Intracellular Calcium Transients Research Articles

Electrical stimulation prevents doxorubicin-induced atrophy and mitochondrial loss in cultured myotubes.

Muscle contraction may protect against the effects of chemotherapy to cause skeletal muscle atrophy, but the mechanisms underlying these benefits are unclear. To address this question, we utilized in vitro modeling of contraction and mechanotransduction in C2C12 myotubes treated with doxorubicin (DOX; 0.2 μM for 3 days). Myotubes expressed contractile proteins and organized these into functional myofilaments, as electrical field stimulation (STIM) induced intracellular calcium (Ca2+) transients and contractions, both of which were prevented by inhibition of membrane depolarization. DOX treatment reduced myotube myosin content, protein synthesis, and Akt (S308) and forkhead box O3a (FoxO3a; S253) phosphorylation and increased muscle RING finger 1 (MuRF1) expression. STIM (1 h/day) prevented DOX-induced reductions in myotube myosin content and Akt and FoxO3a phosphorylation, as well as increases in MuRF1 expression, but did not prevent DOX-induced reductions in protein synthesis. Inhibition of myosin-actin interaction during STIM prevented contraction and the antiatrophic effects of STIM without affecting Ca2+ cycling, suggesting that the beneficial effect of STIM derives from mechanotransductive pathways. Further supporting this conclusion, mechanical stretch of myotubes recapitulated the effects of STIM to prevent DOX suppression of FoxO3a phosphorylation and upregulation of MuRF1. DOX also increased reactive oxygen species (ROS) production, which led to a decrease in mitochondrial content. Although STIM did not alter DOX-induced ROS production, peroxisome proliferator-activated receptor-γ coactivator-1α and antioxidant enzyme expression were upregulated, and mitochondrial loss was prevented. Our results suggest that the activation of mechanotransductive pathways that downregulate proteolysis and preserve mitochondrial content protects against the atrophic effects of chemotherapeutics.

Absence of gravin-mediated signaling inhibits development of high-fat diet-induced hyperlipidemia and atherosclerosis.

Gravin, an A-kinase anchoring protein, is known to play a role in regulating key processes that lead to inflammation and atherosclerosis development, namely, cell migration, proliferation, and apoptosis. We investigated the role of gravin in the development of high-fat diet (HFD)-induced atherosclerosis and hyperlipidemia. Five-week-old male wild-type (WT) and gravin-t/t mice were fed a normal diet or an HFD for 16 wk. Gravin-t/t mice showed significantly lower liver-to-body-weight ratio, cholesterol, triglyceride, and very low-density lipoprotein levels in serum as compared with WT mice on HFD. Furthermore, there was less aortic plaque formation coupled with decreased lipid accumulation and liver damage, as the gravin-t/t mice had lower levels of serum alanine aminotransferase and aspartate aminotransferase. Additionally, gravin-t/t HFD-fed mice had decreased expression of liver 3-hydroxy-3-methyl-glutaryl-CoA reductase, an essential enzyme for cholesterol synthesis and lower fatty acid synthase expression. Gravin-t/t HFD-fed mice also exhibited inhibition of sterol regulatory element binding protein-2 (SREBP-2) expression, a liver transcription factor associated with the regulation of lipid transportation. In response to platelet-derived growth factor receptor treatment, gravin-t/t vascular smooth muscle cells exhibited lower intracellular calcium transients and decreased protein kinase A- and protein kinase C-dependent substrate phosphorylation, notably involving the Erk1/2 signaling pathway. Collectively, these results suggest the involvement of gravin-dependent regulation of lipid metabolism via the reduction of SREBP-2 expression. The absence of gravin-mediated signaling lowers blood pressure, reduces plaque formation in the aorta, and decreases lipid accumulation and damage in the liver of HFD mice. Through these processes, the absence of gravin-mediated signaling complex delays the HFD-induced hyperlipidemia and atherosclerosis.NEW & NOTEWORTHY The gravin scaffolding protein plays a key role in the multiple enzymatic pathways of lipid metabolism. We have shown for the first time the novel role of gravin in regulating the pathways related to the initiation and progression of atherosclerosis. Specifically, an absence of gravin-mediated signaling decreases the lipid levels (cholesterol, triglyceride, and VLDL) that are associated with sterol regulatory element binding protein-2 downregulation.

Abstract 333: Cardiomyocyte Hypercontractility is an Adaptational Response to Stiffness in High-Fat Diet Mice

Diabetic heart disease typically features a restrictive/ hypertrophic cardiac phenotype characterised by diastolic dysfunction with preserved systolic performance. Little is known about the early adaptational mechanisms that underpin maintenance of systolic performance in diabetic diastolic dysfunction and how this may contribute to disease progression. This study aimed to evaluate cardiomyocyte contractility in a high-fat diet (HFD) mouse model of diastolic dysfunction. Echocardiography (GE Vivid 9) was performed in 33 wk old male C57Bl/6 mice fed a high-fat diet (HFD, 43% kcal fat, 24 wks duration). Isolated cardiomyocytes (paced 2Hz, 2.0mM Ca 2+ , 37C) were subjected to progressive axial stretch. Sarcomere length/ shortening, tension and intracellular calcium transients (Fura-2AM, 5μM) were simultaneously measured (Myostretcher, Ionoptix). HFD hearts displayed in vivo (22.2±1.4 vs 16.7±1.0 E/E’; p<0.05) and in vitro diastolic dysfunction (0.244±0.039 vs 0.075±0.017 nN/pl/ %cell stretch; p<0.05). HFD cardiomyocytes exhibited length-dependent hypercontractility with augmented developed tension amplitude at maximal stretch (4.891±0.942 vs 2.484±0.677 nN/pl; p<0.05), but not basal stretch. Hypercontractility could not be attributed to a difference in Ca 2+ transient amplitude but was correlated with length dependence of Ca 2+ sensitivity (n=17 cells, R 2 =0.4039 p<0.05). During progressive cardiomyocyte stretch, systolic tension slopes were strongly correlated with diastolic tension slopes (cardiomyocyte stiffness) (n=30 cells; R 2 =0.8442, p<0.05) while there was no difference in the ratio of systolic/diastolic tension slopes indicating maintained length-dependent activation. These findings provide the first evidence that cardiomyocyte hypercontractility and stiffness are strongly linked. High-fat diet cardiomyocytes may operate near maximal contractile capacity to maintain basal cardiac output, potentially limiting capacity for cardiac output increase in response to physiological stressors. An aberrant myofilament post-translational modification profile represents a potential mechanism through which cardiac dysfunction arises during the development of diabetes.

Interpreting in vivo calcium signals from neuronal cell bodies, axons, and dendrites: a review.

.Calcium imaging is emerging as a popular technique in neuroscience. A major reason is that intracellular calcium transients are reflections of electrical events in neurons. For example, calcium influx in the soma and axonal boutons accompanies spiking activity, whereas elevations in dendrites and dendritic spines are associated with synaptic inputs and local regenerative events. However, calcium transients have complex spatiotemporal dynamics, and since most optical methods visualize only one of the somatic, axonal, and dendritic compartments, a straightforward inference of the underlying electrical event is typically challenging. We highlight experiments that have directly calibrated in vivo calcium signals recorded using fluorescent indicators against electrophysiological events. We address commonly asked questions such as: Can calcium imaging be used to characterize neurons with high firing rates? Can the fluorescent signal report a decrease in spiking activity? What is the evidence that calcium transients in subcellular compartments correspond to distinct presynaptic axonal and postsynaptic dendritic events? By reviewing the empirical evidence and limitations, we suggest that, despite some caveats, calcium imaging is a versatile method to characterize a variety of neuronal events in vivo.

Spontaneous and Acetylcholine Evoked Calcium Transients in the Developing Mouse Utricle.

Spontaneous calcium transients are present during early postnatal development in the mouse retina and cochlea, and play an important role in maturation of the sensory organs and neural circuits in the central nervous system (CNS). It is not known whether similar calcium transients occur during postnatal development in the vestibular sensory organs. Here we demonstrate spontaneous intracellular calcium transients in sensory hair cells (HCs) and supporting cells (SCs) in the murine utricular macula during the first two postnatal weeks. Calcium transients were monitored using a genetically encoded calcium indicator, GCaMP5G (G5), at 100 ms-frame−1 in excised utricle sensory epithelia, including HCs, SCs, and neurons. The reporter line expressed G5 and tdTomato (tdT) in a Gad2-Cre dependent manner within a subset of utricular HCs, SCs and neurons. Kinetics of the G5 reporter limited temporal resolution to calcium events lasting longer than 200 ms. Spontaneous calcium transients lasting 1-2 s were observed in the expressing population of HCs at birth and slower spontaneous transients lasting 10-30 s appeared in SCs by P3. Beginning at P5, calcium transients could be modulated by application of the efferent neurotransmitter acetylcholine (ACh). In mature mice, calcium transients in the utricular macula occurred spontaneously, had a duration 1-2 s, and could be modulated by the exogenous application of acetylcholine (ACh) or muscarine. Long-lasting calcium transients evoked by ACh in mature mice were blocked by atropine, consistent with previous reports describing the role of muscarinic receptors expressed in calyx bearing afferents in efferent control of vestibular sensation. Large spontaneous and ACh evoked transients were reversibly blocked by the inositol trisphosphate receptor (IP3R) antagonist aminoethoxydiphenyl borate (2-APB). Results demonstrate long-lasting calcium transients are present in the utricular macula during the first postnatal week, and that responses to ACh mature over this same time period.

BDNF Controls Bidirectional Endocannabinoid Plasticity at Corticostriatal Synapses.

The dorsal striatum exhibits bidirectional corticostriatal synaptic plasticity, NMDAR and endocannabinoids (eCB) mediated, necessary for the encoding of procedural learning. Therefore, characterizing factors controlling corticostriatal plasticity is of crucial importance. Brain-derived neurotrophic factor (BDNF) and its receptor, the tropomyosine receptor kinase-B (TrkB), shape striatal functions, and their dysfunction deeply affects basal ganglia. BDNF/TrkB signaling controls NMDAR plasticity in various brain structures including the striatum. However, despite cross-talk between BDNF and eCBs, the role of BDNF in eCB plasticity remains unknown. Here, we show that BDNF/TrkB signaling promotes eCB-plasticity (LTD and LTP) induced by rate-based (low-frequency stimulation) or spike-timing-based (spike-timing-dependent plasticity, STDP) paradigm in striatum. We show that TrkB activation is required for the expression and the scaling of both eCB-LTD and eCB-LTP. Using 2-photon imaging of dendritic spines combined with patch-clamp recordings, we show that TrkB activation prolongs intracellular calcium transients, thus increasing eCB synthesis and release. We provide a mathematical model for the dynamics of the signaling pathways involved in corticostriatal plasticity. Finally, we show that TrkB activation enlarges the domain of expression of eCB-STDP. Our results reveal a novel role for BDNF/TrkB signaling in governing eCB-plasticity expression in striatum and thus the engram of procedural learning.

Piezo1 Channels Contribute to Flow‐Induced Intracellular Calcium Transients in Mouse Cortical Collecting Duct (CCD)

Within the CCD, an acute increase in tubular fluid flow rate (TFFR) (i) subjects principal (PC) and intercalated (IC) cells therein to biomechanical forces including hydrostatic pressure/circumferential stretch (CS), fluid shear stress (FSS) and drag/torque on PC cilia, and (ii) leads to a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i) characterized by an immediate high amplitude increase to a peak value within ~10 s due to release of IP3‐sensitive internal Ca2+ stores coupled to extracellular Ca2+ entry at the basolateral membrane, followed by a decay to a plateau elevation in [Ca2+]i that exceeds baseline, sustained by luminal Ca2+ entry (Liu et al. AJP Renal Physiology 2003, 2005; 2007). The latter increase in [Ca2+]i is necessary for flow induced K+ secretion (FIKS) in the microperfused mammalian CCD. The identity of the basolateral Ca2+ entry path is unknown. We sought to examine whether Piezo channels, a family of mechanically‐activated Ca2+ permeable channels, contribute to this response. RNAscope detected single Piezo1 RNA molecules in mouse renal cortex. Immunofluorescence studies in mice expressing Piezo1‐tdTomato construct revealed robust basolateral and faint apical Piezo1 localization in both PC and IC. Flow‐induced [Ca2+]i transients, inferred from fluorescence intensity ratios (FIRs), were measured in microperfused mouse CCDs loaded with the Ca2+ indicator fura‐2 and then subject to low or high TFFRs, in the absence or presence of GsMTx4 (piezo‐blocker) or Yoda1 (piezo1‐activator). IC in CCDs from male and female mice preincubated with luminal or bath GsMTx4 (5 μM) failed to exhibit a flow‐induced increase in [Ca2+]i in response to an acute increase in TFFR. Addition of luminal or bath Yoda 1 (1 μM) to CCDs perfused at a low TFFR led to an increase in [Ca2+]i over 90 s in both PC and IC; addition of Yoda1 (10 μM) to the bath further increased [Ca2+]i. We speculate that mechanosensitive Piezo1 channels, expressed on the basolateral>apical membranes of PC and IC in the mouse CCD, contribute to the increase in [Ca2+]i triggered by an increase in TFFR.Support or Funding InformationNIDDK: R01 DK38470, R01 DK51391 and P30 DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CuZnSOD expressed specifically in neurons rescues mitochondrial function and calcium handling in muscles of Sod1KO mice

BackgroundAging is accompanied by loss of muscle mass and force. The mechanism of the loss is not fully understood, but oxidative stress and mitochondrial dysfunction have been implicated as potential contributors. Mice deficient in CuZnSOD (Sod1−/− mice) show decreased ATP production and increased generation of reactive oxygen species by muscle mitochondria as well as muscle atrophy and weakness early in life that is reminiscent of that observed in old age in wild type (WT) mice. Thus, Sod1−/− mice represent a model of accelerated neuromuscular aging. Surprisingly, SynTgSod1−/− mice, which have Sod1 expression restored specifically in neurons, show none of the neuromuscular deficits observed in Sod1−/− mice. To investigate how neuron‐specific Sod1 expression rescues muscle mass and force in Sod1−/− mice, we examined contractile properties and mitochondrial function in muscles of SynTgSod1−/− mice.MethodLumbrical muscles were removed from 7–9 months old wild‐type, Sod1−/−, and SynTgSod1−/− mice, attached to a force transducer, and placed in an experimental chamber for the measurement of muscle contractile parameters and NADH fluorescence was recorded as a measure of mitochondrial redox status. Because mitochondrial function is regulated by calcium, we also examined intracellular calcium transients (ICT) using the fluorescent dye Mag‐fura2. Immunofluorescence techniques were used to examine neuromuscular junction (NMJ) structure and fiber type composition.ResultCompared with WT muscles, mitochondria of muscles of Sod1−/− mice were in a more reduced state at rest and became transiently more oxidized following a 5s maximum isometric contraction. Consistent with the idea that recovery of NADH may be driven by calcium activation of the TCA cycle, the peak of the ICT was reduced and the width prolonged in muscles of Sod1−/− mice. For all measures, muscles of SynTgSod1−/− and WT mice were not different. A shift in muscle fiber type composition observed in Sod1−/− mice was also restored in SynTgSod1−/− mice. Moreover, NMJ denervation and fragmentation was rescued in SynTg Sod1−/− mice.ConclusionOur finding of improved mitochondrial function in muscles of SynTgSod1−/− compared with Sod1−/− mice is consistent with the hypothesis that the mitochondrial dysfunction is an important contributor to the development of atrophy and weakness in Sod1−/− mice. Since SynTgSod1−/− mice are still lacking CuZnSOD in muscle, we conclude that appropriate neuronal function and intact innervation are key to maintaining muscle mitochondrial function and overall muscle size and strength.Support or Funding InformationSupported by NIH Grant AG051442 and the Second Xiangya Hospital of Central South University, ChinaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Inhibition of RhoA/ROCK signaling pathway ameliorates hypoxic pulmonary hypertension via HIF-1α-dependent functional TRPC channels

Hypoxic pulmonary vasoconstriction (HPV) can be modulated by Rho/Rho kinase signaling, which can alter HPV vascular function via regulating myosin light chain phosphorylation, in a manner generally believed to be Ca2+-independent. We hypothesized that the RhoA/ROCK signaling pathway also can regulate HPV vascular function via a Ca2+-dependent mechanism, signaling through the functional transient receptor potential canonical (TRPC) channels. In this study, male BALB/c mice were exposed to normoxic or 10% oxygen (hypoxic) conditions for six weeks, after which systolic pressure and right ventricular hypertrophy were assessed. Transient intracellular calcium was monitored using a fluorescence imaging system. Muscle tension was measured with a contractile force recording system, and protein expression was assessed by immunoblotting. We found that the expressions of RhoA and ROCK were increased in mouse pulmonary arteries (PAs) under conditions of chronic hypoxia. Inhibition of the RhoA/ROCK signaling pathway prevented the development of hypoxic pulmonary hypertension (HPH), as evidenced by significantly reduced PA remodeling and pulmonary vasoconstriction. Immunoblotting results revealed that inhibition of the RhoA/ROCK signaling pathway significantly decreased the expression of HIF-1α. Knockdown of HIF-1α down-regulated the expression and function of the TRPC1 and TRPC6 channels in PASMCs under conditions of hypoxia. Contraction of the PAs and a Ca2+ influx into PASMCs through either receptor- or store-operated Ca2+ channels were also increased after hypoxia. However, RhoA/ROCK inhibitors markedly attenuated these changes. These results indicate that inhibition of the RhoA/ROCK signaling pathway ameliorates HPH via HIF-1α-dependent functional TRPCs.

Amniotic membrane as novel scaffold for human iPSC-derived cardiomyogenesis.

Recent approaches of using decellularized organ matrices for cardiac tissue engineering prompted us to culture human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) on the human amniotic membrane (hAM). Since hAM has been used lately to patch diseased hearts in patients and has shown anti-inflammatory and anti-fibrotic benefits, it qualifies as a cardiac compatible and clinically relevant heart tissue scaffold. The aim of this study was to test the ability of the hAM to support attachment, differentiation, and maturation of hiPSC-derived CMs in vitro. hAMs were prepared from term placenta. An in-house generated hiPSC line was used for CM derivation. hiPSC-derived cardiac progenitors were cultured on the surface of cryopreserved hAMs and in the presence of cytokines promoting cardiac differentiation. CMs grown on hAM and popular basement membrane matrix (BMM) Matrigel™ were compared for the following aspects of cardiac development: the morphology of cardiomyocytes with respect to shape and cellular alignments, levels of cardiac-related gene transcript expression, functionality in terms of spontaneous calcium fluxes and mitochondrial densities and distributions. hAM is biocompatible with hiPSC-derived CMs. hAM increased cardiac transcription regulator and myofibril protein transcript expression, accelerated intracellular calcium transients, and enhanced cellular mitochondrial complexity of its cardiomyocytes in comparison to cardiomyocytes differentiated on Matrigel™. Our data suggests that hAM supports differentiation and improves cardiomyogenesis in comparison to Matrigel™. hAMs are natural, easily and largely available. The method of preparing hAM cardiac sheets described here is simple with potential for clinical transplantation. Graphical abstract A An outline of the differentiation protocol with stage-specific growth factors and culture media used. B Cell fates from pluripotent stem cells to cardiomyocytes during differentiation on the amniotic membrane. C-FPhotomicrographs of cells at various stages of differentiation. Scale bars represent 100μm.

Caffeine attenuates contraction-induced diminutions of the intracellular calcium transient in mouse lumbrical muscle ex vivo

The amount of calcium released from the sarcoplasmic reticulum in skeletal muscle rapidly declines during repeated twitch contractions. In this study, we test the hypothesis that caffeine can mitigate these contraction-induced declines in calcium release. Lumbrical muscles were isolated from male C57BL/6 mice and loaded with the calcium-sensitive indicator, AM-furaptra. Muscles were then stimulated at 8 Hz for 2.0 s in the presence or absence of 0.5 mM caffeine, at either 30 °C or 37 °C. The amplitude and area of the furaptra-based intracellular calcium transients and force produced during twitch contractions were calculated. For each of these measures, the values for twitch 16 relative to twitch 1 were higher in the presence of caffeine than in the absence of caffeine at both temperatures. We conclude that caffeine can attenuate contraction-induced diminutions of calcium release during repeated twitch contractions, thereby contributing to the inotropic effects of caffeine.

Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation

Upon fertilization or parthenogenesis, zinc is released into the extracellular space through a series of exocytic events termed zinc sparks, which are tightly coordinated with intracellular calcium transients. The zinc spark reduces the total amount of intracellular zinc, and this reduction is necessary and sufficient to induce egg activation even in the absence of calcium transients. In addition, this zinc release contributes to the block to polyspermy through modification of the zona pellucida. The zinc spark has been documented in all organisms examined to date including the mouse, two species of nonhuman primates, and human. Here we determined whether zinc sparks occur in the bovine, an important model of gamete development in mono-ovulatory mammalian species. We obtained metaphase II-arrested (MII) bovine eggs following in vitro maturation. Total zinc, assessed in single cells using X-Ray Fluorescence Microscopy, was significantly more abundant in the bovine egg compared to iron and copper. Studies with intracellular fluorescent probes revealed that labile zinc pools are localized to discrete cytoplasmic punctae enriched at the cortex. To determine whether zinc undergoes dynamic fluxes during egg activation, we parthenogenetically activated bovine eggs using two approaches: ionomycin or bovine phospholipase C zeta (bPlcζ). Both these methods induced zinc sparks coordinately with intracellular calcium transients. The zinc spark was also observed in bovine eggs following intracytoplasmic sperm injection. These results establish that zinc is the most abundant transition metal in the bovine egg, and zinc flux during egg activation - induced by chemical activation or sperm - is a highly conserved event across mammalian species.

Abnormal response of costal chondrocytes to acidosis in patients with chest wall deformity

Costal cartilage is much understudied compared to the load bearing cartilages. Abnormally grown costal cartilages are associated with the inherited chest wall deformities pectus excavatum and pectus carinatum resulting in sunken or pigeon chest respectively. A lack of understanding of the ultrastructural and molecular biology properties of costal cartilage is a major confounder in predicting causes and outcomes of these disorders. Due to the avascular nature of cartilage, chondrocytes metabolize glycolytically, producing an acidic environment. During physical activity hydrogen ions move within cartilage driven by compressive forces, thus at any one time, chondrocytes experience transient changes in pH. A variety of ion channels on chondrocytes plasma membrane equip them to function in the rapidly changing conditions they experience. In this paper we describe reduced expression of the ASIC2 gene encoding the acid sensing ion channel isoform 2 (previously referred to as ACCN1 or ACCN) in patients with chest wall deformities. We hypothesized that chondrocytes from these patients cannot respond normally to changes in pH that are an integral part of the biology of this tissue. Activation of ASICs indirectly creates a cascade ultimately dependent on intracellular calcium transients. The objective of this paper was to compare internal calcium signaling in response to external pH changes in costal chondrocytes from patients with chest wall deformities and healthy individuals. Although the molecular mechanism through which chondrocytes are regulated by acidosis remains unknown, we observed reduced amplitudes of calcium rise in patient chondrocytes exposed to low pH that become further impaired upon repeat exposure.

Concomitant SK current activation and sodium current inhibition cause J wave syndrome.

The mechanisms of J wave syndrome (JWS) are incompletely understood. Here, we showed that the concomitant activation of small-conductance calcium-activated potassium (SK) current (IKAS) and inhibition of sodium current by cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) recapitulate the phenotypes of JWS in Langendorff-perfused rabbit hearts. CyPPA induced significant J wave elevation and frequent spontaneous ventricular fibrillation (SVF), as well as sinus bradycardia, atrioventricular block, and intraventricular conduction delay. IKAS activation by CyPPA resulted in heterogeneous shortening of action potential (AP) duration (APD) and repolarization alternans. CyPPA inhibited cardiac sodium current (INa) and decelerated AP upstroke and intracellular calcium transient. SVFs were typically triggered by short-coupled premature ventricular contractions, initiated with phase 2 reentry and originated more frequently from the right than the left ventricles. Subsequent IKAS blockade by apamin reduced J wave elevation and eliminated SVF. β-Adrenergic stimulation was antiarrhythmic in CyPPA-induced electrical storm. Like CyPPA, hypothermia (32.0°C) also induced J wave elevation and SVF. It facilitated negative calcium-voltage coupling and phase 2 repolarization alternans with spatial and electromechanical discordance, which were ameliorated by apamin. These findings suggest that IKAS activation contributes to the development of JWS in rabbit ventricles.

An Ultrasensitive Calcium Reporter System via CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells

SummaryGenetically encoded calcium indicator (GCaMP) proteins have been reported for imaging cardiac cell activity based on intracellular calcium transients. To bring human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) to the clinic, it is critical to evaluate the functionality of CMs. Here, we show that GCaMP6s-expressing hPSCs can be generated and used for CM characterization. By leveraging CRISPR-Cas9 genome editing tools, we generated a knockin cell line that constitutively expresses GCaMP6s, an ultrasensitive calcium sensor protein. We further showed that this clone maintained pluripotency and cardiac differentiation potential. These knockin hPSC-derived CMs exhibited sensitive fluorescence fluctuation with spontaneous contraction. We then compared the fluorescence signal with mechanical contraction signal. The knockin hPSC-derived CMs also showed sensitive response to isoprenaline treatment in a concentration-dependent manner. Therefore, the GCaMP6s knockin hPSC line provides a non-invasive, sensitive, and economic approach to characterize the functionality of hPSC-derived CMs.

Effect of KN93 on delayed afterdepolarization and calcium ion in ventricular myocytes of rabbits with heart failure

Objective To observe the effect of KN93, a CaMKⅡ inhibitor, on delayed afterdepolarization (DAD) and calcium ion in ventricular myocytes of rabbits with heart failure, and to investigate the effect of CaMKⅡ signaling pathway on trigged arrhythmia after heart failure. Methods Thirty male New Zealand White rabbits were randomized(random number) into the sham operated group (sham group), heart failure group (HF group) and heart failure with KN93 group (HF+KN93 group) (n=10 each group). The rabbit heart failure model was established by abdominal aortic constriction combined with aortic valve regurgitation. The ventricular myocytes were isolated by double enzyme digestion. The action potential and the transient inward current (Iti) were recorded by the whole-cell patch-clamp. The intracellular calcium transient was measured by the ion concentration measurement system. The main calcium transporter protein was detected by Western blotting. Data were analyzed by pCLAMP10.2. Statistical analysis was performed using SPSS 17.0. Comparisons among groups were conducted using ANOVA, and SNK-q multiple comparison procedure was utilized for post-hoc analysis. Results (1) After induction of heart failure, DAD and increment of trigger activity (TA) were observed in rabbit ventricular myocytes. Treatment of KN93 with 1.0 μmol/L reduced the events of DAD and TA. (2) After induction of heart failure, Iti densities were increased from -0.12±0.02 pA/pF to -0.95±0.06 pA/pF at the polarization potential of -50 mV (n=10, P<0.01). The current densities were reduced to -0.44±0.04 pA/pF after application of 1.0 μmol/L of KN93 (n=10, P<0.01). (3) KN93 led to decrement of intracellular calcium ion concentration and calcium transient amplitude, and acceleration of the decay process of calcium transient. (4) KN93 upregulated the expression of pPLN and SERCA2a, increased the uptake of intracellular calcium ion, downregulated the expression of NCX, decreased the Iti, and reduced the occurrence of DAD and TA. Conclusions KN93 can reduce the intracellular calcium ion concentration of the heart failure animal model, and the occurrence of the DAD and TA. CaMKⅡ may be a new therapeutic target for arrhythmias in the heart failure. Key words: KN93; Heart failure; Delayed afterdepolarization; Transient inward current; Calcium ion

High tension in sarcomeres hinders myocardial relaxation: A computational study.

Experiments have shown that the relaxation phase of cardiac sarcomeres during an isometric twitch is prolonged in muscles that reached a higher peak tension. However, the mechanism is not completely understood. We hypothesize that the binding of calcium to troponin is enhanced by the tension in the thin filament, thus contributing to the prolongation of contraction upon higher peak tension generation. To test this hypothesis, we developed a computational model of sarcomere mechanics that incorporates tension-dependence of calcium binding. The model was used to simulate isometric twitch experiments with time dependency in the form of a two-state cross-bridge cycle model and a transient intracellular calcium concentration. In the simulations, peak isometric twitch tension appeared to increase linearly by 51.1 KPa with sarcomere length from 1.9 μm to 2.2 μm. Experiments showed an increase of 47.3 KPa over the same range of sarcomere lengths. The duration of the twitch also increased with both sarcomere length and peak intracellular calcium concentration, likely to be induced by the inherently coupled increase of the peak tension in the thin filament. In the model simulations, the time to 50% relaxation (tR50) increased over the range of sarcomere lengths from 1.9 μm to 2.2 μm by 0.11s, comparable to the increased duration of 0.12s shown in experiments. Model simulated tR50 increased by 0.12s over the range of peak intracellular calcium concentrations from 0.87 μM to 1.45 μM. Our simulation results suggest that the prolongation of contraction at higher tension is a result of the tighter binding of Ca2+ to troponin in areas under higher tension, thus delaying the deactivation of the troponin.

Neutrophils injure gallbladder interstitial Cajal-like cells in a guinea pig model of acute cholecystitis.

Acute cholecystitis is a common disease with gallbladder dysmotility. Disease pathogenesis involves immune cell infiltration as well as changes in gallbladder interstitial Cajal-like cells (ICLCs). However, it remains unclear if or how the immune cells affect ICLC morphology, density, distribution, and function in gallbladder tissue during acute cholecystitis. In this study, we explored the acute cholecystitis-related alterations in gallbladder ICLCs in a guinea pig model, focusing on the effects of neighboring neutrophils. Adult guinea pigs were randomly divided into four groups (control, 24 hr common bile duct ligation [CBDL], 48-hr CBDL, and antipolymorphonuclear neutrophil [PMN] treated) and analyzed using methylene blue staining and immunofluorescence. Gallbladder contractility was also monitored. To culture gallbladder ICLCs, collagenase digestion was performed on tissue from 10- to 15-day-old guinea pigs. Neutrophils isolated from the peripheral blood of experimental animals 48-hr postsurgery were also cocultured with the gallbladder ICLCs. Intracellular calcium was detected with Fluo-4 AM dye. Our results showed that gallbladder ICLC density significantly declined during acute cholecystitis and was accompanied by shortening of the cellular processes and damage to their network-like structure. However, pretreatment with anti-PMN partially prevented these changes. Gallbladder contraction was also significantly decreased during acute cholecystitis, and this appeared to be mediated by the neutrophils. Moreover, ICLCs cocultured with neutrophils also had shortened and reduced processes and impaired network-like structure formation. Intracellular calcium transient was less sensitive to contraction agonists and inhibitors when cocultured with neutrophils. Taken together, neutrophils greatly affect gallbladder ICLCs and dysmotility during acute cholecystitis.

Abstract 342: The Absence of S-nitrosoglutathione Reductase (GSNOR -/- ) Reduces Maturation of iPSC-derived Cardiomyocytes

Introduction: Induced pluripotent stem cells (iPSCs) are a potential source of cardiac regenerative therapy. However, incomplete structural and functional maturation of iPSC-derived cardiomyocytes (iPSC-CMs), including lack of T-tubules, immature sarcoplasmic reticulum (SR), and inefficient Ca 2+ -induced Ca 2+ release remain major limitations. We assessed the influence of nitric oxide (NO) on differentiation and maturation of iPSC-CMs. Objective: We hypothesized that enhanced S-nitrosylation impairs SR calcium (Ca 2+ ) handling (storage and uptake) in iPSC-CMs. Methods: iPSCs were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR -/- ), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into CMs from embryoid bodies via the hanging-drop method. Analyses of cell morphology and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) were performed at an early stage of cardiac development (day 21 of cell culture). The involvement of the SR Ca 2+ ATPase (SERCA2) in iPSC-CM contraction was tested by blocking Ca 2+ re-uptake with 1 μM Thapsigargin and SR Ca 2+ stores were estimated by addition of 20 mM caffeine. The role of the cardiac ryanodine receptor (RyR2) was assessed by blocking the channel with 10 μM ryanodine. The dependence of Ca 2+ transients on L-type Ca 2+ channels (LTCC) was demonstrated using 10 μM nifedipine, which eliminated [Ca 2+ ] i transients and cell contraction. Length of sarcomeres within the contractile apparatus was determined by staining cells with α-actinin. Results: Sarcomeres were more organized in WT iPSC-CMs than in GSNOR -/- iPSC-CMs after 21 days in culture. The loss of GSNOR activity reduced SR Ca 2+ content and sarcomere organization in iPSC-CMs. While iPSC-CMs are relatively immature in terms of ultrastructure and Ca 2+ handling; GSNOR –/– iPSC-CMs were less able to recycle intracellular Ca 2+ . Conclusions: Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that the absence of GSNOR affects CM differentiation. These findings have important implications for regenerative medicine and provide new targets for iPSC-based therapy.