L-type Calcium Channel Research Articles

Renal Function in Hypertensive Patients Receiving Cilnidipine and L-Type Calcium Channel Blockers: A Meta-Analysis of Randomized Controlled and Retrospective Studies

Nearly 65%-95% of chronic kidney disease (CKD) patients have hypertension. Calcium-channel blockers are the first-line drugs for the treatment of hypertension, including hypertension with diabetes. This study aims to estimate the effect of an L-type calcium channel blocker (CCB), cilnidipine, on the renal function of hypertensive patients. Randomized control trials were selected from PubMed, Embase, Google Scholar, China National Knowledge Infrastructure (CNKI), Science Direct, Elton B. Stephens Company (EBSCO), Springer, Ovid, Cochrane Library, Medline, VIP, and Wanfang databases (from the date of databases' establishment till January 2022). Data were independently evaluated following the Cochrane risk-of-bias tool. The changes in serum creatinine (SCr), urinary protein excretion (UPE), urinary protein/creatinine ratio (UPCR), and estimated glomerular filtration rate (eGFR) before and after treatment, in percentages, were extracted for the meta-analysis. The mean difference (MD) and a CI of 95% were determined using RevMan 5.3 software. A total of 11 studies were analyzed. The standardized mean difference (SMD) between cilnidipine and L-type CCBs was -0.022, suggesting a reduced SCr with cilnidipine. For UPCR, the SMD value is 1.28. Although cilnidipine reduced UPCR in all four studies, the L-type CCBs reported a slight increase in UPCR. For eGFR, the SMD value was found to be 0.693. Cilnidipine had a more favorable effect on eGFR when compared to the L-type CCBs. While cilnidipine had similar effects on SCr to that of L-type CCBs, cilnidipine showed greater improvement in UPCR, UPE, and eGFR values.

Abstract P1086: Lrrc10 Is A Multifaceted Modulator Of Cardiac Calcium And Sodium Channels

L-type calcium channels (LTCC) are vital for heart physiology as they allow entry of calcium to the cardiomyocyte, ultimately leading to contraction. These channels are exquisitely modulated by auxiliary subunits and a vast array of regulatory proteins. Heart-specific leucine-rich repeat-containing protein 10 (Lrrc10) plays a key role as a regulator of LTCC. Previous work has shown that Lrrc10 knockdown in zebrafish disrupts development of the cardiac tube, while ablation of Lrrc10 in mice results in heart dysfunction and maladaptation to increased cardiac afterload. Moreover, human mutations in Lrrc10 gene have been linked to dilated cardiomyopathy, and sudden unexplained nocturnal death syndrome (SUNDS). Still, the molecular mechanisms underlying Lrrc10 regulation of LTCC is not fully established. Beyond this, it is unknown whether Lrrc10 modulates other cardiac ion channels. Here, we hypothesized that Lrrc10 serves as a versatile regulator of four-domain voltage-gated ion channels, while mutations in Lrrc10 differentially disrupt channel regulation. Using whole-cell and single-channel electrophysiology, we found that Lrrc10: 1) upregulates LTCC currents by increasing peak open probability; 2) accelerates calcium-dependent inactivation; and 3) evokes a hyperpolarizing-shift in voltage-dependence of activation. Flow cytometric FRET 2-hybrid assay showed that Lrrc10 interacts with LTCC cytoplasmic subunits, specifically the N- and C-termini. We further used FRET 2-hybrid analysis to evaluate the interaction of Lrrc10 with related cardiac ion channels. We observed robust Lrrc10 interaction with Na V 1.5 channels. Multi-channel electrophysiology recordings revealed increased late sodium currents, suggesting that Lrrc10 also modulates Na V 1.5 activity. Additionally, electrophysiological studies showed that Lrrc10 variants linked to dilated cardiomyopathy and SUNDS disparately alter modulation of calcium and sodium channels. In conclusion, Lrrc10 is a multifaceted regulator of four-domain, voltage-gated cardiac ion channels, a finding that will lend critical insights into heart function and disease.

Abstract P1107: Lem2 Is Essential For Cardiac Development By Maintaining Nuclear Integrity

Background: integrity is essential for compartmentalisation of nucleus and cytoplasm. Importantly, mutations in -encoding genes are the second-highest cause of familial dilated cardiomyopathy. One such protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in heart remains poorly understood. Methods: We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue and cellular analyses. High resolution episcopic microscopy was used for 3D reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided physiological assessment of Lem2 iCKO adult mice. Results: We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels. Conversely, Lem2 ablation in adult cardiomyocytes did not lead to overt cardiac dysfunction up to 18 months of age. Conclusions: Our data suggest that Lem2 is critical for integrity at the nascent in fetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by Lem2 loss, perhaps owing to a more established and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general.

Abstract P1076: Phosphodiesterase 1 (pde1) Modulates CAMP Signaling At The Sarcolemmal Membrane

Phosphodiesterases (PDEs) regulate cyclic nucleotide-mediated cell signaling and offer a therapeutic means to leverage cardiac function. FDA-approved PDE3 inhibitors increase myocyte contractility by enhancing L-type calcium channel activity and sarcoplasmic reticulum (SR) calcium (Ca 2+ ) load, but cause fatal arrhythmias in patients with heart failure. The PDE1 inhibitor ITI-214 is a novel inotrope that increases cardiac contractility in animal models and human patients with heart failure. Mechanistically, PDE1 inhibition leads to activation of the L-type Ca 2+ channel without altering the SR Ca 2+ load or myofilament-Ca 2+ sensitivity. The net result is enhanced myocyte contractility with comparatively less Ca 2+ increase and arrhythmias. PDE inhibition increases cAMP levels to activate protein kinase A (PKA), a pathway that regulates Ca 2+ handling and cycling. These results led us to hypothesize that PDEs 1 and 3 differ in localized domains where each controls cAMP-mediated signaling. Live-cell imaging experiments were performed in acutely isolated guinea pig myocytes expressing Förster resonance energy transfer (FRET) sensors to monitor changes in cAMP levels. These sensors were targeted to specific sarcolemmal domains or more generally to the cytosol. Our results showed that PDE1 inhibition is the predominant regulator of cAMP at the non T-tubular sarcolemmal membrane compared to other cAMP-hydrolyzing PDEs (PDE3 and 4). In contrast to PDE1, PDEs 3 and 4 were more indiscriminate and modulated cAMP in the cytosolic as well as in membrane domains. Upon beta-adrenergic stimulation, however, PDE4 but not PDEs 1 or 3 became the predominant cAMP modulator at the T-tubular membrane domain. These results provide evidence that PDE1 is a major regulator of sarcolemmal membrane cAMP, suggesting that PDE1 inhibitory effects upon the L-type Ca 2+ channel is localized to this domain. In future studies, we will examine if PDE1 inhibition selectively activates PKA in this region, and seek to identify the interacting proteins that support PDE1 modulation of sarcolemmal cAMP/PKA signaling. Resolving this cell signaling difference would provide mechanistic insight into a novel inotrope that has already completed a Phase II clinical evaluation in patients.

Abstract P1065: Deletion Of Arginine 14 Increases Phospholamban Oligomerization And Disrupts The Cardiac Force-Frequency Relationship

The Bowditch effect is a physiological phenomenon where accelerated cardiac pacing leads to an increase in the contractile force of the heart. While this has largely been attributed to a frequency-dependent increase in L-type calcium channel influx, evidence suggests intracellular calcium handling proteins must also play an active role. Knock-out of phospholamban (PLB), the inhibitor of the Ca ATPase (SERCA), virtually abolishes the Bowditch effect in mice, suggesting PLB is a major determinant of the cardiac force-frequency relationship. However, no molecular mechanism has explained this frequency-dependent function of PLB. Our lab has previously shown that SERCA loses affinity for PLB when intracellular calcium is elevated. Here, FRET microscopy revealed dynamic decreases in PLB-SERCA binding in response to intracellular calcium elevations. PLB monomers freed from SERCA were rapidly incorporated into PLB pentamers. Interestingly, unbinding of PLB from pentamers was very slow and delayed the recovery of PLB-SERCA complexes by several seconds after calcium elevations. In a computational model of these regulatory binding dynamics, PLB pentamers sequestered inhibitory monomers away from SERCA at fast heart rates. By lowering PLB inhibition of SERCA with increasing cardiac pacing, PLB pentamers serve as a major contributor to the Bowditch effect. Notably, deletion of arginine 14 of PLB causes arrhythmogenic cardiomyopathy and has been thought to destabilize PLB pentamers. Surprisingly, we found R14del-PLB enhanced pentamer stability and slowed the rate of PLB exchange from pentamers to SERCA. We hypothesize that R14del mutation may disrupt frequency-dependent changes in SERCA regulation by PLB during exercise.

CaMKII is a modulator in neurodegenerative diseases and mediates the effect of androgen on synaptic protein PSD95

Androgens rapidly regulate synaptic plasticity in hippocampal neurones, but the underlying mechanisms remain unclear. In this study, we carried out a comprehensive bioinformatics analysis of functional similarities between androgen receptor (AR) and the synaptic protein postsynaptic density 95 (PSD95) to evaluate the effect. Using different measurements and thresholds, we obtained consistent results illustrating that the two proteins were significantly involved in similar pathways. We further identified CaMKII plays a critical role in mediating the rapid effect of androgen and promoting the expression of PSD95. We used mouse hippocampal neurone HT22 cells as a cell model to investigate the effect of testosterone (T) on intracellular Ca2+ levels and the mechanism. Calcium imaging experiments showed that intracellular Ca2+ increased to a peak due to calcium influx in the extracellular fluid through L-type and N-type voltage-gated calcium channels when HT22 cells were treated with 100 nM T for 20 min. Subsequently, we investigated whether the Ca2+/CaMKII signaling pathway mediates the rapid effect of T, promoting the expression of the synaptic protein PSD95. Immunofluorescence cytochemical staining and western blotting results showed that T promoted CaMKII phosphorylation by rapidly increasing extracellular Ca2+ influx, thus increasing PSD95 expression. This study demonstrated that CaMKII acts as a mediator assisting androgen which regulates the synaptic protein PSD95Also, it provides evidence for the neuroprotective mechanisms of androgens in synaptic plasticity and reveals the gated and pharmacological mechanisms of the voltage-gated Ca2+ channel family for androgen replacement therapy.

Extracellular S100B inhibits A-type voltage-gated potassium currents and increases L-type voltage-gated calcium channel activity in dopaminergic neurons.

Parkinson's disease (PD) is associated with an increase in secreted S100B within the midbrain and cerebrospinal fluid. In addition, S100B overexpression in mice accelerates the loss of substantia nigra pars compacta dopaminergic (DA) neurons, suggesting a role for this protein in PD pathogenesis. We found that in the mouse SNc, S100B labeled astrocytic processes completely envelop the somata of tyrosine hydroxylase (TH) expressing DA neurons only in male mice. These data suggest that an increase in S100B secretion by astrocytes within the midbrain could play a role in DA dysfunction during early PD. We therefore asked if acute exposure to extracellular S100B alters the activity of identified TH expressing DA neurons in primary mouse midbrain cultures. Acute exposure to 50 pM S100B specifically inhibited A-type voltage-gated potassium currents in TH+ , but not TH- neurons. This was accompanied by ~2-fold increases in the frequency of both intrinsic firing, as well as L-type voltage-gated calcium channel (VGCC)-mediated calcium fluxes only in TH+ neurons. Further, exposure to 100 μM 4-aminopyridine (4-AP), an A-type voltage-gated potassium channel inhibitor, mimicked the S100B mediated increase in intrinsic firing and L-type VGCC-mediated calcium fluxes in TH+ neurons. Taken together, our finding that extracellular S100B alters the activity of native DA neurons via an inhibition of A-type voltage-gated potassium channels has important implications for understanding the pathophysiology of early PD.

Febrile seizures lead to prolonged epileptiform activity and hyperoxia that when blocked prevents learning deficits

In adult brain tissue, oxygen levels typically remain in the normoxic zone, but status epilepticus results in hyperoxia, whereas brief self-terminating seizures lead to postictal hypoxia. The dynamic changes in oxygen levels and the underlying mechanisms are unknown in juveniles with febrile seizures. Eight-day-old female and male rat pups were implanted with an electrode and oxygen-sensing optode in the hippocampus and then received once daily injections of lipopolysaccharide for 4 days to induce an immune response. Local partial pressure of oxygen (pO2 ) and local field potentials were recorded before, during, and after a heat-induced febrile seizure. Separate groups of pups received injections of vehicle or drugs targeting cyclooxygenase (COX)-1, COX-2, L-type calcium channels (LTCCs), and cannabinoid receptor type 1 (CB1) and transient receptor potential vanilloid-1 (TRPV1) receptors prior to febrile seizure induction to determine pO2 mechanisms. Following febrile seizures, a subset of pups were raised to young adulthood and then tested for learning impairments using the novel object recognition task. Febrile seizures resulted in predictable oxygen dynamics that were related to behavioral seizures and epileptiform activity. During a behavioral seizure, pO2 rapidly increased, rapidly decreased, and then returned to near baseline. When the behavioral seizure terminated, oxygen levels climbed into the hyperoxic zone during a time of prolonged epileptiform activity. When epileptiform activity terminated, oxygen levels slowly returned to baseline. A COX-1 antagonist prevented hyperoxia, whereas a COX-2 antagonist did not. An LTCC antagonist exacerbated hyperoxia. Boosting levels of an endocannabinoid also exacerbated hyperoxia, whereas blocking CB1 receptors and TRPV1 receptors reduced hyperoxia. Inhibiting TRPV1 receptors during a febrile seizure prevented learning deficits in young adult female rats. Brain oxygenation during and following a febrile seizure has a distinct pattern and multiple mechanisms. Brain oxygen dynamics may be an important consideration in the development of treatments for febrile seizures.

Roots of Astragalus propinquus Schischkin Regulate Transmembrane Iron Transport and Ferroptosis to Improve Cerebral Ischemia-Reperfusion Injury.

Background The dried roots of the Astragalus propinquus Schischkin (RAP) plant, as a traditional Chinese medicine, has been widely used to treat stroke, cerebral ischemia, qi deficiency, and hypertension. Buyang Huanwu decoction is traditionally used to treat stroke in China for more than 200 years and has a significant effect on cerebral ischemia, and RAP is monarch medicine of Buyang Huanwu decoction. Therefore, this study was designed to observe the regulatory effect of RAP on transmembrane iron transporters and ferroptosis-related factors in cerebral ischemia-reperfusion injury (CIRI) in rats. Methods Middle cerebral artery occlusion (MCAO) was used to block blood flow in the blood supply area of the middle cerebral artery in seventy male SD rats to induce focal CIRI to establish a rat model of CIRI. RAP was administered to explore the regulatory effect of RAP on iron transmembrane transport under the condition of CIRI. The infarct size was measured using 2,3,5-triphenyl-tetrazolium chloride (TTC) staining, the pathological structure of brain tissue was observed by HE staining, and neuronal injury was evaluated by Nissl staining after treatment. Then, changes in the iron transporters ferritin (Fn), ferritin heavy chain (FHC), ferritin light chain (FLC), transferrin (Tf), transferrin receptor (TfR), divalent metal transporter 1 (DMT1), L-type calcium channel (LTCC), transient receptor potential canonical 6 (TRPC6), and ferroportin 1 (FPN1) were observed by immunohistochemistry staining (IHC) and Western blotting. The expression of key factors of ferroptosis, including the membrane sodium-dependent cystine/glutamate antiporter System Xc− (System Xc−) light chain subunit (XCT) and heavy chain subunit (SLC3A2), glutathione peroxidase 4 (GPX4), nuclear factor erythroid 2-related factor (NRF2), heme oxygenase-1 (HO-1), and iron-responsive element-binding protein 2 (IREB2) in the brain tissues of rats was assessed by Western blotting. RAP decreased the infarct size and neuronal injury after CIRI in rats. Similarly, RAP treatment regulated the expression of iron transporters. As such, RAP was able to reduce the expression of Fn, FHC, FLC, Tf, TfR, DMT1, and TRPC6 and increase the expression of FPN1 through a Tf/TfR-independent pathway after CIRI in rats. Conclusion RAP stimulation inhibited ferroptosis by regulating the expression of the key ferroptosis factors XCT, SLC3A2, GPX4, NRF2, HO-1, and IREB2. In conclusion, RAP regulates transmembrane iron transport and ferroptosis to improve CIRI.

CACNA1C Gene rs1006737 Polymorphism Affects Cognitive Performance in Chinese Han Schizophrenia.

ObjectiveTo investigate the relationship between L-type calcium channel α1C subunit (CACNA1C) gene polymorphism and schizophrenia (SCZ) and cognitive function in the Han nationality, the main nationality in China.MethodsGenotyping of CACNA1C SNP (rs1006737, rs1024582, rs2007044) in SCZ patients (n = 312) and healthy controls (n = 305) was performed. Cognitive function was assessed in the SCZ patients using Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). Then, the correlation between SNP and SCZ, as well as cognition, was calculated.ResultsThere was no significant difference in allele frequency and genotype distribution frequency of the three polymorphic loci of CACNA1C gene between the two groups. In cognitive tests, delayed memory scores in RBANS were significantly lower in rs1006737 “A” risk allele carriers than in non-carriers.ConclusionThere is no significant difference in allele and genotype frequency of CANCA1C Gene rs1006737, rs1024582 and rs2007044 between the schizophrenia patients and healthy controls. The cognitive function of schizophrenia patients is correlated with the rs1006737, and the delayed memory of “A” allele carriers is significantly reduced.

A Preclinical Study on Brugada Syndrome with a CACNB2 Variant Using Human Cardiomyocytes from Induced Pluripotent Stem Cells.

Aims: Some gene variants in the sodium channels, as well as calcium channels, have been associated with Brugada syndrome (BrS). However, the investigation of the human cellular phenotype and the use of drugs for BrS in presence of variant in the calcium channel subunit is still lacking. Objectives: The objective of this study was to establish a cellular model of BrS in the presence of a CACNB2 variant of uncertain significance (c.425C > T/p.S142F) using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and test drug effects using this model. Methods and results: This study recruited cells from a patient with Brugada syndrome (BrS) and recurrent ventricular fibrillation carrying a missense variant in CACNB2 as well as from three healthy independent persons. These cells (hiPSC-CMs) generated from skin biopsies of healthy persons and the BrS patient (BrS-hiPSC-CMs) as well as CRISPR/Cas9 corrected cells (isogenic control, site-variant corrected) were used for this study. The hiPSC-CMs from the BrS patient showed a significantly reduced L-type calcium channel current (ICa-L) compared with the healthy control hiPSC-CMs. The inactivation curve was shifted to a more positive potential and the recovery from inactivation was accelerated. The protein expression of CACNB2 of the hiPSC-CMs from the BrS-patient was significantly decreased compared with healthy hiPSC-CMs. Moreover, the correction of the CACNB2 site-variant rescued the changes seen in the hiPSC-CMs of the BrS patient to the normal state. These data indicate that the CACNB2 gene variant led to loss-of-function of L-type calcium channels in hiPSC-CMs from the BrS patient. Strikingly, arrhythmia events were more frequently detected in BrS-hiPSC-CMs. Bisoprolol (beta-blockers) at low concentration and quinidine decreased arrhythmic events. Conclusions: The CACNB2 variant (c.425C > T/p.S142F) causes a loss-of-function of L-type calcium channels and is pathogenic for this type of BrS. Bisoprolol and quinidine may be effective for treating BrS with this variant.

Epigenetic mechanism of L-type calcium channel β-subunit downregulation in short QT human induced pluripotent stem cell-derived cardiomyocytes with CACNB2 mutation.

A loss-of-function mutation in L-type calcium (Ca2+) channel subunit gene CACNB2 has been reported to cause short QT syndrome subtype 5 (SQT5). However, the mechanism underlying the loss-of-function of the Ca2+ channel has not been clarified. In the present study, we aim to explore the DNA methylation mechanism of L-type Ca2+ channel downregulation in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of SQT5. The hiPSC-CMs were generated from a healthy donor and a SQT5 patient carrying the CACNB2 variant c.1439C > T/p.S480L. The variant was genetically corrected using ribonucleoprotein-based CRISPR/Cas9 technique to obtain an isogenic control cell line. The action potential (AP) and Ca2+ current were measured by patch clamp. Protein expression levels were determined by western blotting. Dot blotting and bisulfite sequence were performed for epigenetic study. Our results showed that AP durations at 10% repolarization (APD10) and 50% repolarization (APD50) were significantly shortened in SQT5 cells and both the expression level of the β-subunit and channel current of L-type Ca2+ channel were reduced. Besides, an increased level of whole-genome DNA methylation and DNA methylation of CpG island in the promoter region of CACNB2 gene was detected. Overexpression of demethylation enzyme could rescue the decreased expression of CACNB2 and the L-type Ca2+ current. In SQT5 hiPSC-CMs carrying the CACNB2-S480L variant, the decreased L-type Ca2+ current resulting from decreased CACNB2 protein expression was caused by enhanced methylation in the promoter region of the CACNB2 gene and upregulation of DNA methyltransferases might be one of the mechanisms.

Hyperinsulinemic Hypoglycemia Associated with a CaV1.2 Variant with Mixed Gain- and Loss-of-Function Effects.

The voltage-dependent L-type calcium channel isoform CaV1.2 is critically involved in many physiological processes, e.g., in cardiac action potential formation, electromechanical coupling and regulation of insulin secretion by beta cells. Gain-of-function mutations in the calcium voltage-gated channel subunit alpha 1 C (CACNA1C) gene, encoding the CaV1.2 α1-subunit, cause Timothy syndrome (TS), a multisystemic disorder that includes autism spectrum disorders and long QT (LQT) syndrome. Strikingly, TS patients frequently suffer from hypoglycemia of yet unproven origin. Using next-generation sequencing, we identified a novel heterozygous CACNA1C mutation in a patient with congenital hyperinsulinism (CHI) and associated hypoglycemic episodes. We characterized the electrophysiological phenotype of the mutated channel using voltage-clamp recordings and in silico action potential modeling experiments. The identified CaV1.2L566P mutation causes a mixed electrophysiological phenotype of gain- and loss-of-function effects. In silico action potential modeling supports that this mixed electrophysiological phenotype leads to a tissue-specific impact on beta cells compared to cardiomyocytes. Thus, CACNA1C variants may be associated with non-syndromic hyperinsulinemic hypoglycemia without long-QT syndrome, explained by very specific electrophysiological properties of the mutated channel. We discuss different biochemical characteristics and clinical impacts of hypoglycemia in the context of CACNA1C variants and show that these may be associated with significant morbidity for Timothy Syndrome patients. Our findings underline that the potential of hypoglycemia warrants careful attention in patients with CACNA1C variants, and such variants should be included in the differential diagnosis of non-syndromic congenital hyperinsulinism.

Abnormal levels of mitochondrial Ca2+ channel proteins in plasma neuron-derived extracellular vesicles of early schizophrenia.

Structural alterations or quantitative abnormalities of some mitochondrial ion channels and exchangers are associated with altered neuronal functions and increased susceptibility to mental illness. Here we have assessed levels of functionally prominent mitochondrial calcium ion channel proteins in plasma neuron-derived extracellular vesicles (NDEVs) of living patients with first episodes of psychosis (FP) and matched controls (Cs). NDEVs were enriched with an established method of precipitation and immunoabsorption by anti-human CD171 neural adhesion protein (L1CAM) antibody and extracted proteins quantified with ELISAs. CD81 exosome marker-normalized NDEV levels of leucine zipper EF-hand containing transmembrane 1 protein (LETM1), transient receptor potential cation channel subfamily M, member 4 (TRPM4), and solute carrier family 8 member B1 (SLC24A6) or mitochondrial Na+ /Ca2+ exchanger (NCLX) were significantly lower for FP patients (n=10) than Cs (n=10), whereas NDEV levels of voltage-dependent L-type calcium channel subunit α-1C (CACNA-1C) were significantly higher for FP patients than Cs. Abnormal structures or mitochondrial levels of LETM1, NCLX, and CACNA-1C have been linked through analyses of individual proteins, genome-wide association studies, and whole exome protein-coding sequence studies to neurodevelopmental disorders, mental retardation, schizophrenia, and major depressive diseases. A greater understanding of the altered calcium homeostasis in schizophrenia, that is attributable to underlying mitochondrial calcium channel abnormalities, will lead to improved diagnosis and treatment.

Gene Dysregulation in the Adult Rat Paraventricular Nucleus and Amygdala by Prenatal Exposure to Dexamethasone.

Fetal programming is the concept that maternal stressors during critical periods of fetal development can alter offspring phenotypes postnatally. Excess glucocorticoids can interact with the fetus to effect genetic and epigenetic changes implicated in adverse developmental outcomes. The present study investigates how chronic exposure to the synthetic glucocorticoid dexamethasone during late gestation alters the expression of genes related to behavior in brain areas relevant to the regulation and function of the hypothalamic–pituitary–adrenal axis. Pregnant Wistar Kyoto rats received subcutaneous injections of dexamethasone (100 μg/kg) daily from gestational day 15–21 or vehicle only as sham controls. The amygdala and paraventricular nucleus (PVN) were micro-punched to extract mRNA for reverse transcription and quantitative polymerase chain reaction for the analysis of the expression of specific genes. In the PVN, the expression of the glucocorticoid receptor NR3C1 was downregulated in female rats in response to programming. The expression of CACNA1C encoding the Cav1.2 pore subunit of L-type voltage-gated calcium channels was downregulated in male and female rats prenatally exposed to dexamethasone. Collectively, the results suggest that prenatal exposure to elevated levels of glucocorticoids plays a role in the dysregulation of the hypothalamic–pituitary–adrenal axis and potentially learning and memory by altering the expression of specific genes within the amygdala and PVN.

Lysophosphatidic Acid Improves Human Sperm Motility by Enhancing Glycolysis and Activating L-Type Calcium Channels.

Until now, the molecular mechanisms underlining sperm motility defect causing male infertility are still poorly understood. Safe and effective compounds or drugs that can improve sperm motility are also very limited. Lysophosphatidic acid (LPA) is a naturally occurring phospholipid and a bioactive intermediate with multiple biological activities. It has been detected in various body fluids such as serum, plasma, saliva, tears, blister fluids, hen egg white, and ascites from patients with ovarian cancer. LPA is also abundant in seminal plasma and follicular fluid. It enhances follicle stimulation, improves oocyte fertilization, and promotes early embryonic development and embryo implantation. However, the physiological role of LPA in the male reproductive system remains unknown. Here, our study showed that LPA significantly improved the motility parameters of human sperm hyperactivation in a dose-dependent manner. The LPA-induced elevation of sperm motility is dependent on bovine serum albumin (BSA) but independent of the classical BSA-induced sAC/cAMP/PKA signaling pathway. The enhancement of sperm motility by LPA could not be blocked by CCCP, a respiratory inhibitor suppressing mitochondrial ATP production. Moreover, LPA improved the activity of triosephosphate isomerase in glycolysis. Meanwhile, LPA treatment significantly increased ATP and phosphoenolpyruvate levels and decreased ADP content during sperm glycolysis. Notably, none of known or identified LPA receptors was detected in human sperm. Further investigations showed that LPA promoted sperm motility through L-type calcium channels. In summary, this study revealed the involvement of LPA in the regulation for human sperm motility by enhancing glycolysis and activating L-type calcium channels. The current findings may shed new light on the understanding of causes of asthenozoospermia, and indicate that LPA could be used as a novel therapeutic agent to improve sperm function and fertilizing capacity.

Solanaceae glycoalkaloids: α-solanine and α-chaconine modify the cardioinhibitory activity of verapamil

Context Solanaceae glycoalkaloids (SGAs) possess cardiomodulatory activity. Objective This study investigated the potential interaction between verapamil and glycoalkaloids. Material and methods The cardioactivity of verapamil and glycoalkaloids (α-solanine and α-chaconine) was tested in adult beetle (Tenebrio molitor) myocardium in vitro using microdensitometric methods. The myocardium was treated with pure substances and mixtures of verapamil and glycoalkaloids for 9 min with saline as a control. Two experimental variants were used: simultaneous application of verapamil and glycoalkaloids or preincubation of the myocardium with one of the compounds followed by perfusion with a verapamil solution. We used 9 × 10−6–5 × 10−5 M and 10−9–10−5 M concentration for verapamil and glycoalkaloids, respectively. Results Verapamil, α-solanine and α-chaconine showed cardioinhibitory activity with IC50 values equal to 1.69 × 10−5, 1.88 × 10−7 and 7.48 × 10−7 M, respectively. When the glycoalkaloids were applied simultaneously with verapamil, an antagonistic effect was observed with a decrease in the maximal inhibitory effect and prolongation of t50 and the recovery time characteristic of verapamil. We also confirmed the expression of two transcript forms of the gene that encodes the α1 subunit of L-type calcium channels in the myocardium and brain with equal transcription levels of both forms in the myocardium and significant domination of the shorter form in the brain of the insect species tested. Discussion and conclusions The results show that attention to the composition of the daily diet during therapy with various drugs is particularly important. In subsequent studies, the nature of interaction between verapamil and SGAs on the molecular level should be checked, and whether this interaction decreases the efficiency of cardiovascular therapy with verapamil in humans.

Inhibition of L-type voltage-gated calcium channel-mediated Ca2+ influx suppresses the collective migration and invasion of ameloblastoma.

Ameloblastoma (AM) has been known as a benign but locally invasive tumour with high recurrence rates. Invasive behaviour of the AM results in destruction of the adjacent jawbone and the non-detectable remnants during surgery, interrupting the complete elimination of cancer cells. To explore novel targets for the tumour cell invasion, a transcriptomic analysis between AM and odontogenic keratocyst were performed through next-generation sequencing in detail. Enrichment of CACNA1C gene (encoding Cav1.2) in AM, a subunit of the L-type voltage-gated calcium channel (VGCC) was observed for the first time. The expression and channel activity of Cav1.2 was confirmed by immunostaining and calcium imaging in the patient samples or primary cells. Verapamil, L-type VGCC blocker revealed suppression of the Ca2+ -induced cell aggregation and collective invasion of AM cells in vitro. Furthermore, the effect of verapamil in suppressing AM invasion into the adjacent bone was confirmed through orthotopic xenograft model specifically. Taken together, Cav1.2 maybe considered to be a therapeutic candidate to decrease the collective migration and invasion of AM.

Silymarin Administration Attenuates Cirrhotic-induced Cardiac Abnormality in the Rats: A Possible Role of β1-adrenergic Receptors and L-type Voltage-Dependent Calcium Channels.

Background: Cirrhotic cardiomyopathy is a well-recognized cardiac dysfunction in cirrhotic patients. Studies have confirmed the protective effects of silymarin in different types of cardiac injury. This study aimed to examine the effectiveness and molecular mechanism of silymarin against myocardial dysfunction and hypertrophy in a rat model of cirrhosis.Methods: The experiment was performed at Alborz University of Medical Sciences (Karaj, Iran) during 2020-2021. Thirty-two male Wistar rats were randomly divided into four groups of Sham-operated (control group for surgical procedures), Bile Duct Ligated (BDL), and two Silymarin extract (SE)-treated groups of 300 and 600 mg/Kg/day. After 28 days, serum levels of AST, ALT, GGT, and ALP, liver histopathological status, as well as cardiac mechanical function, were assessed. Cardiac β1-adrenergic receptors (β1-AR), L-type voltage-dependent calcium channels (L-VDCC), and GATA4 mRNA expression were also determined using real-time RT-PCR. Data analysis was performed using the one-way ANOVA followed by Duncan’s multiple range test. Histological data has been analyzed with Kruskal-Wallis nonparametric test. The analysis was performed at P≤0.05.Results: BDL was associated with a significant elevation in serum AST, ALT, GGT, and ALP, development of necrosis and fibrosis of the liver texture, increased Heart Weight and Heart Weight to Body Weight ratio, enhanced cardiac mechanical function as well as a significant up-regulation of ventricular β1-AR and L-VDCC. Administration of SE600, but not SE300, significantly reduced the serum levels of the enzymes and alleviated signs of liver necrosis and fibrosis. Cirrhotic-induced cardiac dysfunction was also restored by SE600, but not by the lower dose. In addition, cardiac expression of the β1-AR and L-VDCC was down-regulated toward normal values by either higher or lower doses of the SE.Conclusion: Silymarin treatment in higher dose attenuated cirrhosis-associated cardiac remodeling and reduced cardiac mechanical dysfunctions.

Alterations of Dopamine Receptors and the Adaptive Changes of L-Type Calcium Channel Subtypes Regulate Cocaine-Seeking Habit in Tree Shrew.

The putamen (Put) is necessary for habitual actions, while the nucleus caudate (Cd) is critical for goal-directed actions. However, compared with the natural reward (such as sucrose)-seeking habit, how drug-related dysfunction or imbalance between the Put and Cd is involved in cocaine-seeking habit, which is not easy to bias behavior to goal-directed actions, is absent. Therefore, in our present study, in comparison with sucrose-habitual behavior, we evaluated the distinctive changes of the two subtypes of dopamine (DA) receptors (D1R and D2R) in cocaine-seeking habitual behavior animals. Moreover, the adaptive changes of Cav1.2 and Cav1.3, as prime downstream targets of D1R and D2R respectively, were also assessed. Our results showed that a similar percentage of the animals exhibited habitual seeking behavior after cocaine or sucrose variable-interval self-administration (SA) training in tree shrews. In addition, compared with animals with non-habitual behavior, animals with cocaine habitual behavior showed higher D1Rs and Cav1.2 expression in the Put accompanied with lower D2Rs and Cav1.3 expression in the Cd. However, after sucrose SA training, animals with habitual behavior only showed lower membrane expression of D2R in the Put than animals with non-habitual behavior. These results suggested that the upregulation of D1Rs-Cav1.2 signaling may lead to hyper-excitability of the Put, and the inactivation of D2Rs-Cav1.3 signaling may result in depressed activity in the Cd. This imbalance function between the Put and Cd, which causes an inability to shift between habits and goal-directed actions, may underlie the compulsive addiction habit.