Investigation of thin filament near‐neighbour regulatory unit interactions during force development in skinned cardiac and skeletal muscle

Ca2+‐dependent activation of striated muscle involves cooperative interactions of cross‐bridges and thin filament regulatory proteins. We investigated how interactions between individual structural regulatory units (RUs; 1 tropomyosin, 1 troponin, 7 actins) influence the level and rate of demembranated (skinned) cardiac muscle force development by exchanging native cardiac troponin (cTn) with different ratio mixtures of wild‐type (WT) cTn and cTn containing WT cardiac troponin T/I + cardiac troponin C (cTnC) D65A (a site II inactive cTnC mutant). Maximal Ca2+‐activated force (Fmax) increased in less than a linear manner with WT cTn. This contrasts with results we obtained previously in skeletal fibres (using sTnC D28A, D65A) where Fmax increased in a greater than linear manner with WT sTnC, and suggests that Ca2+ binding to each functional Tn activates < 7 actins of a structural regulatory unit in cardiac muscle and > 7 actins in skeletal muscle. The Ca2+ sensitivity of force and rate of force redevelopment (ktr) was leftward shifted by 0.1–0.2 −log [Ca2+] (pCa) units as WT cTn content was increased, but the slope of the force–pCa relation and maximal ktr were unaffected by loss of near‐neighbour RU interactions. Cross‐bridge inhibition (with butanedione monoxime) or augmentation (with 2 deoxy‐ATP) had no greater effect in cardiac muscle with disruption of near‐neighbour RU interactions, in contrast to skeletal muscle fibres where the effect was enhanced. The rate of Ca2+ dissociation was found to be > 2‐fold faster from whole cardiac Tn compared with skeletal Tn. Together the data suggest that in cardiac (as opposed to skeletal) muscle, Ca2+ binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca2+ binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca2+ binding affinity.

[1]  M. Geeves,et al.  Dynamics of the muscle thin filament regulatory switch: the size of the cooperative unit. , 1994, Biophysical journal.

[2]  E. Homsher,et al.  Modulation of Contractile Activation in Skeletal Muscle by a Calcium-insensitive Troponin C Mutant* , 2001, The Journal of Biological Chemistry.

[3]  M. Regnier,et al.  Cardiac troponin C (TnC) and a site I skeletal TnC mutant alter Ca2+versus crossbridge contribution to force in rabbit skeletal fibres , 2005, The Journal of physiology.

[4]  J. Potter.  Preparation of troponin and its subunits. , 1982, Methods in enzymology.

[5]  M J Kushmerick,et al.  Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. , 1988, Biophysical journal.

[6]  J. M. Robinson,et al.  Switching of troponin I: Ca(2+) and myosin-induced activation of heart muscle. , 2004, Journal of molecular biology.

[7]  Kenneth S. Campbell,et al.  Cooperative Mechanisms in the Activation Dependence of the Rate of Force Development in Rabbit Skinned Skeletal Muscle Fibers , 2001, The Journal of general physiology.

[8]  A. M. Gordon,et al.  Familial hypertrophic cardiomyopathy mutations in troponin I (K183D, G203S, K206Q) enhance filament sliding. , 2003, Physiological genomics.

[9]  E. Sonnenblick,et al.  The role of troponin C in the length dependence of Ca(2+)‐sensitive force of mammalian skeletal and cardiac muscles. , 1991, The Journal of physiology.

[10]  Svetlana B Tikunova,et al.  Effect of hydrophobic residue substitutions with glutamine on Ca(2+) binding and exchange with the N-domain of troponin C. , 2002, Biochemistry.

[11]  L. Tobacman,et al.  Cooperative Effect of Calcium Binding to Adjacent Troponin Molecules on the Thin Filament-Myosin Subfragment 1 MgATPase Rate* , 1997, The Journal of Biological Chemistry.

[12]  A. M. Gordon,et al.  Thin filament near‐neighbour regulatory unit interactions affect rabbit skeletal muscle steady‐state force‐Ca2+ relations , 2002, The Journal of physiology.

[13]  A. M. Gordon,et al.  Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C. , 1993, Biophysical journal.

[14]  J. Potter.  [22] Preparation of troponin and its subnits , 1982 .

[15]  D. Martyn,et al.  Cardiac length dependence of force and force redevelopment kinetics with altered cross-bridge cycling. , 2004, Biophysical journal.

[16]  R. Moss,et al.  Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels. , 1983, Analytical biochemistry.

[17]  K. Jakobs,et al.  Dual regulation of adenylate cyclase. A signal transduction mechanism of membrane receptors , 2005, Basic Research in Cardiology.

[18]  R. Moss,et al.  The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers , 1985, The Journal of general physiology.

[19]  J. Johnson,et al.  Modulation of Ca2+ exchange with the Ca(2+)-specific regulatory sites of troponin C. , 1994, The Journal of biological chemistry.

[20]  E. Homsher,et al.  ATP analogs and muscle contraction: mechanics and kinetics of nucleoside triphosphate binding and hydrolysis. , 1998, Biophysical journal.

[21]  R Craig,et al.  Steric-model for activation of muscle thin filaments. , 1997, Journal of molecular biology.

[22]  R. Ludescher,et al.  Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin. , 1999, Biochemistry.

[23]  E. Homsher,et al.  Regulation of the cross-bridge transition from a weakly to strongly bound state in skinned rabbit muscle fibers. , 1995, The American journal of physiology.

[24]  M. Regnier,et al.  2-deoxy-ATP enhances contractility of rat cardiac muscle. , 2000, Circulation research.

[25]  T. Irving,et al.  Myofilament Calcium Sensitivity in Skinned Rat Cardiac Trabeculae: Role of Interfilament Spacing , 2002, Circulation research.

[26]  R. Moss,et al.  Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle , 1989, The Journal of general physiology.

[27]  R J Barsotti,et al.  Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle. , 2004, Biophysical journal.

[28]  A. M. Gordon,et al.  Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition. , 1999, Biophysical journal.

[29]  G. Tibbits,et al.  Increasing mammalian cardiomyocyte contractility with residues identified in trout troponin C. , 2005, Physiological genomics.

[30]  B. Brenner,et al.  The cross-bridge cycle in muscle. Mechanical, biochemical, and structural studies on single skinned rabbit psoas fibers to characterize cross-bridge kinetics in muscle for correlation with the actomyosin-ATPase in solution. , 1986, Basic research in cardiology.

[31]  B. Sykes,et al.  Structural based insights into the role of troponin in cardiac muscle pathophysiology , 2004, Journal of Muscle Research & Cell Motility.

[32]  F. Reinach,et al.  The troponin complex and regulation of muscle contraction , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[33]  A. M. Gordon,et al.  Kinetic Studies of Calcium Binding to the Regulatory Site of Troponin C from Cardiac Muscle (*) , 1996, The Journal of Biological Chemistry.

[34]  R Craig,et al.  Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments. , 2000, Journal of molecular biology.

[35]  M. Regnier,et al.  Regulation of skeletal muscle tension redevelopment by troponin C constructs with different Ca2+ affinities. , 1999, Biophysical journal.

[36]  A. M. Gordon,et al.  A Kinetic Model for the Binding of Ca2+ to the Regulatory Site of Troponin from Cardiac Muscle* , 1997, The Journal of Biological Chemistry.

[37]  E. Homsher,et al.  Regulation of contraction in striated muscle. , 2000, Physiological reviews.

[38]  A. M. Gordon,et al.  Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle. , 2001, Biophysical journal.

[39]  F. Fuchs,et al.  Force, length, and Ca(2+)-troponin C affinity in skeletal muscle. , 1991, The American journal of physiology.

[40]  W. Lehman,et al.  Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy. , 1999, Biophysical journal.

[41]  D. Martyn,et al.  Thin‐filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres , 2007, The Journal of physiology.

[42]  D. Martyn,et al.  Calcium regulation of tension redevelopment kinetics with 2-deoxy-ATP or low [ATP] in rabbit skeletal muscle. , 1998, Biophysical journal.

[43]  B. Sykes,et al.  Structures of the troponin C regulatory domains in the apo and calcium-saturated states , 1995, Nature Structural Biology.

[44]  B. Sykes,et al.  Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C. , 1999, Biochemistry.

[45]  B. Sykes,et al.  Structure of the C-domain of Human Cardiac Troponin C in Complex with the Ca2+ Sensitizing Drug EMD 57033* , 2001, The Journal of Biological Chemistry.

[46]  W. Dong,et al.  Calcium-induced conformational change in cardiac troponin C studied by fluorescence probes attached to Cys-84. , 1996, Biochimica et biophysica acta.

[47]  J. Putkey,et al.  The kinetic cycle of cardiac troponin C: Calcium binding and dissociation at site II trigger slow conformational rearrangements , 1998, Protein science : a publication of the Protein Society.

[48]  A. M. Gordon,et al.  Ca2+ - and cross-bridge-dependent changes in N- and C-terminal structure of troponin C in rat cardiac muscle. , 2001, Biophysical journal.

[49]  E. Eisenberg,et al.  Cooperative binding of myosin subfragment-1 to the actin-troponin-tropomyosin complex. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[50]  Yi Dai,et al.  Phosphorylation of Extracellular Signal-Regulated Kinase in Primary Afferent Neurons by Noxious Stimuli and Its Involvement in Peripheral Sensitization , 2002, The Journal of Neuroscience.

[51]  J. Leiden,et al.  Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant. , 1996, Biophysical journal.