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• W2023486980 abstract "CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The α2δ-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2tm1NCIF null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate α2δ-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the α2δ-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans. CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The α2δ-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2tm1NCIF null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate α2δ-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the α2δ-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans. Voltage-dependent calcium channels (VDCCs)1Berridge MJ Bootman MD Lipp P Calcium: a life and death signal.Nature. 1998; 395: 645-648Crossref PubMed Scopus (1752) Google Scholar play a role in generating the rhythmic membrane potential behavior of many excitable cells and also provide a pathway through which Ca2+ enters cells to serve as a second messenger that regulates a diversity of cellular processes.1Berridge MJ Bootman MD Lipp P Calcium: a life and death signal.Nature. 1998; 395: 645-648Crossref PubMed Scopus (1752) Google Scholar, 2Santella L Carafoli E Calcium signaling in the cell nucleus.EMBO J. 1997; 11: 1091-1109Google Scholar, 3Catterall WA Structure and regulation of voltage-gated Ca2+ channels.Annu Rev Cell Dev Biol. 2000; 16: 521-555Crossref PubMed Scopus (1903) Google Scholar VDCCs consist minimally of an α1 protein that forms the channel pore and voltage-sensor. Core α1 subunits are used for dividing VDCCs into three major phylogenetic types, which are supported by pharmacological and functional classification: T-type (α1G, α1H, and α1I); L-type (α1C, α1D, α1F, and α1S); and the presynaptic channels-type (α1A, α1B, and α1E).4Randall A Benham CD Recent advances in the molecular understanding of voltage-gated Ca2+ channels.Mol Cell Neurosci. 1999; 14: 255-272Crossref PubMed Scopus (72) Google Scholar A variety of auxiliary regulatory subunits, including α2δ, β, and γ, associate with the α1-subunits, forming different heteromeric functional VDCCs.5De Waard M Gurnett CA Campbell KP Structural and functional diversity of voltage-activated calcium channels.Ion Channels. 1996; 4: 41-87Crossref PubMed Scopus (142) Google Scholar, 6Hofmann F Biel M Flockerzi V Molecular basis for Ca2+ channel diversity.Annu Rev Neurosci. 1994; 17: 399-418Crossref PubMed Scopus (450) Google Scholar, 7Walker D De Waard M Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function.Trends Neurosci. 1998; 21: 148-154Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar The members of the α2δ family include α2δ-1, which is ubiquitously expressed; α2δ-2, which is expressed in brain, heart, and other organs; and α2δ-3, whose transcripts are found mostly in the brain.8Klugbauer N Lacinova L Marais E Hobom M Hofmann F Molecular diversity of the calcium channel α2δ subunit.J Neurosci. 1999; 19: 684-691Crossref PubMed Google Scholar, 9Gao B Sekido Y Maximov A Saad M Forgacs E Latif F Wei MH Lerman M Lee JH Perez-Reyes E Bezprozvanny I Minna JD Functional properties of a new voltage-dependent calcium channel α(2)δ auxiliary subunit gene (CACNA2D2).J Biol Chem. 2000; 275: 12237-12242Crossref PubMed Scopus (147) Google Scholar, 10Gong HC Hang J Kohler W Li L Su TZ Tissue-specific expression and gabapentin-binding properties of calcium channel α2δ subunit subtypes.J Membr Biol. 2001; 184: 35-43Crossref PubMed Scopus (123) Google Scholar α2δ subunits consist of two proteins that are encoded by a single gene, the product of which is post-translationally cleaved from a single polypeptide precursor.10Gong HC Hang J Kohler W Li L Su TZ Tissue-specific expression and gabapentin-binding properties of calcium channel α2δ subunit subtypes.J Membr Biol. 2001; 184: 35-43Crossref PubMed Scopus (123) Google Scholar, 11De Jongh KS Warner C Catterall WA Subunits of purified calcium channels: α 2 and δ are encoded by the same gene.J Biol Chem. 1990; 265: 14738-14741Abstract Full Text PDF PubMed Google Scholar The discovery of worm and fly orthologs of α2δ-encoding genes12Lerman MI Minna JD The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes.Cancer Res. 2000; 60: 6116-6133PubMed Google Scholar demonstrates their conservation over billions of years of evolution, suggesting that the α2δ protein is of fundamental importance in cellular function. In humans, α2δ-2 is encoded by CACNA2D2 (GenBank Accession No. AF040709), which is contained, along with seven other genes, in a 120-kb region of chromosome 3p21.3 that is frequently deleted in lung, breast, and other cancers.12Lerman MI Minna JD The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes.Cancer Res. 2000; 60: 6116-6133PubMed Google Scholar Reduction or absence of CACNA2D2 expression in non-small cell lung cancer cell lines suggests that α2δ-2 itself may be a tumor suppressor protein. α2δ-2 is predicted to be a heavily glycosylated 175-kd protein whose single-pass transmembrane δ piece anchors the α2 protein to the membrane.13Gurnett CA De Waard M Campbell KP Dual function of the voltage-dependent Ca2+ channel α 2 δ subunit in current stimulation and subunit interaction.Neuron. 1996; 16: 431-440Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar Electrophysiological studies demonstrated that α2δ-2 might function as an auxiliary component of a variety of different VDCCs: L-type [Cav1.2 (α1C)]; P/Q-type [Cav2.1 (α1A)]; N-type [Cav2.2 (α1B)]; R-type [Cav2.3 (α1E)]; and T-type [Cav3.1 (α1G) and Cav3.2 (α1H)].8Klugbauer N Lacinova L Marais E Hobom M Hofmann F Molecular diversity of the calcium channel α2δ subunit.J Neurosci. 1999; 19: 684-691Crossref PubMed Google Scholar, 9Gao B Sekido Y Maximov A Saad M Forgacs E Latif F Wei MH Lerman M Lee JH Perez-Reyes E Bezprozvanny I Minna JD Functional properties of a new voltage-dependent calcium channel α(2)δ auxiliary subunit gene (CACNA2D2).J Biol Chem. 2000; 275: 12237-12242Crossref PubMed Scopus (147) Google Scholar, 14Hobom M Dai S Marais E Lacinova L Hofmann F Klugbauer N Neuronal distribution and functional characterization of the calcium channel α2δ-2 subunit.Eur J Neurosci. 2000; 12: 1217-1226Crossref PubMed Scopus (102) Google Scholar Co-expression of α2δ-2 with each of these α1 subunits enhanced currents through the heteromeric channels. While these experiments characterized α2δ-2 as electrophysiologically versatile, its biological significance remained uncertain. Here we report that the Cacna2d2 null mice show ataxia, growth retardation, increased susceptibility to seizures, and a reduced life span. Phenotypic similarity with Cacna1a null mice and spontaneous Cacna1a mutants implicates α2δ-2 in regulation of P/Q-type currents as a component of Cav2.1 channels and suggests a possible involvement of the gene in human hereditary cerebellar ataxias and epilepsies. We also provide evidence that α2δ-2 expression in the central nervous system, dorsal sympathetic ganglia, and cardiac conductive tissue may be related to the observed disturbances in null mutants’ heart rate regulation. A replacement-type Cacna2d2-targeting vector was based on a mouse genomic clone isolated from a 129/SvJ lambda FIXII library (Stratagene, La Jolla, California). Cacna2d2 exons were identified by Southern hybridization, partial sequencing, and BLAST analysis. Mouse EST clone represented in GenBank by Accession No. AA000341 (Research Genetics, Huntsville, AL) was used as a probe. A 2629-bp fragment containing eight exonic fragments (positions 2517 to 3184 in the mouse Cacna2d2 mRNA AF247139) and a 6.3-kb fragment representing the 3′ non-coding region of Cacna2d2 were integrated into the construct as 5′ and 3′ flanking sequences, respectively. The neo-gene with the phosphor-glycerol kinase 1 promoter and the bovine growth hormone polyadenylation sequence (pGKneobpA) was used as a positive selectable marker; the pGK-thymidine kinase cassette was used as a negative selectable marker.15Bonin A Reid SW Tessarollo L Isolation, microinjection, and transfer of mouse blastocysts.Methods Mol Biol. 2001; 158: 121-134PubMed Google Scholar Electroporation and selection were performed using CJ7 ES cell line, as described elsewhere.16Tessarollo L Manipulating mouse embryonic stem cells.Methods Mol Biol. 2001; 158: 47-63PubMed Google Scholar DNAs derived from G418/FIAU-resistant ES clones were screened using a diagnostic EcoRI restriction enzyme sites and the 2.0-kb 3′ probe external to the targeting sequence indicated in Figure 1A. This probe detects a ∼20-kb EcoRI fragment in wild-type DNA and an 8.3-kb EcoRI-fragment in a mutant allele. Two independent Cacna2d2-targeted cell lines were isolated and one of those, when injected into C57BL/6J blastocysts, transmitted the mutant allele to the offspring generating chimeras. Chimeras were then identified by the agouti coat color. Male chimeras were mated with C57BL/6J females and agouti-colored offspring were genotyped by tail clip. Chimeras that transmitted the Cacna2d2tm1NCIF allele through the germline were then mated to C57BL/6J females to establish lines. RT-PCR on mouse RNA samples was performed using a SuperScript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Two primer sets were used for RT-PCR. One of them, 5′-GCATAACTATGATGTCACAC-3′ and 5′-GGGGCTTGAAGATATAACCA-3′, represented a Cacna2d2 cDNA fragment with positions 1742–2862. The other pair, 5′-TCTACTCTGTGTCCTCATTG-3′ and 5′-TGAGTCTAGGGACTGTGGG-3′, corresponded to positions 3113–4149 (GenBank Accession No. AF247139). Comparative PCR on human cardiovascular MTN cDNA panel (Clontech, Palo Alto, CA) was performed as suggested by the manufacturer with human primers 5′-GCATAACTATGACGTCACAC-3′ and 5′-GGGGCTTGAAGACATAACCG-3′ corresponding to the first set of mouse primers. Northern analysis and RT-PCR were performed on mouse brain mRNAs isolated with a FastTrack 2.0 kit (Invitrogen). A Cacna2d2 cDNA EST clone (GenBank Accession No. AA000341, positions 3237–5498 in AF042792) purchased from Genome Systems, Inc. (St. Louis, MO) was used as a 3′ probe, while the RT-PCR product obtained with the first set of primers (see above) was used as a 5′ probe. The same mouse EST clone was also used to generate 35S-labeled RNA probes for in situ hybridization on mouse tissues. These probes were synthesized using MAXIscript (Ambion Inc., Austin, TX) in vitro transcription kit and two primers. Primer 5′TAATACGACTCACTATAGGGAGAcctatgactatcaggcag-3′ contained T7 RNA-polymerase promoter shown in uppercase and was used to produce sense probe. Primer 5′AATTAACCCTCACTAAAGGGAGAccacagtctgaggtatct-3′ contained T3 RNA-polymerase promoter and was used to generate anti-sense probe. DNA template used in the RNA synthesis was eliminated with DNase I following the in vitro transcription reaction. For in situ hybridization, tissues of postnatal mice or embryos at stages 13.5 and 15 dpc were perfused and fixed as described.17Shelton JM Lee MH Richardson JA Patel SB Microsomal triglyceride transfer protein expression during mouse development.J Lipid Res. 2000; 41: 532-537Abstract Full Text Full Text PDF PubMed Google Scholar Briefly, the samples were hydrated, paraffin-embedded, and sectioned at 4 mm onto microscope slides. For pre-hybridization, the slides were deparaffinized in xylene, hydrated through a series of graded ethanol/DEPC-saline, put through a microwave RNA retrieval procedure, and the RNA was further unmasked by permeabilization with pronase. For hybridization, a riboprobe 750,000 cpm was added to the hybridization buffer,17Shelton JM Lee MH Richardson JA Patel SB Microsomal triglyceride transfer protein expression during mouse development.J Lipid Res. 2000; 41: 532-537Abstract Full Text Full Text PDF PubMed Google Scholar denatured at 95°C for 5 minutes, and incubated with the slides for 14 hours at 55°C. Slides were washed in the washing buffer, incubated with RNase A, dehydrated in graded ethanol rinses, and dried under vacuum. Dried slides were immersed into twofold-diluted K.5 nuclear emulsion (Polysciences, Warrington, PA), slowly dried at room temperature, and exposed for 14 days. The slides were then developed using D19 reagent (Eastman Kodak, Rochester, NY), and latent images fixed using Kodak Fixer. The slides were finally thoroughly rinsed, counterstained with hematoxylin (Richard-Allen, Kalamazoo, MI), dehydrated, and glass-covered using permanent mounting media. Visualization of signal was done on a Leitz Laborlux-S (Wetzlar, Germany) microscope. Animals used in these studies were maintained in facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and all procedures were performed under protocols approved by the NIH (Publication No. 86–23, 1985) and NCI Animal Care and Use Committees in strict compliance with the Guide for the Care and Use of Laboratory Animals of the National Research Council (National Academy Press, Washington, DC; http://www.nap.edu/readingroom/books/labrats). Twelve- to 24-week-old mice were used. Pentylenetetrazol (PTZ) (Sigma-Aldrich, St. Louis, MO), in 0.9% NaCl solution, was administered by subcutaneous injection into a loose fold of skin on the right belly. Mice were observed for 30 minutes. Animals exhibiting a clonic spasm of at least 5 seconds duration were scored as positive for seizure occurrence. To determine the 50% convulsive dose (CD50) a range of PTZ doses were used spanning the CD50 value. Each mouse received only a single injection and the animal was immediately euthanized after exhibiting convulsions or at the end of the 30-minute observation period. In a separate series of experiments the behavioral seizure score was determined with increasing cumulative doses of PTZ. Mice (18 to 24 weeks old) were injected intraperitoneally with a 10 mg/kg dose of PTZ and observed for 30 minutes. Additional doses of 20, 40, and 60 mg/kg were administered at 30-minute intervals until tonic hind limb extension occurred, at which time the mice were immediately euthanized. PTZ typically induced a sequence of behaviors that were scored in each of the 30-minute intervals after dosing according to the following scale: 0, normal behavior; 1, behavioral arrest; 2, vocalization and limb twitches; 3, sustained fore limb clonus; 4, wild running and jumping; and 5, tonic hind limb extension. A modification of the horizontal screen test was used as a measure of overall motor function.18Coughenour LL Mclean JR Parker RB A new device for the rapid measurement of impaired motor function in mice.Pharmacol Biochem Behav. 1977; 6: 351-353Crossref PubMed Scopus (170) Google Scholar Mice were placed on a horizontally oriented grid (consisting of parallel 1.5-mm diameter rods situated 1 cm apart), and the grid was inverted. The time the animal remained on the underside of the grid was recorded. Mice that fell from the grid within <15 seconds were considered to have reduced strength or coordination. Mice with hind paws dipped in non-toxic ink were placed at one end of a dark tunnel measuring 10 cm × 10 cm × 50 cm, the bottom of which was lined with white paper. Mice walked down the tunnel, and their footprints were used to assess the ability to walk in a straight line, irregularities, and orientation of the hind paws.19Crawley JN What's Wrong with My Mouse? Wiley-Liss, New York1999: 56-57Google Scholar Trans-thoracic echocardiography was performed using an Acuson Sequoia equipped with a 15-MHz linear transducer (15L8) in a phased-array configuration. Animals were placed on a heating pad (37°C) and imaged under isoflurane anesthesia in the left lateral decubitus position. Two-dimensional images were obtained from parasternal long- and short-axis views with optimized gain and depth settings. M-mode images were obtained from short-axis views at the papillary muscle level at a sweep speed of 100 mm/s. Two-dimensional and M-mode images were digitized and stored for off-line analysis. Cardiac measurements were performed off-line from the M-mode images using a leading edge-to-leading edge technique according to the American Society of Echocardiography guidelines.20Schiller NB Shah PM Crawford M DeMaria A Devereux R Feigenbaum H Gutgesell H Reichek N Sahn E Schnittger I Recommendations for quantitation of the left ventricle by two-dimensional echocardiography.J Am Soc Echocardiogr. 1989; 2: 358-367Abstract Full Text PDF PubMed Scopus (49) Google Scholar Measurements from five cardiac cycles were averaged to obtain the following parameters in diastole and systole: interventricular septum (IVSd, IVSs); posterior wall (PWd, PWs); and left ventricular internal diameter (LVIDd, LVIDs). The percentage of LV fractional shortening (LV% FS) was calculated as {(LVIDd − LVIDs/LVIDd} × 100. LV mass was calculated according to the uncorrected cube method using the equation LV mass (mg) = 1.055{(IVSd+LVIDd+PWd)3 − (LVIDd)3}, as previously reported.21Devereux RB Alonso DR Lutas EM Gottlieb GJ Campo E Sachs I Reichek N Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings.Am J Cardiol. 1986; 57: 450-458Abstract Full Text PDF PubMed Scopus (5474) Google Scholar ECGs were recorded in conscious and sedated mice with different Cacna2d2 status using a recently developed noninvasive ECG recording device and software (Mouse Specifics, Boston, MA), as described previously.22Chu V Otero JM Lopez O Sullivan MF Morgan JP Amende I Hampton TG Electrocardiographic findings in mdx mice: a cardiac phenotype of Duchenne muscular dystrophy.Muscle Nerve. 2002; 26: 513-519Crossref PubMed Scopus (53) Google Scholar Conscious or isoflurane-sedated animals were positioned on the ECG platform with gel-coated electrodes embedded in its base. The amplified signals were digitized with 16-bit precision at a sample rate of 2000 samples/second. Only continuous recordings (>10 to 25 ECG complexes) generated from mice with three paws in contact with three electrodes were analyzed. The following parameters were measured: HR and RR interval, PR interval, QRS duration and maximum height, QT and corrected QT intervals. Noninvasive blood pressure and serum electrolytes were measured on each of four null and five wild-type animals by Phenotyping Service of the Jackson Laboratory (West Sacramento, CA). Growth hormone, T3, T4, and IGF-1 serum levels in each of four null and four wild-type animals were assayed by Anilytics, Inc. (Gaithersburg, MD). Cacna2d2+/+, Cacna2d2+/−, and Cacna2d2−/− male and female mice from 22 days to 70 days of age were completely necropsied. All tissues were fixed in neutral buffered formalin, embedded in paraffin, sections cut at 4 to 6 μm, and stained with hematoxylin and eosin (H&E). Selected tissues were stained with luxol fast blue and Bodian stains. For immunohistochemistry of the brain, antibodies to glial fibrillary acidic protein (GFAP) (DAKO, Carpinteria, CA), Ki-67 (polyclonal, Novocastra, Newcastle on Tyne, UK), calbindin (D-28K, Sigma), and caspase-3 (Promega, Madison, WI) were used. The TUNEL method (ApopTag® In Situ Apoptosis Detection kit, Intergen, Purchase, NY) was used to identify apoptotic cells. Data in this study were evaluated using standard analysis of variance and Student's t-tests, repeated measures analysis of variance, profile analysis, analysis of covariance, post-hoc tests, non-parametric methods, survival analysis, and graphical descriptive techniques. Data analyzed with parametric methods were routinely tested for conformance with homogeneity of variance and covariance requirements. Kaplan-Meier plots were constructed for survival, and differences in survival distributions were tested with the log rank test. We constructed a Cacna2d2-targeting vector, in which a 3445-bp XbaI/HindIII fragment (positions 6593434–6596878 on NT_039477) containing seven 3′-terminal exons of the gene was replaced by a neomycin-resistance gene (Figure 1A). This replacement eliminated the sequence encoding the entire δ subunit with a single transmembrane domain plus the 87 C-terminal amino acids of the α2 responsible for binding the δ subunit. Genotyping of 256 F2 offspring by Southern hybridization (Figure 1B) revealed a Cacna2d2+/+: Cacna2d2+/−: Cacna2d2−/− distribution (64:130:62) that was very close to the expected Mendelian distribution. Northern blot analysis on brain mRNAs showed expression of the full-length transcript in heterozygotes but not null-mutant animals (Figure 1, C and D). While Cacna2d2+/− mice appeared healthy, had normal life spans, and were not distinguishable from their Cacna2d2+/+ littermates in any assay, null-mutants could be reliably identified at approximately 4 weeks of age and older by dystonic episodes, ataxic waddling gait typical for cerebellar dysfunction, and reflexive clutching of hind limbs when lifted by their tails (Figure 2). Dystonic episodes usually started with characteristic extension of the hind limbs. During the next stage that lasted from a few minutes to more than one-hour, conscious animals were immobilized on their abdomen or side by whole-body extension. After recovery, the null-mutants walked in a poorly coordinated fashion, dragging their hind limbs and often listing to one side. They frequently showed myoclonic jerking of the limbs or whole body tremor, and tonic extension of the hind limbs. In four animals, generalized clonic seizures that lasted up to 30 minutes and affected the entire body were observed. The null-mutants developed growth delay starting at the age of 4 weeks (P < 0.0001 by analysis of covariance, profile analysis, and post-hoc t-tests). The mean body weights of Cacna2d2−/− mice were ∼1.5 to 2 times less than that of their wild-type age mates (Figure 3A). Cacna2d2−/− mice also had markedly reduced life spans (P < 0.0001 by the log rank test), with a median survival time of 150 days (Figure 3B). Among 50 Cacna2d2−/− mice studied, 23 (46%) died prematurely with no obvious cause of death. Most of these mice died at young ages, and no premature deaths were observed at ages older than 6 months (Figure 3B).Figure 3Growth delay and premature death in Cacna2d2−/− mice. A: Differences in body weights between Cacna2d2−/− (n = 7 for weeks 1 to 4; n = 4 for weeks 5 to 8) and control mice (n = 12) are highly significant (P < 0.0001) from week 4 on. B: Kaplan-Meier curves showing highly significant (P < 0.0001) survival differences between Cacna2d2−/− (n = 50) and control mice (n = 214). Censored observations (life spans of sacrificed animals) are shown with vertical bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Motor impairment in null-mutants was evaluated with the inverted screen test, in which the animals were required to support their body weight by grasping a wire grid. None of 6 Cacna2d2−/− remained on the inverted screen for at least 15 seconds, whereas 3 of 6 of the Cacna2d2+/− and 6 of 7 of the Cacna2d2+/+ remained on the grid. The mean times on the grid were 5.5 ± 1.5 seconds, 56 ± 23 seconds, and 138 ± 83 seconds, respectively. Compared with wild-type control mice, Cacna2d2−/− mice had significant motor impairment (P = 0.006, Kolmogorov-Smirnov test). Heterozygotes were not significantly different (P = 0.713) from wild-type mice. In situ hybridization with mouse embryo and adult cerebellum sections showed that Cacna2d2 is expressed primarily in the central nervous system (Figure 4). In the postnatal cerebellum, a very high level of gene expression was detected in the Purkinje cell layer, while granular produced poor signals (Figure 4, A and B). Remarkably, Cacna2d2 expression was also detected outside the central nervous system, namely, in the dorsal sympathetic ganglia (Figure 4C). While no Cacna2d2 expression was detected in mouse embryonic heart, previous reports indicated that this gene is highly expressed in both mouse and human adult hearts.8Klugbauer N Lacinova L Marais E Hobom M Hofmann F Molecular diversity of the calcium channel α2δ subunit.J Neurosci. 1999; 19: 684-691Crossref PubMed Google Scholar, 9Gao B Sekido Y Maximov A Saad M Forgacs E Latif F Wei MH Lerman M Lee JH Perez-Reyes E Bezprozvanny I Minna JD Functional properties of a new voltage-dependent calcium channel α(2)δ auxiliary subunit gene (CACNA2D2).J Biol Chem. 2000; 275: 12237-12242Crossref PubMed Scopus (147) Google Scholar, 10Gong HC Hang J Kohler W Li L Su TZ Tissue-specific expression and gabapentin-binding properties of calcium channel α2δ subunit subtypes.J Membr Biol. 2001; 184: 35-43Crossref PubMed Scopus (123) Google Scholar Indeed, using the RT-PCR assay we demonstrated that the CACNA2D2 mRNA is expressed mainly in four cardiac regions: auricles, aorta, interventricular septum, and atrioventricular node (Figure 5). These areas represent highly innervated parts of the heart's conductive system. Limited levels of CACNA2D2 expression were also detected in the muscle tissue, such as apex and ventricles.Figure 5PCR analysis of CACNA2D2 expression on human heart cDNA panel. Lane 1, adult heart; lane 2, fetal heart; lane 3, aorta; lane 4, apex of the heart; lane 5, atrium, left; lane 6, atrium, right; lane 7, auricle, dextra; lane 8, auricle, sinistra; lane 9, ventricle, left; lane 10, ventricle, right; lane 11, interventricular septum; lane 12, atrioventricular node. The 35-cycle PCR shows significant differences between heart areas in the CACNA2D2 mRNA content. G3PDH transcripts are used as a control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To explore the possibility of heart failure due to the α2δ-2 deficiency we evaluated null mutants’ heart function. First, histo-pathological study of the null mutants’ hearts revealed no signs of cardiomyopathy, apoptosis, or any other abnormality (data not shown). Second, heart function was assessed in vivo by surface electrocardiography (ECG) and trans-thoracic echocardiography. A trend to bradycardia was revealed in conscious null-mutants as compared to the Cacna2d2+/− and Cacna2d2+/+ control group (747 ± 53 versus 714 ± 73, control versus knockout, P = 0.07, Table 1). No significant differences were observed in other major ECG parameters. Remarkably, isoflurane anesthesia produced different effects on heart rates in two cohorts. While in both cases isoflurane reduced heart rate, the null-mutants’ response was less profound than that of the control cohort (186 ± 37 versus 102 ± 44, control versus knockout, P = 0.01, Table 1). Consistent with the previous histopathological evaluation, echocardiographic study showed no evidence of systolic abnormalities in the null mutants’ echocardiograms, and no significant differences in IVSd, IVSs, PWd, PWs, LVIDd, LVIDs, LV%FS, and LV mass were found between null mutants and wild-type animals (6 null-mutants and 6 wild-type animals; data not shown). No significant differences were detected in blood pressure and serum electrolyte concentrations between Cacna2d2−/− mice and a control group (potassium, sodium, magnesium, and calcium measurements on five wild-type and four homozygous mutant mice; data not shown).Table 1Major ECG Parameters in Wild-Type and Cacna2d2−/− MiceParameterControlNullp-valueHeart rate (bpm)747 (53)714 (73)MS (0.07)RR interval (ms)81 (7)86 (10)MS (0.07)PR interval (ms)27 (3)26 (3)NSQRS duration (ms)11 (1)11 (1)NSQT interval (ms)42 (5)43 (4)NSQTc interval (ms)46 (3)46 (2)NSΔHRiso186 (37)102 (44)0.01ΔRRiso27 (9)14 (7)0.04Mean (standard deviation); p-values computed with Student's t test; QTc, corrected QT interval; for baseline measurements, 29 null-mutants" @default.
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• W2023486980 title "Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene" @default.
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