Muscle Finding Could Lead To New Therapies For Heart Disease And Skeletal Muscle Disorders

Models of how myosin, actin and the elastic scaffold protein work together have largely ignored the possibility that myosin filaments penetrate the Z-disc structure.

In order to identify the proteins that allow the sarcomere to contract, relax, communicate its energy needs, and adapt to exercise, Scientists at the Max Delbrück Center for Molecular Medicine (MDC) in the Helmholtz Association say they have developed a mouse model that enables them to look inside a working muscle.

Their study “Deconstructing sarcomeric structure–function relations in titin-BioID knock-in mice” appears in Nature Communications.

Specifically, they were able to map proteins in defined subregions of the sarcomere, starting from the Z-disc, the boundary between neighboring sarcomeres. This in and of itself was a significant step forward in the study of striated muscle, according to the team.

In the process, they made an unexpected discovery: myosin, one of the three main proteins that make up striated muscle fibers, appears to enter the Z-disc.

Models of how myosin, actin and the elastic scaffold protein work together have largely ignored the possibility that myosin filaments penetrate the Z-disc structure.

Only recently have German scientists theorized that they do, but no experimental evidence has validated the model, until now.

“Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin’s Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism,” write the investigators.

“Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells.”

Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction.

“Extending this proof of concept study to BioID fusion proteins generated with CRISPR/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.”

“This is going to be unexpected even for myosin researchers,” says Michael Gotthardt, PhD, who heads MDC’s Neuromuscular and Cardiovascular Cell Biology Lab and led the research. “It gets to the very basics of how muscles generate force.”

Gotthardt’s team, which worked with colleagues at the MDC and the University of Göttingen, never set out to validate this theory. Their primary goal was to identify the proteins in and near the Z-disc. To do this, they developed a mouse model with an artificial enzyme, called BioID, inserted into the giant protein titin. The Titin-BioID then tagged proteins close to the Z-disc.

Sarcomeres are tiny molecular machines, packed with proteins that tightly interact. Until now it has been impossible to separate proteins specific to the different subregions, especially in live, functioning muscle. “Titin-BioID probes specific regions of the sarcomere structure in vivo,” says Philipp Mertins, PhD, who heads MDC’s Proteomics Lab. “This has not been possible before.”

The team is the first to use BioID in live animals under physiological conditions and identified 450 proteins associated with the sarcomere, of which about half were already known. They found striking differences between heart and skeletal muscle, and adult versus neonatal mice, which relate to sarcomere structure, signaling and metabolism. These differences reflect the need of adult tissue to optimize performance and energy production versus growth and remodeling in neonatal tissue.

“We wanted to know who’s there, know who the players are,” Gotthardt says. “Most were expected, validating our approach.”

The protein that they were not expecting to see in the Z-disc was myosin, which is integrated at the opposite site of the sarcomere. When a muscle is triggered to move, myosin walks along actin bringing neighboring Z-discs closer together. This sliding of actin and myosin filaments creates the force that enables our heart to pump blood or our skeletal muscle to maintain posture, or lift an object.

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