Murali Chandra

Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation A NOVEL MECHANISM FOR INCREASING TENSION AND REDUCING TENSION COST

Nebulin is a giant filamentous F-actin-binding protein (∼800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (−log[Ca2+]) and force-ATPase relations, as well as the rate of tension re-development (ktr) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons−1 mm−1 s−1) and reductions in ktr (4.7 versus 7.3 s−1), calcium sensitivity (pCa50 5.74 versus 5.90), and cooperativity of activation (nH 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.

Cardiac troponin T mutations: correlation between the type of mutation and the nature of myofilament dysfunction in transgenic mice

1 The heterogenic nature of familial hypertrophic cardiomyopathy (FHC) in humans suggests a link between the type of mutation and the nature of patho‐physiological alterations in cardiac myocytes. Exactly how FHC‐associated mutations in cardiac troponin T (cTnT) lead to impaired cardiac function is unclear. 2 We measured steady‐state isometric force and ATPase activity in detergent‐skinned cardiac fibre bundles from three transgenic (TG) mouse hearts in which 50, 92 and 6 % of the native cTnT was replaced by the wild type (WT) cTnT, R92Q mutant cTnT (R92Q) and the C‐terminal deletion mutant of cTnT (cTnTDEL), respectively. 3 Normalized pCa‐tension relationships of R92Q and cTnTDEL fibres demonstrated a significant increase in sensitivity to Ca2+ at short (2.0 μm) and long (2.3 μm) sarcomere lengths (SL). At short SL, the pCa50 values, representing the midpoint of the pCa‐tension relationship, were 5.69 ± 0.01, 5.96 ± 0.01 and 5.81 ± 0.01 for WT, R92Q and cTnTDEL fibres, respectively. At long SL, the pCa50 values were 5.81 ± 0.01, 6.08 ± 0.01 and 5.95 ± 0.01 for WT, R92Q and cTnTDEL fibres, respectively. 4 The difference in pCa required for half‐maximal activation (ΔpCa50) at short and long SL was 0.12 ± 0.01 for the R92Q (92 %) TG fibres, which is significantly less than the previously reported ΔpCa50 value of 0.29 ± 0.02 for R92Q (67 %) TG fibres. 5 At short SL, Ca2+‐activated maximal tension in both R92Q and cTnTDEL fibres decreased significantly (24 and 21 %, respectively; P < 0.005), with no corresponding decrease in Ca2+‐activated maximal ATPase activity. Therefore, at short SL, the tension cost in R92Q and cTnTDEL fibres increased by 35 and 29 %, respectively (P < 0.001). 6 The fibre bundles reconstituted with the recombinant mutant cTnTDEL protein developed only 37 % of the Ca2+‐activated maximal force developed by recombinant WT cTnT reconstituted fibre bundles, with no apparent changes in Ca2+ sensitivity. 7 Our data indicate that an important mutation‐linked effect on cardiac function is the result of an inefficient use of ATP at the myofilament level. Furthermore, the extent of the mutation‐induced dysfunction depends not only on the nature of the mutation, but also on the concentration of the mutant protein in the sarcomere.

Structure of the C-domain of Human Cardiac Troponin C in Complex with the Ca2+ Sensitizing Drug EMD 57033

Ca2+ binding to cardiac troponin C (cTnC) triggers contraction in heart muscle. In heart failure, myofilaments response to Ca2+ are often altered and compounds that sensitize the myofilaments to Ca2+possess therapeutic value in this syndrome. One of the most potent and selective Ca2+ sensitizers is the thiadiazinone derivative EMD 57033, which increases myocardial contractile function bothin vivo and in vitro and interacts with cTnCin vitro. We have determined the NMR structure of the 1:1 complex between Ca2+-saturated C-domain of human cTnC (cCTnC) and EMD 57033. Favorable hydrophobic interactions between the drug and the protein position EMD 57033 in the hydrophobic cleft of the protein. The drug molecule is orientated such that the chiral group of EMD 57033 fits deep in the hydrophobic pocket and makes several key contacts with the protein. This stereospecific interaction explains why the (−)-enantiomer of EMD 57033 is inactive. Titrations of the cCTnC·EMD 57033 complex with two regions of cardiac troponin I (cTnI34–71 and cTnI128–147) reveal that the drug does not share a common binding epitope with cTnI128–147 but is completely displaced by cTnI34–71. These results have important implications for elucidating the mechanism of the Ca2+ sensitizing effect of EMD 57033 in cardiac muscle contraction.

Left Ventricular and Myocardial Function in Mice Expressing Constitutively Pseudophosphorylated Cardiac Troponin I

Rationale: Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction. Objective: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnIPKC-P) expressing mutant cTnI (S43E, S45E, T144E). Methods and Results: Two-dimensional gel analysis showed 7.2±0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model–based analysis for integrative interpretation. Compared to wild-type mice, cTnIPKC-P mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, Fmax). Additionally, cTnIPKC-P mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnIPKC-P mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnIPKC-P mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state. Conclusions: A modest increase in PKC-induced cTnI phosphorylation (≈7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).

Myostatin represses physiological hypertrophy of the heart and excitation–contraction coupling

Although myostatin negatively regulates skeletal muscle growth, its function in heart is virtually unknown. Herein we demonstrate that it inhibits basal and IGF‐stimulated proliferation and differentiation and also modulates cardiac excitation–contraction (EC) coupling. Loss of myostatin induced eccentric hypertrophy and enhanced cardiac responsiveness to β‐adrenergic stimulation in vivo. This was due to myostatin null ventricular myocytes having larger [Ca2+]i transients and contractions and responding more strongly to β‐adrenergic stimulation than wild‐type cells. Enhanced cardiac output and β‐adrenergic responsiveness of myostatin null mice was therefore due to increased SR Ca2+ release during EC coupling and to physiological hypertrophy, but not to enhanced myofilament function as determined by simultaneous measurement of force and ATPase activity. Our studies support the novel concept that myostatin is a repressor of physiological cardiac muscle growth and function. Thus, the controlled inhibition of myostatin action could potentially help repair damaged cardiac muscle by inducing physiological hypertrophy.

Functions of Stretch Activation in Heart Muscle

Stretch activation is an intrinsic length-sensing mechanism that allows muscle to function with an autonomous regulation that reduces reliance on extrinsic regulatory systems. This autonomous regulation is most dramatic in asynchronous insect flight muscle and gives rise to wing beat frequencies

Effects of R92 mutations in mouse cardiac troponin T are influenced by changes in myosin heavy chain isoform

One limitation in understanding how different familial hypertrophic cardiomyopathy (FHC)-related mutations lead to divergent cardiac phenotypes is that such mutations are often studied in transgenic (TG) mouse hearts which contain a fast cycling myosin heavy chain isoform (α-MHC). However, the human heart contains a slow cycling MHC isoform (β-MHC). Given the physiological significance of MHC-troponin interplay effects on cardiac contractile function, we hypothesized that cardiac troponin T (cTnT) mutation-mediated effects on contractile function depend on the type of MHC isoform present in the sarcomere. We tested our hypothesis using two variants of cTnT containing mutations at FHC hotspot R92 (R92L or R92Q), expressed against either an α-MHC or β-MHC background in TG mouse hearts. One finding from our study was that R92L attenuated the length-dependent increase in tension and abolished the length-dependent increase in myofilament Ca(2+) sensitivity only when β-MHC was present. In addition, α- and β-MHC isoforms differentially affected how R92 mutations altered crossbridge (XB) recruitment dynamics. For example, the rate of XB recruitment was faster in R92L or R92Q fibers when β-MHC was present, but was unaffected when α-MHC was present. The R92Q mutation sped XB detachment in the presence of β-MHC, but not in the presence of α-MHC. R92Q affected the XB strain-dependent influence on XB recruitment dynamics, an effect not observed for R92L. Our findings have major implications for understanding not only the divergent effects of R92 mutations on cardiac phenotype, but also the distinct effects of MHC isoforms in determining the outcome of mutations in cTnT.

Model representation of the nonlinear step response in cardiac muscle

Motivated by the need for an analytical tool that can be used routinely to analyze data collected from isolated, detergent-skinned cardiac muscle fibers, we developed a mathematical model for representing the force response to step changes in muscle length (i.e., quick stretch and release). Our proposed model is reasonably simple, consisting of only five parameters representing: (1) the rate constant by which length change–induced distortion of elastic elements is dissipated; (2) the stiffness of the muscle fiber; (3) the amplitude of length-mediated recruitment of stiffness elements; (4) the rate constant by which this length-mediated recruitment takes place; and (5) the magnitude of the nonlinear interaction term by which distortion of elastic elements affects the number of recruited stiffness elements. Fitting this model to a family of force recordings representing responses to eight amplitudes of step length change (±2.0% baseline muscle length in 0.5% increments) enabled four things: (1) reproduction of all the identifiable features seen in a family of force responses to both positive and negative length changes; (2) close fitting of all records from the whole family of these responses with very little residual error; (3) estimation of all five model parameters with a great degree of certainty; and (4) importantly, ready discrimination between cardiac muscle fibers with different contractile regulatory proteins but showing only subtly different contractile function. We recommend this mathematical model as an analytic tool for routine use in studies of cardiac muscle fiber contractile function. Such model-based analysis gives novel insight to the contractile behavior of cardiac muscle fibers, and it is useful for characterizing the mechanistic effects that alterations of cardiac contractile proteins have on cardiac contractile function.

Structural Dynamics of C-domain of Cardiac Troponin I Protein in Reconstituted Thin Filament

Background: The kinetics and dynamics of the C-domain of cTnI were studied. Results: Fluorescence anisotropy data show support for the fly casting model and a fourth state of thin filament activation. Conclusion: The fly casting model holds true in the thin filament, but the presence of S1 modulates the process. Significance: Our study provides information on the role of the cTnI C-domain in thin filament regulation. The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain (MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca2+ binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca2+-bound to the Ca2+-free dynamic states were significantly faster (53.2–116.8 s−1) than that of the IR (14.1 s−1). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca2+ dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca2+ signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation.

Structural mapping of single cysteine mutants of cardiac troponin I.

The global conformation of cardiac muscle troponin I (cTnI) was investigated with single‐cysteine mutants by using a combination of sulfhydryl reactivity and fluorescence resonance energy transfer (FRET) to determine cysteine accessibility and intersite distances. The reactivity was determined with a fluorescent reagent for its reaction with cysteine residues singly located at positions 5, 40, 81, 98, 115, 133, 150, 167, and 192. FRET measurements were made by using the endogenous single Trp‐192 as the energy donor and an acceptor probe covalently attached to the cysteines as energy acceptor. The results suggest an open and extended conformation of cTnI with a large curvature in which the cysteines are highly exposed to the solvent. These conformational features are largely retained in the segment between residues 40 and 192 upon phosphorylation at Ser‐23 and Ser‐24. The sulfhydryl groups of the Cys‐133 and Cys‐150 of the cTnI incorporated into the binary cTnC‐cTnI and fully reconstituted troponin complexes experience large reduced exposure resulting from the binding of Ca2+ to the regulatory site of cTnC, suggesting that key regions of cTnI involved in activation become highly shielded upon activation. In the cTnC‐cTnI complex, every intramolecular distance in the cTnI is lengthened and the overall conformation of the bound cTnI remains elongated with reduced exposure for the cysteines. The global conformation of the troponin C‐troponin I complex from cardiac muscle has an elongated shape with constrained flexibility. The highly flexible nature of the N‐terminal extension of cTnI is preserved in the complex, suggesting that this segment of cTnI is either not bound or only loosely bound to the C‐domain of cTnC. Proteins 2000;41:438–447.