A mechano- and heat-gated two-pore domain K+ channel controls excitability in adult zebrafish skeletal muscle.

TRAAK channels are mechano-gated two-pore-domain K+ channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K+ ions, of 56 pS unitary conductance in the presence of 5 mM external K+, activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K+ channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K+ channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K+ currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development.

Cell differentiation and matrix organization are differentially affected during bone formation in osteogenesis imperfecta zebrafish models with different genetic defects impacting collagen type I structure.

Osteogenesis imperfecta (OI) is a family of rare heritable skeletal disorders associated with dominant mutations in the collagen type I encoding genes and recessive defects in proteins involved in collagen type I synthesis and processing and in osteoblast differentiation and activity. Historically, it was believed that the OI bone phenotype was only caused by abnormal collagen type I fibrils in the extracellular matrix, but more recently it became clear that the altered bone cell homeostasis, due to mutant collagen retention, plays a relevant role in modulating disease severity in most of the OI forms and it is correlated to impaired bone cell differentiation. Despite in vitro evidence, in vivo data are missing. To better understand the physiopathology of OI, we used two zebrafish models: Chihuahua (Chi/+), carrying a dominant p.G736D substitution in the α1 chain of collagen type I, and the recessive p3h1-/-, lacking prolyl 3-hydroxylase (P3h1) enzyme. Both models share the delay of collagen type I folding, resulting in its overmodification and partial intracellular retention. The regeneration of the bony caudal fin of Chi/+ and p3h1-/- was employed to investigate the impact of abnormal collagen synthesis on bone cell differentiation. Reduced regenerative ability was evident in both models, but it was associated to impaired osteoblast differentiation and osteoblastogenesis/adipogenesis switch only in Chi/+. On the contrary, reduced osteoclast number and activity were found in both models during regeneration. The dominant OI model showed a more detrimental effect in the extracellular matrix organization. Interestingly, the chemical chaperone 4-phenylbutyrate (4-PBA), known to reduce cellular stress and increase collagen secretion, improved bone formation only in p3h1-/- by favoring caudal fin growth without affecting bone cell markers expression. Taken together, our in vivo data proved the negative impact of structurally abnormal collagen type I on bone formation but revealed a gene mutation-specific effect on bone cell differentiation and matrix organization in OI. These, together with the distinct ability to respond to the chaperone treatment, underline the need for precision medicine approaches to properly treat the disease.

NRF2 Shortage in Human Skin Fibroblasts Dysregulates Matrisome Gene Expression and Affects Collagen Fibrillogenesis.

NRF2 is a master regulator of the antioxidative response that was recently proposed as a potential regulator of extracellular matrix (ECM) gene expression. Fibroblasts are major ECM producers in all connective tissues, including the dermis. A better understanding of NRF2-mediated ECM regulation in skin fibroblasts is thus of great interest for skin homeostasis maintenance and aging protection. In this study, we investigate the impact of NRF2 downregulation on matrisome gene expression and ECM deposits in human primary dermal fibroblasts. RNA-sequencing‒based transcriptome analysis of NRF2 silenced dermal fibroblasts shows that ECM genes are the most regulated gene sets, highlighting the relevance of the NRF2-mediated matrisome program in these cells. Using complementary light and electron microscopy methods, we show that NRF2 deprivation in dermal fibroblasts results in reduced collagen I biosynthesis and impacts collagen fibril deposition. Moreover, we identify ZNF469, a putative transcriptional regulator of collagen biosynthesis, as a target of NRF2. Both ZNF469 silenced fibroblasts and fibroblasts derived from Brittle Corneal Syndrome patients carrying variants in ZNF469 gene show reduced collagen I gene expression. Our study shows that NRF2 orchestrates matrisome expression in human skin fibroblasts through direct or indirect transcriptional mechanisms that could be prioritized to target dermal ECM homeostasis in health and disease.

FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1-dependent axis that controls VEGFR1 splicing in endothelial cells.

BACKGROUND: Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. RESULTS: By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. CONCLUSIONS: We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression.

Surface charge modulation of rifampicin-loaded PLA nanoparticles to improve antibiotic delivery in Staphylococcus aureus biofilms.

BACKGROUND: After the golden age of antibiotic discovery, bacterial infections still represent a major challenge for public health worldwide. The biofilm mode of growth is mostly responsible for chronic infections that current therapeutics fail to cure and it is well-established that novel strategies must be investigated. Particulate drug delivery systems are considered as a promising strategy to face issues related to antibiotic treatments in a biofilm context. Particularly, poly-lactic acid (PLA) nanoparticles present a great interest due to their ability to migrate into biofilms thanks to their submicronic size. However, questions still remain unresolved about their mode of action in biofilms depending on their surface properties. In the current study, we have investigated the impact of their surface charge, firstly on their behavior within a bacterial biofilm, and secondly on the antibiotic delivery and the treatment efficacy. RESULTS: Rifampicin-loaded PLA nanoparticles were synthetized by nanoprecipitation and characterized. A high and superficial loading of rifampicin, confirmed by an in silico simulation, enabled to deliver effective antibiotic doses with a two-phase release, appropriate for biofilm-associated treatments. These nanoparticles were functionalized with poly-L-lysine, a cationic peptide, by surface coating inducing charge reversal without altering the other physicochemical properties of these particles. Positively charged nanoparticles were able to interact stronger than negative ones with Staphylococcus aureus, under planktonic and biofilm modes of growth, leading to a slowed particle migration in the biofilm thickness and to an improved retention of these cationic particles in biofilms. While rifampicin was totally ineffective in biofilms after washing, the increased retention capacity of poly-L-lysine-coated rifampicin-loaded PLA nanoparticles has been associated with a better antibiotic efficacy than uncoated negatively charged ones. CONCLUSIONS: Correlating the carrier retention capacity in biofilms with the treatment efficacy, positively charged rifampicin-loaded PLA nanoparticles are therefore proposed as an adapted and promising approach to improve antibiotic delivery in S. aureus biofilms.