PurposeStargardt disease (STGD1) is an autosomal recessive retinopathy, caused by mutations in the retina-specific ATP-binding cassette transporter (ABCA4) gene. To establish the mutational spectrum and to assess effects of selected deep intronic and common genetic variants on disease, we performed a comprehensive sequence analysis in a large cohort of German STGD1 patients.MethodsDNA samples of 335 STGD1 patients were analyzed for ABCA4 mutations in its 50 coding exons and adjacent intronic sequences by resequencing array technology or next generation sequencing (NGS). Parts of intron 30 and 36 were screened by Sanger chain-terminating dideoxynucleotide sequencing. An in vitro splicing assay was used to test selected variants for their splicing behavior. By logistic regression analysis we assessed the association of common ABCA4 alleles while a multivariate logistic regression model calculated a genetic risk score (GRS).ResultsOur analysis identified 148 pathogenic or likely pathogenic mutations, of which 48 constitute so far unpublished ABCA4-associated disease alleles. Four rare deep intronic variants were found once in 472 alleles analyzed. In addition, we identified six risk-modulating common variants. Genetic risk score estimates suggest that defined common ABCA4 variants influence disease risk in carriers of a single pathogenic ABCA4 allele.ConclusionsOur study adds to the mutational spectrum of the ABCA4 gene. Moreover, in our cohort, deep intronic variants in intron 30 and 36 likely play no or only a minor role in disease pathology. Of note, our findings demonstrate a possible modifying effect of common sequence variants on ABCA4-associated disease.

No abstract

No abstract

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/CaMKII-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 À 1' χ 2 test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n À 1' χ 2 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.

Increased left ventricular fibrosis has been reported in patients hospitalized with coronavirus disease 2019 (COVID-19). It is unclear whether this fibrosis is a consequence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection or a risk factor for severe disease progression. We observed increased fibrosis in the left ventricular myocardium of deceased COVID-19 patients, compared with matched controls. We also detected increased mRNA levels of soluble interleukin-1 receptor-like 1 (sIL1-RL1) and transforming growth factor β1 (TGF-β1) in the left ventricular myocardium of deceased COVID-19 patients. Biochemical analysis of blood sampled from patients admitted to the emergency department (ED) with COVID-19 revealed highly elevated levels of TGF-β1 mRNA in these patients compared to controls. Left ventricular strain measured by echocardiography as a marker of pre-existing cardiac fibrosis correlated strongly with blood TGF-β1 mRNA levels and predicted disease severity in COVID-19 patients. In the left ventricular myocardium and lungs of COVID-19 patients, we found increased neuropilin-1 (NRP-1) RNA levels, which correlated strongly with the prevalence of pulmonary SARS-CoV-2 nucleocapsid. Cardiac and pulmonary fibrosis may therefore predispose these patients to increased cellular viral entry in the lung, which may explain the worse clinical outcome observed in our cohort. Our study demonstrates that patients at risk of clinical deterioration can be identified early by echocardiographic strain analysis and quantification of blood TGF-β1 mRNA performed at the time of first medical contact.

Rationale: Increased myocardial activity of Ca/calmodulin-dependent kinase II (CaMKII) leads to heart failure (HF) and arrhythmias. In Drosophila neurons, interaction of CaMKII with Ca/CaM-dependent serine protein kinase (CASK) has been shown to inhibit CaMKII activity, but the consequences of this regulation for HF and ventricular arrhythmias are unknown. Objective: We hypothesize that CASK associates with CaMKII in human and mouse hearts thereby limiting CaMKII activity, and that altering CASK expression in mice changes CaMKII activity accordingly, with functional consequences for contractile function and arrhythmias. Methods and Results: Immunoprecipitation revealed that CASK associates with CaMKII in human hearts. CASK expression is unaltered in HF but increased in patients with aortic stenosis. In mice, cardiomyocyte-specific knockout of CASK (CASK-KO) increased CaMKII auto-phosphorylation at the stimulatory T287 site, but reduced phosphorylation at the inhibitory T305/306 site. CASK-KO mice showed increased CaMKII-dependent sarcoplasmic reticulum (SR) Ca leak, reduced SR Ca-content, increased susceptibility to ventricular arrhythmias, greater loss of ejection fraction, and increased mortality after transverse aortic constriction. Intriguingly, stimulation of the cardiac glucagon-like peptide 1-receptor with exenatide increased CASK expression resulting in increased inhibitory CaMKII T305 phosphorylation, reduced CaMKII activity, and reduced SR Ca leak in WT but not CASK KO. Conclusions: CASK associates with CaMKII in the human heart. CASK-KO in mice increases CaMKII activity, leading to contractile dysfunction and arrhythmias. Increasing CASK expression reduces CaMKII activity, improves Ca handling and contractile function.

Reactive oxygen species (ROS) have been shown to prolong cardiac action potential duration resulting in afterdepolarizations, the cellular basis of triggered arrhythmias. As previously shown, protein kinase A type I is readily activated by oxidation of its regulatory subunits. However, the relevance of this mechanism of activation for cardiac pathophysiology is still elusive. In this study, we investigated the effects of oxidation-activated PKA I on cardiac electrophysiology. Ventricular cardiomyocytes were isolated from redox-dead PKA-RI Cys17Ser knock-in (KI) and wild-type (WT) mice and exposed to H2O2 (200 µmol/L) or vehicle (veh) solution. In WT myocytes, exposure to H2O2 significantly increased oxidation of the regulatory subunit I (RI) and thus its dimerization (3-fold increase in PKA RI dimer). Whole cell current-clamp and voltage clamp was used to measure cardiac action potentials (APs), transient outward potassium current (Ito) and inward rectifying potassium current (IK1), respectively. In WT myocytes, H2O2 exposure significantly prolonged AP duration due to significantly decreased Ito and IK1 resulting in frequent early afterdepolarizations (EADs). Pre-incubation with the PKA-specific inhibitor Rp-8-Br-cAMPS (10 µmol/L) completely abolished the H2O2-dependent decrease in Ito and IK1 in WT myocytes. Intriguingly, H2O2 exposure did not prolong AP duration, nor did it decrease Ito, and only slightly enhanced EAD frequency in KI myocytes. Treatment of WT and KI cardiomyocytes with the late INa inhibitor TTX (1µmol/L) completely abolished EAD formation. Our results suggest that redox-activated PKA may be important for H2O2-dependent arrhythmias and could be important for development of specific antiarrhythmic drugs.

No abstract