Abnormal Coronary Function in Mice Deficient in α1H T-type Ca2+ Channels

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Science  21 Nov 2003:
Vol. 302, Issue 5649, pp. 1416-1418
DOI: 10.1126/science.1089268


Calcium ion (Ca2+) influx through voltage-gated Ca2+ channels is important for the regulation of vascular tone. Activation of L-type Ca2+ channels initiates muscle contraction; however, the role of T-type Ca2+ channels (T-channels) is not clear. We show that mice deficient in the α1H T-type Ca2+ channel (α13.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from α13.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni2+ prevented relaxation of wild-type coronary arteries. Thus, Ca2+ influx through α1H T-type Ca2+ channels is essential for normal relaxation of coronary arteries.

Calcium influx through voltage-gated Ca2+ channels initiates many physiological events including neurotransmitter release, muscle contraction, and gene expression. Of the voltage-gated calcium channels, the T-type Ca2+ channels (Cav3) that open at lower membrane potential are the least understood. The poreforming subunits of Cav3 are encoded by at least three genes: Cacna1G, Cacna1H, and Cacna1I13.1, 3.2, and 3.3, respectively) (13). α13.2 is expressed in multiple tissues including brain, heart, liver, testis, zona glomerulosa, skeletal muscle, and spermatids (2, 46). In the heart, Cav3 channels are expressed at high density in the sinoatrial (SA) and atrioventricular (AV) nodes (7, 8), consistent with a potential role for Cav3 channels in the generation of pacemaker potentials.

Cardiac Cav3 channels have been implicated in cardiac hypertrophy. Under normal conditions, T-type Ca2+ currents (T-currents) are not expressed in adult ventricular myocytes; however, T-currents can be reexpressed after development of hypertrophy (9, 10). In cardiomyopathic BIO 14.6 hamsters, an abnormal increase in T-current density may be responsible for Ca2+ overload in the heart (11). These studies suggest that Cav3 channels may function in the remodeling of cardiac myocytes during development of cardiomyopathy. In smooth muscle, Cav3 channels may function in vasoconstriction of rat mesenteric arterioles and renal resistance vessels (12, 13). Expression of T-currents is regulated during the cell cycle, with the highest density of T-currents during G1 and S phases in smooth muscle and neonatal ventricular myocytes (14, 15). Thus, Cav3 channels appear to take part in signaling pathways that control differentiation and proliferation.

To explore the roles of Cav3.2, we have generated mice lacking α13.2 by homologous gene targeting. Homologous recombination resulted in deletion of exon 6, corresponding to amino acid residues 216 to 267 (fig. S1, A and B). Heterozygous mice were bred to generate homozygous mutants (α13.2–/–). Northern blot and Western blot analysis confirmed the loss of α13.2 RNA and protein in α13.2–/– mice (fig. S1, C and D). Because α13.2 is the predominant α1 subunit of T-channels in rat dorsal root ganglia (DRG) (16), we performed whole-cell patch-clamp analysis of voltage-activated Ca2+ currents from neonatal DRG neurons. An averaged low voltage–activated Ca2+ current (IT) was elicited by a voltage change from –90 mV to –30 mV (fig. S1E). The peak current density of IT was –3.7 ± 0.3 pA/pF (n = 5) in wild-type DRG neurons. IT was significantly diminished in the α13.2–/– DRG neurons (n = 8, P < 0.001). Thus, α13.2 channels are the predominant T-channels in neonatal mouse DRG neurons, with no compensation of other α13 proteins in α13.2–/– DRG neurons. There were no significant differences between the high voltage–activated (HVA) Ca2+ currents (elicited from –50 mV to 10 mV) from wild-type or α13.2–/– DRG neurons (fig. S1F). α13.2–/– mice were viable and fertile. Both male and female α13.2–/– mice were smaller than their littermate controls. The body weights of 8-week-old male wild-type and α13.2–/– mice were 26.6 ± 0.92 g (n = 8) and 20.2 ± 2.4 g (n = 9), respectively (t test, P < 0.05). No differences were detected in ratios of specific tissues to total body weight between wild-type and knockout mice.

T-channels are thought to be important in the generation of pacemaker potentials in SA and AV nodes. To investigate whether disruption of α13.2 produces cardiac abnormalities, we compared hearts isolated from wild-type and α13.2–/– mice at various ages histologically. Diffuse regions of cardiac fibrosis were observed in ventricular walls from 10-week-old α13.2–/– mice but not those from wild-type hearts (Fig. 1A). The percentage of cardiac fibrosis area was 0.23 ± 0.06% (n = 4) and 2.16 ± 0.57% (n = 4) in 10-week-old wild-type and α13.2–/– hearts, respectively (P < 0.05). With increasing age, the size of the fibrotic regions increased. At 1 year of age, the α13.2–/– hearts had more severe cardiac pathology (Fig. 1B). Large areas of fibrosis, necrosis, and lymphocyte infiltration were observed only in the α13.2–/– mice. The percentage of cardiac fibrosis area was 0.92 ± 0.3% (n = 4) and 4.04 ± 0.89% (n = 4) in 1-year-old wild-type and α13.2–/– hearts, respectively (P < 0.05). Electrocardiographic (ECG) telemetry and echocardiography in wild-type and α13.2–/– mice (fig. S3; movies S1 and S2) showed no significant differences in heart rate between wild-type (565.6 ± 23 beats/min, n = 16) and α13.2–/– mice (505.2 ± 34.7 beats/min, n = 14). No cardiac arrhythmias were observed and ECG waveform morphologies were normal. On the other hand, α1D11.3) L-type Ca2+ channel–deficient mice have sinus bradycardia and arrhythmia with normal Cav3 currents in the SA node (1719). Thus, we conclude that even though both T-type and α11.3 L-type Ca2+ channels have the intrinsic ability to support pacemaker activity in the SA node, α11.3 L-type Ca2+ channels contribute more toward the generation of pacemaker potential in the heart.

Fig. 1.

Cardiac fibrosis and abnormal cardiac vasculature in α13.2–/– mice. (A) Masson's trichromic staining of transverse heart sections from 10-week-old wild-type (+/+) and α13.2–/– mice. Regions of cardiac fibrosis are shown in green. (B) Larger regions of fibrosis in the α13.2–/– heart at 1 year of age. Scale bar, left panels, 1 mm; right panels, 50 μm. (C) Microfil perfusion of coronary arteries in wild-type, heterozygous (+/–), and α13.2–/– hearts. Spiral vascular constrictions are indicated by arrowheads. Scale bar, 40 μm.

Because T-channels are not expressed in adult mouse ventricular myocytes, we hypothesized that cardiac fibrosis was due to the loss of α13.2 in other cells such as vascular smooth muscle. Abnormal coronary artery constriction can lead to reduced coronary blood flow, cardiac myocyte necrosis, and cardiac fibrosis (20, 21). Thus, we perfused mice with latex Microfil to visualize the architecture of the coronary tree. In wild-type (n = 6) and heterozygous (n = 3) hearts, the vessels appeared smooth and regular, but in hearts from α13.2–/– mice, vessels were constricted and had irregular spiral shapes (n = 10) (Fig. 1C).

We also measured responses of isolated coronary arteries from wild-type and α13.2–/– hearts. Coronary arteries were isolated, cannulated into micropipettes, and pressurized, and the luminal diameter of the artery was measured with an electronic video dimension analyzer (22). Baseline diameters of coronary arteries from wild-type (n = 12) and α13.2–/– mice (n = 18) were 103.3 ± 4.5 μm and 112.5 ± 6.7 μm, respectively. U46619, a thromboxane mimetic, produced a similar dose-dependent constriction of coronary arteries from wild-type and α13.2–/– mice (Fig. 2A). Maximal contraction by U46619 was 41 ± 8% (n = 5) and 36 ± 11% (n = 5) in wild-type and α13.2–/– arteries, respectively. Similar constriction was observed when KCl was used to constrict arteries from wild-type and α13.2–/– hearts. These data suggest that contractile mechanisms in coronary arteries are normal in the α13.2–/– mice.

Fig. 2.

Impaired relaxation response in the coronary artery of α13.2–/– mice. (A to D) Percent change in diameter of coronary arteries from α13.2–/– wild-type and mice treated with U46619 (A), ACh (B), SNP (C), and nifedipine (D). In (C), wild-type arteries were also treated with 100 μM Ni2+. (E) Blockade of Cav3 currents with Ni2+ decreases ACh-induced (10–4 M) relaxation in wild-type arteries.

We also examined relaxation of coronary arteries in response to acetylcholine (ACh) and sodium nitroprusside (SNP). In arteries from wild-type mice that had been constricted with U46619, ACh produced dose-dependent dilation (Fig. 2B). In contrast, ACh caused further constriction in coronary arteries from α13.2–/– mice. Dilation in response to ACh in mouse coronary arteries is mediated predominantly by release of nitric oxide (NO) from endothelial cells (22). We therefore compared responses of coronary arteries from wild-type and α13.2–/– mice to SNP, an NO donor. SNP induced a dose-dependent dilation of coronary arteries from wild-type mice (Fig. 2C). However, relaxation in response to SNP (10–5 M) was reduced to 40 ± 11% (n = 5) in arteries from α13.2–/– α13.2–/– mice. To ensure that arteries can relax in response to other dilators, we tested effects of nifedipine (an L-type Ca2+ channel blocker). Nifedipine produced similar relaxation of arteries isolated from wild-type (n = 8) or α13.2–/– (n = 5) mice (Fig. 2D). Thus, only the NO-mediated relaxation appears to be defective in α13.2–/– coronary arteries.

Ca2+ influx through Ca2+ channels is generally thought to initiate contractile mechanisms in smooth muscle. However, our results suggest that α13.2 is required for NO-mediated relaxation. If Ca2+ influx through T-channels is required for the relaxation of coronary arteries, blockade of T-channels with Ni2+ should decrease relaxation of coronary arteries from wild-type mice. When Ni2+ was added in the presence of U46619 and ACh, ACh-induced dilation was decreased in a dose-dependent manner (Fig. 2E).

To determine whether the defect in relaxation is due to altered endothelial cell NO production or abnormal smooth muscle cell function (or both), we compared responses to SNP in the presence and absence of Ni2+. In the presence of 100 μM Ni2+, relaxation of wild-type arteries in response to SNP was reduced to levels similar to those in α13.2–/– arteries (Fig. 2C). These results suggest that Ni2+ may affect smooth muscle cells directly rather than through inhibition of endothelial release of NO. We detected T-channels by whole-cell patch-clamp analysis of voltage-activated Ca2+ currents on coronary smooth muscle cells isolated from wild-type but not α13.2–/– mice (Fig. 3A). There were no significant differences between the HVA currents from wild-type or α13.2–/– cells (Fig. 3B). Taken together, these results suggest that Ca2+ influx through Cav3.2 channels in coronary smooth muscle is required for normal relaxation of coronary arteries mediated by endogenously released or exogenously administered NO.

Fig. 3.

Expression of α13.2 T-type calcium channels in smooth muscle cells. Left panels: Averaged T-currents (n = 4) (A) and HVA Ca2+ currents (n = 6) (B) recorded from isolated wild-type (+/+) and α13.2–/– coronary smooth muscle cells. Right panels: Peak current density from wild-type and α13.2–/– coronary smooth muscle cell; *P < 0.001 (t test).

The mechanism underlying the involvement of α13.2 in the relaxation of coronary smooth muscle is not clear. Ca2+ influx can activate localized calcium release through ryanodine receptors, which in turn activates the large-conductance Ca2+-sensitive K+ channels (BKCa), causing an outward K+ current and membrane hyperpolarization (23, 24). This closes Ca2+ channels in the plasma membrane and inhibits contraction. Because BKCa channels play an important role in regulating relaxation, it is possible that α13.2 is functionally coupled to the BKCa channel in coronary smooth muscle. Deletion of α13.2 may reduce the activity of the BKCa channel, leading to impaired relaxation of coronary arteries. If one of the physiological roles of α13.2 is to fine-tune the activity of BKCa channels in vascular smooth muscle, activation of BKCa channels should relax constricted α13.2–/– coronary arteries. NS-1619, a BKCa channel opener, induced similar dose-dependent relaxations of wild-type and α13.2–/– coronary arteries (Fig. 4A). Consistent with possible association of α13.2 with the BKCa channel in vivo, the proteins cosedimented on sucrose gradients when isolated from solubilized human embryonic kidney (HEK) cells overexpressing exogenous α13.2 and α subunit of BKCa channels (Fig. 4B). α13.2 and BKCa channels were also coimmunoprecipitated with an antibody to α13.2 from wild-type solubilized brain microsomes but not from α13.2–/– tissues (Fig. 4C).

Fig. 4.

Association of α13.2 T-type calcium channels and α subunit of BKCa channels. (A) Percent change in diameter of coronary arteries from wild-type and α13.2–/– mice treated with NS-1619. (B) Western blot analysis of sucrose gradient fractionation of 1% digitonin-solubilized microsomes from HEK cells expressing α13.2 and BKCa channels. (C) Coimmunoprecipitation of BKCa channels with an antibody to α13.2 T-type calcium channels from wild-type but not α13.2–/– brain microsomes.

Our study provides insights into the role of α1H Cav3 channels in the normal relaxation of coronary vascular smooth muscle. The results suggest that Cacna1H could be a potential target for therapeutic intervention in cardiovascular diseases.

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