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1.
Autophagy ; 6(8): 1078-89, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20861693

ABSTRACT

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50­60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.


Subject(s)
Autophagy , Enzyme Replacement Therapy , Glycogen Storage Disease Type II/therapy , alpha-Glucosidases/therapeutic use , Animals , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Disease Models, Animal , Glycogen/metabolism , Glycogen Storage Disease Type II/pathology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Integrases/metabolism , Mice , Muscle Fibers, Fast-Twitch/enzymology , Muscle Fibers, Fast-Twitch/pathology , Muscle Fibers, Fast-Twitch/ultrastructure , Myosin Light Chains/metabolism , Phosphorylation , Ubiquitin/metabolism , alpha-Glucosidases/deficiency , alpha-Glucosidases/metabolism
2.
J Appl Physiol (1985) ; 108(5): 1383-8, 2010 May.
Article in English | MEDLINE | ID: mdl-20223998

ABSTRACT

Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, is a disorder of glycogen metabolism that can affect infants, children, or adults. In all forms of the disease, there is progressive muscle pathology leading to premature death. The pathology is characterized by accumulation of glycogen in lysosomes, autophagic buildup, and muscle atrophy. The purpose of the present investigation was to determine if myofibrillar dysfunction in Pompe disease contributes to muscle weakness beyond that attributed to atrophy. The study was performed on isolated myofibers dissected from severely affected fast glycolytic muscle in the alpha-glucosidase knockout mouse model. Psoas muscle fibers were first permeabilized, so that the contractile proteins could be directly relaxed or activated by control of the composition of the bathing solution. When normalized by cross-sectional area, single fibers from knockout mice produced 6.3 N/cm2 of maximum Ca2+-activated tension compared with 12.0 N/cm2 produced by wild-type fibers. The total protein concentration was slightly higher in the knockout mice, but concentrations of the contractile proteins myosin and actin remained unchanged. Structurally, X-ray diffraction showed that the actin and myosin filaments, normally arranged in hexagonal arrays, were disordered in the knockout muscle, and a lower fraction of myosin cross bridges was near the actin filaments in the relaxed muscle. The results are consistent with a disruption of actin and myosin interactions in the knockout muscles, demonstrating that impaired myofibrillar function contributes to weakness in the diseased muscle fibers.


Subject(s)
Actin Cytoskeleton/pathology , Glycogen Storage Disease Type II/enzymology , Muscle Contraction , Muscle Fibers, Fast-Twitch/enzymology , Muscle Strength , Muscle Weakness/enzymology , Psoas Muscles/enzymology , alpha-Glucosidases/deficiency , Actin Cytoskeleton/enzymology , Actins/metabolism , Animals , Calcium/metabolism , Disease Models, Animal , Glycogen Storage Disease Type II/genetics , Glycogen Storage Disease Type II/pathology , Glycogen Storage Disease Type II/physiopathology , Mice , Mice, Knockout , Muscle Fibers, Fast-Twitch/pathology , Muscle Weakness/genetics , Muscle Weakness/pathology , Muscle Weakness/physiopathology , Muscular Atrophy/enzymology , Muscular Atrophy/physiopathology , Myosins/metabolism , Psoas Muscles/pathology , Psoas Muscles/physiopathology , alpha-Glucosidases/genetics
3.
Biophys J ; 96(9): 3673-81, 2009 May 06.
Article in English | MEDLINE | ID: mdl-19413972

ABSTRACT

The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction under the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder<-->order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.


Subject(s)
Heterocyclic Compounds, 4 or More Rings/metabolism , Myosins/chemistry , Myosins/metabolism , Actins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Protein Conformation , Psoas Muscles/chemistry , Rabbits , Temperature , Thermodynamics , X-Ray Diffraction
4.
Biophys J ; 91(10): 3768-75, 2006 Nov 15.
Article in English | MEDLINE | ID: mdl-16950853

ABSTRACT

Low angle x-ray diffraction patterns from relaxed permeabilized rabbit cardiac trabeculae and psoas muscle fibers were compared. Temperature was varied from 25 degrees C to 5 degrees C at 200 mM and 50 mM ionic strengths (mu), respectively. Effects of temperature and mu on the intensities of the myosin layer lines (MLL), the equatorial intensity ratio I(1,1)/I(1,0), and the spacing of the filament lattice are similar in both muscles. At 25 degrees C, particularly at mu = 50 mM, the x-ray patterns exhibited up to six orders of MLL and sharp meridional reflections, signifying that myosin heads (cross-bridges) are distributed in a well-ordered helical array. Decreasing temperature reduced MLL intensities but increased I(1,1)/I(1,0). Decreases in the MLL intensities indicate increasing disorder in the distribution of cross-bridges on the thick filaments surface. In the skeletal muscle, order/disorder is directly correlated with the hydrolysis equilibrium of ATP by myosin, [M.ADP.P(i)]/[M.ATP]. Similar effects of temperature on MLL and similar biochemical ATP hydrolysis pathway found in both types of muscles suggest that the order/disorder states of cardiac cross-bridges may well be correlated with the same biochemical and structural states. This implies that in relaxed cardiac muscle under physiological conditions, the unattached cross-bridges are largely in the M.ADP.P(i) state and with the lowering of the temperature, the equilibrium is increasingly in favor of [M.ATP] and [A.M.ATP]. There appear to be some differences in the diffraction patterns from the two muscles, however. Mainly, in the cardiac muscle, the MLL are weaker, the I(1,1)/I(1,0) ratio tends to be higher, and the lattice spacing D(10), larger. These differences are consistent with the idea that under a wide range of conditions, a greater fraction of cross-bridges is weakly bound to actin in the myocardium.


Subject(s)
Actins/ultrastructure , Myocytes, Cardiac/ultrastructure , Myosins/ultrastructure , Sarcomeres/ultrastructure , Animals , Cells, Cultured , Molecular Conformation , Permeability , Protein Conformation , Rabbits , X-Ray Diffraction
5.
Biophys J ; 91(9): 3370-82, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-16905611

ABSTRACT

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A*M*ADP and A*M) and the weakly bound A*M*ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ("stereospecific" attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A*M*ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A*M*ADP*P(i), however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A*M*ADP*P(i) state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M*ATP, M*ADP*P(i) states and the weakly attached A*M*ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A*M*ADP*P(i). The series of experiments presented in this article were carried out under relaxing conditions at 25 degrees C, where approximately 95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A*M*ADP*P(i) state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M*ADP*P(i) with strongly coupled domains may contribute to the unique attachment configuration: the "primed" myosin heads may function as "transient struts" when attached to the thin filaments.


Subject(s)
Actins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Models, Biological , Muscle Fibers, Skeletal/physiology , Myosins/metabolism , Psoas Muscles/physiology , Actins/chemistry , Adenosine Diphosphate/chemistry , Adenosine Triphosphate/chemistry , Binding Sites , Cell Membrane Permeability , Cells, Cultured , Computer Simulation , Models, Chemical , Models, Molecular , Muscle Fibers, Skeletal/chemistry , Myosins/chemistry , Protein Binding , Protein Conformation , Psoas Muscles/chemistry , Structure-Activity Relationship
6.
Biophys J ; 86(2): 1002-11, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14747335

ABSTRACT

Low angle x-ray diffraction measurements of myofilament lattice spacing (D(1,0)) and equatorial reflection intensity ratio (I(1,1)/I(1,0)) were made in relaxed skinned cardiac trabeculae from rats. We tested the hypothesis that the degree of weak cross-bridge (Xbr) binding, which has been shown to be obligatory for force generation in skeletal muscle, is modulated by changes in lattice spacing in skinned cardiac muscle. Altered weak Xbr binding was detected both by changes in I(1,1)/I(1,0) and by measurements of chord stiffness (chord K). Both measurements showed that, similar to skeletal muscle, the probability of weak Xbr binding at 170-mM ionic strength was significantly enhanced by lowering temperature to 5 degrees C. The effects of lattice spacing on weak Xbr binding were therefore determined under these conditions. Changes in D(1,0), I(1,1)/I(1,0), and chord K by osmotic compression with dextran T500 were determined at sarcomere lengths (SL) of 2.0 and 2.35 micro m. At each SL increasing [dextran] caused D(1,0) to decrease and both I(1,1)/I(1,0) and chord K to increase, indicating increased weak Xbr binding. The results suggest that in intact cardiac muscle increasing SL and decreasing lattice spacing could lead to increased force by increasing the probability of initial weak Xbr binding.


Subject(s)
Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Heart/physiology , Myocardium/ultrastructure , Sarcomeres/physiology , Sarcomeres/ultrastructure , Actin Cytoskeleton/drug effects , Animals , Dextrans/pharmacology , Elasticity/drug effects , Heart/drug effects , In Vitro Techniques , Male , Osmotic Pressure , Rats , Rats, Sprague-Dawley , Sarcomeres/drug effects , Stress, Mechanical , Temperature , X-Ray Diffraction/methods
8.
Biophys J ; 82(4): 2123-33, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11916868

ABSTRACT

A model of cross-bridges binding to actin in the weak binding A*M*ATP state is presented. The modeling was based on the x-ray diffraction patterns from the relaxed skinned rabbit psoas muscle fibers where ATP hydrolysis was inhibited by N-phenylmaleimide treatment (S. Xu, J. Gu, G. Melvin, L. C. Yu. 2002. Biophys. J. 82:2111-2122). Calculations included both the myosin filaments and the actin filaments of the muscle cells, and the binding to actin was assumed to be single headed. To achieve a good fit, considerable flexibility in the orientation of the myosin head and the position of the S1-S2 junction is necessary, such that the myosin head can bind to a nearby actin whereas the tail end was kept in the proximity of the helical track of the myosin filament. Hence, the best-fit model shows that the head binds to actin in a wide range of orientations, and the tail end deviates substantially from its lattice position in the radial direction (approximately 60 A). Surprisingly, the best fit model reveals that the detached head, whose location thus far has remained undetected, seems to be located close to the surface of the myosin filament. Another significant requirement of the best-fit model is that the binding site on actin is near the N terminus of the actin subunit, a position distinct from the putative rigor-binding site. The results support the idea that the essential role played by the weak binding states M*ATP <--> A*M*ATP for force generation lies in its flexibility, because the probability of attachment is greatly increased, compared with the weak binding M*ADP*P(i) <--> A*M*ADP*P(i) states.


Subject(s)
Adenosine Monophosphate/chemistry , Psoas Muscles/metabolism , Animals , Binding Sites , Hydrolysis , Models, Chemical , Models, Molecular , Myosins/chemistry , Protein Binding , Rabbits , X-Ray Diffraction
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