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(Circulation Research. 2001;88:403.)
© 2001 American Heart Association, Inc.

Cellular Biology

Identification of the T-Type Calcium Channel (Ca_V3.1d) in Developing Mouse Heart

Leanne L. Cribbs, Beverly L. Martin, Elizabeth A. Schroder, Bradley B. Keller, Brian P. Delisle, Jonathan Satin

From the Cardiovascular Institute (L.L.C., B.L.M.), Loyola University Medical Center, Maywood, Ill, and the Departments of Pediatrics (E.A.S., B.B.K.) and Physiology (B.P.D., J.S.), University of Kentucky College of Medicine, Lexington, Ky.

Correspondence to Jonathan Satin, PhD, Department of Physiology, MS-508, University of Kentucky College of Medicine, Lexington, KY 40536-0298. E-mail jsatin1{at}pop.uky.edu

	Abstract

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Abstract—During cardiac development, there is a reciprocal relationship between cardiac morphogenesis and force production (contractility). In the early embryonic myocardium, the sarcoplasmic reticulum is poorly developed, and plasma membrane calcium (Ca²⁺) channels are critical for maintaining both contractility and excitability. In the present study, we identified the Ca_V3.1d mRNA expressed in embryonic day 14 (E14) mouse heart. Ca_V3.1d is a splice variant of the 1G, T-type Ca²⁺ channel. Immunohistochemical localization showed expression of 1G Ca²⁺ channels in E14 myocardium, and staining of isolated ventricular myocytes revealed membrane localization of the 1G channels. Dihydropyridine-resistant inward Ba²⁺ or Ca²⁺ currents were present in all fetal ventricular myocytes tested. Regardless of charge carrier, inward current inactivated with sustained depolarization and mirrored steady-state inactivation voltage dependence of the 1G channel expressed in human embryonic kidney-293 cells. Ni²⁺ blockade discriminates among T-type Ca²⁺ channel isoforms and is a relatively selective blocker of T-type channels over other cardiac plasma membrane Ca²⁺ handling proteins. We demonstrate that 100 µmol/L Ni²⁺ partially blocked 1G currents under physiological external Ca²⁺. We conclude that 1G T-type Ca²⁺ channels are functional in midgestational fetal myocardium.

Key Words: calcium channel • cardiac development • low-voltage-activated Ca²⁺ channel

	Introduction

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L-type and T-type calcium (Ca²⁺) channels comprise two broadly classified families of voltage-gated Ca²⁺ channels. L-type Ca²⁺ channel expression and function are well characterized in cardiac myocytes, where L-type channels mediate plasma membrane influx of extracellular Ca²⁺. Plasma membrane influx of Ca²⁺ leads to Ca²⁺-induced Ca²⁺ release, which in turn regulates cardiac contractility. The L-type Ca²⁺ channels also are an important therapeutic target for management of a variety of cardiovascular disorders. In contrast to L-type channels, T-type Ca²⁺ channel expression and function are poorly understood, and the therapeutic potential of T-type Ca²⁺ channels in mature myocardium is unknown. Nonetheless, earlier studies showed that de novo expression of T-type Ca²⁺ channels may contribute to the cardiac hypertrophic phenotype.¹ More recently, Nattel and colleagues^{2 3} suggested that T-type Ca²⁺ current might contribute to arrhythmogenesis in patients exhibiting atrial fibrillation.

L-type and T-type Ca²⁺ channels are perhaps most clearly distinguished by their respective voltage range of activation. L-type Ca²⁺ channels activate only with strong depolarizations (>-50 mV), corresponding to the plateau of the action potential. In contrast, T-type Ca²⁺ channels activate with weak depolarizations (>-80 mV). In fact, we recently demonstrated that T-type Ca²⁺ channels activate at slightly more negative potentials than even cardiac Na⁺ channel currents.⁴ This low-voltage-activation range of T-type Ca²⁺ channels allows them to provide a substantial inward, depolarizing current in the late phase of diastole. Thus it is likely that T-type Ca²⁺ current contributes to the initiation of the action potential upstroke in pacemaker cells.^{5 6 7}

Embryonic ventricular myocytes are capable of automatic activity, in contrast to normal adult myocardium. Studies on embryonic chick ventricular myocytes clearly show the expression of T-type Ca²⁺ current^{8 9} ; however, the literature from embryonic mouse heart is controversial. Nuss and Marbán¹⁰ showed substantial T-type Ca²⁺ channel expression, but Davies et al¹¹ were unable to detect T-type current. The recent molecular identification of T-type Ca²⁺ channels^{12 13} facilitates unequivocal detection of channel gene expression. The 1G channel is encoded by the Ca_V3.1 gene,¹² and there are 4 distinct splice variants in the domain III-IV connecting loop in rodents and humans.^{14 15 16} Our recent studies showed that mouse atrial tumor (AT-1) cells functionally express 1G-d (Ca_V3.1d).¹⁷ AT-1 cells have an excitability pattern that parallels embryonic ventricular myocytes, suggesting that developing myocardium may also express 1G channels.

We undertook the present study to evaluate the expression of T-type Ca²⁺ channels in embryonic myocytes. We show that the T-type Ca²⁺ channel splice variant 1G-d is expressed in embryonic day 13 (E13) to E14.5 mouse ventricle, and we present data consistent with the hypothesis that Ca²⁺ entry via the T-type channel may contribute to excitability in the developing myocardium.

	Materials and Methods

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Tissue Harvest
E12.5 to E13 mouse hearts were dissected free of connective tissues, and ventricles were separated from atria. For isolation of total RNA, whole ventricles were immersed in Trizol reagent (GIBCO-BRL) and homogenized. For immunohistochemistry, E13 hearts were removed, embedded in OCT compound, and rapidly frozen in a dry-ice bath. For cell isolation for patch-clamp recordings and immunocytochemistry, ventricles from 3 to 8 embryos were minced and quickly placed into nominally Ca²⁺-free digestion buffer containing 0.5 mg/mL collagenase (type II, Worthington) and 1 mg/mL pancreatin (GIBCO-BRL) for two 15-minute cycles.

Molecular Characterization
Total E13 ventricular RNA was characterized by reverse transcriptase–polymerase chain reaction (RT-PCR), as described elsewhere.¹⁷

Immunostaining
Type-specific antibodies 2xG1 (anti-1G) and 2xH1 (anti-1H), prepared against peptides derived from the 1G and 1H T-type channel sequences^{12 13} (for 2xG1: FVCQGEDTRNITNKSDCAEAS and 2xH1: YYCEGPDTRNISTKAQCRAAH), were immunoaffinity-purified commercially (Bethyl Laboratories). Monoclonal antibody MF 20 (Developmental Studies Hybridoma Bank, University of Iowa) was used to stain myofibrils using a rhodamine-conjugated secondary antibody (Molecular Probes). For additional details, see the online data supplement available at http://www.circresaha.org.

Electrophysiology
Digested tissue yielded a large fraction of single, spontaneously beating cells in culture medium (DMEM with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin). Cells were used within 48 hours of isolation. Cells in culture medium were rinsed with recording buffer for T-type Ca²⁺ current (in mmol/L: CsCl 140, CaCl₂ 2.5, HEPES 10, tetrodotoxin [TTX] 0.036, nifedipine 0.001, and E-4031 0.005; pH 7.4). The pipette contained 140 mmol/L CsCl, 5 mmol/L EGTA, and 5 mg ATP; pH 7.4). All recordings were at room temperature (20°C to 22°C). Mean cell capacitance was 9.1±1.6 pF, n=12. Additional details of recording and data analysis can be found elsewhere.^{17 18}

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Assay of total E12 to E14 ventricular myocyte RNA by RT-PCR and DNA sequencing allows an unequivocal assessment of T-type Ca²⁺ channel mRNA expression. We used a strategy identical to that used by Satin and Cribbs¹⁷ to identify T-type Ca²⁺ channels in fetal mouse ventricle. Mouse E12.5 to E14 ventricular myocytes express the 1G-d splice variant, as shown previously for AT-1 cells (Figure 1). Even though the PCR primers used can amplify all 3 known T-type channels,¹⁹ no other low-voltage-activated channels or splice variants were discovered.

View larger version (47K): [in this window] [in a new window]   Figure 1. RT-PCR of E14 mouse heart RNA. Total RNA (1 µg) was converted to cDNA using Moloney murine leukemia virus–reverse transcriptase, then PCR was done using primers that amplify the III-IV interdomain loop of 1G, 1H, and 1I. The PCR product was run on a 0.8% agarose/TBE gel (lane 2) along with 100-bp ladder (lane M) and negative control reaction, in which reverse transcriptase was omitted (lane 1). DNA sequencing of the PCR product identified it as 1G-d, 1 of 4 known splice variants of this region, shown below the gel.

The expression of mRNA does not always correlate with protein expression. To confirm protein expression, we used antibodies (2xG1 and 2xH1) to evaluate expression and localization of T-type channels in embryonic heart. Figure 2D shows a sagittal section of ventricle with vigorous staining for 1G. We also noted staining of vasculature and atrial walls (Figure 2B). Preimmune controls (Figures 2A and 2C) and anti-1H antibodies did not stain (not shown). The staining of 1G was blocked completely by 2xG1 peptide–absorbed antibody (Figure 2E), whereas 2xH1 peptide had no effect on 2xG1 staining (Figure 2F). To additionally control for antibody specificity, 2xG1 clearly stained human embryonic kidney (HEK) cells stable-transfected with 1G; in contrast, HEK cells expressing 1H or untransfected HEK cells did not show 2xG1 staining (see the online data supplement). We also stained isolated ventricular myocytes using the 2xG1 antibody (Figure 3). Cells were costained with MF 20, a monoclonal antibody to myosin heavy chain, to confirm their identity as myocytes (Figure 3A). Figure 3B shows the expected plasma membrane localization of 1G, a staining pattern distinct from the myofibrillar staining with MF 20. Taken together, the RT-PCR and immunostaining results show that embryonic ventricular myocardium expresses the 1G-d splice variant.

View larger version (168K): [in this window] [in a new window]   Figure 2. Immunostaining of E14 mouse heart. Cryosections (14 µm) of whole heart were stained with either preimmune serum (A and C) or 2xG1 polyclonal antiserum (1:1000, B and D). The VIP substrate produces a positive violet color. Shown are sagittal views of atrial region (x112, A and B) or ventricle (x280, C and D). Absorption of anti-1G antiserum with 50 µmol/L 2xG1 peptide completely blocked staining (E); in contrast, absorption with 50 µmol/L 2xH1 peptide had no effect on 2xG1 staining (F).

View larger version (18K): [in this window] [in a new window]   Figure 3. Immunostain of cultured E13 mouse ventricular myocyte. Isolated cells were stained with either MF 20 (myosin heavy chain monoclonal antibody, rhodamine-conjugated secondary antibody) (A) or 2xG1 (FITC-conjugated secondary antibody) (B). Magnification is x1120.

We performed whole-cell mode patch-clamp recordings of embryonic ventricular myocytes to evaluate 1G-d channel function. To isolate T-type channel current from voltage-gated Na⁺, L-type Ca²⁺, and K⁺ currents, we bathed cells in Na⁺-free bath containing 2.5 mmol/L Ba²⁺, 30 µmol/L TTX, and 100 µmol/L nifedipine. Figure 4 shows representative recordings of T-type Ca²⁺ current. Channel kinetics (Figure 4A) and the steady-state inactivation voltage dependence are consistent with scoring this current as a T-type Ca²⁺ channel current. The voltage dependence of activation is depolarized-shifted (midpoint=-22 mV; Figure 4B), but this is a consistent trend observed for voltage-gated Na⁺ and Ca²⁺ channels in developing myocardium.^{8 20} The presence of inactivating Ba²⁺ current in 100 µmol/L nifedipine (Figures 4A and 4C) is unequivocal evidence that the current is not through L-type Ca²⁺ channels.

View larger version (25K): [in this window] [in a new window]   Figure 4. Inward Ba2+ current is 100 µmol/L DHP–resistant and inactivating in fetal ventricular myocytes. A, Top left, family of currents elicited from Vhold -100 mV to Vtest of -40 and 0 mV demonstrates characteristic crossover of currents. Smooth curves are single exponential fit with =32 and 19 ms at Vtest -40 (blue) and 0 mV (red), respectively. Vtest indicated above currents elicited for -30, -20, and -10 mV. Time to peak and rate of macroscopic current decay increased with increasing depolarization. Bars=20 pA, 20 ms. B, Pooled current-voltage curve fitted with modified Boltzmann distribution. Midpoint=-22 mV, slope=8.4 (n=12). C, Pooled steady-state inactivation curves of Ba2+ current in the presence of 100 µmol/L nifedipine. Midpoint=-67.2 mV, slope=9.7 (n=6). D, Ni2+ sensitivity of IT in fetal ventricular myocytes. Representative steady-state inactivation curves in control and 100 µmol/L Ni2+. Vtest=-10 mV. Midpoints=-67.5 and -74 in control and 100 µmol/L Ni2+, respectively.

We used Ni²⁺ blockade as an indication of T-type isoform expression in fetal cardiomyocytes. Ni²⁺, 200 µmol/L, blocks the 1G Ca²⁺ channel current by 50%; in contrast, 10 µmol/L Ni²⁺ is sufficient for half-block of the 1H T-type Ca²⁺ current.²¹ In E12.5 to E14 ventricular myocytes, 100 µmol/L Ni²⁺ blocks T-type Ca²⁺ current by 38.3±5.8% (Figure 4D; n=3). Ni²⁺-blocked and -unblocked currents have a comparable voltage dependence of inactivation consistent with a uniform dihydropyridine (DHP)-resistant channel population. On the basis of Ni²⁺ blockade and current kinetics coupled with the molecular identification and immunohistochemical data (Figures 1 through 3), we assign the T-type Ca²⁺ current in embryonic ventricular myocardium as an 1G-d current.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca_V3.1d is a splice variant of the 1G, T-type Ca²⁺ channel. In this study we demonstrate for the first time, to our knowledge, the following observations: (1) 1G mRNA and protein are expressed in developing fetal cardiac myocytes; (2) 1G is functionally expressed, as evidenced by T-type Ca²⁺ currents in E12.5 to E14 ventricular myocytes; and (3) 100 µmol/L Ni²⁺ half-blocks this T-type Ca²⁺ current. This report provides an experimental foundation for the hypothesis that T-type Ca²⁺ current, specifically 1G-d, contributes to plasma membrane Ca²⁺ flux for contraction in the developing myocardium.

Our observation that E12.5 to E14 ventricular myocytes express robust T-type Ca²⁺ current is controversial. Nuss and Marbán¹⁰ showed that late fetal and neonatal mouse myocytes express T-type Ca²⁺ current (I_T), but the only other study of murine E12 to E14 ventricle detected no I_T expression.¹¹ Recently, Leranguer et al²² and Pignier and Potreau²³ showed progressive reduction of DHP-resistant current from postnatal day 4 through adulthood. Under conditions of millimolar extracellular Ca²⁺, it may be difficult to separate T- from L-type Ca²⁺ channel current in ventricular myocytes. Nuss and Marbán¹⁰ showed the presence of a 10 µmol/L nitrendipine-resistant Ba²⁺ current in fetal cardiomyocytes. Furthermore, this current inactivated with sustained depolarization and as a function of steady-state holding potential. Similarly, we observed a 100 µmol/L nifedipine-resistant inactivating Ba²⁺ current. Furthermore, the steady-state inactivation voltage dependence was similar to our steady-state inactivation measurements of 1G expressed heterologously in HEK293 cells.^{4 17} We increased the nifedipine dose to 100 µmol/L to completely block L-type Ca²⁺ current^{24 25} and exposed the cells to drug for >5 minutes to prevent confusion of use-dependent blockade of L-type channels with I_T.²⁶ Therefore, the inward Ca²⁺ or Ba²⁺ current that we observe is DHP-resistant, thereby excluding L-type channels. Furthermore, the current we measure inactivates both by sustained depolarization and by steady-state holding potential; these properties exclude the unidentified current in fetal myocytes in the 1C knockout mouse.²⁷ Finally, the activity of this current in the presence of 30 µmol/L TTX excludes a contribution from I_Ca,TTX.²⁸ We also suggest that the novel fetal I_Ca,fe²⁹ is in fact 1G T-type current. Given that heterologously expressed current through 1G is DHP resistant³⁰ and that kinetic and steady-state inactivation properties from fetal ventricular myocytes mirror our earlier studies of 1G current, we conclude that fetal ventricular myocytes express functional T-type Ca²⁺ current. This assignment is based on a combination of the exclusion of known inward current carriers and parallels the reported properties of heterologously expressed T-type channel current.

One procedure common between the present study and the study by Nuss and Marbán¹⁰ is that spontaneously active cells were selected for study. Automatic activity is a well-described property of neonatal ventricular myocytes.^{31 32} We find it interesting to note the ongoing correlation of T-type Ca²⁺ channel expression with pacemaker cells from heart^{5 6 8} and noncardiac tissues.³³ Furthermore, the 1G-d splice variant that we now identify in fetal ventricular myocytes is the same splice variant that we previously identified in the murine AT-1 cell line.¹⁷ As with fetal myocytes, AT-1 cells in culture fire spontaneous action potentials.³⁴ Finally, our present studies and the study by Nuss and Marbán¹⁰ corroborate earlier findings from chick that I_T in early embryonic development has a depolarizing shifted voltage dependence of the macroscopic I-V curve.⁸ This creates an overlap of T- and L-type Ca²⁺ currents in the absence of DHP. In any case, our results unequivocally show that a particular splice variant of T-type Ca²⁺ channels is expressed in E13 ventricle and is a likely pathway for inward, DHP-resistant Ca²⁺ current.

There are 3 major plasma membrane transport mechanisms for inward Ca²⁺ flux in fetal cardiomyocytes: I_Ca,L, I_T, and reverse-mode Na⁺-Ca²⁺ exchange. Unequivocal assessment of the relative contribution of these mechanisms is hindered by the lack of selective pharmacological agents. Ni²⁺ is relatively selective for I_T compared with I_Ca,L and Na⁺-Ca²⁺ exchange, but it may also have subtle effects, particularly on I_Ca,L in the range of 100 µmol/L used in this study.³⁵ Therefore, in the absence of selective T-type channel blockers, establishment of a role for T-type Ca²⁺ channels in developing myocardium may ultimately require a combination of molecular genetic approaches, including their complete ablation using knockout strategies and overexpression of 1G-d in mouse heart.

	Acknowledgments

 
This work was supported by the American Heart Association (grant 0051434Z to L.L.C.) and the National Institutes of Health (National Research Service Award F32-HL 10200-02 to E.S. and grant HL63416 to J.S.). The authors thank Lindsay Burns and Joe Tinney for excellent technical assistance.

	Footnotes

 
Original received October 17, 2000; revision received December 28, 2000; accepted December 28, 2000.

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