Abstract
The giant protein titin performs structure-preserving functions in the sarcomere and is important for the passive stiffness (Fpassive) 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 Fpassive 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. Fpassive 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. Fpassive was elevated in cKO compared to WT cardiomyocytes and PKD administration lowered Fpassive of WT and cKO cardiomyocytes. Cardiomyocytes from hypertrophic cardiomyopathy (HCM) patients showed higher Fpassive compared to control hearts and significantly lower Fpassive 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 Fpassive in HCM cardiomyocytes. These findings establish a previously unknown role for PKDin regulating diastolic passive properties of healthy and diseased hearts.
Highlights
Protein kinase (PK)D is a serine/threonine kinase that belongs to the family of calcium/calmodulin-dependent kinases (CaMKII) due to its catalytic domain structure and substrate specificity
We hypothesized that Prkd1 cKO mouse hearts would show altered titin phosphorylation compared to matched WT hearts
As we found a relatively high level of Protein Kinase D (PKD) expression in failing hypertrophic cardiomyopathy (HCM) hearts using western blot, we wished to determine the cellular distribution of PKD using confocal laser scanning and electron microscopy
Summary
Protein kinase (PK)D is a serine/threonine kinase that belongs to the family of calcium/calmodulin-dependent kinases (CaMKII) due to its catalytic domain structure and substrate specificity. Some substrates that are targets of PKC are not phosphorylated by PKD (Johannes et al, 1994; Valverde et al, 1994; Van Lint et al, 1995), and unlike CaMKII, PKD is not directly activated by Ca2+ or calmodulin (Avkiran et al, 2008). The PKD kinase is involved in the regulation of myocardial contraction by phosphorylating cardiac myosin binding protein C (cMyBPC), cardiac troponin I (cTnI) and the L-type, voltage gated Ca2+ channel (Haworth et al, 2004; Cuello et al, 2007; Aita et al, 2011; Dirkx et al, 2012). PKD alters gene expression leading to hypertrophy and influencing cardiac remodeling processes (Vega et al, 2004; Harrison et al, 2006)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
