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Contribution of endothelial cells to calcium-dependent fluorescence transients in rabbit hearts loaded with indo 1
B H Lorell1, C S Apstein, M J Cunningham
1Cardiovascular Institute, Boston University School of Medicine, Mass.
Insights
Studies using indo 1-AM to measure intracellular calcium in the heart show that nonmyocyte cells, particularly endothelial cells, contribute to fluorescence signals. This finding suggests that observed calcium changes during ischemia may not solely reflect myocyte activity.
Area of Science:
- Cardiovascular Physiology
- Cellular Biology
- Biomedical Imaging
Background:
- Intracellular calcium (Ca2+]i) is crucial for cardiac function.
- Indo 1-AM is a common fluorescent indicator for measuring [Ca2+]i.
- A key assumption is that indo 1 signals accurately reflect myocyte [Ca2+]i.
Purpose of the Study:
- To investigate the source of indo 1-AM fluorescence signals in the intact heart.
- To differentiate contributions from myocytes versus nonmyocytes, specifically endothelial cells.
- To assess the impact of nonmyocyte signals on interpreting [Ca2+]i measurements.
Main Methods:
- Isolated perfused rabbit hearts loaded with indo 1-AM.
- Recording left ventricular surface fluorescence and pressure.
- Stimulating cells with increased extracellular calcium ([Ca2+]o) and bradykinin.
- Comparing responses in intact hearts, isolated myocytes, and cultured endothelial cells.
Main Results:
- Fluorescence transients in intact hearts correlated with contraction, indicating myocyte signals.
- Bradykinin, an endothelial cell agonist, elevated [Ca2+]i-sensitive fluorescence without affecting myocyte contractility.
- Isolated myocytes did not respond to bradykinin, while cultured endothelial cells showed increased [Ca2+]i.
- Microscopy revealed higher indo 1 fluorescence in microvascular endothelial cells than in myocytes.
Conclusions:
- Indo 1-AM fluorescence in whole hearts is partly derived from nonmyocyte sources, including endothelial cells.
- This nonmyocyte contribution can confound the interpretation of [Ca2+]i measurements.
- Observed rapid [Ca2+]i increases during ischemia in indo 1-loaded hearts may originate from endothelial cells, not solely myocytes.
Abstract:
In studies that attempt to measure intracellular calcium [( Ca2+]i) in the intact heart with the calcium indicator indo 1-AM, a fundamental assumption is that the signals report changes in myocyte [Ca2+]i. We studied isolated perfused rabbit hearts loaded with the calcium probe indo 1-AM and recorded surface fluorescence of the left ventricle during continuous excitation at 360 nm. In cells containing indo 1, an increase in [Ca2+]i is associated with an increase in fluorescence intensity at 400 nm, a decrease in intensity at 500 nm, and an increase in the 400:500 nm ratio. Beat-to-beat fluorescence transients were recorded from the surface of the heart coincident with contraction, indicating that a component of the fluorescence signals is derived from beating myocytes. To evaluate the potential contribution of endothelial cells, we compared the response to increases in [Ca2+]o or bradykinin (10(-5) M). In response to an increase of the [Ca2+] in the perfusate from 0.6 to 3.0 mM, left ventricular developed pressure and +dP/dt increased with a simultaneous increase in the [Ca2+]i-sensitive 400:500 nm ratio. Perfusion with the endothelial cell agonist bradykinin caused no change in left ventricular isovolumic peak systolic pressure or left ventricular dP/dt, whereas bradykinin evoked an immediate elevation in both the diastolic and systolic levels of the [Ca2+]i-sensitive 400:500 nm ratio. In additional experiments with indo 1-loaded isolated beating myocytes, superfusion with bradykinin had no effect on either the fluorescence [Ca2+]i transients or contractility. In contrast, superfusion of indo 1-loaded cultured endothelial cells with bradykinin caused the elevation of [Ca2+]i within seconds. Fluorescence microscopy of unstained frozen tissue sections from indo 1-loaded hearts also suggested the presence of more intense microvascular endothelial cell indo 1 fluorescence relative to that observed in myocytes. These experiments provide evidence that a component of [Ca2+]i-sensitive fluorescence of whole hearts loaded with indo 1 is contributed by nonmyocyte sources, including endothelial cells. These results also raise the caution that the abrupt rise of [Ca2+]i that has been observed during the initial phase of ischemia in whole hearts loaded with indo 1 may be partly derived from endothelial cells rather than myocytes.