Potassium Channel KCNH1 Activating Variants Cause Altered Functional and Morphological Ciliogenesis.

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP_002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging.

In vivoorganized neovascularization induced by 3D bioprinted endothelial-derived extracellular vesicles.

Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability to mediate intercellular communication. This feature has been explored and is under constant investigation by researchers, who have demonstrated the important role of EVs in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limitations to overcome before clinical application, including the inability to confine the EVs to strategically defined sites of interest to avoid side effects. In this study, for the first time, EV application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of human umbilical vein endothelial cell-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bioadditives for the formulation of advanced bioinks. Afterin vivosubcutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculaturein situ, consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favour the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischaemic tissues, which represent the fundamental substrate for advanced regenerative medicine applications.

Intermittent ß-adrenergic blockade downregulates the gene expression of ß-myosin heavy chain in the mouse heart.

Expression of the ß-myosin heavy chain (ß-MHC), a major component of the cardiac contractile apparatus, is tightly regulated as even modest increases can be detrimental to heart under stress. In healthy hearts, continuous inhibition of ß-adrenergic tone upregulates ß-MHC expression. However, it is unknown whether the duration of the ß-adrenergic inhibition and ß-MHC expression are related. Here, we evaluated the effects of intermittent ß-blockade on cardiac ß-MHC expression. To this end, the ß-blocker propranolol, at the dose of 15mg/kg, was administered once a day in mice for 14 days. This dosing schedule caused daily drug-free periods of at least 6 h as evidenced by propranolol plasma concentrations and cardiac ß-adrenergic responsiveness. Under these conditions, ß-MHC expression decreased by about 75% compared to controls. This effect was abolished in mice lacking ß1- but not ß2-adrenergic receptors (ß-AR) indicating that ß-MHC expression is regulated in a ß1-AR-dependent manner. In ß1-AR knockout mice, the baseline ß-MHC expression was fourfold higher than in wild-type mice. Also, we evaluated the impact of intermittent ß-blockade on ß-MHC expression in mice with systolic dysfunction, in which an increased ß-MHC expression occurs. At 3 weeks after myocardial infarction, mice showed systolic dysfunction and upregulation of ß-MHC expression. Intermittent ß-blockade decreased ß-MHC expression while attenuating cardiac dysfunction. In vitro studies showed that propranolol does not affect ß-MHC expression on its own but antagonizes catecholamine effects on ß-MHC expression. In conclusion, a direct relationship occurs between the duration of the ß-adrenergic inhibition and ß-MHC expression through the ß1-AR.

Extracellular Vesicles from Skeletal Muscle Cells Efficiently Promote Myogenesis in Induced Pluripotent Stem Cells.

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological "shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

A multi-cellular 3D bioprinting approach for vascularized heart tissue engineering based on HUVECs and iPSC-derived cardiomyocytes.

The myocardium behaves like a sophisticated orchestra that expresses its true potential only if each member performs the correct task harmonically. Recapitulating its complexity within engineered 3D functional constructs with tailored biological and mechanical properties, is one of the current scientific priorities in the field of regenerative medicine and tissue engineering. In this study, driven by the necessity of fabricating advanced model of cardiac tissue, we present an innovative approach consisting of heterogeneous, multi-cellular constructs composed of Human Umbilical Vein Endothelial Cells (HUVECs) and induced pluripotent cell-derived cardiomyocytes (iPSC-CMs). Cells were encapsulated within hydrogel strands containing alginate and PEG-Fibrinogen (PF) and extruded through a custom microfluidic printing head (MPH) that allows to precisely tailor their 3D spatial deposition, guaranteeing a high printing fidelity and resolution. We obtained a 3D cardiac tissue compose of iPSC-derived CMs with a high orientation index imposed by the different defined geometries and blood vessel-like shapes generated by HUVECs which, as demonstrated by in vivo grafting, better support the integration of the engineered cardiac tissue with host's vasculature.

Givinostat reduces adverse cardiac remodeling through regulating fibroblasts activation.

Cardiovascular diseases (CVDs) are a major burden on the healthcare system: indeed, over two million new cases are diagnosed every year worldwide. Unfortunately, important drawbacks for the treatment of these patients derive from our current inability to stop the structural alterations that lead to heart failure, the common endpoint of many CVDs. In this scenario, a better understanding of the role of epigenetics - hereditable changes of chromatin that do not alter the DNA sequence itself - is warranted. To date, hyperacetylation of histones has been reported in hypertension and myocardial infarction, but the use of inhibitors for treating CVDs remains limited. Here, we studied the effect of the histone deacetylase inhibitor Givinostat on a mouse model of acute myocardial infarction. We found that it contributes to decrease endothelial-to-mesenchymal transition and inflammation, reducing cardiac fibrosis and improving heart performance and protecting the blood vessels from apoptosis through the modulatory effect of cardiac fibroblasts on endothelial cells. Therefore, Givinostat may have potential for the treatment of CVDs.