Myosin dilated cardiomyopathy mutation S532P disrupts actomyosin interactions, leading to altered muscle kinetics, reduced locomotion, and cardiac dilation in Drosophila

FIGURE 1:. S532P myosin reduces steady-state actin-activated ATPase activity. (A) The location of the S532P myosin residue within an actin-binding region of the motor domain is shown in the boxed inset. The β-MyHC S532 residue (green) was modeled on the crystal structure of chicken skeletal muscle myosin II in the postrigor configuration (PDB ID: 2MYS). Red: actin-binding sites. (B, C) Full-length myosin isolated from IFMs of S532P mutants (N = 5) and PwMhc2 wild-type transgenic controls (N = 5) was assessed for (B) Mg2+ basal and (C) actin-activated ATPase activities. To determine actin-activated activity, Mg2+ basal ATPase activities were subtracted from measured basal ATPase values over increasing concentrations of F-actin. Values were fitted with the Michaelis–Menten equation to determine the Vmax and Km. (D, E) Myosin subfragment-1 (S1) was isolated in bulk from His-tagged S532P mutants (N = 3) and His-tagged wild-type transgenic controls (N = 3) and assessed for actin-binding affinity using cosedimentation assays. (D) A representative SDS–polyacrylamide gel showing insoluble pellet (P) fractions of F-actin and wild-type S1-containing samples over increasing F-actin concentrations…

FIGURE 2:. The S532P mutation does not affect myofibril stability of S532P IFMs. Transmission electron micrographs of thin-sectioned IFMs in transverse and longitudinal orientations were obtained from 3-wk-old homozygous PwMhc2 control or S532P mutant flies in a homozygous Mhc10 (myosin-null in IFMs and jump muscles) background. (A, B) Low-magnification images of transverse sections show that myofibrillar morphology of IFMs is normal in S532P mutant flies compared with controls. MF: myofibril, M: mitochondrion. Scale bar, 0.5 µm. (C, D) High-magnification images of transverse sections show a regular hexagonal array of thick and thin filaments in controls and mutants. Scale bar, 0.1 µm. (E, F) Low-magnification images of myofibrils from longitudinal sections show that myofibrillar organization is normal in S532P flies compared with controls. MF: myofibril, M: mitochondrion. Scale bar, 2 μm. (G, H) High-magnification images of longitudinal sections show that sarcomere stability is maintained in mutants. Arrowheads: Z-disks. Scale bar, 0.5 μm. (I) Inter–thick filament spacing averages were determined from micrographs of transverse sections using a custom-written Python script. Three flies were…

FIGURE 3:. Fiber mechanics reveal altered viscoelastic muscle properties and reductions in power output of S532P IFMs. (A) Maximum power output and the frequency at which maximum power is generated (fmax) were measured by sinusoidal analysis of IFM fibers from homozygotes. fmax values are indicated by the vertical dashed lines. (B, C) Changes in viscous modulus (B) and elastic modulus (instantaneous stiffness) (C) were plotted as a function of frequency. Dip frequencies are indicated by vertical dashed lines. (D) Sinusoidal analysis was performed at various ATP concentrations. Values for fmax were plotted over changing [ATP] and fitted with a hyperbolic function curve. (E) Viscous moduli vs. elastic moduli were plotted to generate Nyquist plots. The resulting plots were fitted (solid lines) to a three-term equation (Supplemental Figure S1, Eq. 3) to determine exponential rate processes (A, B, and C). Statistical assessments are listed in Table 2. Sample sizes are shown in parentheses: PwMhc2 homozygote (N = 10), S532P-L1 homozygote (N = 8), S532P-L2 homozygote (N = 6), and S532P-L3 homozygote (N = 8). Full genotypes are shown in parentheses: S532P homozygote (Mhc10/Mhc10;…

FIGURE 4:. Small-angle x-ray diffraction experiments of actively beating IFMs reveal reductions in lattice spacing and in the active number of cross-bridges in S532P fibers. The impact of the S532P mutation on lattice spacing and intensity ratio was measured by small-angle x-ray diffraction of thoraces from flies actively beating their wings. (A) Flies were immobilized with an insect pin. (B) Top, equatorial diffraction pattern of a transgenic wild-type fly. Bottom, a one-dimensional intensity trace showing the 1,0 and 2,0 reflection peaks. (C) The interfilament lattice spacing (d10) is reduced in S532P-L1 IFMs (N = 7) compared with PwMhc2 controls (N = 15). (D) The intensity ratios (I2,0/I1,0) associated with the number of active cross-bridges are reduced in S532P-L1 IFMs (N = 7) compared with controls (N = 14). Data are reported as the mean ± SEM. Statistical significance was determined using Student’s t tests, where **p < 0.01 and ****p < 0.0001. Full genotypes are shown in parentheses: S532P-L1 homozygote (Mhc10/Mhc10; P[S532P]/P[S532P]); PwMhc2 homozygote (P[PwMhc2]/P[PwMhc2]; Mhc10/Mhc10).

FIGURE 5:. S532P flies exhibit gene dose–dependent cardiac dilation. (A, B) Cardiac dimensions, (C) fractional shortening, and (D–F) dynamics of 4-d-old PwMhc2 control or mutant S532P heterozygous and homozygous lines (L1 or L2). Values represent mean ± SEM. One-way ANOVAs determined statistical significance compared with controls, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns = nonsignificant. Sample sizes are listed in parentheses: PwMhc2 heterozygote (N = 35), S532P-L1 heterozygote (N = 36), S532P-L2 heterozygote (N = 30), PwMhc2 homozygote (N = 32), S532P-L1 homozygote (N = 33), S532P-L2 homozygote (N = 35). Full genotypes are shown in parentheses, where “-“ indicates that there is no P element on the homologous chromosome: S532P heterozygote (Mhc1/+; P[S532P]/–); PwMhc2 heterozygote (P[PwMhc2]/–; Mhc1/+); S532P homozygote (Mhc1/Mhc1; P[S532P]/P[S532P]); PwMhc2 homozygote (P[PwMhc2]/P[PwMhc2]; Mhc1/Mhc1).

FIGURE 6:. The S532P mutation does not affect the organization and stability of cardiac myofibrils. (A, B) Transmission electron micrographs of hearts of 4-d-old PwMhc2 control or mutant line S532P-L1 homozygotes in a homozygous Mhc1-null background. Micrographs show transverse sections of the heart tube between the third and fourth sets of ostia. The arrows indicate discontinuous Z-disks that are characteristic of Drosophila cardiac myofibrils. MF: myofibril; M: mitochondrion; VL: supportive ventral longitudinal fibers. Scale bar, 0.5 µm. Cardiac thickness of dorsal-side (C) and ventral-side (D) areas of the heart were measured using ImageJ. Three flies were tested for each line. Values represent mean ± SD. Student’s t tests determined statistical significance compared with controls, where ns = nonsignificant. Full genotypes are shown in parentheses: S532P homozygote (Mhc1/Mhc1; P[S532P]/P[S532P]); PwMhc2 homozygote (P[PwMhc2]/P[PwMhc2]; Mhc1/Mhc1).