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Biomechanics

Biomechanics - Poster Session E

Poster Q5 - The Contributions of Myosin Post-Translational Modifications to Sarcopenia

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Sarcopenia, the aging-related decline in skeletal muscle function, reduces force, power, and endurance. This decline leads to decreased mobility, lower quality of life, and significant financial burdens. Previous research identified eight age-specific PTMs in human myosin (Li et al., 2015). Two of these modifications, carbonylation of Pro79 and Asn81, were specifically located within the motor domain of type IIx myosin heavy chain isoforms. The motor domain is a critical myosin region responsible for ATP hydrolysis, which provides the energy for muscle contraction and binding to actin filaments. Carbonylation can lead to structural changes, loss of function, and increased susceptibility to aggregation (Dalle-Donne et al., 2003). The tail region of myosin exhibited a broader spectrum of PTMs, including carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; and deamidation of Gln1164 and Asn1168. However, these PTMs might only accumulate with age and not cause any negative consequences to myosin and muscle function. Thus, we needed a model organism system to determine if and how these PTMS affect muscle and locomotory properties. Fortunately, Drosophila, with its single myosin heavy chain gene and our expertise in measuring the mechanical properties of its indirect flight muscle, enables us to measure how modified myosin alters muscle performance. To determine if these PTMs contribute to sarcopenia, we created transgenic Drosophila expressing myosin with mutations that mimic some of these PTMs in their IFMs and measured if MHC mechanical properties were altered.

Materials and

Methods:  We transgenically modified Drosophila to express mutated myosin that mimic age-related PTMs observed in humans. The modifications included N81T (Hydroxylation mimic), located in the myosin head domain, which potentially affects myosin-actin interactions; R908E (Carbonylation mimic), located in the S2 hinge region, which potentially hinders flexibility and force transmission; and N1168D (Deamidation mimic), located in the rod domain, which potentially disrupts myosin filament assembly or stability.
Flies expressing these myosin PTMs were tested for whole-animal performance using flight and wing beat frequency (WBF) tests. Transmission Electron Microscopy was employed to examine changes in muscle ultrastructure. Single Drosophila Indirect flight muscle (IFM) fibers were dissected from the transgenic flies, skinned, mounted on a mechanics apparatus to a force transducer and length motor, and mechanically tested for potential alterations to force, work, power, and myosin cross-bridge kinetics.

Results, Conclusions, and Discussions: Results
The R908E homozygous mutants initially displayed well-organized sarcomeres until 2 days old but exhibited degeneration by 7 days. Heterozygous PTM mutants, N81T/+ and N1168D/+, showed normal myofibril structure at 2 days but severe maintenance defects by 7-14 days. The R908E/+ mutant showed normal structure at 7 days but significant defects at 14 days. The N1168D and N81T mutations have the most immediate and severe impacts on myofibril assembly and sarcomeric structure, while the R908E mutation only shows effects over a longer timeframe.
Flies with N81T/+ and N1168D/+ mutations had significantly reduced flight index scores compared to wild-type controls. Homozygous N1168D and N81T mutants could not be assessed for flight and wing beat frequency (WBF) due to their inability to fly. Homozygous R908E flies exhibited severe flight impairment. N1168D/+ mutants showed no significant impact on WBF, while N81T/+ and R908E mutants had significant reductions.
Heterozygous N1168D/+ fibers exhibited a severe loss of power production (72% compared to controls). N81T/+ fibers also showed a substantial reduction (81%). R908E fibers produced similar power output to controls.
Conclusions
Our study demonstrates that age-related PTMs in myosin significantly impair muscle function in Drosophila and are likely to contribute to human sarcopenia. PTMs disrupt flight and mechanics, with homozygous N81T and N1168D flies exhibiting the most severe phenotypes. Fiber mechanics experiments showed severe power deficits in heterozygous N1168D and N81T flies. Despite reduced flight performance, young R908E fibers produced similar power output to controls. The defects in heterozygotes and the inability of homozygous N1168D and N81T flies to fly suggest dominant-negative effects on myosin function and muscle structure.
Discussion
Our data support the hypothesis that PTMs contribute to sarcopenia. Targeting these PTMs or their downstream effects could potentially slow age-related muscle decline and improve human healthspan. Antioxidants or compounds inhibiting harmful PTMs could be potential treatments. Continued research in this area is crucial for developing strategies to combat sarcopenia and improve the quality of life for the aging population.

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