Modulation of titin-based stiffness in hypertrophic cardiomyopathy via protein kinase D
- 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 \(\textit {in vivo}\) 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.
Author: | Melissa HerwigORCiDGND, Detmar KolijnGND, Mária LódiORCiDGND, Soraya HölperGND, Árpád KovácsGND, Zoltán PappGND, Kornelia JaquetGND, Peter-Lukas HaldenwangORCiDGND, Cristobal G. Dos RemediosGND, Hans-Peter ReuschGND, Andreas MüggeORCiDGND, Marcus KrügerGND, Jens FielitzGND, Wolfgang LinkeGND, Nazha HamdaniGND |
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URN: | urn:nbn:de:hbz:294-73900 |
DOI: | https://doi.org/10.3389/fphys.2020.00240 |
Parent Title (English): | Frontiers in physiology |
Publisher: | Frontiers Research Foundation |
Place of publication: | Lausanne |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2020/08/06 |
Date of first Publication: | 2020/04/15 |
Publishing Institution: | Ruhr-Universität Bochum, Universitätsbibliothek |
Tag: | Open Access Fonds HCM; Hsp27; PKD; stiffness; titin |
Volume: | 11 |
Issue: | Article 240 |
First Page: | 240-1 |
Last Page: | 240-23 |
Note: | Article Processing Charge funded by the Deutsche Forschungsgemeinschaft (DFG) and the Open Access Publication Fund of Ruhr-Universität Bochum. |
Institutes/Facilities: | St. Josef-Hospital Bochum, Medizinische Klinik II, Klinik für Kardiologie |
Institut für Pharmakologie und Toxikologie, Abteilung für klinische Pharmakologie | |
open_access (DINI-Set): | open_access |
Licence (English): | Creative Commons - CC BY 4.0 - Attribution 4.0 International |