Vijay S. Rao

Direct Regulation of Striated Muscle Myosins by Nitric Oxide and Endogenous Nitrosothiols

Background Nitric oxide (NO) has long been recognized to affect muscle contraction [1], both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins. Principal Findings Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo. Significance These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.

Contractility and kinetics of human fetal and human adult skeletal muscle

•  The contractile properties of human fetal skeletal muscle are unknown. •  Reductionist approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation properties of skeletal muscle from different sources. •  We have tested the contractile properties of human fetal skeletal myofibrils and myosin in comparison with myosin and myofibrils from human adult skeletal muscle and rabbit psoas muscle. •  Human fetal skeletal myofibrils have much slower kinetics of activation and relaxation compared to myofibrils from adult human or rabbit psoas skeletal muscle. •  Investigations using altered substrate and product conditions for both the in vitro motility assay and myofibril mechanics/kinetics indicate that fetal muscle acto‐myosin crossbridges cycle more slowly than, but with similar rate‐limiting steps to, the adult myosin isoforms.

Computational studies of the effect of the S23D/S24D troponin i mutation on cardiac troponin structural dynamics

During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa and pCa50, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1-40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1-161, cTnI 1-172, and cTnT 236-285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca(2+) binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca(2+) binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.

N-terminal phosphorylation of cardiac troponin-I reduces length-dependent calcium sensitivity of contraction in cardiac muscle

•  β‐Adrenergic stimulation is an important control mechanism, matching cardiac output to venous return during increased metabolic demand. •  β‐Adrenergic signalling leads to protein kinase A (PKA) phosphorylation of myofilament proteins cardiac troponin I (cTnI), cardiac myosin binding protein‐C (cMyBP‐C) and titin, but their specific effects on the sarcomeric length (SL) dependence of contraction – which underlies the Frank–Starling Law of the Heart – is debated. •  Recombinant cTnI phosphomimetics were exchanged into cardiac muscle to isolate the effects of cTnI from those of cMyBP‐C/titin phosphorylation on SL‐dependent force–Ca2+ relations and sarcomeric structure. •  Results suggest cTnI or cMyBP‐C/titin phosphorylation, separately or together, eliminate the SL dependence of Ca2+ sensitivity of force, but not maximal force. The reduction occurs particularly at long SL, suggesting effects on thin filament access and crossbridge recruitment. •  The net effect of PKA phosphorylation is to blunt SL dependence of force at submaximal [Ca2+] to maintain elevated systolic function.

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation

Background: R146G and R21C mutations in cardiac TnI are associated with hypertrophic cardiomyopathy. Results: Both mutations blunt PKA-mediated effects on weakening cTnI-cTnC interaction and accelerating myofibril relaxation. Conclusion: Both mutations result in hypercontraction and impaired relaxation, which may contribute to increased risk to traumatic heart failure. Significance: This study increases mechanistic understanding of how single amino acid mutations result in cardiac contractile dysfunction. Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnIR146G or cTnIR21C influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca2+ binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnIWT. cTnIWT, cTnIR146G, and cTnIR21C were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (Tmax) was maintained for cTnIR146G- and cTnIR21C-exchanged myofibrils, and Ca2+ sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnIWT-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnIWT myofibrils, especially at Ca2+ levels that the heart operates in vivo. Importantly, this effect was blunted for cTnIR146G- and cTnIR21C-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnIR146G and cTnIR21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide.

Force Spectroscopy Reveals Multiple “Closed States” of the Muscle Thin Filament

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: “blocked” in the absence of calcium when myosin cannot bind, “closed” when calcium binds troponin and Tm partially covers the myosin binding site, and “open” after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.

Fluorescence Measurements Using Rhodamine-Labeled cTnC Mutants Indicate Little Cooperative Interaction Between Cardiac Thin Filament Regulatory Units

Exchange of mixtures of WT cTnC and mutant cTnC(D65A), which cannot bind Ca2+ at N-terminal site II (“dead” cTnC), reduced maximal Ca2+ activated force (Fmax) with little effect on force-Ca2+ relations and force kinetics in skinned cardiac trabeculae (Gillis et al., J Physiol. 580:561-76, 2007), suggesting interaction between structural regulatory units (RUs; 7 actins, 1 troponin, 1 tropomyosin) along cardiac thin filaments is less than in skeletal muscle (Regnier et al., J Physiol. 540:485-97, 2002). To more directly test that this finding, we exchanged skinned cardiac trabeculae with mixtures of mutant cTnC(C35S) and cTnC(C35S,D65A), with one or the other labeled at Cys 84 with 5’tetramethyl rhodamine (IATR) for dichroism measurements. In trabeculae exchanged with 100% cTnC(C35S)-IATR, dichroism increased in response to both Ca2+ and rigor crossbridges, while trabeculae with 100% Tn containing (cTnC(C35S,D65A)-IATR) had no response to Ca2+, but retained a strong response to rigor crossbridge binding. This response to strong crossbridges allows use of cTnC(C35S,D65A)-IATR to determine if isolated regulatory units containing cTnC(C35S,D65A)-IATR are perturbed by Ca2+-induced active contraction in adjacent “live” RUs. To test this, trabeculae were exchanged with a mixture of 20% functional cTnC(C35S)-IATR and 80% unlabeled cTnC(C35S,D65A), to isolate functional RUs. Fmax decreased but there was little change in the Ca2+-dependence of dichroism compared to trabeculae exchanged with 100% functional cTnC(C35S)-IATR. These data indicate minimal or no apparent spread of activation between adjacent RUs in cardiac muscle, indicating that the apparent cooperativity of force production in cardiac muscle results from interactions between myosin and thin filaments within a thin filament structural regulatory unit. Supported by NIH RO1-HL65497 (Regnier).

Evidence from the Laser Trap for Two Closed States of Tropomyosin

The position of tropomyosin (Tm) on the thin filament is often described by a three state model: 1) blocked, Ca2+ is absent and steric hindrance by Tm blocks myosin from binding to actin, 2) closed, Ca2+ binds to troponin C which unlocks Tm and partially unblocks myosin binding, and 3) open, initial myosin binding shifts Tm further, exposing downstream myosin binding sites and cooperatively activating the thin filament (McKillop and Geeves, 1993). We previously showed that Tm phosphorylation enhances force production by myosin - an effect independent of steric hindrance and thus not predicted by the current three-state model. We therefore tested the hypothesis that Tm phosphorylation affects the on-rate of single actin-myosin bonds. Heavy meromyosin (HMM) was adsorbed to immobilized, nitrocellulose-coated pedestals, and biotinylated actin filaments with natively phosphorylated or dephosphorylated Tm were coupled to streptavidin-coated beads. Using a laser trap, we measured the time necessary for the first bond to form (1/on-rate) between actin and rigor HMM. Measurements were repeated in the presence 10-20 nM N-ethylmaleimide modified myosin-S1 to force Tm from the closed to the open state. Actin-myosin on-rates were increased by Tm, but only in the closed state. Phosphorylation of Tm enhanced this effect. However, the frequency of actin-HMM bond formation was reduced in the closed state in the presence of Tm. Together, these data suggest that there may be at least two closed states of Tm in equilibrium with one another. In the first, Tm hinders myosin binding resulting in a relatively low on-rate, while in the second Tm becomes a “guide” to myosin binding and accelerates the on-rate.