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Updated: Aug 24, 2026
Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes
Published on: September 18, 2017
Spontaneous mitochondrial depolarizations are independent of SR Ca2+ release
Catherine M O'Reilly1, Kevin E Fogarty, Robert M Drummond
1Dept. of Physiology, Univ. of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655, USA.
Abstract:
The mitochondrial membrane potential (DeltaPsi(m)) underlies many mitochondrial functions, including Ca(2+) influx into the mitochondria, which allows them to serve as buffers of intracellular Ca(2+). Spontaneous depolarizations of DeltaPsi(m), flickers, have been observed in isolated mitochondria and intact cells using the fluorescent cationic lipophile tetramethylrhodamine ethyl ester (TMRE), which distributes across the inner mitochondrial membrane in accordance with the Nernst equation. Flickers in cardiomyocytes have been attributed to uptake of Ca(2+) released from the sarcoplasmic reticulum (SR) via ryanodine receptors in focal transients called Ca(2+) sparks. We have shown previously that an increase in global Ca(2+) in smooth muscle cells causes an increase in mitochondrial Ca(2+) and depolarization of DeltaPsi(m). Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca(2+) released focally in Ca(2+) sparks. High-speed three-dimensional imaging was used to monitor DeltaPsi(m) in freshly dissociated myocytes from toad stomach that were simultaneously voltage clamped at 0 mV to ensure the cytosolic TMRE concentration was constant and equal to the low level in the bath (2.5 nM). This approach allows quantitative analysis of flickers as we have previously demonstrated. Depletion of SR Ca(2+) not only failed to eliminate flickers but rather increased their magnitude and frequency somewhat. Flickers were not altered in magnitude or frequency by ryanodine or xestospongin C, inhibitors of intracellular Ca(2+) release, or by cyclosporin A, an inhibitor of the permeability transition pore. Focal Ca(2+) release from the SR does not cause flickers in the cells employed here.
Insights
Mitochondrial membrane potential (DeltaPsi(m)) flickers in smooth muscle cells are not caused by calcium sparks from the sarcoplasmic reticulum. Depleting SR calcium increased flicker magnitude and frequency, suggesting alternative mechanisms.
Area of Science:
- Cellular Physiology
- Mitochondrial Biology
- Smooth Muscle Function
Background:
- Mitochondrial membrane potential (DeltaPsi(m)) is crucial for cellular functions, including calcium buffering.
- Spontaneous DeltaPsi(m) depolarizations, termed flickers, are observed in various cell types.
- In cardiomyocytes, flickers are linked to calcium sparks from the sarcoplasmic reticulum (SR).
Purpose of the Study:
- To investigate if SR calcium release via sparks causes DeltaPsi(m) flickers in smooth muscle cells.
- To elucidate the mechanisms underlying mitochondrial membrane potential fluctuations in smooth muscle.
Main Methods:
- Utilized high-speed 3D imaging to monitor DeltaPsi(m) in toad stomach smooth muscle cells.
- Employed voltage clamp techniques to maintain constant cytosolic tetramethylrhodamine ethyl ester (TMRE) concentration.
- Manipulated SR calcium levels and used specific inhibitors (ryanodine, xestospongin C, cyclosporin A) to assess their impact on flickers.
Main Results:
- Depletion of SR calcium did not abolish flickers; instead, it slightly increased their magnitude and frequency.
- Inhibitors of intracellular calcium release (ryanodine, xestospongin C) and the permeability transition pore (cyclosporin A) did not affect flicker characteristics.
- Findings indicate that focal calcium release from the SR does not drive DeltaPsi(m) flickers in these smooth muscle cells.
Conclusions:
- Focal calcium release from the SR is not the cause of mitochondrial membrane potential flickers in smooth muscle cells.
- Alternative mechanisms, independent of SR calcium sparks, are responsible for DeltaPsi(m) flickers in this cell type.
- Further research is needed to identify the precise triggers and pathways involved in smooth muscle mitochondrial flickers.
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