A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila

Abstract

An "invariant proline" separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138-43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc(5), an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue.

Keywords: Drosophila melanogaster; contraction; electron microscopy; motility; myofibril.

Fig. 1

ATPase and in vitro motility values for wild-type transgenic and P838L myosins. Ca-ATPase (A) and basal Mg-ATPase (B) levels were significantly enhanced (*) for P838L myosin relative to control (p < 0.05). In contrast, V_max (C), K_m (D), and catalytic efficiency (E) showed no statistically significant differences (ns). In vitro motility of actin filaments (F) is significantly increased (*) for the P838L mutant myosin compared to wild-type transgenic myosin (p < 0.05). Statistical comparisons made by Student’s t-test; error bars represent SD.

Fig. 2

Torsional stiffness and S1 head orientation of wild-type transgenic and P838L myosins. (A) Video microscopy was employed to determine the torsional stiffness of wild-type transgenic and P838L myosins by evaluating the rotational motion of fluorescently labeled F-actin bound to surface immobilized myosin. (B) The reduced torsional stiffness (***p < 0.001) for P838L myosin indicates that the mutation yields increased flexibility of the S1 head relative to the S2 region. (C) Orientation of the two myosin heads relative to the S1/S2 junction (outlined in blue) was determined by measuring the angle (θ) from the inner product of two lines made by points connecting the junction of the two head domains with the tail and each S1 head of rotary shadowed molecules. (D) There is a significant increase (***p < 0.001) in the inter S1 angle (θ) in P838L myosin compared to control. Statistical comparisons made by Student’s t-test; error bars represent SD.

Fig. 3

Transmission electron microscopy of IFM from transgenic control and P838L lines during aging. IFMs from control pwMhc2 homozygotes in longitudinal (A) and transverse (A′) orientations at 2 d following eclosion show highly organized sarcomeres with repeating Z- and M-lines, as well as myofibrils with a regular myofilament lattice. Each of the mutant transgenic lines, P838L-6F (B, B′), P838L-1 M (C, C′), and P838L-3 M (D, D′), displays normal myofibril morphology at 2 days. At 3 weeks post-eclosion, pwMhc2 IFM retains normal patterns of sarcomere and myofilament organization (E, E′), while each of the mutants (F, F′: P838L-6F; G, G′: P838L-1 M; H, H′: P838L-3 M) shows abnormal structural features such as sarcomere gaps (F, arrowhead), misshapen myofibrils (F′, asterisk), and Z-line non-linearity (asterisks, G,H). M, mitochondrion. All of these flies lack endogenous IFM MHC, due to being homozygous for the Mhc¹⁰ allele. Bar for A and adjacent sections, 2 μm. Bar for A′ and adjacent sections, 0.5 μm. Bar for E and adjacent sections, 2 μm. Bar for E′ and adjacent sections, 0.5 μm.

Fig. 4

Mechanical power output versus frequency for IFM fibers from transgenic control and P838L lines. Chemically demembranated dorsal longitudinal IFM fibers from a 2–3-day-old flies were assessed for power generation by imposing small amplitude sinusoidal length changes on the fiber over 0.5 to 650 Hz with calcium at pCa 5.0. No statistically significant differences in power output was observed between fibers from pwMhc2 homozygous control and homozygous P838L-1 M (p = 0.223, mixed ANOVA); error bars represent SEM. See Table 2 for details.

Fig. 5

Transmission electron microscopic images of 4-day-old hearts from transgenic control and P838L homozygotes and heterozygotes. Transverse sections were prepared from cardiac tissue located between the second and third abdominal segments. Similar to pwMhc2 homozygous control (A), P838L-6F homozygous mutant hearts (B) contain a normal cardiomyocyte layer thickness with non-degraded myofibrils. CM, cardiomyocyte layer; VL, ventral layer. Both pwMhc2 control homozygote hearts (C, E) and P838L-6F mutant homozygote hearts (D, F) contain non-degraded myofibrils with discontinuous Z-lines (C, D arrows) and normal myofilamentous arrays (E, F). All homozygote transgenics are in the Mhc¹ null background. Sarcomeres from pwMhc2/+ (G) or P838L-6F/+ (H) heterozygotes (in the Mhc¹/+ background) show normal morphology with discontinuous Z-lines (arrows). All bars represent 500 nm.

Fig. 6

Cardiac structure and physiology of 3-week-old transgenic control and P838L homozygotes and heterozygotes. High-speed video image analysis was used to determine various parameters of cardiac structure and function. No statistically significant differences for heart period, systolic interval, diastolic diameter, systolic diameter, and fractional shortening were observed between P{Mhc⁺}/P{Mhc⁺}; Mhc¹/Mhc¹ and P{P838L}/P{P838L}; Mhc¹/Mhc¹ or between P{Mhc⁺}; Mhc¹/+ and P{P838L}; Mhc¹/+ Drosophila. The effects of placing the P838L-6F transgene in combination with Mhc⁵, an allele that causes cardiac constriction in the homozygous state [20] were also examined. P{P838L}; Mhc⁵/Mhc¹ shows a statistically significant (*p < 0.05) increase in systolic diameter and a decrease in fractional shortening compared to P{Mhc⁺}; Mhc⁵/Mhc¹. Statistical significance was assessed by one-way ANOVA followed by a Bonferroni post hoc test; error bars represent SEM.