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, 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

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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
Creators:
CreatorsEmailORCIDORCID Put Code
Herwig, MelissaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kolijn, DetmarUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Lodi, MariaUNSPECIFIEDorcid.org/0000-0002-6533-770XUNSPECIFIED
Hoelper, SorayaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kovacs, ArpadUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Papp, ZoltanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Jaquet, KorneliaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Haldenwang, PeterUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Dos Remedios, CrisUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Reusch, Peter H.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Muegge, AndreasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Krueger, MarcusUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Fielitz, JensUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Linke, Wolfgang A.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Hamdani, NazhaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
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: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
HEAT-SHOCK PROTEINS; ALPHA-B-CRYSTALLIN; CARDIAC TROPONIN-I; SERINE-82 PHOSPHORYLATION; PASSIVE STIFFNESS; MOLECULAR-CLONING; STRESS; EXPRESSION; MUSCLE; TENSIONMultiple languages
PhysiologyMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/33699

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