First high-resolution image of thick filaments of muscle cells

Max Planck Association

Scientists gain insight into the organization of the heart muscle of mammals

An international team, led by Stefan Raunser, director of the Max Planck Institute for Molecular Physiology in Dortmund, in collaboration with Mathias Gautel of King’s College London, has successfully acquired the world’s first high-resolution 3D image of the thick filament in its natural cellular structure. environment, using an advanced technique known as electron cryotomography. This provides a glimpse into the molecular organization and arrangement of the components within the thick filament. The newfound insight is nothing short of a crucial framework for understanding how muscles work in both health and disease.

Often described as the body’s engine, the human heart is a remarkable organ that beats tirelessly to keep us alive. Intricate processes take place in the nucleus of this vital organ as it contracts, with thick and thin protein filaments interacting in the sarcomere, the basic building block of both skeletal and cardiac muscle cells. Any changes in thick filament proteins can have serious consequences for our health, leading to conditions such as hypertrophic cardiomyopathy and various other heart and muscle diseases.

Atrial fibrillation, heart failure and stroke – hypertrophic cardiomyopathy can lead to many serious health problems and is a leading cause of sudden cardiac death in people under the age of 35. “The heart muscle is a central engine of the human body. Of course, it is easier to repair a broken engine if you know how it is built and functions,” says Stefan Raunser. “At the beginning of our muscle research, we successfully visualized the structure of the essential muscle building blocks and their interaction using electron cryomicroscopy. However, these were static images of proteins extracted from the living cell. They tell us very little about how the highly variable, dynamic interplay of muscle components moves the muscle in its native environment,” says Raunser.

Through thick and thin

Skeletal and cardiac muscles contract due to the interaction of two types of parallel protein filaments in the sarcomere: thin and thick. The sarcomere is divided into different areas, called zones and bands, in which these filaments are arranged in different ways. The thin filament consists of F-actin, troponin, tropomyosin and nebulin. The thick filament consists of myosin, titin and myosin binding protein C (MyBP-C). The latter can form connections between the filaments, while myosin, the so-called motor protein, interacts with the thin filament to generate force and muscle contraction. Changes in the thick filament proteins are associated with muscle diseases. A detailed view of the thick filament would be of enormous importance for developing therapeutic strategies to cure these diseases, but has so far been lacking.

“To fully understand how muscle works at the molecular level, you need to image its components in their natural environment – ​​one of the biggest challenges in biological research today that cannot be addressed with traditional experimental approaches,” says Raunser. To overcome this obstacle, his team developed an electron cryo-tomography workflow specifically tailored to the examination of muscle samples: the scientists freeze mammalian cardiac muscle samples, produced by the Gautel group in London, at a very low temperature ( -175°C). ). This preserves their hydration and fine structure and therefore their original state. A focused ion beam (FIB milling) is then applied to thin the samples to an ideal thickness of approximately 100 nanometers for the transmission electron microscope, which acquires multiple images as the sample is tilted along an axis. Finally, computational methods reconstruct a high-resolution three-dimensional image.

In recent years, Raunser’s group has successfully applied the tailor-made workflow, resulting in two recent groundbreaking publications: They produced the first high-resolution images of the sarcomere and of a previously vague muscle protein called nebulin. Both studies provide unprecedented insights into the 3D organization of muscle proteins in the sarcomere, for example how myosin binds to actin to control muscle contraction and how nebulin binds to actin to stabilize it and determine its length.

Complete the painting

In their current study, the scientists produced the first high-resolution image of the thick cardiac filament stretching across several regions in the sarcomere. “With a length of 500 nanometers, this makes for the longest and largest structure ever solved by cryo-ET,” says Davide Tamborrini of the Max Planck Institute in Dortmund, first author of the study. Even more impressive are the newly acquired insights into the molecular organization of the thick filament and therefore into its function. The arrangement of the myosin molecules depends on their position in the filament. The scientists suspect that this allows the thick filament to sense and process numerous muscle-regulating signals and thus regulate the force of muscle contraction, depending on the sarcomere area. They also revealed how titin chains run along the filament. Titin chains are intertwined with myosin, acting as a scaffold for its assembly and likely orchestrating a length-dependent activation of the sarcomere.

“Our goal is to one day provide a complete picture of the sarcomere. The image of the thick filament in this study is ‘just’ a snapshot of the relaxed state of the muscle. To fully understand how the sarcomere functions and how it is regulated, we want to analyze it in different states, for example during contraction,” says Raunser. Comparison with samples from patients with muscle diseases will ultimately contribute to a better understanding of diseases such as hypertrophic cardiomyopathy and to the development of innovative therapies.