium and phosphate increase the risk of cardiovascular calcification. However several factors can promote or inhibit the natural process of pathological calcification. This cross-sectional study evaluated the relationship between physiological levels of urinary phytate and heart valve calcification in a population of elderly out subjects. A population of 188 elderly subjects (mean age: 68 years) was studied. Valve calcification was measured by echocardiography. Phytate determination was performed from a urine sample and data on blood chemistry, end-systolic volume, concomitant diseases, cardiovascular risk factors, medication usage and food were obtained. The study population was classified in three tertiles according to level of urinary phytate: low (<0.610 µM), intermediate (0.61-1.21 µM), and high (>1.21 µM). Subjects with higher levels of urinary phytate had less mitral annulus calcification and were less likely to have diabetes and hypercholesterolemia. In the multivariate analysis, age, serum phosphorous, leukocytes total count and urinary phytate excretion appeared as independent factors predictive of presence of mitral annulus calcification. There was an inverse correlation between urinary phytate content and mitral annulus calcification in our population of elderly out subjects. These results suggest that consumption of phytate-rich foods may help to prevent cardiovascular calcification evolution.

Urinary 8-hydroxy-2'-deoxyguanosine as a novel biomarker of inflammatory activity in patients with cardiac sarcoidosis.

BACKGROUND: Inflammation and oxidative stress play a crucial role in the pathogenesis of cardiac sarcoidosis (SAR). We investigated whether urinary (U) 8-hydroxy-2'-deoxyguanosine (8-OHdG)--an oxidative DNA damage marker--was related to SAR inflammatory activity. METHODS: U-8-OHdG levels were measured in 31 SAR patients, classified as active (n=17) or non-active (n=14) based on (18)F-fluorodeoxyglucose positron emission tomography-computed tomography ((18)F-FDG-PET/CT), 28 dilated cardiomyopathy (DCM) patients, and 30 controls. In active SAR patients, U-8-OHdG levels were reexamined and compared with (18)F-FDG-PET/CT results at 6 months after corticosteroid treatment to assess therapeutic response. RESULTS: Immunohistochemical examination of left ventricle (LV) autopsy samples from SAR patients revealed positive 8-OHdG staining in cardiomyocyte nuclei from LV sections showing (18)F-FDG accumulation on PET/CT, while serum 8-OHdG levels were significantly higher in the coronary sinus than in the aortic root only in active SAR patients. U-8-OHdG levels in SAR patients were higher than those in controls, and significantly higher in active SAR patients than in non-active SAR and DCM patients. U-8-OHdG was a powerful predictor of active SAR in receiver operating characteristic curve analysis (AUC, 0.98; 95% CI, 0.94-1.02; optimal cutoff value, 13.1 ng/mg creatinine), with a sensitivity of 88.2% and a specificity of 92.9%. U-8-OHdG levels in responders significantly decreased at 6 months after corticosteroid treatment initiation, in proportion with the decrease in the focal cardiac uptake of (18)F-FDG. CONCLUSIONS: U-8-OHdG is a potentially clinically useful biomarker for evaluating inflammatory activity and monitoring the effectiveness of corticosteroid therapy in SAR patients.

Biochemical and genetic analysis of 3-methylglutaconic aciduria type IV: a diagnostic strategy.

The heterogeneous group of 3-methylglutaconic aciduria type IV consists of patients with various organ involvement and mostly progressive neurological impairment in combination with 3-methylglutaconic aciduria and biochemical features of dysfunctional oxidative phosphorylation. Here we describe the clinical and biochemical phenotype in 18 children and define 4 clinical subgroups (encephalomyopathic, hepatocerebral, cardiomyopathic, myopathic). In the encephalomyopathic group with neurodegenerative symptoms and respiratory chain complex I deficiency, two of the children, presenting with mild Methylmalonic aciduria, Leigh-like encephalomyopathy, dystonia and deafness, harboured SUCLA2 mutations. In children with a hepatocerebral phenotype most patients presented with complex I deficiency and mtDNA-depletion, three of which carried POLG1-mutations. In the cardiomyopathic subgroup most patients had complex V deficiency and an overlapping phenotype with that previously described in isolated complex V deficiency, in three patients a TMEM70 mutation was confirmed. In one male with a pure myopathic form and severe combined respiratory chain disorder, based on the pathogenomic histology of central core disease, RYR1 mutations were detected. In our patient group the presence of the biochemical marker 3-methylglutaconic acid was indicative for nuclear coded respiratory chain disorders. By delineating patient-groups we elucidated the genetic defect in 10 out of 18 children. Depending on the clinical and biochemical phenotype we suggest POLG1, SUCLA2, TMEM70 and RYR1 sequence analysis and mtDNA-depletion studies in children with 3-methylglutaconic aciduria type IV.

Duchenne's cardiomyopathy: two case reports.

We describe two cases of Duchenne's cardiomyopathy with severe cardiac dysfunction, sporadic episodes of myoglobinuria induced by effort and increased levels of serum creatine kinase. Very mild signs of skeletal myopathy were clinically evident. Left ventriculography showed diffuse severe hypokinesia. Skeletal muscle biopsy demonstrated a dystrophic process. The patients had no familial background of the disease. These 2 patients might have a sporadic inheritance pattern with severe cardiac involvement.

Biochemical markers of cardiovascular damage from tobacco smoke.

There is growing evidence that several biochemical constituents of cigarette smoking play a significant role in the development and progression of heart and blood vessel damage, especially atherosclerotic lesions. Some biochemical markers of tobacco smoke may be determined in blood and urine samples. They are also the main responsible factors of cardiovascular harm. Nicotine and its major metabolite, cotinine, carbon monoxide, and thiocyanate seem to be specific markers. Ischaemic heart disease, cardiac arrhythmias, and endothelial dysfunction are the most common evidence of both active and passive smoking exposure. Dosage of cotinine in urine is of easier determination than that of other metabolites in assessing exposure to smoking, although carboxyhaemoglobin levels seem to be a qualitative, but not quantitative factor to estimate either the degree of cardiovascular damage or the level of exposure. Cigarette smoking is addictive because of nicotine, and it is nicotine withdrawal that causes many side effects of quitting smoking as well as the nicotine itself may exacerbate cardiac lesions. Also haematologic changes are a consequence of cigarette smoking exposure. Increased white blood cells, platelet aggregation and adhesiveness, fibrinogen level, and changes in serum lipids characterise the response to smoking. Anatomical and ultrastructural alterations of the heart and blood vessels are also described as a consequence of negative effects of biochemical markers of cigarette smoking. These alterations are known as "Smoke cardiomyopathy" in experimental pathology.

Human amyloidogenic light chains directly impair cardiomyocyte function through an increase in cellular oxidant stress.

Primary amyloidosis is a systemic disorder characterized by the clonal production and tissue deposition of immunoglobulin light chain (LC) proteins. Congestive heart failure remains the greatest cause of death in primary amyloidosis, due to the development of a rapidly progressive amyloid cardiomyopathy. Amyloid cardiomyopathy is largely unresponsive to current heart failure therapies, and is associated with a median survival of less than 6 months and a 5-year survival of less than 10%. The mechanisms underlying this disorder, however, remain unknown. In this report, we demonstrate that physiological levels of human amyloid LC proteins, isolated from patients with amyloid cardiomyopathy (cardiac-LC), specifically alter cellular redox state in isolated cardiomyocytes, marked by an increase in intracellular reactive oxygen species and upregulation of the redox-sensitive protein, heme oxygenase-1. In contrast, vehicle or control LC proteins isolated from patients without cardiac involvement did not alter cardiomyocyte redox status. Oxidant stress imposed by cardiac-LC proteins further resulted in direct impairment of cardiomyocyte contractility and relaxation, associated with alterations in intracellular calcium handling. Cardiomyocyte dysfunction induced by cardiac-LC proteins was independent of neurohormonal stimulants, vascular factors, or extracellular fibril deposition, and was prevented through treatment with a superoxide dismutase/catalase mimetic. This study suggests that cardiac dysfunction in amyloid cardiomyopathy is directly mediated by LC protein-induced cardiomyocyte oxidant stress and alterations in cellular redox status, independent of fibril deposition. Antioxidant therapies or treatment strategies aimed at eliminating circulating LC proteins may therefore be beneficial in the treatment of this fatal disease.

D-2-Hydroxyglutaric aciduria in a patient with a severe clinical phenotype and unusual MRI findings.

We report an infant with intermittent urinary excretion of D-2-hydroxyglutaric (D-2-OHG) acid who died at the age of 10 months from cardiogenic shock due to cardiomyopathy. High urinary concentrations of D-2-OHG and succinic acid, as well as increased levels of lactic acid were detected on three different occasions, whereas a normal urinary profile of organic acids was found on one occasion. The clinical findings of our patient consisted of generalized hypotonia, irritability, developmental delay, generalized tonic seizures, lethargy, cardiomyopathy, and respiratory distress. Cerebral MRI revealed bilateral lesions in the substantia nigra, the periaqueductal area, the medial part of the thalamus, the hypothalamus, the caudate nucleus, putamen and globus pallidus. This pattern is suggestive of a mitochondriopathy. However, respiratory chain enzyme activities were normal in fibroblasts. Exogenous supplementation of D-2-OHG acid strongly inhibited cytochrome-c oxidase activity in fibroblasts from the patient and from normal controls in vitro. The results suggest that our patient has an unusual form of D-2-hydroxyglutaric aciduria (D-2-OHGA), different from the patients published so far, and that the increase of lactic acid and some citric acid cycle intermediates encountered in some patients with D-2-OHGA may be due to a functional defect of the respiratory chain caused by D-2-OHG acid.

A new case of malonyl coenzyme A decarboxylase deficiency presenting with cardiomyopathy.

UNLABELLED: A new case of mitochondrial malonyl coenzyme A decarboxylase deficiency is described. The patient presented with an initial episode of metabolic acidosis, seizures, hypoglycemia, and cardiac failure at 2 months of age which slowly resolved. Subsequent evaluations at 4 years of age for developmental delay revealed a prominent elevation of malonic acid in urine. Malonyl carnitine was also elevated. The activity of Malonyl CoA decarboxylase in cultured fibroblasts was 7% of normal. CONCLUSION: Malonyl CoA decarboxylase deficiency may result in inhibition of fatty acid oxidation, which may account for the cardiomyopathy.

Increased concentrations of transferrin in the urine and serum of cattle with cardiomyopathy.

Bovine cardiomyopathy affects adult cattle of the Simmental/red Holstein and Holstein-Friesian breeds and is characterised clinically by signs of congestive heart failure. Animals with cardiomyopathy suffered a marked renal loss of transferrin (Tf). The urinary concentration of Tf discriminated very well between healthy and affected cattle, 93 per cent of the affected and 97 per cent of the healthy cows being identified correctly. In spite of the severe liver congestion and renal loss of Tf, cattle with cardiomyopathy had considerably higher concentrations of serum Tf than healthy cattle. This increase may help to compensate for the low serum concentration of iron in the affected animals. The pronounced changes in the concentrations of iron and transferrin in the serum had little effect on the haemogram of the diseased animals.

Cardiac amyloidosis in the presence of Bence-Jones proteinuria and normal serum immunoglobulins.

We describe a case of cardiac amyloidosis, which is an uncommon cause of heart failure. This case is unusual, as the patient presented with symptoms of angina in the presence of normal coronary arteries and subsequently developed heart failure. Amyloidosis was secondary to myeloma with Bence-Jones proteinuria alone, which is rare.

Urinary protein analysis in cardiomyopathy-affected and healthy cattle by SDS-polyacrylamide gel electrophoresis.

Urinary proteins of cardiomyopathy (CMP)-affected (n = 31) and of healthy cattle (n = 35) were analyzed by a combination of SDS-polyacrylamide gel electrophoresis and silver stain. The following results were obtained: 1) Out of the various urinary proteins found in CMP-affected and healthy cattle, IgG, Tf, Alb, alpha 1-acid GP, Apo A1, IgG heavy and light chain could be identified. 2) In CMP-affected cattle, all the proteins mentioned above were detected in increased amounts in the urine. The quantities of Alb, Tf, and IgG were especially large. 3) In the case of CMP, the permeability of the glomerular filter was highly increased for large serum proteins. The tubular protein reabsorption of small proteins was less altered by the disease. 4) Although the electrophoretic pattern of the urinary proteins was abnormal in all the examined CMP-affected animals, the urinary protein concentrations were increased (> 230 mg/l) in only 42% of the cases. Consequently, the urinary protein concentration is not a reliable parameter for detecting renal diseases. 5) For both CMP-affected and healthy cattle the urinary protein pattern depended neither on the urine sampling technique (catheterization, spontaneous urine) nor on the reproductive stage.

Catecholamine heart muscle disease in pheochromocytoma.

Many clinical symptoms and signs in patients with pheochromocytoma are evoked by the influence of catecholamines on the heart muscle and on the coronary circulation. In our work besides clinical features the ECG pictures of the patients were studied where the elevation of the ST segment with a negative coronary T wave but without a Q wave were observed. In the echocardiographic picture our patients showed a rather hyperkinetic heart action in the florid phase of the illness. For several years after surgery a nonhomogenous structure of the interventricular septum persisted. Hypertrophy of the septum was a less frequent finding. In patients with pheochromocytoma we suppose the signs mentioned above to be the manifestation of the catecholamine heart muscle disease (CHMD).

Morphometric study of myocardial changes during doxorubicin-induced cardiomyopathy in mice.

Doxorubicin (DOX) is one of the most effective anti-cancer drugs in oncology, but may cause a cumulative dose-dependent cardiomyopathy in a number of cancer patients. The effect of DOX on the heart was studied in mice treated with i.v. injections of 2 mg/kg by measuring morphometric parameters, including nuclear index (number of non-myocytes/number of myocyte nuclei), reticulin index (reticulin area/number of myocyte transsections), nuclear transsectional area, myocyte transsectional area, capillary index (number of capillaries/number of myocyte transsections) and capillary transsectional area. The highest significant difference between control mice and DOX-treated mice was observed immediately after the 12th dose of DOX except for the two capillary parameters. The highest level of significance for these two parameters was obtained 12 weeks after the end of DOX treatment. In contrast to the observations in rats, mice did not develop a nephrotic syndrome during treatment with DOX. The morphometric analysis of myocardial changes in mice, as a quantitative and objective method, seems to be a good model for comparative studies on cardiomyopathy induced by anthracycline analogues.

Psychological stress and silent myocardial ischemia.

Episodes of transient myocardial ischemia during daily life were investigated in 30 patients on two separate occasions, by ambulatory Holter ST monitoring. The first occasion was at a time of uncertainty in the patients' lives, when the results of coronary angiography and the need for surgery were to be discussed. The second was at a later date, when there had been time to adjust to the decision-making process. There were 515 episodes of myocardial ischemia of which 174 were associated with pain and 341 were asymptomatic. Silent ischemia was significantly more frequent during the first period of monitoring compared to the second (p less than 0.02). Patients who had more silent ischemia on the first occasion also entered more self reports of "emotional upset" (tension, worry, etc.,) in their diaries compared to the second occasion. The level of urinary cortisol was taken as a measure of uncertainty and worry, and was significantly higher on the first occasion (p less than 0.03). Differences in urinary noradrenaline excretion were taken as a measure of subjective stress. Patients who excreted more noradrenaline on the first compared to the second occasion had significantly more silent ischemia (p less than 0.007) and longer total ischemic time (p less than 0.01). We suggest that psychological stress may exacerbate myocardial ischemia which is frequently painless.