Systematic evaluation of the adaptability of the non-radioactive SUnSET assay to measure cardiac protein synthesis.

Heart is a dynamic organ that undergoes remodeling in response to both physiological and pathological stimuli. One of the fundamental cellular processes that facilitates changes in the size and shape of this muscular organ is the protein synthesis. Traditionally changes in cardiac protein synthesis levels were measured by radiolabeled tracers. However, these methods are often cumbersome and suffer from radioactive risk. Recently a nonradioactive method for detecting protein synthesis under in vitro conditions called the Surface Sensing of Translation (SUnSET) was described in cell lines of mouse dendrites and T cells. In this work, we provide multiple lines of evidence that the SUnSET assay can be applied to reliably detect changes in protein synthesis both in isolated neonatal primary cardiomyocytes and heart. We successfully tracked the changes in protein synthesis by western blotting as well as immunohistochemical variants of the SUnSET assay. Applying the SUnSET assay, we measured the cardiac protein synthesis during the different ages of mice. Further, we successfully tracked the increase in cardiac protein synthesis during different stages of a well-established model for pathological hypertrophy. Overall, we propose SUnSET assay as a simple, reliable and robust method to measure protein synthesis in the cardiac milieu.

SIRT6 deacetylase transcriptionally regulates glucose metabolism in heart.

Sirtuins are a family of enzymes, which govern a number of cellular processes essential for maintaining physiological balance. SIRT6, a nuclear sirtuin, is implicated in the development of metabolic disorders. The role of SIRT6 in regulation of cardiac metabolism is unexplored. Although glucose is not the primary energy source of heart, defects in glucose oxidation have been linked to heart failure. SIRT6+/- mice hearts exhibit increased inhibitory phosphorylation of PDH subunit E1α. SIRT6 deficiency enhances FoxO1 nuclear localization that results in increased expression of PDK4. We show that SIRT6 transcriptionally regulates the expression of PDK4 by binding to its promoter. SIRT6+/- hearts show accumulation of lactate, indicating compromised mitochondrial oxidation. SIRT6 deficiency results in decreased oxygen consumption rate and concomitantly lesser ATP production. Mechanistically, SIRT6 deficiency leads to increased FoxO1-mediated transcription of PDK4. Our findings establish a novel link between SIRT6 and cardiac metabolism, suggesting a protective role of SIRT6 in maintaining cardiac homeostasis.