PROJECT SUMMARY Heart failure is a result of aberrant matrix remodeling in the left ventricle; male patients experience ventricular wall thinning, whereas females experience ventricular wall thickening. While current pharmacologic agents palliate symptoms associated with heart failure, the morbidity of the disease remains high as these treatments do not provide a universal cure, necessitating a sex-specific understanding of heart failure. Mechanical cues and gonadal hormones regulate the sex-specific cardiac remodeling observed during heart failure through changes in the extracellular matrix (ECM). Resident cardiac fibroblasts that reside in the ventricular wall largely regulate aberrant ECM remodeling during heart failure, but the sex-specific regulation of cell-mediated ECM remodeling remains poorly understood. Both sex hormones and sex chromosomes have been implicated in regulating sex differences in cardiac ECM remodeling, but current methods do not decouple their synergistic effects. My proposal seeks to decouple the effects of sex chromosomes and sex hormones using gonadectomized animal models and biomaterials-based cell culture tools that recapitulate the cardiac ECM during fibrotic stiffening. Our laboratory tools enable our central hypothesis: sex chromosome combination and gonadal hormones synergistically regulate myofibroblast phenotypes, and X-chromosome linked genes and estrogen dosage will promote myofibroblast matrix remodeling. The hypothesis will be investigated through the following aims: Aim 1 will improve our understanding of myofibroblast populations that drive sex-specific matrix remodeling independent of gonadal hormones; Aim 2 will use engineered hydrogels to mimic sex-specific myofibroblast phenotypes in vitro; Aim 3 will investigate the role of estrogen and estrogen receptor expression on mediating the myofibroblast phenotype independent of sex chromosome combination. By leveraging animal models and biomaterial technologies, this study will unveil a robust understanding of the myofibroblast phenotype by decoupling the effects of sex chromosomes and sex hormones. If successful, the proposed research will significantly advance the understanding of sex-specific biology in myocardial fibrosis.