Alicia Ortega

Cholesterol effect on enzyme activity of the sarcolemmal (Ca2+ + Mg2+)-ATPase from cardiac muscle

The effect of cholesterol incorporation and depletion of the cardiac sarcolemmal sacs on (Ca2+ + Mg2+)-ATPase activity was examined. Cholesterol incorporation to the sarcolemmal sacs was achieved utilizing an in vivo and an in vitro procedure. Cholesterol depleted membranes were obtained in vitro after incubation of the sarcolemmal sacs with inactivated plasma. Arrhenius plots of the (Ca2+ + Mg2+)-ATPase activity showed a triphasic curve when the assays were carried out using a temperature range between 0 and 40 degrees C. The sarcolemmal (Ca2+ + Mg2+)-ATPase activity was shown to be inversely proportional to the cholesterol concentration of the membranes, showing a low ATPase activity with a high cholesterol content and a high ATPase activity when the cholesterol concentration was low. Although the (Ca2+ + Mg2+)-ATPase activity was found to be inhibited in the cholesterol incorporated sarcolemmal sacs, the withdrawal of small amounts of cholesterol from the membranes produced an important stimulatory effect. Changes in (Ca2+ + Mg2+)-ATPase activity due to variation in the membrane cholesterol concentration were shown to be reversible. Our results indicate the possibility of a slow exchange of cholesterol between the tightly bound lipid surrounding the (Ca2+ + Mg2+)-ATPase and the bulk lipid of the sarcolemma.

Direct regulatory effect of cholesterol on the calmodulin stimulated calcium pump of cardiac sarcolemma

A great controversy has been set for several years related to an indirect versus a direct effect of cholesterol upon the muscle calcium pump. Employing an enriched cardiac sarcolemma preparation and a (Ca2+, Mg2+)-ATPase fraction isolated from this preparation, this study demonstrates that cholesterol directly interacts with the sarcolemmal calcium pump importantly inhibiting its enzyme activity. It was discovered that this inhibition can be in part explained by a total sensitivity loss of the pump to calmodulin. These results can be considered of importance in the correlation of plasma membrane cholesterol levels with deficiencies in calcium transport and cardiac muscle cell damage.

Biomechanics of the sarcolemma and costameres in single skeletal muscle fibers from normal and dystrophin-null mice.

We studied the biomechanical properties of the sarcolemma and its links through costameres to the contractile apparatus in single mammalian myofibers of Extensor digitorum longus muscles isolated from wild (WT) and dystrophin-null (mdx) mice. Suction pressures (P) applied through a pipette to the sarcolemma generated a bleb, the height of which increased with increasing P. Larger increases in P broke the connections between the sarcolemma and myofibrils and eventually caused the sarcolemma to burst. We used the values of P at which these changes occurred to estimate the tensions and stiffness of the system and its individual elements. Tensions of the whole system and the sarcolemma, as well as the maximal tension sustained by the costameres, were all significantly lower (1.8–3.3 fold) in muscles of mdx mice compared to WT. Values of P at which separation and bursting occurred, as well as the stiffness of the whole system and of the isolated sarcolemma, were ~2-fold lower in mdx than in WT. Our results indicate that the absence of dystrophin reduces muscle stiffness, increases sarcolemmal deformability, and compromises the mechanical stability of costameres and their connections to nearby myofibrils.

Cholesterol increases the thermal stability of the Ca2+/Mg2+-ATPase of cardiac microsomes

The effect of membrane cholesterol on the thermal inactivation of Ca2+/Mg(2+)-ATPase activity of bovine cardiac microsome was measured and compared to the thermal denaturation profiles of the microsomes as measured by differential scanning calorimetry (DSC). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Both thermal inactivation of Ca2+/Mg(2+)-ATPase activity and thermal denaturation were shifted to higher temperatures in microsomes enriched with cholesterol (37 +/- 5 micrograms cholesterol/mg protein, cholesterol/phospholipid molar ratio 0.31) compared to control microsomes (15 +/- 3 micrograms cholesterol/mg protein, molar ratio 0.12). Thermal inactivation was measured by two methods: first, measuring activity at room temperature as a function of heating to elevated temperatures at 1 K/min, where inactivation temperatures (T1, temperature of half activity) were 58.9 +/- 0.3 degrees C for control membranes and 59.9 +/- 0.1 degrees C for cholesterol-enriched membranes, respectively. Second, measuring ATPase activity as a function of time at constant temperature, where T1 values of 57.6 +/- 0.5 degrees C and 59.2 +/- 0.5 degrees C were determined for control and cholesterol-enriched membranes, respectively. DSC profiles of microsomal membranes consisting of a number of overlapping peaks were obtained. A well resolved component (transition C) was observed with a transition temperature (T 1/2) of 58.2 degrees C. This T 1/2, which is a measure of conformational stability, correlates with the T1 for Ca2+/Mg(2+)-ATPase activity and is 1.9 +/- 0.6 K higher in cholesterol-enriched membranes. Thus, the increased resistance to inactivation appears to be due to increased conformational stability of the protein induced by cholesterol, demonstrating that a change in lipid composition can influence the stability of an integral membrane protein in a natural membrane. The increased stability is of sufficient magnitude to account for the previously observed correlation between cholesterol content and resistance to heat shock in several cell lines.

Cholesterol Depletion Uncouples β-dystroglycans from Discrete Sarcolemmal Domains, Reducing the Mechanical Activity of Skeletal Muscle

Background/Aims: β-Dystroglycan (β-DG) is a transmembrane glycoprotein that links the intracellular cytoskeleton to the extracellular matrix and is crucial for the molecular pathway of lateral force transmission in muscle. We aimed to investigate the effect of decreasing sarcolemmal cholesterol on the distribution of β-DG, its interaction with dystrophin and the impact on the contraction efficiency of muscle. Methods: Isolated rat extensor digitorum longus muscles were incubated with methyl β-cyclodextrin (MβCD) to deplete cholesterol and with MβCD-cholesterol to restore cholesterol. Electric stimulation protocols were used to determine muscle force and fatigue. Detergent-resistant membranes (lipid rafts) were separated from isolated skeletal muscle sarcolemma. The distribution and interactions of β-DG, caveolin-3 and dystrophin were determined by an immunoreactivity analysis. Results: Cholesterol depletion in muscle results in a weakened force of contraction, which recovers after cholesterol restoration. The rate of fatigue is unaffected, but fatigue recovery is dependent upon cholesterol restoration. MβCD modifies the structures of lipid rafts obtained from MβCD-treated muscles by, displacing the membrane proteins β-DG and caveolin-3 f from the lipid raft, thus reducing the interaction of β-DG with dystrophin. Conclusion: Cholesterol depletion weakens the muscle contractile force by disturbing the sarcolemmal distribution of β-dystroglycan and its interaction with dystrophin, two key proteins in the alignment of lateral force transmission pathway.

KV10.1 K+-channel plasma membrane discrete domain partitioning and its functional correlation in neurons ☆

KV10.1 potassium channels are implicated in a variety of cellular processes including cell proliferation and tumour progression. Their expression in over 70% of human tumours makes them an attractive diagnostic and therapeutic target. Although their physiological role in the central nervous system is not yet fully understood, advances in their precise cell localization will contribute to the understanding of their interactions and function. We have determined the plasma membrane (PM) distribution of the KV10.1 protein in an enriched mouse brain PM fraction and its association with cholesterol- and sphingolipid-rich domains. We show that the KV10.1 channel has two different populations in a 3:2 ratio, one associated to and another excluded from Detergent Resistant Membranes (DRMs). This distribution of KV10.1 in isolated PM is cholesterol- and cytoskeleton-dependent since alteration of those factors changes the relationship to 1:4. In transfected HEK-293 cells with a mutant unable to bind Ca(2+)/CaM to KV10.1 protein, Kv10.1 distribution in DRM/non-DRM is 1:4. Mean current density was doubled in the cholesterol-depleted cells, without any noticeable effects on other parameters. These results demonstrate that recruitment of the KV10.1 channel to the DRM fractions involves its functional regulation.

Altered calcium pump and secondary deficiency of γ-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from δ-sarcoglycan knockout mice

Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and delta-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of delta-SG isoforms in TT and SR results in a secondary deficiency of gamma-SG and microSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the delta-SG isoforms may stabilize the expression of gamma-SG and microSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle.

Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T m = 55 °C) most likely corresponds to the receptor/translocation domain, peak B (T m = 59 °C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 °C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T m = 65 °C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B (≈40%) or its pore-forming fragment (≈80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T m= 54 °C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.

Gestational Undernourishment Modifies the Composition of Skeletal Muscle Transverse Tubule Membranes and the Mechanical Properties of Muscles in Newborn Rats

Backgroud/Aims: Skeletal muscle (SM) constitutes more than 40% of the body weight in adulthood. Transports dietary glucose mainly through the insulin-dependent glucose transporter (Glut-4) located in the Transverse tubule membrane system (TT). The TT development ends shortly after birth. The TT membrane hosts the proteins involved in excitation-contraction coupling and glucose uptake. Glycaemic regulation through movement is a key function of fully developed skeletal muscle. In this study, we aimed to characterize the effect of gestational undernourishment (GUN) in rats GLUT-4 expression and on the protein/lipid content of the TT membranes. We also examined the effect of GUN on the mechanical properties of muscles as an indication of the metabolic condition of the SM at birth. Methods: Isolated TT membrane from SM of GUN rats were used to study lipid/protein content and protein stability by differential scanning calorimetry. The effect of GUN on the SM mechanical properties was determined in isolated Extensor Digitorum Longus (EDL) muscle. Results: We demonstrate that compared to control, GUN in the new-born produces; i) decreases body weight; ii) diminution in SM mass; iii) decreases the formation of TT membranes; iv) expresses TT membrane proteins with higher thermal stability. The TT membrane expression of GLUT-4 in GUN offspring was twice that of controls. The isolated EDL of GUN offspring was 20% stronger as measured by contractile force and more resistant to fatigue relative to controls. Conclusion; These results provide the first evidence of adaptive changes of the SM in new-borns exposed to severe gestational food restriction. The effects of GUN on muscle at birth are the first step toward detrimental SM metabolic function, contributing to the physiopathology of metabolic diseases in adulthood.

Elastic Properties of the Sarcolemma‐Costamere Complex of Muscle Cells in Normal Mice

Costameres at the sarcolemma of skeletal myofibers are thought to mediate the lateral transmission of force from the myofibrils to the extracellular matrix. We applied elastimetry to single myofibers from the Extensor digitorum longus muscles of mice to measure the biomechanical properties of the sarcolemma and the costameres as a function of sarcomere length. Suction pressure (P) applied by the elastimeter to the sarcolemma generated a bleb of variable height, which depended on P and sarcomere length. Connections between the sarcolemma and nearby myofibrils broke as P increased. Pressure, tension, force and stiffness (k) were calculated using the displacement pressure curves. With Laplace’s and Hooke’s equations, we estimated tensions generated by the sarcolemma attached to the myofibrils (Tc+s) and the sarcolemma after detachment (Ts). Tc+s − Ts = Tc, the maximal tension sustained by the costameres. The sarcolemma‐costamere complex was represented as a lumped elastic model, in which the elasticity of ea...