Intracellular Ca2+ signalling: unexpected new roles for the usual suspect

Cytosolic Ca2+ signals are organized in complex spatial and temporal patterns that underlie their unique ability to regulate multiple cellular functions. Changes in intracellular Ca2+ concentration ([Ca2+](i)) are finely tuned by the concerted interaction of membrane receptors and ion channels that introduce Ca2+ into the cytosol, Ca2+-dependent sensors and effectors that translate the elevation in [Ca2+](i) into a biological output, and Ca2+-clearing mechanisms that return the [Ca2+](i) to pre-stimulation levels and prevent cytotoxic Ca2+ overload. The assortment of the Ca2+ handling machinery varies among different cell types to generate intracellular Ca2+ signals that are selectively tailored to subserve specific functions. The advent of novel high-speed, 2D and 3D time-lapse imaging techniques, single-wavelength and genetic Ca2+ indicators, as well as the development of novel genetic engineering tools to manipulate single cells and whole animals, has shed novel light on the regulation of cellular activity by the Ca2+ handling machinery. A symposium organized within the framework of the 72nd Annual Meeting of the Italian Society of Physiology, held in Bari on 14-16th September 2022, has recently addressed many of the unexpected mechanisms whereby intracellular Ca2+ signalling regulates cellular fate in healthy and disease states. Herein, we present a report of this symposium, in which the following emerging topics were discussed: 1) Regulation of water reabsorption in the kidney by lysosomal Ca2+ release through Transient Receptor Potential Mucolipin 1 (TRPML1); 2) Endoplasmic reticulum-to-mitochondria Ca2+ transfer in Alzheimer's disease-related astroglial dysfunction; 3) The non-canonical role of TRP Melastatin 8 (TRPM8) as a Rap1A inhibitor in the definition of some cancer hallmarks; and 4) Non-genetic optical stimulation of Ca2+ signals in the cardiovascular system

The physics of plasma membrane photostimulation

Cell membrane perturbation is a common way to stimulate cells by using external actuators. Recently, nanotechnology has added a number of new strategies for doing this, enlarging the scope and the range of mechanisms involved. Here, we describe a number of possible perturbation actions that are driven by light, and we try to capture the underlying phenomena. The discussion is based on the simple equivalent circuit model for the cell membrane

Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation

The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC–CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal‐like hiPSC‐CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC‐CMs maturity, spanning from long‐term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.Fil: Lodola, Francesco. Università degli Studi di Milano; ItaliaFil: de Giusti, Verónica Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Maniezzi, Claudia. Università degli Studi di Milano; ItaliaFil: Martone, Daniele. Università degli Studi di Milano; ItaliaFil: Stadiotti, Ilaria. Università degli Studi di Milano; ItaliaFil: Sommariva, Elena. Università degli Studi di Milano; ItaliaFil: Maione, Angela Serena. Università degli Studi di Milano; Itali

VEGF-induced intracellular Ca2+ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells.

Endothelial colony forming cells (ECFCs) represent a population of truly endothelial precursors that promote the angiogenic switch in solid tumors, such as breast cancer (BC). The intracellular Ca2+ toolkit, which drives the pro-angiogenic response to VEGF, is remodelled in tumor-associated ECFCs such that they are seemingly insensitive to this growth factor. This feature could underlie the relative failure of anti-VEGF therapies in cancer patients. Herein, we investigated whether and how VEGF uses Ca2+ signalling to control angiogenesis in BC-derived ECFCs (BCECFCs). Although VEGFR-2 was normally expressed, VEGF failed to induce proliferation and in vitro tubulogenesis in BC-ECFCs. Likewise, VEGF did not trigger robust Ca2+ oscillations in these cells. Similar to normal cells, VEGF-induced intracellular Ca2+ oscillations were triggered by inositol-1,4,5-trisphosphate-dependent Ca2+ release from the endoplasmic reticulum (ER) and maintained by store-operated Ca2+ entry (SOCE). However, InsP3-dependent Ca2+ release was significantly lower in BC-ECFCs due to the down-regulation of ER Ca2+ levels, while there was no remarkable difference in the amplitude, pharmacological profile and molecular composition of SOCE. Thus, the attenuation of the pro-angiogenic Ca2+ response to VEGF was seemingly due to the reduction in ER Ca2+ concentration, which prevents VEGF from triggering robust intracellular Ca2+ oscillations. However, the pharmacological inhibition of SOCE prevented BC-ECFC proliferation and in vitro tubulogenesis. These findings demonstrate for the first time that BC-ECFCs are insensitive to VEGF, which might explain at cellular and molecular levels the failure of anti-VEGF therapies in BC patients, and hint at SOCE as a novel molecular target for this disease

Ca2+ dysregulation in cardiac stromal cells sustains fibro-adipose remodeling in Arrhythmogenic Cardiomyopathy and can be modulated by flecainide

BACKGROUND: Cardiac mesenchymal stromal cells (C-MSC) were recently shown to differentiate into adipocytes and myofibroblasts to promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM). A calcium (Ca(2+)) signaling dysfunction, mainly demonstrated in mouse models, is recognized as a mechanism impacting arrhythmic risk in ACM cardiomyocytes. Whether similar mechanisms influence ACM C-MSC fate is still unknown. Thus, we aim to ascertain whether intracellular Ca(2+) oscillations and the Ca(2+) toolkit are altered in human C-MSC obtained from ACM patients, and to assess their link with C-MSC-specific ACM phenotypes. METHODS AND RESULTS: ACM C-MSC show enhanced spontaneous Ca(2+) oscillations and concomitant increased Ca(2+)/Calmodulin dependent kinase II (CaMKII) activation compared to control cells. This is manly linked to a constitutive activation of Store-Operated Ca(2+) Entry (SOCE), which leads to enhanced Ca(2+) release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors. By targeting the Ca(2+) handling machinery or CaMKII activity, we demonstrated a causative link between Ca(2+) oscillations and fibro-adipogenic differentiation of ACM C-MSC. Genetic silencing of the desmosomal gene PKP2 mimics the remodelling of the Ca(2+) signalling machinery occurring in ACM C-MSC. The anti-arrhythmic drug flecainide inhibits intracellular Ca(2+) oscillations and fibro-adipogenic differentiation by selectively targeting SOCE. CONCLUSIONS: Altogether, our results extend the knowledge of Ca(2+) dysregulation in ACM to the stromal compartment, as an etiologic mechanism of C-MSC-related ACM phenotypes. A new mode of action of flecainide on a novel mechanistic target is unveiled against the fibro-adipose accumulation in ACM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-022-03742-8

PixFEL: development of an X-ray diffraction imager for future FEL applications

A readout chip for diffraction imaging applications at new generation X-ray FELs (Free Electron Lasers) has been designed in a 65 nm CMOS technology. It consists of a 32 × 32 matrix, with square pixels and a pixel pitch of 110 µm. Each cell includes a low-noise charge sensitive amplifier (CSA) with dynamic signal compression, covering an input dynamic range from 1 to 104 photons and featuring single photon resolution at small signals at energies from 1 to 10 keV. The CSA output is processed by a time-variant shaper performing gated integration and correlated double sampling. Each pixel includes also a small area, low power 10-bit time-interleaved Successive Approximation Register (SAR) ADC for in-pixel digitization of the amplitude measurement. The channel can be operated at rates up to 4.5 MHz, to be compliant with the rates foreseen for future X-ray FEL machines. The ASIC has been designed in order to be bump bonded to a slim/active edge pixel sensor, in order to build the first demonstrator for the PixFEL (advanced X-ray PIXel cameras at FELs) imager

Optical modulation of excitation-contraction coupling in human-induced pluripotent stem cell-derived cardiomyocytes

Non-genetic photostimulation is a novel and rapidly growing multidisciplinary field that aims to induce light-sensitivity in living systems by exploiting exogeneous phototransducers. Here, we propose an intramembrane photoswitch, based on an azobenzene derivative (Ziapin2), for optical pacing of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The light-mediated stimulation process has been studied by applying several techniques to detect the effect on the cell properties. In particular, we recorded changes in membrane capacitance, in membrane potential (V-m), andmodulation of intracellular Ca2+ dynamics. Finally, cell contractility was analyzed using a custom MATLAB algorithm. Photostimulation of intramembrane Ziapin2 causes a transient V-m hyperpolarization followed by a delayed depolarization and action potential firing. The observed initial electrical modulation nicely correlates with changes in Ca2+ dynamics and contraction rate. This work represents the proof of principle that Ziapin2 can modulate electrical activity and contractility in hiPSC-CMs, opening up a future development in cardiac physiology

Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation

The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC–CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal-like hiPSC-CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC-CMs maturity, spanning from long-term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.Centro de Investigaciones Cardiovasculare