Upregulation of the MAGUK Sap97 Enhances Protein Expression in Stem Cell Derived Myocytes

Abstract

Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) hold promise for personalized disease modeling and therapy development, however use of hiPSC-CMs is limited by problems of cell immaturity. Studies show that hiPSC-CMs have inadequate expression of proteins necessary for normal structure and function. Membrane-associated guanylate kinases (MAGUKs) are important for protein targeting/clustering needed for signal transduction. Synapse associated protein 97, the archetypical MAGUK, interacts with cell adhesion molecules, receptors/ion channels, and other MAGUKs, which necessary for cell differentiation and maturation. Preliminary evidence shows that Sap97 protein levels are marginal or undetectable in hiPSC-CMs. We tested the hypothesis that upregulation of Sap97 will increase protein expression and thereby enhance hiPSC-CMs maturation. Methods and Results: hiPSC-CMs were infected with control adenoviral constructs (Adv-GFP) or with Sap97 constructs (Adv-Sap97), and 6-well plates of cells were collected after 72 hours for biochemical analyses. Control hiPSC-CMs had marginal to undetectable levels of Sap97 expression. Infection with Adv-Sap97 increased Sap97 expression by 0.35+/- 0.01 (10 MOI; n=4), and by 0.70 +/- 0.01 (50 MOI; n=4). Furthermore, Adv-Sap97 (50 MOI) enhanced expression of N-cadherin and myosin VI, respectively, from 0.48+/- 0.01 to 0.78 +/- 0.01 (p=0.0003; n=5), and from 0.48 +/-0.4 to 1.04 +/-0.1 p=0.0027; n=5). Moreover, Adv-Sap97 (50 MOI) increased expression of HCN4 and CX43 proteins, respectively, from 1.05+/- 0.01 to 2.5 +/- 0.01 p= 0.0425; n=4) and from 0.43+/- 0.01 to 0.9 +/- 0.01 p=0.0001; n=5). In optically measured action potentials, Adv-Sap97 treated hiPSC-CM monolayers had slower spontaneous depolarization rate (1.1±0.22 Hz, n=7) than control (Adv-GFP, 1.68±0.12 Hz, n=6). Conclusions: Upregulation of MAGUK protein, Sap97, enhances expression of proteins that are important for structural and electrical maturation of hiPSC-CMs.