Long-term hypoxia modulates depolarization activation of BKCa currents in fetal sheep middle cerebral arterial myocytes

Abstract

Introduction: Previous evidence indicates that gestational hypoxia disrupts cerebrovascular development, increasing the risk of intracranial hemorrhage and stroke in the newborn. Due to the role of cytosolic Ca²⁺ in regulating vascular smooth muscle (VSM) tone and fetal cerebrovascular blood flow, understanding Ca²⁺ signals can offer insight into the pathophysiological disruptions taking place in hypoxia-related perinatal cerebrovascular disease. This study aimed to determine the extent to which gestational hypoxia disrupts local Ca²⁺ sparks and whole-cell Ca²⁺ signals and coupling with BK_Ca channel activity.

Methods: Confocal imaging of cytosolic Ca²⁺ and recording BK_Ca currents of fetal sheep middle cerebral arterial (MCA) myocytes was performed. MCAs were isolated from term fetal sheep (∼140 days of gestation) from ewes held at low- (700 m) and high-altitude (3,801 m) hypoxia (LTH) for 100+ days of gestation. Arteries were depolarized with 30 mM KCl (30K), in the presence or absence of 10 μM ryanodine (Ry), to block RyR mediated Ca²⁺ release.

Results: Membrane depolarization increased Ry-sensitive Ca²⁺ spark frequency in normoxic and LTH groups along with BK_Ca activity. LTH reduced Ca²⁺ spark and whole-cell Ca²⁺ activity and induced a large leftward shift in the voltage-dependence of BK_Ca current activation. The influence of LTH on the spatial and temporal aspects of Ca²⁺ sparks and whole-cell Ca²⁺ responses varied.

Discussion: Overall, LTH attenuates Ca²⁺ signaling while increasing the coupling of Ca²⁺ sparks to BK_Ca activity; a process that potentially helps maintain oxygen delivery to the developing brain.

Keywords: Ca2+ oscillation; Ca2+ sparks; calcium; high altitude; hypoxia; ion channel; smooth muscle.

FIGURE 1

Representative image of a Fluo-4 loaded middle cerebral arterial segment from a fetal normoxic sheep recorded en face in the presence of 30 mM K⁺. Shown is the grayscale maximum intensity projection from 476 images recorded over 371 s. Scale bar is 10 microns. Recording was made with a 1.2 NA ×63 water immersion objective. Image brightness and contrast were adjusted to improve visualization of cells.

FIGURE 2

Threshold for identifying Ca²⁺ spark events in line-scan recordings impacts the sensitivity, positive predictive value, and false discovery rate. (A) Representative fluo-4 fluorescence line scan recording in a single fetal cerebral arterial myocyte. Recording was performed in a myocyte from an artery of a hypoxic fetus recorded en face under control conditions. Numbers indicate Ca²⁺ sparks identified by a trained observer whereas neighboring-colored dots indicate the Ca²⁺ sparks identified at various thresholds by Sparklab 5.8 as denoted in the adjoining legend. (B) The sensitivity of identifying a Ca²⁺ spark as a function of the positive predictive value (PPV) along with the false discovery rate (FDR) at various thresholds. Scale Bar is 5 microns. A total of 214 Ca²⁺ spark events from 19 line scan recordings from 2 hypoxic animals recorded under control conditions were made to determine the sensitivity, PPV and FDR. Line scan recording was made with a 1.2 NA ×63 water immersion objective at 529 Hz. Image brightness and contrast were adjusted to improve visualization of cells.

FIGURE 3

Long-term hypoxia reduces the activity of Ca²⁺ sparks in middle cerebral arterial myocytes of fetal sheep. (A) Percentage of line scans with detectable Ca²⁺ sparks. (B) Ca²⁺ spark firing frequency in each line scan. Ca²⁺ sparks of myocytes were recorded using line scan approaches in the absence (control) and presence of 30 mM K⁺ (30K) or 30K with 10 μM ryanodine (Ry). Bars represent mean ± 95% CI. Data were analyzed either with a chi square test (A) or with a Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparison test (B). **p < 0.01, ***p < 0.001 ****p < 0.0001. For normoxic animals, 240 line scans were performed under each recording condition. For hypoxic animals, a total of 241 control, 240 30K and 220 30K + 10 μM ryanodine line scan recordings were made.

FIGURE 4

Frequency distribution and data filtering methods for spatial and temporal aspects of Ca²⁺ sparks in middle cerebral arterial myocytes of fetal sheep. Histogram plots of Ca²⁺ spark (A) amplitude, (B) full width at half maximum (FWHM), (C) full duration at half maximum (FDHM), and (D) Tau. Values are numbers of events with responses within each bin. Gray vertical lines provide upper and lower IQR 1.5 limits. Responses were compiled across all 12 animals and recording conditions, this being a total of 16,992 events made in 1,421-line scan recordings from middle cerebral arterial segments of 6 normoxic and 6 hypoxic animals.

FIGURE 5

Spatial-temporal aspects to Ca²⁺ sparks were minimally influenced by LTH, membrane depolarization, or ryanodine. (A) amplitude, (B) full width at half-maximum, (C) full duration at half-maximum, and (D) tau exposed to control, 30K, or 30K with 10 μM ryanodine (Ry) for Ca²⁺ spark events of arterial myocytes from fetal sheep under normoxic and hypoxic conditions. Bars represent mean ± 95% CI for each parameter. Data were analyzed by a Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparison test based on ranks for each group *P < 0.05, ****P < 0.0001. Responses were examined in 1,421-line scan recordings from middle cerebral arterial segments of 6 normoxic and 6 hypoxic animals. N values for each parameter, condition, and group are provided in Table 1.

FIGURE 6

The voltage dependence of activation for spontaneous transient outward currents (STOC) in fetal middle cerebral arterial myocytes experiences a leftward shift following LTH. Raw tracings of STOC activity at various membrane potentials for myocytes isolated from MCA of (A) normoxic and (B) hypoxic fetal sheep. (C) frequency and (D) amplitude of STOC activity at various membrane potentials. Bars represent mean ± 95% CI for STOC frequency or amplitude at each membrane voltage. Data were analyzed by a two-way repeated measures ANOVA with a Bonferroni’s multiple comparison test for each group *P < 0.05, **P < 0.01, ***P < 0.001. Recordings were made in myocytes isolated from 5 normoxic, and 6 hypoxic fetuses.

FIGURE 7

Iberiotoxin and tetraethylammonium inhibit spontaneous transient outward currents in fetal hypoxic middle cerebral arterial myocytes. Raw tracings of STOC activity at a membrane potential of +10 mV in the absence and presence of (A) 100 nM IBTX or (D) 1 mM TEA in myocytes isolated from MCA of hypoxic fetal sheep. The (B,E) frequency and (C,F) amplitude of STOC activity in the absence or presence of IBTX or TEA. Bars represent mean ± 95% CI for each parameter. Data were analyzed by a paired t-test in the absence and presence of the channel blockers for each group **P < 0.01, **P < 0.001, ****P< 0.0001. Recordings were made in myocytes isolated from hypoxic fetuses in the presence and absence of IBTX (N = 5) and TEA (N = 5).

FIGURE 8

Representative spontaneous whole-cell Ca²⁺ oscillatoions in middle cerebral arterial myocytes from a fetal normoxic sheep recorded en face in the presence of 30 mM K⁺. (A) Maximum intensity projection for Fluo-4 fluorescence of recorded cells using laser scanning confocal microscopy. Red and black arrows point to regions of interest in two individual myocytes for (B) fluorescence intensity tracing showing spontaneous Ca²⁺ oscillations in the two ROIs denoted by the arrows. Scale bar is 10 microns. Recording was made with a 1.2 NA ×63 water immersion objective. Image brightness and contrast were adjusted to improve visualization of cells.

FIGURE 9

Long-term hypoxia has little impact on the percentage of middle cerebral arterial myocytes with Ca²⁺ oscillations in fetal sheep. Each dot represents the firing rate of cells with Ca²⁺ oscillations under control or with treatment of 30K in the presence or absence of 10 μM ryanodine (Ry) based on an examination of myocytes in 1,094 μm² regions of interest. Replicates were performed in 3 separate regions per recording for fetal normoxic (6 animals with 6 control, 6 30K, and 6 30K + ryanodine recordings), fetal hypoxic (6 animals with 14 control, 7 30K, and 9 30K + ryanodine recordings). ***P < 0.001, ****P< 0.0001 indicates significance based on a Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparison.

FIGURE 10

Frequency distribution and data filtering methods for spatial and temporal aspects of Ca²⁺ oscillations recorded from regions of interest in middle cerebral arterial myocytes of fetal sheep. Histogram plots of calcium oscillation (A) amplitude, (B) area under the curve (AUC), (C) rise time, (D) duration, and (E) decay. Values are numbers of oscillatory events within each bin. Gray vertical lines along the abscissa provide upper and lower IQR 1.5 limits for each examined parameter. Responses were compiled across all animals and conditions; this being obtained from 3,499 oscillatory events from recordings made in myocytes of arterial segments of 6 normoxic and 6 hypoxic animals.

FIGURE 11

Long-term hypoxia reduces Ca²⁺ oscillations in middle cerebral arterial myocytes of fetal sheep. Effects of membrane depolarization with 30K in the presence and absence of ryanodine (Ry) and long-term hypoxia on (A) amplitude of the fractional fluorescence, (B) area under the curve, (C) rise time, (D) duration, and (E) decay of the event. Bars represent mean ± 95% CI for each parameter; circles specify individual responses in each condition. *P < 0.05, ***P < 0.001, ****P < 0.0001 indicates significance based on a Kruskal–Wallis ANOVA with a Dunn’s multiple comparisons test based on ranks. The various oscillatory responses were measured in recordings made in myocytes of arterial segments of 6 normoxic and 6 hypoxic animals. Numbers of observations for each parameter, recording condition, and group are provided in Table 2.

FIGURE 12

Spatial and temporal correlations of spontaneous Ca²⁺ oscillations in middle cerebral arterial myocytes from a normoxic fetal sheep recorded en face under control conditions. (A) Maximum intensity projection for Fluo-4 fluorescence of recorded cells using laser scanning confocal microscopy. Highly correlated temporal events are colored and plotted around the center (reference) region of interest (ROI, white dot). Gray dots show ROIs of spontaneous Ca²⁺ oscillations that had less than an 80% temporal correlation with the reference (white) ROI. The large white open circle shows a 26 μm diameter that was used to determine the spatial correlation measurements depicted in Figure 12. (B) Fluorescence intensity tracing showing spontaneous Ca²⁺ oscillations. The black line is the reference tracing, and the orange lines are the other individual ROIs, with correlation coefficients greater than 80% temporal correlation plotted surrounding the reference ROI (white dot in A). Key shows the degree of correlation of each ROI relative to the reference ROI. Recording was made with a 1.2 NA ×63 water immersion objective. Image brightness and contrast were adjusted to improve visualization of cells.

FIGURE 13

Long-term hypoxia impairs the influence of membrane depolarization on the friendship and neighborliness of Ca²⁺ oscillations in MCA myocytes of fetal sheep. (A–E) Arteries were analyzed for (A) number of correlated ROIs (friends), (B) number of nearby ROIs (neighbors), (C) distance between correlated ROIs, (D) percentage of correlated ROIs that were nearby, and (E) percentage of nearby ROIs that were correlated. Show are the individual data points for each ROI (gray dots) along with the mean ± 95% CI for control, 30K, and 30K + Ry as denoted on the graph. *P < 0.05, **P < 0.01, ***P < 0.001 indicates significance based on a Kruskal–Wallis ANOVA with a Dunn’s multiple comparisons test based on ranks. Numbers of observations for each recording condition and group are provided in Table 2.