Mutations in the cardiac Ryanodine Receptor gene (
) cause dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals ...exposed to emotions or physical exercise.
We investigated the efficacy of allele-specific silencing by RNA interference to prevent CPVT phenotypic manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in
.
We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant
-R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 (Ryanodine Receptor type 2 protein) function after in vivo delivery by intraperitoneal injection in neonatal and adult RyR2
(mice heterozygous for the R4496C mutation in the RyR2) heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10, the ratio between wild-type and mutant
mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit
-R4496C mRNA, whereas protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively (1) reduced isoproterenol-induced delayed afterdepolarizations and triggered activity in infected cells, (2) reduced adrenergically mediated ventricular tachycardia in treated mice, (3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and transverse tubules, and (4) attenuated mitochondrial abnormalities.
The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice, suggesting that the reduction of mutant RyR2 may be a novel therapeutic approach for CPVT.
Cardiovascular diseases are the leading cause of mortality worldwide. Such a widespread diffusion makes the conditions affecting the heart and blood vessels a primary medical and economic burden. It, ...therefore, becomes mandatory to identify effective treatments that can alleviate this global problem. Among the different solutions brought to the attention of the medical-scientific community, therapeutic angiogenesis is one of the most promising. However, this approach, which aims to treat cardiovascular diseases by generating new blood vessels in ischemic tissues, has so far led to inadequate results due to several issues. In this perspective, we will discuss cutting-edge approaches and future perspectives to alleviate the potentially lethal impact of cardiovascular diseases. We will focus on the consolidated role of resident endothelial progenitor cells, particularly endothelial colony forming cells, as suitable candidates for cell-based therapy demonstrating the importance of targeting intracellular Ca
signaling to boost their regenerative outcome. Moreover, we will elucidate the advantages of physical stimuli over traditional approaches. In particular, we will critically discuss recent results obtained by using optical stimulation, as a novel strategy to drive endothelial colony forming cells fate and its potential in the treatment of cardiovascular diseases.
Optical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. In this respect, azobenzene-based photoswitches are promising ...nanoscale tools for neuronal photostimulation. Here we engineered a light-sensitive azobenzene compound (Ziapin2) that stably partitions into the plasma membrane and causes its thinning through trans-dimerization in the dark, resulting in an increased membrane capacitance at steady state. We demonstrated that in neurons loaded with the compound, millisecond pulses of visible light induce a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. These effects are persistent and can be evoked in vivo up to 7 days, proving the potential of Ziapin2 for the modulation of membrane capacitance in the millisecond timescale, without directly affecting ion channels or local temperature.
Cytosolic Ca
2+
signals are organized in complex spatial and temporal patterns that underlie their unique ability to regulate multiple cellular functions. Changes in intracellular Ca
2+
concentration ...(Ca
2+
i
) are finely tuned by the concerted interaction of membrane receptors and ion channels that introduce Ca
2+
into the cytosol, Ca
2+
-dependent sensors and effectors that translate the elevation in Ca
2+
i
into a biological output, and Ca
2+
-clearing mechanisms that return the Ca
2+
i
to pre-stimulation levels and prevent cytotoxic Ca
2+
overload. The assortment of the Ca
2+
handling machinery varies among different cell types to generate intracellular Ca
2+
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 Ca
2+
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 Ca
2+
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 Ca
2+
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 Ca
2+
release through Transient Receptor Potential Mucolipin 1 (TRPML1); 2) Endoplasmic reticulum-to-mitochondria Ca
2+
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 Ca
2+
signals in the cardiovascular system.
Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart ...failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca
handling machinery, focusing on the available data supporting this hypothesis. The Ca
toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca
, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca
signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.
Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca
ATP-ase (SERCA2a). Heterozygous PLN p.Arg14del mutation is associated with an arrhythmogenic dilated cardiomyopathy ...(DCM), whose pathogenesis has been attributed to SERCA2a "superinhibition".
To test in cardiomyocytes (hiPSC-CMs) derived from a PLN p.Arg14del carrier whether (1) Ca
dynamics and protein localization were compatible with SERCA2a superinhibition and (2) if functional abnormalities could be reverted by pharmacological SERCA2a activation (PST3093).
Ca
transients (CaT) were recorded at 36 °C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN where immunolabeled in single hiPSC-CMs. Mutant preparations (MUT) were compared to isogenic wild-type ones (WT), obtained by mutation reversal.
WT and MUT differed for the following properties: (1) CaT time to peak (t
) and half-time of CaT decay were shorter in MUT; (2) several CaT profiles were identified in WT, "hyperdynamic" ones largely prevailed in MUT; (3) whereas t
rate-dependently declined in WT, it was shorter and rate-independent in MUT; (4) diastolic Ca
rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 turned all the above properties to resemble those of MUT; when applied to MUT, PST3093 had a smaller or negligible effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs.
Functional data converge to argue for PLN p.Arg14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, possibly including dysregulation of Ca
-dependent transcription.
Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be ...repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca
signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca
concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca
-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.
Abstract
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.
Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC ...proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca(2+) entry (SOCE), which is activated by a depletion of the intracellular Ca(2+) pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+)-sensor, Stim1, and the plasmalemmal Ca(2+) channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+) influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.
The present study employed Ca(2+) imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La(3+) and Gd(3+). Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca(2+) release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca(2+) buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.
SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.
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