The Dark Side of Actin: Cardiac Actin Variants Highlight the Role of Allostery in Disease Development

Archives of Biochemistry and Biophysics 695 (2020) 108624

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Archives of Biochemistry and Biophysics

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The Dark Side of Actin: Cardiac actin variants highlight the role of allostery in disease development

Grace Zi Teng, John F. Dawson *

Department of Molecular & Cellular Biology and Centre for Cardiovascular Investigations, University of Guelph, Guelph, ON, N1G 2W1, Canada

ARTICLE INFO ABSTRACT

Keywords: Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are Actin the result of changes in protein interactions between ACTC protein and force-generating β-myosin or the calcium- Allostery dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall Cardiomyopathy contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin Calcium sensitivity pathway relative to the myosin or regulatory protein binding sites on what we call the “dark side” of actin. The T126I Altered force pathway change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomy opathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development.

1. Introduction (2008) [9]. The effects of the S271F and T126I variants on ACTC and their interactions with myosin have not been determined. For the past decade, cardiovascular diseases (CVD) have been the Allostery is a key aspect of the function of actin and is thought to be a number one cause of death worldwide [1]. A significantportion of CVD major contributor to the extreme conservation of the actin protein is due to heritable gene mutations [2]. Cardiomyopathy is the most sequence throughout eukaryotes [10,11]. Within the filament,subunits commonly inherited CVD [3,4] with hypertrophic cardiomyopathy communicate with each other through structural changes dictated by (HCM) and dilated cardiomyopathy (DCM) being two main forms. HCM their nucleotide state. Those allosteric changes are sensed or influenced is characterized by thick ventricular and septal walls, decreasing the by actin binding protein interactions. volume of the left chamber [5]. In contrast, DCM is characterized by thin Disease-related actin variants have been linked to a pathogenic helix and weak ventricular walls, resulting in enlarged ventricles [6]. that extends from residues K113 to T126 of actin molecules [12]. For Mutations in genes encoding sarcomere proteins can lead to HCM or example, K118 N γ-cytoplasmic actin (ACTG) is linked to early-onset DCM. The human ACTC1 gene encoding α-cardiac actin (ACTC) is one of deafness [13], and E117K β-actin (ACTB) was identified in patients the few genes where mutations are linked to both HCM and DCM [7]. with severe cases of Baraitser-Winter syndrome [14]. However, the During heart contractions, the interactions between filamentouscardiac position of the amino acid changes on the actin molecule are far actin and β-myosin are regulated by cardiac-tropomyosin (cTm) and removed from sites of major actin binding protein interactions, sug cardiac troponin (cTn) complex in a calcium dependent manner. gesting that allosteric changes are responsible for functional alterations. To date, 18 missense mutations in the ACTC1 gene have been iden Both the T126I and S271F ACTC amino acid substitutions are located tifiedby genetic screening of HCM and DCM patients. In genetic testing on the back side of the conventional figureof actin distal to the myosin on patients diagnosed with DCM by Lakdawala et al. (2014), one patient or regulatory protein binding sites or what we call the “dark side” of was identifiedwith a cytosine to thymine transition at nucleotide 383 of actin (Fig. 1). The T126I change is located in the pathogenic helix, while ACTC1 that resulted in the T126I substitution mutation [8]. The S271F recent structural studies showed that the hydrophobic plug of actin is ACTC variant was identified in a patient with HCM by Olivotto et al. part of a tripartite interaction involving residues E270, R39 and D286 of

Abbreviations: HCM, hypertrophic cardiomyopathy; ACTC, cardiac actin; RTF, regulated thin filament;IVM, in vitro motility; cTn, cardiac troponin; cTm, cardiac tropomyosin; HMM, heavy meromyosin. * Corresponding author. Department of Molecular & Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada. E-mail address: [email protected] (J.F. Dawson). https://doi.org/10.1016/j.abb.2020.108624 Received 30 June 2020; Received in revised form 24 September 2020; Accepted 5 October 2020 Available online 10 October 2020 0003-9861/© 2020 Elsevier Inc. All rights reserved. G.Z. Teng and J.F. Dawson Archives of Biochemistry and Biophysics 695 (2020) 108624 two neighbouring protomers in the actin filament. During actomyosin glycerol, 1.3 M β-mercaptoethanol (BME), and 0.1% bromophenol blue) interactions, residue E270 is repositioned due to allosteric effects [15]. It [18]. Gels were run for 1 h at 150 mV with a 1 × running buffer (25 mM is hypothesized that the substitution of serine with phenylalanine at Tris, 250 mM glycine, and 0.1% SDS), stained with Coomassie blue, and residue 271 would impact this allosteric change and alter actomyosin destained in 40% methanol and 10% acetic acid. interactions [3]. Models of CVD development with ACTC variants include changes in 2.2. Protein purification the intrinsic properties of the actin molecule or changes in contractility due to what we call the calcium sensitivity pathway, where hypersen As described by Teng et al.(2019) [19], recombinant human ACTC sitivity to calcium in the sarcomere leads to HCM and hyposensitivity proteins were purified from Sf9 cells infected with baculovirus and results in DCM. These hypotheses are not mutually exclusive, since full-length myosin and heavy meromyosin (HMM) were isolated from changes in the intrinsic properties of actin can impact the F-actin con rabbit soleus muscle. formations accessible to different actin binding proteins (ABPs), Regulatory proteins cardiac tropomyosin and cardiac troponin changing their affinities for different F-actin conformations [16,17]. complex were purifiedfrom bovine cardiac ether powder adapted from Therefore, to test these hypotheses, we biochemically characterized the the protocols of Greaser and Gergely (1971) [20] and Adelstein and S271F and T126I ACTC variants, examining the protein stability, acto Tobacman (1986) [21]. myosin interactions and troponin/tropomyosin (Tn/Tm)-dependent calcium sensitivity of the two variants. The T126I ACTC variant dis 2.3. Actin intrinsic properties assays played calcium hyposensitivity, aligning with changes in the calcium sensitivity pathway. Alternatively, S271F ACTC variant did not exhibit Thermal Shift Assay –0.4 mg/ml of recombinant ACTC protein stored major changes in calcium sensitivity but had slower IVM filamentsliding in G buffer (2 mM Tris pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM beta- speeds, suggesting altered force production. Our work supports the mercaptoethanol, 0.002% NaN3) was mixed with 10 × SYPRO Orange model that altered force development at physiological calcium levels are Dye in a 9:1 ratio. The sample was aliquoted into RT-PCR 96 Well Plates the molecular cause of cardiomyopathies, whether the result of changed in triplicate. The StepOne Plus Software 2.3 was used to measure fluo ◦ calcium sensitivity or the maximum force possible in the system. The rescence with readings starting at 4 C and increasing one degree every ◦ location of the T126I and S271F ACTC changes further reinforces the minute until reaching 100 C. role of actin allostery in the regulation of force and the development of Polymerization Assay – The fluorescence of 5 μM sample containing disease. 85% recombinant ACTC protein and 15% pyrene-labeled α-skeletal actin was measured using Cary Eclipse Fluorescence Spectrophotometer with 2. Materials and methods an excitation wavelength of 347 nm and an emission wavelength at 407 nm. The baseline intensity was measured for 30 min, then the poly 2.1. Reagents and basic protocols merization reaction was initiated with the addition of 10 × polymeri zation buffer (finalconcentration 25 mM Tris, pH 8.0, 50 mM KCl, 1 mM Unless stated otherwise, all reagents were from ThermoFisher Sci EGTA, 2 mM MgCl2 and 0.1 mM ATP). The reaction ran for 400 min with entificor Sigma–Aldrich. Spodoptera frugiperda (Sf9) cells were cultured intensity measurements every 5 min. in I-Max media (Wisent Bioproducts, Toronto, ON) supplemented with 1% Penicillin/Streptomycin (PenStrep) mix were obtained from Gibco 2.4. Actomyosin activity assay (Life Technologies, Mississauga, ON). The concentration of recombinant ACTC protein was determined Unregulated Actin Activated Myosin ATPase Assay – Unregulated with the Bradford colourmetric assay using Protein Assay Dye Reagent myosin ATPase activity was measured with an molybdate-based col (Bio-Rad, Hercules, CA). Additionally, polyacrylamide gels with 10% ourimeteric Pi release assay from Trybus (2000) and adapted to a 96- resolving gels and 5% stacking gel were used. Samples were mixed in a well format as described [22]. Samples of 15 μM recombinant ACTC 1:1 ratio with 2 × Laemmli buffer (50 mM Tris (pH 6.8), 2% SDS, 10% protein were polymerized with 10 × AB buffer (75 mM KCl, 10 mM

Fig. 1. The Dark Side of Actin. Shown is the structure of three actin subunits (A1, A2, and A3) from Mentes et al., 2018 [15] (PDB 6C1D) from the perspective of the customary front (left), side (middle), and “dark side” (left) of the filament.T126 (blue) is located in subdomain 1 on the outer surface of the filament,while S271 (red) is in the hydrophobic plug located between subdomain 3 and 4 of the large domain of actin in the interior of the filament.At left, the A2 subunit of one F-actin strand ◦ is rotated 180 away from subunits A1 and A3 of the opposite strand, revealing the location of S271 on A2 and its site of interaction between subunits A1 and A3 (dotted circles). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2 G.Z. Teng and J.F. Dawson Archives of Biochemistry and Biophysics 695 (2020) 108624 imidazole (pH 8), 10 mM dithiothreitol (DTT), 1 mM EGTA, 1 mM 2.5. Calcium sensitivity assays MgCl2, and 1 mM NaN3) at varying concentrations of ATP. 0.125 mg/ml HMM in 1 × AB buffer was added to each sample of ACTC in a 1:1 ratio Regulated Actin Activated Myosin ATPase Assay and Regulated In Vitro to initiate the reaction. Every 2.5 min for 10 min, one fourth of the re Motility Assay – The regulated actin activated myosin ATPase assay and action mixture was added to stop solution (60 mM EDTA, pH 6.5, 6.6% regulated IVM assay were performed as per Teng et al. (2019) [19]. SDS) in a 1:1 ratio. In triplicates, the reaction and stop solution mixture was added to colouring solution (0.5% ferrous sulfate, 25% of 2% 2.6. Statistical tests ammonium molybdate, 4 N H2SO4) in a 1:2 ratio and intensity was read at 750 nm. A standard curve was used to determine the concentration of Paired t-tests were performed using GraphPad Prism 6 (GraphPad Pi in each assay and the rate was calculated by fitting the linear data. Software, San Diego, CA) to test the significance between the mean Unregulated In Vitro Motility Assay – The setup of the nitrocellulose difference of the ACTC variants compared to WTrec. In each assay, three flowcells and the removal of inactive HMM followed the protocol by Lui biological samples for the WTrec control and the two variants were et al. The motility buffers used varying ATP concentrations in assay tested, represented as n = 3 with each data point performed in tripli buffer (25 mM KCl, 25 mM imidazole (pH 7.5), 10 mM DTT, 4 mM cates. For each graph, the error bars show standard error of the mean MgCl2, and 1 mM EGTA) contained an oxygen-scavenging system (25 (SEM). μg/mL glucose oxidase, 45 μg/mL catalase, and 1% glucose). The movement of the rhodamine-phalloidin-labeled recombinant ACTC fil aments were captured for each motility buffer using the upright Leica DM 5000B with the camera Hamamatsu Orca-Flash4.0.

Fig. 2. A) Thermal shift curves comparing the two variants with WTrec ACTC. B) Polymerization curves comparing the two variants with WTrec ACTC. C) Michaelis- Menten curves generated from unregulated ATPase assays comparing the two variants with WTrec ACTC. D) Michaelis-Menten curves generated from unregulated IVM as says comparing the two variants with WTrec ACTC. E) pCa curves generated from regu lated ATPase assays comparing the two variants with WTrec ACTC. F) pCa curves generated from regulated IVM assays comparing the two variants with WTrec ACTC.

3 G.Z. Teng and J.F. Dawson Archives of Biochemistry and Biophysics 695 (2020) 108624

3. Results Table 2 Actomyosin parameters from unregulated ATPase and IVM assays (n = 3 for 3.1. Protein stability and Polymerization Assays each, values are ± SEM, *p < 0.05 comparing the mean of variant ACTC protein measurements to the mean of WT measurements). Catalytic efficiency is re Some ACTC variants related to heart disease exhibit changes in ported as a percentage of the WTrec efficiency. protein stability and actin polymerization [23,24]. To determine if Variant Myosin ATPase Activity In vitro Motility altered protein stability results from the T126I or S271F ACTC amino Vmax (μM/ KM (mM) Catalytic vmax (μm/ KM (μM) acid changes, we expressed and purified these protein variants and re min) efficiency sec) combinant WT ACTC protein (WTrec) using a baculovirus expression WTrec 16.97 ± 0.28 ± 100% 2.00 ± 45.3 ± system. We found the melting temperatures and the rate of polymeri 0.29 0.017 0.023 2.61 zation of the S271F and T126I ACTC variants were similar to WTrec T126I 11.20 ± 0.21 ± 88% 1.71 ± 41.2 ± ACTC (Fig. 2A and Table 1). The change in maximal pyrene fluorescence 0.19* 0.013* 0.061* 3.17 S271F 14.18 ± 0.17 ± 138% 2.01 ± 65.5 ± for T126I ACTC was about the same as WTrec, while S271F ACTC was 0.44* 0.017* 0.036 12.11* lower than WTrec ACTC, suggesting a slight difference in maximal F-actin concentration or the quenching of fluorescenceas a result of the amino acid change on actin (Fig. 2B and Table 1). each variant were plotted with data from myosin ATPase and IVM assays including RTFs. The resulting pCa curves showed no significantchange in the pCa50 with the S271F ACTC variant while the maximum gliding 3.2. Actomyosin assays velocity of S271F RTFs in IVM assays was significantly lower than WTrec. Alternatively, the T126I ACTC variant displayed a decrease in To ensure that differences in actomyosin activity observed is most calcium sensitivity compared to WTrec ACTC (p < 0.0010) and higher likely due to the amino acid change in the ACTC and not due to bundling regulated myosin ATPase activity (see Table 3). or filamentlength changes, we ensured that filamentsin our IVM video analysis of fluorescence microscopy showed the appearance of long 4. Discussion regular filaments with the T126I or S271F ACTC proteins (data not shown). In addition, negative staining electron micrographs revealed F-actin is a dynamic polymer, with changes in twist along the fila filament morphology that was similar to WTrec F-actin (data not ment accompanying conformational changes in individual protomers, shown). potentially resulting in multiple different states along the length of a Work examining mutations of genes encoding sarcomere proteins single filament [10,25,26]. Actin binding proteins (ABPs) have varying generally support a model of cardiomyopathy development where hy binding affinitiesfor different conformational states of F-actin, allowing persensitivity to calcium in the sarcomere leads to HCM and hypo ABPs to “sense” the conformational states of F-actin [16,17]. Our work sensitivity results in DCM. To determine if the S271F (related to HCM) or provides insight into potential changes in actin that translate to changes T126I (related to DCM) ACTC variants follow this model, we measured in myosin binding and force generating activity as a result of mutations the activity of myosin with the ACTC variants in the presence or absence in the ACTC gene. of regulatory proteins using myosin ATPase and in vitro motility (IVM) Polymerization assays describe how well the ACTC proteins form assays. The ATPase assay reports on the hydrolysis of ATP by myosin in filaments. Changes in polymerization kinetics might reflect changes in the presence of F-actin, while the IVM assay measures the velocity (v ) of 0 interactions between actin protomers that foreshadow changes in ABP the myosin. Relative catalytic efficiencies of the ATPase activity of interactions, including myosin or the regulatory Tn/Tm proteins. Un myosin for different ACTC variants are represented by the ratio of V / max regulated myosin ATPase assays report on the interactions specific to K since the myosin concentration is constant in all assays. M actin and myosin in the actomyosin cycle. The unregulated ATPase assay Michaelis-Menten curves for the unregulated ATPase activity of determines the rate of myosin ATP hydrolysis, while the velocities of the myosin at increasing concentrations of ATP revealed a significant unregulated IVM assay report on the rate of myosin lever arm move decrease in V and K compared to WTrec ACTC for both ACTC var max M ment. Finally, the addition of the regulatory Tm/Tn proteins provides iants (Fig. 2C and Table 2). The myosin ATPase catalytic efficiencywith insight into the impact of ACTC changes in force generation of the T126I ACTC was 88% that with WTrec ACTC owing mainly to the much regulated actomyosin system. When viewed in the context of our poly lower V from the Michaelis-Menten curves. Alternatively, the relative max merization and unregulated data, regulated assays provide a fuller pic myosin ATPase catalytic efficiency with S271F ACTC was 138% that ture of the role of stabilization from Tm/Tn binding in the system. with WTrec; in this case, the lower KM influenced the determination. Unregulated IVM assays at increasing ATP concentrations revealed a 4.1. Intrinsic properties lower maximum velocity (vmax) for the T126I ACTC variant compared to WTrec, while the S271F ACTC variant had a very similar vmax compared For both the S271F and T126I ACTC variants, there was no major to WTrec, but exhibited a higher in KM (Fig. 2D and Table 2). The dif ferences in these catalytic efficiencies in light of the IVM velocity data provides insight into the mechanisms of changes in the presence of ACTC Table 3 = variants, as discussed below. Compiled values from regulated ATPase assay and regulated IVM assay (n 3 ± < To examine the impact of the S271F and T126I ACTC variants on for each, values are SEM, *p 0.05 comparing the mean of variant ACTC protein measurements to the mean of WT measurements). calcium sensitivity of regulated thin filaments (RTFs), pCa curves for Variant Myosin ATPase Activity In vitro Motility

Table 1 pCa50 Hill Max Activity pCa50 Hill vmax Values from thermal shift assay and polymerization assay (n = 3 for each, values n (uM/min) n (μm/ are ± SEM, *p < 0.05 comparing the mean of variant ACTC protein measure sec) ments to the mean of WT measurements). WTrec 6.96 ± 5.1 15.9 ± 1.27 6.64 ± 2.0 3.61 ± ◦ 0.039 0.024 0.27 Variant Melting Temperature ( C) Change in Fluorescence (a.u.) T126I 6.74 ± 2.8 19.9 ± 0.52* 6.48 ± 1.9 3.88 ± WTrec 60.03 ± 0.17 512.43 ± 2.25 0.011* 0.024* 0.23 T126I 60.56 ± 0.32 510.90 ± 2.07* S271F 7.02 ± 2.2 13.4 ± 0.84 6.58 ± 2.8 2.88 ± S271F 59.09 ± 0.26* 464.02 ± 1.44* 0.047 0.027 0.17*

4 G.Z. Teng and J.F. Dawson Archives of Biochemistry and Biophysics 695 (2020) 108624 change in the intrinsic properties of the actin proteins aside from a slight filament, stabilizing the conformations of the subunits into a more WT reduction in steady state pyrene fluorescence as F-actin with S271F conformation. ACTC. This reduction may be the result of a lower concentration of F- With the T126I ACTC variant, the vmax of IVM remained similar to actin formed. Alternatively, maximal pyrene fluorescence might be that of WTrec, but the maximum regulated ATPase activity at very high reduced as consequence of the amino acid substitution on actin since the concentrations of calcium (low pCa) was significantly higher than that S271F change is involved in interactions with the C-terminus of actin with WTrec. While a higher maximum ATPase activity suggests higher where the pyrene fluorophore is located. Since there are no major overall contractile force with T126I, that increase in force occurs only at changes in the intrinsic properties of the variant ACTC proteins, diseases very high non-physiological calcium concentrations and does not over resulting from these variants is more likely due to change in force gen come the calcium hyposensitivity observed with this variant. eration with myosin. While the primary change with T126I is related to calcium sensi tivity, the change on the actin molecule is distal to the binding site of 4.2. Unregulated myosin interactions regulatory Tn and Tm proteins; rather, the change is closer to the binding position of the L50 domain of myosin [15,27]. However ACTC The ATPase and IVM assays measure different processes in the variants in the myosin binding site (M-class ACTC variants [3]) lead to actomyosin cycle. The ATPase assay measures on the hydrolysis of ATP calcium hypersensitivity and HCM [19]. in myosin during the actomyosin cycle, while the IVM assay measures How does the T126I change lead to DCM? The T126I change is the velocity of the myosin. Assuming the step size of myosin remains located at the C-terminus of the pathogenic helix, hypothesized to be constant, the velocity of myosin reports on the rate of lever arm move part of an allosteric network within and between F-actin subunits that ment which in turn reflectsthe time myosin is strongly bound to F-actin. impacts F-actin polymerization and stability [12]. Changes in this region The two processes of ATP hydrolysis and lever arm movement are linked might create or limit a range of F-actin conformations accessible to actin by structural changes in myosin where ATP is hydrolysed, leading to binding proteins, thereby modifying affinities of binding proteins for atomic changes in the nucleotide binding pocket that are amplified F-actin [16,17] Specifically, modification of that allosteric network through the myosin molecule, resulting in large conformational changes might alter Tn/Tm binding interactions resulting in the change in cal in the lever arm. Actin conformational changes also contribute to cium sensitivity. Similar changes were seen in recent in silico modeling movement of the myosin lever arm, but the details of that contribution of the cardiomyopathy-related M305L ACTC variant [28]. are not clear. Decreases in Vmax and KM were observed with unregulated myosin 4.4. Regulated changes seen with S271F ACTC ATPase assays at varying ATP concentrations for both the T126I and S271F ACTC variants. Myosin ATPase in the presence of T126I ACTC While not directly in the myosin binding site, the S271F substitution had a lower relative catalytic efficiency of 88% compared to WTrec was hypothesized to negatively impact actomyosin interactions [3] ACTC, reflecting the IVM vmax with T126I ACTC that was 86% of that owing to its location near a tripartite interaction in the core of the with WTrec ACTC. The similar reductions in catalytic efficiencyand IVM F-actin filament [27] as part of the hydrophobic plug of actin first velocities with T126I ACTC suggest that the processes of myosin ATP described by Holmes et al. [29]. hydrolysis and lever arm movement remain closely coupled with this The IVM vmax of S271F RTFs was significantly lower than WTrec variant. RTFs, while the myosin ATPase activity with RTFs was about the same as Conversely, the myosin catalytic efficiency of ATP hydrolysis with that with WTrec. Assuming that the myosin step size remains un S271F ACTC is 138% that with WTrec ACTC while the IVM vmax was the changed, the impact of the S271F variant under regulated conditions same as that with WTrec ACTC. These data suggest that myosin ATP would be to increase the duty ratio of myosin [30], resulting in higher hydrolysis and lever arm movement in the presence of S271F ACTC are contractile force in the sarcomere. While the pCa50 of S271F ACTC was not as efficiently coupled, further suggesting allosteric or F-actin inter similar to that of WTrec ACTC, producing more force overall matches protomer changes modify myosin conformational changes and sup development of HCM in the patient where the S271F mutation was porting the hypothesis that the S271F ACTC substitution modifies found. The lack of major change in the pCa50 with the S271F suggests movement of the actin hydrophobic plug observed in high resolution that HCM development in this case is due primarily to changes in structures of actomyosin [15,27] that are involved in actomyosin func maximum force produced in the sarcomere. tion [3]. Earlier work with a substitution mutation in the hydrophobic plug Previously, we reported variable changes of other ACTC variants tested the Holmes model that the plug inserted into a pocket between (E99K, R95C, H88Y, and F90Δ) on unregulated myosin activity at two subunits of the opposite strand, resulting in a cold-sensitive poly varying ACTC protein concentrations [22] that became consistent with merization mutant [31]. Perhaps the introduction of a large hydropho the related disease state in the presence of regulatory proteins [19]. In bic residue at residue 271 in the hydrophobic plug changes the some cases, the presence of regulatory proteins in the ACTC variant thin conformational flexibility of the filament, resulting in lower myosin filaments compensated for an actomyosin dysfunction seen with un maximum ATPase and IVM velocities in presence of calcium-bound regulated F-actin. Therefore, while unregulated activity might reveal Tn/Tm. Such changes in filament flexibility are the result of some potentially fundamental dysfunction such as lower actomyosin long-range allosteric effects transmitted between the core of the filament activity with T126I ACTC, analysis of regulated ACTC variant thin fil and the bound Tn/Tm and myosin at the exterior of the filament. aments is necessary to test the calcium sensitivity pathway of cardio While much attention has been given to the calcium sensitivity myopathy development. pathway for cardiomypathy development, with several compounds targeting regulatory proteins in muscle [32], ultimately the cause of 4.3. Regulated changes seen with T126I ACTC disease is the change in force developed at physiological calcium levels. While the T126I ACTC variant exhibited changes aligning with the The T126I ACTC variant displayed changes in the calcium sensitivity calcium hyposensitivity pathway of DCM development, the S271F ACTC of actomyosin interactions aligning with the calcium sensitivity only exhibited altered force generation at physiological calcium levels. pathway where calcium hyposensitivity leads to DCM. As observed with The development of drugs targeting the regulatory proteins or myosin other ACTC variants [19], the decreased unregulated actomyosin itself are designed to modulate the force production in sarcomeres, ATPase activity and IVM motility speeds with variant ACTC proteins are either by shifting the pCa-force curve or by changing the maximum force largely compensated for in the presence of Tm and Tn. This compensa produced by myosin. Understanding the changes at the molecular level tion may be due to the binding of tropomyosin along the F-actin is needed to determine the best approach for correcting the problem.

5 G.Z. Teng and J.F. Dawson Archives of Biochemistry and Biophysics 695 (2020) 108624

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