The aim of WP 08 will be the identification and the characterization of functional, ultrastructural and molecular changes in skeletal muscle tissue (SMT) in chronic heart failure that could potentially be considered as early markers of worsening clinical evolution of the disease. We aim to gain insight into the cross-talk between muscle, fat and the immune system in these pathological conditions.
In adult life, skeletal muscle is a highly plastic tissue, responding to different environmental signals that determine muscle growth, muscle fibre type differentiation, innervation and degeneration after damaging. More importantly, in response to muscle injury, adult muscle stem cells, also known as satellite cells, are activated for adult muscle maintenance and repair. In heart failure, functional capacity and clinical status are also related to the anatomical and functional properties of the skeletal muscle. In patients with similar left ventricular function, functional capacity is related more to muscle strength than to heart function.
The aim of this study is (i) to show that the muscle is a pivotal player in the pathophysiology of the deterioration process related to chronic heart failure (CHF) and (ii) to try to identify novel molecular targets able to improve the outcome of disease.
Samples from patients affected by heart failure in the presence of different co-morbidities such as type-2 diabetes, obesity or cachexia, will be provided by WP03. We will take advantage of the availability of frozen SMT samples from a bio-bank of over 1000 healthy donors.
Workpackage Leader: IRCCS
Involved Partners: Moscow CRC
Task 1: Collection of SMT samples
IRCCS Rome will participate in the enrollement of CHF patients in the context of WP03 with at least 50 patients recruited over the course of 2 years. Skeletal muscle samples will be obtained by the soleous and/or by the femoral quadriceps muscles, after anesthetizing the skin and underlying tissues with lidocaine, by using Tilley-Henkel forceps (Sontec Instruments, Inc; Centennial, CO) inserted through a small (0.5 cm) skin incision. Tissues will be immediately frozen in liquid nitrogen for RNA preparation, following exactly the same standard procedures among Institutes, or differently processed for cell culture and ultrasctructural studies.
Task 2: Ultrastructural analysis of SMT samples
Fresh biopsies of approximately 50 mm3 will be frozen in OCT and sections of 10 um will be obtained at the criostate. Size and architecture of the fibers, together with the characterization of all parameters indicating the regenerative and inflammatory status of muscle will be examined. Immunohistochemical analysis of muscle specific markers (i.e. myosin heavy chain, etc.) will be also carried out.
Task 3: Microarray analysis and gene expression profile of SMT samples
RNA will be prepared from all samples, then purified, reversed transcribed and subjected to genome wide expression analysis by microarrays (Affimetrix). We will examine the expression of a wide series of genes, including those related to the ultrastructural architecture (i.e. myosin) of adult skeletal muscle, to the myogenic network (MRFs, ID and MSY-3) and those that normally are involved in atrophy, hypertrophy and muscle degeneration (atrogin- 1, MURF-1, FOXO, etc.) and muscle metabolism (muscle creatine kinase, aldolase, etc).
Task 4: Establishment of myogenic primary cultures for functional studies
In a small subset of patients (at least 10, of the same age and gender), a larger amount of tissue will be isolated for in vitro studies (at least 2 grams per patient). We will establish myogenic primary cultures in order to evaluate several parameters of proliferation (life span) and myogenic differentiation, as size of myotubes, correct expression of the muscle specific regulatory factors, cell cycle regulators and terminal differentiation markers.
Task 5: Establishment of autologous co-cultures of myotubes with mature adipocytes and peripheral blood mononuclear cells (PBMC) for cellular cross-talk studies
It is well established that muscle, fat and immunity cells are untimely connected in vivo, with adipose tissue infiltrating skeletal muscle in the context of ectopic deposition of fat, and immune cells (especially macrophages) residing in the context of fat. For this specific reason, once obtained mature myotubes in vitro from SMC samples, they will be co-cultured for 24, 48 and 96 hours with mature adipocytes previously purified from fat tissue biopsies (using the same procedures described in WP09), and with PBMC isolated from heparinised venous blood sample of the same patient, by density gradient centrifugation over Ficoll-Paque. Subsequently RNA from myotubes will be extracted and processed for gene expression analysis of selected myogenic differentiation and functional markers by Real Time PCR. Aim of this task is to study the potential cross talk between muscle and fat, and muscle and immune system, in order to create a simple model ex vivo where the potential effects of comorbidities such as obesity and cachexia on muscle function can be reproduced.
Task 6: Investigation of signalling pathways potentially involved in the observed alterations of muscular function
On the basis of the information coming from the above studies, cellular functions potentially affected in CHF or by comorbidities will be further investigated in vitro, using C2C12, a murine miogenic cell line, which represents the most valuable model to study in vitro muscle differentiation. We will induce overexpression or knock-down of genes potentially implicated in structural and functional abnormalities of muscle during CHF, to get more insights in molecular pathways involved. These could potentially represent new molecular targets for future therapeutical strategies.
We expect that our efforts may lead to find new pharmacological targets for the control of gene expression in order to manipulate muscle growth, innervation and regeneration to contrast muscle degeneration, under physiological and pathological conditions.
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