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

Enhanced cardiomyocyte function in hypertensive rats with diastolic dysfunction and human heart failure patients after acute treatment with soluble guanylyl cyclase (sGC) activator

$\textbf {Aims:}$ Our aim was to investigate the effect of nitric oxide (NO)-independent activation of soluble guanylyl cyclase (sGC) on cardiomyocyte function in a hypertensive animal model with diastolic dysfunction and in biopsies from human heart failure with preserved ejection fraction (HFpEF). $\textbf {Methods:}$ Dahl salt-sensitive (DSS) rats and control rats were fed a high-salt diet for 10 weeks and then acutely treated $\textit {in vivo}$ with the sGC activator BAY 58-2667 (cinaciguat) for 30 min. Single skinned cardiomyocyte passive stiffness ($F_{passive}$) was determined in rats and human myocardium biopsies before and after acute treatment. Titin phosphorylation, activation of the NO/sGC/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade, as well as hypertrophic pathways including NO/sGC/cGMP/PKG, PKA, calcium–calmodulin kinase II (CaMKII), extracellular signal-regulated kinase 2 (ERK2), and PKC were assessed. In addition, we explored the contribution of pro-inflammatory cytokines and oxidative stress levels to the modulation of cardiomyocyte function. Immunohistochemistry and electron microscopy were used to assess the translocation of sGC and connexin 43 proteins in the rat model before and after treatment. $\textbf {Results:}$ High cardiomyocyte $F_{passive}$ was found in rats and human myocardial biopsies compared to control groups, which was attributed to hypophosphorylation of total titin and to deranged site-specific phosphorylation of elastic titin regions. This was accompanied by lower levels of PKG and PKA activity, along with dysregulation of hypertrophic pathway markers such as CaMKII, PKC, and ERK2. Furthermore, DSS rats and human myocardium biopsies showed higher pro-inflammatory cytokines and oxidative stress compared to controls. DSS animals benefited from treatment with the sGC activator, as $F_{passive}$, titin phosphorylation, PKG and the hypertrophic pathway kinases, pro-inflammatory cytokines, and oxidative stress markers all significantly improved to the level observed in controls. Immunohistochemistry and electron microscopy revealed a translocation of sGC protein toward the intercalated disc and t-tubuli following treatment in both control and DSS samples. This translocation was confirmed by staining for the gap junction protein connexin 43 at the intercalated disk. DSS rats showed a disrupted connexin 43 pattern, and sGC activator was able to partially reduce disruption and increase expression of connexin 43. In human HFpEF biopsies, the high $F_{passive}$, reduced titin phosphorylation, dysregulation of the NO–sGC–cGMP–PKG pathway and PKA activity level, and activity of kinases involved in hypertrophic pathways CaMKII, PKC, and ERK2 were all significantly improved by sGC treatment and accompanied by a reduction in pro-inflammatory cytokines and oxidative stress markers. $\textbf {Conclusion:}$ Our data show that sGC activator improves cardiomyocyte function, reduces inflammation and oxidative stress, improves sGC–PKG signaling, and normalizes hypertrophic kinases, indicating that it is a potential treatment option for HFpEF patients and perhaps also for cases with increased hypertrophic signaling