The hypertrophic cardiomyopathy-associated A331P actin variant enhances basal contractile activity and elicits resting muscle dysfunction

Figure 1. The ACTC A331P HCM mutation lies proximal to residues that form critical salt bridges with Tpm (A) F-actin in the absence of thin filament regulatory proteins. Individual actin protomers are presented in different shades of gray to help visualize helical packing. The 320–334 surface strand of each actin protomer is displayed in yellow. The loci of K326/328 are shown in blue, and A331 in black, along the surface strand. (B) Thin filament, replete with Tn (light blue) and Tpm (red/salmon) regulatory components. Inset: a single actin subunit depicting Tpm in its inhibitory, B-state position. K326/328 (blue), located along actin’s 320–334 surface strand (yellow), establish highly favorable, stabilizing electrostatic contacts with Tpm.6,9,19,20,28,30 These recurring salt bridges, formed between each actin protomer and every pseudo-repeat binding module along Tpm’s entire length, are required for proper B-state positioning and muscle relaxation. A331 is displayed in black. Models are based on PDB 6KN8, and rendered in ChimeraX.34

Figure 2. A331P actin causes Drosophila IFM hypercontraction and reduces thin filament length (A) Flight indices of w[1118] wild-type, WT▼/+; Act88F-null/+ control, A331P▼/+; Act88F-null/+ heterozygous mutant, WT▼; Act88F-null control, A331P▼; Act88F-null homozygous mutant, and Act88F-null Drosophila. Relative to w[1118], WT▼/+; Act88F-null/+ control Drosophila exhibited a slight, but significant reduction in flight ability. A331P▼/+; Act88F-null/+ heterozygous mutants displayed a significantly lower flight index vs. WT▼/+; Act88F-null/+ control flies. WT▼; Act88F-null, A331P▼; Act88F-null, and Act88F-null Drosophila were flightless. Data are presented as mean ± SEM. Significant differences in flight ability were assessed using the Kruskal–Wallis test (n = 173–657 flies/genotype). (B) Fluorescent micrographs of Alexa 568-phalloidin-stained dorsal longitudinal IFMs (DLMs) of two-day-old w[1118] wild-type, Act88F-null, WT▼/+; Act88F-null/+ control, and A331P▼/+; Act88F-null/+ heterozygous mutant Drosophila. The cuticle from each hemi-thorax is outlined to illustrate orientation. Numbers in brackets indicate transgenic vs. endogenous Act88F allele copy number. w[1118] wild-type, WT▼/+;…

Figure 3. Expression of A331P actin results in restrictive physiology and impaired relaxation in Drosophila heart tubes (A) Heart-restricted expression of Act57B A331P mutant actin significantly altered several indices of cardiac function relative to wild-type Act57B. WT = Hand>Act57BWT; A331P = Hand>Act57BA331P. Hand>Act57BA331PDrosophila displayed significantly decreased heart rates and cardiac diameters, that worsened with age, relative to controls. Data are presented as mean ± SEM. Significant differences between genotype and age, and interaction effects, were determined using two-way ANOVAs (n = 20–59 flies/age/genotype). (B) Significant, stepwise increases in cardiac diameters were observed in Hand>Act57BWT and Hand>Act57BA331P flies following extra- and intra-cellular Ca2+ chelation and, again, upon exposure to blebbistatin. Data are presented as mean ± SEM. Increases in cardiac dimensions due to EGTA/EGTA,AM and to blebbistatin were evaluated using repeated measures ANOVAs followed by Tukey’s multiple comparison tests of the matched groups (n = 22–26 flies/genotype). (C) Left: the change in cardiac diameter in response to EGTA/EGTA,AM was similar between Hand>Act57BWT and…

Figure 4. Cryo-EM reconstruction and MD analysis of human wild-type and mutant ACTC filaments reveal decreased A331P actin flexibility (A) Final cryo-EM maps for wild-type (green) and A331P (blue) filaments. Adjacent actin subunits are shaded differently in order to display the filament assembly and protomer arrangement. Density corresponding to the surface strands (residues 320–334) is rendered in yellow in the central subunits to highlight the general locus of residue 331. (B) Atomic models of the wild-type (green) and A331P (blue) mutant surface strands, spanning residues 320–335, are shown within their respective cryo-EM densities. Both maps fit well into the cryo-EM densities. No overt structural disparities were detected between the wild-type and mutant surface strands. (C) MD analyses of human wild-type and A331P ACTC. MD simulations were run on periodic wild-type and A331P ACTC filaments until the RMSD to the starting coordinates of the backbone atoms stabilized around a single value. Each actin monomer in the system was then superimposed on a central actin fitted using all actin backbone atoms. To compare the dynamic motions of the surface strand containing the A331P mutation,…

Figure 5. The A331P mutation decreases attractive F-actin-Tpm vdW forces and results in greater Tpm inhibitory positional variance (A) vdW interaction energy between Tpm (in the B-state or C-state) and ACTC F-actin (including all residues, residues 320–334, or just 331–334) was measured over the last 20–30 ns of MD simulation time. Bar graphs represent the average values ±SD. All energetic differences in vdW forces between wild-type actin and Tpm vs. A331P actin and Tpm were highly significant (p < 0.0001). Significance was assessed via unpaired t-tests (n = 135–200 total frames/genotype). (B) RMSF was calculated over the same MD simulation time frames as in (A) for the superhelix of Tpm as determined by Twister, in either the B- or C-state position along wild-type (500 frames, green) or A331P mutant (1000 frames, cyan) thin filament models. Prior to calculation, frames were aligned to the actin underlying the Tpm superhelical residue. Note, the greater degree of fluctuation of B-state Tpm along A331P vs. wild-type thin filaments, whilst the C-state RMSF values were considerably less disparate between the two models. (C) Cut away view showing Tpm (red/salmon ribbons) near residue 90 in the…