Fig 2: T-type CaV3.2 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+ (BKCa) channels to produce a negative feedback effect on vasoconstriction. L-type Cav1.2 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 CaV3.2 channels sufficiently close to RyRs for extracellular Ca2+ influx to trigger (~25%) Ca2+ sparks. In aged mice, this CaV3.2-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

Fig 3: Age attenuates the role of CaV3.2 channels in Ca2+ spark generation and decreases CaV3.2 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 CaV3.2 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

Fig 4: Proposed mechanisms of NmbR signalling on Cav3.2 channels in TG neurons. The binding of Nmb triggers NmbR to activate Gq protein and releases the Gß? subunits. The released Gß? dimer then stimulates AMPK activity, induces the activation of PKA and subsequently phosphorylates Cav3.2 channels to increase IT. Neither PLC/PKC nor direct binding of Gß? to Cav3.2 channels is required for T-type channel response mediated by NmbR.

Fig 5: Defective CaV3.2-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