Project Summary Cardiac performance declines with age, but genetic variation within a population makes it difficult to identify the conserved aging mechanisms that negatively impact mechanical function, i.e. mechanogenetics. Over the past 5 years, we have studied cardiac function of the rapidly aging Drosophila melanogaster across many laboratory fruit fly strains. We found that fly strains with age-dependent and heart-specific vinculin up- regulation use it to reinforce and stiffen their costameres and intercalated discs, which helps maintain the crystallinity of their sarcomere lattice and improves contractility. At the cardiomyocyte level, maintaining inter- myofilament spacing prolongs wall shortening velocity and fractional shortening, and systemically, it extends lifespan. Fly strains with decreased basement membrane (BM) protein expression, e.g. laminin, also exhibit similar systemic benefits but due to thinner extracellular matrix, which improves cell-cell coupling between adjacent myocyte layers; importantly, both vinculin up-regulation and BM down-regulation with age appear conserved up through non-human primates. Together these data suggest a new inside-outside aging paradigm that prolongs heart function, i.e. a robust internal contractile apparatus with limited extracellular connections to BM. In this grant application, we propose further aims to determine how these intracellular and extracellular heart perturbations act combinatorially to alter function across cellular-, tissue-, and organ-levels. Preliminary assessments in transgenic fly strains suggest that more efficient cardiomyocyte contraction, may improve substrate utilization and oxygen consumption within fly hearts, and subsequently organ perfusion and systemic metabolism, and better health- and lifespan. In light of this paradigm and the challenges that mechanogenetics present, we have developed new biological and analytical tools for this grant that will improve the feasibility of assessing the age-related mechanical differences of Drosophila myocardium between wild-type and transgenic lines. Thus, this on-going work will continue to improve our understanding of the inside-outside aging paradigm.