Bioactive adrenomedullin and interleukin-6 in COVID-19: potential biomarkers of acute kidney injury and critical illness.

BACKGROUND: The aim of this study was to investigate whether bioactive adrenomedullin (bio-ADM) and interleukin-6 (IL-6) are related to acute kidney injury (AKI) and severe illness in COVID-19 patients. METHODS: 153 patients with COVID-19 admitted to the emergency department (ED) were included. Blood samples were collected from each patient at admission. Bio-ADM and IL-6, as well as DPP3 and routinely measured markers were evaluated regarding the endpoints AKI (22/128 hospitalized patients) and a composite endpoint of admission to intensive care unit and/or in-hospital death (n = 26/153 patients). RESULTS: Bio-ADM and IL-6 were significantly elevated in COVID-19 patients with AKI compared to COVID-19 patients without AKI (each p < 0.001). According to ROC analyses IL-6 and bio-ADM had the largest AUC (0.84 and 0.81) regarding the detection of AKI. Furthermore, bio-ADM and IL-6 were significantly elevated in COVID-19 patients reaching the composite endpoint (each p < 0.001). Regarding the composite endpoint ROC analysis showed an AUC of 0.89 for IL-6 and 0.83 for bio-ADM in COVID-19 patients. In the multivariable logistic model bio-ADM and IL-6 presented as independent significant predictors regarding both endpoints AKI and the composite endpoint in COVID-19 patients (as well as creatinine regarding the composite endpoint; each p < 0.05), opposite to leukocytes, C-reactive protein (CRP) and dipeptidyl peptidase 3 (DPP3; each p = n.s.). CONCLUSION: Elevated levels of bio-ADM and IL-6 are associated with AKI and critical illness in patients with COVID-19. Therefore, both biomarkers may be potential tools in risk stratification in COVID-19 patients at presentation in the ED.

Dipeptidyl-peptidase 3 and IL-6: potential biomarkers for diagnostics in COVID-19 and association with pulmonary infiltrates.

Coronavirus SARS-CoV-2 spread worldwide, causing a respiratory disease known as COVID-19. The aim of the present study was to examine whether Dipeptidyl-peptidase 3 (DPP3) and the inflammatory biomarkers IL-6, CRP, and leucocytes are associated with COVID-19 and able to predict the severity of pulmonary infiltrates in COVID-19 patients versus non-COVID-19 patients. 114 COVID-19 patients and 35 patients with respiratory infections other than SARS-CoV-2 were included in our prospective observational study. Blood samples were collected at presentation to the emergency department. 102 COVID-19 patients and 28 non-COVID-19 patients received CT imaging (19 outpatients did not receive CT imaging). If CT imaging was available, artificial intelligence software (CT Pneumonia Analysis) was used to quantify pulmonary infiltrates. According to the median of infiltrate (14.45%), patients who obtained quantitative CT analysis were divided into two groups (> median: 55 COVID-19 and nine non-COVID-19, ≤ median: 47 COVID-19 and 19 non-COVID-19). DPP3 was significantly elevated in COVID-19 patients (median 20.85 ng/ml, 95% CI 18.34-24.40 ng/ml), as opposed to those without SARS-CoV-2 (median 13.80 ng/ml, 95% CI 11.30-17.65 ng/ml; p < 0.001, AUC = 0.72), opposite to IL-6, CRP (each p = n.s.) and leucocytes (p < 0.05, but lower levels in COVID-19 patients). Regarding binary logistic regression analysis, higher DPP3 concentrations (OR = 1.12, p < 0.001) and lower leucocytes counts (OR = 0.76, p < 0.001) were identified as significant and independent predictors of SARS-CoV-2 infection, as opposed to IL-6 and CRP (each p = n.s.). IL-6 was significantly increased in patients with infiltrate above the median compared to infiltrate below the median both in COVID-19 (p < 0.001, AUC = 0.78) and in non-COVID-19 (p < 0.05, AUC = 0.81). CRP, DPP3, and leucocytes were increased in COVID-19 patients with infiltrate above median (each p < 0.05, AUC: CRP 0.82, DPP3 0.70, leucocytes 0.67) compared to infiltrate below median, opposite to non-COVID-19 (each p = n.s.). Regarding multiple linear regression analysis in COVID-19, CRP, IL-6, and leucocytes (each p < 0.05) were associated with the degree of pulmonary infiltrates, as opposed to DPP3 (p = n.s.). DPP3 showed the potential to be a COVID-19-specific biomarker. IL-6 might serve as a prognostic marker to assess the extent of pulmonary infiltrates in respiratory patients.

A Pilot Trial to Compare the Long-Term Efficacy of Pulmonary Vein Isolation with High-Power Short-Duration Radiofrequency Versus Laser Energy with Rapid Ablation Mode.

BACKGROUND: Pulmonary vein (PV) reconnection is the major cause of atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI). The probability of reconnection is higher if the primary lesion is not sufficiently effective, which can be unmasked with an adenosine provocation test (APT). High-power short-duration radiofrequency energy (HPSD) guided with ablation index (AI) and the third generation of the visually guided laser balloon (VGLB) are new methods for PVI. METHODS: A total of 70 participants (35 in each group) who underwent a PVI with either AI-guided HPSD (50 W; AI 500 for the anterior and 400 for the posterior wall, respectively) or VGLB ablation were included in this observational pilot trial. Twenty minutes after each PVI, an APT was performed. The primary endpoint was the event-free survival from AF after three years. RESULTS: A total of 137 (100%) PVs in the HPSD arm and 131 PVs (98.5%) in the VGLB arm were initially successfully isolated (p = 0.24). The overall procedure duration was similar in both arms (155 ± 39 in HPSD vs. 175 ± 58 min in VGLB, p = 0.191). Fluoroscopy time, left atrial dwelling time and duration from the first to the last ablation were longer in the VGLB arm (23 ± 8 vs. 12 ± 3 min, p < 0.001; 157 (111-185) vs. 134 (104-154) min, p = 0.049; 92(59-108) vs. 72 (43-85) min, p = 0.010). A total of 127 (93%) in the HPSD arm and 126 (95%) PVs in the VGLB arm remained isolated after APT (p = 0.34). The primary endpoint was met 1107 ± 68 days after ablation in 71% vs. 66% in the VGLB and HPSD arms, respectively (p = 0.65). CONCLUSIONS: HPSD and VGLB did not differ with respect to long-term outcome of PVI. A large, randomized study should be conducted to compare clinical outcomes with respect to these new ablation techniques.

Arterial and venous vascular complications in patients requiring peripheral venoarterial extracorporeal membrane oxygenation.

Introduction: The aim of this study was to investigate the prevalence of arterial and venous complications in patients requiring peripheral venoarterial extracorporeal membrane oxygenation (VA ECMO) and its risk factors at the time of cannulation and during extracorporeal membrane oxygenation (ECMO) support and to assess vascular complications in association with decannulation. Material and methods: Between January 2010 to January 2020, out of 1,030 eligible patients requiring VA-ECMO, 427 with analyzable vascular screening were included. Duplex sonography and/or CT scan after decannulation were used to screen for thrombosis and pulmonary embolism as well as arterial complications. Near-infrared spectrometry (NIRS) was established at the time of cannulation and was continuously monitored during the ECMO therapy. Results: The prevalence of venous complications was 27%. Thrombosis and pulmonary embolism were observed in 21 and 7% of patients, respectively. Pulmonary embolism was more frequently diagnosed in patients with thrombosis (22 vs. 3%, p < 0.001). In multivariate analysis, cannulation in the jugular vein was determined as a risk factor for venous thrombosis in contrast to the extent of anticoagulation. The prevalence of arterial complications was 37%, mainly ischemia followed by bleeding, dissection, and compartment syndrome. Vascular surgery was necessary for 19% of the patients, of whome 1% required major amputations. A distal perfusion cannula (DPC) was implanted at cannulation in 24% of patients and secondarily in 16% of patients after cannulation as required during ECMO support. In the multivariate analysis, risk factors for leg ischemia at the time of cannulation were elevated D-dimers, lower NIRS on the cannulated leg, and lack of a DPC. The best discriminative parameter was the difference in NIRS between the non-cannulated leg and the cannulated leg. In contrast, during ECMO support, only the lack of a DPC was associated with leg ischemia. A similar rate of complications associated with decannulation, mainly arterial thrombosis, ischemia, or bleeding, was seen with percutaneous and surgical approaches (18 vs. 17%, p = 0.295). Conclusion: Patients requiring VA ECMO should be routinely screened for vascular complications. The decision to insert a DPC should be evaluated individually. However, NIRS monitoring of the cannulated leg and the non-cannulated leg is essential to identify the legs at risk for critical ischemia. As complications associated with decannulation were equally distributed between percutaneous and surgical approaches, the applied method may be chosen according to local experience.

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes.

BACKGROUND: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. OBJECTIVE: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. METHODS AND RESULTS: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. CONCLUSION: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.

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.

Reduction of radiation exposure during transcatheter edge-to-edge mitral valve repair.

BACKGROUND: Transcatheter mitral valve repair is an increasingly used therapy for mitral regurgitation which requires fluoroscopic guidance. Limiting radiation exposure during lengthy procedures is important for both patient and operator safety. This study aimed to investigate radiation dose during contemporary use of MitraClip implantation and the effects of a dose reduction program. METHODS: A total of 115 patients who underwent MitraClip implantation were prospectively enrolled in a single-center observational study. During the inclusion period, our institution adopted a radiation dose reduction program, comprising lowering of fluoroscopy pulse rate and image target dose. The first 58 patients were treated with conventional fluoroscopy settings, while the following 57 patients underwent the procedure with the newly implemented low dose protocol. RESULTS: Radiation dose area product significantly decreased after introduction of the low dose protocol (693 [366-1231] vs. 2265 [1517-3914] cGy·cm2 , p < 0.001). After correcting for fluoroscopy time, gender and body mass index, the low dose protocol emerged as a strong negative predictor of radiation dose (p < 0.001), reducing dose area product by 64% (95% confidence interval [57-70]). Device time, device success, and procedural safety did not differ between the normal dose and low dose group. Furthermore, the low dose protocol was not associated with an increased incidence of a combined endpoint consisting of death, repeat intervention, or heart surgery during 12 months follow-up. CONCLUSION: Reduction of radiation exposure during transcatheter mitral valve repair by 64% is feasible without affecting procedural success or safety.

Enhanced Heart Failure in Redox-Dead Cys17Ser PKARIα Knock-In Mice.

Background PKARIα (protein kinase A type I-α regulatory subunit) is redox-active independent of its physiologic agonist cAMP. However, it is unknown whether this alternative mechanism of PKARIα activation may be of relevance to cardiac excitation-contraction coupling. Methods and Results We used a redox-dead transgenic mouse model with homozygous knock-in replacement of redox-sensitive cysteine 17 with serine within the regulatory subunits of PKARIα (KI). Reactive oxygen species were acutely evoked by exposure of isolated cardiac myocytes to AngII (angiotensin II, 1 µmol/L). The long-term relevance of oxidized PKARIα was investigated in KI mice and their wild-type (WT) littermates following transverse aortic constriction (TAC). AngII increased reactive oxygen species in both groups but with RIα dimer formation in WT only. AngII induced translocation of PKARI to the cell membrane and resulted in protein kinase A-dependent stimulation of ICa (L-type Ca current) in WT with no effect in KI myocytes. Consequently, Ca transients were reduced in KI myocytes as compared with WT cells following acute AngII exposure. Transverse aortic constriction-related reactive oxygen species formation resulted in RIα oxidation in WT but not in KI mice. Within 6 weeks after TAC, KI mice showed an enhanced deterioration of contractile function and impaired survival compared with WT. In accordance, compared with WT, ventricular myocytes from failing KI mice displayed significantly reduced Ca transient amplitudes and lack of ICa stimulation. Conversely, direct pharmacological stimulation of ICa using Bay K8644 rescued Ca transients in AngII-treated KI myocytes and contractile function in failing KI mice in vivo. Conclusions Oxidative activation of PKARIα with subsequent stimulation of ICa preserves cardiac function in the setting of acute and chronic oxidative stress.

Beneficial effect of voluntary physical exercise in Plakophilin2 transgenic mice.

Arrhythmogenic right ventricular cardiomyopathy is a hereditary, rare disease with an increased risk for sudden cardiac death. The disease-causing mutations are located within the desmosomal complex and the highest incidence is found in plakophilin2. However, there are other factors playing a role for the disease progression unrelated to the genotype such as inflammation or exercise. Competitive sports have been identified as risk factor, but the type and extend of physical activity as cofactor for arrhythmogenesis remains under debate. We thus studied the effect of light voluntary exercise on cardiac health in a mouse model. Mice with a heterozygous PKP2 loss-of-function mutation were given the option to exercise in a running wheel which was monitored 24 h/d. We analyzed structural and functional development in vivo by echocardiography which revealed that neither the genotype nor the exercise caused any significant structural changes. Ejection fraction and fractional shortening were not influenced by the genotype itself, but exercise did cause a drop in both parameters after 8 weeks, which returned to normal after 16 weeks of training. The electrophysiological analysis revealed that the arrhythmogenic potential was slightly higher in heterozygous animals (50% vs 18% in wt littermates) and that an additional stressor (isoprenaline) did not lead to an increase of arrhythmogenic events pre run or after 8 weeks of running but the vulnerability was increased after 16 weeks. Exercise-induced alterations in Ca handling and contractility of isolated myocytes were mostly abolished in heterozygous animals. No fibrofatty replacements or rearrangement of gap junctions could be observed. Taken together we could show that light voluntary exercise can cause a transient aggravation of the mutation-induced phenotype which is abolished after long term exercise indicating a beneficial effect of long term light exercise.

Mapping genetic changes in the cAMP-signaling cascade in human atria.

AIM: To obtain a quantitative expression profile of the main genes involved in the cAMP-signaling cascade in human control atria and in different cardiac pathologies. METHODS AND RESULTS: Expression of 48 target genes playing a relevant role in the cAMP-signaling cascade was assessed by RT-qPCR. 113 samples were obtained from right atrial appendages (RAA) of patients in sinus rhythm (SR) with or without atrium dilation, paroxysmal atrial fibrillation (AF), persistent AF or heart failure (HF); and left atrial appendages (LAA) from patients in SR or with AF. Our results show that right and left atrial appendages in donor hearts or from SR patients have similar expression values except for AC7 and PDE2A. Despite the enormous chamber-dependent variability in the gene-expression changes between pathologies, several distinguishable patterns could be identified. PDE8A, PI3Kγ and EPAC2 were upregulated in AF. Different phosphodiesterase (PDE) families showed specific pathology-dependent changes. CONCLUSION: By comparing mRNA-expression patterns of the cAMP-signaling cascade related genes in right and left atrial appendages of human hearts and across different pathologies, we show that 1) gene expression is not significantly affected by cardioplegic solution content, 2) it is appropriate to use SR atrial samples as controls, and 3) many genes in the cAMP-signaling cascade are affected in AF and HF but only few of them appear to be chamber (right or left) specific. TOPIC: Genetic changes in human diseased atria. TRANSLATIONAL PERSPECTIVE: The cyclic AMP signaling pathway is important for atrial function. However, expression patterns of the genes involved in the atria of healthy and diseased hearts are still unclear. We give here a general overview of how different pathologies affect the expression of key genes in the cAMP signaling pathway in human right and left atria appendages. Our study may help identifying new genes of interest as potential therapeutic targets or clinical biomarkers for these pathologies and could serve as a guide in future gene therapy studies.

Extracorporeal life support in patients with acute myocardial infarction complicated by cardiogenic shock - Design and rationale of the ECLS-SHOCK trial.

BACKGROUND: In acute myocardial infarction complicated by cardiogenic shock the use of mechanical circulatory support devices remains controversial and data from randomized clinical trials are very limited. Extracorporeal life support (ECLS) - venoarterial extracorporeal membrane oxygenation - provides the strongest hemodynamic support in addition to oxygenation. However, despite increasing use it has not yet been properly investigated in randomized trials. Therefore, a prospective randomized adequately powered clinical trial is warranted. STUDY DESIGN: The ECLS-SHOCK trial is a 420-patient controlled, international, multicenter, randomized, open-label trial. It is designed to compare whether treatment with ECLS in addition to early revascularization with percutaneous coronary intervention or alternatively coronary artery bypass grafting and optimal medical treatment is beneficial in comparison to no-ECLS in patients with severe infarct-related cardiogenic shock. Patients will be randomized in a 1:1 fashion to one of the two treatment arms. The primary efficacy endpoint of ECLS-SHOCK is 30-day mortality. Secondary outcome measures such as hemodynamic, laboratory, and clinical parameters will serve as surrogate endpoints for prognosis. Furthermore, a longer follow-up at 6 and 12 months will be performed including quality of life assessment. Safety endpoints include peripheral ischemic vascular complications, bleeding and stroke. CONCLUSIONS: The ECLS-SHOCK trial will address essential questions of efficacy and safety of ECLS in addition to early revascularization in acute myocardial infarction complicated by cardiogenic shock.

Secondary hemophagocytic lymphohistiocytosis and severe liver injury induced by hepatic SARS-CoV-2 infection unmasking Wilson's disease: Balancing immunosuppression.

A 21-year-old woman was hospitalized due to coronavirus disease 2019 (COVID-19)-associated respiratory and hepatic impairment concomitant with severe hemolytic anemia. Upon diagnosis of secondary hemophagocytic lymphohistiocytosis, immunosuppression with anakinra and steroids was started, leading to a hepatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and viremia. Subsequent liver biopsy revealed virus particles in hepatocytes by electron microscopy and SARS-CoV-2 virus could be isolated and cultured. Immunosuppression was stopped and convalescent donor plasma given. In the differential diagnosis, an acute crisis of Wilson's disease was raised by laboratory and genetic testing. This case highlights the complexity of balancing immunosuppression to control hyperinflammation versus systemic SARS-CoV-2 dissemination.

Angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker treatment and haemodynamic factors are associated with increased cardiac mRNA expression of angiotensin-converting enzyme 2 in patients with cardiovascular disease.

AIMS: Coronavirus disease 2019 (COVID-19) is a widespread pandemic with an increased morbidity and mortality, especially for patients with cardiovascular diseases. Angiotensin-converting enzyme 2 (ACE2) has been identified as necessary cell entry point for SARS-CoV-2. Previous animal studies have demonstrated an increased ACE2 expression following treatment with either angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II receptor blockers (ARB) that have led to a massive precariousness regarding the optimal cardiovascular therapy during this pandemic. METHODS AND RESULTS: We have measured ACE2 mRNA expression using real-time quantitative polymerase chain reaction in atrial biopsies of 81 patients undergoing coronary artery bypass grafting and we compared 62 patients that received ACEi/ARB vs. 19 patients that were not ACEi/ARB-treated. We found atrial ACE2 mRNA expression to be significantly increased in patients treated with an ACEi or an ARB, independent of potential confounding comorbidities. Interestingly, the cardiac ACE2 mRNA expression correlated significantly with the expression in white blood cells of 22 patients encouraging further evaluation if the latter may be used as a surrogate for the former. Similarly, analysis of 18 ventricular biopsies revealed a significant and independent increase in ACE2 mRNA expression in patients with end-stage heart failure that were treated with ACEi/ARB. On the other hand, cardiac unloading with a left ventricular assist device significantly reduced ventricular ACE2 mRNA expression. CONCLUSION: Treatment with ACEi/ARB is independently associated with an increased myocardial ACE2 mRNA expression in patients with coronary artery disease and in patients with end-stage heart failure. Further trials are needed to test whether this association is deleterious for patients with COVID-19, or possibly protective. Nevertheless, haemodynamic factors seem to be equally important for regulation of cardiac ACE2 mRNA expression.

The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.

AIMS: Excessive activation of Ca/calmodulin-dependent kinase II (CaMKII) is of critical importance in heart failure (HF) and atrial fibrillation. Unfortunately, lack of selectivity, specificity, and bioavailability have slowed down development of inhibitors for clinical use. We investigated a novel CaMKIIδ/CaMKIIÉ£-selective, ATP-competitive, orally available CaMKII inhibitor (RA608) on right atrial biopsies of 119 patients undergoing heart surgery. Furthermore, we evaluated its oral efficacy to prevent deterioration of HF in mice after transverse aortic constriction (TAC). METHODS AND RESULTS: In human atrial cardiomyocytes and trabeculae, respectively, RA608 significantly reduced sarcoplasmic reticulum Ca leak, reduced diastolic tension, and increased sarcoplasmic reticulum Ca content. Patch-clamp recordings confirmed the safety of RA608 in human cardiomyocytes. C57BL6/J mice were subjected to TAC, and left ventricular function was monitored by echocardiography. Two weeks after TAC, RA608 was administered by oral gavage for 7 days. Oral RA608 treatment prevented deterioration of ejection fraction. At 3 weeks after TAC, ejection fraction was 46.1 ± 3.7% (RA608) vs. 34.9 ± 2.6% (vehicle), n = 9 vs. n = 12, P < 0.05, ANOVA, which correlated with significantly less CaMKII autophosphorylation at threonine 287. Moreover, a single oral dose significantly reduced inducibility of atrial and ventricular arrhythmias in CaMKIIδ transgenic mice 4 h after administration. Atrial fibrillation was induced in 6/6 mice for vehicle vs. 1/7 for RA608, P < 0.05, 'n - 1' χ2 test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n - 1' χ2 test. CONCLUSIONS: RA608 is the first orally administrable CaMKII inhibitor with potent efficacy in human myocytes. Moreover, oral administration potently inhibits arrhythmogenesis and attenuates HF development in mice in vivo.

Alterations of the renin angiotensin system in human end-stage heart failure before and after mechanical cardiac unloading by LVAD support.

Heart transplantation is often an unrealizable therapeutic option for end-stage heart failure, which is why mechanical left ventricular assist devices (LVADs) become an increasingly important therapeutic alternative. Currently, there is a lack of information about molecular mechanisms which are influenced by LVADs, particularly regarding the pathophysiologically critical renin angiotensin system (RAS). We, therefore, determined regulation patterns of key components of the RAS and the ß-arrestin signaling pathways in left ventricular (LV) tissue specimens from 8 patients with end-stage ischemic cardiomyopathy (ICM) and 12 patients with terminal dilated cardiomyopathy (DCM) before and after LVAD implantation and compared them with non-failing (NF) left ventricular tissue samples: AT1R, AT2R, ACE, ACE2, MasR, and ADAM17 were analyzed by polymerase chain reaction. ERK, phosphorylated ERK, p38, phosphorylated p38, JNK, phosphorylated JNK, GRK2, ß-arrestin 2, PI3K, Akt, and phosphorylated Akt were determined by Western blot analysis. Angiotensin I and Angiotensin II were quantified by mass spectrometry. Patients were predominantly middle-aged (53 ± 10 years) men with severely impaired LV function (LVEF 19 ± 8%), when receiving LVAD therapy for a mean duration of 331 ± 317 days. Baseline characteristics did not differ significantly between ICM and DCM patients. By comparing failing with non-failing left ventricles, i.e., before LVAD implantation, a downregulation of AT1R, AT2R, and MasR and an upregulation of ACE, ACE2, GRK, ß-arrestin, ERK, PI3K, and Akt were seen. Following LVAD support, then angiotensin I, ACE2, GRK, and ß-arrestin were downregulated and AT2R, JNK, and p38 were upregulated. ACE, angiotensin II, AT1R, ADAM17, MasR, ERK, PI3K, and Akt remained unchanged. Some regulation patterns were influenced by the underlying etiology of heart failure, the severity of LV dysfunction at baseline, and the duration of LVAD therapy. Key components of the RAS and ß-arrestin signaling pathways were divergently altered in failing left ventricles both before and after LVAD implantation, whereas a remarkable fraction remained unchanged. This indicates a rather incomplete molecular reverse remodeling, whose functional relevance has to be further evaluated.

Design of the SILICOFCM study: Effect of sacubitril/valsartan vs lifestyle intervention on functional capacity in patients with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease with a broad spectrum of disease severity. HCM ranges from a benign course to a progressive disorder characterized by angina, heart failure, malignant arrhythmia, syncope, or sudden cardiac death. So far, no medical treatment has reliably shown to halt or reverse progression of HCM or to alleviate its symptoms. While the angiotensin receptor neprilysin inhibitor sacubitril/valsartan has shown to reduce mortality and hospitalization in heart failure with reduced ejection fraction, data on its effect on HCM are sparse. HYPOTHESIS: A 4-month pharmacological (sacubitril/valsartan) or lifestyle intervention will significantly improve exercise tolerance (ie, peak oxygen consumption) in patients with nonobstructive HCM compared to the optimal standard therapy (control group). METHODS: SILICOFCM is a prospective, multicenter, open-label, randomized, controlled, three-arm clinical trial (NCT03832660) that will recruit 240 adult patients with a confirmed diagnosis of nonobstructive HCM. Eligible patients are randomized to sacubitril/valsartan, lifestyle intervention (physical activity and dietary supplementation with inorganic nitrate), or optimal standard therapy alone (control group). The primary endpoint is the change in functional capacity (ie, peak oxygen consumption). Secondary endpoints include: (a) Change in cardiac structure and function as assessed by transthoracic echocardiography and cardiac magnetic resonance (MRI imaging), (b) change in biomarkers (ie, CK, CKMB, and NT-proBNP), (c) physical activity, and (d) quality of life. RESULTS: Until December 2019, a total of 41 patients were recruited into the ongoing SILICOFCM study and were allocated to the study groups and the control group. There was no significant difference in key baseline characteristics between the three groups. CONCLUSION: The SILICOFCM study will provide novel evidence about the effect of sacubitril/valsartan or lifestyle intervention on functional capacity, clinical phenotype, injury and stretch activation markers, physical activity, and quality of life in patients with nonobstructive HCM.

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.

Ca2+/calmodulin-dependent protein kinase II is essential in hyperacute pressure overload.

BACKGROUND: Activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is established as a central intracellular trigger for various cardiac pathologies such as hypertrophy, heart failure or arrhythmias in animals and humans suggesting CaMKII as a promising target protein for future medical treatments. However, the physiological role of CaMKII is scarcely well defined. AIM & METHODS: To investigate the role of CaMKII in hyperacute pressure overload, we evaluated the effects of pressure overload induced by transverse aortic constriction (TAC) on survival, cardiac function, protein expression and excitation-contraction coupling (ECC) in female WT littermate vs. AC3-I mice 2 days after TAC (2d post TAC). AC3-I mice express the CaMKII inhibitor autocamtide-3 related inhibitory peptide (AiP) under the control of the α-myosin heavy chain promotor in the heart. RESULTS: CaMKII activation is significantly increased in WT TAC vs. sham mice 2d post TAC. Interestingly, survival is significantly reduced in AC3-I animals within the first five days after TAC compared to WT TAC littermates, while systolic cardiac function is markedly reduced in AC3-I TAC vs. AC3-I sham mice, but preserved in WT TAC vs. WT sham mice. Proteins regulating ECC such as ryanodine receptors (RyR2) and phospholamban (PLB) are hypophosphorylated at their CaMKII phosphorylation site in AC3-I TAC mice, but hyperphosphorylated in WT TAC mice compared to controls. In isolated cardiomyocytes fractional shortening is significantly impaired in AC3-I compared to WT mice 2d post TAC, and CaMKII incubation with AiP mimics the AC3-I phenotype in cardiomyocytes from WT TAC mice in vitro. In summary, this suggests cardiac dysfunction due to CaMKII inhibition as a potential cause of increased mortality in AC3-I TAC mice. However, proarrhythmic spontaneous Ca2+ release events (SCR) appear less frequent in cardiomyocytes from AC3-I TAC mice than in WT TAC mice. CONCLUSIONS: Our data indicate that excessive CaMKII inhibition as present in AC3-I transgenic mice leads to an impaired adaptation of ECC to hyperacute pressure overload resulting in diminished cardiac contractility and increased death. Thus, our data suggest that in pressure overload the activation of CaMKII is a pivotal, but previously unknown part of hyperacute stress physiology in the heart, while CaMKII inhibition, albeit potentially antiarrhythmic, can be detrimental. This should be taken into account for future studies with CaMKII inhibitors as therapeutic agents.

Skeletal muscle alterations in tachycardia-induced heart failure are linked to deficient natriuretic peptide signalling and are attenuated by RAS-/NEP-inhibition.

BACKGROUND: Heart failure induced cachexia is highly prevalent. Insights into disease progression are lacking. METHODS: Early state of left ventricular dysfunction (ELVD) and symptomatic systolic heart failure (HF) were both induced in rabbits by tachypacing. Tissue of limb muscle (LM) was subjected to histologic assessment. For unbiased characterisation of early and late myopathy, a proteomic approach followed by computational pathway-analyses was performed and combined with pathway-focused gene expression analyses. Specimen of thoracic diaphragm (TD) served as control for inactivity-induced skeletal muscle alterations. In a subsequent study, inhibition of the renin-angiotensin-system and neprilysin (RAS-/NEP) was compared to placebo. RESULTS: HF was accompanied by loss of protein content (8.7±0.4% vs. 7.0±0.5%, mean±SEM, control vs. HF, p<0.01) and a slow-to-fast fibre type switch, establishing hallmarks of cachexia. In ELVD, the enzymatic set-up of LM and TD shifted to a catabolic state. A disturbed malate-aspartate shuttle went well with increased enzymes of glycolysis, forming the enzymatic basis for enforced anoxic energy regeneration. The histological findings and the pathway analysis of metabolic results drew the picture of suppressed PGC-1α signalling, linked to the natriuretic peptide system. In HF, natriuretic peptide signalling was desensitised, as confirmed by an increase in the ratio of serum BNP to tissue cGMP (57.0±18.6pg/ml/nM/ml vs. 165.8±16.76pg/ml/nM/ml, p<0.05) and a reduced expression of natriuretic peptide receptor-A. In HF, combined RAS-/NEP-inhibition prevented from loss in protein content (8.7±0.3% vs. 6.0±0.6% vs. 8.3±0.9%, Baseline vs. HF-Placebo vs. HF-RAS/NEP, p<0.05 Baseline vs. HF-Placebo, p = 0.7 Baseline vs. HF-RAS/NEP). CONCLUSIONS: Tachypacing-induced heart failure entails a generalised myopathy, preceding systolic dysfunction. The characterisation of "pre-cachectic" state and its progression is feasible. Early enzymatic alterations of LM depict a catabolic state, rendering LM prone to futile substrate metabolism. A combined RAS-/NEP-inhibition ameliorates cardiac-induced myopathy independent of systolic function, which could be linked to stabilised natriuretic peptide/cGMP/PGC-1α signalling.