Molecular effects of cardiac contractility modulation in patients with heart failure of ischemic aetiology uncovered by transcriptome analysis.

Cardiac contractility modulation (CCM) is based on electrical stimulation of the heart without alteration of action potential and mechanical activation, the data on its fundamental molecular mechanisms are limited. Here we demonstrate clinical and physiological effect of 12 months CCM in 29 patients along with transcriptomic molecular data. Based on the CCM effect the patients were divided into two groups: responders (n = 13) and non-responders (n = 16). RNA-seq data were collected for 6 patients before and after CCM including 3 responders and 3 non-responders. The overall effect of CCM on gene expression was mainly provided by samples from the responder group and included the upregulation of the genes involved in the maintenance of proteostasis and mitochondrial structure and function. Using pathway enrichment analysis, we found that baseline myocardial tissue samples from responder group were characterized by upregulation of mitochondrial matrix-related genes, Z disc-protein encoding genes and muscle contraction-related genes. In summary, twelve months of ССM led to changes in signaling pathways associated with cellular respiration, apoptosis, and autophagy. The pattern of myocardial remodeling after CCM is associated with initial expression level of myocardial contractile proteins, adaptation reserves associated with mitochondria and low expression level of inflammatory molecules.

Desmin mutations impact the autophagy flux in C2C12 cell in mutation-specific manner.

Desmin is the main intermediate filament of striated and smooth muscle cells and plays a crucial role in maintaining the stability of muscle fiber during contraction and relaxation cycles. Being a component of Z-disk area, desmin integrates autophagic pathways, and the disturbance of Z-disk proteins' structure negatively affects chaperone-assisted selective autophagy (CASA). In the present study, we focused on alteration of autophagy flux in myoblasts expressing various Des mutations. We applied Western blotting, immunocytochemistry, RNA sequencing, and shRNA approach to demonstrate that DesS12F, DesA357P, DesL345P, DesL370P, and DesD399Y mutations. Mutation-specific effect on autophagy flux being most severe in aggregate-prone Des mutations such as DesL345P, DesL370P, and DesD399Y. RNA sequencing data confirmed the most prominent effect of these mutations on expression profile and, in particular, on autophagy-related genes. To verify CASA contribution to desmin aggregate formation, we suppressed CASA by knocking down Bag3 and demonstrated that it promoted aggregate formation and lead to downregulation of Vdac2 and Vps4a and upregulation of Lamp, Pink1, and Prkn. In conclusion, Des mutations showed a mutation-specific effect on autophagy flux in C2C12 cells with either a predominant impact on autophagosome maturation or on degradation and recycling processes. Aggregate-prone desmin mutations lead to the activation of basal autophagy level while suppressing the CASA pathway by knocking down Bag3 can promote desmin aggregate formation.

[Practical guidelines for the diagnosis and treatment of transthyretin amyloid cardiomyopathy (ATTR-CM or transthyretin cardiac amyloidosis)].

This paper summarizes the data from updated international protocols and guidelines for diagnosis of transthyretin amyloid cardiomyopathy (ATTR-CM). The invasive and non-invasive diagnosis techniques and their combinations are briefly reviewed; the evidentiary foundations for each diagnostic option and tool are analyzed. The paper describes a customized algorithm for sequential diagnosis and differential diagnosis of patients with suspected ATTR-CM with allowance for the combination of clinical signs and diagnostic findings. Along with the awareness of primary care providers about the red flags of the disease and visualization criteria, as well as providing information to the patients about the possibility of performing therapy of ATTR amyloidosis and the risks of delayed diagnosis, the proposed algorithm enables timely patient routing and prescribing specific treatment.

Rare clinical phenotype of filaminopathy presenting as restrictive cardiomyopathy and myopathy in childhood.

BACKGROUND: FLNC is one of the few genes associated with all types of cardiomyopathies, but it also underlies neuromuscular phenotype. The combination of concomitant neuromuscular and cardiac involvement is not often observed in filaminopathies and the impact of this on the disease prognosis has hitherto not been analyzed. RESULTS: Here we provide a detailed clinical, genetic, and structural prediction analysis of distinct FLNC-associated phenotypes based on twelve pediatric cases. They include early-onset restrictive cardiomyopathy (RCM) in association with congenital myopathy. In all patients the initial diagnosis was established during the first year of life and in five out of twelve (41.7%) patients the first symptoms were observed at birth. RCM was present in all patients, often in combination with septal defects. No ventricular arrhythmias were noted in any of the patients presented here. Myopathy was confirmed by neurological examination, electromyography, and morphological studies. Arthrogryposes was diagnosed in six patients and remained clinically meaningful with increasing age in three of them. One patient underwent successful heart transplantation at the age of 18 years and two patients are currently included in the waiting list for heart transplantation. Two died due to congestive heart failure. One patient had ICD instally as primary prevention of SCD. In ten out of twelve patients the disease was associated with missense variants and only in two cases loss of function variants were detected. In half of the described cases, an amino acid substitution A1186V, altering the structure of IgFLNc10, was found. CONCLUSIONS: The present description of twelve cases of early-onset restrictive cardiomyopathy with congenital myopathy and FLNC mutation, underlines a distinct unique phenotype that can be suggested as a separate clinical form of filaminopathies. Amino acid substitution A1186V, which was observed in half of the cases, defines a mutational hotspot for the reported combination of myopathy and cardiomyopathy. Several independent molecular mechanisms of FLNC mutations linked to filamin structure and function can explain the broad spectrum of FLNC-associated phenotypes. Early disease presentation and unfavorable prognosis of heart failure demanding heart transplantation make awareness of this clinical form of filaminopathy of great clinical importance.

[AL-amyloidosis with cardiac involvement. Diagnostic capabilities of non-invasive methods].

There are presented the literature data and a description of the clinical course of the disease in isolated/predominant cardiac amyloidosis. Amyloid cardiomyopathy is the most common phenocopy of hypertrophic cardiomyopathy. The modern possibilities of non-invasive diagnostics using osteoscintigraphy for the differential diagnosis between amyloid cardiomyopathy caused by AL- and transthyretin amyloidosis are described in detail.

R482L Mutation of the LMNA Gene Affects Dynamics of C2C12 Myogenic Differentiation and Stimulates Formation of Intramuscular Lipid Droplets.

Mutations in the LMNA gene resulting in the substitution of the highly conserved arginine 482 residue in the globular C-terminal domain of lamin A/C are associated with the Dunnigan-type familial partial lipodystrophy (FPLD2) often accompanied by impairments in the muscle tissue development. The mechanisms underlying these impairments remain unknown. The purpose of our work was to investigate the effects of the LMNA gene mutation R482L on the muscle differentiation and intramuscular fat accumulation using C2C12 mouse myoblasts transduced with the lentiviral constructs carrying the wild-type human LMNA gene (LMNA-WT) or the LMNA-R482L mutant gene. After stimulation of myogenesis and adipogenesis in the transduced cell, expression of muscle and adipose tissue differentiation markers, morphology of differentiated myotubes, and formation of intramuscular lipid droplets were analyzed. C2C12 cells carrying the LMNA-R482L construct exhibited upregulated desmin expression at all stages of muscle differentiation and transformed into hypertrophied myotubules (in comparison with C2C12 myoblasts transduced with LMNA-WT). Reduced expression levels of the myogenic transcription factor Myf6 in the cells with the LMNA-R482L mutant indicated delayed maturation of muscle fibers. These cells more actively accumulated fat in response to the stimulation of adipose differentiation than myoblasts modified with the wild-type LMNA; they also expressed the markers of lipid droplets, such as FABP4 (fatty acid-binding protein 4), ATGL (adipose triglyceride lipase), and PLIN2 (perilipin 2). Therefore, the R482L mutation of the LMNA gene affects the dynamics of C2C12 myoblast differentiation into myotubules and stimulates formation of fat deposits in the myoblasts and myotubules in a tissue-specific manner.

Extracellular MicroRNAs and Mitochondrial DNA as Potential Biomarkers of Arrhythmogenic Cardiomyopathy.

Differential diagnosis of arrhythmogenic cardiomyopathy (ACM) during the disease latent phase is a challenging clinical problem that requires identification of early ACM biomarkers. Because extracellular nucleic acids are stable, specific, and can be easily detected, they can be used as reliable biomarkers of various diseases. In this study, we analyzed the levels of extracellular microRNAs and mitochondrial DNA in the conditioned medium collected from cardiomyocytes differentiated from induced pluripotent stem cells of ACM patients and healthy donor. Several microRNAs were expressed differently by the affected and healthy cardiomyocytes; therefore, they could be considered as potential ACM biomarkers.

Skeletal Muscle Resident Progenitor Cells Coexpress Mesenchymal and Myogenic Markers and Are Not Affected by Chronic Heart Failure-Induced Dysregulations.

BACKGROUND AND PURPOSE: In heart failure (HF), metabolic alterations induce skeletal muscle wasting and decrease of exercise capacity and quality of life. The activation of skeletal muscle regeneration potential is a prospective strategy to reduce muscle wasting; therefore, the aim of this project was to determine if functional properties of skeletal muscle mesenchymal progenitor cells (SM-MPC) were affected by HF-induced functional and metabolic dysregulations. METHODS: Gastrocnemius muscle biopsy samples were obtained from 3 healthy donors (HD) and 12 HF patients to purify mRNA for further analysis and to isolate SM-MPC. Cells were expanded in vitro and characterized by immunocytochemistry and flow cytometry for expression of mesenchymal (CD105/CD73/CD166/CD146/CD140b/CD140a/VIM) and myogenic (Myf5/CD56/MyoG) markers. Cells were induced to differentiate and were then analyzed by immunostaining and Q-PCR to verify the efficiency of differentiation. The expression of genes that control muscle metabolism and development was compared for HD/HF patients in both muscle biopsy and in vitro-differentiated myotubes. RESULTS: The upregulation of MYH3/MYH8/Myf6 detected in HF skeletal muscle along with metabolic alterations indicates chronic pathological activation of the muscle developmental program. SM-MPC isolated from HD and HF patients represented a mixed population that coexpresses both mesenchymal and myogenic markers and differs from AD-MMSC, BM-MMSC, and IMF-MSC. The functional properties of SM-MPC did not differ between HD and HF patients. CONCLUSION: In the present work, we demonstrate that the metabolic and functional alterations we detected in skeletal muscle from HF patients do not dramatically affect the functional properties of purified and expanded in vitro SM-MPC. We speculate that skeletal muscle progenitor cells are protected by their niche and under beneficial circumstances could contribute to muscle restoration and prevention and treatment of muscle wasting. The potential new therapeutic strategies of HF-induced skeletal muscle wasting should be targeted on both activation of SM-MPC regeneration potential and improvement of skeletal muscle metabolic status to provide a favorable environment for SM-MPC-driven muscle restoration.

[Left ventricular non - compaction: contemporary view of genetic background, clinical course, diagnostic and treatment].

This review highlights and discusses recent advances in understanding left ventricular non - compaction (LVNC). Clinical profile, prognosis and even diagnosis are still a great challenge faced by the world. The population prevalence of left ventricular non - compaction remains unknown. High variability of clinical manifestations, genetic heterogenity with overlap of different phenotypes, variability of hereditary patterns suggests that LVNC seems to be rather an isolated trait or a part of phenotypic expression of different cardiac diseases or complex genetic syndromes.

[Hypertrophic cardiomyopathy: modern aspects of pharmacologic treatment].

This article discusses recent advances in understanding genetic basis and classification of hypertrophic cardiomyopathy. Here, we review pharmacologic treatment strategies and new developments in disease - specific management of HCM.

A Randomised, Controlled Study of Different Glycaemic Targets during Gestational Diabetes Treatment: Effect on the Level of Adipokines in Cord Blood and ANGPTL4 Expression in Human Umbilical Vein Endothelial Cells.

Our aim was to study the expression of adipokine-encoding genes (leptin, adiponectin, and angiopoietin-like protein 4 (ANGPTL4)) in human umbilical vein endothelial cells (HUVECs) and adipokine concentration in cord blood from women with gestational diabetes mellitus (GDM) depending on glycaemic targets. GDM patients were randomised to 2 groups per target glycaemic levels: GDM1 (tight glycaemic targets, fasting blood glucose < 5.1 mmol/L and <7.0 mmol/L postprandial, N = 20) and GDM2 (less tight glycaemic targets, <5.3 mmol/L and < 7.8 mmol/L, respectively, N = 21). The control group included 25 women with normal glucose tolerance. ANGPTL4 expression was decreased in the HUVECs from GDM patients versus the control group (23.11 ± 5.71, 21.47 ± 5.64, and 98.33 ± 20.92, for GDM1, GDM2, and controls; p < 0.001) with no difference between GDM1 and GDM2. The level of adiponectin gene expression was low and did not differ among the groups. Leptin gene expression was undetectable in HUVECs. In cord blood, leptin/adiponectin ratio (LAR) was increased in GDM2 compared to controls and GDM1 (p = 0.038) and did not differ between GDM1 and controls. Tight glycaemic targets were associated with normalisation of increased LAR in the cord blood. ANGPTL4 expression was downregulated in HUVECs of newborns from GDM mothers and was not affected by the intensity of glycaemic control.

Cellular Mechanisms of Aortic Valve Calcification.

Comparative in vitro study examined the osteogenic potential of interstitial cells of aortic valve obtained from the patients with aortic stenosis and from control recipients of orthotopic heart transplantation with intact aortic valve. The osteogenic inductors augmented mineralization of aortic valve interstitial cells (AVIC) in patients with aortic stenosis in comparison with the control level. Native AVIC culture of aortic stenosis patients demonstrated overexpression of osteopontin gene (OPN) and underexpression of osteoprotegerin gene (OPG) in comparison with control levels. In both groups, AVIC differentiation was associated with overexpression of RUNX2 and SPRY1 genes. In AVIC of aortic stenosis patients, expression of BMP2 gene was significantly greater than the control level. The study revealed an enhanced sensitivity of AVIC to osteogenic inductors in aortic stenosis patients, which indicates probable implication of OPN, OPG, and BMP2 genes in pathogenesis of aortic valve calcification.

Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves.

AIMS: Calcific aortic valve disease is the most common heart valve disease in the Western world. Bicuspid and tricuspid aortic valve calcifications are traditionally considered together although the dynamics of the disease progression is different between the two groups of patients. Notch signaling is critical for bicuspid valve development and NOTCH1 mutations are associated with bicuspid valve and calcification. We hypothesized that Notch-dependent mechanisms of valve mineralization might be different in the two groups. METHODS AND RESULTS: We used aortic valve interstitial cells and valve endothelial cells from patients with calcific aortic stenosis with bicuspid or tricuspid aortic valve. Expression of Notch-related genes in valve interstitial cells by qPCR was different between bicuspid and tricuspid groups. Discriminant analysis of gene expression pattern in the interstitial cells revealed that the cells from calcified bicuspid valves formed a separate group from calcified tricuspid and control cells. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notch-activated endothelial-to-mesenchymal transition and the corresponding expression of HEY1 and SLUG were also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves. CONCLUSION: Early signaling events including Notch-dependent mechanisms that are responsible for the initiation of aortic valve calcification are different between the patients with bicuspid and tricuspid aortic valves.

Lamin A/C mutation associated with lipodystrophy influences adipogenic differentiation of stem cells through interaction with Notch signaling.

Lamins A and C are involved in many cellular functions, owing to its ability to bind chromatin and transcription factors and affect their properties. Mutations of the LMNA gene encoding lamin A/C affect differentiation capacity of stem cells. However, the signaling pathways involved in interactions with lamins during cellular differentiation remain unclear. Lipodystrophy associated with LMNA mutation R482L causes loss of fat tissue. In this study we investigated the role of LMNA mutation R482L in modulating Notch signaling activity in the adipogenic differentiation of mesenchymal stem cells. Notch was activated using lentiviral Notch intracellular domain. Activation of Notch was estimated through the expression of Notch-responsive genes by qPCR and by activation of a luciferase CSL-reporter construct. The effect of LMNA mutation on Notch activation and adipogenic differentiation was analyzed in cells bearing lentiviral LMNA WT or LMNA R482L. We show that, when Notch is activated, LMNA R482L contributes to down-regulation of Notch activation in undifferentiated and differentiated cells, and decreases adipogenic differentiation. Thus, lamin A/C interacts with Notch signaling, thereby influencing cellular differentiation, and point mutation in LMNA could halt this interaction.

EXTRACELLULAR VESICLES FROM BLOOD PLASMA STUDIED BY LOW VOLTAGE SCANNING ELECTRON MICROSCOPY.

Extracellular vesicles are subspherical membranous structures secreted by cells and enriched with different types of biological molecules. The number and the molecular content of these structures depend on pathological conditions and the physiological state of the organism. Extracellular vesicles play an important role in intercellular communication and represent potential disease biomarkers. However, mechanisms of formation, functions and morphological characteristics of extracellular vesicles are still studied insufficiently. Low voltage scanning electron microscopy is a promising method to investigate extracellular vesicles, since it does not require conductive coating and therefore enables a high-resolution visualization of morphological details of nanosized objects. This paper presents the results of low voltage scanning electron microscopy study of morphology and size of objects from blood plasma fractions.

THE ROLE OF LMNA MUTATIONS IN MYOGENIC DIFFERENTIATION OF PRIMARY SATELLITE CELLS AND C2C12 CELLS.

Nuclear lamins form nuclear lamina localized under the inner nuclear membrane. It was previously considered that the nuclear lamina predominantly plays a structural role, however, its involvement have been recently described in the regulatory processes such as chromatin organization and gene transcription. It is known that mutations in the LMNA gene lead to the development of a large number of diseases, laminopathies, which mainly affect mesenchymal tissue. Nowadays, the mechanisms by which the lamina can regulate cell differentiation remain incompletely understood. In the present work, we have studied the effect of LMNA gene mutations on the process of muscle differentiation of primary satellite cells and Ñ2Ñ12 cell line. The genome of satellite cells and Ñ2Ñ12 cell line was modified by the introduction of lentiviral constructs encoding LMNA G232E associated with the development of muscular dystrophy Emery­Dreyfus and LMNA R571S associated with the development of dilated cardiomyopathy. The morphology of the cells was estimated using immunofluorescence, the expression level of myogenic genes were analyzed by qPCR. We have shown that the analyzed mutations reduce the ability of cells to differentiate, to fuse and to form myotubes. We have suggested that it is due to enhanced expression of markers at the early stages and to reduced expression markers at the late stages of myogenesis. Therefore, mutations in nuclear lamins can influence the process of muscle differentiation.

[Possibilities of cryopreservation of ovarian tissue for fertility preservation in cancer patients].

One of the effective methods of fertility preservation is an autologous transplantation of cryopreserved ovarian tissue. Currently, according to the world literature, after orthotopic autotransplantation of ovarian tissue 37 healthy children were born. In 2014 at the North-West Federal Medical Research Center it was established Cryobank of ovarian tissue, which is now kept 50 samples of ovarian tissue of man. Cryoconservation is performed by standard slow freezing. Autotransplantation of cryopreserved ovarian tissue has unique advantages over other methods of fertility preservation. This method does not lead to the postponement of anticancer therapy, safe for hormone-dependent cancer and can be performed regardless of the day of menstrual cycle and it is the only option for fertility preservation in prepubertal girls. The use of this method in clinical practice leads to restoration of endocrine function of the ovaries as well as of fertility in the future.

[PROTEIN KINASE C EXPRESSION FOLLOWING REMOTE ISCHEMIC PRECONDITIONING IN CARDIAC SURGERY].

OBJECTIVE: To evaluate cardioprotective effects of remote ischemic preconditioning (RIPC) in cardiac surgery patients undergoing aortic valve replacement depending on the type of anesthesia and investigate the level of myocardial protein kinase C epsilon (PKC-ε) expression after RIPC. METHODS: In prospective randomized trial, forty eight patients aging from 50 to 75 years old (64 (56 ;69)) were included All patients were scheduled for aortic valve replacement using cardiopulmonary bypass (CPB). The patients were randomized into 4 groups: 1) RIPC applied during propofol anesthesia (RIPC prop, n = 12), 2) RIPC applied during sevoflurane anesthesia (RIPC sev, n = 12), 3) propofol anesthesia without RIPC (CONTROL prop, n = 12), 4) sevoflurane anesthesia without RIPC (CONTROL sev, n = 12). There was no difference found between the groups as to the baseline patient's data. RIPC protocol consisted of 3 simultaneous ischemic episodes of both lower limbs (5 minutes) with 5-min reperfusion intervals. PKC-ε expression in right atrial myocardium was assessed using Western blotting. Troponin I (cTnI) was estimated before anesthesia induction, after 30 min, 6, 12, 24, 48 hours after CPB completion. Also we calculated area under curve of cTnI (cTnI AUC). According to nonparametric distribution, data were assessed by the Mann-Whitney U-test and Newman-Keuls methodfor multigroup comparison. p < 0.05 was considered signifcant. The data are presented as median (25th; 75th percentile). RESULTS: Cardioprotective effects of RIPC were observed only after sevoflurane anesthesia: cTnI AUC was 134,8 (122,3; 232.4) ng/ml/48 h in CONTROL sev group and only 74.3 (64.7; 85.0) ng/ml/48 h in RIPC sev group (p < 0.05). RIPC applied during propofol anesthesia was not associated with cTnIAUC decrease: 93.8 (74.1; 246.8) ng/ml/48 h in CONTROL prop group and 122.5 (74.1; 185.0) ng/ml/48 h in RIPC prop group (p = 0.37). RIPC applied during sevoflurane anesthesia significantly increased PKC-ε expression: 1221 (921; 1438) U in CONTROL sev group vs 1882 (1564; 2131) U in RIPC sev group 6 (p < 0.05). RIPC implication during propofol anesthesia was not associated with any significant difference in PKC-ε expression in comparison with control group: 620 (436; 782) U in CONTROL prop group versus 788 (574;1063) U in RIPC prop group. In control groups, PKC-ε expression was significantly higher in sevoflurane anesthesia in comparison with propofol anesthesia. CONCLUSION: RIPC was only effective when it was applied during sevofiurane anesthesia. This was confirmed by PKC-ε expression increase and lower value of cTnI. There were no evidence of preconditioning and cardioprotection when MPG was initiated during propofol anesthesia.

[THE EFFECT OF PLAKOPHILIN-2 GENE MUTATIONS ON ACTIVITY OF THE CANONICAL Wnt SIGNALING PATHWAY].

Plakophilin-2 is a desmosomal protein encoded by PKP2 gene. Desmosomal proteins are usually considered as structural proteins with the main function of maintaining intercellular interactions. Genetic studies revealed that mutations in desmosomal genes could lead to arrhythmogenic right ventricular cardiomyopathy, heart disease characterized by substitution of cardiomyocytes by adipose and fibrotic tissue predominantly in right ventricle. Wnt signaling pathway is one of the signal transduction pathways which could be involved in the formation of the pathology. The purpose of this study was to investigate Wnt activity changes caused by PKP2 mutations during adipogenic and cardiomyogenic differentiation. We used multipotent mesenchymal stromal cells and iPS cells generated from patient carrying PKP2 gene mutation. We show that Wnt activity is lower in the cells with mutant PKP2. This data indicate a possible signaling role of plakophilin-2 by regulating Wnt activity.