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PGK1 (phosphoglycerate kinase-1) is required for ATP production in the body. Mutation in the PGK1 gene causes a rare, inherited metabolic disorder causing deficiency of enzyme PGK1, leading to hemolytic anemia, neurological symptoms, and muscle weakness. We generated induced pluripotent stem cells (iPSCs) from a patient carrying a PGK1 variant by isolating fibroblasts from skin punch biopsy and reprogramming using CytoTune iPS 2.0 Sendai reprogramming kit. The resulting iPSCs had normal karyotype, expressed pluripotent markers, and differentiated into three germ layers in vitro. The iPSC line NIMHi016-A can be used to model neuromuscular disorders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Nandeesh BN reports financial support was provided by Indian Council of Medical Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

We report the iPSC line NIMHi014-A, generated from the PBMCs of an epileptic girl child. The proband harbours a de novo, pathogenic variant of the SCN1A gene, which encodes the Na V 1.1 subtype of the voltage gated sodium channel. The cell line NIMHi014-A displayed typical iPSC-like morphology, expressed stemness markers and possessed trilineage differentiation potency. The cells presented a normal karyotype and had no mycoplasma contamination. This cell line will be used to study mechanism of epilepsy and develop patient specific therapies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Yogananda S. Markandeya reports financial support was provided by Indian Council of Medical Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper]., (Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.)

The iPSC line NIMHi013-A was generated from peripheral blood mononuclear cells of a paediatric patient with drug resistant epilepsy. The proband was found to have a likely pathogenic missense variant in the SCN1A gene in heterozygous state, which segregated in the affected in dominant fashion. The variant is in the Nav1.1 subunit of the voltage gated sodium ion channel. The iPSCs were generated using Sendai virus-based reprogramming. These iPSCs express pluripotent markers, present a normal karyotype and could differentiate into three germ layers. The iPSC line NIMHi013-A can be used to investigate epileptogenesis in vitro., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yogananda S. Markandeya reports financial support was provided by Indian Council of Medical Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Human induced pluripotent stem cells provide an exceptional platform for studying pathogenesis in vitro. We, therefore, have generated and characterized human induced pluripotent stem cell (iPSC) line NIMHi009-A derived from peripheral blood mononuclear cells (PBMCs) of healthy adult male control for an epileptic patient carrying voltage gated sodium channel mutation, using Sendai virus-based reprogramming. The generated iPSCs express pluripotency genes and can spontaneously differentiate into three germ layers. These cells display a normal karyotype and are free of mycoplasma. The iPSC line NIMHi009-A can be used as healthy control for modelling various diseases and screening for drugs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yogananda S. Markandeya reports financial support was provided by Indian Council of Medical Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In this study, we have established human induced pluripotent stem cell (hiPSC) line, NIMHi010-A of a 42-year-old healthy donor. The iPSC line was generated from human dermal fibroblasts using Sendai viruses carrying reprogramming factors c-MYC, SOX2, KLF4, and OCT4 under a feeder-free culture system. The generated hiPSC line expressed typical pluripotency markers, displayed a normal karyotype, and demonstrated the potential to differentiate into the three germ layers. This hiPSC line will serve as a healthy control model for physiological processes and drug screening of Asian origin from Indian population., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

We report the generation and characterisation of a human induced pluripotent stem cell (iPSC) line, NIMHi007-A, derived from peripheral blood mononuclear cells (PBMCs) of a healthy female adult individual. PBMCs were reprogrammed using the non-integrating Sendai virus consisting of Yamanaka reprogramming factors- SOX2, cMYC, KLF4, and OCT4. The iPSCs displayed a normal karyotype, express pluripotency markers, and could generate into three germ layers, endoderm, mesoderm, and ectoderm, in-vitro. This iPSC line, NIMHi007-A, can be used as a healthy control for various in-vitro disease models and study their underlying pathophysiological mechanisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Rationale: Flask-shaped invaginations of the cardiomyocyte sarcolemma called caveolae require the structural protein caveolin-3 (Cav-3) and host a variety of ion channels, transporters, and signaling molecules. Reduced Cav-3 expression has been reported in models of heart failure, and variants in CAV3 have been associated with the inherited long-QT arrhythmia syndrome. Yet, it remains unclear whether alterations in Cav-3 levels alone are sufficient to drive aberrant repolarization and increased arrhythmia risk., Objective: To determine the impact of cardiac-specific Cav-3 ablation on the electrophysiological properties of the adult mouse heart., Methods and Results: Cardiac-specific, inducible Cav3 homozygous knockout (Cav-3KO) mice demonstrated a marked reduction in Cav-3 expression by Western blot and loss of caveolae by electron microscopy. However, there was no change in macroscopic cardiac structure or contractile function. The QT c interval was increased in Cav-3KO mice, and there was an increased propensity for ventricular arrhythmias. Ventricular myocytes isolated from Cav-3KO mice exhibited a prolonged action potential duration (APD) that was due to reductions in outward potassium currents (I to , I ss ) and changes in inward currents including slowed inactivation of I Ca,L and increased I Na,L . Mathematical modeling demonstrated that the changes in the studied ionic currents were adequate to explain the prolongation of the mouse ventricular action potential. Results from human iPSC-derived cardiomyocytes showed that shRNA knockdown of Cav-3 similarly prolonged APD., Conclusion: We demonstrate that Cav-3 and caveolae regulate cardiac repolarization and arrhythmia risk via the integrated modulation of multiple ionic currents., Competing Interests: Declaration of Competing Interest TJK is a consultant for Fujifilm Cellular Dynamics Incorporated., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Excitotoxicity is known to associate with neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis and Huntington's disease, as well as aging, stroke, trauma, ischemia and epilepsy. Excessive release of glutamate, overactivation of glutamate receptors, calcium overload, mitochondrial dysfunction and excessive reactive oxygen species (ROS) formation are a few of the suggested key mechanisms. Astaxanthin (AST), a carotenoid, is known to act as an antioxidant and protect neurons from excitotoxic injuries. However, the exact molecular mechanism of AST neuroprotection is not clear. Thus, in this study, we investigated the role of AST in neuroprotection in excitotoxicity. We utilized primary cortical neuronal culture and live cell fluorescence imaging for the study. Our results suggest that AST prevents neuronal death, reduces ROS formation and decreases the abnormal mitochondrial membrane depolarization induced by excitotoxic glutamate insult. Additionally, AST modulates intracellular calcium levels by inhibiting peak and irreversible secondary sustained calcium levels in neurons. Furthermore, AST regulates the ionotropic glutamate subtype receptors NMDA, AMPA, KA and mitochondrial calcium. Moreover, AST decreases NMDA and AMPA receptor protein expression levels, while KA remains unaffected. Overall, our results indicate that AST protects neurons from excitotoxic neuronal injury by regulating ionotropic glutamate receptors, cytosolic secondary calcium rise and mitochondrial calcium buffering. Hence, AST could be a promising therapeutic agent against excitotoxic insults in neurodegenerative diseases.

Lead (Pb 2+ ), a ubiquitously present heavy metal toxin, has various detrimental effects on memory and cognition. However, the molecular processes affected by Pb 2+ causing structural and functional anomalies are still unclear. To explore this, we employed behavioral and proteomic approaches using rat pups exposed to lead acetate through maternal lactation from postnatal day 0 (P0) until weaning. Behavioral results from three-month-old rats clearly emphasized the early life Pb 2+ exposure induced impairments in spatial cognition. Further, proteomic analysis of synaptosomal fractions revealed differential alteration of 289 proteins, which shows functional significance in elucidating Pb 2+ induced physiological changes. Focusing on the association of Small Ubiquitin-like MOdifier (SUMO), a post-translational modification, with Pb 2+ induced cognitive abnormalities, we identified 45 key SUMO target proteins. The significant downregulation of SUMO target proteins such as metabotropic glutamate receptor 3 (GRM3), glutamate receptor isoforms 2 and 3 (GRIA 2 and GRIA3) and flotilin-1 (FLOT1) indicates SUMOylation at the synapses could contribute to and drive Pb 2+ induced physiological imbalance. These findings identify SUMOylation as a vital protein modifier with potential roles in hippocampal memory consolidation and regulation of cognition., Data Availbility: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034212&#34., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Engineering cellular microenvironment on a functional platform using various biophysical cues to modulate stem cell fate has been the central theme in regenerative engineering. Among the various biophysical cues to direct stem cell differentiation, the critical role of physiologically relevant electric field (EF) stimulation was established in the recent past. The present study is the first to report the strategy to switch EF-mediated differentiation of human mesenchymal stem cells (hMSCs) between neuronal and glial pathways, using tailored functional properties of the biomaterial substrate. We have examined the combinatorial effect of substrate functionalities (conductivity, electroactivity, and topography) on the EF-mediated stem cell differentiation on polyvinylidene-difluoride (PVDF) nanocomposites in vitro , without any biochemical inducers. The functionalities of PVDF have been tailored using conducting nanofiller (multiwall-carbon nanotube, MWNT) and piezoceramic (BaTiO 3 , BT) by an optimized processing approach (melt mixing-compression molding-rolling). The DC conductivity of PVDF nanocomposites was tuned from ∼10 -11 to ∼10 -4 S/cm and the dielectric constant from ∼10 to ∼300. The phenotypical changes and genotypical expression of hMSCs revealed the signatures of early differentiation toward neuronal pathway on rolled-PVDF/MWNT and late differentiation toward glial lineage on rolled-PVDF/BT/MWNT. Moreover, we were able to distinguish the physiological properties of differentiated neuron-like and glial-like cells using membrane depolarization and mechanical stimulation. The excitability of the EF-stimulated hMSCs was also determined using whole-cell patch-clamp recordings. Mechanistically, the roles of intracellular reactive oxygen species (ROS), Ca 2+ oscillations, and synaptic and gap junction proteins in directing the cellular fate have been established. Therefore, the present work critically unveils complex yet synergistic interaction of substrate functional properties to direct EF-mediated differentiation toward neuron-like and glial-like cells, with distinguishable electrophysiological responses.

Mitochondrial membrane potential (Ψ m ) is a critical parameter that can be used to determine cellular well-being. As it is a direct measure of the cell's ATP generating capability, in recent years, this key component in cell biology has been the subject of thousands of biochemical and biophysical investigations. Membrane-permeant fluorescent dyes, like tetramethylrhodamine ethyl ester (TMRE), have been predominantly employed to monitor ΔΨ m in cells. These dyes are typically lipophilic cationic compounds that equilibrate across membranes in a Nernstian fashion, thus accumulating into the mitochondrial membrane matrix space in inverse proportion to Ψ m . However, the bath loading method practiced for labelling tissue slices with these cationic dyes poses limitations in the form of non-specificity and low signal to noise ratio, which compromises the precision of the results. Therefore, we introduce an alternative way for TMRE loading to image the ΔΨ m in tissue slices by utilizing a low resistance glass pipette attached to a pressure injector. This method shows highly precise fluorescent dye labelling of the mitochondria and offers maximum output intensity, in turn enhancing signal to noise ratio., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Native myocardium has limited regenerative potential post injury. Advances in lineage reprogramming have provided promising cellular sources for regenerative medicine in addition to research applications. Recently we have shown that adult mouse fibroblasts can be reprogrammed to expandable, multipotent, induced cardiac progenitor cells (iCPCs) by employing forced expression of five cardiac factors along with activation of canonical Wnt and JAK/STAT signaling. Here we aim to further characterize iCPCs by highlighting their safety, ease of attainability, and functionality within a three-dimensional cardiac extracellular matrix scaffold. Specifically, iCPCs did not form teratomas in contrast to embryonic stem cells when injected into immunodeficient mice. iCPC reprogramming was achieved in wild type mouse fibroblasts without requiring a cardiac-specific reporter, solely utilizing morphological changes to identify, clonally isolate, and expand iCPCs, thus increasing the versatility of this technology. iCPCs also show the ability to repopulate decellularized native heart scaffolds and differentiated into organized structures containing cardiomyocytes, smooth muscle, and endothelial cells. Optical mapping of recellularized scaffolds shows field-stimulated calcium transients that propagate across islands of reconstituted tissue and bipolar local stimulation demonstrates cell-cell coupling within scaffolds. Overall, iCPCs provide a readily attainable, scalable, safe, and functional cell source for a variety of application including drug discovery, disease modeling, and regenerative therapy., Competing Interests: Declaration of competing interest TJK is a consultant for FujiFilm Cellular Dynamics Inc. RAA is a consultant for Cell Reprogramming and Therapeutics LLC. Pending patent applications are related to iCPC generation (TJK, PAL). EGS and ANR have ownership in Cellular Logistics Inc., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A number of bioengineering strategies, using biophysical stimulation, are being explored to guide the human mesenchymal stem cells (hMScs) into different lineages. In this context, we have limited understanding on the transdifferentiation of matured cells to another functional-cell type, when grown with stem cells, in a constrained cellular microenvironment under biophysical stimulation. While addressing such aspects, the present work reports the influence of the electric field (EF) stimulation on the phenotypic and functionality modulation of the coculture of murine myoblasts (C2C12) with hMScs [hMSc:C2C12=1:10] in a custom designed polymethylmethacrylate (PMMA) based microfluidic device with in-built metal electrodes. The quantitative and qualitative analysis of the immunofluorescence study confirms that the cocultured cells in the conditioned medium with astrocytic feed, exhibit differentiation towards neural-committed cells under biophysical stimulation in the range of the endogenous physiological electric field strength (8 ± 0.06  mV/mm). The control experiments using similar culture protocols revealed that while C2C12 monoculture exhibited myotube-like fused structures, the hMScs exhibited the neurosphere-like clusters with SOX2, nestin, βIII-tubulin expression. The electrophysiological study indicates the significant role of intercellular calcium signalling among the differentiated cells towards transdifferentiation. Furthermore, the depolarization induced calcium influx strongly supports neural-like behaviour for the electric field stimulated cells in coculture. The intriguing results are explained in terms of the paracrine signalling among the transdifferentiated cells in the electric field stimulated cellular microenvironment. In summary, the present study establishes the potential for neurogenesis on-chip for the coculture of hMSc and C2C12 cells under tailored electric field stimulation, in vitro., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

While much progress has been made in the resolution of the cellular hierarchy underlying cardiogenesis, our understanding of chamber-specific myocardium differentiation remains incomplete. To better understand ventricular myocardium differentiation, we targeted the ventricle-specific gene, Irx4, in mouse embryonic stem cells to generate a reporter cell line. Using an antibiotic-selection approach, we purified Irx4 + cells in vitro from differentiating embryoid bodies. The isolated Irx4 + cells proved to be highly proliferative and presented Cxcr4, Pdgfr-alpha, Flk1, and Flt1 on the cell surface. Single Irx4 + ventricular progenitor cells (VPCs) exhibited cardiovascular potency, generating endothelial cells, smooth muscle cells, and ventricular myocytes in vitro. The ventricular specificity of the Irx4 + population was further demonstrated in vivo as VPCs injected into the cardiac crescent subsequently produced Mlc2v + myocytes that exclusively contributed to the nascent ventricle at E9.5. These findings support the existence of a newly identified ventricular myocardial progenitor. This is the first report of a multipotent cardiac progenitor that contributes progeny specific to the ventricular myocardium. Stem Cells 2016;34:2875-2888., (© 2016 AlphaMed Press.)

Background: Lamin A and C are nuclear filament proteins encoded by the LMNA gene. Mutations in the LMNA gene cause many congenital diseases known as laminopathies, including Emery-Dreifuss muscular dystrophy, Hutchinson-Gilford progeria syndrome, and familial dilated cardiomyopathy (DCM) with conduction disease. A missense mutation (N195K) in the A-type lamins results in familial DCM and sudden arrhythmic death., Objective: The purpose of this study was to investigate the ion current mechanism of arrhythmia and DCM caused by the LaminA-N195K variant., Methods: A homozygous mouse line expressing the Lmna-N195K mutation (Lmna N195K/N195K ) that exhibited arrhythmia, DCM, and sudden death was used. Using whole cell patch-clamp technique, we measured action potential duration (APD), Na + currents (I Na ) in ventricular myocytes isolated from Lmna N195K/N195K , and wild-type mice., Results: Both peak and late I Na were significantly (P <.05) increased in Lmna N195K/N195K ventricular myocytes. Similarly, Lmna N195K/N195K ventricular myocytes exhibited significant (P <.005) prolongation of APD (time to 50% [APD 50 ] and 90% [APD 90 ] repolarization) and triggered activity. Acute application of ranolazine inhibited late I Na , shortened APD, and abolished triggered activity in Lmna N195K/N195K ventricular myocytes., Conclusion: Inhibition of late I Na may be an effective therapy in preventing arrhythmia in patients with LmnaN195K mutation-related DCM., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently available induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) do not ideally model cellular mechanisms of human arrhythmic disease due to lack of a mature action potential (AP) phenotype. In this study, we create and characterize iPS-CMs with an electrically mature AP induced by potassium inward rectifier (IK1) enhancement. The advantages of IK1-enhanced iPS-CMs include the absence of spontaneous beating, stable resting membrane potentials at approximately -80 mV and capability for electrical pacing. Compared with unenhanced, IK1-enhanced iPS-CMs calcium transient amplitudes were larger (P < 0.05) with a typical staircase pattern. IK1-enhanced iPS-CMs demonstrated a twofold increase in cell size and membrane capacitance and increased DNA synthesis compared with control iPS-CMs (P < 0.05). Furthermore, IK1-enhanced iPS-CMs expressing the F97C-CAV3 long QT9 mutation compared with wild-type CAV3 demonstrated an increase in AP duration and late sodium current. IK1-enhanced iPS-CMs represent a more mature cardiomyocyte model to study arrhythmia mechanisms., (Copyright © 2016 the American Physiological Society.)

Several studies have reported reprogramming of fibroblasts into induced cardiomyocytes; however, reprogramming into proliferative induced cardiac progenitor cells (iCPCs) remains to be accomplished. Here we report that a combination of 11 or 5 cardiac factors along with canonical Wnt and JAK/STAT signaling reprogrammed adult mouse cardiac, lung, and tail tip fibroblasts into iCPCs. The iCPCs were cardiac mesoderm-restricted progenitors that could be expanded extensively while maintaining multipotency to differentiate into cardiomyocytes, smooth muscle cells, and endothelial cells in vitro. Moreover, iCPCs injected into the cardiac crescent of mouse embryos differentiated into cardiomyocytes. iCPCs transplanted into the post-myocardial infarction mouse heart improved survival and differentiated into cardiomyocytes, smooth muscle cells, and endothelial cells. Lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for drug discovery, disease modeling, and cardiac regenerative therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca(2+) cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca(2+) signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca(2+) current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.