D-cis-Diltiazem Can Produce Oxidative Stress in Healthy Depolarized Rods In Vivo

PURPOSE: New perspectives are needed to understand decades of contradictory reports on the neuroprotective effects of the Cav1.2 L-type calcium channel blocker d-cis-diltiazem in retinitis pigmentosa (RP) models. Here, we address, in vivo, the following two knowledge gaps regarding d-cis-diltiazem's actions in the murine outer retina: (1) do normal mouse rods contain d-cis-diltiazem-insensitive Cav1.2 L-type calcium channels? (2) Can d-cis-diltiazem modify the normal rod redox environment? METHODS: First, transretinal Cav1.2 L-type calcium channels were noninvasively mapped with manganese-enhanced magnetic resonance imaging (MRI) following agonist Bay K 8644 in C57BL/6 (B6) and in Cav1.2 L-type calcium channel BAY K 8644–insensitive mutant B6 mice. Second, d-cis-diltiazem–treated oxidative stress–vulnerable (B6) or –resistant [129S6 (S6)] mice were examined in vivo (QUEnch-assiSTed [QUEST] MRI) and in whole retina ex vivo (lucigenin). Retinal thickness was measured using MRI. RESULTS: The following results were observed: (1) manganese uptake patterns in BAY K 8644–treated controls and mutant mice identified in vivo Cav1.2 L-type calcium channels in inner and outer retina; and (2) d-cis-diltiazem induced rod oxidative stress in dark-adapted B6 mice but not in light-adapted B6 mice or dark-adapted S6 mice (QUEST MRI). Oxidative stress in vivo was limited to inferior outer retina in dark-adapted B6 mice approximately 1-hour post d-cis-diltiazem. By approximately 4 hours post, only superior outer retina oxidative stress was observed and whole retinal superoxide production was supernormal. All groups had unremarkable retinal thicknesses. CONCLUSIONS: D-cis-diltiazem's unexpectedly complex spatiotemporal outer retinal oxidative stress pattern in vivo was dependent on genetic background and rod membrane depolarization, but not apparently dependent on Cav1.2 L-type calcium channels, providing a potential rationale for contradictory results in different RP models.