Parker Stephens

Nanoscale Characterization of Contractile Architecture in iPSC-derived Cardiomyocytes

Faculty PI: Dorit Hanein

Cardiomyocytes generate contractile force through interactions between actin filaments and the motor protein myosin. Cardiac myosin exists predominantly as two myosin heavy chain (MHC) isoforms, α-MHC and β-MHC, which differ in their force- and velocity-generating properties [1,2]. The thyroid hormone triiodothyronine (T3) regulates cardiac MHC isoform expression and promotes α-MHC expression, but whether this shift can be reliably induced and characterized in individual hiPSC-derived cardiomyocytes (hiPSC-CMs) on mechanically defined, cryo-ET-compatible substrates remains unclear [1,3].

Here, we show that T3 treatment induces α-MHC expression in individual hiPSC-CMs. We developed a device using contactless UV-photopatterning of cryo-EM-compatible grids placed on a polyacrylamide gel to reproduce physiologically relevant mechanical environments while enabling subsequent cellular cryo-electron tomography [4,5]. This approach increased usable cell coverage on patterned grids from 25% to 74%.

We cultured cardiomyocytes under three conditions- T3, vehicle, and no treatment- and assessed MHC isoform expression by immunofluorescence, with cell health confirmed by Rapid Cell Integrity Assessment [6]. T3-treated cardiomyocytes expressed α-MHC, while both controls predominantly expressed β-MHC. ELISA analysis showed that atrial natriuretic peptide (ANP), expected to decrease following T3 treatment, was reduced in T3-treated cells relative to both controls. Similar ANP levels between vehicle and untreated groups, independent of substrate type, further support T3 as the driver of these changes.

Together, these results establish the application of T3 treatment to individual hiPSC-CMs on mechanically defined, cryo-ET-compatible substrates. Future work will quantify MHC isoform composition using isoform-specific ELISAs, determine isoform-dependent functional differences using traction force microscopy [7], and apply cellular cryo-electron tomography [8,5] to resolve how MHC isoform composition and mechanical environment shape the 3D nanoscale organization of the contractile apparatus.