Regulation of systemic PCO
is a life-preserving homeostatic mechanism. In the medulla oblongata, the retrotrapezoid nucleus (RTN) and rostral medullary Raphe are proposed as CO
chemosensory nuclei ...mediating adaptive respiratory changes. Hypercapnia also induces active expiration, an adaptive change thought to be controlled by the lateral parafacial region (pF
). Here, we use GCaMP6 expression and head-mounted mini-microscopes to image Ca
activity in these nuclei in awake adult mice during hypercapnia. Activity in the pF
supports its role as a homogenous neuronal population that drives active expiration. Our data show that chemosensory responses in the RTN and Raphe differ in their temporal characteristics and sensitivity to CO
, raising the possibility these nuclei act in a coordinated way to generate adaptive ventilatory responses to hypercapnia. Our analysis revises the understanding of chemosensory control in awake adult mouse and paves the way to understanding how breathing is coordinated with complex non-ventilatory behaviours.
Force measurements in ex vivo and engineered heart tissues are well established. Analysis of calcium transients (CaT) is complementary to force, and the combined analysis is meaningful to the study ...of cardiomyocyte biology and disease. This article describes a model of human induced pluripotent stem cell cardiomyocyte-derived engineered heart tissues (hiPSC-CM EHTs) transduced with the calcium sensor GCaMP6f followed by sequential analysis of force and CaT. Average peak analysis demonstrated the temporal sequence of the CaT preceding the contraction twitch. The pharmacological relevance of the test system was demonstrated with inotropic indicator compounds. Force-frequency relationship was analyzed in the presence of ivabradine (300 nM), which reduced spontaneous frequency and unmasked a positive correlation of force and CaT at physiological human heart beating frequency with stimulation frequency between 0.75 and 2.5 Hz (force +96%; CaT +102%). This work demonstrates the usefulness of combined force/CaT analysis and demonstrates a positive force-frequency relationship in hiPSC-CM EHTs.
•Analysis of calcium transients and force in engineered heart tissues•Accurate replications of drug effects on calcium transients and force analysis•Positive force- and calcium transients-frequency relationship•Reverse correlation between omecamtiv mecarbil's inotropic effect and frequency
In this article, Hansen and colleagues establish a system using human induced pluripotent stem cell-derived cardiomyocytes to sequentially analyze force and calcium transients (CaTs) in an engineered heart tissue model by using genetically encoded calcium indicator (GCaMP6f) and demonstrate a positive force- and CaT-frequency relationship at physiological human heart beat frequencies.
Loss of vision due to progressive retinal degeneration is a hallmark of neuronal ceroid lipofuscinoses (NCL), a group of fatal neurodegenerative lysosomal storage diseases. Enzyme substitution ...therapies represent promising treatment options for NCLs caused by dysfunctions of soluble lysosomal enzymes. Here, we compared the efficacy of a cell-based enzyme substitution strategy and a gene therapy approach to attenuate the retinal pathology in cathepsin D- (CTSD) deficient mice, an animal model of CLN10 disease. Levels of enzymatically active CTSD in mutant retinas were significantly higher after an adeno-associated virus vector-mediated CTSD transfer to retinal glial cells and retinal pigment epithelial cells than after intravitreal transplantations of a CTSD overexpressing clonal neural stem cell line. In line with this finding, the gene therapy treatment restored the disrupted autophagy-lysosomal pathway more effectively than the cell-based approach, as indicated by a complete clearance of storage, significant attenuation of lysosomal hypertrophy, and normalized levels of the autophagy marker sequestosome 1/p62 and microtubule-associated protein 1 light chain 3-II. While the cell-based treatment did not prevent the rapidly progressing loss of various retinal cell types, the gene therapy approach markedly attenuated retinal degeneration as demonstrated by a pronounced rescue of photoreceptor cells and rod bipolar cells.
•A gene therapy approach restored CTSD enzymatic activity close to wild-type levels.•The treatment fully restored the disrupted autophagy-lysosomal pathway.•The therapy markedly promoted the survival of photoreceptors and rod bipolar cells.
Abstract
Aims
Chronic tachypacing is commonly used in animals to induce cardiac dysfunction and to study mechanisms of heart failure and arrhythmogenesis. Human induced pluripotent stem cells (hiPSC) ...may replace animal models to overcome species differences and ethical problems. Here, 3D engineered heart tissue (EHT) was used to investigate the effect of chronic tachypacing on hiPSC-cardiomyocytes (hiPSC-CMs).
Methods and results
To avoid cell toxicity by electrical pacing, we developed an optogenetic approach. EHTs were transduced with lentivirus expressing channelrhodopsin-2 (H134R) and stimulated by 15 s bursts of blue light pulses (0.3 mW/mm2, 30 ms, 3 Hz) separated by 15 s without pacing for 3 weeks. Chronic optical tachypacing did not affect contractile peak force, but induced faster contraction kinetics, shorter action potentials, and shorter effective refractory periods. This electrical remodelling increased vulnerability to tachycardia episodes upon electrical burst pacing. Lower calsequestrin 2 protein levels, faster diastolic depolarization (DD) and efficacy of JTV-519 (46% at 1 µmol/L) to terminate tachycardia indicate alterations of Ca2+ handling being part of the underlying mechanism. However, other antiarrhythmic compounds like flecainide (69% at 1 µmol/L) and E-4031 (100% at 1 µmol/L) were also effective, but not ivabradine (1 µmol/L) or SEA0400 (10 µmol/L).
Conclusion
We demonstrated a high vulnerability to tachycardia of optically tachypaced hiPSC-CMs in EHT and the effective termination by ryanodine receptor stabilization, sodium or hERG potassium channel inhibition. This new model might serve as a preclinical tool to test antiarrhythmic drugs increasing the insight in treating ventricular tachycardia.
Graphical Abstract
Graphical Abstract
Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into ...systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.
Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. ...Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.
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•ChREBP controls de novo lipogenesis (DNL) in BAT•DNL-derived lipids are enriched in BAT of mice in response to thermoneutral housing•Thermoneutrality causes loss of mitochondria and UCP1 by mitophagy in BAT•DNL-linked lipid remodeling and BAT involution are prevented by ChREBP deficiency
Schlein et al. show that carbohydrate-response element-binding protein (ChREBP) controls de novo lipogenesis (DNL) in brown adipose tissue (BAT) and determines BAT whitening in response to thermoneutral housing. ChREBP deficiency prevents enrichment of DNL-derived lipids and mitophagy during BAT involution, which is associated with higher thermogenic capacity.
The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, ...isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+‐load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow‐up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end‐stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non‐failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca2+‐binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+‐scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.
Synopsis
The disease mechanism linking the phospholamban (PLN) p.Arg14del mutation to dilated cardiomyopathy is incompletely understood. In this study, patient‐derived human induced pluripotent stem cell‐cardiomyocytes were used to elucidate this molecular mechanism.
Sarcoplasmic reticulum function remained unaltered in PLN p.Arg14del human cardiomyocytes.
Impairment of the interface between endoplasmic reticulum and mitochondria was discovered as a novel disease phenotype.
Cytoplasmic calcium‐scavenging improved the cardiomyopathy phenotype and revealed the role for cytoplasmic calcium in disease development.
The disease mechanism linking the phospholamban (PLN) p.Arg14del mutation to dilated cardiomyopathy is incompletely understood. In this study, patient‐derived human induced pluripotent stem cell‐cardiomyocytes were used to elucidate this molecular mechanism.
A limiting factor for the use of adeno-associated viruses (AAVs) as vectors in gene therapy is the broad tropism of AAV serotypes, i.e., the parallel infection of several cell types. Nanobodies are ...single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Their small size and high solubility allow easy reformatting into fusion proteins. Herein we show that a membrane protein-specific nanobody can be inserted into a surface loop of the VP1 capsid protein of AAV2. Using three structurally distinct membrane proteins—a multispan ion channel, a single-span transmembrane protein, and a glycosylphosphatidylinositol (GPI)-anchored ectoenzyme—we show that this strategy can dramatically enhance the transduction of specific target cells by recombinant AAV2. Moreover, we show that the nanobody-VP1 fusion of AAV2 can be incorporated into the capsids of AAV1, AAV8, and AAV9 and thereby effectively redirect the target specificity of other AAV serotypes. Nanobody-mediated targeting provides a highly efficient AAV targeting strategy that is likely to open up new avenues for genetic engineering of cells.
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