Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Oxidative stress and inflammation distinctly drive molecular mechanisms of diastolic dysfunction and remodeling in female and male heart failure with preserved ejection fraction rats.

Heart failure with preserved ejection fraction (HFpEF) is a complex cardiovascular insufficiency syndrome presenting with an ejection fraction (EF) of greater than 50% along with different proinflammatory and metabolic co-morbidities. Despite previous work provided key insights into our understanding of HFpEF, effective treatments are still limited. In the current study we attempted to unravel the molecular basis of sex-dependent differences in HFpEF pathology. We analyzed left ventricular samples from 1-year-old female and male transgenic (TG) rats homozygous for the rat Ren-2 renin gene (mRen2) characterized with hypertension and diastolic dysfunction and compared it to age-matched female and male wild type rats (WT) served as control. Cardiomyocytes from female and male TG rats exhibited an elevated titin-based stiffness (Fpassive), which was corrected to control level upon treatment with reduced glutathione indicating titin oxidation. This was accompanied with high levels of oxidative stress in TG rats with more prominent effects in female group. In vitro supplementation with heat shock proteins (HSPs) reversed the elevated Fpassive indicating restoration of their cytoprotective function. Furthermore, the TG group exhibited high levels of proinflammatory cytokines with significant alterations in apoptotic and autophagy pathways in both sexes. Distinct alterations in the expression of several proteins between both sexes suggest their differential impact on disease development and necessitate distinct treatment options. Hence, our data suggested that oxidative stress and inflammation distinctly drive diastolic dysfunction and remodeling in female and male rats with HFpEF and that the sex-dependent mechanisms contribute to HF pathology.

SARS-CoV-2 infects human cardiomyocytes promoted by inflammation and oxidative stress.

INTRODUCTION: The respiratory illness triggered by severe acute respiratory syndrome virus-2 (SARS-CoV-2) is often particularly serious or fatal amongst patients with pre-existing heart conditions. Although the mechanisms underlying SARS-CoV-2-related cardiac damage remain elusive, inflammation (i.e. 'cytokine storm') and oxidative stress are likely involved. METHODS AND RESULTS: Here we sought to determine: 1) if cardiomyocytes are targeted by SARS-CoV-2 and 2) how inflammation and oxidative stress promote the viral entry into cardiac cells. We analysed pro-inflammatory and oxidative stress and its impact on virus entry and virus-associated cardiac damage from SARS-CoV-2 infected patients and compared it to left ventricular myocardial tissues obtained from non-infected transplanted hearts either from end stage heart failure or non-failing hearts (donor group). We found that neuropilin-1 potentiates SARS-CoV-2 entry into human cardiomyocytes, a phenomenon driven by inflammatory and oxidant signals. These changes accounted for increased proteases activity and apoptotic markers thus leading to cell damage and apoptosis. CONCLUSION: This study provides new insights into the mechanisms of SARS-CoV-2 entry into the heart and defines promising targets for antiviral interventions for COVID-19 patients with pre-existing heart conditions or patients with co-morbidities.

Ca2+/calmodulin-dependent protein kinase II and protein kinase G oxidation contributes to impaired sarcomeric proteins in hypertrophy model.

AIMS: Volume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression. METHODS AND RESULTS: Therefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO-induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age-matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4- and 8-month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated. RESULTS: The model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4- and 8-month, which indicates VO-induced hypertrophy. In addition, 8-months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity-time index compared with their age-matched controls. These changes were accompanied by increased Ca2+ sensitivity and titin-based cardiomyocyte stiffness in 8-month VO rats compared with other groups. The altered cardiomyocyte mechanics was associated with phosphorylation deficit of sarcomeric proteins cardiac troponin I, myosin binding protein C and titin, also accompanied with impaired signalling pathways involved in phosphorylation of these sarcomeric proteins in 8-month VO rats compared with age-matched control group. Impaired protein phosphorylation status and dysregulated signalling pathways were associated with significant alterations in the oxidative status of both kinases CaMKII and PKG explaining by this the elevated Ca2+ sensitivity and titin-based cardiomyocyte stiffness and perhaps the development of hypertrophy. CONCLUSIONS: Our findings showed VO-induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy.

Stress activated signalling impaired protein quality control pathways in human hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a complex myocardial disorder with no well-established disease-modifying therapy so far. Our study aimed to investigate how autophagy, oxidative stress, inflammation, stress signalling pathways, and apoptosis are hallmark of HCM and their contribution to the cardiac dysfunction. Demembranated cardiomyocytes from patients with HCM display increased titin-based stiffness (Fpassive), which was corrected upon antioxidant treatment. Titin as a main determinant of Fpassive was S-glutathionylated and highly ubiquitinated in HCM patients. This was associated with a shift in the balance of reduced and oxidized forms of glutathione (GSH and GSSG, respectively). Both heat shock proteins (HSP27 and α-ß crystalline) were upregulated and S-glutathionylated in HCM. Administration of HSPs in vitro significantly reduced HCM cardiomyocyte stiffness. High levels of the phosphorylated monomeric superoxide anion-generating endothelial nitric oxide synthase (eNOS), decreased nitric oxide (NO) bioavailability, decreased soluble guanylyl cyclase (sGC) activity, and high levels of 3-nitrotyrosine were observed in HCM. Many regulators of signal transduction pathways that are involved in autophagy, apoptosis, cardiac contractility, and growth including the mitogen-activated protein kinase (MAPK), protein kinase B (AKT), glycogen synthase kinase 3ß (GSK-3ß), mammalian target of rapamycin (mTOR), forkhead box O transcription factor (FOXO), c-Jun N-terminal protein kinase (JNK), and extracellular-signal-regulated kinase (ERK1/2) were modified in HCM. The apoptotic factors cathepsin, procaspase 3, procaspase 9 and caspase 12, but not caspase 9, were elevated in HCM hearts and associated with increased proinflammatory cytokines (Interleukin 6 (IL-6), interleukin 18 (IL-18), intercellular cell adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1), the Toll-like receptors 2 (TLR2) and the Toll-like receptors 4 (TLR4)) and oxidative stress (3-nitrotyrosine and hydrogen peroxide (H2O2)). Here we reveal stress signalling and impaired PQS as potential mechanisms underlying the HCM phenotype. Our data suggest that reducing oxidative stress can be a viable therapeutic approach to attenuating the severity of cardiac dysfunction in heart failure and potentially in HCM and prevent its progression.

The Interplay between S-Glutathionylation and Phosphorylation of Cardiac Troponin I and Myosin Binding Protein C in End-Stage Human Failing Hearts.

Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase in oxidative stress, cell damage occurs, due to protein, DNA and lipid oxidation. Here, we investigated the oxidative modifications of myofilament proteins, and their role in modulating cardiomyocyte function in end-stage human failing hearts. We found altered maximum Ca2+-activated tension and Ca2+ sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by the increased oxidation of troponin I and myosin binding protein C, and decreased levels of protein kinases A (PKA)- and C (PKC)-mediated phosphorylation of both proteins. The Ca2+ sensitivity and maximal tension correlated strongly with the myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters that were measured in all the samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes, which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing hearts compared to non-failing hearts, and corrected upon treatment with the anti-oxidant GSH enzyme. Here, we provide evidence that the altered mechanical properties of failing human cardiomyocytes are partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to the development of heart failure.

Interventricular Differences of Signaling Pathways-Mediated Regulation of Cardiomyocyte Function in Response to High Oxidative Stress in the Post-Ischemic Failing Rat Heart.

Standard heart failure (HF) therapies have failed to improve cardiac function or survival in HF patients with right ventricular (RV) dysfunction suggesting a divergence in the molecular mechanisms of RV vs. left ventricular (LV) failure. Here we aimed to investigate interventricular differences in sarcomeric regulation and function in experimental myocardial infarction (MI)-induced HF with reduced LV ejection fraction (HFrEF). MI was induced by LAD ligation in Sprague-Dawley male rats. Sham-operated animals served as controls. Eight weeks after intervention, post-ischemic HFrEF and Sham animals were euthanized. Heart tissue samples were deep-frozen stored (n = 3-5 heart/group) for ELISA, kinase activity assays, passive stiffness and Ca2+-sensitivity measurements on isolated cardiomyocytes, phospho-specific Western blot, and PAGE of contractile proteins, as well as for collagen gene expressions. Markers of oxidative stress and inflammation showed interventricular differences in post-ischemic rats: TGF-ß1, lipid peroxidation, and 3-nitrotyrosine levels were higher in the LV than RV, while hydrogen peroxide, VCAM-1, TNFα, and TGF-ß1 were increased in both ventricles. In addition, nitric oxide (NO) level was significantly decreased, while FN-1 level was significantly increased only in the LV, but both were unchanged in RV. CaMKII activity showed an 81.6% increase in the LV, in contrast to a 38.6% decrease in the RV of HFrEF rats. Cardiomyocyte passive stiffness was higher in the HFrEF compared to the Sham group as evident from significantly steeper Fpassive vs. sarcomere length relationships. In vitro treatment with CaMKIIδ, however, restored cardiomyocyte passive stiffness only in the HFrEF RV, but had no effect in the HFrEF LV. PKG activity was lower in both ventricles in the HFrEF compared to the Sham group. In vitro PKG administration decreased HFrEF cardiomyocyte passive stiffness; however, the effect was more pronounced in the HFrEF LV than HFrEF RV. In line with this, we observed distinct changes of titin site-specific phosphorylation in the RV vs. LV of post-ischemic rats, which may explain divergent cardiomyocyte stiffness modulation observed. Finally, Ca2+-sensitivity of RV cardiomyocytes was unchanged, while LV cardiomyocytes showed increased Ca2+-sensitivity in the HFrEF group. This could be explained by decreased Ser-282 phosphorylation of cMyBP-C by 44.5% in the RV, but without any alteration in the LV, while Ser-23/24 phosphorylation of cTnI was decreased in both ventricles in the HFrEF vs. the Sham group. Our data pointed to distinct signaling pathways-mediated phosphorylations of sarcomeric proteins for the RV and LV of the post-ischemic failing rat heart. These results implicate divergent responses for oxidative stress and open a new avenue in targeting the RV independently of the LV.

Empagliflozin improves endothelial and cardiomyocyte function in human heart failure with preserved ejection fraction via reduced pro-inflammatory-oxidative pathways and protein kinase Gα oxidation.

AIMS: Sodium-glucose-cotransporter-2 inhibitors showed favourable cardiovascular outcomes, but the underlying mechanisms are still elusive. This study investigated the mechanisms of empagliflozin in human and murine heart failure with preserved ejection fraction (HFpEF). METHODS AND RESULTS: The acute mechanisms of empagliflozin were investigated in human myocardium from patients with HFpEF and murine ZDF obese rats, which were treated in vivo. As shown with immunoblots and ELISA, empagliflozin significantly suppressed increased levels of ICAM-1, VCAM-1, TNF-α, and IL-6 in human and murine HFpEF myocardium and attenuated pathological oxidative parameters (H2O2, 3-nitrotyrosine, GSH, lipid peroxide) in both cardiomyocyte cytosol and mitochondria in addition to improved endothelial vasorelaxation. In HFpEF, we found higher oxidative stress-dependent activation of eNOS leading to PKGIα oxidation. Interestingly, immunofluorescence imaging and electron microscopy revealed that oxidized PKG1α in HFpEF appeared as dimers/polymers localized to the outer-membrane of the cardiomyocyte. Empagliflozin reduced oxidative stress/eNOS-dependent PKGIα oxidation and polymerization resulting in a higher fraction of PKGIα monomers, which translocated back to the cytosol. Consequently, diminished NO levels, sGC activity, cGMP concentration, and PKGIα activity in HFpEF increased upon empagliflozin leading to improved phosphorylation of myofilament proteins. In skinned HFpEF cardiomyocytes, empagliflozin improved cardiomyocyte stiffness in an anti-oxidative/PKGIα-dependent manner. Monovariate linear regression analysis confirmed the correlation of oxidative stress and PKGIα polymerization with increased cardiomyocyte stiffness and diastolic dysfunction of the HFpEF patients. CONCLUSION: Empagliflozin reduces inflammatory and oxidative stress in HFpEF and thereby improves the NO-sGC-cGMP-cascade and PKGIα activity via reduced PKGIα oxidation and polymerization leading to less pathological cardiomyocyte stiffness.

The molecular mechanisms associated with the physiological responses to inflammation and oxidative stress in cardiovascular diseases.

The complex physiological signal transduction networks that respond to the dual challenges of inflammatory and oxidative stress are major factors that promote the development of cardiovascular pathologies. These signaling networks contribute to the development of age-related diseases, suggesting crosstalk between the development of aging and cardiovascular disease. Inhibition and/or attenuation of these signaling networks also delays the onset of disease. Therefore, a concept of targeting the signaling networks that are involved in inflammation and oxidative stress may represent a novel treatment paradigm for many types of heart disease. In this review, we discuss the molecular mechanisms associated with the physiological responses to inflammation and oxidative stress especially in heart failure with preserved ejection fraction and emphasize the nature of the crosstalk of these signaling processes as well as possible therapeutic implications for cardiovascular medicine.