A new method to detect rapid oxygen changes around cells: How quickly do calcium channels sense oxygen in cardiomyocytes?

Abstract

Acute hypoxia is thought to trigger protective responses that, in tissues like heart and carotid body, include rapid (5-10 s) suppression of Ca(2+) and K(+) channels. To gain insight into the mechanism for the suppression of the cardiac l-type Ca(2+) channel, we measured O2-dependent fluorescence in the immediate vicinity of voltage-clamped cardiac cells subjected to rapid exchange of solutions with different O2 tensions. This was accomplished with an experimental chamber with a glass bottom that was used as a light guide for excitation of a thin ruthenium-based O2-sensitive ORMOSIL coating. Fluorescence imaging showed that steady-state Po2 was well controlled within the entire stream from an electromagnetically controlled solution "puffer" but that changes were slower at the periphery of the stream (τ1/2 ∼ 500 ms) than immediately around the voltage-clamped myocyte (τ1/2 ∼ 225 ms) where, in turn, firmly attached cells produced an additional local delay of 50-100 ms. Performing simultaneous voltage clamp and O2 measurements, we found that acute hypoxia gradually and reversibly suppressed the Ca(2+) channel (CaV1.2). Using Ba(2+) as charge carrier, the suppression was significant after 1.5 s, reached ∼10% after 2.5 s, and was nearly completely reversible in 5 s. The described fluorescence measurements provide the means to check and fine tune solution puffers and suggest that changes in Po2 can be accomplished within ∼200 ms. The rapid and reversible suppression of barium current under hypoxia is consistent with the notion that the cardiac Ca(2+) channel is directly modulated by O2.

Regulation of Ca2+ signaling by acute hypoxia and acidosis in rat neonatal cardiomyocytes

“…The rapid and reversible suppression of I Ba with hypoxia (Fig. 5A, B), as also reported for adult rat cardiomyocytes [36, 58], is consistent with the idea that L-type cardiac Ca 2+ channel can directly sense O 2 by a mechanisms somewhat independent of slower alterations of cellular constituents such as ROS, ADP or ATP. Although the nature of the rapid sensing of O 2 remains somewhat elusive, the finding that heme-oxygenase inhibitors block the suppressive effects of hypoxia on I Ca is consistent with the idea that CaM/CaMKII binding motif of C-carboxyl terminal of calcium channel may bind to heme-oxgenase, thereby allowing the channel to also sense O 2 [36].…”

A Dynamical Threshold for Cardiac Delayed Afterdepolarization-Mediated Triggered Activity

“…For experiments involving Na þ channel blockade, patchclamped myocytes were rapidly superfused with either normal Tyrode's solution or Tyrode's solution containing 20 mmol/L tetrodotoxin (TTX) (Tocris Bioscience, Bristol, UK) using a rapid solution exchange device (19) positioned near the myocyte under the control of Axopatch software (Axon Instruments).…”

Multiscale Determinants of Delayed Afterdepolarization Amplitude in Cardiac Tissue

“…To measure SR Ca content, patch-clamped myocytes were rapidly superfused with Tyrode's solution containing 10 mmol/L caffeine, 2.7 mmol/L Ca, and 0.25 mmol/L ISO using a rapid solution exchange device (20) positioned near the myocyte under the control of the software pCLAMP (Molecular Devices).…”