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

During ß-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.

Contractility and kinetics of human fetal and human adult skeletal muscle.

Little is known about the contraction and relaxation properties of fetal skeletal muscle, and measurements thus far have been made with non-human mammalian muscle. Data on human fetal skeletal muscle contraction are lacking, and there are no published reports on the kinetics of either fetal or adult human skeletal muscle myofibrils. Understanding the contractile properties of human fetal muscle would be valuable in understanding muscle development and a variety of muscle diseases that are associated with mutations in fetal muscle sarcomere proteins. Therefore, we characterised the contractile properties of developing human fetal skeletal muscle and compared them to adult human skeletal muscle and rabbit psoas muscle. Electron micrographs showed human fetal muscle sarcomeres are not fully formed but myofibril formation is visible. Isolated myofibril mechanical measurements revealed much lower specific force, and slower rates of isometric force development, slow phase relaxation, and fast phase relaxation in human fetal when compared to human adult skeletal muscle. The duration of slow phase relaxation was also significantly longer compared to both adult groups, but was similarly affected by elevated ADP. F-actin sliding on human fetal skeletal myosin coated surfaces in in vitro motility (IVM) assays was much slower compared with adult rabbit skeletal myosin, though the Km(app) (apparent (fitted) Michaelis-Menten constant) of F-actin speed with ATP titration suggests a greater affinity of human fetal myosin for nucleotide binding. Replacing ATP with 2 deoxy-ATP (dATP) increased F-actin speed for both groups by a similar amount. Titrations of ADP into IVM assays produced a similar inhibitory affect for both groups, suggesting ADP binding may be similar, at least under low load. Together, our results suggest slower but similar mechanisms of myosin chemomechanical transduction for human fetal muscle that may also be limited by immature myofilament structure.

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

Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for ß-adrenergic modulation of cardiac contractility. In myofilaments PKA targets troponin I (cTnI), myosin binding protein-C (cMyBP-C) and titin. We studied how this affects the sarcomere length (SL) dependence of force-pCa relations in demembranated cardiac muscle. To distinguish cTnI from cMyBP-C/titin phosphorylation effects on the force-pCa relationship, endogenous troponin (Tn) was exchanged in rat ventricular trabeculae with either wild-type (WT) Tn, non-phosphorylatable cTnI (S23/24A) Tn or phosphomimetic cTnI (S23/24D) Tn. PKA cannot phosphorylate either cTnI S23/24 variant, leaving cMyBP-C/titin as PKA targets. Force was measured at 2.3 and 2.0 µm SL. Decreasing SL reduced maximal force (F(max)) and Ca(2+) sensitivity of force (pCa(50)) similarly with WT and S23/24A trabeculae. PKA treatment of WT and S23/24A trabeculae reduced pCa(50) at 2.3 but not at 2.0 µm SL, thus eliminating the SL dependence of pCa(50). In contrast, S23/24D trabeculae reduced pCa(50) at both SL values, primarily at 2.3 µm, also eliminating SL dependence of pCa(50). Subsequent PKA treatment moderately reduced pCa(50) at both SLs. At each SL, F(max) was unaffected by either Tn exchange and/or PKA treatment. Low-angle X-ray diffraction was performed to determine whether pCa(50) shifts were associated with changes in myofilament spacing (d(1,0)) or thick-thin filament interaction. PKA increased d(1,0) slightly under all conditions. The ratios of the integrated intensities of the equatorial X-ray reflections (I(1,1)/I(1,0)) indicate that PKA treatment increased crossbridge proximity to thin filaments under all conditions. The results suggest that phosphorylation by PKA of either cTnI or cMyBP-C/titin independently reduces the pCa(50) preferentially at long SL, possibly through reduced availability of thin filament binding sites (cTnI) or altered crossbridge recruitment (cMyBP-C/titin). Preferential reduction of pCa(50) at long SL may not reduce cardiac output during periods of high metabolic demand because of increased intracellular Ca(2+) during ß-adrenergic stimulation.

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.

Direct regulation of striated muscle myosins by nitric oxide and endogenous nitrosothiols.

BACKGROUND: Nitric oxide (NO) has long been recognized to affect muscle contraction, 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.

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit.

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.

Alterations to myofibrillar protein function in nonischemic regions of the heart early after myocardial infarction.

Remote-zone left ventricular dysfunction (LVD) contributes to global reductions in contractile function after localized myocardial infarction (MI). However, the molecular mechanisms underlying this form of LVD are not clear. This study tested the hypothesis that myofibrillar protein function is directly affected in remote-zone LVD early after MI. Cardiac myosin and native thin filaments were purified from mouse myocardium taken from both the nonnecrotic zone adjacent to and the nonischemic zone remote from an infarct induced by 1 h of coronary occlusion followed by 24 h of reperfusion. Thin filament velocities were measured using the in vitro motility assay. Results showed that overall function was significantly reduced in samples from both the adjacent (43 +/- 12% of control, n = 7) and remote (53 +/- 8% of control, n = 13) zones when compared with control proteins (P < 0.05). Myosin from the remote zone propelled control thin filaments at reduced velocities similar to those measured above. In contrast, the Ca(2+) sensitivity of remote-zone thin filaments over control myosin was unchanged from control thin filaments (half-maximal at pCa 6.32 +/- 0.08 and 6.27 +/- 0.06, respectively) but showed a 20% increase in velocity at saturating Ca(2+) that parallels an increase in tropomyosin phosphorylation. Myosin dysfunction may be related to oxidation of cysteines in the myosin heavy chains or carbonylation of myosin binding protein-C. We hypothesize that phosphorylation of tropomyosin may serve a compensatory role, augmenting contraction during periods of oxidative stress when myosin function is compromised.