Proteomics based mapping of cardiac extracellular matrix to define sex and age-dependent changes. - PROJECT SUMMARY The extracellular matrix (ECM) provides a scaffold for cardiac structural integrity and plays an active role in modulating cellular responses. ECM consists of a network of fibrillar proteins and proteoglycans binding cyto- kines, growth factors, and ECM-modifying enzymes. The aging heart develops interstitial fibrosis, and prelimi- nary data demonstrate that different proteins are enriched in ECM in the male and female heart. An altered ECM can affect cellular phenotypes. The ECM-cell communication occurs via cell surface receptors such as integrins, through outside-in signaling, where integrin-binding to ECM protein domains translates into prolifera- tion, differentiation, or gene expression. On the other hand, activation of the intracellular integrin tail increases their ECM-binding affinity, promoting adhesion, migration, or ECM assembly (by inside-out signaling). There- fore, ECM dictates a cellular phenotype, but cells can modify ECM in response by changing its composition or assembly. Because of profound differences in ECM composition in males and females, we hypothesize that the quality of repair assembled after injury and subsequent adverse remodeling in males and females will also be differ- ent, and the differences will be accentuated by aging. In SA1, we will map the differences in ECM dependent on age (young and old animals will be studied), sex hormone level (animals will be subjected to chemical ovary failure or castration) or level of inflammation (CCL2KO animals will be used; these mice have reduced leuko- cyte infiltration and therefore reduced fibrosis). The cellular phenotype of fibroblasts, endothelial cells (ECs), and smooth muscle cells (SMCs) isolated from all these animals will be studied ex vivo and in 3D culture. The effect of ECM on cell phenotype will be tested in 3D cultures using the matrix swap approach, where i.e. young cells will be cultured on ECM from the old hearts and vice versa. And the effect of matrix stiffness on the pro- duction of ECM protein will be examined as well. Similarly, the physiology of human cells will be studied. In SA2, we will examine ECM-integrin dependent phenotypic changes in mouse and human cells. We will first examine the effect of sex hormones, age, and inflammation on the levels of integrin expression in fibroblasts, ECs, and SMCs. Then we will manipulate the integrin expression level (via downregulation or overexpression), and examine the cellular phenotype. Finally, we will manipulate estrogen and androgen receptors and deter- mine the effect of these manipulations on integrin levels and ECM protein synthesis. In SA3, we will examine how sex hormone levels and age affect scar formation and adverse remodeling after ischemia/reperfusion inju- ry. Changes in ECM will be examined via ELISA, mass spectrometry, and western blot. The approach is inno- vative because it examines the role of three superimposed variables (age, sex hormone, and inflammation) on ECM and cellular phenotypes. Successful completion of these studies will provide a mechanistic link for future therapeutics targeting insufficient reparative and excessive adverse fibrosis.
