Abstract
We studied the contractile properties of the MYH7 G256E mutation using engineered human stem cell derived cardiomyocytes (hSC-CMs). When hSC-CMs were cultured to 40-45 days post-differentiation and Western blot analysis demonstrated near 100% β-myosin (MYH7). Single sub-cellular contractile organelles (myofibrils) were mounted in a custom built apparatus between a force transducer and linear motor. Rapid solutions switching to maximal activation solution (pCa 4.0) resulted in a doubling of steady-state force and rate of force development for G256E myofibrils compared with wild type (WT) controls. Upon switching to relaxation solution (pCa 9.0), the slope of the initial linear, slow phase relaxation was significantly slower (∼4-fold) for G256E vs WT myofibrils, suggesting the mutation slows the crossbridge detachment rate in loaded contractions. Crossbridge detachment rate can be reduced by slower ADP release or slower ATP binding to myosin. Analysis using stopped flow kinetics showed the ATP binding rate was slightly, but significantly reduced for G256E vs WT myofibrils. Preliminary data from myofibril mechanical analysis suggests that ADP release is also slower with the G256E mutation. Molecular dynamics (MD) simulations of the rigor and myosin.ATP states showed that G256E led to reorganization of hydrogen bonding networks within the transducer region. Enhanced sampling MD simulations of the rigor to post-rigor (ATP-bound) structural transition indicated G256E myosin surmounts a greater energetic barrier describing the binding of switch 1 to ATP, rotation of the transducer backbone, and opening of the actin binding cleft. This increased barrier is attributed to salt bridges formed by the mutant Glu residue. Combined, our results suggest that G256E leads to hypercontraction and delayed relaxation phenotype via modulation of the transducer structure that alters communication between the nucleotide binding pocket and the actin binding surface during the chemo-mechanical cycle.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.