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Single-molecule biochemical characterization of human β-cardiac myosin purified from non-virally transfected C2C12 cells
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. (The molecular motors and bionano-group)ORCID iD: 0000-0003-0225-8227
2025 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Myosin II is a molecular motor that primarily functions in muscle contraction where it generates force and motion through cyclic interaction with actin filaments, driven by ATP turnover. Gaining mechanistic insights into actomyosin energy transduction is essential, particularly in the context of mutations associated with hypertrophic cardiomyopathy (HCM), a genetic condition linked to altered cardiac function. The current approach for studying mutations in the myosin motor domain relies on protein expression in C2C12 cells using an adenovirus-based transfection system. However, this method is constrained by slow turnaround time and labor-intensive protocols. This thesis presents a virus-free transfection method to express human β-cardiac myosin subfragment-1 (denoted as S1L) in C2C12 cells using commercially available chemical reagents – JetPrime and GenJet.  The purified S1L proteins exhibited actin-activated ATPase and sliding velocities (using in vitro motility assay) were comparable to those obtained using the virus-based system. Our new alternative method provides a faster and less complex approach for screening a wide range of HCM mutations. Furthermore, a highly miniaturized single-molecule ATPase assay was developed to leverage the advantage of the virus-free expression system. Using total internal reflection fluorescence microscopy (TIRF), fluorescent ATP turnover rate constants for myosin were determined for both basal and actin-activated conditions. The latter was obtained by crosslinking S1L proteins to surface-immobilized actin filaments via EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) crosslinker. The observed turnover rates were consistent with those obtained from solution-based kinetic assays. By integrating the virus-free expression system with the single-molecule ATPase assay, several S1L point mutations, including R243E, R243E+E466R, R243C, and R243H were biochemically characterized alongside the classical HCM mutations R403Q and R453C. The R243 mutants are of particular interest, as this residue plays an important role in the secondary Pi binding site and may influence the multistep process of inorganic phosphate release. Initial findings suggest that the R243C mutation could serve as a promising model for investigating orthophosphate release, potentially offering deeper insights into the molecular mechanisms underlying HCM pathogenesis.

Place, publisher, year, edition, pages
Linnaeus University Press, 2025. , p. 89
Series
Linnaeus University Dissertations ; 556/2025
Keywords [en]
Human β-cardiac myosin, mutations, hypertrophic cardiomyopathy, C2C12, virus-free transfection, expression, single-molecule ATPase, actin-activated ATPase, crosslinking, basal ATPase, total internal reflection fluorescence microscopy, phosphate release.
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-134689ISBN: 9789180822558 (print)ISBN: 9789180822565 (electronic)OAI: oai:DiVA.org:lnu-134689DiVA, id: diva2:1929498
Public defence
2025-02-07, Vi 2166 (Azur), Linnaeus University, Building Vita, Kalmar, 16:28 (English)
Opponent
Supervisors
Available from: 2025-01-21 Created: 2025-01-20 Last updated: 2025-01-21Bibliographically approved
List of papers
1. Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays
Open this publication in new window or tab >>Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays
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2023 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, no 1, article id 4101Article in journal (Refereed) Published
Abstract [en]

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5-1.7 mu m/s; 25 degrees C) in the in vitro motility assay as well as maximum actin activated catalytic activity (k(cat); 8-9 s(-1)) and actin concentration for half maximal activity (K-ATPase; 70-80 mu M) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

Place, publisher, year, edition, pages
Nature Publishing Group, 2023
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-120565 (URN)10.1038/s41598-023-30576-1 (DOI)000949110500003 ()36907906 (PubMedID)2-s2.0-85150205206 (Scopus ID)
Available from: 2023-05-05 Created: 2023-05-05 Last updated: 2025-02-04Bibliographically approved
2. Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence
Open this publication in new window or tab >>Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence
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2024 (English)In: Cytoskeleton, ISSN 1949-3584, Vol. 81, no 12, p. 883-901Article in journal (Refereed) Published
Abstract [en]

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (k(cat)). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Place, publisher, year, edition, pages
John Wiley & Sons, 2024
Keywords
actin, actin-activated ATPase, Alexa 647 ATP, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, single molecule fluorescence, beta-Cardiac myosin II
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-129124 (URN)10.1002/cm.21858 (DOI)001203081500001 ()38623952 (PubMedID)2-s2.0-85191016814 (Scopus ID)
Available from: 2024-05-02 Created: 2024-05-02 Last updated: 2025-02-04Bibliographically approved
3. Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent
Open this publication in new window or tab >>Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent
2024 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 25, no 12, article id 6747Article in journal (Refereed) Published
Abstract [en]

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac beta-myosin heavy chain (beta-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current similar to 4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Place, publisher, year, edition, pages
MDPI, 2024
Keywords
virus-free gene delivery, C2C12, cell transfection, protein expression, protein purification, human cardiac myosin II, in vitro motility assay, single-molecule assays
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-131772 (URN)10.3390/ijms25126747 (DOI)001257630300001 ()38928453 (PubMedID)2-s2.0-85197301116 (Scopus ID)
Available from: 2024-08-15 Created: 2024-08-15 Last updated: 2025-02-04Bibliographically approved

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