Identification of the calcium channel alpha 1E (Ca(v)2.3) isoform expressed in atrial myocytes.

Antisense oligonucleotides targeting the calcium channel alpha 1E (Ca(v)2.3) subunit significantly inhibit the insulin-like growth factor-1 (IGF-1)-stimulated increase in low voltage-activated (LVA) (T-type) calcium current in cultured rat atrial myocytes [Proc. Natl. Acad. Sci. U.S.A. 94(1997) 14936]. As part of a continuing effort to understand the regulation of LVA current expression in the heart, we have identified the specific alpha 1E isoform that is expressed in atrial tissue. Through reverse transcription-polymerase chain reaction (RT-PCR), nine overlapping partial clones spanning the entire coding region of the cardiac alpha 1E mRNA were obtained. The predominate isoform in atrial tissue was identified and found to be highly homologous to the alpha 1E isoform previously isolated from kidney and the islets of Langerhans [Eur. J. Biochem. 257(1998) 274]. The expression of alpha 1E in the heart occurs specifically in cardiac myocytes and not in smooth muscle or fibroblasts as demonstrated by RT-PCR performed on isolated atrial myocytes and by in situ hybridization.

Disruption of growth hormone secretion alters Ca2+ current density and expression of Ca2+ channel and insulin-like growth factor genes in rat atria.

The influence of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis on expression of low-voltage-activated (LVA) Ca2+ current in atrial tissue was investigated using spontaneous dwarf (SpDwf) rats, a mutant strain that lacks GH. Atrial myocytes from SpDwf rats express LVA and high-voltage-activated (HVA) Ca2+ currents and the Ca2+ channel alpha1-subunit genes CaV1.2, CaV2.3, CaV3.1, and CaV3.2. LVA current density decreases significantly beginning at, or shortly after, birth in normal animals; however, its density is maintained in SpDwf rats at 1 pA/pF for > or =12 wk after birth. The abundance of mRNAs encoding CaV2.3 and CaV3.2 declines with advancing age in normal atrial development, yet expression of CaV2.3 mRNA remains significantly elevated in older SpDwf animals. Quantitation of local transcript levels for mRNAs encoding IGF-I and IGF-I receptor (IGF-IR) also reveals significant differences in expression of these transcripts in atrial tissue of SpDwf animals compared with controls. In SpDwf rats, the abundance of IGF-IR mRNA remains elevated at many postnatal ages, whereas mRNA encoding IGF-I is maintained only in older animals. Physiological concentrations of IGF-I cause two- to threefold increases in LVA current density in primary cultures of atrial myocytes, and this effect is blocked by an antisense oligonucleotide targeting the IGF-IR. Thus disruption of GH production in SpDwf animals alters expression of atrial LVA Ca2+ channel and IGF genes as well as postnatal regulation of LVA Ca2+ current density, most likely acting through compensatory mechanisms via the local IGF-IR.

Quantitative analysis of the expression and distribution of calcium channel alpha 1 subunit mRNA in the atria and ventricles of the rat heart.

Two distinct calcium currents are present in mammalian cardiac myocytes. Utilizing quantitative RT-PCR methods, we have analysed the expression patterns and abundance of four calcium channel alpha 1 subunit mRNAs in different regions of the rat heart and compared them to the known density of calcium currents recorded from rat atria. Our results show that Ca(V)1.2 is the most abundant of the four alpha 1 subunit transcripts in the rat heart. The Ca(V)1.2 message is more abundant in ventricle than in atria and does not vary in expression as a function of developmental age. Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNAs are 10-100 times less abundant than Ca(V)1.2. Interestingly, Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 are expressed in both atria and ventricle. The abundance of atrial Ca(V)3.1 mRNA does not change significantly during development and remains high in older animals. In contrast, levels of atrial Ca(V)3.2 mRNA are high in embryonic tissue and at 3- and 4-weeks postnatal but become undetectable at 5 weeks. Expression of atrial Ca(V)2.3 mRNA is highest at 4-weeks postnatal and then declines gradually. We have previously documented that the LVA calcium current density is highest within 4-5 weeks after birth and then declines gradually reaching less than 30% of its maximal value at 12-14 weeks. The complex relationship between atrial LVA current density and the abundance of Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNA suggests that their contribution to the cardiac LVA current may vary as a function of postnatal age.

Distribution and relative expression levels of calcium channel beta subunits within the chambers of the rat heart.

Calcium channel beta subunits expressed in rat atria and atrial myocytes are identified and their expression quantified and compared to beta subunit expression in the ventricle. mRNAs encoding all four know beta subunits are expressed in atrial myocytes including the following splice variants: beta1a, beta2b, beta2c, beta2e and beta4d. The specific beta2 splice variants expressed in the atria (beta2b, beta2c, beta2e) differ from those previously reported from rat ventricle. Beta2 isoform is the most abundant beta mRNA expressed in the heart and the amount of both beta2 subunit mRNA and beta2 subunit protein is significantly greater in the ventricles than in the atria. The expression of individual beta2 splice variants varies with age and within different chambers of the heart. The beta2b splice variant appears in both atria and ventricle in both young (4.5 week) and old (16 week) animals, the beta2c isoform is more highly expressed in young as compared to old animals and the beta2e splice variant is robustly expressed only in 4.5 week ventricle. Beta4 mRNA expression is higher in atrial tissue than in ventricles and its expression decreases in older animals. In contrast, the abundance of the beta3 mRNA does not significantly change as a function of postnatal age. Antisense oligonucleotides targeting sequence common to all the beta isoforms as well as that specific for beta2 significantly reduced HVA calcium current density in isolated atrial cells confirming that the beta2 subunits contribute to the regulation of HVA calcium current expression in the rat atria. The complexity of beta isoform expression within the heart may provide a mechanism for functional fine-tuning of the cardiac HVA current.

Calcium channel gamma6 subunits are unique modulators of low voltage-activated (Cav3.1) calcium current.

The calcium channel gamma (gamma) subunit family consists of eight members whose functions include modulation of high voltage-activated (HVA) calcium currents in skeletal muscle and neurons, and regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propanoic acid (AMPA) receptor targeting. Cardiac myocytes express at least three gamma subunits, gamma(4), gamma(6) and gamma(7), whose function(s) in the heart are unknown. Here we compare the effects of the previously uncharacterized gamma(6) subunit with that of gamma(4) and gamma(7) on a low voltage-activated calcium channel (Cav3.1) that is expressed in cardiac myocytes. Co-expression of both the long and short gamma(6) subunit isoforms, gamma(6L) and gamma(6S), with Cav3.1 in HEK-293 cells significantly decreases current density by 49% and 69%, respectively. Two other gamma subunits expressed in cardiac myocytes, gamma(4) and gamma(7), have no significant effect on Cav3.1 current. Neither gamma(6L), gamma(6S), gamma(4) nor gamma(7) significantly affect the voltage dependency of activation or inactivation or the kinetics of Cav3.1 current. Transient expression of gamma(6L) in an immortalized atrial cell line (HL-1) significantly reduces the endogenous low voltage-activated current in these cells by 63%. Green fluorescent protein tagged gamma(6L) is localized primarily in HEK-293 cell surface membranes where it is evenly distributed. Expression of gamma(6L) does not affect the level of Cav3.1 mRNA or the amount of total Cav3.1 protein in transfected HEK-293 cells. These results demonstrate that the gamma(6) subunit has a unique ability to inhibit Cav3.1 dependent calcium current that is not shared with the gamma(4) and gamma(7) isoforms and is thus a potential regulator of cardiac low voltage-activated calcium current.