L-type Calcium Channel alpha1C Research Articles

Astragalus Granule Prevents Ca2+ Current Remodeling in Heart Failure by the Downregulation of CaMKII.

Background Astragalus was broadly used for treating heart failure (HF) and arrhythmias in East Asia for thousands of years. Astragalus granule (AG), extracted from Astragalus, shows beneficial effect on the treatment of HF in clinical research. We hypothesized that administration of AG prevents the remodeling of L-type Ca2+ current (ICa-L) in HF mice by the downregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Methods HF mice were induced by thoracic aortic constriction (TAC). After 4 weeks of AG treatment, cardiac function and QT interval were evaluated. Single cardiac ventricular myocyte was then isolated and whole-cell patch clamp was used to record action potential (AP) and ICa-L. The expressions of L-type calcium channel alpha 1C subunit (Cav1.2), CaMKII, and phosphorylated protein kinase A (p-PKA) were examined by western blot. Results The failing heart manifested distinct electrical remodeling including prolonged repolarization time and altered ICa-L kinetics. AG treatment attenuated this electrical remodeling, supported by AG-related shortened repolarization time, decreased peak ICa-L, accelerated ICa-L inactivation, and positive frequency-dependent ICa-L facilitation. In addition, AG treatment suppressed the overexpression of CaMKII, but not p-PKA, in the failing heart. Conclusion AG treatment protected the failing heart against electrical remodeling and ICa-L remodeling by downregulating CaMKII.

Upregulation of SERCA2a following short-term ACE inhibition (by enalaprilat) alters contractile performance and arrhythmogenicity of healthy myocardium in rat.

Chronic angiotensin-converting enzyme inhibitor (ACEIs) treatment can suppress arrhythmogenesis. To examine whether the effect is more immediate and independent of suppression of pathological remodelling, we tested the antiarrhythmic effect of short-term ACE inhibition in healthy normotensive rats. Wistar rats were administered with enalaprilat (ENA, i.p., 5 mg/kg every 12 h) or vehicle (CON) for 2 weeks. Intraarterial blood pressure in situ was measured in A. carotis. Cellular shortening was measured in isolated, electrically paced cardiomyocytes. Standard 12-lead electrocardiography was performed, and hearts of anaesthetized open-chest rats were subjected to 6-min ischemia followed by 10-min reperfusion to examine susceptibility to ventricular arrhythmias. Expressions of calcium-regulating proteins (SERCA2a, cardiac sarco/endoplasmic reticulum Ca(2+)-ATPase; CSQ, calsequestrin; TRD, triadin; PLB, phospholamban; Thr(17)-PLB-phosphorylated PLB at threonine-17, FKBP12.6, FK506-binding protein, Cav1.2-voltage-dependent L-type calcium channel alpha 1C subunit) were measured by Western blot; mRNA levels of L-type calcium channel (Cacna1c), ryanodine receptor (Ryr2) and potassium channels Kcnh2 and Kcnq1 were measured by qRT-PCR. ENA decreased intraarterial systolic as well as diastolic blood pressure (by 20%, and by 31%, respectively, for both P < 0.05) but enhanced shortening of cardiomyocytes at basal conditions (by 34%, P < 0.05) and under beta-adrenergic stimulation (by 73%, P < 0.05). Enalaprilat shortened QTc interval duration (CON 78 ± 1 ms vs. ENA 72 ± 2 ms; P < 0.05) and significantly decreased the total duration of ventricular fibrillations (VF) and the number of VF episodes (P < 0.05). Reduction in arrhythmogenesis was associated with a pronounced upregulation of SERCA2a (CON 100 ± 20 vs. ENA 304 ± 13; P < 0.05) and complete absence of basal Ca(2+)/calmodulin-dependent phosphorylation of PLB at Thr(17). Short-term ACEI treatment can provide protection against I/R injury-induced ventricular arrhythmias in healthy myocardium, and this effect is associated with increased SERCA2a expression.

Altered expression of L-type calcium channel alpha1C and alpha1D subunits in colon of rats with diarrhea-predominant irritable bowel syndrome

To investigate the molecular identities of L-type calcium channel alpha1C subunit (Cav1.2) and alpha1D subunit (Cav1.3) responsible for motor dysfunction of diarrhea-predominant irritable bowel syndrome (D-IBS). A total of 30 male Wistar rats were randomly divided into two groups. The traditional method for irritable bowel syndrome in a cold environment and intragastric administration of Folium Cassiae were combined to develop the D-IBS model. The fecal particles of rats and the water content in feces were measured. Then the expression of Cav1.2 and Cav1.3 mRNA was detected by reverse transcription polymerase chain reaction. The expressions of Cav1.2 and Cav1.3 were examined by immunohistochemistry in colonic tissues from D-IBS model rats and matched tissues. The fecal particles and the water content in feces of D-IBS model rats significantly increased as compared with the normal rats (6.8 +/- 1.4 vs 3.2 +/- 0.8, P = 0.032, 80% +/- 4% vs 47% +/- 5%, P = 0.018). Cav1.2 and Cav1.3 were positively expressed in colon of both model group and control group rats. The immunohistochemical scores of Cav1.2 and Cav1.3 expression increased in colon of D-IBS model rats as compared with those in normal control rats (3.43 +/- 0.92 vs 2.82 +/- 0.60, P = 0.034, 4.32 +/- 0.51 vs 3.75 +/- 1.05, P = 0.039). The immunohistochemical scores of Cav1.3 expression were significantly higher than Cav1.2 in colon of both two group rats (P = 0.003, 0.005). Similarly, the expression of Cav1.2 and Cav1.3 mRNA increased in colon of D-IBS model rats as compared with those in normal control rats (1.18 +/- 0.15 vs 1.06 +/- 0.12, P = 0.023, 1.32 +/- 0.13 vs 1.23 +/- 0.13, P = 0.033). The expression of Cav1.3 mRNA was significantly higher than Cav1.2 in colon of both two group rats (P = 0.038, 0.012). The traditional modeling of irritable bowel syndrome in rats alters the expression of Cav1.2 and Cav1.3. It may be directly related to the generation of enhanced colonic contraction in D-IBS. In addition, Cav1.3 may play a more important role than Cav1.2 in colonic contractility. It offers another potential therapeutic target in the treatment of irritable bowel syndrome.

Bepridil Reverses Atrial Electrical Remodeling and L‐Type Calcium Channel Downregulation in a Canine Model of Persistent Atrial Tachycardia

This study tested whether bepridil, a multichannel blocker, would reverse electrical remodeling induced by persistent atrial tachycardia. Fourteen dogs were subjected to rapid atrial pacing at 400 bpm for 6 weeks after atrioventricular block was created to control the ventricular rate. During the study period, seven dogs were given placebo for 6 weeks (Control group), and seven were given placebo for 3 weeks, followed by 3 weeks of bepridil (10 mg/kg/day, Bepridil group). The atrial effective refractory period (ERP) and the inducibility and duration of atrial fibrillation (AF) were determined on a weekly basis. After 6 weeks, expression of L-type calcium channel alpha1C messenger ribonucleic acid (mRNA) was quantified by real-time reverse transcription-polymerase chain reaction. In the Control group, ERP was shortened and the inducibility and duration of AF increased through the 6-week period. In the Bepridil group, the same changes occurred during the first 3 weeks, but were gradually reversed with bepridil. After 6 weeks, ERP was longer, AF inducibility was lower, and AF duration was shorter in Bepridil group than in the Control group. Expression of alpha1C mRNA was decreased by 64% in the Control group (P < 0.05 vs sham), but in the Bepridil group, it was not different compared with the sham dogs. As a whole group of dogs, ERP was positively correlated with alpha1C mRNA expression. Bepridil reverses the electrophysiological consequences of atrial remodeling to some extent and L-type calcium channel downregulation in a canine model of atrial tachycardia.

New insights into the function and regulation of vitamin D target proteins

Calbindin-D28k has been reported to be a facilitator of calcium diffusion and to protect against apoptotic cell death. Most recently, we found that the presence of calbindin-D28k results in reduced calcium influx through voltage-dependent L-type Ca2+ channels and enhanced sensitivity of the channels to calcium dependent inactivation. Co-immunoprecipitation and GST pull down assays indicate that calbindin-D28k interacts with the C-terminus of the L-type calcium channel alpha1c subunit (Cav1.2). This is the first report of the binding of calbindin to a calcium channel and provides new insight concerning mechanisms by which calbindin acts to modulate intracellular calcium. Besides calbindin, another major target of 1,25(OH)2D3 is 24(OH)ase, which is involved in the catabolism of 1,25(OH)2D3. We reported that C/EBPβ is a major transcriptional activator of 24(OH)ase that cooperates with CBP/p300 in regulating VDR mediated 24(OH)ase transcription. Recently, we found, in addition to p160 coactivators, that SWI/SNF complexes (that facilitate transcription by remodeling chromatin using the energy of ATP hydrolysis) are also involved in VDR mediated 24(OH)ase transcription and functionally cooperate with C/EBPβ in regulating 24(OH)ase. These findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)2D3 mediates its multiple biological effects.

Negative Transcriptional Regulation of Human Colonic Smooth Muscle Cav1.2 Channels by p50 and p65 Subunits of Nuclear Factor-κB

The expression of Cav1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappaB subunits mediates these effects. Primary cultures of human colonic circular smooth muscle cells and muscle strips were used. The messenger RNA and protein expression of the pore-forming alpha1C subunit of Cav1.2 channels decreased time dependently in response to tumor necrosis factor alpha. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha1C. Three putative kappaB binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha1C gene. Progressive 5' deletions of the promoter and point mutations of the kappaB binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha1C. Transient transfection of human colonic circular muscle strips with antisense oligonucleotides to p50 and p65 decreased expression of the 2 nuclear factor-kappaB units and reversed the suppression of alpha1C, as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha. The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha1C subunit of Cav1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappaB must bind concurrently to the two 5' kappaB motifs on the promoter of alpha1C to produce this effect.

The L-type calcium channel alpha 1C subunit gene undergoes extensive, uncoordinated alternative splicing

The alpha1C subunit is the pore-forming protein for the L-type calcium channel. Previous studies indicate that there is possible tissue-specific alternative splicing of this gene. In this study we cloned the entire open reading frame of the alpha1C subunit cDNA from adult rat cardiac myocytes in a single piece (6.64 kb). Using 75 positive clones that were identified by restriction enzyme mapping, we tested the alternative splicing patterns of the Ca(v) 1.2 gene that encodes the alpha1C subunit protein and focused on five loci: IS6, post-IS6, IIIS2, IVS3, and the c-terminus. The results indicate that: (1) alternative splicing occurs in most of the loci, giving rise to two or three different isoforms at those sites; (2) there is a predominant form for each splicing site, (3) there does not appear to be consistent coordination of splicing at multiple loci of this gene. Alternative splicing is not tissue-specific in most regions.

Characterization of auto-regulation of the human cardiac alpha1 subunit of the L-type calcium channel: importance of the C-terminus.

The carboxyl terminal of the L-type calcium channel alpha1C subunit comprises approximately one third of the primary structure of the alpha1 subunit (> 700 amino acids residues). This region is sensitive to limited posttranslational processing. In heart and brain the alpha1C subunits are found to be truncated but the C-terminal domain remains functionally present. Based on our previous data we hypothesized that the distal C-terminus (approximately residues 1650-1950) harbors an important, predominantly inhibitory domain. We generated C-terminal-truncated alpha1C mutants, and after expressing them in combination with a beta3 subunit in HEK-293 cells, electrophysiological experiments were carried out. In order to dissect the important inhibitory part of the C-terminus, trypsin was dialyzed into the cells. The data provide evidence that there are multiple residues within the inhibitory domain that are crucial to the inhibitory process as well as to the enhancement of expressed current by intracellular application of proteases. In addition, the expression of the chimeric mutant alpha(1C)delta1673-DRK1 demonstrated that the C-terminal is specific for the heart channel.

Developmental regulation of the L-type calcium channel alpha1C subunit expression in heart.

We used Northern analyses, RNase protection assays and immunoblot analyses to examine the relationship among developmental age of the heart, abundance of mRNA and L-type calcium channel alpha1C subunit protein, and to establish the size of the native protein in heart. Northern analysis, RNase protection assays, and immunoblots were used to study RNA and protein from rat heart of various ages. In fetal and adult ventricles there was a predominant 8.3-kb transcript for the alpha1C subunit with no change in transcript size during development. RNase protection assays demonstrated a 2-fold increase in abundance of the DHP receptor message during postnatal development. Immunoblots identified a 240 kD protein, corresponding to the predicted molecular mass of the full length alpha1C subunit. No change in size of protein for the alpha1C subunit was observed at any developmental stage and there was no evidence for a truncated isoform. There was an approximate 2-fold increase in alpha1C subunit protein in ventricular homogenates during postnatal development. Thus, in the developing rat heart, alterations in calcium channel properties during development appear to result neither from alternative splicing that produces a smaller transcript for the alpha1C subunit nor from expression of a truncated protein, but at least in part from transcriptionally-regulated expression of the 240 kDa polypepde.