Abnormal Ca2 Research Articles

Connexin 43 dephosphorylation contributes to arrhythmias and cardiomyocyte apoptosis in ischemia/reperfusion hearts.

Connexin 43 (Cx43)-associated gap junctions form electrical and mechanical conduits between adjacent ventricular cardiomyocytes, ensuring coordinate electrical excitation and synchronic contraction for each heartbeat. Cx43 dephosphorylation is a characteristic of ischemia, arrhythmia, and a failing and aging myocardium, but the exact phosphosite(s) triggering myocardial apoptosis and electrical disturbance and its underlying mechanisms are unclear. We previously found that Cx43-serine 282 phosphorylation (pS282) can regulate cardiomyocyte survival and electrical stability. Here, we investigated the hypothesis that S282 dephosphorylation occurs in and contributes to ischemia/reperfusion (I/R)-induced cardiac injury. We found enhanced Cx43-pS262 and Cx43-pS368 but decreased Cx43-pS282 in rat hearts subjected to I/R (30min/2h). I/R rats had ventricular arrhythmias and myocardial apoptosis with activation of the p38 mitogen-activated protein kinase (p38)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway. Similarly, S282 dephosphorylation, abnormal Ca2+ transients, cell apoptosis and p38/Fas/FADD activation also occurred in neonatal rat ventricular myocytes exposed to anoxia/reoxygenation (12/6h). To confirm the causative role of S282 dephosphorylation in cardiac injury, rat hearts were intramyocardially injected with a virus carrying the S282 mutant substituted with alanine (S282A), thus causing arrhythmias and reducing cardiac output and myocardial apoptosis with p38/Fas/FADD pathway activation. Moreover, Cx43-S282A+/- mice displayed arrhythmias and impaired cardiac output with global myocardial apoptosis. Our findings revealed that Cx43 dephosphorylation at S282 triggers arrhythmias and, at least partly, contributes to cardiomyocyte death upon I/R by activating the p38/Fas/FADD pathway, providing a novel molecular mechanism and potential target for protecting against cardiac I/R injury.

Anti-Osteoclastic Effect of Zinc Studied in MCF-7 Induced Osteoclastogenesis

Bone which a key structural support of the body, undergoes dynamic micro structural remodelling all over life to control automatic stress and calcium requirement in the body. A number of risk factors including oxidative stress, apoptosis and abnormal intracellular Ca2+ metabolism have been postulated to play a function in the inception and progress of bone osteolysis. Cancer cells establish a tight relationship with the host tissue, secreting factors that stimulate or inhibit bone cells, receiving signals generated from the bone remodelling activity, and displaying some features of bone cells. This interplay between tumour and bone cells alters the physiological bone remodelling, leading to the generation of a vicious cycle that promotes bone metastasis growth. Zinc is one of the most relevant minerals to human health, because of its antioxidant properties. The present study was aimed to investigate protective role of zinc against bone metastasis. In the present study, TRAP positive multinucleated cell count was low compared to CM treated cells. Zinc treatment suppressed MCF-7 induced mRNA levels of cytoplasmic 1 (Nfatc1), TRAP and Cathepsin-K. Hence, it can be concluded that zinc decreases osteoclastogenesis induced by MCF-7 cells.
 Keywords: Oxidative stress; Metastasis; Bone remodelling; Zinc.

α1-Na/K-ATPase inhibition rescues aberrant dendritic calcium dynamics and memory deficits in the hippocampus of an Angelman syndrome mouse model

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of function of the maternal copy of the UBE3A gene. Previous studies reported an increase in α1-Na/K-ATPase (α1-NaKA) expression in the AS hippocampus at the age of 2 weeks as the initial and isolated molecular alteration. This increase was further implied upon actuating much of the hippocampal-related deficits in an AS mouse model, although the underlying mechanism was never investigated. Here, we showed that enhanced α1-NaKA expression resulted in increased pump activity that reduced activity-dependent dendritic Ca2+ dynamics in the AS hippocampus, as well as selective inhibition of α1-NaKA by marinobufagenin (MBG) to normalize these aberrant Ca2+ dynamics. In addition, we demonstrated that selective α1-NaKA inhibition corrected impaired hippocampal synaptic plasticity and hippocampal-dependent cognitive deficits. Furthermore, we showed that the isolated increase in hippocampal α1-NaKA expression in AS mice at 2 weeks of age was accompanied by an unexpected enhancement in excitability. Altogether, our study implicates the modification of Ca2+ dynamics as one of the major underlying mechanisms by which enhanced α1-NaKA expression induces deleterious effects in the hippocampus of AS model mice. Finally, we propose a therapeutic approach for AS and possibly other neurodevelopmental disorders that entail aberrant NaKA expression or abnormal Ca2+ dynamics.

Abstract 652: Increased Gα o Expression Plays a Pivotal Role in the Progression of Heart Failure by Impairing Ca 2+ Handling

Background: A transcriptional repressor, neuron restrictive silencer factor (NRSF), maintains normal cardiac function and electrical stability. Transgenic mice expressing a dominant-negative mutant of NRSF in their hearts (dnNRSF-Tg) exhibit systolic dysfunction and premature death due to lethal arrhythmias like human dilated cardiomyopathy(DCM). Underlining mechanisms remain to be elucidated, however. Purpose: To clarify how NRSF maintains normal cardiac homeostasis. Methods and Results: cDNA microarray analysis of dnNRSF-Tg and newly generated cardiac-specific NRSF knockout mice (NRSFcKO), which show similar cardiac phenotypes to those of dnNRSF-Tg, revealed that cardiac gene expression of GNAO1 that encodes Gα o , a member of inhibitory G protein Gα i family, is increased in both dnNRSF-Tg and NRSFcKO ventricles. ChIP-seq analysis, reporter assay and electrophoretic mobility shift assay identified that GNAO1 is a direct target of NRSF. In dnNRSF-Tg, pharmacological inhibition of Gα o by pertussis toxin improved systolic dysfunction and knockdown of Gα o by crossing with GNAO1 knockout mice improved not only systolic function but also frequency of ventricular arrhythmias and survival rates. Electrophysiological analysis in ventricular myocytes obtained from dnNRSF-Tg demonstrated that genetic reduction of Gα o ameliorated abnormalities in Ca 2 + handling, which include increased current density in surface sarcolemmal L-type Ca 2 + channel, reduced content of sarcoplasmic reticulum Ca 2+ and lowered peak of Ca 2+ transient. In addition, genetic reduction of Gα o attenuated increased phosphorylation levels of CAMKII and RyR2 in dnNRSF-Tg ventricles, which presumably underlies the improvement in Ca 2 + handling. Furthermore, we identified increased Gα o expression in ventricles of heart failure model mice induced by transverse aortic constriction and cardiac troponin T mutant DCM model mice, in both of which, genetic reduction of Gα o ameliorated cardiac dysfunction. Conclusions: We found that increased expression of Gα o , induced by attenuation of NRSF-mediated repression, plays a pivotal role in the progression of heart failure by evoking Ca 2+ handling abnormality. Gα o is a potential therapeutic target for heart failure.

Abstract 331: Loss of Myosin Binding Protein H-Like Causes Cardiac Conduction Abnormalities

A premature truncation (R255X) in MYBPHL associates with human dilated cardiomyopathy (DCM) and arrhythmias. Loss of Mybphl in mice causes DCM and arrhythmia. MYBPHL encodes myosin binding protein H-like (MyBP-HL) and is expressed highly in the atria. We hypothesize that MyBP-HL is required for proper conduction system function. Immunofluorescence microscopy on normal human atria showed MyBP-HL staining in all atrial cardiomyocytes with a sarcomere A-band pattern. Atria from the heterozygous (het) MYBPHL R255X mutant carrier lacked MyBP-HL staining. Human induced pluripotent stem cell-derived cardiomyocytes from the het MYBPHL R255X carrier and control cell lines were also examined. MyBP-HL was found in a subset of control cardiomyocytes, whereas R255X cells showed no MyBP-HL, suggesting that the R255X allele exerts a dominant-negative effect on the normal MYBPHL allele. Immunofluorescence microscopy in wild-type (WT) mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl het ventricles have 10% as many MyBP-HL-positive cells compared to WT. Surface telemetry revealed atrioventricular block and atrial bigeminy and intracardiac pacing revealed a shorter atrial relative refractory period and inducible atrial tachycardia in Mybphl -null mice. Ca 2+ transients measured with confocal microscopy revealed that isolated Mybphl -null atrial cardiomyocytes had an increased occurrence of triggered Ca 2+ waves and more heterogenous Ca 2+ release than WT controls. Super-resolution microscopy revealed ryanodine receptor disorganization in Mybphl het and null atrial cardiomyocytes compared to WT controls. Abnormal Ca 2+ release, shorter atrial refractory period, and dilated atria could account for the observed atrial arrhythmias, bigeminy, and atrial tachycardia.

Abstract 766: SGLT-Mediated Na + Overload Results in Oxidative Stress and Abnormal SR Ca 2+ Release in Diabetic Hearts

Intracellular Na + concentration ([Na + ] i ) regulates Ca 2+ cycling, oxidative state and electrical stability of the heart. [Na + ] i is linked to glucose uptake through the Na + -glucose cotransporter (SGLT). The SGLT1 isoform is present in the heart and its overexpression causes hypertrophy and left-ventricular dysfunction. Here, we hypothesized that cardiac SGLT activity is increased in type-2 diabetes (T2D), which causes myocyte Na + overload and results in oxidative stress and exacerbated sarcoplasmic reticulum (SR) Ca 2+ leak. To test this hypothesis, we analyzed myocardial tissue from humans with/without T2D and compared rats with late-onset T2D that display diabetic cardiomyopathy similar to that seen in humans (HIP rats) with their non-diabetic littermates (WT). SGLT1 expression was increased in hearts from T2D patients compared to non-diabetic individuals and in hearts from HIP vs . WT rats. SGLT inhibition significantly decreased the uptake of both glucose and Na + in myocytes from HIP rats but not in WT, which indicates that SGLT function is increased in T2D hearts. While SGLT1 upregulation may partially compensate for the reduced insulin-dependent glucose uptake, the ensuing raise in Na + influx resulted in elevated [Na + ] i in HIP myocytes (by 3 mM compared to WT). Higher [Na + ] i causes oxidative stress by activating the mitochondrial Na + /Ca 2+ exchanger (mitoNCX), which lowers mitochondrial [Ca 2+ ] and thus slows down regeneration of the antioxidant NADPH. In agreement with a role for elevated [Na + ] i in causing oxidative stress in T2D hearts, H 2 O 2 production was increased in HIP myocytes vs . WT and mitoNCX inhibition, which uncouples mitochondria from [Na + ] i , significantly reduced H 2 O 2 production in HIP hearts. Oxidative stress enhances the SR Ca 2+ leak directly through oxidation of ryanodine receptors (RyRs) and indirectly via CaMKII activation and consequent RyR phosphorylation. SR Ca 2+ leak was augmented in myocytes from HIP vs . WT rats and mitoNCX inhibition significantly reduced the leak in HIP myocytes, but not in the WT. Thus, our data indicate that SGLT activity is enhanced in myocytes from T2D rats, which increases Na + influx and causes Na + overload. Elevated [Na + ] i contributes to oxidative stress and abnormal SR Ca 2+ leak in diabetic hearts.

Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice.

Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Developmental exposure to polychlorinated biphenyls (PCBs) in the maternal diet causes host-microbe defects in weanling offspring mice

The gut microbiota is important for maintaining homeostasis of the host. Gut microbes represent the initial site for toxicant processing following dietary exposures to environmental contaminants. The diet is the primary route of exposure to polychlorinated biphenyls (PCBs), which are absorbed via the gut, and subsequently interfere with neurodevelopment and behavior. Developmental exposures to PCBs have been linked to increased risk of neurodevelopmental disorders (NDD), including autism spectrum disorder (ASD), which are also associated with a high prevalence of gastrointestinal (GI) distress and intestinal dysbiosis. We hypothesized that developmental PCB exposure impacts colonization of the gut microbiota, resulting in GI pathophysiology, in a genetically susceptible host. Mouse dams expressing two heritable human mutations (double mutants [DM]) that result in abnormal Ca2+ dynamics and produce behavioral deficits (gain of function mutation in the ryanodine receptor 1 [T4826I-RYR1] and a human CGG repeat expansion [170–200 CGG repeats] in the fragile X mental retardation gene 1 [FMR1 premutation]). DM and congenic wild type (WT) controls were exposed to PCBs (0–6 mg/kg/d) in the diet starting 2 weeks before gestation and continuing through postnatal day 21 (P21). Intestinal physiology (Ussing chambers), inflammation (qPCR) and gut microbiome (16S sequencing) studies were performed in offspring mice (P28–P30). Developmental exposure to PCBs in the maternal diet caused significant mucosal barrier defects in ileum and colon (increased secretory state and tight junction permeability) of juvenile DM mice. Furthermore, PCB exposure increased the intestinal inflammatory profile (Il6, Il1β, and Il22), and resulted in dysbiosis of the gut microbiota, including altered β-diversity, in juvenile DM mice developmentally exposed to 1 mg/kg/d PCBs when compared to WT controls. Collectively, these findings demonstrate a novel interaction between PCB exposure and the gut microbiota in a genetically susceptible host that provide novel insight into environmental risk factors for neurodevelopmental disorders.

Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients.

Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. However, its cellular mechanisms are not fully understood, and presently there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD in HCM and as a platform for drug discovery. In the present study, beating iPSC-CMs were generated from healthy controls and HCM patients with DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, decreased relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca2+ imaging indicated elevated diastolic [Ca2+]i and abnormal Ca2+ handling in HCM iPSC-CMs, which were exacerbated by β-adrenergic challenge. Combining Ca2+ imaging and traction force microscopy, we observed enhanced myofilament Ca2+ sensitivity (measured as dF/Δ[Ca2+]i) in HCM iPSC-CMs. These results were confirmed with genome-edited isogenic iPSC lines that carry HCM mutations, indicating that cytosolic diastolic Ca2+ overload, slowed [Ca2+]i recycling, and increased myofilament Ca2+ sensitivity, collectively impairing the relaxation of HCM iPSC-CMs. Treatment with partial blockade of Ca2+ or late Na+ current reset diastolic Ca2+ homeostasis, restored diastolic function, and improved long-term survival, suggesting that disturbed Ca2+ signalling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type Ca2+channel (LTCC) and transient receptor potential cation channels (TRPC) in HCM iPSC-CMs compared with control iPSC-CMs, which likely contributed to diastolic [Ca2+]i overload. In summary, this study recapitulated DD in HCM at the single-cell level, and revealed novel cellular mechanisms and potential therapeutic targets of DD using iPSC-CMs.

TRP Channels Mediated Pathological Ca2+-Handling and Spontaneous Ectopy.

Ion channel biology offers great opportunity in identifying and learning about cardiac pathophysiology mechanisms. The discovery of transient receptor potential (TRP) channels is an add-on to the opportunity. Interacting with numerous signaling pathways, being activated multimodally, and having prescribed signatures underlining acute hemodynamic control and cardiac remodeling, TRP channels regulate cardiac pathophysiology. Impaired Ca2+-handling cause contractile abnormality. Modulation of intracellular Ca2+ concentration ([Ca2+]i) is a major part of Ca2+-handling processes in cardiac pathophysiology. TRP channels including TRPM4 regulate [Ca2+]i, Ca2+-handling and cardiac contractility. The channels modulate flux of divalent cations, such as Ca2+ during Ca2+-handling and cardiac contractility. Seminal works implicate TRPM4 and TRPC families in intracellular Ca2+ homeostasis. Defective Ca2+-homeostasis through TRP channels interaction with Ca2+-dependent regulatory proteins such as sodium calcium exchanger (NCX) results in abnormal Ca2+ handling, contractile dysfunction and in spontaneous ectopy. This review provides insight into TRP channels mediated pathological Ca2+-handling and spontaneous ectopy.

Nonlinear Dynamic Modeling of 2-Dimensional Interdependent Calcium and Inositol 1,4,5-Trisphosphate in Cardiac Myocyte

Calcium (Ca2+) and inositol 1,4,5-trisphosphate (IP3) is critically important actors for a vast array of cellular processes. The most significant of the functions is One of the main functions is communication in all parts of the body which is achieved through cell signaling. Abnormalities in Ca2+signaling have been implicated in clinically important conditions such as heart failure and cardiac arrhythmias. We propose a mathematical model which systematically investigates complex Ca2+and IP3dynamics in cardiac myocyte. This two dimensional model is based on calcium-induced calcium release via inositol 1,4,5-trisphosphate receptors and includes calcium modulation of IP3levels through feedback regulation of degradation and production. Forward-Time Centered-Space method has been used to solve the coupled equations. We were able to reproduce the observed oscillatory patterns in Ca2+as well as IP3signals. The model predicts that calcium-dependent production and degradation of IP3is a key mechanism for complex calcium oscillations in cardiac myocyte. The impact and sensitivity of source, leak, diffusion coefficients on both Ca2+and IP3dynamics have been investigated. The results show that the relationship between Ca2+and IP3dynamics is nonlinear.

Protective roles of carbonic anhydrase 8 in Machado-Joseph Disease.

Machado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is an inherited neurodegenerative disease that can lead to a regression of motor coordination and muscle control in the extremities. It is known that expansion of CAG repeats encodes abnormally long polyQ in mutant ataxin-3, the disease protein. It is also noted that mutant ataxin-3 interacts with 1,4,5-trisphosphate receptor type 1 (IP3R1) and induces abnormal Ca2+ release. Previously, we have shown a significant increase in the expression of carbonic anhydrase VIII (CA8) in SK-N-SH-MJD78 cells, which are human neuroblastoma cells overexpressing mutant ataxin-3 with 78 glutamine repeats. In the current study, we showed the presence of significantly increased CA8 expression in MJD mouse cerebellum in either early or late disease stage, with a gradual decrease in CA8 expression as the MJD mice naturally aged. By immunofluorescence and immunoprecipitation analysis, we also found that CA8 co-localized and interacted with mutant ataxin-3 in SK-N-SH-MJD78 cells harboring overexpressed CA8 (SK-MJD78-CA8). In addition, we found that SK-MJD78-CA8 cells, as well as cerebellar granule neurons (CGNs) of MJD transgenic (Tg) mouse with overexpressed CA8, were more resistant to reactive oxygen species (ROS) stress than the control cells. Importantly, overexpression of CA8 in SK-MJD78-CA8 cells and in MJD CGNs rescued abnormal Ca2+ release and caused an increase in cell survival. In summary, we demonstrate the protective function of CA8 in MJD disease models and speculate that the declining expression of CA8 following an initial increased expression may be related to the late onset phenomenon of MJD.

Role of TRPC1 channels in pressure-mediated activation of airway remodeling

BackgroundBronchoconstriction and cough, a characteristic of the asthmatic response, leads to development of compressive stresses in the airway wall. We hypothesized that progressively pathological high mechanical stress could act on mechanosensitive cation channels, such as transient receptor potential channel 1 (TRPC1) and then contributes to airway remodeling.MethodsWe imitate the pathological airway pressure in vitro using cyclic stretch at 10 and 15% elongation. Ca2+ imaging was applied to measure the activity of TRPC1 after bronchial epithelial cells exposed to cyclic stretch for 0, 0.5, 1, 1.5, 2, 2.5 h. To further clarify the function of channnel TRPC1 in the process of mechano-transduction in airway remodeling, the experiment in vivo was implemented. The TRPC1 siRNA and budesonide were applied separately to asthmatic models. The morphological changes were measured by HE and Massion method. The expression levels of TRPC1 were evaluated by real-time PCR, western blot and immunohistochemistry. The protein expression level of IL-13, TGF-β1 and MMP-9 in BALF were measured by ELISA.ResultsThe result showed that cyclic stretch for 15% elongation at 1.5 h could maximize the activity of TRPC1 channel. This influx in Ca2+ was blocked by TRPC1 siRNA. Higher TRPC1 expression was observed in the bronchial epithelial layer of ovalbumin induced asthmatic models. The knockdown of TRPC1 with TRPC1 siRNA was associated with a hampered airway remodeling process, such as decreased bronchial wall thickness and smooth muscle hypertrophy/hyperplasia, a decreased ECM deposition area and inflammation infiltration around airway wall. Meantime, expression of IL-13, TGF-β1 and MMP-9 in OVA+TRPC1 siRNA also showed reduced level. TRPC1 intervention treatment showed similar anti-remodeling therapeutic effect with budesonide.ConclusionsThese results demonstrate that most TRPC1 channels expressed in bronchial epithelial cells mediate the mechanotransduction mechanism. TRPC1 inducing abnormal Ca2+ signal mediates receptor-stimulated and mechanical stimulus-induced airway remodeling. The inhibition of TRPC1 channel could produce similar therapeutic effect as glucocortisteroid to curb the development of asthmatic airway remodeling.

Mechanisms of Calcium Leak from Cardiac Sarcoplasmic Reticulum Revealed by Statistical Mechanics

Heart muscle contraction is normally activated by a synchronized Ca release from sarcoplasmic reticulum (SR), a major intracellular Ca store. However, under abnormal conditions, Ca leaks from the SR, decreasing heart contraction amplitude and increasing risk of life-threatening arrhythmia. The mechanisms and regimes of SR operation generating the abnormal Ca leak remain unclear. Here, we employed both numerical and analytical modeling to get mechanistic insights into the emergent Ca leak phenomenon. Our numerical simulations using a detailed realistic model of the Ca release unit reveal sharp transitions resulting in Ca leak. The emergence of leak is closely mapped mathematically to the Ising model from statistical mechanics. The system steady-state behavior is determined by two aggregate parameters: the analogs of magnetic field (h) and the inverse temperature (β) in the Ising model, for which we have explicit formulas in terms of SR [Ca] and release channel opening and closing rates. The classification of leak regimes takes the shape of a phase β-h diagram, with the regime boundaries occurring at h = 0 and a critical value of β (β∗) that we estimate using a classical Ising model and mean field theory. Our theory predicts that a synchronized Ca leak will occur when h > 0 and β >β∗, and a disordered leak occurs when β <β∗ and h is not too negative. The disorder leak is distinguished from synchronized leak (in long-lasting sparks) by larger Peierls contour lengths, an output parameter reflecting degree of disorder. Thus, in addition to our detailed numerical model approach, we also offer an instantaneous computational tool using analytical formulas of the Ising model for respective ryanodine receptor parameters and SR Ca load that describe and classify phase transitions and leak emergence.

Cardiac electrical and contractile disorders promoted by anabolic steroid overdose are associated with late autonomic imbalance and impaired Ca2+ handling

AimInvestigate cardiac electrical and mechanical dysfunctions elicited by chronic anabolic steroid (AS) overdose. MethodsMale Wistar rats were treated with nandrolone decanoate (DECA) or vehicle (CTL) for 8 weeks. Electrocardiography and heart rate variability were assessed at weeks 2, 4, and 8. Cardiac reactivity to isoproterenol was investigated in isolated rat hearts. Action potential duration (APD) was measured from left ventricular (LV) muscle strips. L-type Ca2+ current (ICaL), and transient outward potassium current (Ito) were recorded by whole-cell patch-clamp in LV cardiomyocytes. Sarcoplasmic reticulum (SR) Ca2+ mobilization and Ca2+-induced contractile response sensitivity were evaluated in skinned cardiac fibers. Muscarinic type 2 receptor (M2R), β1-adrenergic receptor (β1AR), sarcoplasmic Ca2+ ATPase (SERCA-2a), type 2 ryanodine receptor (RyR2), L-type Ca2+ channel (CACNA1), Kv4.2 (KCND2), and Kv4.3 (KCND3) mRNA expression levels were measured by quantitative RT-PCR. ResultsCompared with CTL group, DECA group exhibited decreased high frequency band power density (HF) and increased low frequency power density (LF), Cardiac M2R mRNA level was decreased. QTc interval at 2nd, 4th, and 8th week as well as APD30 and APD90 were increased by DECA. Ito density was decreased, while ICaL density was increased by DECA. SR Ca2+ loading and release were decreased by DECA, while contractile sensitivity to Ca2+ was increased versus CTL group. ConclusionDECA overdose induced cardiac rhythmic and mechanical abnormalities that can be associated with autonomic imbalance, up-regulated ICaL and down-regulated Ito, abnormal SR Ca2+ mobilization, and increased contractile sensitivity to Ca2+.

Quantification of Protein Expression Changes in the PKC‐epsilon/Ca2+/CaMKII‐alpha Pathway after Early and Late Onset of Ischemic‐reperfusion Injury in Adult Male Rats

BackgroundAccording to 2018 AHA/ASA guidelines, stroke remains a leading cause of death and long‐term disability in the U.S. and worldwide. Despite prevention efforts, risk factors such as hypertension attributes to worse clinical outcomes. The therapeutic window for stroke is limited to fibrinolytic therapy. Protein Kinase C‐epsilon activity provides resistance against oxidative stress.ObjectiveThe objective of this study was to test the hypothesis that the cell‐specific Protein Kinase C‐epsilon /Ca2+/calmodulin‐dependent protein kinase II‐alpha (CaMKII‐alpha) system contributes to homeostatic dysfunction, glial hypersensitivity, and cell death mechanisms. Here, we aimed to investigate key protein expression changes in tissue after middle cerebral artery (MCA) onset in animals. Our primary and secondary endpoints assess significant expression differences for astrocyte‐specific glial fibrillary acidic protein and microglia/macrophage‐specific ionized calcium adaptor molecule 1.MethodsWe used a transient MCA occlusion model to quantify cell and tissue‐type‐specific expression changes by Western blot after oxygen deprivation of N2:O2 (99:1) and ischemic‐reperfusion intervals of 4, 14, 24, 48, 72‐hrs after a 90‐min occlusion. Neuroprotective potential is determined by Protein Kinase C‐epsilon expression after reperfusion onset. IHC and TTC staining is used to evaluate lesion volume and metabolic activity. All experiments were randomized and double‐blinded.ResultsOxygen‐deprived cell lines and tissue were confirmed by HIF1‐alpha expression and distribution. Global expression changes reveal an inverse correlation between Protein Kinase C‐epsilon and glial immunoreactivity. Calcium‐independent Protein Kinase C‐epsilon expression levels are most significant at 24‐hrs after MCA reperfusion onset in cortical and subcortical tissue.ConclusionOur data provides new evidence of a tissue‐ and cell‐type‐specific mechanism of protein regulation in the Protein Kinase C‐epsilon /Ca2+/CaMKII‐alpha pathway after stroke. The findings suggest an inverse relationship between glial activation and Protein Kinase C‐epsilon expression. Reduced Protein Kinase C‐epsilon expression in cells and tissue may exacerbate gliosis. Significant changes to Protein Kinase C‐epsilon and CaMKII‐alpha activities in tissue often results in abnormal Ca2+ homeostasis, dysfunctional calcium‐dependent potassium conductance, and cytoskeletal deformation. Additional studies are needed to further elucidate the complex role of Ca2+ and its regulation in the context of acute and chronic neurodegenerative conditions.Support or Funding InformationThis project was funded by the U.S. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (Grant K01 DK099617; to A.A.A.) and the University of Florida College of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Large number tracking of depth and mechanics dependent calcium signaling in chondrocytes of articular cartilage

Purpose: Moderate loading of cartilage stimulates chondrocytes to synthetize matrix proteins, but excessive loading can lead to apoptosis. It is well known that macroscale strains affect cell health, but the mechanism behind how tissue level strain causes cellular and subcellular biological response is poorly understood. Previous work done by our group showed impact-induced tissue strain resulting in mitochondrial depolarization in chondrocytes, with correlation between strain and chondrocyte death, demonstrating the importance of microscale mechanics driving chondrocyte fate after impact. The correlation between strain and mitochondrial dysfunction indicates the mitochondria’s role in mechanosensing. However, the mechanotransduction and cell signaling pathway driving this process has not been fully characterized. In traumatic brain injury, Ca2+ signaling is known to produce mitochondrial swelling, while in other tissues Ca2+ is buffered by mitochondria. However, if too much Ca2+ is taken up, the mitochondrial permeability transition pore will open, leading to depolarization and cell death. In brain injury, this process can be averted if the mitochondrial swelling is addressed immediately. This indicates an opportunity to introduce mitoprotective therapies in a similar vein in articular cartilage. To better understand chondrocyte fate and investigate the process of mitochondrial depolarization after strain, an understanding of the Ca2+ signaling during mechanical stimulation is required. We predict that chondrocytes in different regions of cartilage will show different Ca2+ signal responses, in relation to the depth dependent compressive and shear properties of the tissue. In order to test this prediction, we compare changes in Ca2+ signaling under various magnitudes of compressive and shear strain, and compare the signaling response with previous work that determined impact-induced strain thresholds for mitochondrial dysfunction and chondrocyte death. Methods: Explants of articular cartilage were sterilely dissected from the femoral condyles of neonatal calves. The explants were cultured in a solution of DMEM and Antibiotic-Antimycotic for 24 hours. After removal from culture, the explants were vertically bisected into hemicylinders. They were stained for Ca2+ ions in 5μM Fluo-8 AM for one hour at 37° C, and rinsed in pure DMEM three times for ten minutes each to remove excess stain. The bisected samples were affixed to a custom-built Tissue Deformation Imaging Stage (TDIS) that can apply compressive and shear strain while directly imaging the tissue on a Zeiss LSM 510 inverted confocal microscope [Fig. 1]. Each sample was subjected to compression and shear. Recordings were made for 1000s while uncompressed, immediately following compression, post-relaxation, and immediately following shear. From these videos we were able to extract cell locations as a function of time through a centroid tracking algorithm [Fig. 2]. We extracted location and intensity information for all cells framed within a 636× 636μm frame (∼1000 cells per video), and identified cells showing Ca2+ transients. Local strain was calculated using a 2D digital image correlation program and the intensity curve of each cell was matched with the corresponding local strain. Results: Cell tracking showed different families of curves in Ca2+ signal intensity for cells located in the superficial (0 to 200μm depth) and middle (> 200μm depth) regions [Fig. 3], as well as multiple different families of cell signals present for uncompressed, compressed, and sheared samples [Fig. 4]. Increased proportion of cells showing Ca2+signaling was observed at greater compressive strain and in comparison with unstrained samples, indicating a threshold at which abnormal Ca2+ signaling occurs. Conclusions: The development of a technique to track a large number of cells in a non-equilibrium state is an improvement upon previous methods. This method allows for the study of transient signals despite changes in location and brightness, allows for large-scale tracking of cells for population statistics and provides the basis to directly study the relationship between Ca2+ signaling and mitochondrial response. The observation of differences in cell Ca2+ signaling between the surface and middle zones before any tissue strain was applied reinforces the idea that the superficial layer has a protective role for articular cartilage. This is in agreement with previous work showing that removal of the superficial layer allowed strain to penetrate deeper into the tissue. The identification of strain thresholds for increased Ca2+ signaling aligns with previous work showing strain thresholds for mitochondrial depolarization and cell death, further promoting the role that Ca2+ has in the cellular response pathway to strain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

ER stress mediated degradation of diacylglycerol acyltransferase impairs mitochondrial functions in TMCO1 deficient cells

In eukaryotic cells, Endoplasmic Reticulum (ER) is an interconnected membranous organelle and plays important roles in protein synthesis and lipid metabolism. We have previously demonstrated that TMCO1 is an ER Ca2+ channel actively preventing ER Ca2+ overloading. Recently, we also found that TMCO1 deficiency in mouse granulosa cells (GCs) caused abnormal Ca2+ signaling, ER stress and enhanced reactive oxygen species (ROS). In this study, we further examined the roles of TMCO1 in lipid metabolism and mitochondrial functions. Intriguingly, we found that TMCO1 deletion reduced the number of lipid droplets (LDs) and the content of triglyceride (TG), which was due to ER stress associated degradation (ERAD) of the important enzyme in catalyzing TG synthesis, diacylglycerol acyltransferase 2 (DGAT2). Hypofunction in transforming non-esterification fatty acid (NEFA) to TG caused NEFA deposit, a potential risk of mitochondrial dysfunction. Furthermore, in TMCO1 deficient cells, mitochondria volume decreased and inefficient oxidative phosphorylation was detected, which underlined enhanced mitophagy and impaired mitochondrial functions. Taken these data together, we for the first time revealed the role of TMCO1 in regulating lipid-metabolism and mitochondrial function. This study may provide new insights into understanding TMCO1 defect syndrome.

Single-Cell RNA Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development.

SummaryNicotine, the main chemical constituent of tobacco, is highly detrimental to the developing fetus by increasing the risk of gestational complications and organ disorders. The effects of nicotine on human embryonic development and related mechanisms, however, remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of human embryonic stem cell (hESC)-derived embryoid body (EB) in the presence or absence of nicotine. Nicotine-induced lineage-specific responses and dysregulated cell-to-cell communication in EBs, shedding light on the adverse effects of nicotine on human embryonic development. In addition, nicotine reduced cell viability, increased reactive oxygen species (ROS), and altered cell cycling in EBs. Abnormal Ca2+ signaling was found in muscle cells upon nicotine exposure, as verified in hESC-derived cardiomyocytes. Consequently, our scRNA-seq data suggest direct adverse effects of nicotine on hESC differentiation at the single-cell level and offer a new method for evaluating drug and environmental toxicity on human embryonic development in utero.

The Impact and Mechanism of a Novel Allosteric AMPA Receptor Modulator LCX001 on Protection Against Respiratory Depression in Rodents.

Analgesics and sedative hypnotics in clinical use often give rise to significant side effects, particularly respiratory depression. For emergency use, specific antagonists are currently administered to counteract respiratory depression. However, antagonists are often short-lasting and eliminate drug generated analgesia. To resolve this issue, novel positive AMPA modulators, LCX001, was tested to alleviate respiratory depression triggered by different drugs. The acetic acid writhing and hot-plate test were conducted to evaluate analgesic effect of LCX001. Binding assay, whole-cell recording, live cell imaging, and Ca2+ imaging were used to clarify mechanism and impact of LCX001 on respiratory protection. Results showed that LCX001 effectively rescued and prevented opioid (fentanyl and TH-030418), propofol, and pentobarbital-induced respiratory depression by strengthening respiratory frequency and minute ventilation. The acetic acid writhing test and hot-plate test revealed potent anti-nociceptive efficacy of LCX001, in contrast to other typical ampakines that did not affect analgesia. Furthermore, LCX001 potentiated [3H]AMPA and L-glutamate binding affinity to AMPA receptors, and facilitated glutamate-evoked inward currents in HEK293 cells stably expressing GluA2(R). LCX001 had a typical positive modulatory impact on AMPAR-mediated function. Importantly, application of LCX001 generated a significant increase in GluA2(R) surface expression, and restrained opioid-induced abnormal intracellular Ca2+ load, which might participate in breathing modulation. Our study improves therapeutic interventions for the treatment of drug induced respiratory depression, and increases understanding of potential mechanism of AMPA receptor modulators.