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The role of Calcium/calmodulin-dependent kinase and Calcineurin pathways in activity-dependent gene regulation in skeletal muscle

Motor neuron activity is a fundamental controller of skeletal muscle growth and differentiation. Excitation-transcription coupling is the process whereby activity causes specific changes in gene transcription through an increase in the concentration of sarcolemmal Ca2+. The molecular mechanisms that relay plasma membrane depolarization to activity-dependent gene transcription is not yet fully understood. Two main Ca2+-dependent pathways are known to transduce Ca2+ signals into changes in gene expression, the Cn-NFAT and CaMK pathways. We have focused on Cn-NFAT and CaMKII, which is the major CaMK isoform in skeletal muscle. We have studied the effects of the inhibition of these pathways on the expression of Ca2+-dependent genes involved in muscle metabolism, such as glucose transporter 4 (GLUT4), mitochondrial transcription factor A (TFAM) and citrate synthase (CS), or in muscle contraction and specific of slow or fast IIB fibers, such as myosin heavy chain-slow and -2B (MyHC-slow and -2B). To this purpose we have used a loss-of-function approach by co-transfecting vectors, coding for natural peptide inhibitors of Cn and CaMKII pathways, together with Luciferase (LUC) reporters in rat skeletal muscle, through in vivo electroporation. We have analyzed the effects of these inhibitors on activity-dependent gene expression. Our results indicate that CaMKII controls not only muscle metabolism, by regulating the expression of GLUT4, TFAM and CS, but also the expression of contractile proteins, such as MyHC-slow and -2B. The Cn-NFAT signalling pathway has been widely demonstrated to be a fundamental regulator of muscle fiber slow program. Interestingly our data indicate that Cn also controls the expression of genes involved in muscle metabolism such as CS and GLUT4. A question remains open as to whether CaMKII and Cn pathways can synergistically control the expression of shared activity-dependent genes. If synergy exists it will be interesting to investigate which is the molecular target underlying CaMKII and Cn synergistic cooperation.

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5 1. INTRODUCTION 1.1 Skeletal muscle fiber properties In vertebrates three different muscular tissues can be distinguished: smooth, cardiac and skeletal. Smooth muscle surrounds internal organs and major blood vessels, is innervated by the autonomous nervous system and it is made of mono-nucleated cells (called myocytes). The cells of cardiac muscle are mono-nucleated, and together with skeletal muscles form the complex of striated muscles. The functional units of skeletal muscles are the muscle fibers, long cylindrical multinucleated, with about 100 nuclei cells, 10-50 µm of diameter, 1-10 mm of length in rat muscles. The great majority of the cytoplasm of these cells is occupied by fascicles of longitudinal filaments called myofibrils, subdivided in light and dark bands, called I band and A band respectively. The basic structural and functional unit of skeletal muscle is called sarcomer: this repeated element has a length of 2 µm. Adjacent sarcomeric units are perfectly aligned, giving rise to the characteristic striated morphology that can be seen with the microscope. Each sarcomer contains two kinds of filaments: thin filaments, made of actin (that constitutes the light I band), thick filaments, made of myosin (dark A band). Contraction speed is related to the ATPase activity of myosin, and to the enzymes of the sarcoplasmic reticulum (SR) that sequester calcium (Ca 2+ ). Myosins are composed of three couple of subunits: two heavy chains (MyHC), two light chains (MLC) and two essential or alkaline chains. The two C-terminal domains of the MyHC subunits are coiled to form an α-helix tails, responsible for the association of many other MyHC dimers in a single filament. ATPasic activity and actin binding site are localized within the globular region of the protein (head). 1.1.1 Myofiber diversity Muscle fibers vary considerably with respect to their morphological, biochemical and physiological properties, enabling different muscles to fulfill a variety of functions, from maintaining the body posture to performing a wide range of movements and motions. Mammalian skeletal muscles are composed of two major fiber-types (I and II), basically distinguished on the basis of the myosin heavy chain isoform (MyHC) that they express, and differ in terms of size, metabolism, and contractile properties. MyHCs

Tesi di Dottorato

Dipartimento: Dipartimento di Scienze Biomediche Sperimentali

Autore: Marzia Cusinato Contatta »

Composta da 89 pagine.


Questa tesi ha raggiunto 31 click dal 21/12/2011.

Disponibile in PDF, la consultazione è esclusivamente in formato digitale.