Cryo-EM Research reveals Zebrafish as a Promising Model for Studying Human Heart Disease

Summary of cardiac thin filament preparation and cryoEM images

An excised adult Zebrafish heart (A) used to extract cardiac thin filaments and image via negative stain TEM (B) and cryo-TEM (C). 2D class averages from cryo-EM data reveal pairs of troponin labelling the two strands of actin (D).

Structure of cardiac thin filament and troponin.

Zebrafish high Ca2+ thin filament model, depicting atomic models of full-length actin (grey) and troponin complex (green, yellow, orange, and red) docked into the electron density calculated from cryo-TEM data.

23 July 2023

In a recent breakthrough, researchers at the University of Bristol have utilized cryo-electron microscopy (cryo-EM) to investigate the three-dimensional structure of the cardiac thin filament in zebrafish, shedding light on its similarity to the human counterpart. The study establishes zebrafish as a valuable model for human heart diseases, particularly hypertrophic cardiomyopathy (HCM).

The thin filament in cardiac muscle plays a vital role in the contraction process. It consists of three key proteins: actin, tropomyosin, and troponin. Understanding the structure and function of these proteins is crucial in unravelling the mechanisms behind heart diseases.

By employing cryo-EM in conjunction with a novel approach known as non-helical single-particle analysis, the team of researchers led by PhD candidate Marston Bradshaw and Dr. Danielle Paul achieved high-resolution 3D structures of zebrafish cardiac thin filaments. This innovative methodology, which was pioneered by Dr. Paul herself, was necessitated by the unique asymmetry of the troponin pairs periodically spaced along the filament. Dr. Paul's work on this method has since been embraced by numerous research groups employing cryo-EM to study the intricate details of the thin filament. Notably, their findings revealed a striking resemblance between the zebrafish and human cardiac thin filament structures, extending to the critical troponin-T region, which harbors a "hotspot" linked to hypertrophic cardiomyopathy.

The study addresses previous limitations in understanding the structural intricacies of the thin filament. Cryo-EM allowed researchers to capture detailed structural information, highlighting the conserved regions between zebrafish and humans while also revealing subtle differences.

"We found that the Zebrafish thin filament structure strongly resembles the Human structure. However, we also discovered some significant differences; additional density was observed at the base of the troponin cores. Further structural data obtained by our lab supports this finding. Notably, this density was not identified in Human models and may be attributed to the native isolation process or an unidentified protein within the complex."

One of the key takeaways from this research is the potential of zebrafish as a model for studying heart diseases. Zebrafish's genetic similarity to humans, combined with their amenability to gene-editing techniques, makes them valuable tools for investigating cardiomyopathies and other heart-related conditions. The study underscores the importance of zebrafish models in advancing our understanding of heart diseases and potentially developing new therapies.

However, the research also acknowledges certain limitations, such as the need for higher resolution reconstructions of the full thin filament to accurately assess the effects of disease-causing mutations. Additionally, zebrafish may have a more specialised mechanism for calcium activation and regulation which requires further exploration.

The next steps involve a collaborative effort to create zebrafish models with specific, pathogenic mutations associated with Hypertrophic Cardio Myopathy (HCM). Through equivalent cryo-EM studies, the researchers aim to delve into the effects of these mutations on the cardiac thin filament structure. This exploration holds the potential to unlock critical insights into the molecular mechanisms underlying HCM and other heart diseases. Moreover, the knowledge gained from these studies may pave the way for the development of targeted treatments, offering new hope for individuals affected by these conditions.

In summary, this groundbreaking study leverages cryo-EM technology to reveal the structural similarities between zebrafish and human cardiac thin filaments. Zebrafish is emerging as a promising model for studying heart diseases, offering potential insights into the molecular mechanisms underlying conditions like hypertrophic cardiomyopathy. This research represents a significant step forward in our quest to understand and combat heart diseases.

Further information

Bradshaw, M., Squire, J.M., Morris, E. et al. Zebrafish as a model for cardiac disease; Cryo-EM structure of native cardiac thin filaments from Danio Rerio. J Muscle Res Cell Motil 44, 179–192 (2023).

Paul, D.M., Squire, J.M., Morris, E.P. A novel approach to the structural analysis of partially decorated actin based filaments. J Struct Biol., 170(2):278-85 (2010).