Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G)
Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the <i>baculovirus/Sf21</i> insect cell system. The c-actin variants inhibited DNase I, indicating maintenance...
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MDPI AG
2022-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/8/4465 |
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| author | Roua Hassoun Constanze Erdmann Sebastian Schmitt Setsuko Fujita-Becker Andreas Mügge Rasmus R. Schröder Matthias Geyer Mina Borbor Kornelia Jaquet Nazha Hamdani Hans Georg Mannherz |
| author_facet | Roua Hassoun Constanze Erdmann Sebastian Schmitt Setsuko Fujita-Becker Andreas Mügge Rasmus R. Schröder Matthias Geyer Mina Borbor Kornelia Jaquet Nazha Hamdani Hans Georg Mannherz |
| author_sort | Roua Hassoun |
| collection | DOAJ |
| description | Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the <i>baculovirus/Sf21</i> insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca<sup>2+</sup>-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca<sup>2+</sup>-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca<sup>2+</sup>-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca<sup>2+</sup>-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca<sup>2+</sup>-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm. |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T04:33:50Z |
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| spelling | doaj.art-49d3b78b6e394695a0092d242d4034f72023-12-03T13:31:12ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-04-01238446510.3390/ijms23084465Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G)Roua Hassoun0Constanze Erdmann1Sebastian Schmitt2Setsuko Fujita-Becker3Andreas Mügge4Rasmus R. Schröder5Matthias Geyer6Mina Borbor7Kornelia Jaquet8Nazha Hamdani9Hans Georg Mannherz10Institut für Forschung und Lehre (IFL), Molecular and Experimental Cardiology, Medical Faculty, Ruhr University Bochum, D-44791 Bochum, GermanyDepartment of Anatomy and Molecular Embryology, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, GermanyInstitute of Structural Biology, University of Bonn, D-53127 Bonn, GermanyCryoelectron Microscopy, BioQuant, Medical Faculty, University of Heidelberg, D-69120 Heidelberg, GermanyInstitut für Forschung und Lehre (IFL), Molecular and Experimental Cardiology, Medical Faculty, Ruhr University Bochum, D-44791 Bochum, GermanyCryoelectron Microscopy, BioQuant, Medical Faculty, University of Heidelberg, D-69120 Heidelberg, GermanyInstitute of Structural Biology, University of Bonn, D-53127 Bonn, GermanyDepartment of Neurology, University Hospital Essen, D-45147 Essen, GermanyInstitut für Forschung und Lehre (IFL), Molecular and Experimental Cardiology, Medical Faculty, Ruhr University Bochum, D-44791 Bochum, GermanyInstitut für Forschung und Lehre (IFL), Molecular and Experimental Cardiology, Medical Faculty, Ruhr University Bochum, D-44791 Bochum, GermanyInstitut für Forschung und Lehre (IFL), Molecular and Experimental Cardiology, Medical Faculty, Ruhr University Bochum, D-44791 Bochum, GermanyHuman wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the <i>baculovirus/Sf21</i> insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca<sup>2+</sup>-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca<sup>2+</sup>-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca<sup>2+</sup>-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca<sup>2+</sup>-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca<sup>2+</sup>-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.https://www.mdpi.com/1422-0067/23/8/4465cardiac actincalcium sensitivitycardiomyopathieslevosimendanmyosin subfragment 1myosin binding protein C |
| spellingShingle | Roua Hassoun Constanze Erdmann Sebastian Schmitt Setsuko Fujita-Becker Andreas Mügge Rasmus R. Schröder Matthias Geyer Mina Borbor Kornelia Jaquet Nazha Hamdani Hans Georg Mannherz Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) International Journal of Molecular Sciences cardiac actin calcium sensitivity cardiomyopathies levosimendan myosin subfragment 1 myosin binding protein C |
| title | Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) |
| title_full | Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) |
| title_fullStr | Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) |
| title_full_unstemmed | Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) |
| title_short | Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) |
| title_sort | functional characterization of cardiac actin mutants causing hypertrophic p a295s and dilated cardiomyopathy p r312h and p e361g |
| topic | cardiac actin calcium sensitivity cardiomyopathies levosimendan myosin subfragment 1 myosin binding protein C |
| url | https://www.mdpi.com/1422-0067/23/8/4465 |
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