Herwig, Melissa, Kolijn, Detmar, Lodi, Maria 
ORCID: 0000-0002-6533-770X, Hoelper, Soraya, Kovacs, Arpad, Papp, Zoltan, Jaquet, Kornelia, Haldenwang, Peter, Dos Remedios, Cris, Reusch, Peter H., Muegge, Andreas, Krueger, Marcus ORCID: 0000-0003-2008-4582, Fielitz, Jens, Linke, Wolfgang A. and Hamdani, Nazha
(2020).
Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D.
Front. Physiol., 11.
LAUSANNE:
FRONTIERS MEDIA SA.
ISSN 1664-042X
Abstract
The giant protein titin performs structure-preserving functions in the sarcomere and is important for the passive stiffness (F-passive) of cardiomyocytes. Protein kinase D (PKD) enzymes play crucial roles in regulating myocardial contraction, hypertrophy, and remodeling. PKD phosphorylates myofilament proteins, but it is not known whether the giant protein titin is also a PKD substrate. Here, we aimed to determine whether PKD phosphorylates titin and thereby modulates cardiomyocyte F-passive in normal and failing myocardium. The phosphorylation of titin was assessed in cardiomyocyte-specific PKD knock-out mice (cKO) and human hearts using immunoblotting with a phosphoserine/threonine and a phosphosite-specific titin antibody. PKD-dependent site-specific titin phosphorylation in vivo was quantified by mass spectrometry using stable isotope labeling by amino acids in cell culture (SILAC) of SILAC-labeled mouse heart protein lysates that were mixed with lysates isolated from hearts of either wild-type control (WT) or cKO mice. F-passive of single permeabilized cardiomyocytes was recorded before and after PKD and HSP27 administration. All-titin phosphorylation was reduced in cKO compared to WT hearts. Multiple conserved PKD-dependent phosphosites were identified within the Z-disk, A-band and M-band regions of titin by quantitative mass spectrometry, and many PKD-dependent phosphosites detected in the elastic titin I-band region were significantly decreased in cKO. Analysis of titin site-specific phosphorylation showed unaltered or upregulated phosphorylation in cKO compared to matched WT hearts. F-passive was elevated in cKO compared to WT cardiomyocytes and PKD administration lowered F-passive of WT and cKO cardiomyocytes. Cardiomyocytes from hypertrophic cardiomyopathy (HCM) patients showed higher F-passive compared to control hearts and significantly lower F-passive after PKD treatment. In addition, we found higher phosphorylation at CaMKII-dependent titin sites in HCM compared to control hearts. Expression and phosphorylation of HSP27, a substrate of PKD, were elevated in HCM hearts, which was associated with increased PKD expression and phosphorylation. The relocalization of HSP27 in HCM away from the sarcomeric Z-disk and I-band suggested that HSP27 failed to exert its protective action on titin extensibility. This protection could, however, be restored by administration of HSP27, which significantly reduced F-passive in HCM cardiomyocytes. These findings establish a previously unknown role for PKDin regulating diastolic passive properties of healthy and diseased hearts.
| Item Type: | Journal Article | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| URN: | urn:nbn:de:hbz:38-336994 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| DOI: | 10.3389/fphys.2020.00240 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Journal or Publication Title: | Front. Physiol. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Volume: | 11 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Date: | 2020 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Publisher: | FRONTIERS MEDIA SA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Place of Publication: | LAUSANNE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| ISSN: | 1664-042X | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Language: | English | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Faculty: | Faculty of Mathematics and Natural Sciences | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Divisions: | Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Subjects: | no entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Refereed: | Yes | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| URI: | http://kups.ub.uni-koeln.de/id/eprint/33699 |
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