Montenegro, G. C. | Search bioRxiv

bioRxiv2023

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy an optimized cryo-ET platform that enables cellular-structural biology in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using this platform, we reconstructed sub-nanometer resolution structures of the human cardiac muscle thin filament, a central component of the contractile machinery. Reconstructing the troponin complex, a regulatory component of the thin filament, from within cells, we identified previously unobserved conformations that highlight the structural flexibility of this regulatory complex. We next measured the impact of chemical and genetic perturbations associated with cardiovascular disease on the structure of troponin. In both cases, we found changes in troponin structure that are consistent with known disease phenotypes--highlighting the value of our approach for dissecting complex disease mechanisms in the cellular context.

biophysics↗

bioRxiv2023

Evaluating Study Design Rigor in Preclinical Cardiovascular Research: A Replication Study

BackgroundMethodological rigor remains a priority in preclinical cardiovascular research to ensure experimental reproducibility and high-quality research. Limited reproducibility diminishes the translation of preclinical discoveries into medical practice. In addition, lack of reproducibility fosters uncertainty in the publics acceptance of reported research results. MethodsWe evaluated the reporting of methodological practices in preclinical cardiovascular research studies published in leading scientific journals by screening articles for the inclusion of the following study design elements (SDEs): considering sex as a biological variable, randomization, blinding, and sample size power estimation. We screened for these SDEs across articles regarding preclinical cardiovascular research studies published between 2011 and 2021. We replicated and extended a study published in 2017 by Ramirez et al. We hypothesized a higher SDE inclusion across preclinical studies over time, that preclinical studies that include human and animal substudies within the same study will exhibit greater SDE inclusion than animal-only preclinical studies, and that a difference exists in SDE usage between large and small animal models. ResultsSDE inclusion was low; with 15.2% of animal-only studies including both sexes as a biological variable, 30.4% including randomization, 32.1% including blinding, and 8.2% including sample size estimation. The incorporation of SDEs did not significantly increase over the ten-year timeframe in the screened articles. Randomization and sample size estimation differed significantly between animal and human substudies (corrected p=1.85e-05 and corrected p=3.81e-07, respectively.) ConclusionsEvidence of methodological rigor varies depending on the study type and model organisms used. From 2011-2021, SDE reporting within preclinical studies has not increased, suggesting more work is needed to foster the inclusion of rigorous study design elements in cardiovascular research.

systems biology↗