Explore the vast range of titles available.

An interplay between Ca 2+ /calmodulin-dependent protein kinase IIδc (CaMKIIδc) and late Na + current (I NaL ) is known to induce arrhythmias in the failing heart. Here, we elucidate the role of the sodium channel isoform Na V 1.8 for CaMKIIδc-dependent proarrhythmia. In a CRISPR-Cas9-generated human iPSC-cardiomyocyte homozygous knock-out of Na V 1.8, we demonstrate that Na V 1.8 contributes to I NaL formation. In addition, we reveal a direct interaction between Na V 1.8 and CaMKIIδc in cardiomyocytes isolated from patients with heart failure (HF). Using specific blockers of Na V 1.8 and CaMKIIδc, we show that Na V 1.8-driven I NaL is CaMKIIδc-dependent and that Na V 1.8-inhibtion reduces diastolic SR-Ca 2+ leak in human failing cardiomyocytes. Moreover, increased mortality of CaMKIIδc-overexpressing HF mice is reduced when a Na V 1.8 knock-out is introduced. Cellular and in vivo experiments reveal reduced ventricular arrhythmias without changes in HF progression. Our work therefore identifies a proarrhythmic CaMKIIδc downstream target which may constitute a prognostic and antiarrhythmic strategy. In heart failure, increased CaMKII activity is decisively involved in arrhythmia formation. Here, the authors introduce the neuronal sodium channel Na V 1.8 as a CaMKII downstream target as its specific knock-out reduces arrhythmias and improves survival in a CaMKII-overexpressing mouse model.

Single cell multi-omics analysis has the potential to yield a comprehensive understanding of the cellular events that underlie the basis of human diseases. The cardinal feature to access this information is the technology used for single-cell isolation, barcoding, and sequencing. Most currently used single-cell RNA-sequencing platforms have limitations in several areas including cell selection, documentation and library chemistry. In this study, we describe a novel high-throughput, full-length, single-cell RNA-sequencing approach that combines the CellenONE isolation and sorting system with the ICELL8 processing instrument. This method offers substantial improvements in single cell selection, documentation and capturing rate. Moreover, it allows the use of flexible chemistry for library preparations and the analysis of living or fixed cells, whole cells independent of sizing and morphology, as well as of nuclei. We applied this method to dermal fibroblasts derived from six patients with different segmental progeria syndromes and defined phenotype associated pathway signatures with variant associated expression modifiers. These results validate the applicability of our method to highlight genotype-expression relationships for molecular phenotyping of individual cells derived from human patients.

While excessive production of reactive oxygen species (ROS) is a characteristic hallmark of numerous diseases, clinical approaches that ameliorate oxidative stress have been unsuccessful. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes, mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidative defense mechanism. Paradoxically reduced expression of IDH2 associated with ventricular eccentric hypertrophy is counterbalanced by an increase in the enzyme activity. We unveil redox-dependent sex dimorphism, and extensive mutual regulation of the antioxidative activities of IDH2 and NRF2 by a feedforward network that involves 2-oxoglutarate and L-2-hydroxyglutarate and mediated in part through unconventional hydroxy-methylation of cytosine residues present in introns. Consequently, conditional targeting of ROS in a murine model of heart failure improves cardiac function in sex- and phenotype-dependent manners. Together, these insights may explain why previous attempts to treat heart failure with antioxidants have been unsuccessful and open new approaches to personalizing and, thereby, improving such treatment. Efforts to treat heart failure with antioxidants have failed. Here, authors reveal a robust sex-dependent endogenous defense against oxidative damage and demonstrate antioxidative treatment’s efficacy solely in subjects with inadequate redox capacity.

Background Pressure overload first leads to compensated hypertrophy and secondary to heart failure. m6A-RNA methylation is a fast process for the adaptation of cell composition. m6A-RNA-methylation is regulated by the demethylase, fat mass and obesity-associated protein ( FTO), and FTO protein levels are diminished in heart failure. Cardiomyocyte-specific FTO-transgenic/knockout-mice have shown the relevance of FTO in pressure overload remodeling. However, its functional downstream regulatory mechanisms are still unclear. In this study, we discover the harmful signaling pathways that are triggered by m6A imbalance and FTO loss, which eventually lead to adverse cardiac remodeling and heart failure. Methods FTOcKO animals were generated by crossing FTO fl/fl mice with - MHC Cre mice using Cre-lox system. Control and the FTOcKO animals groups were subjected to TAC (transverse aortic constriction) surgery. Echocardiography was performed 1-week post-TAC surgery. MeRIP (m6A RNA immunoprecipitation) sequencing was performed from the heart tissues of mice after one week TAC surgery. Additionally, the mechanistical interrelation between the signaling pathways during FTO loss and adverse cardiac remodeling were investigated in human iPS-CMs (hiPS-CMs). Results One week post-TAC surgery, FTOcKO mice showed impaired cardiac function (EF: CreC TAC (45%) vs. FTOcKO TAC (25%), p  < 0.0001) and increased LVID (CreC TAC(3.9 mm) vs. FTOcKO TAC (4.8 mm), p  < 0.0001), indicating a lack of adaption to pressure overload. Knockdown of FTO in hiPS-cardiomyocytes also reduced endothelin-induced hypertrophic response. MeRIP-seq data of FTOcKO mice showed that the differentially hypermethylated transcripts were associated with cardiac apoptosis inhibition (CDK1, CFLAR), mTORC1 signaling pathway (AKT1S1) and autophagy regulation (TFEB). mTORC1 was identified as a central player of dysregulation with hyperactivation of its canonical substrates phospho-S6K1 (Thr 389) and phospho-S6 (ser235/236) ex-vivo (FTOcKO) and in-vitro (FTO-KD-hiPS-CMs). Moreover, FTO-deficient cardiomyocytes cause autophagic flux impairment and defective autophagy. The effect of atrophy and induced apoptosis upon FTO-m6A imbalance could be rescued by pharmacological inhibiton of the mTORC1-S6K1 pathway. Conclusions Downregulation of FTO leads to mTORC1-S6K1 hyperactivation that shift the compensative hypertrophic response to atrophy and apoptosis leading to progressive heart failure. These findings might pave the way for the development of novel therapeutic targets for the early phases of heart failure treatments.

A multimodal therapeutic approach involving radiotherapy is required when treating head and neck squamous cell carcinoma. However, radiotherapy is restricted due to its high risk for damages to the surrounding healthy tissue of the treated area. Tissue regeneration and wound healing is promoted by the survival and regenerative capacities of tissue-resident or invading stem cells. Mesenchymal stem cells (MSCs) exhibit a promising therapeutic potential in the field of cell-based tissue engineering and regenerative medicine due to their immunomodulatory properties and differentiation capacity. However, the generation of MSCs for therapeutic applications is still a major challenge. We aimed to produce highly homogeneous induced pluripotent stem cell-derived mesenchymal stem cells (iP-MSCs) in an autologous manner from initially isolated human mucosa mesenchymal stem cells (mMSCs) of the upper respiratory tract. Therefore, mMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) by non-integrative chromosomal technologies and differentiated into corresponding iP-MSCs. We demonstrated that mMSCs and iP-MSCs show similar cell characteristics in terms of morphology, clonogenic potential, differentiation, and surface phenotype. Moreover, iP-MSCs demonstrated related immunosuppressive capacity as mMSCs including the secretion of cytokines, and T cell inhibition. Therefore, generating iP-MSCs in an autologous manner may be a novel personalized treatment option in regenerative medicine.

“Medical scientists” are postgraduate investigators who are engaged in biomedical research, and either hold a biomedical PhD or are qualified in medicine but do not participate in patient care. Medical scientists constitute ~40% of staff at medical faculties and >90% at nonuniversity medical research institutions in Germany. However, medical scientists in Germany face limited long‐term career prospects and a lack of dedicated training and support programmes. They also face time limits on their career progression arising from national academic employment legislation, and imminent reforms by the German government are likely to make this worse. Nevertheless, recent developments in the educational landscape including the introduction of increasingly focused MSc, pre‐PhD, and doctoral programmes to train medically aware basic scientists, as well as improved general recognition of the roles and relevance of medical scientists in health research, are encouraging. Physiologists have taken essential steps to improve the recognition of medical scientists in Germany by introducing a “specialist physiologist” qualification; this initiative could be applied to support medical scientists in other fields and countries. In this review, we describe the particular challenges facing medical scientists in Germany and make recommendations that may apply to other academic systems.

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30±0.18, Sendai virus: 1.37±0.01, and episomal plasmids: 0.04±0.02; PBMCs: Sendai virus: 0.002±0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32±1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.

Pharmacologic approaches for the treatment of atrial arrhythmias are limited due to side effects and low efficacy. Thus, the identification of new antiarrhythmic targets is of clinical interest. Recent genome studies suggested an involvement of SCN10A sodium channels (NaV1.8) in atrial electrophysiology. This study investigated the role and involvement of NaV1.8 (SCN10A) in arrhythmia generation in the human atria and in mice lacking NaV1.8. NaV1.8 mRNA and protein were detected in human atrial myocardium at a significant higher level compared to ventricular myocardium. Expression of NaV1.8 and NaV1.5 did not differ between myocardium from patients with atrial fibrillation and sinus rhythm. To determine the electrophysiological role of NaV1.8, we investigated isolated human atrial cardiomyocytes from patients with sinus rhythm stimulated with isoproterenol. Inhibition of NaV1.8 by A-803467 or PF-01247324 showed no effects on the human atrial action potential. However, we found that NaV1.8 significantly contributes to late Na+ current and consequently to an increased proarrhythmogenic diastolic sarcoplasmic reticulum Ca2+ leak in human atrial cardiomyocytes. Selective pharmacological inhibition of NaV1.8 potently reduced late Na+ current, proarrhythmic diastolic Ca2+ release, delayed afterdepolarizations as well as spontaneous action potentials. These findings could be confirmed in murine atrial cardiomyocytes from wild-type mice and also compared to SCN10A−/− mice (genetic ablation of NaV1.8). Pharmacological NaV1.8 inhibition showed no effects in SCN10A−/− mice. Importantly, in vivo experiments in SCN10A−/− mice showed that genetic ablation of NaV1.8 protects against atrial fibrillation induction. This study demonstrates that NaV1.8 is expressed in the murine and human atria and contributes to late Na+ current generation and cellular arrhythmogenesis. Blocking NaV1.8 selectively counteracts this pathomechanism and protects against atrial arrhythmias. Thus, our translational study reveals a new selective therapeutic target for treating atrial arrhythmias.

Introduction: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. Methods: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. Results: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. Conclusion: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.

The SGLT2 inhibitor empagliflozin improved cardiovascular outcomes in patients with diabetes. As the cardiac mechanisms remain elusive, we investigated the long-term effects (up to 2 months) of empagliflozin on excitation-contraction (EC)-coupling in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) in a blinded manner. IPSC from 3 donors, differentiated into pure iPSC-CM (4 differentiations), were treated with a clinically relevant concentration of empagliflozin (0.5 μmol/l) or vehicle control. Treatment, data acquisition, and analysis were conducted externally blinded. Epifluorescence microscopy measurements in iPSC-CM showed that empagliflozin has neutral effects on Ca2+ transient amplitude, diastolic Ca2+ levels, Ca2+ transient kinetics, or sarcoplasmic Ca2+ load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca2+ release events showed that in iPSC-CM, Ca2+ spark frequency and leak was not altered after chronic treatment with empagliflozin. Finally, in patch-clamp experiments, empagliflozin did not change action potential duration, amplitude, or resting membrane potential compared with vehicle control after long-term treatment. Next-generation RNA sequencing (NGS) and mapped transcriptome profiles of iPSC-CMs untreated and treated with empagliflozin for 8 weeks showed no differentially expressed EC-coupling genes. In line with NGS data, Western blots indicate that empagliflozin has negligible effects on key EC-coupling proteins. In this blinded study, direct treatment of iPSC-CM with empagliflozin for a clinically relevant duration of 2 months did not influence cardiomyocyte EC-coupling and electrophysiology. Therefore, it is likely that other mechanisms independent of cardiomyocyte EC-coupling are responsible for the beneficial treatment effect of empagliflozin.Key messagesThis blinded study investigated the clinically relevant long-term effects (up to 2 months) of empagliflozin on cardiomyocyte excitation-contraction (EC)-coupling.Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) were used to study a human model including a high repetition number of experiments.Empagliflozin has neutral effects on cardiomyocyte Ca2+ transients, sarcoplasmic Ca2+ load, and diastolic sarcoplasmic Ca2+ leak.In patch-clamp experiments, empagliflozin did not change the action potential.Next-generation RNA sequencing, mapped transcriptome profiles, and Western blots of iPSC-CM untreated and treated with empagliflozin showed no differentially expressed EC-coupling candidates.