Ranganath Mamidi

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.

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.

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.

Identification of two new regions in the N‐terminus of cardiac troponin T that have divergent effects on cardiac contractile function

To elucidate the cardiac‐specific role of the highly acidic extended N‐terminus of cardiac troponin T (cTnT), the following deletions were made in the N‐terminus of mouse cTnT (McTnT): McTnT1‐44Δ and McTnT45‐74Δ. Thin‐filament activation was assessed after reconstituting the deletion proteins into skinned non‐transgenic mouse cardiac fibres expressing α‐tropomyosin (Tm). Because the N‐terminus of cTnT interacts with the overlapping ends of Tm, we also sought to understand how Tm isoforms modulate the functional effects of the N‐terminus of cTnT. Thus, the deletion proteins were reconstituted into skinned transgenic mouse cardiac fibres expressing β‐Tm. Maximal activation was decreased by McTnT1‐44Δ irrespective of the type of Tm background. Cooperativity was decreased by McTnT45‐74Δ, an effect that was more pronounced under β‐Tm background. We provide the first explicit evidence to show that the cardiac‐specific extended N‐terminus of cTnT contains two distinct regions that have divergent physiological roles in modulating cardiac thin‐filament activation.

Divergent effects of α- and β-myosin heavy chain isoforms on the N terminus of rat cardiac troponin T

Divergent effects of α– and β–myosin heavy chain (MHC) isoforms on contractile behavior arise mainly because of their impact on thin filament cooperativity. The N terminus of cardiac troponin T (cTnT) also modulates thin filament cooperativity. Our hypothesis is that the impact of the N terminus of cTnT on thin filament activation is modulated by a shift from α- to β-MHC isoform. We engineered two recombinant proteins by deleting residues 1–43 and 44–73 in rat cTnT (RcTnT): RcTnT1–43Δ and RcTnT44–73Δ, respectively. Dynamic and steady-state contractile parameters were measured at sarcomere length of 2.3 µm after reconstituting proteins into detergent-skinned muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rat hearts. α-MHC attenuated Ca2+-activated maximal tension (∼46%) in RcTnT1–43Δ fibers. In contrast, β-MHC decreased tension only by 19% in RcTnT1–43Δ fibers. Both α- and β-MHC did not affect tension in RcTnT44–73Δ fibers. The instantaneous muscle fiber stiffness measurements corroborated the divergent impact of α- and β-MHC on tension in RcTnT1–43Δ fibers. pCa50 (-log of [Ca2+]free required for half-maximal activation) decreased significantly by 0.13 pCa units in α-MHC + RcTnT1–43Δ fibers but remained unaltered in β-MHC + RcTnT1–43Δ fibers, demonstrating that β-MHC counteracted the attenuating effect of RcTnT1–43Δ on myofilament Ca2+ sensitivity. β-MHC did not alter the sudden stretch–mediated recruitment of new cross-bridges (ER) in RcTnT1–43Δ fibers, but α-MHC attenuated ER by 36% in RcTnT1–43Δ fibers. The divergent impact of α- and β-MHC on how the N terminus of cTnT modulates contractile dynamics has implications for heart disease; alterations in cTnT and MHC are known to occur via changes in isoform expression or mutations.

Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

The functional significance of the molecular swivel at the head‐to‐tail overlapping ends of contiguous tropomyosin (Tm) dimers in striated muscle is unknown. Contractile measurements were made in muscle fibers from transgenic (TG) mouse hearts that expressed a mutant α‐Tm (TmH276N). We also reconstituted mouse cardiac troponin T (McTnT) N‐terminal deletion mutants, McTnT1–44Δ and McTnT45–74Δ, into muscle fibers from TmH276N. For controls, we used the wild‐type (WT) McTnT because altered effects could be correlated with the mutant forms of McTnT. TmH276N slowed crossbridge (XB) detachment rate (g) by 19%. McTnT1‐44Δ attenuated Ca2+‐activated maximal tension against TmWT (36%) and TmH276N (38%), but sped g only against TmH276N by 35%. The rate of tension redevelopment decreased (17%) only in McTnT1–44Δ + TmH276N fibers. McTnT45–74Δ attenuated tension (19%) and myofilament Ca2+ sensitivity (pCa50=5.93 vs. 6.00 in the control fibers) against TmH276N, but not against TmWT background. Thus, altered XB cycling kinetics decreased the fraction of strongly bound XBs in McTnT1‐44Δ + TmH276N fibers, whereas diminished thin‐filament cooperativity attenuated tension in McTnT45‐74Δ + TmH276N fibers. In summary, our study is the first to show that the interplay between the N terminus of cTnT and the overlapping ends of contiguous Tm effectuates different states of Tm on the actin filament. —Mamidi, R., Michael, J. J., Muthuchamy, M., Chandra, M., Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin. FASEB J. 27, 3848–3859 (2013). www.fasebj.org

Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~36% decrease in Ca(2+)-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics.

Structural and Kinetic Effects of PAK3 phosphorylation mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament

Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca(2+), but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca(2+)-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca(2+)-induced changes in cTnI-cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC-cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca(2+) by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca(2+) dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI.