Eavesdropping on the social lives of Ca(2+) sparks

Ca²⁺ sparks arise from the stochastic opening of spatially discrete clusters of ryanodine receptors called a Ca²⁺ release unit (CRU). If the RyR clusters were not spatially separated, then Ca²⁺ released from one RyR would immediately diffuse to its neighbor and lead to uncontrolled, runaway Ca²⁺ release throughout the cell. While physical separation provides some isolation from neighbors, CRUs are not incommunicado. When inter-neighbor interactions become large enough, Ca²⁺ waves spontaneously emerge. A more circumscribed interaction shows up in high-speed two-dimensional confocal images as jumping Ca²⁺ sparks that seem to be sequentially activated along the Z-line and across Z-lines. However, since Ca²⁺ sparks are stochastic events how can we tell whether two sparks occurring close together in space and time are causally related or appeared simply by coincidence? Here we develop a mathematical method to disentangle cause and coincidence in a statistical sense. From our analysis we derive three fundamental properties of Ca²⁺ spark generation: 1), the “intrinsic” spark frequency, the spark frequency one would observe if the CRUs were incommunicado; 2), the coupling strength, which measures how strongly one CRU affects another; and 3), the range over which the communication occurs. These parameters allow us to measure the effect RyR regulators have on the intrinsic activity of CRUs and on the coupling between them.     

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP?/? Mice

Background

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.

Principal Findings

We used intact olfactory epithelium obtained from WT and OMP^−/− mice to monitor the Ca²⁺ dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca²⁺ channels, or Ca²⁺ stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca²⁺-homeostasis in these neurons by influencing the activity of the plasma membrane Na⁺/Ca²⁺-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca²⁺ elevation by stimulating the reverse mode of NCX in both WT and OMP^−/− mice. The resulting Ca²⁺ responses indicate that OMP facilitates NCX activity and allows rapid Ca²⁺ extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).

Conclusions

Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions.     

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes

Background

The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca²⁺-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/Principal Findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca²⁺-handling proteins. Detailed laser confocal Ca²⁺ imaging demonstrated spontaneous whole-cell [Ca²⁺]_i transients. These transients required Ca²⁺ influx via L-type Ca²⁺ channels, as demonstrated by their elimination in the absence of extracellular Ca²⁺ or by administration of the L-type Ca²⁺ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca²⁺ store, contributing to [Ca²⁺]_i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca²⁺) and ryanodine (decreasing [Ca²⁺]_i). Similarly, the importance of Ca²⁺ reuptake into the SR via the SR Ca²⁺ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca²⁺]_i transients elimination. Finally, the presence of an IP3-releasable Ca²⁺ pool in hiPSC-CMs and its contribution to whole-cell [Ca²⁺]_i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusions/Significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca²⁺ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca²⁺]_i transients in hiPSC-CMs on both sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and intracellular store Ca²⁺ release.     

Inhibitory control over Ca(2+) sparks via mechanosensitive channels is disrupted in dystrophin deficient muscle but restored by mini-dystrophin expression

Background

In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca²⁺ release events (ECRE) in high Ca²⁺ external environments. Such ‘uncontrolled’ Ca²⁺ sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition.

Methodology/Principal Findings

We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca²⁺ concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 µM), spider peptide GsMTx-4 (5 µM) and Gd³⁺ (20 µM) that block unspecific, specific cationic and Ca²⁺ selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation.

Conclusions/Significance

Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca²⁺ homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

PI3Ks maintain the structural integrity of T-tubules in cardiac myocytes

Background

Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.

Methods and Results

Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca²⁺ channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca²⁺ transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.

Conclusions

PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca²⁺-induced Ca²⁺ release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.     

Enhancement effects of martentoxin on glioma BK channel and BK channel (α+β1) subtypes

Background

BK channels are usually activated by membrane depolarization and cytoplasmic Ca²⁺. Especially,the activity of BK channel (α+β4) can be modulated by martentoxin, a 37 residues peptide, with Ca²⁺-dependent manner. gBK channel (glioma BK channel) and BK channel (α+β1) possessed higher Ca²⁺ sensitivity than other known BK channel subtypes.

Methodology and Principal Findings

The present study investigated the modulatory characteristics of martentoxin on these two BK channel subtypes by electrophysiological recordings, cell proliferation and Ca²⁺ imaging. In the presence of cytoplasmic Ca²⁺, martentoxin could enhance the activities of both gBK and BK channel (α+β1) subtypes in dose-dependent manner with EC₅₀ of 46.7 nM and 495 nM respectively, while not shift the steady-state activation of these channels. The enhancement ratio of martentoxin on gBK and BK channel (α+β1) was unrelated to the quantitive change of cytoplasmic Ca²⁺ concentrations though the interaction between martentoxin and BK channel (α+β1) was accelerated under higher cytoplasmic Ca²⁺. The selective BK pore blocker iberiotoxin could fully abolish the enhancement of these two BK subtypes induced by martentoxin, suggesting that the auxiliary β subunit might contribute to the docking for martentoxin. However, in the absence of cytoplasmic Ca²⁺, the activity of gBK channel would be surprisingly inhibited by martentoxin while BK channel (α+β1) couldn''t be affected by the toxin.

Conclusions and Significance

Thus, the results shown here provide the novel evidence that martentoxin could increase the two Ca²⁺-hypersensitive BK channel subtypes activities in a new manner and indicate that β subunit of these BK channels plays a vital role in this enhancement by martentoxin.     

Differential regulation and recovery of intracellular Ca2+ in cerebral and small mesenteric arterial smooth muscle cells of simulated microgravity rat

Background

The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca²⁺ determined by the alterations in the functions of plasma membrane Ca_L channels and ryanodine-sensitive Ca²⁺ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.

Methodology/Principal Findings

Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of Ca_L channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca²⁺ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of Ca_L channels and ryanodine-sensitive Ca²⁺ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.

Conclusions

The differential regulation of Ca_L channels and ryanodine-sensitive Ca²⁺ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca²⁺, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.     

Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery

Background

The rate-limiting step that determines the dominant time constant (τ_D) of mammalian rod photoresponse recovery is the deactivation of the active phosphodiesterase (PDE6). Physiologically relevant Ca²⁺-dependent mechanisms that would affect the PDE inactivation have not been identified. However, recently it has been shown that τ_D is modulated by background light in mouse rods.

Methodology/Principal Findings

We used ex vivo ERG technique to record pharmacologically isolated photoreceptor responses (fast PIII component). We show a novel static effect of calcium on mouse rod phototransduction: Ca²⁺ shortens the dominant time constant (τ_D) of saturated photoresponse recovery, i.e., when extracellular free Ca²⁺ is decreased from 1 mM to ∼25 nM, the τ_D is reversibly increased ∼1.5–2-fold.

Conclusions

We conclude that the increase in τ_D during low Ca²⁺ treatment is not due to increased [cGMP], increased [Na⁺] or decreased [ATP] in rod outer segment (ROS). Also it cannot be due to protein translocation mechanisms. We suggest that a Ca²⁺-dependent mechanism controls the life time of active PDE.     

Fructose modulates cardiomyocyte excitation-contraction coupling and Ca²⁺ handling in vitro

Background

High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated.

Objective

The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting.

Methods and Results

The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca²⁺ handling were evaluated under physiological conditions (37°C, 2 mM Ca²⁺, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca²⁺ transient delay induced by exposure to 2 DG (time to peak Ca²⁺ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR.

Conclusion

This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function.     

Carbenoxolone blocks the light-evoked rise in intracellular calcium in isolated melanopsin ganglion cell photoreceptors

Background

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca²⁺ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca²⁺ signals in ipRGCs independent of gap junction blockade.

Methodology/Principal Findings

To test the possibility that carbenoxolone directly inhibits light-evoked Ca²⁺ responses in ipRGCs, the light-evoked rise in intracellular Ca²⁺ ([Ca²⁺]_i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca²⁺]_i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.

Conclusions/Significance

We demonstrate that the light-evoked rise in [Ca²⁺]_i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca²⁺]_i in isolated ipRGCs is almost entirely due to Ca²⁺ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca²⁺]_i in ipRGCs by blocking L-type voltage-gated Ca²⁺ channels. The ability of carbenoxolone to block evoked Ca²⁺ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca²⁺]_i is the output being measured.     

Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and S-glutathionylation of nitric oxide synthetase

Background

PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood.

Methodology/Principal Findings

We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca²⁺ release and causing auto-regulation of eNOS through S-glutathionylation.

Conclusions/Significance

The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca²⁺ flux and calmodulin (CaM) activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNOS activation.     

Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin

Background

Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood.

Methodology/Principal Findings

Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca²⁺/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca²⁺/CaM formed a 1∶1 complex with an equilibrium association constant around 10⁵ M⁻¹, whereas no binding reaction of K-RasB-(DESGPC) with Ca²⁺/CaM is detected. Furthermore, the interaction of K-RasB with Ca²⁺/CaM is found to be enhanced by the farnesylation of K-RasB.

Conclusions/Significance

We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca²⁺/CaM, but also defines its isoform specific interaction with Ca²⁺/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca²⁺/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.     

Fibro-vascular coupling in the control of cochlear blood flow

Background

Transduction of sound in the cochlea is metabolically demanding. The lateral wall and hair cells are critically vulnerable to hypoxia, especially at high sound levels, and tight control over cochlear blood flow (CBF) is a physiological necessity. Yet despite the importance of CBF for hearing, consensus on what mechanisms are involved has not been obtained.

Methodology/Principal Findings

We report on a local control mechanism for regulating inner ear blood flow involving fibrocyte signaling. Fibrocytes in the super-strial region are spatially distributed near pre-capillaries of the spiral ligament of the albino guinea pig cochlear lateral wall, as demonstrably shown in transmission electron microscope and confocal images. Immunohistochemical techniques reveal the inter-connected fibrocytes to be positive for Na+/K+ ATPase β1 and S100. The connected fibrocytes display more Ca²⁺ signaling than other cells in the cochlear lateral wall as indicated by fluorescence of a Ca²⁺ sensor, fluo-4. Elevation of Ca²⁺ in fibrocytes, induced by photolytic uncaging of the divalent ion chelator o-nitrophenyl EGTA, results in propagation of a Ca²⁺ signal to neighboring vascular cells and vasodilation in capillaries. Of more physiological significance, fibrocyte to vascular cell coupled signaling was found to mediate the sound stimulated increase in cochlear blood flow (CBF). Cyclooxygenase-1 (COX-1) was required for capillary dilation.

Conclusions/Significance

The findings provide the first evidence that signaling between fibrocytes and vascular cells modulates CBF and is a key mechanism for meeting the cellular metabolic demand of increased sound activity.     

Calpain-catalyzed proteolysis of human dUTPase specifically removes the nuclear localization signal peptide

Background

Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca²⁺-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.

Methodology/Principal Findings

Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca²⁺-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca²⁺-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues ⁴SE⁵; ⁷TP⁸; and ³¹LS³²). The cleavage between the ³¹LS³² peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.

Conclusions/Significance

Results argue for a mechanism where Ca²⁺-calpain may regulate nuclear availability and degradation of dUTPase.     

Bisphenol A and 17β-estradiol promote arrhythmia in the female heart via alteration of calcium handling

Background

There is wide-spread human exposure to bisphenol A (BPA), a ubiquitous estrogenic endocrine disruptor that has been implicated as having potentially harmful effects on human heart health. Higher urine BPA concentrations have been shown to be associated with cardiovascular diseases in humans. However, neither the nature nor the mechanism(s) of BPA action on the heart are understood.

Methodology/Principal Findings

The rapid (<7 min) effects of BPA and 17β-estradiol (E2) in the heart and ventricular myocytes from rodents were investigated in the present study. In isolated ventricular myocytes from young adult females, but not males, physiological concentrations of BPA or E2 (10⁻⁹ M) rapidly induced arrhythmogenic triggered activities. The effects of BPA were particularly pronounced when combined with estradiol. Under conditions of catecholamine stimulation, E2 and BPA promoted ventricular arrhythmias in female, but not male, hearts. The cellular mechanism of the female-specific pro-arrhythmic effects of BPA and E2 were investigated. Exposure to E2 and/or BPA rapidly altered myocyte Ca²⁺ handling; in particular, estrogens markedly increased sarcoplasmic reticulum (SR) Ca²⁺ leak, and increased SR Ca²⁺ load. Ryanodine (10⁻⁷ M) inhibition of SR Ca²⁺ leak suppressed estrogen-induced triggered activities. The rapid response of female myocytes to estrogens was abolished in an estrogen receptor (ER) β knockout mouse model.

Conclusions/Significance

Physiologically-relevant concentrations of BPA and E2 promote arrhythmias in a female-specific manner in rat hearts; the pro-arrhythmic actions of estrogens are mediated by ERβ-signaling through alterations of myocyte Ca²⁺ handling, particularly increases in SR Ca²⁺ leak. Our study provides the first experimental evidence suggesting that exposure to estrogenic endocrine disrupting chemicals and the unique sensitivity of female hearts to estrogens may play a role in arrhythmogenesis in the female heart.     

Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells

Background

The present study investigates the effects of high external calcium concentration ([Ca²⁺]_o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.

Methodology/Principal Findings

A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca²⁺]_o (0.37 mM); 2) high [Ca²⁺]_o (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca²⁺]_o and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca²⁺]_o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca²⁺]_o was not effective in this cell line. In small cells, both higher [Ca²⁺]_o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca²⁺]_o and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.

Conclusions/Significance

In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.     

Receptor Activated Ca2+ Release Is Inhibited by Boric Acid in Prostate Cancer Cells

Background

The global disparity in cancer incidence remains a major public health problem. We focused on prostate cancer since microscopic disease in men is common, but the incidence of clinical disease varies more than 100 fold worldwide. Ca²⁺ signaling is a central regulator of cell proliferation, but has received little attention in cancer prevention. We and others have reported a strong dose-dependent reduction in the incidence of prostate and lung cancer within populations exposed to boron (B) in drinking water and food; and in tumor and cell proliferation in animal and cell culture models.

Methods/Principal Findings

We examined the impact of B on Ca²⁺ stores using cancer and non-cancer human prostate cell lines, Ca²⁺ indicators Rhod-2 AM and Indo-1 AM and confocal microscopy. In DU-145 cells, inhibition of Ca²⁺ release was apparent following treatment with Ringers containing RyR agonists cADPR, 4CmC or caffeine and respective levels of BA (50 µM), (1, 10 µM) or (10, 20, 50,150 µM). Less aggressive LNCaP cancer cells required 20 µM BA and the non-tumor cell line PWR1E required 150 µM BA to significantly inhibit caffeine stimulated Ca²⁺ release. BA (10 µM) and the RyR antagonist dantroline (10 µM) were equivalent in their ability to inhibit ER Ca²⁺ loss. Flow cytometry and confocal microscopy analysis showed exposure of DU-145 cells to 50 µM BA for 1 hr decreased stored [Ca²⁺] by 32%.

Conclusion/Significance

We show B causes a dose dependent decrease of Ca²⁺ release from ryanodine receptor sensitive stores. This occurred at BA concentrations present in blood of geographically disparate populations. Our results suggest higher BA blood levels lower the risk of prostate cancer by reducing intracellular Ca²⁺ signals and storage.     

Heart failure-inducible gene therapy targeting protein phosphatase 1 prevents progressive left ventricular remodeling

Background

The targeting of Ca²⁺ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca²⁺ ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca²⁺ uptake in the SR among the three PP1 isoforms, thereby contributing to Ca²⁺ downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice.

Methods

We created an adeno-associated virus 9 (AAV9) vector encoding PP1β short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×10¹¹ GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months.

Results

In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months.

Conclusion

Heart failure-inducible molecular targeting of PP1β has potential as a novel therapeutic strategy for heart failure.