Age-dependent impact of CaV 3.2 T-type calcium channel deletion on myogenic tone and flow-mediated vasodilatation in small arteries

Abstract

Key points: Blood pressure and flow exert mechanical forces on the walls of small arteries, which are detected by the endothelial and smooth muscle cells, and lead to regulation of the diameter (basal tone) of an artery. Ca_V 3.2 T-type calcium channels are expressed in the wall of small arteries, although their function remains poorly understood because of the low specificity of T-type blockers. We used mice deficient in Ca_V 3.2 channels to study their role in pressure- and flow-dependent tone regulation and the possible impact of ageing on this role. In young mice, Ca_V 3.2 channels oppose pressure-induced vasoconstriction and participate in endothelium-dependent, flow-mediated dilatation. These effects were not seen in mature adult mice. The results of the present study demonstrate an age-dependent impact of Ca_V 3.2 T-type calcium channel deletion in rodents and suggest that the loss of Ca_V 3.2 channel function leads to more constricted arteries, which is a risk factor for cardiovascular disease.

Abstract: The myogenic response and flow-mediated vasodilatation are important regulators of local blood perfusion and total peripheral resistance, and are known to entail a calcium influx into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), respectively. Ca_V 3.2 T-type calcium channels are expressed in both VSMCs and ECs of small arteries. The T-type channels are important drug targets but, as a result of the lack of specific antagonists, our understanding of the role of Ca_V 3.2 channels in vasomotor tone at various ages is scarce. We evaluated the myogenic response, flow-mediated vasodilatation, structural remodelling and mRNA + protein expression in small mesenteric arteries from Ca_V 3.2 knockout (Ca_V 3.2KO) vs. wild-type mice at a young vs. mature adult age. In young mice only, deletion of Ca_V 3.2 led to an enhanced myogenic response and a ∼50% reduction of flow-mediated vasodilatation. Ni²⁺ had both Ca_V 3.2-dependent and independent effects. No changes in mRNA expression of several important K⁺ and Ca²⁺ channel genes were induced by Ca_V 3.2KO However, the expression of the other T-type channel isoform (Ca_V 3.1) was reduced at the mRNA and protein level in mature adult compared to young wild-type arteries. The results of the present study demonstrate the important roles of the Ca_V 3.2 T-type calcium channels in myogenic tone and flow-mediated vasodilatation that disappear with ageing. Because increased arterial tone is a risk factor for cardiovascular disease, we conclude that Ca_V 3.2 channels, by modulating pressure- and flow-mediated vasomotor responses to prevent excess arterial tone, protect against cardiovascular disease.

Figure 1. Myogenic responsiveness in young vs. mature WT and Ca_V3.2KO mice

Myogenic response (and effects of 30 μm Ni²⁺) in young WT (A), young Ca_V3.2KO (B), mature adult WT (C) and mature adult Ca_V3.2KO (D) mouse mesenteric artery. Myogenic tone (MT, %) in young WT vs. Ca_V3.2KO mice (E, same mice as in A and B), and mature adult WT vs. Ca_V3.2KO mice (F, same mice as in C and D). Effects of paxilline on MT in young WT (G) vs. Ca_V3.2KO mice (H). ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001 at individual pressures.

Figure 2. Flow‐mediated vasodilatation in rat small arteries

Original recording showing reversibility of FMVD (%) in SMAs induced by a longitudinal pressure gradient (ΔP = 20 mmHg) at a mean pressure of 60 mmHg in a cannulated vessel preconstricted using U46619 (A). Effects of l‐Name (100 μm) (B); apamin (50 nm) + Tram‐34 (1 μm) (C); and NiCl₂ (100 μm) (D) on FMVD responses in rat mesenteric artery.

Figure 3. FMVD measurements in WT vs. Ca_V3.2KO mice

FMVD (and effects of 30 μm Ni²⁺) in young WT vs. Ca_V3.2KO mouse mesenteric arteries (A) and in mature adult WT vs. Ca_V3.2KO arteries (B).

Figure 4. Endothelium‐dependent (A, B) and ‐ independent (C, D) vasodilatation in WT vs. Ca_V3.2KO mice

Dilatations (%) to ACh (ACh, 10 μm) in young WT vs. Ca_V3.2KO mouse mesenteric arteries (A) and in mature adult WT vs. Ca_V3.2KO arteries (B). C, concentration‐dependent dilatations to NO‐donor SNAP in age‐matched WT vs. Ca_V3.2KO arteries. D, dilatations to elevated bath [KCl] = 9.5 mM in age‐matched WT vs. Ca_V3.2KO arteries.

Figure 5. Acute vasoconstriction to NiCl₂

Acute vasoconstriction upon exposure to 30 μM NiCl₂ in pressurized (60 mmHg) mesenteric arteries in young WT vs. Ca_V3.2KO arteries and in mature adult WT vs. Ca_V3.2KO arteries.

Figure 6. Passive structural properties of SMAs

Passive lumen diameter (A), media/lumen‐ratio (B) and media CSA (C) in WT vs. Ca_V3.2KO arteries for both young and mature adult mice.

Figure 7. Expression of Ca_V3.2 at the mRNA and protein level in young mouse SMAs

A, mouse SMA (young) stained with primary antibody against Ca_V3.2 (red colour). The internal and external elastic laminas are visible as faint green bands using the inherent autofluorescence of the tissue. Nuclei are stained blue with 4′,6‐diamidino‐2‐phenylindole. An endothelial cell nucleus is marked with an asterisk in the vessel lumen. Clear Ca_V3.2‐specific red staining is present in the media layer and faint red staining is visible in the intima layer on the luminal side of the green internal elastic lamina. B, negative peptide pre‐absorption control staining for Ca_V3.2 primary antibody of mouse mesenteric artery (young). Note the absence of a positive red signal. An endothelial cell nucleus is marked with an asterisk in the vessel lumen. C, image of isolated endothelial tube from a young mouse SMA. The image was acquired with a Axiovert S‐100 microscope (Carl Zeiss, Oberkochen, Germany) through a Neofluar 20× objective using a USB camera and Myoview II software. D, agarose gel showing electrophoresis of Cacna1H qPCR products from mouse brain (n = 1), mouse whole SMA (n = 1) and SMA endothelial tubes isolated from four young WT mice (EC1–EC4). Horizontal arrows depict the product size corresponding to Cacna1H (169 bp). Note the similar product size in brain, whole SMA and EC tubes.

Figure 8. Age‐dependent intensity of Ca_V3.1‐specific staining in mouse SMAs

Immunostaining of the Ca_V3.1 T‐type isoform (red colour) in young (A) vs. mature adult (B) WT mouse mesenteric arteries, and in staining without primary antibody (C). Note the lower fluorescence intensity visible in the mature adult artery compared to the young artery. Images were acquired using same settings of microscope, camera and software. Quantitation of background‐corrected total tissue fluorescence in young vs. mature adult arteries using ImageJ (D).