Age attenuates the T-type CaV 3.2-RyR axis in vascular smooth muscle

Abstract

Caveolae position Ca_V 3.2 (T-type Ca²⁺ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca²⁺ influx to trigger Ca²⁺ sparks and large-conductance Ca²⁺ -activated K⁺ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca²⁺ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Ca_v 3.2 channel inhibition by Ni²⁺ (50 µM) and caveolae disruption by methyl-ß-cyclodextrin or genetic abolition of Eps15 homology domain-containing protein (EHD2) inhibited Ca²⁺ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Ca_v 3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Ca_v 1.2^-/- mice, caffeine (RyR activator) and thapsigargin (Ca²⁺ transport ATPase inhibitor), we found that sufficient SR Ca²⁺ load is a prerequisite for the Ca_V 3.2-RyR axis to generate Ca²⁺ sparks. We identified a fraction of Ca²⁺ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd³⁺ (100 µM), but insensitive to Ca_V 1.2 and Ca_V 3.2 channel blockade. Our data demonstrate that the VSMC Ca_V 3.2-RyR axis is down-regulated by aging. This defective Ca_V 3.2-RyR coupling is counterbalanced by a Gd³⁺ sensitive Ca²⁺ pathway providing compensatory Ca²⁺ influx for triggering Ca²⁺ sparks in aged VSMCs.

Keywords: T-type calcium channels; aging; calcium sparks; caveolae; ryanodine receptors; vascular smooth muscle.

Figure 1

Age attenuates the role of Ca_V3.2 channels in Ca²⁺ spark generation and decreases Ca_V3.2 protein expression in VSMC. (a), Ca²⁺ fluorescence images of a Fluo‐4‐AM–loaded VSMC from a young mouse and time course of Ca²⁺ fluorescence changes in the cellular ROI (upper panel). Cell boundary is marked with dashed line. (b), same as (a) but in the presence of Ni²⁺ (50 µM). (c), same as (a) but in a VSMC from an old mouse. (d), same as (c) but in the presence of Ni²⁺ (50 µM). (e, f), summary of the results. Ca²⁺ spark frequency (e) and fraction of cells producing Ca²⁺ sparks (f) in VSMCs from young mice (n = 102), in VSMCs from young mice cells incubated with Ni²⁺ (n = 85), in VSMCs from aged mice (n = 129), and in VSMCs from aged mice cells incubated with Ni²⁺ (n = 127). Cells were isolated from 4 mice in each group; 25–40 cells were recorded and analyzed from each mouse. VSMC, vascular smooth muscle cell. (g), Western blot analysis of Ca_V3.2 proteins in mesenteric arteries of young versus old mice. (h), quantification of Western blot results. Mesenteric arteries were taken from 9 mice in each group. *, p < .05. n.s., not significant

Figure 2

Role of luminal SR calcium on T‐type Ca_V3.2‐RyR axis. Effects of different concentrations of thapsigargin on Ca²⁺ spark frequency (a, left) and fraction of cells producing Ca²⁺ sparks (a, right) in Ca_v1.2^+/+ VSMCs from young mice. Effects of different concentrations of thapsigargin on Ca²⁺ spark frequency (b, left) and fraction of cells producing Ca²⁺ sparks (b, right) in VSMCs from Ca_v1.2^−/− (SMAKO) mice. (c), overlay of the data for Ca²⁺ spark frequency (left) and fraction of cells producing Ca²⁺ sparks (right). Cells were isolated from 4 mice in each group; 30–35 cells were recorded and analyzed from each mouse. (d), time course of Ca²⁺ fluorescence changes in the cellular ROI in a wild‐type (Ca_V1.2^+/+) Fluo‐4‐AM–loaded VSMC induced by 10 mM caffeine (upper panel) and Ca²⁺ fluorescence plots (lower panel). (e), the same as (d), but in Ca_V1.2^−/− VSMC. (f), summary of the 10 mM caffeine‐induced Ca²⁺ peaks in wild‐type versus Ca_V1.2^−/− VSMCs. n = 7 cells from 3 mice, 2–3 cells were recorded and analyzed from each mouse. *, p < .05. n.s., not significant

Figure 3

Defective Ca_V3.2‐RyR axis in aged VSMC result from alterations in the ultrastructure of caveolae. (a), Ca²⁺ fluorescence images of a Fluo‐4‐AM–loaded VSMC from a young mouse and time course of Ca²⁺ fluorescence changes in the cellular ROI (upper panel). Cell boundary is marked with dashed line. (b), same as (a) but with a cell incubated with methyl‐ß‐cyclodextrin (10 mM, 90 min at room temperature) to disrupt caveolae. (c), same as (a) but with VSMCs from old mice. (d), same as (c) but with a cell incubated with methyl‐ß‐cyclodextrin. (e, f), summary of the results. Ca²⁺ spark frequency (e) and fraction of cells producing Ca²⁺ sparks (f) in VSMCs from young mice (n = 98), in VSMCs from young mice cells incubated with methyl‐ß‐cyclodextrin (n = 111), in VSMCs from old mice (n = 121), and in VSMCs from old mice cells incubated with methyl‐ß‐cyclodextrin (n = 128). Cells were isolated from 4 mice in each group; 25–40 cells were recorded and analyzed from each mouse. (g), Electron microscopy image of a young VSMC. (h), same as (g) but from old VSMC. (i), summary of the results. Caveolae density, diameter of caveolae neck, caveolae size in VSMCs from young versus old mice (10–20 cells from each mouse, 4 mice in each group). *, p < .05. n.s., not significant

Figure 4

EHD2 knockout (EHD2 del/del) alters the ultrastructure of caveolae and decrease Ca_V3.2 expression, resulting in Ca_V3.2‐RyR axis malfunction. (a), Electron microscopy image of a EHD2 del/+ VSMC and a EHD2 del/del VSMC. (b, left), Ca_V3.2 immuno‐staining in BAT cryostat sections from EHD2 del/+ and del/del mice. (b, right), summary of the results, n (del/+)=46/5 mice and n (del/del)=53/5 mice. (c), Ca²⁺ fluorescence images of a Fluo‐4‐AM–loaded VSMC from EHD2 del/del mouse and time course of Ca²⁺ fluorescence changes in the cellular ROI (upper panel). Cell boundary is marked with dashed line. (d), same as (c) but in the presence of Ni²⁺ (50 µM). (e), same as (c) but in the presence of Cd²⁺ (200 µM). (f), same as (e) but in the presence of Ni²⁺ (50 µM). (g, h), summary of the results. Ca²⁺ spark frequency (g) and fraction of cells producing Ca²⁺ sparks (h) in VSMCs from EHD2 del/+ mice (n = 99), in VSMCs from EHD2 del/+ mice cells incubated with Ni²⁺ (n = 96), in VSMCs from EHD2 del/del mice (n = 144), and in VSMCs from EHD2 del/del mice cells incubated with Ni²⁺ (n = 125). Cells were isolated from 4 mice in each group; 25–40 cells were recorded and analyzed from each mouse. (I, j), summary of the results. Ca²⁺ spark frequency (i) and fraction of cells producing Ca²⁺ sparks (j) in VSMCs from EHD2 del/+ mice incubated with Cd²⁺ (n = 56), in VSMCs from EHD2 del/+ mice cells incubated with Ni²⁺+Cd²⁺ (n = 56), in VSMCs from EHD2 del/del mice incubated with Cd²⁺ (n = 75), and in VSMCs from EHD2 del/del mice cells incubated with Ni²⁺+Cd²⁺ (n = 68). Cells were isolated from 4 mice in each group; 15–20 cells were recorded and analyzed from each mouse. *, p < .05. n.s., not significant

Figure 5

Gd³⁺ sensitive (TRP) cation channels generate Ca²⁺ sparks in old VSMCs. (a), Ca²⁺ fluorescence images of a Fluo‐4‐AM–loaded VSMC from an old mouse and time course of Ca²⁺ fluorescence changes in the cellular ROI (upper panel). Cell boundary is marked with dashed line. (b), same as (a) but with a cell incubated with Cd²⁺ (200 µM). (c), same as (a) but with Ni²⁺ (50 µM) following Cd²⁺ treatment. (d), same as (a) but with Gd³⁺ (100 µM) following Cd²⁺+ Ni²⁺ treatment. (e, f), summary of the results. Ca²⁺ spark frequency (e) and fraction of cells producing Ca²⁺ sparks (f) in cells (n = 66), in cells incubated with Cd²⁺ (n = 69), in cells incubated with Cd²⁺+ Ni²⁺ (n = 61), and in cells incubated with Cd²⁺+ Ni²⁺+ Gd³⁺ (n = 86). Cells were isolated from 4 old mice in each group; 15–30 cells were recorded and analyzed from each mouse. *, p < .05. n.s., not significant

Figure 6

T‐type Ca_V3.2 blockade does not constrict mesenteric arteries from old mice. (a, b), representative traces and summary data show the effect of Ni²⁺ (50 µM) on mesenteric arteries pressurized to 60–100 mmHg from young and old mice, respectively. (c), vasoconstriction evoked by 60 mM K⁺ was similar in young and old pressurized (15 mmHg) arteries. (d, e), summary of myogenic tone measurements in pressurized mesenteric arteries from young and old mice (n = 5 arteries from 5 mice, one artery was recorded and analyzed from each mouse). Experiments were performed in the absence and presence of 50 µM Ni²⁺. *, p < .05. n.s., not significant. (f), schematic illustration of major Ca²⁺ influx pathways regulating Ca²⁺ sparks in VSMCs during aging. Ca²⁺ sparks, which result from opening of clustered RyRs in the SR, activate large‐conductance Ca²⁺‐activated K⁺ (BK_Ca) channels to produce a negative feedback effect on vasoconstriction. L‐type Ca_v1.2 channels contribute to global cytosolic [Ca²⁺], which in turn influences luminal SR calcium (via SERCA) and thus generates the majority (75%) of Ca²⁺ sparks. Caveolae position Ca_V3.2 channels sufficiently close to RyRs for extracellular Ca²⁺ influx to trigger (~25%) Ca²⁺ sparks. In aged mice, this Ca_V3.2‐RyR pathway loses importance. Instead, a gadolinium‐sensitive Ca²⁺ influx pathway is upregulated to trigger (20%) Ca²⁺ sparks. This pathway may compromise nonselective TRP channels. RyRs, ryanodine receptors; SERCA, sarcoplasmic/endoplasmic calcium pump; SR, sarcoplasmic reticulum; VSMC, mesenteric artery vascular smooth muscle cell