Heparan Sulfate Deficiency in Cartilage: Enhanced BMP-Sensitivity, Proteoglycan Production and an Anti-Apoptotic Expression Signature after Loading.

Osteoarthritis (OA) represents one major cause of disability worldwide still evading efficient pharmacological or cellular therapies. Severe degeneration of extracellular cartilage matrix precedes the loss of mobility and disabling pain perception in affected joints. Recent studies showed that a reduced heparan sulfate (HS) content protects cartilage from degradation in OA-animal models of joint destabilization but the underlying mechanisms remained unclear. We aimed to clarify whether low HS-content alters the mechano-response of chondrocytes and to uncover pathways relevant for HS-related chondro-protection in response to loading. Tissue-engineered cartilage with HS-deficiency was generated from rib chondrocytes of mice carrying a hypomorphic allele of Exostosin 1 (Ext1), one of the main HS-synthesizing enzymes, and wildtype (WT) littermate controls. Engineered cartilage matured for 2 weeks was exposed to cyclic unconfined compression in a bioreactor. The molecular loading response was determined by transcriptome profiling, bioinformatic data processing, and qPCR. HS-deficient chondrocytes expressed 3-6% of WT Ext1-mRNA levels. Both groups similarly raised Sox9, Col2a1 and Acan levels during maturation. However, HS-deficient chondrocytes synthesized and deposited 50% more GAG/DNA. TGFß and FGF2-sensitivity of Ext1gt/gt chondrocytes was similar to WT cells but their response to BMP-stimulation was enhanced. Loading induced similar activation of mechano-sensitive ERK and P38-signaling in WT and HS-reduced chondrocytes. Transcriptome analysis reflected regulation of cell migration as major load-induced biological process with similar stimulation of common (Fosl1, Itgα5, Timp1, and Ngf) as well as novel mechano-regulated genes (Inhba and Dhrs9). Remarkably, only Ext1-hypomorphic cartilage responded to loading by an expression signature of negative regulation of apoptosis with pro-apoptotic Bnip3 being selectively down-regulated. HS-deficiency enhanced BMP-sensitivity, GAG-production and fostered an anti-apoptotic expression signature after loading, all of which may protect cartilage from load-induced erosion.

Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease.

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.

Verapamil inhibits Kir2.3 channels by binding to the pore and interfering with PIP2 binding.

The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at - 120 mV (73.2 ± 3.7%), - 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.

Cannabinoid 1 receptor signaling on GABAergic neurons influences astrocytes in the ageing brain.

Astrocytes, key regulators of brain homeostasis, interact with neighboring glial cells, neurons and the vasculature through complex processes involving different signaling pathways. It is not entirely clear how these interactions change in the ageing brain and which factors influence astrocyte ageing. Here, we investigate the role of endocannabinoid signaling, because it is an important modulator of neuron and astrocyte functions, as well as brain ageing. We demonstrate that mice with a specific deletion of CB1 receptors on GABAergic neurons (GABA-Cnr1-/- mice), which show a phenotype of accelerated brain ageing, affects age-related changes in the morphology of astrocytes in the hippocampus. Thus, GABA-Cnr1-/- mice showed a much more pronounced age-related and layer-specific increase in GFAP-positive areas in the hippocampus compared to wild-type animals. The number of astrocytes, in contrast, was similar between the two genotypes. Astrocytes in the hippocampus of old GABA-Cnr1-/- mice also showed a different morphology with enhanced GFAP-positive process branching and a less polarized intrahippocampal distribution. Furthermore, astrocytic TNFα levels were higher in GABA-Cnr1-/- mice, indicating that these morphological changes were accompanied by a more pro-inflammatory function. These findings demonstrate that the disruption of endocannabinoid signaling on GABAergic neurons is accompanied by functional changes in astrocyte activity, which are relevant to brain ageing.

Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups, cPEG, and the PEG/fumaric acid copolymer OPF. After incubation in Ca-GP solution, FTIR, EDS, SEM, XRD, SAED, ICP-OES, and von Kossa staining confirm CaP formation. The amount of mineral formed decreases in the order cPEG > collagen > OPF. The mineral:polymer ratio decreases in the order collagen > cPEG > OPF. Mineralization increases Young's modulus, most profoundly for cPEG. Such enzymatically mineralized hydrogel/CaP composites may find application as bone regeneration materials.

Enzymatically induced mineralization of platelet-rich fibrin.

Membranes of the autologous blood-derived biomaterial platelet-rich fibrin (PRF) were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, and subsequently incubated in calcium glycerophosphate (CaGP) solution to induce PRFs mineralization with calcium phosphate (CaP) to improve PRFs suitability as a material for bone replacement. Incorporated ALP retained its bioactivity and induced formation of CaP material within PRF membranes, as confirmed by SEM, EDS, FTIR, and von Kossa staining. The mass percentage attributable to CaP was quantified by lyophilization and measurement of the remaining mass fraction as well as by TGA. Cytocompatibility tests (LDH, MTT, and WST) with SAOS-2 cells showed that mineralized PRF did not release substances detrimental to cell vitality. Live/dead staining and SEM showed that mineralized PRF was colonized by cells. The results show that hydrogel biomaterials such as PRF can be mineralized through functionalization with ALP.