Rationale: Atrial fibrillation (AF) and heart failure often coexist, but their interaction is poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to left ventricular (LV) dysfunction. Objective: This study investigates the effects and molecular mechanisms of AF on the human LV. Methods and Results: Ventricular myocardium from patients with aortic stenosis and preserved LV function with sinus rhythm or rate-controlled AF was studied. LV myocardium from patients with sinus rhythm and patients with AF showed no differences in fibrosis. In functional studies, systolic Ca ²⁺ transient amplitude of LV cardiomyocytes was reduced in patients with AF, while diastolic Ca ²⁺ levels and Ca ²⁺ transient kinetics were not statistically different. These results were confirmed in LV cardiomyocytes from nonfailing donors with sinus rhythm or AF. Moreover, normofrequent AF was simulated in vitro using arrhythmic or rhythmic pacing (both at 60 bpm). After 24 hours of AF-simulation, human LV cardiomyocytes from nonfailing donors showed an impaired Ca ²⁺ transient amplitude. For a standardized investigation of AF-simulation, human iPSC-cardiomyocytes were tested. Seven days of AF-simulation caused reduced systolic Ca ²⁺ transient amplitude and sarcoplasmic reticulum Ca ²⁺ load likely because of an increased diastolic sarcoplasmic reticulum Ca ²⁺ leak. Moreover, cytosolic Na ⁺ concentration was elevated and action potential duration was prolonged after AF-simulation. We detected an increased late Na ⁺ current as a potential trigger for the detrimentally altered Ca ²⁺ /Na ⁺ -interplay. Mechanistically, reactive oxygen species were higher in the LV of patients with AF. CaMKII (Ca ²⁺ /calmodulin-dependent protein kinase IIδc) was found to be more oxidized at Met281/282 in the LV of patients with AF leading to an increased CaMKII activity and consequent increased RyR2 phosphorylation. CaMKII inhibition and ROS scavenging ameliorated impaired systolic Ca ²⁺ handling after AF-simulation. Conclusions: AF causes distinct functional and molecular remodeling of the human LV. This translational study provides the first mechanistic characterization and the potential negative impact of AF in the absence of tachycardia on the human ventricle.

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B–mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B–KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser ₄₀₈₀ , the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B–KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser ₄₀₈₀ phosphorylation and reverted passive stiffness of GC-B–deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

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 (F_passive), which was corrected upon antioxidant treatment. Titin as a main determinant of F_passive 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.

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.

Patients diagnosed with both aortic stenosis (AS) and diabetes mellitus (DM) encounter a distinctive set of challenges due to the interplay between these two conditions. This study aimed to investigate the effects of DM on the left ventricle in AS patients, specifically focusing on the inflammatory response, oxidative stress, and their implications for cardiomyocyte function, titin phosphorylation, and the nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway. Left ventricular myocardial biopsies (in total: n = 28) were obtained from patients with diabetic AS (n = 11) and compared with those from non-diabetic AS patients (n = 17). Enzyme-linked immunosorbent assay (ELISA) demonstrated significantly elevated levels of pro-inflammatory mediators, including high mobility group box protein 1 (HMGB1) and calprotectin, as well as receptors associated with the inflammatory response, such as Toll-like receptor 2 (TLR2), 4 (TLR4), and receptor for advanced glycation endproducts (RAGE). These were correlated with an enhanced NOD-like receptor protein 3 (NLRP3) inflammasome and the release of interleukins (IL) 1, 6, and 18 in diabetic AS patients compared to their non-diabetic counterparts. Additionally, in the diabetic AS cohort, there was an increase in oxidative stress markers (hydrogen peroxide (H2O2), 3-nitrotyrosine, lipid peroxidation (LPO), oxidative glutathione (GSSG)/reduced glutathione (GSH) ratio) within the myocardium and mitochondria, accompanied by impaired NO-sGC-cGMP-PKG signaling, decreased titin phosphorylation, and increased passive stiffness (Fpassive) of cardiomyocytes relative to non-diabetic AS patients. In vitro anti-inflammatory treatment with an IL-6 inhibitor and antioxidant treatment with GSH effectively normalized the elevated Fpassive observed in AS patients with DM to levels comparable to the non-diabetic group. Furthermore, treatment with PKG and the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin also resulted in a reduction of Fpassive in cardiomyocytes from diabetic AS patients, although not to the levels observed in non-diabetic AS patients. DM exacerbates inflammation and oxidative stress in AS patients, leading to impaired NO-sGC-cGMP-PKG signaling and increased cardiomyocyte Fpassive. These conditions are reminiscent of the pathophysiology of heart failure with preserved ejection fraction (HFpEF). These alterations can be ameliorated through anti-inflammatory and antioxidant therapies, indicating potential therapeutic strategies for diabetic patients suffering from AS.

BACKGROUND: Cardiometabolic heart failure with preserved ejection fraction (cHFpEF) is a highly prevalent and deadly condition. Histone 3 trimethylation at lysine 36 (H3k36me3)—a chromatin signature induced by the histone methyltransferase SETD2 (SET domain containing 2)—correlates with changes in gene expression in human failing hearts; however, its role remains poorly understood. This study investigates the role of SETD2 in cHFpEF. METHODS: Chromatin immunoprecipitation sequencing and RNA sequencing were used to investigate H3k36me3-related transcriptional regulation. Mice with cardiomyocyte-specific deletion of SETD2 (c-SETD2 ^−/− ) were generated and subjected to high-fat diet feeding and L-NAME treatment for 15 weeks to induce cHFpEF. Cardiac function and exercise tolerance were assessed by echocardiography and treadmill exhaustion test. A selective pharmacological inhibitor of SETD2, EZM0414, was also tested in cHFpEF mice. Mechanistic experiments were performed in cultured cardiomyocytes exposed to palmitic acid. SETD2 signaling and the effects of EZM0414 were also investigated in cardiomyocytes from patients with cHFpEF and control donors. RESULTS: SETD2 was upregulated in cHFpEF mouse hearts, and its chromatin mark H3k36me3 was involved in lipid metabolism and highly enriched on the promoter of the Srebf1 gene, encoding for SREBP1 (sterol regulatory binding protein 1). SETD2 activation in cHFpEF led to SREBP1 upregulation, triglyceride accumulation, and lipotoxic damage. Of note, cardiomyocyte-specific deletion of SETD2 in mice prevented heart failure with preserved ejection fraction–related hypertrophy, diastolic dysfunction, and lung congestion while improving exercise tolerance. SETD2 deletion blunted H3K36me3 enrichment on Srebf1 promoter, thus leading to a marked rewiring of the cardiac lipidome and restoration of autophagic flux. In vivo treatment with the SETD2 inhibitor EZM0414 recapitulated the effects of SETD2 deletion. Silencing of SETD2 in palmitic acid–treated cardiomyocytes prevented SREBP1 upregulation, whereas SETD2 overexpression mirrored lipotoxic damage. Finally, SETD2 was upregulated in left ventricle specimens from patients with cHFpEF while EZM0414 attenuated cardiomyocyte stiffness. CONCLUSIONS: Targeting SETD2 might prevent lipotoxic injury in cHFpEF.