Elimination of CaMKIIδ Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice.

BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic ß-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.

Enhanced CaMKII-Dependent Late INa Induces Atrial Proarrhythmic Activity in Patients With Sleep-Disordered Breathing.

RATIONALE: Sleep-disordered breathing (SDB) is frequently associated with atrial arrhythmias. Increased CaMKII (Ca/calmodulin-dependent protein kinase II) activity has been previously implicated in atrial arrhythmogenesis. OBJECTIVE: We hypothesized that CaMKII-dependent dysregulation of Na current (INa) may contribute to atrial proarrhythmic activity in patients with SDB. METHODS AND RESULTS: We prospectively enrolled 113 patients undergoing elective coronary artery bypass grafting for cross-sectional study and collected right atrial appendage biopsies. The presence of SDB (defined as apnea-hypopnea index ≥15/h) was assessed with a portable SDB monitor the night before surgery. Compared with 56 patients without SDB, patients with SDB (57) showed a significantly increased level of activated CaMKII. Patch clamp was used to measure INa. There was a significantly enhanced late INa, but reduced peak INa due to enhanced steady-state inactivation in atrial myocytes of patients with SDB consistent with significantly increased CaMKII-dependent cardiac Na channel phosphorylation (NaV1.5, at serine 571, Western blotting). These gating changes could be fully reversed by acute CaMKII inhibition (AIP [autocamtide-2 related inhibitory peptide]). As a consequence, we observed significantly more cellular afterdepolarizations and more severe premature atrial contractions in atrial trabeculae of patients with SDB, which could be blocked by either AIP or KN93 (N-[2-[[[(E)-3-(4-chlorophenyl)prop-2-enyl]-methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide). In multivariable linear regression models incorporating age, sex, body mass index, existing atrial fibrillation, existing heart failure, diabetes mellitus, and creatinine levels, apnea-hypopnea index was independently associated with increased CaMKII activity, enhanced late INa and correlated with premature atrial contraction severity. CONCLUSIONS: In atrial myocardium of patients with SDB, increased CaMKII-dependent phosphorylation of NaV1.5 results in dysregulation of INa with proarrhythmic activity that was independent from preexisting comorbidities. Inhibition of CaMKII may be useful for prevention or treatment of arrhythmias in SDB. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02877745. Visual Overview: An online visual overview is available for this article.

Dantrolene reduces CaMKIIδC-mediated atrial arrhythmias.

AIMS: In atrial fibrillation (AF), an increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) mediated by calcium/calmodulin-dependent-protein-kinaseIIδC (CaMKII) can serve as a substrate for arrhythmia induction and persistence. Dantrolene has been shown to stabilize the cardiac ryanodine-receptor. This study investigated the effects of dantrolene on arrhythmogenesis in human and mouse atria with enhanced CaMKII activity. METHODS AND RESULTS: Human atrial cardiomyocytes (CMs) were isolated from patients with AF. To investigate CaMKII-mediated arrhythmogenesis, atrial CMs from mice overexpressing CaMKIIδC (TG) and the respective wildtype (WT) were studied using confocal microscopy (Fluo-4), patch-clamp technique, and in vivo atrial catheter-based burst stimulations. Dantrolene potently reduced Ca2+ spark frequency (CaSpF) and diastolic SR Ca2+ leak in AF CMs. Additional CaMKII inhibition did not further reduce CaSpF or leak compared to dantrolene alone. While the increased SR CaSpF and leak in TG mice were reduced by dantrolene, no effects could be detected in WT. Dantrolene also potently reduced the pathologically enhanced frequency of diastolic SR Ca2+ waves in TG without having effects in WT. As an increased diastolic SR Ca2+ release can induce a depolarizing transient inward current, we could demonstrate that the incidence of afterdepolarizations in TG, but not in WT, mice was significantly diminished in the presence of dantrolene. To translate these findings into an in vivo situation we could show that dantrolene strongly suppressed the inducibility of AF in vivo in TG mice. CONCLUSION: Dantrolene reduces CaMKII-mediated atrial arrhythmogenesis and may therefore constitute an interesting antiarrhythmic drug for treating patients with atrial arrhythmias driven by an enhanced CaMKII activity, such as AF.

Predictors of delirium after cardiac surgery in patients with sleep disordered breathing.

INTRODUCTION: Delirium ranks among the most common complications after cardiac surgery. Although various risk factors have been identified, the association between sleep disordered breathing (SDB) and delirium has barely been examined so far. Here, our objectives were to determine the incidence of post-operative delirium and to identify the risk factors for delirium in patients with and without SDB. METHODS: This subanalysis of the ongoing prospective observational study CONSIDER-AF (ClinicalTrials.gov identifier NCT02877745) examined risk factors for delirium in 141 patients undergoing cardiac surgery. The presence and type of SDB were assessed with a portable SDB monitor the night before surgery. Delirium was prospectively assessed with the validated Confusion Assessment Method for the Intensive Care Unit on the day of extubation and for a maximum of 3 days. RESULTS: Delirium was diagnosed in 23% of patients: in 16% of patients without SDB, in 13% with obstructive sleep apnoea and in 49% with central sleep apnoea. Multivariable logistic regression analysis showed that delirium was independently associated with age ≥70 years (OR 5.63, 95% CI 1.79-17.68; p=0.003), central sleep apnoea (OR 4.99, 95% CI 1.41-17.69; p=0.013) and heart failure (OR 3.3, 95% CI 1.06-10.35; p=0.039). Length of hospital stay and time spent in the intensive care unit/intermediate care setting were significantly longer for patients with delirium. CONCLUSIONS: Among the established risk factors for delirium, central sleep apnoea was independently associated with delirium. Our findings contribute to identifying patients at high risk of developing post-operative delirium who may benefit from intensified delirium prevention strategies.

Improvement of cardiomyocyte function by a novel pyrimidine-based CaMKII-inhibitor.

OBJECTIVE: Pathologically increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the associated Ca2+-leak from the sarcoplasmic reticulum are recognized to be important novel pharmacotherapeutic targets in heart failure and cardiac arrhythmias. However, CaMKII-inhibitory compounds for therapeutic use are still lacking. We now report on the cellular and molecular effects of a novel pyrimidine-based CaMKII inhibitor developed towards clinical use. METHODS AND RESULTS: Our findings demonstrate that AS105 is a high-affinity ATP-competitive CaMKII-inhibitor that by its mode of action is also effective against autophosphorylated CaMKII (in contrast to the commonly used allosteric CaMKII-inhibitor KN-93). In isolated atrial cardiomyocytes from human donors and ventricular myocytes from CaMKIIδC-overexpressing mice with heart failure, AS105 effectively reduced diastolic SR Ca2+ leak by 38% to 65% as measured by Ca2+-sparks or tetracaine-sensitive shift in [Ca2+]i. Consistent with this, we found that AS105 suppressed arrhythmogenic spontaneous cardiomyocyte Ca2+-release (by 53%). Also, the ability of the SR to accumulate Ca2+ was enhanced by AS105, as indicated by improved post-rest potentiation of Ca2+-transient amplitudes and increased SR Ca2+-content in the murine cells. Accordingly, these cells had improved systolic Ca2+-transient amplitudes and contractility during basal stimulation. Importantly, CaMKII inhibition did not compromise systolic fractional Ca2+-release, diastolic SR Ca2+-reuptake via SERCA2a or Ca2+-extrusion via NCX. CONCLUSION: AS105 is a novel, highly potent ATP-competitive CaMKII inhibitor. In vitro, it effectively reduced SR Ca2+-leak, thus improving SR Ca2+-accumulation and reducing cellular arrhythmogenic correlates, without negatively influencing excitation-contraction coupling. These findings further validate CaMKII as a key target in cardiovascular disease, implicated by genetic, allosteric inhibitors, and pseudo-substrate inhibitors.

SR Ca2+-leak and disordered excitation-contraction coupling as the basis for arrhythmogenic and negative inotropic effects of acute ethanol exposure.

AIMS: Ethanol has acute negative inotropic and arrhythmogenic effects. The underlying mechanisms, however, are largely unknown. Sarcoplasmic reticulum Ca2+-leak is an important mechanism for reduced contractility and arrhythmias. Ca2+-leak can be induced by oxidative stress and Ca2+/Calmodulin-dependent protein kinase II (CaMKII). Therefore, we investigated the influence of acute ethanol exposure on excitation-contraction coupling in atrial and ventricular cardiomyocytes. METHODS AND RESULTS: Isolated human atrial and murine atrial or ventricular cardiomyocytes were preincubated for 30 min and then superfused with control solution or solution containing ethanol. Ethanol had acute negative inotropic and positive lusitropic effects in human atrial muscle strips and murine ventricular cardiomyocytes. Accordingly, Ca2+-imaging indicated lower Ca2+-transient amplitudes and increased SERCA2a activity, while myofilament Ca2+-sensitivity was reduced. SR Ca2+-leak was assessed by measuring Ca2+-sparks. Ethanol induced severe SR Ca2+-leak in human atrial cardiomyocytes (calculated leak: 4.60 ±â€¯0.45 mF/F0 vs 1.86 ±â€¯0.26 in control, n ≥ 80). This effect was dose-dependent, while spontaneous arrhythmogenic Ca2+-waves increased ~5-fold, as investigated in murine cardiomyocytes. Delayed afterdepolarizations, which can result from increased SR Ca2+-leak, were significantly increased by ethanol. Measurements using the reactive oxygen species (ROS) sensor CM-H2DCFDA showed increased ROS-stress in ethanol treated cells. ROS-scavenging with N-acetylcysteine prevented negative inotropic and positive lusitropic effects in human muscle strips. Ethanol-induced Ca2+-leak was abolished in mice with knockout of NOX2 (the main source for ROS in cardiomyocytes). Importantly, mice with oxidation-resistant CaMKII (Met281/282Val mutation) were protected from ethanol-induced Ca2+-leak. CONCLUSION: We show for the first time that ethanol acutely induces strong SR Ca2+-leak, also altering excitation-contraction coupling. Acute negative inotropic effects of ethanol can be explained by reduced systolic Ca2+-release. Mechanistically, ROS-production via NOX2 and oxidative activation of CaMKII appear to play central roles. This provides a mechanism for the arrhythmogenic and negative inotropic effects of ethanol and suggests a druggable target (CaMKII).

CaMKIIδ-dependent dysregulation of atrial Na+ homeostasis promotes pro-arrhythmic activity in an obstructive sleep apnea mouse model.

Background: Obstructive sleep apnea (OSA) has been linked to various pathologies, including arrhythmias such as atrial fibrillation. Specific treatment options for OSA are mainly limited to symptomatic approaches. We previously showed that increased production of reactive oxygen species (ROS) stimulates late sodium current through the voltage-dependent Na+ channels via Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ), thereby increasing the propensity for arrhythmias. However, the impact on atrial intracellular Na+ homeostasis has never been demonstrated. Moreover, the patients often exhibit a broad range of comorbidities, making it difficult to ascertain the effects of OSA alone. Objective: We analyzed the effects of OSA on ROS production, cytosolic Na+ level, and rate of spontaneous arrhythmia in atrial cardiomyocytes isolated from an OSA mouse model free from comorbidities. Methods: OSA was induced in C57BL/6 wild-type and CaMKIIδ-knockout mice by polytetrafluorethylene (PTFE) injection into the tongue. After 8 weeks, their atrial cardiomyocytes were analyzed for cytosolic and mitochondrial ROS production via laser-scanning confocal microscopy. Quantifications of the cytosolic Na+ concentration and arrhythmia were performed by epifluorescence microscopy. Results: PTFE treatment resulted in increased cytosolic and mitochondrial ROS production. Importantly, the cytosolic Na+ concentration was dramatically increased at various stimulation frequencies in the PTFE-treated mice, while the CaMKIIδ-knockout mice were protected. Accordingly, the rate of spontaneous Ca2+ release events increased in the wild-type PTFE mice while being impeded in the CaMKIIδ-knockout mice. Conclusion: Atrial Na+ concentration and propensity for spontaneous Ca2+ release events were higher in an OSA mouse model in a CaMKIIδ-dependent manner, which could have therapeutic implications.

CRISPR-Cas9 base editing of pathogenic CaMKIIδ improves cardiac function in a humanized mouse model.

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.

Insights into the Interaction of Heart Failure with Preserved Ejection Fraction and Sleep-Disordered Breathing.

Heart failure with preserved ejection fraction (HFpEF) is emerging as a widespread disease with global socioeconomic impact. Patients with HFpEF show a dramatically increased morbidity and mortality, and, unfortunately, specific treatment options are limited. This is due to the various etiologies that promote HFpEF development. Indeed, cluster analyses with common HFpEF comorbidities revealed the existence of several HFpEF phenotypes. One especially frequent, yet underappreciated, comorbidity is sleep-disordered breathing (SDB), which is closely intertwined with the development and progression of the "obese HFpEF phenotype". The following review article aims to provide an overview of the common HFpEF etiologies and phenotypes, especially in the context of SDB. As general HFpEF therapies are often not successful, patient- and phenotype-individualized therapeutic strategies are warranted. Therefore, for the "obese HFpEF phenotype", a better understanding of the mechanistic parallels between both HFpEF and SDB is required, which may help to identify potential phenotype-individualized therapeutic strategies. Novel technologies like single-cell transcriptomics or CRISPR-Cas9 gene editing further broaden the groundwork for deeper insights into pathomechanisms and precision medicine.

CaMKII-Dependent Contractile Dysfunction and Pro-Arrhythmic Activity in a Mouse Model of Obstructive Sleep Apnea.

Left ventricular contractile dysfunction and arrhythmias frequently occur in patients with sleep-disordered breathing (SDB). The CaMKII-dependent dysregulation of cellular Ca homeostasis has recently been described in SDB patients, but these studies only partly explain the mechanism and are limited by the patients' heterogeneity. Here, we analyzed contractile function and Ca homeostasis in a mouse model of obstructive sleep apnea (OSA) that is not limited by confounding comorbidities. OSA was induced by artificial tongue enlargement with polytetrafluorethylene (PTFE) injection into the tongue of wildtype mice and mice with a genetic ablation of the oxidative activation sites of CaMKII (MMVV knock-in). After eight weeks, cardiac function was assessed with echocardiography. Reactive oxygen species (ROS) and Ca transients were measured using confocal and epifluorescence microscopy, respectively. Wildtype PTFE mice exhibited an impaired ejection fraction, while MMVV PTFE mice were fully protected. As expected, isolated cardiomyocytes from PTFE mice showed increased ROS production. We further observed decreased levels of steady-state Ca transients, decreased levels of caffeine-induced Ca transients, and increased pro-arrhythmic activity (defined as deviations from the diastolic Ca baseline) only in wildtype but not in MMVV PTFE mice. In summary, in the absence of any comorbidities, OSA was associated with contractile dysfunction and pro-arrhythmic activity and the inhibition of the oxidative activation of CaMKII conveyed cardioprotection, which may have therapeutic implications.

Ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease.

CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIIδ, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIIδ gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIIδ editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIIδ gene editing may thus represent a permanent and advanced strategy for heart disease therapy.

Sleep-Disordered Breathing Is Associated With Reduced Left Atrial Strain Measured by Cardiac Magnetic Resonance Imaging in Patients After Acute Myocardial Infarction.

AIMS: Sleep disordered breathing (SDB) is known to cause left atrial (LA) remodeling. However, the relationship between SDB severity and LA dysfunction is insufficiently understood and may be elucidated by detailed feature tracking (FT) strain analysis of cardiac magnetic resonance images (CMR). After myocardial infarction (MI), both the left ventricle and atrium are subjected to increased stress which may be substantially worsened by concomitant SDB that could impair consequential healing. We therefore analyzed atrial strain in patients at the time of acute MI and 3 months after. METHODS AND RESULTS: 40 patients with acute MI underwent CMR and polysomnography (PSG) within 3-5 days after MI. Follow-up was performed 3 months after acute MI. CMR cine data were analyzed using a dedicated FT software. Atrial strain (ε) and strain rate (SR) for atrial reservoir ([εs]; [SRs]), conduit ([εe]; [SRe]) and booster function ([εa]; [SRa]) were measured in two long-axis views. SDB was defined by an apnea-hypopnea-index (AHI) ≥15/h. Interestingly, LA εs and εe were significantly reduced in patients with SDB and correlated negative with AHI as a measure of SDB severity at both baseline and follow-up. Intriguingly, patients that exhibited a reduced AHI at follow-up were more likely to have developed improved atrial reservoir and conduit strain (linear regression, p=0.08 for εs and εe). Patients with improved SDB (ΔAHI < -5/h) exhibited a mean improvement of LA reservoir strain of +7.2 ± 8.4% whereas patients with SDB deterioration (ΔAHI> + 5/h) showed a mean decrease of -5.3 ± 11.0% (p = 0.0131). Similarly, the difference for LA conduit function was +4.8 ± 5.9% (ΔAHI < -5/h) vs -3.6 ± 8.8% (ΔAHI> +5/h). Importantly, conventional volumetric parameters for atrial function (LA area, LA volume index) did not correlate with AHI at baseline or follow-up. CONCLUSION: Our results show that LA function measured by CMR strain but not by volumetry is impaired in patients with SDB during acute cardiac injury. Consistent with a mechanistic association, improvement of SBD at follow-up resulted in improved LA strain. LA strain measurement might thus provide insight into atrial function in patients with SDB.

Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing.

BACKGROUND: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. METHODS: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. RESULTS: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm-1s-1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [-0.1; 0.03], p = 0.387). CONCLUSIONS: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB.

The Effect of Gender and Sex Hormones on Cardiovascular Disease, Heart Failure, Diabetes, and Atrial Fibrillation in Sleep Apnea.

Sleep apnea is a highly prevalent disorder with increasing impact on healthcare systems worldwide. Previous studies have been conducted primarily with male subjects, and prevalence and severity of sleep apnea in women are underestimated. Recent clinical and basic science evidence increasingly points to different mechanisms in men and women with sleep-disordered breathing (SDB). SDB is associated with a variety of comorbidities, including cardiovascular disease, heart failure, diabetes, and atrial fibrillation. In this review, we discuss sex-dependent mechanisms of SDB in select associated conditions to sharpen our clinical understanding of these sex-dependent inherent differences.

Female Patients With Sleep-Disordered Breathing Display More Frequently Heart Failure With Preserved Ejection Fraction.

Objective: Sleep-disordered breathing (SDB) is a widespread disease that is often associated with heart failure (HF) with preserved ejection fraction (HFpEF). HFpEF is more frequent in women than in men, but detailed pathomechanisms remain unclear. We investigated HFpEF in women and men in a high-risk cohort with SDB monitoring. Methods and Results: Three hundred twenty-seven patients (84.4% men) undergoing elective coronary artery bypass grafting were prospectively subjected to SDB monitoring, and an apnea-hypopnea index (AHI) ≥15/h defined SDB. HF was classified according to current guidelines. HFpEF was significantly more frequent in SDB patients compared to those without SDB (28 vs. 17%, P = 0.016). This distribution was driven by an increased frequency of HFpEF in female SDB patients (48% vs. only 25% in male, P = 0.022). In accordance, female patients with SDB exhibited significantly more impaired diastolic left ventricular filling compared to men (echocardiographic E/e'). In contrast to men, in women, minimum oxygen saturation (O2min, measured by polygraphy, R 2 = 0.470, P < 0.001) and time of oxygen saturation <90% (R 2 = 0.165, P = 0.044) were significantly correlated with E/e'. Moreover, the correlation between O2min and E/e' was significantly different in women compared to men (P < 0.001). Intriguingly, this association remained independent of clinical covariates in women [age, body mass index, systolic contractile dysfunction, diabetes mellitus, and glomerular filtration rate (GFR), R 2 = 0.534, P = 0.042, multivariate regression analysis]. Since angiotensin II signaling has been mechanistically linked to HF, we measured protein expression of its cleavage enzyme ACE2 in human right atrial appendage biopsies (Western blot). Intriguingly, we found a significantly decreased ACE2 expression preferentially in women with SDB (2.66 ± 0.42 vs. 4.01 ± 2.47 in men with SDB, P = 0.005). In accordance, left ventricular mass index was significantly increased in women with SDB compared to women without SDB. Conclusion: In patients with SDB, HFpEF and diastolic dysfunction were more frequent in women compared to men. In contrast to men, the severity of SDB was associated with the degree of diastolic dysfunction in women. These insights might help to find sex-specific therapies for patients with sleep-disordered breathing and heart failure. Clinical Trial Registration: Unique identifier: NCT02877745, URL: http://www.clinicaltrials.gov.

Sleep-disordered breathing is independently associated with reduced atrial connexin 43 expression.

BACKGROUND: Patients with atrial fibrillation (AF) exhibit decreased atrial expression of connexin (Cx), which has been causally linked to a proarrhythmogenic substrate. Interestingly, patients with sleep-disordered breathing (SDB) are at increased risk of AF, but the mechanisms remain unclear. OBJECTIVE: We tested the hypothesis that patients with SDB have reduced atrial Cx expression independent of important comorbidities. METHODS: We analyzed right atrial appendage biopsies from 77 patients undergoing coronary artery bypass grafting. Patients were tested for SDB by polygraphy before surgery. Expression of Cx40 and Cx43 messenger RNA was quantified using real-time quantitative polymerase chain reaction and Western blot (Cx43). Structural atrial remodeling was investigated histologically and by quantitative polymerase chain reaction. Postoperative AF was assessed by 12-lead electrocardiography. RESULTS: Patients were stratified according to apnea-hypopnea index (SDB if apnea-hypopnea index ≥15 per hour, n = 32 vs n = 45). Patients with SDB had significantly lower atrial Cx43 expression, which was negatively correlated with apnea-hypopnea index and oxygen desaturation index. No significant increase in atrial fibrosis or expression of hypertrophy and inflammatory markers was observed. Interestingly, SDB remained the strongest independent predictor of decreased atrial Cx43 expression in a multivariate logistic regression model including age, sex, diabetes, and heart failure with reduced ejection fraction (odds ratio 7.58; 95% confidence interval 1.891-30.375; P = .004). Moreover, reduced atrial Cx43 expression was strongly associated with the occurrence of postoperative AF (odds ratio 15.749; 95% confidence interval 1.072-231.472; P = .044). CONCLUSION: Patients with SDB exhibited decreased atrial Cx43 expression, which correlated with the severity of SDB. This correlation was independent of several concomitant diseases and may be linked to an increased risk of AF after cardiac surgery.

Abnormal P-wave terminal force in lead V1 is a marker for atrial electrical dysfunction but not structural remodelling.

AIMS: There is a lack of diagnostic and therapeutic options for patients with atrial cardiomyopathy and paroxysmal atrial fibrillation. Interestingly, an abnormal P-wave terminal force in electrocardiogram lead V1 (PTFV1 ) has been associated with atrial cardiomyopathy, but this association is poorly understood. We investigated PTFV1 as a marker for functional, electrical, and structural atrial remodelling. METHODS AND RESULTS: Fifty-six patients with acute myocardial infarction and 13 kidney donors as control cohort prospectively underwent cardiac magnetic resonance imaging to evaluate the association between PTFV1 and functional remodelling (atrial strain). To further investigate underlying pathomechanisms, right atrial appendage biopsies were collected from 32 patients undergoing elective coronary artery bypass grafting. PTFV1 was assessed as the product of negative P-wave amplitude and duration in lead V1 and defined as abnormal if ≥4000 ms*µV. Activity of cardiac Ca/calmodulin-dependent protein kinase II (CaMKII) was determined by a specific HDAC4 pull-down assay as a surrogate for electrical remodelling. Atrial fibrosis was quantified using Masson's trichrome staining as a measure for structural remodelling. Multivariate regression analyses were performed to account for potential confounders. A total of 16/56 (29%) of patients with acute myocardial infarction, 3/13 (23%) of kidney donors, and 15/32 (47%) of patients undergoing coronary artery bypass grafting showed an abnormal PTFV1 . In patients with acute myocardial infarction, left atrial (LA) strain was significantly reduced in the subgroup with an abnormal PTFV1 (LA reservoir strain: 32.28 ± 12.86% vs. 22.75 ± 13.94%, P = 0.018; LA conduit strain: 18.87 ± 10.34% vs. 10.17 ± 8.26%, P = 0.004). Abnormal PTFV1 showed a negative correlation with LA conduit strain independent from clinical covariates (coefficient B: -7.336, 95% confidence interval -13.577 to -1.095, P = 0.022). CaMKII activity was significantly increased from (normalized to CaMKII expression) 0.87 ± 0.17 to 1.46 ± 0.15 in patients with an abnormal PTFV1 (P = 0.047). This increase in patients with an abnormal PTFV1 was independent from clinical covariates (coefficient B: 0.542, 95% confidence interval 0.057 to 1.027, P = 0.031). Atrial fibrosis was significantly lower with 12.32 ± 1.63% in patients with an abnormal PTFV1 (vs. 20.50 ± 2.09%, P = 0.006), suggesting PTFV1 to be a marker for electrical but not structural remodelling. CONCLUSIONS: Abnormal PTFV1 is an independent predictor for impaired atrial function and for electrical but not for structural remodelling. PTFV1 may be a promising tool to evaluate patients for atrial cardiomyopathy and for risk of atrial fibrillation.