Endostatin inhibits T-type Ca2+ channel current in guinea pig ventricular myocyte.

Endostatin, a fragment of collagen XVIII, is known as an endogenous angiogenesis inhibitor, and its serum concentration increases in various cardiovascular diseases. T-type Ca(2+) channel, low voltage-activated Ca(2+) channel, is not expressed in adult ventricular myocytes. Re-expression of T-type Ca(2+) channels in cardiac myocytes is thought to be involved in the development of cardiac hypertrophy. We examined the effects of endostatin on T-type Ca(2+) channel current by whole-cell patch clamp technique in freshly isolated adult guinea pig ventricular myocytes, which exceptionally express T-type Ca(2+) channels. Although endostatin 300 ng/ml had no effect on L-type Ca(2+) current, it significantly inhibited T-type Ca(2+) current. These data indicate that endostatin can be an endogenous inhibitor of T-type Ca(2+) channels in the cardiac myocytes.

Endostatin, a non-collagenous C-terminal fragment cleaved from collagen XVIII, is an endogenous angiogenesis inhibitor [13]. A number of clinical trials of endostatin have been conducted for a new anti-cancer therapy development. Recent studies have revealed that the serum concentration and the expression levels of endostatin increased in various cardiovascular diseases, such as cardiac hypertrophy, myocardial infarction and heart failure [5, 8]. Therefore, endostatin might be involved in the development of cardiac diseases. However, the effect of endostatin on heart has not been clarified.

In cardiac myocytes, two types of voltage-gated Ca2+ channels are expressed in the cell membrane [9]. High-voltage-activated L-type Ca2+ channels are responsible for the main Ca2+ entry into the cardiac myocytes associated with the depolarization, which triggers the cardiac contraction [16]. On the contrary, T-type Ca2+ channels are low-voltage-activated Ca2+ channels and open after small depolarization of the membrane. In the adult mammal heart, T-type Ca2+ channels are found in the pacemaker cells located in sinoatrial node [1]. T-type Ca2+ channels are also expressed in the embryonic ventricular myocytes and are involved in the development of the cells [19]. Many studies demonstrated that T-type Ca2+ channels were absent in the adult ventricular myocytes of rats, cats and dogs [15]. Although T-type Ca2+ channels disappear during maturation of cardiac myocytes, re-expression of T-type Ca2+ channels was observed in the cardiac myocytes of mice, rats and cats with pathological conditions, such as cardiac hypertrophy and post-myocardial infarction [4, 11, 15]. Re-expression of T-type Ca2+ channels is thought to play an important role in the enlargement of cardiac myocytes [3]. It has been reported that endostatin inhibits cell proliferation and migration through the inhibition of T-type Ca2+ channels in human glioblastoma cells [20]. However, the influence of endostatin on voltage-gated Ca2+ channels in cardiac myocytes has not been clarified. Therefore, we investigated the effect of endostatin on T-type Ca2+ channel current by using whole-cell patch clamp technique in the adult guinea pig ventricular myocytes, which exceptionally express T-type Ca2+ channels [12].

The present study was conducted in accordance with the Guide for Care and Use of Laboratory Animals as adopted by Kitasato University. Isolation of ventricular myocytes was performed as described previously [14]. Briefly, the heart of adult guinea pig (248–602 g body weight) was isolated under sodium pentobarbital anesthesia (70 mg/kg, i.p.) and perfused by a modified Langendorff apparatus. The perfused heart was digested by 0.02% collagenase, and ventricular myocytes were isolated. Electrophysiological recording of membrane currents was performed by whole-cell patch clamp method as described previously [7]. The external solution contained the following compositions: 137 mM Tris, 1 mM MgCl2, 5.4 mM CaCl2, 20 mM CsCl and 5 mM glucose adjusted to pH of 7.4 with HCl. The pipette solution contained the following compositions: 125 mM CsOH, 5 mM ATP-Mg, 15 mM EGTA, 20 mM TEA-Cl and 10 mM HEPES adjusted to pH 7.2 with CsOH [21]. Ca2+ currents were elicited by 300 ms depolarizing test pulses from a holding potential (HP) of −90 and −50 mV. Whole Ca2+ channel current (ICa) was elicited by 15 steps depolarizing pulses of each +10 mV from a HP of −90 mV. L-type Ca2+ channel current (ICaL) was elicited by 11 steps depolarizing pulses of each +10 mV from a HP of −50 mV [17]. The current density (pA/pF) was calculated by normalizing current amplitude by cellular membrane capacitance. Results are expressed as mean ± S.E.M. Statistical analysis was performed by Student’s t-test. A value of P<0.05 was considered as statistically different.

T-type Ca2+ channel current (ICaT) was obtained by subtracting ICaL from ICa. In the present study, the peak inward current of ICaT was observed at −20 mV. It was consistent with the previous report [21]. Lee et al. reported that NiCl2 inhibited ICaT in guinea pig ventricular myocyte [10]. This study confirmed that NiCl2 100 µM significantly inhibited the ICaT (Fig. 2A, at −20 mV, control: −1.15 ± 0.20 pA/pF, NiCl2: −0.15 ± 0.17 pA/pF, n=4). First, we investigated the effect of endostatin on ICaL. Endostatin (300 ng/ml) was bath applied 5 min before the stimulation of depolarizing pulse. It was reported that blood concentration of endostatin in healthy volunteers is about 40 ng/ml [18]. On the other hand, the concentration in patients of cardiovascular disease reached about 250–300 ng/ml [6]. Therefore, the concentration of endostatin 300 ng/ml was within the pathological range. In the present study, endostatin 300 ng/ml had no effect on ICaL (Fig. 1, at +10 mV, control: −15.2 ± 2.0 pA/pF, endostatin: −14.8 ± 1.5 pA/pF, n=11). Zhang et al. reported that endostatin had no effect on ICaL in glioblastoma cells [20]. Our results were consistent with this report. Next, we investigated the effect of endostatin on ICaT. Endostatin 300 ng/ml significantly inhibited ICaT at −20 mV (Fig. 2B, control: −1.18 ± 0.12 pA/pF, endostatin: −0.75 ± 0.10 pA/pF, n=11, P<0.05), while endostatin 300 ng/ml did not inhibit it at −30 mV (Fig. 2, control: −0.99 ± 0.12 pA/pF, endostatin: −0.90 ± 0.10 pA/pF, n=11) and −40 mV (Fig. 2, control: −0.66 ± 0.11 pA/pF, endostatin: −0.62 ± 0.10 pA/pF, n=11). Bladen and Zamponi reported that a Nav1.8 blocker 5-(4-chlorophenyl-N (3, 5-dimethoxyphenyl) furan-2-carboxamide (A803467) inhibited T-type Ca2+ channel in tsA-201 cells. Hyperpolarizing shift of both half-activation voltage and peak current of ICaT was observed in A803467-treated cells [2]. We suppose that similar hyperpolarizing shift of peak current of ICaT might be occurred in endostatin-treated cardiomyocytes, which would blunt the inhibition of ICaT at −30 and −40 mV. We confirmed that low dose endostatin (30 ng/ml) did not significantly inhibit ICaT (at −20 mV: control: −0.85 ± 0.17 pA/pF, endostatin: −0.71 ± 0.16 pA/pF, n=6, data not shown), indicating its role in the pathogenesis of cardiovascular diseases. Three types of α1-subunit genes encode T-type Ca2+ channels: Cav3.1, 3.2 and 3.3. In mammals, Cav3.1 and 3.2 are known to be expressed in cardiac cells [15]. Zhang et al. reported that endostatin inhibited Cav3.1 and Cav3.2, but not Cav3.3 in human glioblastoma cell [20]. Therefore, endostatin might inhibit Cav3.1 and/or Cav3.2 in ventricular myocytes. The present study did not clarify how endostatin inhibits T-type Ca2+ channels. It was suggested in glioblastoma cells that endostatin inhibited T-type Ca2+ channels directly rather than via other pathways including G-protein and protein tyrosine kinase [20]. Further investigations are needed to clarify the inhibitory mechanisms of endostatin on T-type Ca2+ channels.

Fig. 2.

Endostatin inhibited T-type Ca2+ channel current in isolated guinea pig ventricular myocytes. Representative T-type Ca2+ channel current (ICaT) records (arrows) calculated by subtracting the current of a VHP-40 (holding potential: −40 mV) from the current of a VHP-90 (holding potential: −90 mV) at −20 mV in the same cell are shown (A, control: left, NiCl2: right; B, control: upper left, endostatin: lower left). Endostatin (300 ng/ml) was pre-treated for 5 min. Current-Voltage relations for the peak currents of ICaT (−40–0 mV) in the absence (closed symbols) or presence (open symbols) of endostatin are shown (right). The current density (pA/pF) was calculated by normalizing current amplitude by cellular membrane capacitance. Results were expressed as means ± S.E.M (n=11). *P<0.05 vs. control.

Fig. 1.

Endostatin had no effect on L-type Ca2+ channel current in isolated guinea pig ventricular myocytes. Representative L-type Ca2+ channel current (ICaL) records (arrows; holding potential: −40 mV) at 10 mV in the same cell are shown (control: left, endostatin: middle). Current-Voltage relations for the peak currents of ICaL (−20–60 mV) in the absence (closed symbols) or presence (open symbols) of endostatin (300 ng/ml, 5 min-pretreatment) are shown (right). The current density (pA/pF) was calculated by normalizing current amplitude by cellular membrane capacitance. Results were expressed as means ± S.E.M (n=11).

In conclusion, we for the first time demonstrated that endostatin can inhibit T-type Ca2+ channels in cardiac myocytes. It has been reported that Ca2+ signaling pathway activated through the T-type Ca2+ channels has an important role in the development of cardiac hypertrophy. Therefore, endostatin can be a new pharmaceutical target for the treatment of cardiac hypertrophy.