Voltage-gated calcium channels have been introduced for over half century. Growing evidences show that voltage-gated calcium channels play roles in calcium influx involve in cellular processes including membrane depolarization and regulate excitability, contraction, hormonal secretion and possibly gene transcription (cardiac, Ono). Voltage-gated calcium channels can be classified by pharmacological; L (long lasting), T (transient), N (neuronal), P/Q (Purkinje), and R (residual-drug resistant) and electrophysiological; high-voltage (L-, N-, P/Q-, and R-type) and low-voltage (T-type). Nevertheless, only L- and T-type calcium channels can be found in cardiac myocytes.
Figure 1 Voltage-gated Calcium Channel
L-type calcium channels, long-lasting high-voltage activated channels, are ubiquitous in mature cardiac myocytes and play crucial role in excitation-contraction coupling. L-type calcium channels consist of 4 subunits; α1, γ, β and α2δ. The α1 subunit, however, is the most important because of their pore forming property which calcium can pass through. There are two isoforms that α1 encoded in L-type calcium channels; CaV1.2 (α1C) and CaV1.3 (α1D)
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T-type calcium channels are characterized by a transient low-voltage activated channels. Unlike L-type calcium channels, functional roles of T-type calcium channels depend on age and diseases. Under physiological condition, T-type calcium channels are teeming finding in embryonic myocytes. However, they are markedly downregulate in postnatal ventricle. In adult heart, T-type calcium channels are undetectable in ventricular myocytes and restricted to pacemaker cell in conducting system. Once pathological condition occur such as cardiac hypertrophy, myocardial infarction, and heart failure, T-type calcium channels have been found to re-express in ventricular myocytes, leading to altered cardiac functions and arrhythmogenicity. There is only α1 subunit that encodes 3 isoforms in T-type calcium channels; CaV3.1 (α1G), CaV3.2 (α1H), and CaV3.3 (α1I). In the heart, only CaV3.1 and CaV3.2 are the major subunits for T-type calcium channels. CaV3.3, however, was reported to be expressed in brain and Purkinje fiber. The CaV3.1 and CaV3.2 can be distinguished by Nickel (Ni2+) sensitivity property. CaV3.2 current can be blocked with lower level of Ni2+ than CaV3.1 current by the half maximal inhibitory concentration (IC50) 13 µM and 216 µM, respectively.
Beside the genetic difference, T-type calcium channels can be differentiated from L-type calcium channels by kinetics and conductance properties. T-type calcium channels open at the more negative membrane potential. The threshold for activation of L-type calcium current (ICaL) is positive to -40 mV and fully activates at 0 to + 10 mV, and tends to reverse at +60 to +70 mV at physiological calcium concentration. On the other hand, the T-type calcium current (ICaT) activates at potentials of -70 to -60 mV, peaks at -30 to -10 mV. ICaT is completely inactivated at membrane depolarization more positive than -40 mV. Furthermore, Inactivation time of ICaT is more rapidly than ICaL.
T-type calcium channels during development
The alteration of subtype expression and their currents during developmental stage has been investigated in different species. In the embryonic stage, ventricular myocytes are capable of automatic activity, in contrast to physiological adult myocardium. T-type calcium channels are proposed to underlying this automatic activity. Cribbs et al. have studied on mouse ventricular myocytes at midgestational stage. They identified the CaV3.1d mRNA expression, which is a splice variant of the α1G T-type calcium channels, underlying the functional T-type calcium in embryonic day 14 (E14). Moreover, dihydropyridine-resistant inward Ba2+ or Ca2+ current was presented in these cells. Nuss and Marban have shown the exhibition of T-type calcium current on primary culture of the late fetal and neonatal (1-to 3-day old) mouse cardiac myocytes. Leuranguer et al. have suggested the alteration of voltage-gated calcium channels current by T-type calcium current density decreased during development, while L-type calcium current density still constant in both rat atrial and ventricular myocytes. Although ICaT in ventricular myocytes was undetectable at 3 weeks after birth, genes encoded T-type calcium channels were express in the same manner. Interestingly, ICaT reported in this study is closer to the α1G by the Ni2+ sensitivity test. Suggesting that α1G expresses in neonatal stage. In addition, they suggested that dihydropyridine-resistant current, which indicate L-type calcium channels, was gradually increase from postnatal through adulthood. In a molecular biological study, Huang et al. also suggested that α1G mRNA diminishes in normal adult rats approximately 80% from perinatal stage. Ferron et al. have studied on the expression of T-type gene underlies ICaT at middle embryonic stage (fetuses at 16-, 18-day of gestation), perinatal stage (1-, 5-, 21- day old) and adult rat. With development, not only the current density of T-type calcium channels decreased, but also T-type calcium genes. ICaT was no longer detected after 21-day of age. Both α1G and α1H participating in the functional T-type Ca2+ channels express in difference proportion. The expression of α1G mRNA was higher than α1H during the fetal stage to neonate. In adult, however, the proportion significantly changed to α1H over α1G. In contrast to the study of Niwa et al in mice, the results were opposite. They showed that CaV3.2 mRNA, which is an α1H, is prominent subtype during mid-embryonic state (embryonic day 9.5). During fetal development, CaV3.2 mRNA was downregulate, while CaV3.1 mRNA, which is an α1G, was upregulate. CaV3.2 mRNA, however, is still dominant. In the adulthood, in conversely, CaV3.1 mRNA expression was higher than CaV3.2. Theses suggest that CaV3.2 is the main subtype of functional T-type calcium current under in embryonic heart. According to the study of Mizuta et al. on the mouse embryonic stem cell, the CaV3.2 underlies the functional T-type calcium channels during embryonic stage and switch to CaV3.1 after birth.
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The growing evidences have been obviously shown that ICaT is predominant in the embryonic state. Afterward, ICaT gradually decreases with development and is almost undetectable in adulthood. In conversely, the contribution of genes encoded T-type calcium channels is still controversial. This controversy might due to species difference and specific gene primers. In some studies, the expression of T-type calcium channels genes without ICaT. This may suggest that there is threshold level of expression of T-type calcium channels gene which necessary for the ability to detect ICa-T in isolated myocytes. However, these observations need to be investigated.
Table 1 T-Type Calcium Channels during development
Mouse ventricular myocytes
CaV3.1d (α1G) expressed in embryonic day 14
Cribbs et al
Mouse primary culture (1-3 days old)
Nuss and Marban
Rat atrial and ventricular myocytes (8 day, 3 week, adult)
ICaTdecreases with development, while ICaL iof both atrial and ventricular myocytes.
ICaTis found in adult atium, whereas ventricle doesn't.
CaV3.1 (α1G) may underlies the ICaTat neonatal stage
Expression of α1G mRNA and α1HmRNA didn't change before and after birth as well as α1C
Leuranguer et al.
Rat ventricular myocytes
(E16, E18, D1, D5, D21 and adult)
ICaTdecreases with development, no longer detected in 21-day old
Fetal to neonatal α1G >α1H
Adult α1H >α1G
Ferron et al.
Rat ventricular myocytes
(D3, D7, W12)
α1G mRNA diminishes in normal adult rats approximately 80% from perinatal stage
ICaT is not found in adult rats.
Huang et al.
CaV3.2 (α1H) mRNA is predominant in early stage
CaV3.1 (α1G) mRNA and CaV1.2 (α1C) mRNA were upregulated in the late stage
Mizuta et al.
Mouse ventricular myocytes (E9.5, E18 and 10-week old)
ICaTwas observed during E9.5 and E18, but was undetectable in adult
CaV3.2 (α1H) mRNA is predominant subtype at both E9.5 and E18, but CaV3.1 (α1G) mRNA is predominant in adult
Niwa et al.
E = Early embryonic day
W = Week of age
ICaT = T-type Calcium Current
ICaL = L-type Calcium Current
T-Type Calcium channels and pathologic conditions
Normally, T-type calcium channels are down-regulated in adult ventricular myocytes. However, cardiac T-type calcium channels have been reported to reappear under pathological conditions for instance cardiac hypertrophy and heart failure as well as chronic exposure to growth promoting substances such as endothelin, angiotesin II, aldosterone and growth hormone-secreting tumors.
T-type calcium current has been reported under the ongoing of pathological condition. In 1990, Xu and Best have studied in volume overload and cardiomegaly rat induced by subcutaneous implantation of growth hormone secreting tumors (GH3). They found that after 8 weeks after implantation, tumor-bearing rats re-entered active growth phase which increasing not only in heart weight, body weight, and size of myocytes, but also atrial ICaT. Furthermore, the voltage dependency of activation and inactivation of T-type current was not change. This may suggest that ICaT may involve in growth process. Three years later, Nuss and Houser studied in long-standing pressure overload-induced cardiac hypertrophied feline using aortic banding. The results showed ICaT modestly soared, while ICaL fell down to 77% of normal cat. A year later, 200- to 300-day old cardiomyopathic Syrian hamsters, which develop progressive and ultimately fatal congestive heart failure, have been shown both ICaT and ICaL in their ventricular myocytes. As current density of T-type calcium markedly escalated, L-type calcium density is comparable to normal. In addition, Sen and Smith reported that the voltage dependencies of inactivation and activation were more negative. Martinez et al. have studied in aortic banding induced cardiac hypertrophy rats. After 11 weeks of this procedure, hypertrophied rats have shown both ICaT and ICaL, whereas sham operated rats showed only ICaL. The ICaL level was no difference in both groups. The occurrence of this ICaT was highly Ni2+ sensitive, suggesting that the CaV3.2 or α1H underlies the expression of T-type calcium channels. Nonetheless, the study of Yasui et al. have found that CaV3.2 mRNA was comparable to control but CaV3.1 mRNA significantly decreased in aortic banding mouse ventricular septum. They also showed that CaV3.1 significantly decreased, but CaV3.2 significant increase in ventricular septum of post-myocardial heart. Moreover, they found negative correlation between neuron-restrictive silencer factor (NRSF) and CaV3.2, which suggests that NRSF might regulate CaV3.2 expression. Huang et al., however, have shown that CaV3.1 is increased approximately 158% in rat with myocardial infarction in same way of ICaT. On the other hand, expression of both CaV3.1 mRNA and CaV3.2 mRNA increased in rats with monocrotaline-induced right ventricular hypertrophy by 264% and 191%, respectively. Whereas, CaV1.2 (α1C) and neuronal CaV3.3 (α1I) was not changed.
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Many mechanisms underlying the expression of T-type calcium channels have been proposed. Firstly, Calcineurin-NFAT singnaling cascade has been described that prolong excessive intracellular calcium triggers for cardiac hypertrophy via calcineurin, a calcium-activated phosphatase. Increasing calcineurin activity will translocate cytoplasmic phosphorylated-NFAT into nucleus by dephosphorylation. Several studies showed that TRPC1, TRPC3, and TRPC6 are involved in calcineurin/NFAT activation and cardiac hypertrophy in rodent models. Furthermore, the promoter region of TRPC6 contains 2 NFAT binding sites, which suggests that positive feedback is involved in the regulation of the Calcineurin-NFAT-TRPC6 signaling pathways. Chiang et al.'s study has supported this idea. They found that pressure overload-induced hypertrophy of CaV3.2 gene deleted transgenic mice (CaV3.2-/-) were suppressed, while CaV3.1 gene deleted transgenic mice (CaV3.1-/-) weren't. In addition, cultured neonatal myocytes isolated from CaV3.2-/- mice fail to respond hypertrophic stimulation by exposure with angiotensin II. More importantly, transgenic mice CaV3.2-/-/NFAT-Luciferase reporter mice failed to increase NFAT-luciferase reporter activity after pressure overload. Together, these results demonstrate the activation of Cav3.2 channels is essential to the development of cardiac hypertrophy via Calcineurin-NFAT pathway both in vivo and in vitro study. Horiba et al. have emphasized the substantial role of calcium overload through T-type calcium channels induced cardiac hypertrophy via calcineurin-mediated NFAT activation by pharmacological intervention using kurtoxin, efonidine, and nisodipine, which are specific T-type calcium channels blocker, both T- and L-type calcium channels blocker, and selective L-type calcium channels blocker, respectively. They suggested that although blockade of calcium via T-type calcium channels may block pathophysiological signaling pathways leading to hypertrophy as well as via L-type calcium channel, damage of cardiomyocytes by L-type calcium channels blocker was more severe than selective blocking of T-type calcium channels.
The other mechanism is neuron-restrictive silencer factor-neuron-restrictive silencer element (NRSF-NRSE) system. NRSF-NRSE plays role in repression of fetal gene program transcription, for example, CaV3.2 (α1H), atrial natriuretic hormone (ANP), B-type natriuretic hormone (BNP). Transgenic mice without NRSF or dominant-negative of NRSF (dnNRSF) in their hearts showed progressive cardiomyopathy and sudden death at 8 weeks old. The hearts from dnRSF-Tg mice showed increased expression of CaV3.2 mRNA and their current.
As mention above, T-Type calcium current obviously has been played role in the active growth process. Expression of T-type calcium channels may cause calcium overload. Although, amount of calcium influx through T-type calcium channels is small when compared with L-type calcium channels, this increasing may prone to spontaneous action potential leading to arrhythmias. Many mechanisms have been postulated that reappearance of T-type calcium channels genes and their currents are underlying pathogenesis of cardiac diseases. Understanding these mechanisms will provide a worthwhile strategy for drug development.
Rat (Atrial and ventricular free wall)
3 folds increased ICaTin tumor-bearing atrial myocytes compared with normal rat
Dependancy of activation and inactivation of both ICaTand ICaLcalcium current were not change
Xu and Best
Feline (Left ventricular myocytes)
ICaLdecreased 23% compared with normal
ICaTincreased in hypertrophied heart
Nuss and Houser
(Left ventricular myocytes)
200 - 300 days old
ICaTand ICaLin both cardiomyopathy and normal
ICaT significantly increased in cardiomyopathy, however, voltage dependencies of inactivation and activation was more negative
ICaLwas the same in both cardiomyopathy and normal
Sen and Smith
Rat (Left ventricular myocytes and septum)
CaV3.2 underlies the ICaT after hypertrophy
Martinez et al.
(AOB and MI)
Mouse (Ventricular septum)
In AOB; CaV3.1 decrease, CaV3.2 comparable to control
In MI; CaV3.1 decrease, CaV3.2 increase
Yasui et al.
Rat (Left ventricular myocytes)
α1G mRNA was re-expressed in MI by 158% compared to adult sham operated rat
ICaTwas undetectable in adult, but was recorded after MI
Negative correlation between NRSF and CaV3.2 in MI rat
Huang et al.
Rat (Right ventricular myocytes)
α1G and α1H increased, but α1C and α1I was not changed
Mibefradil, T-type calcium channels blocker, strongly inhibited twitch tension of right ventricular muscle
Takebayashi et al.
Mice and cell culture (left ventricular myocytes)
Pressure overload-induced hypertrophy was suppressed in CaV3.2-/-, but not in CaV3.1-/-
Angiotensin II-induced hypertrophy was suppressed in CaV3.2-/-and cultured neonatal myocytes as well
Transgenic mice CaV3.2-/-/NFAT-Luciferase reporter mice failed to increase NFAT-luciferase reporter activity after pressure overload
Chiang et al.
(Fetal bovine serum)
Mouse primary cell culture (Ventricle)
Cell surface area was reduced in KT, abolished in ED and ND as well as BNP mRNA expression
Translocation of NFAT into nucleus and calcineurin phophatase activity were inhibited by KT, ED and ND
Horiba et al.
Table 2 T-type calcium channels under pathological conditions
AOB = Aortic banding KT = Kurtoxin
MI = Myocardial infarction ED = Efonidipine
ICaT = T-type calcium current ND = Nisoldipine
ICaL = L-type calcium current BND = B-type Natriuretic Peptide
NRSF = Neuron-Restrictive Silencer Factor dnNRSF = dominant-negative form of neuron-restrictive silencer factor
NRSE = Neuron-Restrictive Silencer Element
NFAT = Nuclear Factor of Activated T-cells
T-type calcium channels and pharmacological intervention and clinical aspect
Under pathological conditions such as cardiac hypertrophy, heart failure, and myocardial infarction, T-type calcium channels and their ICaT have been recorded. Therefore, development of T-type calcium channels should be an effective treatment of cardiac diseases.
Mibefradil is a selective T-type calcium channels. In post-myocardial infarction induced heart failure, mibefradil improved the survival rate to the same extent as cilazapril (ACE ihibitor) without impairing left ventricular function and also reduced left ventricular weight and fibrosis. Furthermore, mibefradil showed more effective than L-type calcium channels blocker, verapamil and amlodipine, in the aspect of inotropic effect and intracellular calcium modulating effect. Beneficial effect of mibefradil not only found in heart failure from chronic myocardial infarction but also in dilated cardiomyopathy. (Kinoshita) In the contrary, when used mibefradil as adjunct therapy, it did not show different outcome of usual congestive heart failure compared with placebo group. Moreover, drug interaction with amiodarone, antiarrhythmic drugs, may cause worsen outcome within first 3 months. Mibefradil had been used as drug for treatment of hypertension, angina pectoris, and also congestive heart failure. In 1997, however, mibefradil had to withdraw from that market because of it inhibited cytochrome p450 which led to drugs interaction.
Efonidipine hydrochloride is a dihydrpyridine with dual blocking T- and L-type calcium channels. Blocking activity on T- and L-type calcium channels are about 40% and 50%, respectively. Efonidipine has been showed the excellent in treatment in both cardiac hypertrophy and myocardial infarction. Efonidipine improved hemodynamic function and decreased T-type calcium function in a dose-dependent manner in UM-X7.1 cardiomyopathic hamster. Moreover, mibefradil improved cardiac autonomic nervous system and reduced arrhytmogenicity which led to increase survival rate of cardiac hypertrophied mice, R(-)-efonidipine as well. In addition, efonidipine had been reported that prevent sudden death in mice with acute myocardial infarction.
Accumulation studies have been proved that T-type calcium channels blockers have prior benefits than L-type calcium channels blockers in both cardiac hypertrophy and myocardial infarction by increase survival rate, reduce arrhythmogenicity, improve cardiac autonomic nervous system and hemodynamic parameter as well as prevent sudden death. The development of highly specific T-type calcium channels blocker will provide more powerful and safer therapeutic drugs for treatment cardiac diseases.
Duration of Drug administration