Poster abstracts

Poster number 12 submitted by Casey Beard

Sarcomere generation is localized to intercalated disc and dependent on local protein synthesis in cardiac hypertrophy

Casey Beard (Biophysics), Vladimir Bogdanov (Biophysics), Galina Sakuta (Physiology and Cellular Biology), Svetlana Tikunova (Physiology and Cellular Biology), Jonathan Davis (Physiology and Cellular Biology), Sandor Gyorke (Physiology and Cellular Biology)

Abstract:
Changes to cardiomyocyte (CM) cellular geometry correspond to different types of cardiac remodeling such as concentric and eccentric hypertrophy; thus, knowing where and how sarcomeres incorporate within CMs is an important steppingstone to developing therapeutic interventions for cardiac remodeling. Recent work revealing mRNA of various protein types distributed throughout CMs1 implicates local protein synthesis as a crucial determinant of sarcomeric growth during hypertrophy. Using fluorescent labeling techniques to detect nascent proteins in a variety of in vivo mouse models of hypertrophy, we investigated our hypothesis that the intercalated disc serves as a hotspot for cellular growth. We developed a myofibril-integrated biomarker using adenovirus to acutely express GFP-tagged troponin C. We detected sarcomeric proteins synthesized within a 4 hour window by introducing the methionine-analog azidohomoalanine in culture, then labeling with click chemistry and a proximity ligation assay (PLA)2 in order to visualize nascent proteins of interest in CMs undergoing hypertrophy. To evaluate growth in a variety of induced hypertrophic conditions, we used the following models: 1) a troponin C mutation (D73N) that desensitizes the thin filament to calcium3, 2) an aortocaval shunt surgery that induces volume overload4, and 3) a diet high in branched chain amino acids fed during the last 4 hours of the active period that induces cardiac hypertrophy5. These models were selected because of the rapidity of the hypertrophy they induce, which allows us to discriminate protein production triggered by hypertrophic signaling. Our results indicate that nascent protein synthesis and genesis of new sarcomeres during hypertrophy primarily occur at cell peripheries, including intercalated disc regions.

References:
1. Bogdanov, V., et al. Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes. Basic Research in Cardiology. 116, 63 (2021).
2. tom Dieck, S., et al. Direct visualization of newly synthesized target proteins in situ. Nature Methods. 12, 411 (2015).
3. Shettigar, V., et al. Rationally engineered troponin c modulates in vivo cardiac function and performance in health and disease. Nat Commun. 7, 10794 (2016).
4. Karram, T., et al. Induction of cardiac hypertrophy by a controlled reproducible sutureless aortocaval shunt in the mouse. J Invest Surg. 18, 325 (2005).
5. Latimer, M. N., et al. Branched chain amino acids selectively promote cardiac growth at the end of the awake period. J Mol Cell Cardiol. 157, 31 (2021).

Keywords: Cardiac hypertrophy