Sex-specific cardiovascular remodeling leads to a divergent sex-dependent development of heart failure in aged hypertensive rats.

INTRODUCTION: The prevalence of heart failure with preserved ejection fraction (HFpEF) is continuously rising and predominantly affects older women often hypertensive and/or obese or diabetic. Indeed, there is evidence on sex differences in the development of HF. Hence, we studied cardiovascular performance dependent on sex and age as well as pathomechanisms on a cellular and molecular level. METHODS: We studied 15-week- and 1-year-old female and male hypertensive transgenic rats carrying the mouse Ren-2 renin gene (TG) and compared them to wild-type (WT) controls at the same age. We tracked blood pressure and cardiac function via echocardiography. After sacrificing the 1-year survivors we studied vascular smooth muscle and endothelial function. Isolated single skinned cardiomyocytes were used to determine passive stiffness and Ca2+-dependent force. In addition, Western blots were applied to analyse the phosphorylation status of sarcomeric regulatory proteins, titin and of protein kinases AMPK, PKG, CaMKII as well as their expression. Protein kinase activity assays were used to measure activities of CaMKII, PKG and angiotensin-converting enzyme (ACE). RESULTS: TG male rats showed significantly higher mortality at 1 year than females or WT male rats. Left ventricular (LV) ejection fraction was specifically reduced in male, but not in female TG rats, while LV diastolic dysfunction was evident in both TG sexes, but LV hypertrophy, increased LV ACE activity, and reduced AMPK activity as evident from AMPK hypophosphorylation were specific to male rats. Sex differences were also observed in vascular and cardiomyocyte function showing different response to acetylcholine and Ca2+-sensitivity of force production, respectively cardiomyocyte functional changes were associated with altered phosphorylation states of cardiac myosin binding protein C and cardiac troponin I phosphorylation in TG males only. Cardiomyocyte passive stiffness was increased in TG animals. On a molecular level titin phosphorylation pattern was altered, though alterations were sex-specific. Thus, also the reduction of PKG expression and activity was more pronounced in TG females. However, cardiomyocyte passive stiffness was restored by PKG and CaMKII treatments in both TG sexes. CONCLUSION: Here we demonstrated divergent sex-specific cardiovascular adaptation to the over-activation of the renin-angiotensin system in the rat. Higher mortality of male TG rats in contrast to female TG rats was observed as well as reduced LV systolic function, whereas females mainly developed HFpEF. Though both sexes developed increased myocardial stiffness to which an impaired titin function contributes to a sex-specific molecular mechanism. The functional derangements of titin are due to a sex-specific divergent regulation of PKG and CaMKII systems.

Reorganization of the actin cytoskeleton during the formation of neutrophil extracellular traps (NETs).

We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA). Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of the cortical F-actin at spatially confined areas, the NET extrusion sites, effected by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself appeared to be proteolyzed by neutrophil elastase possibly to confine its activity to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma membrane of the remaining cell body. A similar distribution was observed for myosin that concentrated together with cortical F-actin along the periphery of the remaining cell body suggesting force production by acto-myosin interactions supporting NET expulsion as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin. Isolated human neutrophils displayed differences in their content of certain cytoskeletal proteins. After stimulation neutrophils with high gelsolin content preferentially formed "cloud"-like NETs, whereas those with low or no gelsolin formed long "filamentous" NETs.

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.

Altered Cellular Protein Quality Control System Modulates Cardiomyocyte Function in Volume Overload-Induced Hypertrophy.

Volume-induced hypertrophy is one of the risk factors for cardiac morbidity and mortality. In addition, mechanical and metabolic dysfunction, aging, and cellular redox balance are also contributing factors to the disease progression. In this study, we used volume overload (VO), which was induced by an aortocaval fistula in 2-month-old male Wistar rats, and sham-operated animals served as control. Functional parameters were measured by transthoracic echocardiography at termination 4- or 8-months after VO. The animals showed hypertrophic remodeling that was accompanied by mechanical dysfunction and increased cardiomyocyte stiffness. These alterations were reversible upon treatment with glutathione. Cardiomyocyte dysfunction was associated with elevated oxidative stress markers with unchanged inflammatory signaling pathways. In addition, we observed altered phosphorylation status of small heat shock proteins 27 and 70 and diminished protease expression caspases 3 compared to the matched control group, indicating an impaired protein quality control system. Such alterations might be attributed to the increased oxidative stress as anticipated from the enhanced titin oxidation, ubiquitination, and the elevation in oxidative stress markers. Our study showed an early pathological response to VO, which manifests in cardiomyocyte mechanical dysfunction and dysregulated signaling pathways associated with enhanced oxidative stress and an impaired protein quality control system.

Current Understanding of Molecular Pathophysiology of Heart Failure With Preserved Ejection Fraction.

Heart Failure (HF) is the most common cause of hospitalization in the Western societies. HF is a heterogeneous and complex syndrome that may result from any dysfunction of systolic or diastolic capacity. Abnormal diastolic left ventricular function with impaired relaxation and increased diastolic stiffness is characteristic of heart failure with preserved ejection fraction (HFpEF). HFpEF accounts for more than 50% of all cases of HF. The prevalence increases with age: from around 1% for those aged <55 years to >10% in those aged 70 years or over. Nearly 50% of HF patients have HFrEF and the other 50% have HFpEF/HFmrEF, mainly based on studies in hospitalized patients. The ESC Long-Term Registry, in the outpatient setting, reports that 60% have HFrEF, 24% have HFmrEF, and 16% have HFpEF. To some extent, more than 50% of HF patients are female. HFpEF is closely associated with co-morbidities, age, and gender. Epidemiological evidence suggests that HFpEF is highly represented in older obese women and proposed as 'obese female HFpEF phenotype'. While HFrEF phenotype is more a male phenotype. In addition, metabolic abnormalities and hemodynamic perturbations in obese HFpEF patients appear to have a greater impact in women then in men (Sorimachi et al., European J of Heart Fail, 2022, 22). To date, numerous clinical trials of HFpEF treatments have produced disappointing results. This outcome suggests that a "one size fits all" approach to HFpEF may be inappropriate and supports the use of tailored, personalized therapeutic strategies with specific treatments for distinct HFpEF phenotypes. The most important mediators of diastolic stiffness are the cardiomyocytes, endothelial cells, and extracellular matrix (ECM). 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 HFpEF pathologies. These signalling networks contribute to the development of the diseases. Inhibition and/or attenuation of these signalling networks also delays the onset of disease. In this review, we discuss the molecular mechanisms associated with the physiological responses to inflammation and oxidative stress and emphasize the nature of the contribution of most important cells to the development of HFpEF via increased inflammation and oxidative stress.

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.