T-type calcium channel blockade improves survival and cardiovascular function in thalassemic mice

Objectives: Iron‐overload cardiomyopathy is a major cause of morbidity and mortality in patients with thalassemia. However, the precise mechanisms of iron entry and sequestration in the heart are still unclear. Our previous study showed that Fe²⁺ uptake in thalassemic cardiomyocytes are mainly mediated by T‐type calcium channels (TTCC). Nevertheless, the role of TTCC as well as other transporters such as divalent metal transporter1 (DMT1) and L‐type calcium channels (LTCC) as possible portals for iron entry into the heart in in vivo thalassemic mice under an iron‐overload condition has not been investigated. Methods: An iron‐overload condition was induced in genetically altered β‐thalassemic mice and adult wild‐type mice by feeding them with an iron diet (0.2% ferrocene w/w) for 3 months. Then, blockers for LTCC (verapamil and nifedipine), TTCC (efonidipine), and DMT1 (ebselen) as well as iron chelator desferoxamine (DFO) were given for 1 month with continuous iron feeding. Results: Treatment with LTCC, TTCC, DMT1 blockers, and DFO reduced cardiac iron deposit, cardiac malondialdehyde (MDA), plasma non‐transferrin‐bound iron, and improved heart rate variability and left ventricular (LV) function in thalassemic mice with iron overload. Only TTCC and DMT1 blockers and DFO reduced liver iron accumulation, liver MDA, plasma MDA, and decreased mortality rate in iron‐overloaded thalassemic mice. Conclusions: DMT1, LTCC, and TTCC played important roles for iron entry in the thalassemic heart under an iron‐overloaded condition. Unlike LTCC blocker, TTCC blocker provided all benefits including attenuating iron deposit in both the heart and liver, reduced oxidative stress, and decreased mortality in iron‐overloaded mice.     

Calcium channels and iron uptake into the heart

Iron overload can lead to iron deposits in many tissues,particularly in the heart.It has also been shown to be associated with elevated oxidative stress in tissues.Elevated cardiac iron deposits can lead to iron overload cardiomyopathy,a condition which provokes mortality due to heart failure in iron-overloaded patients.Currently,the mechanism of iron uptake into cardiomyocytes is still not clearly understood.Growing evidence suggests L-type Ca2+channels(LTCCs)as a possible pathway for ferrous iron(Fe2+)uptake into cardiomyocytes under iron overload conditions.Nevertheless,controversy still exists since some findings on pharmacological interventions and those using different cell types do not support LTCC’s role as a portal for iron uptake in cardiac cells.Recently,T-type Ca2+channels (TTCC)have been shown to play an important role in the diseased heart.Although TTCC and iron uptake in cardiomyocytes has not been investigated greatly,a recent finding indicated that TTCC could be an important portal in thalassemic hearts.In this review,comprehensive findings collected from previous studies as well as a discussion of the controversy regarding iron uptake mechanisms into cardiomyocytes via calcium channels are presented with the hope that understanding the cellular iron uptake mechanism in cardiomyocytes will lead to improved treatment and prevention strategies,particularly in iron-overloaded patients.     

L型钙通道与心房重构

近年来的研究表明心房颤动引起 L 型钙通道变化,后者可能是引起以电重构、缝隙连接及收缩功能重构为主要形式的心房重构的中心环节。相应的药物干预可能是心房颤动治疗的新靶点。     

T-type calcium current in electrical activity of cardiomyocytes isolated from rabbit pulmonary vein

INTRODUCTION: Pulmonary veins (PVs) are known to initiate paroxysmal atrial fibrillation. T-type calcium current (I(Ca-T)) has a role in normal and abnormal automaticity of cardiomyocytes. The aim of this study was to evaluate whether I(Ca-T) contributes to PV electrical activity. METHODS AND RESULTS: By whole-cell clamp techniques in rabbit myocytes, I(Ca-T) was identified in 12 (39%) of 31 PV cardiomyocytes with pacemaker activity, 2 (9%) of 23 PV cardiomyocytes without pacemaker activity, and 2 (15%) of 13 atrial myocytes (P < 0.05). Maximum I(Ca-L) and I(Ca-T) densities from PV cardiomyocytes with pacemaker activity were 6.87 +/- 2.17 pA/pF and 1.38 +/- 0.69 pA/pF, respectively. Nickel (40 microM) decreased the spontaneous activity in 5 (36%) of 14 PV cardiomyocytes (3.1 +/- 0.6 Hz vs 2.2 +/- 0.5 Hz, P < 0.05), reduced the amplitudes of delayed after depolarization from 13 +/- 1 mV to 7 +/- 1 mV (n = 4, P < 0.05) and inhibited transient inward currents from 1.2 +/- 0.2 pA/pF to 0.7 +/- 0.1 pA/pF (n = 11, P < 0.01). CONCLUSIONS: We conclude that I(Ca-T) contributes to PV pacemaker activity and triggered activity, which are of functional importance in PV arrhythmogenesis.     

Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.Voltage-gated calcium channels (L-, N-, P/Q-, R-, and T-types) mediate calcium entry in many different cell types in response to membrane depolarization and action potentials. Calcium influx through these channels serves as an important second messenger of electrical signaling, initiating a variety of cellular events and physiological functions (1, 2). Among the family of voltage-gated calcium channels, T-type calcium channels (Cav3 family) have unique electrophysiological properties, because they display low voltage-activated calcium currents with rapid activation/inactivation kinetics. In neurons, small changes in the membrane potential near the resting potential can activate T-type channels, favoring further membrane depolarization and repetitive firing of action potentials (3–5). These unique gating properties of T-type channels make them important in many different processes, including neuronal spontaneous firing and pacemaker activities, rebound burst firing, sleep rhythms, sensory processing, and neuronal differentiation, as well as in pathological conditions, such as epilepsy and neuropathic pain (6).To properly assure this plurality of physiological functions, a tight control of T-type calcium channels is necessary. An important regulatory mechanism is phosphorylation, the fastest and most frequent posttranslational modification for a protein. Ion channels, especially voltage-gated channels, are critically regulated by phosphorylation. Voltage-dependent sodium and potassium channels have been shown to be the target of multiple phosphorylation events, regulating different channel functions and being involved in pathological states, like epilepsy (7–11). Also, several studies have shown the crucial role of the L-type/Cav1 phosphorylation in important physiological functions, like the fight or flight response (12–15).Regarding T-type channels, phosphorylation remains poorly understood. There are three Cav3 pore-forming proteins (Cav3.1, Cav3.2, and Cav3.3 subunits) all displaying typical properties of T-type channels when expressed in heterologous cell systems (3, 16). Among them, the Cav3.2 channel seems to be particularly sensitive to various types of regulation, including phosphorylation. To date, several serine/threonine kinases, like PKA, PKC, or CamKII, have been shown to regulate Cav3.2 activity (reviewed in refs. 17–20); however, in most cases, this regulation is tissue-dependent, and little is still known about its molecular basis. Cav3.2 channels bear more than 100 multiple intracellular serine and threonine residues that are predicted to be phosphorylated by common prediction algorithms, like NetPhos2.0 (21). However, which of these residues are actually phosphorylated and what functional impact this phosphorylation will have remain to be determined.In this study, we investigate the phosphorylation pattern of the Cav3.2 isoform of the T-type channels and its role in Cav3.2 channel properties. Using an MS approach, we have established the first, to our knowledge, phosphorylation map of the Cav3.2 channel in a mammalian brain and a human cell line. Then, by using alkaline phosphatase (AP) and dephosphomimetic mutants in patch-clamp experiments, we reveal the importance of phosphorylation in modulating Cav3.2 gating properties. We have also identified a phosphorylation hot spot situated in the loop connecting domains I and II of the channel that plays a crucial role in this regulation. Altogether, this study provides important insights regarding how phosphorylation regulates Cav3.2 channels.     

A marked increase of calcium uptake in the ATP-depleted red cells of patients with iron deficiency

Calcium (Ca) uptake was markedly increased in ATP-depleted red cells of patients with iron deficiency anemia (IDA) compared to ATP-depleted normal red cells. The extent of increased Ca uptake was related to the severity of iron deficiency as judged by decreased mean cell volume. Moreover, the increased Ca uptake returned to normal levels after oral iron supplementation therapy. The net calcium content of fresh red cells from iron-deficient individuals was the same as in red cells from normal subjects. Sodium influx and ferric ion uptake appeared to be virtually unaffected in the iron deficient red cells.     

High expression of N-type calcium channel indicates a favorable prognosis in gliomas

For the diagnosis and prognosis of glioma, the development of prognostic biomarkers is critical. The N-type calcium channel, whose predominant subunit is encoded by calcium voltage-gated channel subunit alpha1 B (CACNA1B), is mostly found in the nervous system and is closely associated with neurosensory functions. However, the link between the expression of CACNA1B and glioma remains unknown. We used ONCOMINE to explore the differences in CACNA1B expression among different cancers. We then conducted survival analysis and COX analysis using TCGA_LGG and TCGA_GBM datasets, which were divided into CACNA1B^high and CACNA1B^low based on the median. We examined the differences in other favorable prognostic markers or clinical characteristics between CACNA1B^high and CACNA1B^low using t tests. Differentially expressed genes were identified, and KEGG pathway enrichment was performed. We compared the expression of methyltransferases and analyzed the differentially methylated regions. Immunohistochemistry results were retrieved from the Human Protein Atlas database for validation purposes. CACNA1B was expressed at lower levels in gliomas, and, for the first time, we found that high expression of CACNA1B in gliomas predicts a good prognosis. Other favorable prognostic markers, such as isocitrate dehydrogenase mutation, 1p/19q codeletion, and O6-methylguanine-DNA methyltransferase promoter methylation, were increased in tandem with high expression of CACNA1B. Differentially expressed genes were enriched in multiple pathways related to cancer progression and aberrant epigenetic alterations were significantly associated with CACNA1B. High expression of N-type calcium channels indicates a favorable prognosis for gliomas. This study provides a better understanding of the link between gliomas and N-type calcium channels and may offer guidance for the future treatment of gliomas.     

溶血磷脂酸对胚胎干细胞分化心肌细胞L型钙通道电流的影响

目的诱导小鼠胚胎干(ES)细胞向心肌细胞分化并观察溶血磷脂酸(LPA)对分化心肌细胞L型钙通道电流(ICa-L)的影响。方法采用悬滴培养法诱导小鼠ES细胞向心肌细胞分化,逆转录-聚合酶链式反应及免疫荧光检测心肌细胞特异性标志物,全细胞膜片钳记录LPA0.1,1.0和10μmol/L对小鼠ES细胞分化心肌细胞ICa-L的影响。结果小鼠ES细胞成功向心肌细胞分化。0.1,1.0和10.0μmol/LLPA使分化心肌细胞ICa-L峰电流密度分别由用药前-6.8±0.7pA/pF增加到-8.9±1.2,-12.6±2.9和-16.6±3.5pA/pF(P<0.01或P<0.05)。结论LPA呈浓度依赖性促进小鼠ES细胞分化的心肌细胞ICa-L。     

Diabetes-induced changes in calcium homeostasis and the effects of calcium channel blockers in rat and mice nociceptive neurons

Aims/hypothesis: Distal neuropathy is the most common complication of diabetes mellitus, making it important to reveal the cellular mechanisms leading to its development, one of which might be the alteration in intracellular calcium homeostasis in primary and secondary nociceptive neurons. We aimed to investigate these possible changes. Methods: Control and streptozotocin-treated diabetic rats and mice were used. Changes in intracellular free calcium concentrations ([Ca²⁺]_i) were measured fluorometrically in primary nociceptive neurons from dorsal root ganglia and in secondary nociceptive neurons from substantia gelatinosa of spinal dorsal horn slices. Results: Measurements of [Ca²⁺]_i increases induced in dorsal root ganglion and dorsal horn neurons by membrane depolarization did not show any substantial difference in their peak amplitudes in control and diabetic animals. However, a definite prolongation of the decay phase of the transients was observed under diabetic conditions. Caffeine application to dorsal root ganglion and dorsal horn neurons induced a transient elevation of [Ca²⁺]_i which was less prominent in cells from diabetic animals. Short-term application of a calcium channel blocker nifedipine showed a substantial amplification of its action in diabetic neurons. However, chronic administration of nimodipine induced a clear increase in the peak values of transients in dorsal root ganglion neurons of diabetic animals compared with those of untreated animals. Conclusion/interpretation: The described changes of calcium signalling in nociceptive neurons could be the reason for the development of distal polyneuropathy and its symptoms in the early stages of diabetes mellitus. [Diabetologia (2001) 44: 1302–1309] Received: 28 December 2000 and in revised form: 25 May 2001     

Structure and regulation of L-type calcium channels a current assessment of the properties and roles of channel subunits

L-type Ca channels are complex heteromultimeric proteins that play important roles in the cardiovascular system. Recent studies have revealed new insights into how the pore-forming ₁ subunits interact with accessory subunits to produce functional Ca channels. The function of L-type Ca channels is often regulated by receptor-mediated signal transduction events that are thought to result in the phosphorylation of proteins that comprise the Ca channels. Although the molecular events underlying phosphorylation based regulation have been intensely investigated with the use of electrophysiological approaches, surprisingly few details are known about the biochemical events involved, and many questions remain unanswered. © 1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:265–273).     

Urine biochemical markers of early renal dysfunction are associated with iron overload in β‐thalassaemia

Summary Renal dysfunction in thalassemia patients can be attributed to chronic anemia, and iron overload as well as to desferioxamine (DFO) toxicity. We analyzed the urine of 91 well‐maintained homozygous β‐thalassemia patients, with no evidence of renal disease, for early evidence of kidney dysfunction by means of electrophoresis and quantitative biochemical tests. Measurement of liver magnetic resonance imaging (MRI) T2 values and serum ferritin concentration was used to estimate iron overload. In 55 of the 91 patients, urine analysis indicated signs of tubular dysfunction. The urine concentration of albumin and beta 2‐microglobulin, as well as the activity of n ‐acetyl‐beta‐d ‐glucosaminidase (NAG), correlated positively with serum ferritin concentration and liver iron deposition, as detected by MRI T2 values. This suggested that the cause of renal dysfunction in homozygous β‐thalassemia is iron overload. On the other hand, the same urine markers did not correlate with age, indicating that chronic anemia or desferrioxamine (DFO) treatment are not related to renal dysfunction in thalassemia.     

Effect of a calcium channel blocker and antispasmodic in diarrhoea-predominant irritable bowel syndrome

BACKGROUND: Irritable bowel syndrome (IBS) is a colonic function disorder. Both pinaverlum bromide (a selective calcium channel blocker) and mebeverine (an antispasmodic) are reported to be effective in the long-term (12-16 weeks) treatment of IBS patients. Their efficacy in the short-term treatment of IBS patients and colonic transit time is unclear. Furthermore, substance P and neuropeptide Y have either excitatory or inhibitory effects on colonic motility. Whether the efficacy of both drugs is mediated through these neuropeptides remains unknown. METHODS AND RESULTS: A clinical trial was conducted with 91 patients with diarrhoea-predominant IBS. After basal measurement of the total colonic transit time, IBS patients were randomized to receive either pinaverlum bromide (50 mg, t.i.d.) or mebeverine (100 mg, t.i.d.) for 2 weeks. The symptomatic scores regarding defaecation, total colonic transit time and serum levels of substance P and neuropeptide Y were measured before and after treatments. The daily defaecation frequency was markedly decreased after treatment (pinaverlum bromide, 2.9+/-1.2 vs 2.0+/-1.0, P< 0.05; mebeverine, 2.7+/-1.1 vs 2.1+/-1.0, P< 0.05). The stool consistency became well formed after both treatments (P< 0.05). Both drugs similarly improved the global well-being in these IBS patients (pinaverlum bromide vs mebeverine 73.4 vs 71.8%, P> 0.05). The total colonic transit time was significantly prolonged only after pinaverlum bromide treatment (21.4+/-15.5 vs 30.8+/-14.8 h, P< 0.01). Neither substance P nor neuropeptide Y serum level was significantly changed after either treatments. CONCLUSION: Pinaverlum bromide and mebeverine have similar therapeutic efficacies on diarrhoea-predominant IBS patients. Prolonged colonic transit time may be one of the factors responsible for the efficacy of pinaverlum bromide on the IBS patients. Substance P and neuropeptideY appear less important in the pathogenesis of diarrhoea-predominant IBS.     

The effect of an L/N-type calcium channel blocker on intradialytic blood pressure in intradialytic hypertensive patients

Background: Intradialytic hypertension (HTN), which is one of the poor prognostic markers in patients undergoing hemodialysis, may be associated with sympathetic overactivity. The L/N-type calcium channel blocker, cilnidipine, has been reported to suppress sympathetic nerves activity in vivo. Therefore, we hypothesized that cilnidipine could attenuate intradialytic systolic blood pressure (SBP) elevation. Methods: Fifty-one patients on chronic hemodialysis who had intradialytic-HTN (SBP elevation ≥10 mmHg during hemodialysis) and no fluid overload were prospectively randomized into two groups: control and cilnidipine groups. Cilnidipine group patients took cilnidipine (10 mg/day) for 12 weeks. The primary endpoint was the change in the intradialytic SBP elevation before and after the 12-week intervention. Results: Before the intervention, no differences were observed in age, sex or pre-dialytic SBP (148.5 ± 12.9 vs. 148.3 ± 19.3 mmHg) between the two groups. Intradialytic SBP elevation was unchanged in the control group. Cilnidipine significantly lowered the post-dialytic SBP with an attenuation of the intradialytic SBP elevation from 12.0 ± 15.4 mmHg to 4.8 ± 10.1 mmHg. However, the observed difference in the intradialytic SBP elevation by cilnidipine did not reach statistical significance (group×time interaction effect p = 0.25). Cathecolamine levels were unaffected by the intervention in both groups. Conclusion: Cilnidipine lowers both the pre- and post-dialytic SBP and might attenuate intradialytic SBP elevation. Therefore, cilnidipine may be effective in lowering SBP during HD in patients with intradialytic-HTN.