Subunit Of L-type Calcium Channel Research Articles

Functional Mechanisms of Novel G Protein Inhibitor Ligands As Vasodilators

G‐protein coupled receptor (GPCR) signaling plays an essential role in physiologic homeostasis and cardiovascular health. Dysfunction of GPCR pathway is implicated in hypertension, a leading risk factor for cardiovascular diseases. Here, we examined the functional properties of novel Gq inhibitor ligands, YM‐254890, FR900359, and WU‐07047, and determined their effectiveness at blocking GPCR signaling in the resistance vasculature. Under ex‐vivo pressurized vessel preparation, we measured vascular reactivity of small mesenteric arteries isolated from 3–4 month old wild type mice. Vasoconstriction of mesenteric arteries was evoked by applying increasing concentrations of the α1‐adrenergic receptor agonist, phenylephrine (PE), in the presence or absence of Gq inhibitors. We found that WU‐07047 had the least effect on maximal contractile response to PE (FR[0.01 μM]: 0.8 ± 0.6% vs. YM[0.01 μM]: 12.8 ± 2.8% vs. WU[10 μM]: 34.8 ± 3.2% %), whereas FR was the most potent (FR[0.01uM]: LogIC50 −0.008 ± 0 vs. YM[0.01uM]: LogIC50 −0.49 ± 0 vs. WU: LogIC50 −64.95 ± 6.4). WU‐07047 and YM‐254890 inhibited vasoconstriction through G protein‐independent pathway by blocking L‐type calcium channels (LTCCs). Bioinformatics analysis revealed a high sequence similarity between binding sites for dihydropyridines in the pore‐forming subunit of LTCCs and the binding pocket for the novel Gq inhibitor ligands in Gqα subunit. We conclude that the novel Gq inhibitors decrease vascular tone by blocking G protein‐dependent and G protein‐independent signaling mechanisms mediating resistance artery vasoconstriction. These findings suggest that targeting signaling downstream of GPCRs may be a more effective therapeutic approach to managing hypertension and associated cardiovascular diseases.Support or Funding InformationPennsylvania Dept. of Health (CURE) Grant (PO)Margaret Q. Landenberger Research Grant (PO)AHA Scientist Development Grant (PO)National Science Foundation (CBET 1262176) (KDM)

Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell-autonomous fashion.

Ca2+ is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha-1C subunit (i.e., Cav 1.2) of the voltage-dependent l-type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Cav 1.2 channels located on astrocyte-like stem cells and their descendants in the development of new granule neurons, we created TgGLAST-CreERT2 /Cacna1cfl/fl /RCE:loxP mice, a transgenic tool that allows cell-type-specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type-1 cells. FACS-sorted Cacna1c-deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c-deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type-1 cells decreased type-1 cell proliferation and reduced the neuronal fate-choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type-1 cells, indicating that there must be Cav 1.2-independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L-type 1.2 channels on type-1 cells. Cav 1.2 channels promote type-1 cell proliferation and push the glia-to-neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Cav 1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell-autonomous fashion.

A Novel Human CAMK2A Mutation Disrupts Dendritic Morphology and Synaptic Transmission, and Causes ASD-Related Behaviors.

Characterizing the functional impact of novel mutations linked to autism spectrum disorder (ASD) provides a deeper mechanistic understanding of the underlying pathophysiological mechanisms. Here we show that a de novo Glu183 to Val (E183V) mutation in the CaMKIIα catalytic domain, identified in a proband diagnosed with ASD, decreases both CaMKIIα substrate phosphorylation and regulatory autophosphorylation, and that the mutated kinase acts in a dominant-negative manner to reduce CaMKIIα-WT autophosphorylation. The E183V mutation also reduces CaMKIIα binding to established ASD-linked proteins, such as Shank3 and subunits of l-type calcium channels and NMDA receptors, and increases CaMKIIα turnover in intact cells. In cultured neurons, the E183V mutation reduces CaMKIIα targeting to dendritic spines. Moreover, neuronal expression of CaMKIIα-E183V increases dendritic arborization and decreases both dendritic spine density and excitatory synaptic transmission. Mice with a knock-in CaMKIIα-E183V mutation have lower total forebrain CaMKIIα levels, with reduced targeting to synaptic subcellular fractions. The CaMKIIα-E183V mice also display aberrant behavioral phenotypes, including hyperactivity, social interaction deficits, and increased repetitive behaviors. Together, these data suggest that CaMKIIα plays a previously unappreciated role in ASD-related synaptic and behavioral phenotypes.SIGNIFICANCE STATEMENT Many autism spectrum disorder (ASD)-linked mutations disrupt the function of synaptic proteins, but no single gene accounts for >1% of total ASD cases. The molecular networks and mechanisms that couple the primary deficits caused by these individual mutations to core behavioral symptoms of ASD remain poorly understood. Here, we provide the first characterization of a mutation in the gene encoding CaMKIIα linked to a specific neuropsychiatric disorder. Our findings demonstrate that this ASD-linked de novo CAMK2A mutation disrupts multiple CaMKII functions, induces synaptic deficits, and causes ASD-related behavioral alterations, providing novel insights into the synaptic mechanisms contributing to ASD.

The Effect of Substrate Stiffness on Cardiomyocyte Action Potentials.

The stiffness of myocardial tissue changes significantly at birth and during neonatal development, concurrent with significant changes in contractile and electrical maturation of cardiomyocytes. Previous studies by our group have shown that cardiomyocytes generate maximum contractile force when cultured on a substrate with a stiffness approximating native cardiac tissue. However, effects of substrate stiffness on the electrophysiology and ion currents in cardiomyocytes have not been fully characterized. In this study, neonatal rat ventricular myocytes were cultured on the surface of flat polyacrylamide hydrogels with elastic moduli ranging from 1 to 25 kPa. Using whole-cell patch clamping, action potentials and L-type calcium currents were recorded. Cardiomyocytes cultured on hydrogels with a 9 kPa elastic modulus, similar to that of native myocardium, had the longest action potential duration. Additionally, the voltage at maximum calcium flux significantly decreased in cardiomyocytes on hydrogels with an elastic modulus higher than 9 kPa, and the mean inactivation voltage decreased with increasing stiffness. Interestingly, the expression of the L-type calcium channel subunit α gene and channel localization did not change with stiffness. Substrate stiffness significantly affects action potential length and calcium flux in cultured neonatal rat cardiomyocytes in a manner that may be unrelated to calcium channel expression. These results may explain functional differences in cardiomyocytes resulting from changes in the elastic modulus of the extracellular matrix, as observed during embryonic development, in ischemic regions of the heart after myocardial infarction, and during dilated cardiomyopathy.

Cross-talk between macrophages and atrial myocytes in atrial fibrillation.

Increased macrophage accumulation occurs in the atria of patients with atrial fibrillation (AF). However, the phenotype and functions of the macrophages in AF remain unclear. We investigated the macrophage-atrial myocyte interaction in AF patients and found that the increased macrophages were mainly pro-inflammatory macrophages (iNOS+, Arg1−). Tachypacing of HL-1 atrial myocytes also led to pro-inflammatory macrophage polarization. In addition, lipopolysaccharide (LPS)-stimulated pro-inflammatory macrophages-induced atrial electrical remodeling, evidenced by increased AF incidence and decreased atrial effective refractory period and L-type calcium currents (I Ca-L) in both canine and mouse AF models. Depletion of macrophages relieved LPS-induced atrial electrical remodeling, confirming the role of pro-inflammatory macrophages in the pathogenesis of AF. We also found that the effect of LPS-stimulated macrophages on atrial myocytes was mediated by secretion of interleukin 1 beta (IL-1β), which inhibited atrial myocyte quaking protein (QKI) expression. IL-1β knockout in macrophages restored the LPS-stimulated macrophage-induced inhibition of QKI and CACNA1C (α1C subunit of L-type calcium channel) in atrial myocytes. Meanwhile, QKI overexpression in atrial myocytes restored the LPS-stimulated macrophage-induced electrical remodeling through enhanced binding of QKI to CACNA1C mRNA, which upregulated the expression of CACNA1C as well as I Ca-L. In contrast, QKI knockout inhibited CACNA1C expression. Finally, using transcription factor activation profiling plate array and chromatin immunoprecipitation, we revealed that special AT-rich sequence binding protein 1 activated QKI transcription. Taken together, our study uncovered the functional interaction between macrophages and atrial myocytes in AF. AF induced pro-inflammatory macrophage polarization while pro-inflammatory macrophages exacerbated atrial electrical remodeling by secreting IL-1β, further inhibiting QKI expression in atrial myocytes, which contributed to I Ca-L downregulation. Our study demonstrates a novel molecular mechanism underlying the pathogenesis and progression of AF and suggests that QKI is a potential therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0584-z) contains supplementary material, which is available to authorized users.

Vascular mineralocorticoid receptor regulates microRNA-155 to promote vasoconstriction and rising blood pressure with aging.

Hypertension is nearly universal yet poorly controlled in the elderly despite proven benefits of intensive treatment. Mice lacking mineralocorticoid receptors in smooth muscle cells (SMC-MR-KO) are protected from rising blood pressure (BP) with aging, despite normal renal function. Vasoconstriction is attenuated in aged SMC-MR-KO mice, thus they were used to explore vascular mechanisms that may contribute to hypertension with aging. MicroRNA (miR) profiling identified miR-155 as the most down-regulated miR with vascular aging in MR-intact but not SMC-MR-KO mice. The aging-associated decrease in miR-155 in mesenteric resistance vessels was associated with increased mRNA abundance of MR and of predicted miR-155 targets Cav1.2 (L-type calcium channel (LTCC) subunit) and angiotensin type-1 receptor (AgtR1). SMC-MR-KO mice lacked these aging-associated vascular gene expression changes. In HEK293 cells, MR repressed miR-155 promoter activity. In cultured SMCs, miR-155 decreased Cav1.2 and AgtR1 mRNA. Compared to MR-intact littermates, aged SMC-MR-KO mice had decreased systolic BP, myogenic tone, SMC LTCC current, mesenteric vessel calcium influx, LTCC-induced vasoconstriction and angiotensin II-induced vasoconstriction and oxidative stress. Restoration of miR-155 specifically in SMCs of aged MR-intact mice decreased Cav1.2 and AgtR1 mRNA and attenuated LTCC-mediated and angiotensin II-induced vasoconstriction and oxidative stress. Finally, in a trial of MR blockade in elderly humans, changes in serum miR-155 predicted the BP treatment response. Thus, SMC-MR regulation of miR-155, Cav1.2 and AgtR1 impacts vasoconstriction with aging. This novel mechanism identifies potential new treatment strategies and biomarkers to improve and individualize antihypertensive therapy in the elderly.

Comparison of pathogenicity prediction tools on missense variants in RYR1 and CACNA1S associated with malignant hyperthermia

BackgroundMalignant hyperthermia (MH) is a pharmacogenetic disorder that has been linked to the skeletal muscle calcium release channel (RYR1) and the α1S subunit of the voltage-dependent L-type calcium channel (CACNA1S). Genomic DNA capture and next generation sequencing are becoming the preferred method to identify mutations in these genes. Bioinformatic pathogenicity prediction of identified variants may help to determine if these variants are in fact disease causing. MethodsEight pathogenicity prediction programmes freely available on the web were used to determine their ability to correctly predict the impact of a missense variant on RyR1 or dihydropyridine receptor (DHPR) protein function. We tested MH-causative variants, variants that had been shown to alter calcium release in cells, and common sequence variants in RYR1 and CACNA1S. ResultsNone of the prediction programmes was able to identify all of the variants tested correctly as either ‘damaging’ (MH-causative variants, variants that had been shown to alter calcium release in cells) or as ‘benign’ (common sequence variants). The overall sensitivity of predictions ranged from 84% to 100% depending on the programme used, with specificity from 25% to 83%. ConclusionsIn this study we determined the sensitivity and specificity of bioinformatic pathogenicity prediction tools for RYR1 and CACNA1S. We suggest that the prediction results should be treated with caution, as none of the programmes tested predicted all the variants correctly and should only be used in combination with other available data (functional assays, segregation analysis).

The Neuropsychiatric Disease-Associated Gene cacna1c Mediates Survival of Young Hippocampal Neurons.

Genetic variations in CACNA1C, which encodes the Cav1.2 subunit of L-type calcium channels (LTCCs), are associated with multiple forms of neuropsychiatric disease that manifest high anxiety in patients. In parallel, mice harboring forebrain-specific conditional knockout of cacna1c (forebrain-Cav1.2 cKO) display unusually high anxiety-like behavior. LTCCs in general, including the Cav1.3 subunit, have been shown to mediate differentiation of neural precursor cells (NPCs). However, it has not previously been determined whether Cav1.2 affects postnatal hippocampal neurogenesis in vivo. Here, we show that forebrain-Cav1.2 cKO mice exhibit enhanced cell death of young hippocampal neurons, with no change in NPC proliferation, hippocampal size, dentate gyrus thickness, or corticosterone levels compared with wild-type littermates. These mice also exhibit deficits in brain levels of brain-derived neurotrophic factor (BDNF), and Cre recombinase-mediated knockdown of adult hippocampal Cav1.2 recapitulates the deficit in young hippocampal neurons survival. Treatment of forebrain-Cav1.2 cKO mice with the neuroprotective agent P7C3-A20 restored the net magnitude of postnatal hippocampal neurogenesis to wild-type levels without ameliorating their deficit in BDNF expression. The role of Cav1.2 in young hippocampal neurons survival may provide new approaches for understanding and treating neuropsychiatric disease associated with aberrations in CACNA1C. Visual Abstract.

Genetic findings are challenging the symptom-based diagnostic classification system of mental disorders.

The present diagnostic classification of mental illnesses is primarily based on symptomatology. A recent cross-disorder genome-wide association study revealed that there were genetic similarities between multiple clinically defined diagnoses (including schizophrenia, bipolar disorder, depression, attention deficit hyperactivity disorder, and autism spectrum disorder) on regions of chromosomes 3p21 and 10q24 and single-nucleotide polymorphisms (SNPs) within two L-type voltage-gated calcium channel subunits of CACNA1C and CACNB2. These findings suggest that the pathogenesis of these five independent disorders are related. Such cross-disorder genetic studies challenge the current symptom-based diagnostic classification of mental disorders. Researchers need to identify creative ways to bridge the gap between these two approaches to understanding and labelling mental disorders.

Abstract 15700: L-type Calcium Channels Localization and Function are Affected by Microdomain Remodelling in Human Failing Cardiomyocytes

Introduction: L-type calcium channels (LTCCs) are essential in determining the electrical and mechanical properties of the cardiac muscle. LTCCs are located in specialized membrane microdomains in cardiac cells. Disruption of their microdomain localization in heart failure may contribute to the pathophysiology of cardiac disease. Methods: We studied isolated ventricular cardiomyocytes from patients with dilated cardiomyopathy with or without Left Ventricular Assistance Device (DCM or DCM+LVAD) versus those with normal ventricular function (nonfailing). Super-resolution scanning patch clamp provided topography images of the cell surface which enabled us to record LTCC current in distinct cellular compartments (T-tubules openings and areas between them, crests). Confocal microscopy was used to score T-tubular density and regularity in Di-8-ANEPPS stained cells. Expression of β2 subunit of LTCC was detected by immunofluorescence, Western blot and qPCR. Results: In failing cells T-tubular density is lower than in normal. The loss of cellular micro-architecture in failing cardiomyocytes leads to a redistribution of functional LTCCs from their usual location (T-tubules) to non-native crests of the sarcolemma. These relocated LTCCs in the crest of DCM cardiomyocytes have increased open probability and lower amplitude as compared with the channels located on the T-tubules. Interestingly, in cardiomyocytes isolated from patients with LVAD the open probability of LTCC is reduced dramatically and the amplitude recovers to the normal level, as does the T-tubular density. Immunostaining of DCM cardiomyocytes shows an increased amount of LTCC β2 subunit in the crest, and Western blot/qPCR shows more abundant expression of LTCC β2 subunit. Conclusion: In DCM cardiomyocytes a considerable amount of LTCCs improperly located on the crest microdomain display a pathologically high activity; this study suggests that the increased expression of LTCC β2-subunit could be responsible. Although LVAD cannot reverse the remodelling in DCM cardiomyocytes it helps to restore part of the microarchitecture of the cells and to normalize the function of the LTCCs.

Variation in CACNA1C is Associated with Amygdala Structure and Function in Adolescents.

Genome-wide association studies have identified allelic variation in CACNA1C as a risk factor for multiple psychiatric disorders associated with limbic system dysfunction, including bipolar disorder, schizophrenia, and depression. The CACNA1C gene codes for a subunit of L-type voltage-gated calcium channels, which modulate amygdala function. Although CACNA1C genotype appears to be associated with amygdala morphology and function in adults with and without psychopathology, whether genetic variation influences amygdala structure and function earlier in development has not been examined. In this first investigation of the neural correlates of CACNA1C in young individuals, we examined associations between two single nucleotide polymorphisms in CACNA1C (rs1006737 and rs4765914) with amygdala volume and activation during an emotional processing task in 58 adolescents and young adults 13-20 years of age. Minor (T) allele carriers of rs4765914 exhibited smaller amygdala volume than major (C) allele homozygotes (β=-0.33, p=0.006). Furthermore, minor (A) allele homozygotes of rs1006737 exhibited increased blood-oxygen-level-dependent (BOLD) signal in the amygdala when viewing negative (vs. neutral) stimuli (β=0.29, p=0.040) and decreased BOLD signal in the amygdala when instructed to downregulate their emotional response to negative stimuli (β=-0.38, p=0.009). Follow-up analyses indicated that childhood trauma did not moderate the associations of CACNA1C variation with amygdala structure and function (ps>0.170). Findings indicate that CACNA1C-related differences in amygdala structure and function are present by adolescence. However, population stratification is a concern, given the racial/ethnic heterogeneity of our sample, and our findings do not have direct clinical implications currently. Nevertheless, these results suggest that developmentally informed research can begin to shed light on the time course by which genetic liability may translate into neural differences associated with vulnerability to psychopathology.

Mechanisms of Intracellular Calcium Homeostasis in MC3T3-E1 Cells and Bone Tissues of Sprague-Dawley Rats Exposed to Fluoride

Calcium homeostasis of osteoblasts (OBs) has an important role in the physiology and pathology of bone tissue. In order to study the mechanisms of intracellular calcium homeostasis, MC3T3-E1 cells and Sprague-Dawley rats were treated with different concentrations of fluoride. Then, we examined intracellular-free calcium ion ([Ca(2+)]i) in MC3T3-E1 cells as well as mRNA and protein levels of Cav1.2, the main subunit of L-type voltage-dependent calcium channels (VDCCs), Na(+)/Ca(2+) exchange carriers (NCS), and plasma membrane Ca(2+)-ATPase (PMCA), inositol 1,4,5-trisphosphate receptor (IP3R) channels, sarco/endoplasmic reticulum calcium ATPase 2b (SERCA2b)/ATP2A2 in vitro, and rat bone tissues in vivo. Our results showed that [Ca(2+)]i of fluoride-treated OBs increased in a concentration-dependent manner with an increase in the concentration of fluoride. We also found that the low dose of fluoride led to high expression levels of Cav1.2, NCS-1, and PMCA and low expression levels of IP3R and SERCA2b/ATP2A2, while the high dose of fluoride induced an increase in SERCA2b/ATP2A2 levels and decrease in Cav1.2, PMCA, NCS-1, and IP3R levels. These results demonstrate that calcium channels and calcium pumps of plasma and endoplasmic reticulum (ER) membranes keep intracellular calcium homeostasis by regulating Cav1.2, NCS-1, PMCA, IP3R, and SERCA2b/ATP2A2 expression.

Spontaneous activity and stretch-induced contractile differentiation are reduced in vascular smooth muscle of miR-143/145 knockout mice.

Stretch is essential for maintaining the contractile phenotype of vascular smooth muscle cells, and small non-coding microRNAs are known to be important in this process. Using a Dicer knockout model, we have previously reported that microRNAs are essential for stretch-induced differentiation and regulation of L-type calcium channel expression. The aim of this study was to investigate the importance of the smooth muscle-enriched miR-143/145 microRNA cluster for stretch-induced differentiation of the portal vein. Contractile force and depolarization-induced calcium influx were determined in portal veins from wild-type and miR-143/145 knockout mice. Stretch-induced contractile differentiation was investigated by determination of mRNA expression following organ culture for 24h under longitudinal load by a hanging weight. In the absence of miR-143/145, stretch-induced mRNA expression of contractile markers in the portal vein was reduced. This was associated with decreased amplitude of spontaneous activity and depolarization-induced contractile and intracellular calcium responses, while contractile responses to 5-HT were largely maintained. We found that these effects correlated with a reduced basal expression of the pore-forming subunit of L-type calcium channels and an increased expression of CaMKIIδ and the transcriptional repressor DREAM. Our results suggest that the microRNA-143/145 cluster plays a role in maintaining stretch-induced contractile differentiation and calcium signalling in the portal vein. This may have important implications for the use of these microRNAs as therapeutic targets in vascular disease.

CACNA1C risk variant is associated with increased amygdala volume.

Genome-wide association studies suggest that genetic variation within L-type calcium channel subunits confer risk to psychosis. The single nucleotide polymorphism at rs1006737 in CACNA1C has been associated with both schizophrenia and bipolar disorder and with several intermediate phenotypes that may serve as neurobiological antecedents, linking psychosis to genetic aetiology. Amongst others, it has been implicated in alterations in amygdala structure and function. In the present study, we show that the risk allele (A) is associated with increased amygdala volume in healthy individuals (n=258). This observation reinforces a hypothesis that genetic variation may confer risk to psychosis via alterations in limbic structures. Further study of CACNA1C using intermediate phenotypes for psychosis will determine the mechanisms by which variation in this gene confers risk.

MiR-103 inhibits osteoblast proliferation mainly through suppressing Cav1.2 expression in simulated microgravity

Emerging evidence indicates that microRNAs (miRNAs) play important roles in modulating osteoblast function and bone formation. However, the influence of miRNA on osteoblast proliferation and the possible mechanisms underlying remain to be defined. In this study, we aimed to investigate whether miR-103 regulates osteoblast proliferation under simulated microgravity condition through regulating Cav1.2, the primary subunit of L-type voltage sensitive calcium channels (LTCCs). We first investigated the effect of simulated microgravity on osteoblast proliferation and the outcomes clearly demonstrated that the mechanical unloading inhibits MC3T3-E1 osteoblast-like cell proliferation. Using quantitative Real-Time PCR (qRT-PCR), we provided data showing that miR-103 was up-regulated in response to simulated microgravity. In addition, we observed that up-regulation of miR-103 inhibited and down-regulation of miR-103 promoted osteoblast proliferation under simulated microgravity condition. Furthermore, knocking-down or over-expressing miR-103, respectively, up- or down-regulated the level of Cav1.2 expression and LTCC currents, suggesting that miR-103 acts as an endogenous attenuator of Cav1.2 in osteoblasts under simulated microgravity condition. More importantly, we showed that the effect of miR-103 on osteoblast proliferation was diminished in simulated microgravity, when co-transfecting miR-103 mimic or inhibitor with Cav1.2 siRNA. Taken together, our data suggest that miR-103 inhibits osteoblast proliferation mainly through suppression of Cav1.2 expression under simulated microgravity condition. This work may provide a novel mechanism of microgravity-induced detrimental effects on osteoblast proliferation, identifying miR-103 as a novel possible therapeutic target in bone remodeling disorders in this mechanical unloading.

Mechanism of Aging‐associated Hypertension: Role of Smooth Muscle Cell Mineralocorticoid Receptor Regulation of Vascular L‐type Calcium Channels

Hypertension (HTN), the most prevalent cardiovascular disease risk factor, increases with age. We found that mice with SMC-specific deletion of the mineralocorticoid receptor (MR KO) lack the aging-associated rise in blood pressure (BP). We hypothesize that SMC MR contributes to BP regulation with aging by regulating vascular L-type calcium channels (LTCC). Patch clamp studies on mesenteric resistance vessel (MRV) SMC from young (3-4 mo.) and aged (9-12 mo.) MR intact and MR KO reveal reduced LTCC current density in aged MR KO (p<0.05 vs. MR intact). Fura-2 photometry studies reveal decreased MRV Ca flux in aged MR KO (p<0.05 vs. MR intact) in response to BayK (LTCC agonist). Contraction to BayK is blunted in aged MR KO MRV (p<0.05 vs. MR intact). RNA expression of Cav1.2, the pore-forming subunit of LTCC, is reduced by 60% in aged MR KO vs. MR intact MRV (p<0.05). Micro-RNA expression profiling identified miR-155 as being modulated by SMC MR with aging. MRV miR-155 expression is increased in aged MR KO (p<0.05 vs. MR intact). Ingenuity pathway analysis identified Cav1.2 as a potential target of miR-155. Overexpression of miR-155 in mouse MRV SMC reduced Cav1.2 expression by 45% (p<0.05 vs. ctrl). These data suggest that SMC MR contributes to HTN through regulating Cav1.2 expression/function, potentially via miR-155. These results enhance our basic understanding of BP control with aging, and provide support for innovative therapeutic targets to treat aging-associated HTN. Supported by HL095590-05.

Atrial fibrillation complicated by heart failure induces distinct remodeling of calcium cycling proteins.

Atrial fibrillation (AF) and heart failure (HF) are two of the most common cardiovascular diseases. They often coexist and account for significant morbidity and mortality. Alterations in cellular Ca2+ homeostasis play a critical role in AF initiation and maintenance. This study was designed to specifically elucidate AF-associated remodeling of atrial Ca2+ cycling in the presence of mild HF. AF was induced in domestic pigs by atrial burst pacing. The animals underwent electrophysiologic and echocardiographic examinations. Ca2+ handling proteins were analyzed in right atrial tissue obtained from pigs with AF (day 7; n = 5) and compared to sinus rhythm (SR) controls (n = 5). During AF, animals exhibited reduction of left ventricular ejection fraction (from 73% to 58%) and prolonged atrial refractory periods. AF and HF were associated with suppression of protein kinase A (PKA)RII (-62%) and Ca2+-calmodulin-dependent kinase II (CaMKII) δ by 37%, without changes in CaMKIIδ autophosphorylation. We further detected downregulation of L-type calcium channel (LTCC) subunit α2 (-75%), sarcoplasmic reticulum Ca2+-ATPase (Serca) 2a (-29%), phosphorylated phospholamban (Ser16, -92%; Thr17, -70%), and phospho-ryanodine receptor 2 (RyR2) (Ser2808, -62%). Na+-Ca2+ exchanger (NCX) levels were upregulated (+473%), whereas expression of Ser2814-phosphorylated RyR2 and LTCCα1c subunits was not significantly altered. In conclusion, AF produced distinct arrhythmogenic remodeling of Ca2+ handling in the presence of tachycardia-induced mild HF that is different from AF without structural alterations. The changes may provide a starting point for personalized approaches to AF treatment.

Dysregulation of miR-34a links neuronal development to genetic risk factors for bipolar disorder.

Bipolar disorder (BD) is a heritable neuropsychiatric disorder with largely unknown pathogenesis. Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD. Here we show that levels of miR-34a, which is predicted to target multiple genes implicated as genetic risk factors for BD, are increased in postmortem cerebellar tissue from BD patients, as well as in BD patient-derived neuronal cultures generated by reprogramming of human fibroblasts into induced neurons (iNs) or into induced pluripotent stem cells (iPSCs) subsequently differentiated into neurons. Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets. Using human iPSC-derived neuronal progenitor cells (hNPCs), we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration. Taken together, we propose that miR-34a serves as a critical link between multiple etiological factors for BD and its pathogenesis through the regulation of a molecular network essential for neuronal development and synaptogenesis.

The α2δ Subunit Efficiently Couples VSDs Activation to Pore Opening in Human Cav1.2 Channels

The α2δ-1 auxiliary subunit binds to the pore-forming α1c subunit of voltage-gated L-type calcium channels (CaV1.2) and facilitates both membrane trafficking and voltage-dependent activation. Using voltage-clamp fluorometry to study human CaV1.2 channels expressed in Xenopus oocytes, we have recently found that α2δ-1 association, in the presence of β3 subunits, causes voltage sensor domains (VSDs) I, II and III, but not IV, to activate at more hyperpolarized potentials (ΔVhalf= −39mV, −32mV, −18mV for VSDs I-III respectively) and with steeper voltage-dependence (z fold increase with α2δ-1: 1.9, 2.2 and 1.6 for VSDs I-III respectively). Thus, the α2δ-1-induced facilitation of CaV1.2 voltage-dependent activation seems to be mediated through the remodeling of three VSDs.We analyzed our voltage-clamp fluorometry data with a 32-state allosteric model for CaV1.2 activation, consisting of five gating particles (one pore, four VSDs). By simultaneously fitting kinetic and steady-state data from the pore (ionic currents) and from the individual VSDs (fluorescence), we estimated the energetic contribution of each VSD to pore opening in the presence and in the absence of α2δ-1. The model predicts that, in channels not associated with α2δ-1, all VSDs were poorly energetically-coupled to pore opening. The association of α2δ-1 with α1c+β3 specifically increased the coupling energy of VSDs II and III to the pore. In agreement with the model prediction, we found that without α2δ-1 subunits the ionic current deactivation is largely voltage-independent.A low-resolution structure of CaV1.2 channel complexes shows that the α2δ-1 subunit forms a cap that embraces ∼¾ of the extracellular surface of the α1c subunit (Walsh et al., JBC 2009). Based on our results, we propose that the exposed ¼ of the α1c subunit is VSD IV, which does not show evidence of functional interaction with α2δ-1 subunits.

Identification and characterization of novel protein-derived arginine-rich cell-penetrating peptides

Cell-penetrating peptides (CPPs) have proven their potential as an efficient delivery system due to their intrinsic ability to traverse biological membranes and transport various cargoes into the cells. In the present study, we have identified novel natural protein-derived CPPs using an integrated (in silico and experimental) approach. First, using bioinformatics approach, arginine-rich peptide segments were extracted from SwissProt proteins and their cell-penetrating properties were predicted. Finally, eight peptides were selected and their internalization was validated using experimental techniques. Laser scanning confocal microscopy and flow cytometry confirmed that seven out of eight peptides were internalized into live cells with varying efficiencies without significant cytotoxicity. Three peptides have shown higher internalization efficacy than TAT peptide, the most widely used CPP. Among these three peptides, one peptide (P8), derived from voltage-dependent L-type calcium channel subunit alpha-1D, was able to accumulate inside in a variety of cell types very efficiently through a rapid dose-dependent process. Further, experiments involving inhibition with various endocytic inhibitors along with co-localization studies indicate that the uptake mechanism of P8 is macropinocytosis, a fluid-phase endocytosis process. In addition, competitive inhibition with heparin revealed the involvement of cell-surface proteoglycans in P8 uptake. In summary, the present study provides evidence that an integrated in silico and experimental approach is an effective strategy for the identification of novel CPPs and CPPs identified in the present study have promising perspectives for future drug delivery applications.