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PEP 8302 Module 2 Topics Covered Membrane Structure and Function. - Membrane structure and components. - Membrane biophysical properties. - Mechanical loading effects on membrane properties. - Role of cholesterol in membrane protein function. PEP 8302 Module 2 MEMBRANE FUNCTION • To serve as a physical barrier between the external and internal cellular environment • To serve as a three-dimensional scaffold to support a variety of signaling proteins embedded in the three dimensional matrix of the membrane PEP 8302 Module 2 1 Mammalian Plasma Membrane Cholesterol Receptor Phospholipid Bilayer G Ion Channel Kinase (RYR-1, DHP, SERCA) Lipid Raft (CRMD) Fluid Mosaic Model http://www.youtube.com/watch?v=owEgqrq51zY - Cholesterol is the major structural lipid of the mammalian membrane - some proteins require lipid raft localization to function correctly PEP 8302 Module 2 Biomechanical Properties of the Living Cell Membrane -rigidity, elasticity, fluidity, tensile strength (all physical parameters) - MEMBRANE ORDER (MO) - cholesterol enrichment of large vessel endothelial cell membranes results in both an increase in MO and an increase in susceptibility to mechanically-induced membrane wounding (Clarke et al., 1995; Endothelium 4:127-139). - reversed by the acute (i.e. 2 min) addition of a synthetic membrane disordering agent (i.e. decreases MO). PEP 8302 Module 2 Biomechanical Changes In Myofiber Membranes Associated with Mechanical Loading/Exercise - exercise-induced hypertrophy includes the assembly of large amounts of new membrane components required to service the new contractile components. - myofiber membranes are more resistant to damage than those in unexercised muscle as evidenced by reduced levels of circulating myofiber damage markers after a period of regular exercise. - appears to be a “training effect” not only on contractile level but also the membrane level. - mechanical loading induces membrane wounding and release of the muscle growth factor, fibroblast growth factor (FGF). - this may explain the requirement for increasing resistive loads to maintain muscle hypertrophic response during training regime. PEP 8302 Module 2 2 Mechanically-Induced, Myofiber Wound-Mediated FGF Release - FGF has no signal transduction peptide sequence released via a nonsecretory pathway. - release of FGF from myofiber cytoplasm via transient disruptions or “membrane wounds” of the sarcolemma - myofiber wounding increases as mechanical load increases. - FGF release increases as mechanical load increases - both acidic and basic isoforms of FGF released in this manner - this phenomenon first shown indirectly in rat and mouse skeletal muscle after exercise in vivo. Similar results obtained in rat myocardium. - phenomenon exacerbated in Duchenne’s muscular dystrophy as mechanical strength of the myofiber is reduced. (?) PEP 8302 Module 2 Myofiber Wounding In Eccentrically Loaded Mdx Mouse Muscle FDx FGF Linear correlation between wounding and FGF release PEP 8302 Module 2 Biomechanical Changes In Myofiber Membranes Associated with Mechanical Unloading/Microgravity Exposure - myofiber is more susceptible to reloading-induced mechanical damage than control muscle - rat hindlimb suspension (HLS) muscles and space flown rat muscle (Krippendorf and Riley, 1993; Riley et al., 1996). - human subjects after 14 days of bedrest (Clarke et al., 1998). - damage is associated with mechanical disruption of the myofiber membranes as evidenced by increased circulating damage markers and membrane lesions at the EM level. PEP 8302 Module 2 3 Creatine Kinase (MM) as A Marker of Myofiber Wounding During Bedrest CK (mm) specific to skeletal muscle, released upon myofiber damage as a consequence of mechanical load Clarke et al., (1998) JAP 85: 593-600. PEP 8302 Module 2 In Vivo Correlation Between Myofiber Wounding and Myofiber CrossCross-Sectional Area Linear Correlation Between Myofiber Wounding And Myofiber CSA Atrophy Hypertrophy Clarke et al., (1998) JAP 85: 593-600. PEP 8302 Module 2 Creatine Kinase (MM) as A Marker of Myofiber Wounding During Bedrest Unexpected observation that unloaded muscle membranes are susceptible to mechanical damage than normal membranes Clarke et al., (1998) JAP 85: 593-600. PEP 8302 Module 2 4 Effect of Mechanical Unloading On Muscle Membrane Cholesterol Content (Atrophic) Human Bedrest (Hypertrophic) Rat Hindlimb Suspension PEP 8302 Module 2 Electro-mechanical activation of the SR membrane Ryanodine Channel (RyR1) via the dihydropyridine receptor (DHP) results in Ca2+ efflux initiating contraction. SR. Ca2+ reuptake into the SR brings about relaxation. Ca2+ RyR1 DHP CaATPase PEP 8302 Module 2 Membrane Localization of Cholesterol In Skeletal Muscle Clarke et al., (2000) J. App. Physiol. Physiol. 89: 731731-741 PEP 8302 Module 2 5 Membrane Localization of Cholesterol In Skeletal Muscle Clarke et al., (2000) J. App. Physiol. Physiol. 89: 731731-741 PEP 8302 Module 2 Cholesterol Immunostaining In Skeletal Muscle Control Atrophied Rat Soleus Muscle (14 days of HLS) Human MVL (Young vs Old) Young (35 yrs) Old (70 yrs) PEP 8302 Module 2 Cholesterol Enrichment and Membrane Function •increased cholesterol content affects skeletal muscle membrane protein function –increased calcium flux through DHP-sensitive calcium channels –inhibition of sarcoplasmic reticulum (Ca2+)-ATPase activity in cardiac myocte membranes –decreased muscle sensitivity to circulating insulin and IGF-1 •increased levels of free sarcoplasmic calcium ?? • increased cytosolic calcium is a catabolic stimulus PEP 8302 Module 2 6 Relationship Between Myofiber Cholesterol and Myofiber Free Calcium Levels Calcium Green -1 Staining Cholesterol vs. Calcium PEP 8302 Module 2 Effect of Modifying Membrane Cholesterol Content on SERCA-1 and SERCA-2 Ca2+ATPase Activity SERCA-1 SERCA-2 PEP 8302 Module 2 Effect of Cholesterol Modulation on Ca2+ ATPase Activity Rates in Rat Muscle Membranes (N=4 per group, p < 0.05 between all groups) Crude Membrane Fraction of Rat Soleus Muscle CaATPase Rate Assay. PEP 8302 Module 2 7 Effect of Modulating Membrane Cholesterol Content On RyR1 Activity in a Lipid Bilayer Model (1) Addition of MβC-CHOL (2) Remove MβC-CHOL, add MβC alone (3) Remove MβC, add MβC-CHOL again PEP 8302 Module 2 The Effect of Dynamic Foot Pressure (DFP) on Myofiber Cross Sectional Area in Rat Soleus Muscle With and Without HLS. HLS Induces approx. 50% reduction in CSA. DFP applied intermittently for 4 hr per day during HLS. DFP completely abolishes myofiber atrophy in the soleus of HLS rats. PEP 8302 Module 2 Insulin Signaling in Skeletal Muscle PEP 8302 Module 2 8 Vesicle Fusion PEP 8302 Module 2 GLUT-4 Mobilization in Response to Insulin Signaling • GLUT-4 (glucose transporter) • http://www.bristol.ac.uk/biochemistry/tavare/r esearch/glut4.html PEP 8302 Module 2 Diabetes and Skeletal Muscle • Alteration in sarcolemma function – Same number of sarcolemmasarcolemma-bound insulin receptors as nonnondiabetic muscle – Same number of intracellular GLUTGLUT-4 (glucose channels) as nonnon-diabetics – Less GLUTGLUT-4 located in the sarcolemma of diabetic than nonnon-diabetic muscle – The result of less GLUTGLUT-4 in the sarcolemma of those with T2DM is that the muscle is less able to uptake glucose than those without T2DM – The mechanism behind this reduction of GLUTGLUT-4 in the sarcolemma is unknown PEP 8302 Module 2 9 Immuno-localization of Cholesterol in Rat Soleus Muscle Of Ambulatory Controls, HLS and HLS-DFP Animals PEP 8302 Module 2 Immuno-localization of Inducible GLUT-4 in Rat Soleus Muscle Of Ambulatory Controls, HLS and HLS-DFP Animals PEP 8302 Module 2 10