Cardiac Tropomyosin Research Articles

Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation.

We characterized a novel genetic variant c.292G > A (p.E98K) in the TPM1 gene encoding cardiac tropomyosin 1.1 isoform (Tpm1.1), found in a proband with a phenotype of complex cardiomyopathy with conduction dysfunction and slow progressive neuromuscular involvement. To understand the molecular mechanism by which this mutation impairs cardiac function, we produced recombinant Tpm1.1 carrying an E98K substitution and studied how this substitution affects the structure of the Tpm1.1 molecule and its functional properties. The results showed that the E98K substitution in the N-terminal part of the Tpm molecule significantly destabilizes the C-terminal part of Tpm, thus indicating a long-distance destabilizing effect of the substitution on the Tpm coiled-coil structure. The E98K substitution did not noticeably affect Tpm's affinity for F-actin but significantly impaired Tpm's regulatory properties. It increased the Ca2+ sensitivity of the sliding velocity of regulated thin filaments over cardiac myosin in an in vitro motility assay and caused an incomplete block of the thin filament sliding at low Ca2+ concentrations. The incomplete motility block in the absence of Ca2+ can be explained by the loosening of the Tpm interaction with troponin I (TnI), thus increasing Tpm mobility on the surface of an actin filament that partially unlocks the myosin binding sites. This hypothesis is supported by the molecular dynamics (MD) simulation that showed that the E98 Tpm residue is involved in hydrogen bonding with the C-terminal part of TnI. Thus, the results allowed us to explain the mechanism by which the E98K Tpm mutation impairs sarcomeric function and myocardial relaxation.

De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), caused by mutations in thin filament proteins, manifests as moderate cardiac hypertrophy and is associated with sudden cardiac death (SCD). We identified a new de novo variant, c.656A>T (p.D219V), in the TPM1 gene encoding cardiac tropomyosin 1.1 (Tpm) in a young SCD victim with post-mortem-diagnosed HCM. We produced recombinant D219V Tpm1.1 and studied its structural and functional properties using various biochemical and biophysical methods. The D219V mutation did not affect the Tpm affinity for F-actin but increased the thermal stability of the Tpm molecule and Tpm-F-actin complex. The D219V mutation significantly increased the Ca2+ sensitivity of the sliding velocity of thin filaments over cardiac myosin in an in vitro motility assay and impaired the inhibition of the filament sliding at low Ca2+ concentration. The molecular dynamics (MD) simulation provided insight into a possible molecular mechanism of the effect of the mutation that is most likely a cause of the weakening of the Tpm interaction with actin in the "closed" state and so makes it an easier transition to the “open” state. The changes in the Ca2+ regulation of the actin-myosin interaction characteristic of genetic HCM suggest that the mutation is likely pathogenic.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Differentiation of Mouse Embryonic Fibroblasts (MEFs) into Cardiomyocytes Using Human-Derived Cardiac Inducing RNA (CIR)

The present study explores an RNA we have discovered in human heart that induces differentiation of mouse embryonic stem cells and human induced pluripotent stem cells into cardiomyocytes in vitro. We have designated this RNA as Cardiac Inducing RNA or CIR. We now find that CIR also induces mouse embryonic fibroblasts (MEF) to form cardiomyocytes in vitro. For these studies, human-derived CIR is transfected into MEF using lipofectamine. The CIR-transfected mouse fibroblasts exhibit spindle-shaped cells, characteristic of myocardial cells in culture and express cardiac-specific troponin-T and cardiac tropomyosin. As such, the CIR-induced conversion of the fibroblasts into cardiomyocytes in vitro appears to take place without initial dedifferentiation into pluripotent stem cells. Instead, after CIR transfection using a lipofectamine transfection system, over the next 8 days there appears to be a direct transdifferentiation of ˃80% of the cultured fibroblasts into definitive cardiomyocytes. Fewer than ˂7% of the untreated controls using non-active RNA or lipofectamine by itself show cardiomyocyte characteristics. Thus, discovery of CIR may hold significant potential for future use in repair/regeneration of damaged myocardial tissue in humans after myocardial infarction or other disease processes such that affected patients may be able to return to pre-heart-disease activity levels.

Qualitative and quantitative evaluation of TPM transcripts and proteins in developing striated chicken muscles indicate TPM4α is the major sarcomeric cardiac tropomyosin from early embryonic life to adulthood.

The chicken has been used since the 1980s as an animal model for developmental studies regarding tropomyosin isoform diversity in striated muscles, however, the pattern of expression of transcripts as well as the corresponding TPM proteins of various tropomyosin isoforms in avian hearts are not well documented. In this study, using conventional and qRT-PCR, we report the expression of transcripts for various sarcomeric TPM isoforms in striated muscles through development. Transcripts of both TPM1α and TPM1κ, the two sarcomeric isoforms of the TPM1 gene, are expressed in embryonic chicken hearts but disappear in post hatch stages. TPM1α transcripts are expressed in embryonic and adult skeletal muscle. The sarcomeric isoform of the TPM2 gene is expressed mostly in embryonic skeletal muscles. As reported earlier, TPM3α is expressed in embryonic heart and skeletal muscle but significantly lower in adult striated muscle. TPM4α transcripts are expressed from embryonic to adult chicken hearts but not in skeletal muscle. Our 2D Western blot analyses using CH1 monoclonal antibody followed by mass spectra evaluations found TPM4α protein is the major sarcomeric tropomysin expressed in embryonic chicken hearts. However, in 7-day-old embryonic hearts, a minute quantity of TPM1α or TPM1κ is also expressed. This finding suggests that sarcomeric TPM1 protein may play some important role in cardiac contractility and/or cardiac morphogenesis during embryogenesis. Since only the transcripts of TPM4α are expressed in adult chicken hearts, it is logical to presume that TPM4α is the only sarcomeric TPM protein produced in adult cardiac tissues.

Mechanism of Cardiac Tropomyosin Transitions on Filamentous Actin As Revealed by All-Atom Steered Molecular Dynamics Simulations.

The three-state model of tropomyosin (Tm) positioning along filamentous actin allows for Tm to act as a gate for myosin head binding with actin. The blocked state of Tm prevents myosin binding, while the open state allows for strong binding. Intermediate to this transition is the closed state. The details of the transition from the blocked to the closed state and then finally to the open state by Tm have not been fully accessible to experiment. Utilizing steered molecular dynamics, we investigate the work required to move the Tm strand through the extant set of proposed transitions. We find that an azimuthal motion around the actin filament by Tm is most probable in spite of increased initial energy barrier from the topographical landscape of actin.

Structural and Functional Effects of Cardiomyopathy-Causing Mutations in the Troponin T-Binding Region of Cardiac Tropomyosin.

Hypertrophic cardiomyopathy (HCM) is a severe heart disease caused by missense mutations in genes encoding sarcomeric proteins of cardiac muscle. Many of these mutations are identified in the gene encoding the cardiac isoform of tropomyosin (Tpm), an α-helical coiled-coil actin-binding protein that plays a key role in Ca2+-regulated contraction of cardiac muscle. We employed various methods to characterize structural and functional features of recombinant human Tpm species carrying HCM mutations that lie either within the troponin T-binding region in the C-terminal part of Tpm (E180G, E180V, and L185R) or near this region (I172T). The results of our structural studies show that all these mutations affect, although differently, the thermal stability of the C-terminal part of the Tpm molecule: mutations E180G and I172T destabilize this part of the molecule, whereas mutation E180V strongly stabilizes it. Moreover, various HCM-causing mutations have different and even opposite effects on the stability of the Tpm-actin complexes. Studies of reconstituted thin filaments in the in vitro motility assay have shown that those HCM-associated mutations that lie within the troponin T-binding region of Tpm similarly increase the Ca2+ sensitivity of the sliding velocity of the filaments and impair their relaxation properties, causing a marked increase in the sliding velocity in the absence of Ca2+, while mutation I172T decreases the Ca2+ sensitivity and has no influence on the sliding velocity under relaxing conditions. Finally, our data demonstrate that various HCM mutations can differently affect the structural and functional properties of Tpm and cause HCM by different molecular mechanisms.

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca2+-sensitivity and suppress the modulation of Ca2+-sensitivity by troponin I phosphorylation

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca2+-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca2+-sensitivity when compared with donor heart troponin and the Ca2+-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca2+-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca2+-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Expression of sarcomeric tropomyosin in striated muscles in axolotl treated with shz-1, a small cardiogenic molecule.

We evaluated the effect of shz-1, a cardiogenic molecule, on the expression of various tropomyosin (TM) isoforms in the Mexican axolotl (Ambystoma mexicanum) hearts. qRT-PCR data show a ~1.5-fold increase in cardiac transcripts of the Nkx2.5 gene, which plays a crucial role in cardiogenesis in vertebrates. Shz-1 augments the expression of transcripts of the total sarcomeric TPM1 (both TPM1α & TPM1κ) and sarcomeric TPM4α. In order to understand the mechanism by which shz-1 augments the expression of sarcomeric TPM transcription in axolotl hearts, we transfected C2C12 cells with pGL3.axolotl. We transfected C2C12 cells with pGL3-axolotl TPM4 promoter constructs containing the firefly luciferase reporter gene. The transfected C2C12 cells were grown in the absence or presence of shz-1 (5μM). Subsequently, we determined the firefly luciferase activity in the extracts of transfected cells. The results suggest that shz-1 activates the axolotl TPM4 promoter-driven ectopic expression in C2C12 cells. Also, we transfected C2C12 cells with a pGL3.1 vector containing the promoter of the mouse skeletal muscle troponin-I and observed a similar increase in the luciferase activity in shz-1-treated cells. We conclude that shz-1 activates the promoters of a variety of genes including axolotl TPM4. We have quantified the expression of the total sarcomeric TPM1 and observed a 1.5-fold increase in treated cells. Western blot analyses with CH1 monoclonal antibody specific for sarcomeric isoforms show that shz-1 does not increase the expression of TM protein in axolotl hearts, whereas it does in C2C12 cells. These findings support our hypothesis that cardiac TM expression in axolotl undergoes translational control.

Using baculovirus/insect cell expressed recombinant actin to study the molecular pathogenesis of HCM caused by actin mutation A331P

Recombinant WT human cardiac actin (WT actin) was expressed using the baculovirus/insect cell expression system, purified, and used to reconstitute the thin-filament of bovine cardiac muscle fibers, together with bovine cardiac tropomyosin (Tm) and troponin (Tn). Effects of [Ca2+], [ATP], [phosphate] and [ADP] on tension and tension transients were studied at 25°C by using sinusoidal analysis, and the results were compared with those of native fibers and fibers reconstituted with purified bovine cardiac actin (BVC actin). In actin filament reconstituted fibers (without Tm/Tn), those reconstituted with WT actin showed exactly the same active tension as those reconstituted with purified BVC actin (WT: 0.75±0.06 T0, N=11; BVC: 0.73±0.07 T0, N=12, where T0 is the tension of original fibers before extraction). After Tm/Tn reconstitution, fibers reconstituted with WT actin generated 0.85±0.06 T0 (N=11) compared to 0.98±0.04 T0 (N=12) recovered by those reconstituted with BVC actin. In the presence of Tm/Tn, WT actin reconstituted fibers showed exactly the same Ca2+ sensitivity as those of the native fibers and BVC actin reconstituted fibers (pCa50: native fibers: 5.69±0.01, N=10; WT: 5.69±0.02, N=11; BVC: 5.68±0.02, N=12). Sinusoidal analysis showed that the cross-bridge kinetics were the same among native fibers, BVC actin reconstituted fibers and WT actin reconstituted fibers, followed by reconstitution of Tm/Tn. These results demonstrate that baculovirus/insect cell expressed actin has no significant differences from tissue purified actin and can be used for thin-filament reconstitution assays. One hypertrophic cardiomyopathy (HCM) causing actin mutant A331P actin was also expressed and studied similarly, and the results were compared to those of the WT actin. In the reconstituted fibers, A331P significantly decreased the tension both in the absence of Tm/Tn (0.55±0.03 T0, N=13) and in their presence (0.65±0.02 T0, N=13) compared to those of the WT (0.75±0.06 T0 and 0.85±0.06 T0, respectively, N=11). A331P also showed decreased pCa50 (5.57±0.03, N=13) compared to that of WT (5.69±0.02, N=11). The cross-bridge kinetics and its distribution were similar between WT and A331P actin reconstituted fibers, indicating that force/cross-bridge was decreased by A331P. In conclusion, A331P causes a weakened cross-bridge force, which leads to a decreased active tension, reduces left-ventricular ejection fraction, and eventually results in the HCM phenotype.

Significant role of female sex hormones in cardiac myofilament activation in angiotensin II-mediated hypertensive rats.

Ovariectomy leads to suppression of cardiac myofilament activation in healthy rats implicating the physiological essence of female sex hormones on myocardial contraction. However, the possible function of these hormones during pathologically induced myofilament adaptation is not known. In this study, sham-operated and ovariectomized female rats were chronically exposed to angiotensin II (AII), which has been shown to cause myocardial adaptation. In the shams, AII induced cardiac adaptation by increasing myofilament Ca(2+) sensitivity. Interestingly, this hypersensitivity was further enhanced in AII-infused ovariectomized rats. Ovariectomy increased the phosphorylation levels of cardiac tropomyosin, which may underlie the mechanism of hypersensitivity. On the other hand, AII infusion did not alter maximal tension that was suppressed after ovariectomy. This finding coincided with a comparable increase in β-isoform of myosin heavy chains in both ovariectomized groups. Together, it is conceivable that female sex hormones serve as predominant factors that regulate cardiac myofilament activation. Furthermore, they may prevent stress-induced myofilament maladaptation.

Thermal Activation of Cardiac Thin Filaments Induces Contraction without Intracellular Ca2+ Changes: Studies with Cardiomyocytes and an In Vitro Motility Assay

It is well established that heart beat is triggered by electrical excitation, followed by an increase in [Ca2+]i and ensuing activation of myofilaments in cardiomyocytes. This property is the basis for the regulation of the heart with electric stimulators or chemical compounds that mediate Ca2+ signaling. Recently, we found that microscopic heat pulses induce contractions in isolated rat cardiomyocytes without a rise in [Ca2+]i (Oyama, K. et al., Biochem. Biophys. Res. Commun., 417, 607-612, 2012). Microscopic heat pulses produced by focusing infrared laser beam increase temperature of the extracellular solution near cardiomyocytes. In our experiments, a microscopic heat pulse (ΔT = 5°C for 0.5 sec) induced contractions at a basal temperature of 36°C. When heat pulses were repeatedly applied at 2.5 Hz, oscillatory contractions were observed. In contrast to the electric field stimulation, Ca2+ transients were not detected during the course of contraction. Moreover, we confirmed that skinned cardiomyocytes are thermally activated in the Ca2+-free solution. To elucidate the molecular mechanism of thermal activation, microscopic heat pulses were applied to an in vitro motility assay system utilizing reconstituted thin filaments (rabbit skeletal actin filaments reconstituted with human cardiac tropomyosin and bovine cardiac troponin). Thin filaments started to move during heating in the absence of Ca2+, and most of filaments immediately stopped after switching the laser off, demonstrating that thin filaments can be thermally activated, presumably due to the weakening of the binding affinity of the troponin-tropomyosin complex with actin (Ishiwata, S., Biochim. Biophys. Acta, 534, 350-357, 1978). These results suggest that heat pulses can regulate cardiac contractions by shifting the “on-off” equilibrium in the thin filament toward the “on” state, without accompanying intracellular Ca2+ transients.

Tropomyosin de-phosphorylation in the heart: What are the consequences?

The focus of this review is on the very recent work we have conducted that addresses the molecular, morphological, and physiological significance of cardiac tropomyosin phosphorylation in the heart. We employ transgenic mice to address questions of how cardiomyocytes and the whole heart respond when the tropomyosin phosphorylation site (Ser283) is converted to a non-phosphorylatable amino acid (Ala). We address the phenotype of these mice during normal development and in response to acute cardiac stress (transaortic coarctation). In addition, we also examined how transgenic mice encoding the altered tropomyosin phosphorylation site (Ser283Ala) would respond to chronic cardiac stress through an encoded hypertrophic cardiomyopathy mutation (Glu180Gly). These studies are the first to address the in vivo significance of tropomyosin phosphorylation in the heart. In this review manuscript, we report the recent findings of these investigations.

Injection of Vessel-Derived Stem Cells Prevents Dilated Cardiomyopathy and Promotes Angiogenesis and Endogenous Cardiac Stem Cell Proliferation in mdx/utrn−/− but Not Aged mdx Mouse Models for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. DMD patients lack dystrophin protein and develop skeletal muscle pathology and dilated cardiomyopathy (DCM). Approximately 20% succumb to cardiac involvement. We hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent DCM in animal models of DMD. ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and α-actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn(-/-) mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin(+) cardiac stem cells were detected in ADM-injected heart. Nestin(+) striated cells were also detected in four of five mdx/utrn(-/-) hearts injected with ADMs. In contrast, when ADMs were injected into the heart of aged mdx mice with advanced fibrosis, no functional improvement was detected by echocardiography. Instead, ADMs exacerbated some features of DCM. No dystrophin protein, increase in CD31 expression, or increase in Nestin(+) cell proliferation was detected following ADM injection in aged mdx heart. Dystrophin was observed following transplantation of ADMs into the hearts of young mdx mice, however, suggesting that pathology in aged mdx heart may alter the fate of donor stem cells. In summary, ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.

Structural Dynamics of Troponin I during Ca2+-Activation of Cardiac Thin Filaments: A Multi-Site Förster Resonance Energy Transfer Study

A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca2+-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm. Unlike earlier FRET analyses of protein dynamics within the thin filament, this study considered the effects of non-random distribution of dipoles for the donor and acceptor probes. The major conclusion drawn from this study is that Ca2+ and myosin S1-binding to the thin filament results in movement of the C-terminal domain of cTnI from the outer domain of F-actin towards the inner domain, which is associated with the myosin-binding. A hinge-linkage model is used to best-describe the finding of a Ca2+-induced movement of the C-terminus of cTnI with a stationary N-terminus. This dynamic model of the activation of the thin filament is discussed in the context of other structural and biochemical studies on normal and mutant cTnI found in hypertrophic cardiomyopathies.

A Phosphomimetic Mutation of TnI Partially Rescues a Disease Causing Mutation of TnT

Cardiomyopathy causing mutations of troponin and mutations that simulate phosphorylation of troponin can alter the distribution of states of actin-tropomyosin-troponin. The active state of actin filaments containing the Δ14 TnT mutant is stabilized whereas filaments containing the S45E TnI mutation are more difficult to activate. We show that actin filaments containing both [[Unsupported Character - ]]Δ14 TnT and S45E TnI were somewhat normalized. However, because the Δ14 TnT mutation was far from the norm its effects dominated those of the S45E TnI mutation. ATPase activities of actin filaments at high Ca2+ followed the pattern: Δ14 > double mutant > wild type > S45E. In EGTA the activities were: Δ14 > double mutant > wild type ≥ S45E. At high concentrations of the activator, NEM-S1 all types of actin filaments had about the same rate. The rate of transition from the active state to the inactive state was measured using acrylodan labeled cardiac tropomyosin. Following rapid dissociation of S1 with ATP, wild type filaments exhibited a rapid decrease in fluorescence (as the intermediate state population increased) followed by a slower fluorescence redevelopment (formation of the inactive state). The rapid fluorescence decrease had a similar rate to that of S1-ATP dissociation. The slow fluorescence redevelopment was affected by mutations. Filaments containing S45E TnI were similar to wild type. Filaments containing the Δ14 TnT mutation alone or together with the S45E TnI mutation did not exhibit a slow fluorescence redevelopment although the S45E mutant partially restored normal ATPase activity. The rescue of disease causing mutations of troponin is possible but this particular pairing is not optimal for rescuing either mutation.

Effects of a Disulfide Crosslink (XL) on the Trypsin Cleavage Pattern of Rabbit Cardiac Tropomyosin (TM)

A specific tryptic cleavage site at R133 with a subsequent site at R182 in Tm (1-284) was identified by Pato, et al, 1981. Ueno reported (1984) that a disulfide XL at C190 increased the rate of cleavage at higher temperatures (35o-40o). Here we redetermined the affect of the XL on the cleavage pattern at 26o, where Tm is >95% helical, and at 40o, where the middle region of Tm is unfolded. We used SDS-gels, N-terminal sequencing, and MALDI, to identify the products of cleavage. We found that the rate of Tm cleavage at R133 was unaffected by the crosslink at 26oC, but was slightly increased at 40oC. XL cleavage intermediates containing one cleaved and one uncleaved chain were observed. At 26o, cleavage of XL Tm at R133 produced a 51K XL heterodimer, (134-284/1-284), and a 35K XL homodimer, (134-284)2. At longer times, a second cleavage site at R168, produced a smaller 27K XL homodimer (169-284)2. At 40o, The XL increased the cleavage rate at K168 to produce a 47K XL heterodimer (169-284/1-284) and the 51K XL heterodimer. A shorter 31K XL heterodimer, (169-284/134-284) was also produced. The homodimer, (169-284)2 was also present early . The 35K homodimer seen at 26o was not produced. This study detected cleavage intermediates that could not be seen for uncrosslinked Tm. A new cleavage at K168 was seen which dominated at 40o. Cleavage at R183 after the initial cleavage at R133, (Pato et al, 1981), was not observed. This shows that: 1) both chains are not cleaved simultaneously; 2) locally unstable regions near R133 and R168 exhibit increased flexibility especially at physiological temperatures. Supported by NIH HL 22461-29 and an ARRA student summer grant. to D.Y.

Electron Microscopy and Persistence Length Analysis of Semi-Rigid Smooth Muscle Tropomyosin Strands

The structural mechanics of tropomyosin are essential determinants of its affinity and positioning on F-actin. Thus, tissue-specific differences among tropomyosin isoforms may influence both access of actin-binding proteins along the actin filaments and the cooperativity of actin-myosin interactions. Here, 40 nm long smooth and striated muscle tropomyosin molecules were rotary-shadowed and compared by means of electron microscopy. Electron microscopy shows that striated muscle tropomyosin primarily consists of single molecules or paired molecules linked end-to-end. In contrast, smooth muscle tropomyosin is more a mixture of varying-length chains of end-to-end polymers. Both isoforms are characterized by gradually bending molecular contours that lack obvious signs of kinking. The flexural stiffness of the tropomyosins was quantified and evaluated. The persistence lengths along the shaft of rotary-shadowed smooth and striated muscle tropomyosin molecules are equivalent to each other (approximately 100 nm) and to values obtained from molecular-dynamics simulations of the tropomyosins; however, the persistence length surrounding the end-to-end linkage is almost twofold higher for smooth compared to cardiac muscle tropomyosin. The tendency of smooth muscle tropomyosin to form semi-rigid polymers with continuous and undampened rigidity may compensate for the lack of troponin-based structural support in smooth muscles and ensure positional fidelity on smooth muscle thin filaments.

Comparing Pumps

Comparing Pumps

Smooth Muscle Tropomyosin Forms Semi-Rigid End to End Polymers

Variation in the structural mechanics of tropomyosin isoforms may govern differences in their affinity and positioning on F-actin. Such differences may influence the access of actin-binding proteins along the sides of actin filaments and also the cooperativity of actin-myosin interactions. Here, smooth and striated muscle tropomyosin were rotary shadowed and compared by electron microscopy. EM shows that cardiac and skeletal tropomyosin primarily consist of 40 nm long single molecules, whereas smooth muscle tropomyosin is a mixture of varying length chains of end-to-end linked molecules found together with single molecules. The tendency of smooth muscle tropomyosin to polymerize reflects greater end-to-end interaction, possibly required on smooth muscle thin filaments, which lack troponin to stabilize this interaction. Measurement of the apparent persistence length (PL) of single smooth muscle tropomyosin molecules and the chain-like polymers yield indistinguishable values, which are comparable to those that we find for cardiac tropomyosin. The semi-rigidity of smooth muscle tropomyosin polymers may ensure a high degree of positional fidelity of tropomyosin on smooth muscle thin filaments, despite the lack of troponin (cf. Lehman et al., 2009). It is unlikely, however, that stiff, polymerized superhelical chains of tropomyosin can bind directly to F-actin. However, in vitro an equilibrium may yield sufficient single smooth muscle tropomyosin molecules or short chains to bind. In vivo, actin and smooth muscle (or cytoskeletal) tropomyosin may copolymerize or, alternatively, G-actin may polymerize on a scaffold of tropomyosin chains. Thus differing mechanisms of thin filaments assembly may be related to tropomyosin end-to-end binding strength.