Long-Term Hypoxia Negatively Influences Ca2+ Signaling in Basilar Arterial Myocytes of Fetal and Adult Sheep

Abstract

Cerebral arterial vasoreactivity is vital to the regulation of cerebral blood flow. Depolarization of arterial myocytes elicits whole-cell Ca2+ oscillations as well as subcellular Ca2+ sparks due to activation of ryanodine receptors on the sarcoplasmic reticulum. Previous evidence illustrates that contraction of cerebral arteries from sheep and underlying Ca2+ signaling pathways are modified by age and that long-term hypoxia (LTH) causes aberrations in Ca2+ signaling pathways and downstream effectors impacting vasoregulation. We hypothesize that age and LTH affect the influence of membrane depolarization on whole-cell intracellular Ca2+ oscillations and sub-cellular Ca2+ spark activity in cerebral arteries. To test this hypothesis, we examined Ca2+ oscillatory and spark activities using confocal fluorescence imaging techniques of Fluo-4 loaded basilar arterial myocytes of low- and high-altitude term fetal (∼145 days of gestation) and adult sheep, where high-altitude pregnant and non-pregnant sheep were placed at 3,801 m for >100 days. Ca2+ oscillations and sparks were recorded using an in situ preparation evaluated in the absence or presence of 30 mM K+ (30K) to depolarize myocytes. Myocytes from adult animals tended to have a lower basal rate of whole-cell Ca2+ oscillatory activity and 30K increased the activity within cells. LTH decreased the ability of myocytes to respond to depolarization independent of age. These observations illustrate that both altitude and age play a role in affecting whole-cell and localized Ca2+ signaling, which are important to arterial vasoreactivity and cerebral blood flow.

Improved Workflow for Analysis of Vascular Myocyte Time-Series and Line-Scan Ca2+ Imaging Datasets

“…The watershed algorithms used in currently available open-source and commercial programs have difficulties handling cylindrical as opposed to round objects. The issues with faithfully generating ROIs that properly outline smooth muscle cells that are elongated as opposed to round underlies sampling errors associated with LCPro [7,14]. The issues with ROI detection currently lead to oversampling, with multiple ROIs being generated in individual cells.…”

“…Analysis worked toward the goal of optimizing the threshold used for Ca 2+ spark detection and generating critical cutoffs for automated analysis. We initially evaluated data mining techniques based on critical evaluation of over 20,000 Ca 2+ spark events that were published in previous studies performed in pulmonary [7,37], uterine [12], and basilar arterial smooth muscle [14]. However, that data mining approach was not incorporated into this manuscript, as the boundary limits were based on earlier versions of Sparklab, which had different background subtraction approaches that affected spatial-temporal responses.…”

“…Instead, we performed a robust evaluation of the threshold for Ca 2+ spark responses above background noise using various thresholds for spark detection including 0.5, 0.6, 0.7, 1.0, and 1.5 above background noise. Notably, our previous projects using various versions of Sparklab have used a threshold of 0.5 for the detection of Ca 2+ sparks [7,12,14,38], with visually guided extraction of false positive events. The resultant data derived from various thresholds were compared to visual guided analysis of selected line scans by a single observer with verification by a secondary observer.…”

“…Over the past number of years our imaging and analysis workflow has become far more robust. Manuscripts are now based on hundreds if not thousands of data traces and events [7,[12][13][14]. Analysis of these larger datasets is now performed using customized semi-automated analysis routines.…”