The ubiquitin-proteasome system and nonsense-mediated mRNA decay in hypertrophic cardiomyopathy

Cardiomyopathies represent an important cause of cardiovascular morbidity and mortality due to heart failure, arrhythmias, and sudden death. Most forms of hypertrophic cardiomyopathy (HCM) are familial with an autosomal-dominant mode of inheritance. Over the last 20 years, the genetic basis of the disease has been largely unravelled. HCM is considered as a sarcomeropathy involving mutations in sarcomeric proteins, most often β-myosin heavy chain and cardiac myosin-binding protein C. ‘Missense’ mutations, more common in the former, are associated with dysfunctional proteins stably integrated into the sarcomere. ‘Nonsense’ and frameshift mutations, more common in the latter, are associated with low mRNA and protein levels derived from the diseased allele, leading to haploinsufficiency of the remaining healthy allele. The two quality control systems responsible for the removal of the affected mRNAs and proteins are the nonsense-mediated mRNA decay (NMD) and the ubiquitin-proteasome system (UPS), respectively. This review discusses clinical and genetic aspects of HCM and the role of NMD and UPS in the regulation of mutant proteins, evidence for impairment of UPS as a pathogenic factor, as well as potential therapies for HCM

Resting membrane potential is less negative in trabeculae from right atrial appendages of women, but action potential duration does not shorten with age

Aims: The incidence of atrial fibrillation (AF) increases with age. Women have a lower risk. Little is known on the impact of age, sex and clinical variables on action potentials (AP) recorded in right atrial tissue obtained during open heart surgery from patients in sinus rhythm (SR) and in longstanding AF. We here investigated whether age or sex have an impact on the shape of AP recorded in vitro from right atrial tissue. Methods: We performed multivariable analysis of individual AP data from trabeculae obtained during heart surgery of patients in SR (n = 320) or in longstanding AF (n = 201). AP were recorded by sharp microelectrodes at 37 °C at 1 Hz. Impact of clinical variables were modeled using a multivariable mixed model regression. Results: In SR, AP duration at 90% repolarization (APD90) increased with age. Lower ejection fraction and higher body mass index were associated with smaller action potential amplitude (APA) and maximum upstroke velocity (Vmax). The use of beta-blockers was associated with larger APD90. In tissues from women, resting membrane potential was less negative and APA as well as Vmax were smaller. Besides shorter APD20 in elderly patients, effects of age and sex on atrial AP were lost in AF. Conclusion: The higher probability to develop AF at advanced age cannot be explained by a shortening in APD90. Less negative RMP and lower upstroke velocity might contribute to lower incidence of AF in women, which may be of clinical relevance.</p

Valvular cardiomyopathy in aortic valve regurgitation correlates with myocardial fibrosis

Objective: At the tissue level, disruption of the extracellular matrix network leads to irreversible cardiac fibrosis, which contributes to myocardial dysfunction. At the myocyte level, downregulation of beta-adrenoceptors (beta-AR) reduces adaptation to increased workload. The aim of our study was to analyse the correlation between myocardial fibrosis and beta-AR sensitivity in patients with aortic valve (AV) disease. Methods: A total of 92 consecutive patients who underwent elective AV surgery between 2017–2019 were included in our study (51 with aortic regurgitation (AR-group); 41 with aortic stenosis (AS-group) and left ventricular (LV) biopsies were obtained intraoperatively. In vitro force contractility testing was performed by measuring beta-AR sensitivity (−log EC50[ISO]). In parallel, a quantitative analysis of myocardial fibrosis burden was performed. Results: Mean age at the time of AV surgery was not statistically different in both groups (AR: 53.3 ± 15.3 years vs. AS: 58.7 ± 17.0 years; p = 0.116). The LV end-diastolic diameter was significantly enlarged in the AR-group when compared to the AS-group (59.4 ± 15.6 vs. 39.7 ± 21.2; p < 0.001). Analysis of beta-AR sensitivity (AR: −6.769 vs. AS: −6.659; p = 0.316) and myocardial fibrosis (AR: 8.9% vs. AS: 11.3%; p = 0.284) showed no significant differences between patients with AS and AR. There was no correlation between myocardial fibrosis and beta-AR sensitivity in the whole study cohort (R = 0.1987; p = 0.100) or in the AS-subgroup (R = 0.009; p = 0.960). However, significant correlation of fibrosis and beta-AR sensitivity was seen in AR-patients (R = 0.363; p = 0.023). Conclusion: More severe myocardial fibrosis was associated with reduced beta-AR sensitivity in patients presenting with AR but not with AS. Therefore, our results suggest that in patients with AR, cellular myocardial dysfunction is present and correlates with the extent of myocardial fibrosis in the myocardium

Proteomic analysis in valvular cardiomyopathy: aortic regurgitation vs. aortic stenosis

Left ventricular (LV) reverse remodeling after aortic valve (AV) surgery is less predictable in chronic aortic regurgitation (AR) than in aortic stenosis (AS). We aimed to disclose specific LV myocardial protein signatures possibly contributing to differential disease progression. Global protein profiling of LV myocardial samples excised from the subaortic interventricular septum in patients with isolated AR or AS undergoing AV surgery was performed using liquid chromatography–electrospray ionization–tandem mass spectrometry. Based on label-free quantitation protein intensities, a logistic regression model was calculated and adjusted for age, sex and protein concentration. Web-based functional enrichment analyses of phenotype-associated proteins were performed utilizing g:Profiler and STRING. Data are available via ProteomeXchange with identifier PXD039662. Lysates from 38 patients, including 25 AR and 13 AS samples, were analyzed. AR patients presented with significantly larger LV diameters and volumes (end-diastolic diameter: 61 (12) vs. 48 (13) mm, p < 0.001; end-diastolic volume: 180.0 (74.6) vs. 92.3 (78.4), p = 0.001). A total of 171 proteins were associated with patient phenotype: 117 were positively associated with AR and the enrichment of intracellular compartment proteins (i.e., assigned to carbohydrate and nucleotide metabolism, protein biosynthesis and the proteasome) was detected. Additionally, 54 were positively associated with AS and the enrichment of extracellular compartment proteins (i.e., assigned to the immune and hematopoietic system) was observed. In summary, functional enrichment analysis revealed specific AR- and AS-associated signatures of LV myocardial proteins

Human Engineered Heart Tissue as a Versatile Tool in Basic Research and Preclinical Toxicology

Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research

Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits

Hypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations

Thymosin beta 4 Improves Differentiation and Vascularization of EHTs

Induced pluripotent stem cells (iPSC) constitute a powerful tool to study cardiac physiology and represents a promising treatment strategy to tackle cardiac disease. However, iPSCs remain relatively immature after differentiation. Additionally, engineered heart tissue (EHT) has been investigated as a therapy option in preclinical disease models with promising results, although their vascularization and functionality leave room for improvement. Thymosin beta 4 (T beta 4) has been shown to promote the differentiation of progenitor cell lines to cardiomyocytes while it also induces angiogenic sprouting and vascular maturation. We examined the potential impact of T beta 4 to enhance maturation of cardiomyocytes from iPSCs. Assessing the expression of transcription factors associated with cardiac differentiation, we were able to demonstrate the increased generation of cells displaying cardiomyocyte characteristics in vitro. Furthermore, we demonstrated, in a zebrafish model of embryonic vascular development, that T beta 4 is crucial for the proper execution of lymphatic and angiogenic vessel sprouting. Finally, utilizing T beta 4-transduced EHTs generated from mice genetically engineered to label endothelial cells in vitro, we show that treatment with T beta 4 promotes vascularization and contractility in EHTs, highlighting T beta 4 as a growth factor improving the formation of cardiomyocytes from iPSC and enhancing the performance of EHTs generated from neonatal cardiomyocytes