The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-microm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titin's elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titin's Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.
␣B-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. ␣B-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309 -319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of ␣B-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. ␣B-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, ␣B-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between ␣B-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by ␣B-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with ␣B-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.␣B-crystallin is one of several crystallins in the vertebrate lens. An important function of the protein is to act as a chaperone preventing partially unfolded proteins from forming aggregates that would make the lens opaque (1, 2). ␣B-crystallin is a member of the family of small heat shock proteins (sHSPs)1 . These are chaperones in multisubunit complexes that bind to proteins in the early stages of denaturation, holding them in a folding-competent state. ␣B-crystallin is also found in tissues other than the lens, including cardiac and skeletal muscle (3). The ␣B-crystallin content of cardiac muscle is 3-5% of the total soluble protein (4), and it protects the fibers from the effects of ischemia, preventing extensive structural damage (5-7). ␣B-crystallin moves from general distribution in the cytosol to myofibrils following ischemia (8, 9). Recent immunoelectron microscopy of rat heart has shown that ␣B-crystallin is in a narrow region of the I-band rather than in the Z-disc, as was previously thought, and is also associated with desmin filaments connecting neighboring myofibrils (10). When actin was extracted from pig heart myofibrils, ␣B-crystallin remained, suggesting that the protein was associated with titin in the I-band rather than with actin; and this was supported by co-purification of ␣B-crystallin with titin (10).Titin is a large (3 MDa) protein in striated muscle that spans half the sarcomere, forming an extens...
Abstract-We have tested the hypothesis that decreased functioning of creatine kinase (CK) at sites of energy production and utilization may contribute to alterations in energy fluxes and calcium homeostasis in congestive heart failure (CHF). Heart failure was induced by aortic banding in 3-week-old rats. Myofilaments, sarcoplasmic reticulum (SR), mitochondrial functions, and CK compartmentation were studied in situ using selective membrane permeabilization of left ventricular fibers with detergents (saponin for mitochondria and SR and Triton X-100 for myofibrils). Seven months after surgery, animals were in CHF. A decrease in total CK activity could be accounted for by a 4-fold decrease in activity and content (Western blots) of mitochondrial CK and a 30% decrease in M isoform of CK (MM-CK) activity. In myofibrils, maximal force, crossbridge kinetics, and ␣-myosin heavy-chain expression decreased, whereas calcium sensitivity of tension development remained unaltered. Myofibrillar CK efficacy was unchanged. Calcium uptake capacities of SR were estimated from the surface of caffeine-induced tension transient (S Ca ) after loading with different substrates. In CHF, S Ca decreased by 23%, and phosphocreatine was 2 times less efficient in enhancing calcium uptake.Oxidative capacities of the failing myocardium measured as oxygen consumption per gram of fiber dry weight decreased by 28%. Moreover, the control of respiration by creatine, ADP, and AMP was severely impaired. Our observations provide evidence that alterations in CK compartmentation may contribute to alterations of energy fluxes and calcium homeostasis in CHF. (Circ Res. 1999;85:68-76.)Key Words: mitochondrial respiration Ⅲ myofibril Ⅲ compartmentation Ⅲ sarcoplasmic reticulum Ⅲ skinned fiber T he mechanisms underlying the decline in cardiac pump function in heart failure are incompletely understood. They lead to a gradual increase in left ventricular (LV) end diastolic pressure and a decrease in systolic pressure. In the past decade attention has been focused on the alterations of the various steps in excitation-contraction coupling and intracellular calcium homeostasis, 1-3 whereas the possible involvement of a mismatch in energy supply and demand has received less attention. 4 -6 It has been shown recently that in human heart failure, there is a generalized alteration of the creatine kinase (CK) system with a decrease in total enzyme activity and velocity and alteration in the isoenzyme pattern, which could contribute to the pathogenesis of heart failure. 5 Moreover, in patients with dilated cardiomyopathy, the phosphocreatine (PCr)/ATP ratio, governed by CK activity, may be a predictor of both total and cardiovascular mortality. 7 However, the precise cellular mechanisms by which altered CK may compromise energy fluxes and contractility are not well understood.CK is an important enzyme involved in energy maintenance and energy transfer in muscle and brain cells. It catalyzes the reversible transfer of a phosphate moiety between ATP and creatine. Four d...
Abstract-Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads. Key Words: mitochondria Ⅲ sarcoplasmic reticulum Ⅲ myofibrils Ⅲ creatine kinase Ⅲ knockout mice D ifferentiation and maturation of adult mammalian muscle cells lead to complex specialization and organization. In cardiac cells, specialized cellular functions are highly organized within structural and functional compartments. Energy-consuming processes are localized to the sarcoplasmic reticulum (SR) and myofibrillar compartments, while energy production occurs mainly within mitochondria. Muscle cells contain complex and specialized energy transfer systems, which efficiently link energy production and utilization. One such system is the family of creatine kinase (CK) isoenzymes that catalyze the reversible transfer of a phosphate moiety between creatine (Cr) and ATP. The mitochondrial sarcomeric isoenzyme (mi-CK) is bound to the outer surface of the inner mitochondrial membrane so that ATP generated by oxidative phosphorylation is transphosphorylated to phosphocreatine (PCr). 1-3 On the other hand, the cytosolic isoenzyme (MM-CK) that is structurally associated with myofibrils and SR membranes can use PCr to rephosphorylate all of the ADP produced by the ATPases and thus provide enough energy for normal contractile kinetics or SR calcium uptake. 4 ...
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