L-type Calcium Channel Expression Research Articles

Hippocampal hyperphosphorylated tau-induced deficiency is rescued by L-type calcium channel blockade.

Aging and Alzheimer's disease are associated with chronic elevations in neuronal calcium influx via L-type calcium channels. The hippocampus, a primary memory encoding structure in the brain, is more vulnerable to calcium dysregulation in Alzheimer's disease. Recent research has suggested a link between L-type calcium channels and tau hyperphosphorylation. However, the precise mechanism of L-type calcium channel-mediated tau toxicity is not understood. In this study, we seeded a human tau pseudophosphorylated at 14 amino acid sites in rat hippocampal cornu ammonis 1 region to mimic soluble pretangle tau. Impaired spatial learning was observed in human tau pseudophosphorylated at 14 amino acid sites-infused rats as early as 1-3 months and worsened at 9-10 months post-infusion. Rats infused with wild-type human tau exhibited milder behavioural deficiency only at 9-10 months post-infusion. No tangles or plaques were observed in all time points examined in both human tau pseudophosphorylated at 14 amino acid sites and human tau-infused brains. However, human tau pseudophosphorylated at 14 amino acid sites-infused hippocampus exhibited a higher amount of tau phosphorylation at S262 and S356 than the human tau-infused rats at 3 months post-infusion, paralleling the behavioural deficiency observed in human tau pseudophosphorylated at 14 amino acid sites-infused rats. Neuroinflammation indexed by increased Iba1 in the cornu ammonis 1 was observed in human tau pseudophosphorylated at 14 amino acid sites-infused rats at 1-3 but not 9 months post-infusion. Spatial learning deficiency in human tau pseudophosphorylated at 14 amino acid sites-infused rats at 1-3 months post-infusion was paralleled by decreased neuronal excitability, impaired NMDA receptor-dependent long-term potentiation and augmented L-type calcium channel-dependent long-term potentiation at the cornu ammonis 1 synapses. L-type calcium channel expression was elevated in the soma of the cornu ammonis 1 neurons in human tau pseudophosphorylated at 14 amino acid sites-infused rats. Chronic L-type calcium channel blockade with nimodipine injections for 6 weeks normalized neuronal excitability and synaptic plasticity and rescued spatial learning deficiency in human tau pseudophosphorylated at 14 amino acid sites-infused rats. The early onset of L-type calcium channel-mediated pretangle tau pathology and rectification by nimodipine in our model have significant implications for preclinical Alzheimer's disease prevention and intervention.

Structural and electro-anatomical characterization of the equine pulmonary veins: implications for atrial fibrillation

Introduction/objectivesSpontaneous pulmonary vein (PV) activity triggers atrial fibrillation (AF) in humans. Although AF frequently occurs in horses, the origin remains unknown. This study investigated the structural and electro-anatomical properties of equine PVs to determine the potential presence of an arrhythmogenic substrate. Animals, materials and methodsEndocardial three-dimensional electro-anatomical mapping (EnSite Precision) using high-density (HD) catheters was performed in 13 sedated horses in sinus rhythm. Left atrium (LA) access was obtained retrogradely through the carotid artery. Post-mortem, tissue was harvested from the LA, right atrium (RA), and PVs for histological characterization and quantification of ion channel expression using immunohistochemical analysis. ResultsGeometry, activation maps, and voltage maps of the PVs were created and a median of four ostia were identified. Areas of reduced conduction were found at the veno-atrial junction. The mean myocardial sleeve length varied from 28 ± 13 to 49 ± 22 mm. The PV voltage was 1.2 ± 1.4 mV and lower than the LA (3.4 ± 0.9 mV, P < 0.001). The fibrosis percentage was higher in PV myocardium (26.1 ± 6.6%) than LA (14.5 ± 5.0%, P = 0.003). L-type calcium channel (CaV1.2) expression was higher in PVs than LA (P = 0.001). T-type calcium channels (CaV3.3), connexin-43, ryanodine receptor-2, and small conductance calcium-activated potassium channel-3 was expressed in PVs. ConclusionsThe veno-atrial junction had lower voltages, increased structural heterogeneity and areas of slower conduction. Myocardial sleeves had variable lengths, and a different ion channel expression compared to the atria. Heterogeneous properties of the PVs interacting with the adjacent LA likely provide the milieu for re-entry and AF initiation.

Aging impairs the clustering and reduces the activity of the L-type calcium channels in the heart's pacemaker

Every heartbeat is initiated by a spontaneous electrical signal generated inside the cardiac pacemaker. In mammals, including humans, aging leads to the slowdown of the pacemaker rate, which can lead to pathological dysfunction and death. However, the mechanisms behind the age-associated slowdown of the pacemaker are not well understood. The automaticity of the pacemaker relies on a finely tuned balance of ionic currents, where L-type calcium channels play a central role. CaV1.2 and CaV1.3 channels are key players involved in the diastolic depolarization phase and action potential upstroke. In this project, we studied the effects of aging on CaV1.2 and CaV1.3 calcium channel function and regulation. Here we recorded the pacemaker activity and calcium currents on isolated spindle pacemaker cells with patch clamp. The clustering of LTCC has been highlighted with Super-resolution microscopy (GSD). We found that in mouse pacemaker cells, aging causes a reduction of calcium entry through L-type calcium channels, which is not accompanied by a reduction of protein expression. In turn, we found that aging disrupts calcium channel function at different levels. First, aging reduces the expression of L-type calcium channels at the plasma membrane. Second, aging reduces the clustering and nano-organization of L-type calcium channels. And finally, aging reduces the open channel probability, a sign of decreased functional coupling. Moreover, aging also alters the association between L-type calcium channels and beta-adrenergic receptors, altering the adrenergic modulation of the pacemaker cells. Next, we tested the hypothesis that reduced open probability of L-type calcium channels was responsible for a slower firing frequency. Interestingly, we found that the application of BayK was enough to restore intrinsic pacemaker rates in old cells to those levels observed in young cells. Overall, our findings provide evidence that the proper organization and function of L-type calcium channels is affected by aging and that this dysfunction contributes to the slowdown of pacemaker cells in old animals.

Effects of Danshensu Sodium Combined with Tongxinluo on Cardiac Function and Calcium Channels in a Rat Model of Heart Failure

The study aimed to investigate the protective effects of Danshensu sodium and Tongxinluo on cardiac function, apelin-13, and L-type calcium channel in a rat model of heart failure. Thirty-two rats were divided into three groups: Danshensu Sodium, Tongxinluo, and Experimental (combination of Danshensu Sodium and Tongxinluo). Twenty-four rat models of heart failure were treated with Danshensu Sodium, Tongxinluo, or Danshensu Sodium+Tongxinluo. Cardiac function, apelin-13, and L-type calcium channel expression were compared among the four groups. The injury and repair of myocardial tissue were observed by HE staining, and the fibrosis of myocardial tissues was observed by Masson staining. The results showed that the combination of Danshensu sodium and Tongxinluo significantly improved cardiac function, decreased apelin-13 levels, decreased L-type calcium channel current density, and increased Cav1.2 protein expression compared to the Danshensu Sodium group and Tongxinluo group. Histological observations showed that the morphology and structure of cardiomyocytes were better, and the fibrosis of myocardium was lower in the experimental group compared to the other two groups. In conclusion, Danshensu sodium and Tongxinluo combined effectively improved cardiac function and had a positive effect on the expression of apelin-13 and L-type calcium channel in rats with heart failure.

Cholecalciferol affects cardiac proteins regulating malonyl-CoA availability and intracellular calcium level.

Cholecalciferol improves insulin signaling and glucose metabolism in the heart and reduces circulating non-esterified fatty acids. Cholecalciferol effects on the cardiac fatty acid (FA) metabolism and the consequences on calcium handling were examined. Blood lipid profile was determined. Western blot and qRT-PCR were used to examine protein and mRNA expression. Cholecalciferoltreated rats had increased acetyl CoA carboxylase 2 protein expression and decreased expression of malonyl CoA decarboxylase. In addition, the expression of uncoupling protein 3 was elevated. Also, the level of peroxisome proliferator-activated receptor-gamma coactivator in the nucleus of heart cells was increased along with the level of sarcoplasmic/endoplasmic reticulum Ca2+ATPase in the microsomal fraction. In parallel, the L-type calcium channel and ryanodine receptor expression was reduced. In the heart of healthy rats, cholecalciferol affects proteins regulating malonyl CoA availability and intracellular Ca2+ handling proteins.

Excitation and contraction of cardiac muscle and coronary arteries of brain-dead pigs.

Excitability and contraction of cardiac muscle from brain-dead donors critically influence the success of heart transplantation. Membrane physiology, Ca2+-handling, and force production of cardiac muscle and the contractile properties of coronary arteries were studied in hearts of brain-dead pigs. Cardiac muscle and vascular function after 12 h brain death (decapitation between C2 and C3) were compared with properties of fresh tissue. In both isolated cardiomyocytes (whole-cell patch clamp) and trabecular muscle (conventional microelectrodes), action potential duration was shorter in brain dead, compared to controls. Cellular shortening and Ca2+ transients were attenuated in the brain dead, and linked to lower mRNA expression of L-type calcium channels and a slightly lower ICa,L, current, as well as to a lower expression of phospholamban. The current-voltage relationship and the current above the equilibrium potential of the inward K+ (IK1) channel were altered in the brain-dead group, associated with lower mRNA expression of the Kir2.2 channel. Delayed K+ currents were detected (IKr, IKs) and were not different between groups. The transient outward K+ current (Ito) was not observed in the pig heart. Coronary arteries exhibited increased contractility and sensitivity to the thromboxane analogue (U46619), and unaltered endothelial relaxation. In conclusion, brain death involves changes in cardiac cellular excitation which might lower contractility after transplantation. Changes in the inward rectifier K+ channel can be associated with an increased risk for arrhythmia. Increased reactivity of coronary arteries may lead to increased risk of vascular spasm, although endothelial relaxant function was well preserved.

L‐Type calcium channel antagonist isradipine decreases area of plaque associated dystrophic neurites in 5XFAD mouse model

AbstractBackgroundEpidemiological studies suggest that L‐type calcium channel (LTCC) antagonists may reduce the incidence of neurodegenerative diseases including Alzheimer’s disease (AD). However, the neuroprotective mechanism of LTCC antagonists is unknown. Amyloid‐β (aβ) increases intracellular calcium signaling, which regulates lysosomal processing and microglial inflammatory responses, both of which are altered in AD. Neurons near aβ plaques develop dystrophic neurites, which are abnormal axonal swellings that accumulate lysosomes. Further, microglia accumulate around aβ plaques and secrete inflammatory cytokines. We hypothesized that antagonism of LTCCs with isradipine would reduce aβ plaque associated dystrophic neurites and inflammatory microglia in the 5XFAD mouse model of AD by restoring normal intracellular calcium regulation.MethodWe treated 6‐ and 9‐month‐old 5XFAD mice with isradipine (3 mg/kg/day) or vehicle via subcutaneous pellets for 30 days. Following three weeks of treatment we tested locomotion and cognitive behavior via open field, novel object recognition and Morris water maze. We measured gene expression of inflammatory mediators (IL1β, TNF, CD68) and LTCC subunits (Cav1.2, Cav1.3) using qPCR. Using immunohistochemistry (IHC), we examined aβ plaque number and area (D54D2 and ThioS), microglia area (Iba1), dystrophic neurites area and particle number (LAMP1) normalized to total brain area.ResultIHC revealed that isradipine treatment in 9‐month‐old 5XFADs decreased the total area of LAMP1 positive dystrophic neurites (p = 0.0310) compared to vehicle‐treated 5XFADs; this change was not observed in the 6‐month‐old group. Isradipine treatment in the 9‐month‐old 5XFADs also led to a decreasing trend (p = 0.0552) in plaque load compared to vehicle‐treated 5xFADs as measured by IHC. Isradipine treatment did not modulate plaque associated microglia as measured by IHC or inflammatory gene expression as measured by qPCR, regardless of age. There was no treatment effect on behavior or expression of LTCC subunits.ConclusionOur data show that isradipine reduces aβ plaque associated dystrophic neurite pathology in 9‐month‐old 5XFAD mice, potentially showing LTCC play a role in mediating this specific phenotype. Our future studies will focus on understanding why antagonizing LTCCs leads to a decrease in LAMP1 and whether this is due to changes in lysosomal development and processing or other possible mechanisms.

Effects of bisphenol A on human umbilical arteries.

Bisphenol A (BPA) is an endocrine-disrupting chemical widely used in the plastics industry, including food container, toys, and medical equipment. We analyzed the effect of BPA in human umbilical artery contractility and expression of some proteins modulating this function, such as ionic channels and proteins involved in the cGMP pathway. Using standard organ bath technique, rings of human umbilical arteries without endothelium were contracted by 5-HT (1μM) and histamine (10μM) and the effect of different concentrations of BPA (1nM-100μM) was analyzed. The results showed that BPA is a vasodilator of these arteries in a concentration-dependent way. Besides, qPCR studies on human umbilical smooth muscle cells (HUSMC) allowed to analyze the effects of BPA on gene expression. Thus, 12-h exposition to BPA induced reduction of expression of L-type calcium channels (LTCC), alpha subunit of BKCa channels, and Kvβ1 and Kvβ3 from Kv channels. BPA also decreased the expression of soluble guanylate cyclase (sGC) and natriuretic peptide receptor type A (NPRA), meanwhile increasing that of PKG, proteins involved in vasodilation of human umbilical arteries (HUA) by cGMP. Further studies will be necessary to increase knowledge about the implications of these changes induced by BPA exposure.

P1513: LOW VITAMIN D LEVELS ARE ASSOCIATED WITH INCREASED CARDIAC IRON UPTAKE IN THALASSEMIA MAJOR.

Background: Decreased vitamin D (VitD) levels stimulate the expression of the transmembrane L-type voltage-dependent calcium channels (LVDCC), which, in addition to calcium, absorb iron. However, there is little and controversial data on the correlation of VitD levels with cardiac iron. Aims: We investigated the association between vitamin D levels and cardiac iron and function in thalassemia major (TM) patients. Methods: We considered 278 TM patients (39.04±8.58 years, 56.8% females), consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia Network. Myocardial iron overload (MIO) was quantified using a T2* segmental approach. Left ventricular (LV) function parameters were quantified by cine images. Measurement of serum 25(OH)D was carried out using a chemiluminescent immunoassay. Results: Vitamin D supplements were taken by 61.4% of our patients. VitD levels were deficient (<20 ng/dl) in 107 (38.5%) patients, insufficient (20–30 ng/dl) in 96 (34.5%) patients and adequate (>30 ng/dl) in 75 (27.0%) patients. Patients with deficient vitD levels were significantly younger than patients with adequate (36.96±7.64 yrs vs 41.26±8.19 yrs; P<0.0001) as well as insufficient vitD levels (36.96±7.64 yrs vs 39.63±9.39 yrs; P=0.042) while no difference was detected in male to female ratio. MIO (global heart T2*<20 ms) was significantly more frequent in patients with deficient VitD than in patients with adequate and insufficient VitD levels (Figure). Patients with deficient vitD levels had a significant higher risk of MIO than patients with adequate vitD levels (odds ratio-OR=20.62, 95%CI=2.67-153.72; P=0.004) and patients with insufficient VitD levels (OR=8.49, 95%CI=2.46-29.29; P=0.001). At receiver operating characteristic curve analysis, a vitD level≤ 17.3 ng/dl predicted MIO with a sensitivity of 81.5% and a specificity of 75.3% (P<0.0001). The area under the curve was 0.79 (95% confidence interval: 0.74–0.84). Compared to patients with normal VitD levels, patients with deficient vitamin levels showed significantly higher LV end-diastolic volume index (87.61±19.19 ml/m2 vs 79.17±14.27 ml/m2; P=0.003) and LV mass index (63.39±13.55 g/m2 vs 57.45±12.90 g/m2; P=0.006). The LV ejection fraction tended to be lower in patients with VitD deficiency, but no statistical difference versus the other groups was detected. Image:Summary/Conclusion: In thalassemia major vitamin D deficiency is associated with and increased risk of cardiac iron overload. Periodic and regular assessment of VitD levels can be useful in the prevention not only of bone disorders but also of cardiac iron accumulation and subsequent overt dysfunction.

Modulation of LTCC Pathways by a Melusin Mimetic Increases Ventricular Contractility During LPS-Induced Cardiomyopathy.

Sepsis-induced cardiomyopathy is commonplace and carries an increased risk of death. Melusin, a cardiac muscle-specific chaperone, exerts cardioprotective function under varied stressful conditions through activation of the AKT pathway. The objective of this study was to determine the role of melusin in the pathogenesis of lipopolysaccharide (LPS)-induced cardiac dysfunction and to explore its signaling pathway for the identification of putative therapeutic targets. Prospective, randomized, controlled experimental study in a research laboratory. Melusin overexpressing (MelOV) and wild-type (MelWT) mice were used. MelOV and MelWT mice were injected intraperitoneally with LPS. Cardiac function was assessed using trans-thoracic echocardiography. Myocardial expression of L-type calcium channel (LTCC), phospho-Akt and phospho-Gsk3-b were also measured. In separate experiments, wild-type mice were treated post-LPS challenge with the allosteric Akt inhibitor Arq092 and a mimetic peptide (R7W-MP) targeting the LTCC. The impact of these therapies on protein-protein interactions, cardiac function, and survival was assessed. MelOV mice had limited derangement in cardiac function after LPS challenge. Protection was associated with higher Akt and Gsk3-b phosphorylation and restored LTCC density. Pharmacological inhibition of Akt activity reversed melusin-dependent cardiac protection. Treatment with R7W-MP preserved cardiac function in wild-type mice after LPS challenge and significantly improved survival. This study identifies AKT / Melusin as a key pathway for preserving cardiac function following LPS challenge. The cell-permeable mimetic peptide (R7W-MP) represents a putative therapeutic for sepsis-induced cardiomyopathy.

Alzheimer's Disease Risk Gene BIN1 Modulates Neural Network Activity Through the Regulation of L-Type Calcium Channel Expression in Human Induced Neurons

Alzheimer's Disease Risk Gene BIN1 Modulates Neural Network Activity Through the Regulation of L-Type Calcium Channel Expression in Human Induced Neurons

Tachypacing-induced CREB/CD44 signaling contributes to the suppression of L-type calcium channel expression and the development of atrial remodeling.

Atrial fibrillation (AF), a common arrhythmia in clinics, is characterized as downregulation of L-type calcium channel (LTCC) and shortening of atrial action potential duration (APD). Our prior studies have shown the association of CD44 with AF genesis. The purpose of this study was to explore the potential role of CD44 and its related signaling in tachypacing-induced downregulation of LTCC. Invitro, tachypacing in atrium-derived myocytes (HL-1 cell line) induced activation (phosphorylation) of cyclic adenosine monophosphate response element-binding protein (CREB). Furthermore, tachypacing promoted an association between CREB and CD44 in HL-1 myocytes, which was documented in atrial tissues from patients with AF. Deletion and mutational analysis of the LTCC promoter along with chromatin immunoprecipitation revealed that cyclic adenosine monophosphate response element is essential for tachypacing-inhibited LTCC transcription. Tachypacing also hindered the binding of p-CREB to the promoter of LTCC. Blockade of CREB/CD44 signaling in HL-1 cells attenuated tachypacing-triggered downregulation of LTCC and shortening of APD. Atrial myocytes isolated from CD44-/- mice exhibited higher LTCC current and longer APD than did those from wild-type mice. Exvivo, tachypacing caused less activation of CREB in CD44-/- mice than in wild-type mice. Invivo, burst atrial pacing stimulated less inducibility of AF in CREB inhibitor-treated mice than in controls. Tachypacing-induced CREB/CD44 signaling contributes to the suppression of LTCC, which provides valuable information about the pathogenesis of atrial modeling and AF.

Antidepressant Actions of Ketamine: Potential Role of L-Type Calcium Channels.

Recently, the United States Food and Drug Administration approved esketamine, the S-enantiomer of ketamine, as a fast-acting therapeutic drug for treatment-resistant depression. Although ketamine is known as an N-methyl-d-aspartate (NMDA) receptor antagonist, the underlying mechanisms of how it elicits an antidepressant effect, specifically at subanesthetic doses, are not clear and remain an advancing field of research interest. On the other hand, high-dose (more than the anesthetic dose) ketamine-induced neurotoxicity in animal models has been reported. There has been progress in understanding the potential pathways involved in ketamine-induced antidepressant effects, some of which include NMDA-receptor antagonism, modulation of voltage-gated calcium channels, and brain-derived neurotrophic factor (BDNF) signaling. Often these pathways have been shown to be linked. Voltage-gated L-type calcium channels have been shown to mediate the rapid-acting antidepressant effects of ketamine, especially involving induction of BDNF synthesis downstream, while BDNF deficiency decreases the expression of L-type calcium channels. This review focuses on the reported studies linking ketamine's rapid-acting antidepressant actions to L-type calcium channels with an objective to present a perspective on the importance of the modulation of intracellular calcium in mediating the effects of subanesthetic (antidepressant) versus high-dose ketamine (anesthetic and potential neurotoxicant), the latter having the ability to reduce intracellular calcium by blocking the calcium-permeable NMDA receptors, which is implicated in potential neurotoxicity.

In utero hypoxia altered Ang II-induced contraction via PKCβ in fetal cerebral arteries.

Cerebral circulation is important in fetal brain development, and angiotensin II (Ang II) plays vital roles in regulation of adult cerebral circulation. However, functions of Ang II in fetal cerebral vasculature and influences of in utero hypoxia on Ang II-mediated fetal cerebral vascular responses are largely unknown. This study investigated the effects and mechanisms of in utero hypoxia on fetal middle cerebral arteries (MCA) via Ang II. Near-term ovine fetuses were exposed to in utero hypoxia, and fetal MCA responses to Ang II were tested for vascular tension, calcium transient, and molecular analysis. Ang II caused significant dose-dependent contraction in control fetal MCA. Ang II-induced MCA constriction was decreased significantly in hypoxic fetuses. Neither losartan (AT1R antagonist, 10-5 mol/L) nor PD123,319 (AT2R antagonist, 10-5 mol/L) altered Ang II-mediated contraction in fetal MCA. Phenylephrine-mediated constriction was also significantly weaker in hypoxic fetuses. Bay K8644 caused similar contractions between the two groups. Protein expression of L-type voltage-dependent calcium channels was unchanged. There were no differences in caffeine-mediated vascular tension or calcium transients. Contraction induced by PDBu (PKC agonist) was obviously weaker in hypoxic MCA. Protein expression of PKCβ was reduced in the hypoxic compared with the control, along with no differences in phosphorylation levels. The results showed that fetal MCA was functionally responsive to Ang II near term. Intrauterine hypoxia reduced the vascular agonist-mediated contraction in fetal MCA, probably via decreasing PKCβ and its phosphorylation, which might play protective effects on fetal cerebral circulation against transient hypoxia.

Ca2+ channel subunit a 1D inhibits endometriosis cell apoptosis and mediated by prostaglandin E2.

Endometriosis is considered as a chronic pelvic inflammatory disease and prostaglandin E2(PGE2) (a kind of the inflammatory cytokines) was increased in the endometriosis patient's peritoneal fluid . Ca2+ signal and Ca2+ channels play an important role in cell apoptosis. This study was to explore the L-type calcium channel (Cav1.3) expression and its biological function in endometriosis. Furthermore the molecular mechanism between Cav1.3 and PGE2 was also clarified. The real-time PCR and immunohistochemical were used to detect the expression of Cav1.3. Apoptosis was detected by Flow cytometry assay and Western blot assay. Cav1.3 was high expression in endometriosis tissue and primary endometrial stromal cells (hEM15A). Treatment with PGE2 rapidly inhibited apoptosis and increased Cav1.3 expression in hEM15A . The silencing of Cav1.3 promoted apoptosis, which was unchanged after PGE2 treatment. Moreover, the inhibition of Cav1.3 by shRNA transfection activated cleaved PARP and cleaved caspase-3. These available evidences suggest that Cav1.3 is required for PGE2 induction apoptosis and relates to the pathophysiology of endometriosis. Interference with Cav1.3 may offer a neo-therapeutic window in endometriosis treatment.

Sleep deprivation of rats increases postsurgical expression and activity of L-type calcium channel in the dorsal root ganglion and slows recovery from postsurgical pain

Perioperative sleep disturbance is a risk factor for persistent pain after surgery. Clinical studies have shown that patients with insufficient sleep before and after surgery experience more intense and long-lasting postoperative pain. We hypothesize that sleep deprivation alters L-type calcium channels in the dorsal root ganglia (DRG), thus delaying the recovery from post-surgical pain. To verify this hypothesis, and to identify new predictors and therapeutic targets for persistent postoperative pain, we first established a model of postsurgical pain with perioperative sleep deprivation (SD) by administering hind paw plantar incision to sleep deprivation rats. Then we conducted behavioral tests, including tests with von Frey filaments and a laser heat test, to verify sensory pain, measured the expression of L-type calcium channels using western blotting and immunofluorescence of dorsal root ganglia (an important neural target for peripheral nociception), and examined the activity of L-type calcium channels and neuron excitability using electrophysiological measurements. We validated the findings by performing intraperitoneal injections of calcium channel blockers and microinjections of dorsal root ganglion cells with adeno-associated virus. We found that short-term sleep deprivation before and after surgery increased expression and activity of L-type calcium channels in the lumbar dorsal root ganglia, and delayed recovery from postsurgical pain. Blocking these channels reduced impact of sleep deprivation. We conclude that the increased expression and activity of L-type calcium channels is associated with the sleep deprivation-mediated prolongation of postoperative pain. L-type calcium channels are thus a potential target for management of postoperative pain.

NAKα2 inhibits fibrosis formation and protects against cardiomyocyte hypertrophy by suppressing hypertrophy associated molecules and activating LTCC/NCX signaling pathway.

Cardiomyocyte hypertrophy is considered to be a compensatory process of heart suffering from pathological damages. This study aimed to evaluate effects of Na+/K+ APTaseα2 (NAKα2) on isoprenaline (ISO) induced cardiomyocyte hypertrophy. Mouse atrial cardiomyocytes were cultured and treated with ISO to establish cardiomyocyte hypertrophy model. NAKα2 over-expression and small interfere RNA (siRNA) plasmids were constructed and transfected to cardiomyocytes. Influx Ca2+ ([Ca2+]i) was measured using flow cytometry method. Fibrosis formation was examined with Masson staining. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was used to examine apoptosis. Major histocompatibility complex β (β-MHC), atrial natriuretic peptides (ANP), B-type natriuretic peptides (BNP) were evaluated with quantitative Real-time PCR (qRT-PCR). Western blot was used to detect β-MHC, ANP, BNP, Na+/Ca2+ exchanger (NCX) and L-type calcium channel (LTCC). NAKα2 significantly inhibited NCX and LTCC expression compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly suppressed expression of β-MHC, ANP and BNP compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly alleviated fibrosis formation and inhibited apoptosis compared to that in ISO-treated cardiomyocytes (p<0.05). NAKα2 reduced intracellular calcineurin and activated phosphorylation of calcineurin-nuclear factor of activated T cells (NFAT) compared to ISO-treated cardiomyocytes (p<0.05). NAKα2 significantly strengthened effects of Klotho on ISO-induced up-regulation of hypertrophy associated molecules (p<0.05) by activating LTCC and NCX. Comparing to ISO-treated cardiomyocytes, NAKα2 combining Klotho treatment exhibited significantly better improvement of Ca2+ influx, alleviation of fibrosis and reduction of apoptosis by triggering LTCC/NCX signaling pathway. Over-expression of NKAα2 suppressed fibrosis formation and protected against cardiomyocyte hypertrophy by inhibiting hypertrophy associated molecules, alleviating apoptosis and activating LTCC/NCX signaling pathway.

Matrix Stiffening and EGFR Cooperate to Promote the Collective Invasion of Cancer Cells.

In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg Cancer Res; 78(18); 5229-42. ©2018 AACR.

Abstract 243: Modeling of Diastolic Dysfunction in Induced Pluripotent Stem Cell-derived Cardiomyocytes From Hypertrophic Cardiomyopathy Patients

Aims: Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. Yet 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 and as a platform for drug discovery. Methods and results: In the present study, beating iPSC-CMs were generated from healthy recruits and HCM patients with evident DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, retarded relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca 2+ imaging indicated elevated diastolic [Ca 2+ ] i and abnormal Ca 2+ handling in HCM iPSC-CMs, which were exacerbated by β-adrenergic challenge. Combining Ca 2+ imaging and traction force microscopy (TFM), we observed enhanced myofilament Ca 2+ sensitivity (measured as dF/Δ[Ca 2+ ] i ) in HCM iPSC-CMs. These results indicated that cytosolic diastolic Ca 2+ overload, slowed [Ca 2+ ] i decline and increased myofilament Ca 2+ sensitivity, collectively impair the relaxation of HCM cells. Treating HCM iPSC-CMs with partial blockade of Ca 2+ or late Na + current reset diastolic Ca 2+ homeostasis, restored diastolic function and improved long-term survival, suggesting disturbed Ca 2+ signaling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type calcium channel (LTCC) and transient receptor potential channels (TRPCs) in HCM iPSC-CMs, which likely contribute to diastolic [Ca 2+ ] i overload. Conclusion: In summary, this study recapitulated DD at the single cell level, and revealed novel mechanisms and potential therapeutic targets of DD using HCM iPSC-CMs.

LRP5 controls cardiac QT interval by modulating the metabolic homeostasis of L-type calcium channel

BackgroundLow-density lipoprotein receptor-related protein 5 (LRP5) has been intensively studied as a co-receptor for β-catenin-dependent Wnt signaling. Emerging evidences have demonstrated β-catenin-independent functions of LRP5. However, the biological role of LRP5 in the mammalian heart is largely unknown. Methods and resultsConditional cardiac-specific Lrp5 knockout (Lrp5-CKO) mice were generated by crossing Lrp5flox/flox mice with αMHC/MerCreMer mice. Lrp5-CKO mice consistently displayed normal cardiac structure and function. Telemetric electrocardiogram recordings revealed a short QT interval in Lrp5-CKO mice, which was tightly linked to the striking abbreviation of action potential duration (APD) in ventricular myocytes. The analysis of whole-cell currents indicated that a reduction in activity and protein expression of L-type calcium channel (LTCC), rather than other ion channels, contributed to the abnormality in APD. Furthermore, we showed that Lrp5 ablation induced a significant convergence of CaV1.2α1c proteins to the endoplasmic reticulum. Consequently, increased proteasomal degradation of these proteins was observed, which was independent of the Wnt/β-catenin signaling pathway. ConclusionsLRP5 directly modulates the degradation of LTCC to control cardiac QT interval. These findings provide compelling evidence for the potential role of LRPs in cardiac electrophysiology.