Cardiomyopathy Ctnt Mutation in Patient Derived Cardiomyocytes from Induced Pluripotent Stem Cells Affects Sarcomere Structure and Function

Abstract

Cardiomyocytes derived from human induced pluripotent stem cells (hIPS-CM) are useful to understand basic structural and functional characteristics of normal and diseased human heart cells. We investigated mechanical properties of hIPS-CM derived from unaffected and affected members of a family harboring a dilated cardiomyopathy (DCM) mutation in cardiac troponin T (cTnT), the tropomyosin binding unit of troponin. Patients with a cTnT point mutation (R173W) develop DCM, which commonly leads to diastolic and systolic dysfunction and progressive heart failure. To study the shortening and shortening velocity of normal and cTnT mutant hIPS-CM cells, cells were plated on substrates with a relatively soft stiffness (160 kPa) molded from polydimethylsiloxane and measured using a line scan method. Data were collected using a Zeiss 710 confocal microscope. Visual observation of cells after 5 days maturity on the PDMS substrates indicate a smaller percent of mutant (8%) compared to normal (87%) hIPS-CM were spontaneously beating. Immunohistochemistry showed that myofibril structure was better developed in normal compared to cTnT mutation cells. Day 5 line scans revealed normal hIPS-CM cells shorten more (0.47 μm) than mutant cTnT cells (0.32 μm); shortening velocity was faster in normal (1.42 μm/s) compared to mutant cTnT cells (0.67 μm/s). Acute treatment (1 dose with a 10 min activation dwell time) with omecamtiv mecarbil (200nM), a cardiac myosin activator, increased the shortening and shortening velocity of normal (0.78 μm at 1.85 μm/s) but not cTnT mutant (0.32 μm at 0.70 μm/s) hIPS-CM cells. Although more studies are necessary, these results suggest that cTnT may affect development of sarcomeres and the regulation of contractility. Furthermore, the myosin activator omecamtiv mecarbil may not be sufficient to rescue dysfunction in the cTnT mutation R173W. [Funding: NIH T32HL07692, PO1HL62426]