Maternal high-altitude hypoxia and suppression of ryanodine receptor-mediated Ca2+ sparks in fetal sheep pulmonary arterial myocytes

Abstract

Ca(2+) sparks are fundamental Ca(2+) signaling events arising from ryanodine receptor (RyR) activation, events that relate to contractile and dilatory events in the pulmonary vasculature. Recent studies demonstrate that long-term hypoxia (LTH) can affect pulmonary arterial reactivity in fetal, newborn, and adult animals. Because RyRs are important to pulmonary vascular reactivity and reactivity changes with ontogeny and LTH we tested the hypothesis that RyR-generated Ca(2+) signals are more active before birth and that LTH suppresses these responses. We examined these hypotheses by performing confocal imaging of myocytes in living arteries and by performing wire myography studies. Pulmonary arteries (PA) were isolated from fetal, newborn, or adult sheep that lived at low altitude or from those that were acclimatized to 3,801 m for > 100 days. Confocal imaging demonstrated preservation of the distance between the sarcoplasmic reticulum, nucleus, and plasma membrane in PA myocytes. Maturation increased global Ca(2+) waves and Ca(2+) spark activity, with sparks becoming larger, wider, and slower. LTH preferentially depressed Ca(2+) spark activity in immature pulmonary arterial myocytes, and these sparks were smaller, wider, and slower. LTH also suppressed caffeine-elicited contraction in fetal PA but augmented contraction in the newborn and adult. The influence of both ontogeny and LTH on RyR-dependent cell excitability shed new light on the therapeutic potential of these channels for the treatment of pulmonary vascular disease in newborns as well as adults.

Fig. 1.

Ontogeny and long-term hypoxia (LTH) do not affect the spatial relationships between the sarcoplasmic-endoplasmic reticulum and the plasma membrane or nucleus. A: representative micrograph and associated intensity profiles of the plasma membrane (red), sarcoplasmic-endoplasmic reticulum (green), and nucleus (blue) of myocytes in a living pulmonary artery from normoxic adult, newborns, and fetuses. Bars indicate means ± SE for the distance between the sarcoplasmic endoplasmic reticulum and the plasma membrane (B) as well as nucleus (C–D). The difference in the distances for the various compartments in myocytes of fetal and adult pulmonary arteries of sheep shown in B, C, and D were within the calculated point-spread function for the Zeiss 710. Images were made in arterial segments from 4 fetal, 3 newborn, and 4 adult normoxic sheep and from 7 fetal, 4 newborn, and 6 adult long-term hypoxic animals with a ×63 water immersion c-Apochromat objective. F/F₀: difference between peak spark fluorescence and background fluorescence. Scale bar = 5 μm.

Fig. 2.

Ca²⁺ sparks from sheep pulmonary arterial myocytes. A–F: pseudo color filtered line scan images and associated time series profiles recorded in individual myocytes for pulmonary arteries from normoxic and long-term hypoxic adults, newborns, and fetuses. Line plots show the relative fluorescent intensity profile for the line (yellow) shown in each micrograph. Calcium spark and wave recordings for Figs. 2–8 were obtained from recordings performed in a total of 12 fetal, 8 newborn, and 8 adult normoxic sheep and from 9 fetal, 9 newborn, and 7 adult long-term hypoxic sheep with a ×60 oil immersion Apochromat objective. Scale bars = 5 μm.

Fig. 3.

Ca²⁺ spark activity in pulmonary arterial myocytes and the influence of ontogeny and LTH. A, C, and E: bars are % of myocytes with sparks. B, D, and F: bars are number of sparks/100 μm/s. Ca²⁺ spark activity was determined by visual inspection of line scans. Each trial was ∼10 s. Measurements were made in 58–262 records from a minimum of 3 animals in each condition. §,†,#P < 0.05; **,§§,††,##P < 0.01; ***,§§§,†††,###P < 0.001 denotes significant difference within (*) groups based on the treatment using a Kruskal-Wallis 1-way ANOVA on ranks with a Dunn's multiple comparison test and (†) between groups based on their altitude using a Mann-Whitney U-test. The symbol § shows difference within groups and the symbol #, between groups based on a χ² test.

Fig. 4.

Ontogeny increases, while LTH reduces, Ca²⁺ spark amplitude. A–F: histograms of Ca²⁺ spark amplitudes for recordings made from fetal, newborn, and adult sheep pulmonary arterial myocytes housed in normoxic or long-term hypoxic conditions. G: bars are for fetal, newborn, and adult normoxic (open) and long-term hypoxic (solid) conditions. ***, ††† P < 0.001 denotes significant difference within (*) groups based on their age using a Kruskal-Wallis 1-way ANOVA on ranks with a Dunn's multiple comparison test and between (†) groups based on their altitude using a Mann-Whitney U-test.

Fig. 5.

Ontogeny and LTH increase the full width at half maximum (FWHM) amplitude for Ca²⁺ sparks. A–F: histograms of Ca²⁺ spark FWHM for SparkMaster analyses of recordings made from fetal, newborn, and adult sheep pulmonary arterial myocytes housed in normoxic or long-term hypoxic conditions. G: bars are for fetal, newborn, and adult normoxic (open) and long-term hypoxic (solid) conditions. *P < 0.05 and ***,†††P < 0.001 denote significant difference within (*) groups based on their age using a Kruskal-Wallis 1-way ANOVA on ranks with a Dunn's multiple comparison test and between (†) groups based on their altitude using a Mann-Whitney U-test.

Fig. 6.

Ontogeny and LTH increase the FDHM amplitude for Ca²⁺ sparks. A–F: histograms of Ca²⁺ spark FDHM for SparkMaster analyses of recordings made from fetal, newborn, and adult sheep pulmonary arterial myocytes housed in normoxic or long-term hypoxic conditions. G: bars are for fetal, newborn, and adult normoxic (open) and long-term hypoxic (solid) conditions. ***,††† P < 0.001 denotes significant difference within (*) groups based on their age using a Kruskal-Wallis 1-way ANOVA on ranks with a Dunn's multiple comparison test and between (†) groups based on their altitude using a Mann-Whitney U-test.

Fig. 7.

Ontogeny increases the time to the peak of Ca²⁺ sparks. A–F: histograms of time to the peak from Ca²⁺ spark events for SparkMaster analyses of recordings made from fetal, newborn, and adult sheep pulmonary arterial myocytes housed in normoxic or long-term hypoxic conditions. G: bars are for fetal, newborn, and adult normoxic (open) and long-term hypoxic (solid) conditions. ††P < 0.01; ***,†††P < 0.001 denotes significant difference within (*) groups based on their age using a Kruskal-Wallis 1-way ANOVA on ranks with a Dunn's multiple comparison test and between (†) groups based on their altitude using a Mann-Whitney U-test.

Fig. 8.

Effect of 30 mM K⁺ on whole-cell spatial and temporal Ca²⁺ signaling characteristics recorded in situ. A: baseline-subtracted fractional fluorescence (F/F_o) traces for Fluo-4 fluorescence are shown for individual pulmonary arterial myocytes from a normoxic adult sheep. Recordings were made in the absence (control) or presence of 30 mM K⁺ (30K) with or without 10 μM ryanodine (30K RY). Bars indicate means ± SE for normoxic (open) and long-term hypoxic (solid) conditions. B, D, and F: number of myocytes with Ca²⁺ responses each minute in a 1,000 μm² area. C, E, and G: frequency of Ca²⁺ events *,§,†P < 0.05; **,§§,††P < 0.01; or ***,§§§P < 0.001, where the symbol * denotes significant difference within groups based on treatment using a Kruskal-Wallis 1-way ANOVA on ranks with a Dunn's multiple comparison procedure, the symbol § denotes significant difference within groups based on a 1-way ANOVA and an SNK multiple comparison procedure, and the symbol † denotes between groups based on their altitude using a Mann-Whitney U-test. Images were made with a ×60 Apochromat oil immersion objective.

Fig. 9.

Caffeine causes whole cell Ca²⁺ responses in pulmonary arterial myocytes. A: representative fluorescent images at time points (a, b, c, and d) corresponding to the lower case italicized letters for the traces (B) of fractional Fluo-4 fluorescence shown for four pulmonary arterial myocyte from a LTH adult sheep. Colored arrows on images in A correspond to the placement of regions of interest for the traces of the same color in B. The 10 mM caffeine (CAF) was present during the time period denoted by the horizontal bar. C: number of myocytes in a 1,000 μm² area measured in the multicolor images for Fig. 1 (open bars) and the number of myocytes with Ca²⁺ responses in the presence (solid bars) of 10 mM caffeine. Bars indicate means ± SE. No significant differences were noted between the number of myocytes responding to caffeine in each recording vs. the total number of myocytes in the arterial wall. Caffeine-elicited calcium responses were obtained in 3 fetal, 4 newborn, and 4 adult normoxic sheep and in 4 fetal, 4 newborn, and 3 adult LTH sheep. Images were made with a ×20 nonimmersion Apochromat or a ×63 water immersion c-Apochromat objective. Images in A were made with a ×63 objective. Scale bar = 20 μm.

Fig. 10.

Caffeine causes transient contraction of pulmonary arterial rings in fetus, newborn, and adult. A–F: representative traces of arterial tension relative to %T_Kmax in response to 10 mM caffeine for arterial rings from normoxic and LTH sheep.

Fig. 11.

Contraction of pulmonary arterial rings to high K and caffeine are affected by maturation and LTH. Average tension generated by 125 mM K⁺ (A), 10 mM caffeine (B), and the normalized (C) contraction response relative to %T_Kmax in vessels from fetal, newborn, and adult sheep housed in normoxic (open) and LTH (solid) conditions. Bars indicate means ± SE. §P < 0.05; ‡‡P < 0.01; ***,†††,###,‡‡‡P < 0.001 denote significant difference (*) within and (†) between groups based on their age or altitude using a 2-way ANOVA and a Bonferroni multiple comparison procedure, (§) within groups using a 1-way ANOVA and a Newman-Keuls multiple comparison procedure, and (‡) using a Kruskal-Wallis 1-way ANOVA based on ranks and a Dunn's multiple comparison procedure, and (#) between groups using a Mann-Whitney U-test. Caffeine-induced contractile responses were examined in 4 fetal, 3 newborn, and 3 adult normoxic and in 4 fetal, 4 newborn, and 4 adult LTH sheep.