Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo

Abstract

Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca(2+) signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca(2+) signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca(2+) signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca(2+)-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca(2+) signals in ECs was reduced by ∼85% in old (24-26 mo) vs. young (3-6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/aging-impairs-endothelial-ca2-signaling/.

Keywords: aging; calcium; endothelium; internal elastic lamina; myoendothelial signaling.

Fig. 1.

Advanced age decreases spontaneous local endothelial Ca²⁺ signals in vivo. A: representative image depicting placement of regions of interest (ROIs; 1–6) over active sites on MA endothelium. Each FOV segment contained ∼15 ECs and was recorded for 10 s. Scale bar, 100 μm. B: graph depicting Ca²⁺ signal amplitudes over time from ROIs in A. This record was selected to illustrate the range of signal amplitudes within a FOV; representative traces include boxcar averaging of 3 frames. C–F: analysis of local Ca²⁺ signal properties from MAs in young and old mice. Summary data are means ± SE for active signaling sites per observed FOV (C); frequency of Ca²⁺ events per active site in each FOV (D); amplitude of Ca²⁺ signaling events (E); and full duration at half-maximal amplitude of Ca²⁺ events (FDHM; F). These data represent 88 active sites in 20 MAs of 9 young mice and 9 active sites in 14 MAs of 6 old mice. *P < 0.05, old vs. young.

Fig. 2.

Decreased IEL holes with advanced age. A and B: representative images of IEL in MAs of young (A) and old (B) mice stained with Alexa Fluor 633 hydrazide. Scale bars, 20 μm. C–E: summary data are means ± SE for average number of holes per area (mm²) of IEL (C); individual hole areas (D); and % IEL area occupied by holes (E). F: frequency histograms summarizing distribution of IEL hole areas across all MAs studied from young and old mice. Old MAs: 1,080 holes analyzed from 30 vessel segments; young MAs: 1,634 holes analyzed from 34 vessel segments. For clarity, F excludes holes > 8 μm² (26 holes for young, 20 holes for old). These data represent 8 MAs from 4 mice per age group. *P < 0.05, old vs. young.