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Study of the Expression Transition of Cardiac Myosin Using Polarization-Dependent SHG Microscopy.

ABSTRACT: Detection of the transition between the two myosin isoforms ?- and ?-myosin in living cardiomyocytes is essential for understanding cardiac physiology and pathology. In this study, the differences in symmetry of polarization spectra obtained from ?- and ?-myosin in various mammalian ventricles and propylthiouracil-treated rats are explored through polarization-dependent second harmonic generation microscopy. Here, we report for the, to our knowledge, first time that ?- and ?-myosin, as protein crystals, possess different symmetries: the former has C6 symmetry, and the latter has C3v. A single-sarcomere line scan further demonstrated that the differences in polarization-spectrum symmetry between ?- and ?-myosin came from their head regions: the head and neck domains of ?- and ?-myosin account for the differences in symmetry. In addition, the dynamic transition of the polarization spectrum from C6 to C3v line profile was observed in a cell culture in which norepinephrine induced an ?- to ?-myosin transition.

SUBMITTER: Yuan C

PROVIDER: S-EPMC7063480 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Study of the Expression Transition of Cardiac Myosin Using Polarization-Dependent SHG Microscopy.

Yuan Cai C Zhao Xiaolei X Wang Zhonghai Z Borg Thomas K TK Ye Tong T Khalpey Zain I ZI Runyan Raymond B RB Shao Yonghong Y Gao Bruce Z BZ

Biophysical journal 20200103 5

Detection of the transition between the two myosin isoforms α- and β-myosin in living cardiomyocytes is essential for understanding cardiac physiology and pathology. In this study, the differences in symmetry of polarization spectra obtained from α- and β-myosin in various mammalian ventricles and propylthiouracil-treated rats are explored through polarization-dependent second harmonic generation microscopy. Here, we report for the, to our knowledge, first time that α- and β-myosin, as protein c ...[more]

PMID: 31995740

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Project description:Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.

Dataset Information

Study of the Expression Transition of Cardiac Myosin Using Polarization-Dependent SHG Microscopy.

Publications

Study of the Expression Transition of Cardiac Myosin Using Polarization-Dependent SHG Microscopy.

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