SummaryTo harness the potential of human pluripotent stem cells (hPSCs), an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation.

Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound's therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme.

Key points• ClC channels are double-barrelled channels with two ion conduction pathways per individual channel.• Substituting cysteine 277 by serine constitutively opens the common gate suggesting that this residue plays a major role in joint openings/closings of both protopores of ClC-1.• We studied here the functional consequences of two novel myotonia-associated mutations, C277R and C277Y, of human ClC-1 chloride channels.• C277Y not only modified the common gate, but also protopore gating.• C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates and thus decreases the absolute open probabilities of homodimeric hClC-1 channels to values below 3%.• C277Y reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence.• Our results explain the disease-causing effects and provide novel insights into the molecular processes underlying normal and pathologically altered function of muscle chloride channels.Abstract Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants' effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I − = NO 3 − > Br − > Cl − . Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects

Background: CLC-K channels and their subunit barttin are crucial for urinary concentration and hearing. Results: Non-palmitoylated barttin mutants reduce CLC-K current amplitudes without modifying unitary channel properties. Conclusion: Palmitoylation of barttin switches CLC-K channels into an active state. Significance: Palmitoylation/depalmitoylation of CLC-K/barttin channels pre-inserted in epithelial plasma membranes might regulate renal chloride absorption.