Effects of Ca²⁺-channel blockers on photoreceptor Ca²⁺-levels and activity of calpains

Abstract

Purpose : Retinitis pigmentosa (RP) is a rare hereditary retinopathy which leads to primary rod degeneration followed by secondary cone loss. RP mutations often lead to accumulation of cGMP and over-activation of Cyclic Nucleotide Gated Channels (CNGC) in the photoreceptor outer segments. CNGC in turn mediate Ca²⁺-influx. High Ca²⁺ concentrations are believed to cause rod photoreceptor degeneration by activating Ca²⁺-dependent calpain-type proteases. An additional source of Ca²⁺ in rods is the Voltage Gated Calcium Channels (VGCC). Genetic knockout of CNGC showed improved rod viability and cone function, whereas VGCC knockout showed no improvement. Here, we further explore the role of CNGC and VGCC in photoreceptor degeneration using specific pharmacological inhibitors.

Methods : We used L-cis-diltiazem and D-cis-diltiazem, two enantiomers that are known to selectively block CNGC and VGCC, respectively. Their efficacy, at a concentration of 100 µM, was tested on organotypic retinal explant cultures derived from rd1 and wild-type (wt) animals, and cultured under controlled, serum-free conditions. This was followed by histological processing and assays for calpain activity and cell death (TUNEL). Furthermore, the effects of the compounds on Ca²⁺ dynamics in cone photoreceptors were tested using two-photon imaging.

Results : Our initial results showed 330% higher numbers of calpain activity positive cells per 1000 µm² in the rd1 ONL (1.18 ± 0.08, n=4)^, as compared to wt (0.36 ± 0.03, n=4). With L-cis-diltiazem treatment, calpain activity positive cells decreased by 30% in rd1 ONL (0.91 ± 0.07, n=3). The treatment with D-cis-diltiazem decreased calpain activity positive cells in rd1 by 34% (0.88 ± 0.07, n=2). Yet, D-cis-diltiazem increased the calpain positive cell count in wt ONL by 45% (0.66 ± 0.06, n=2). Ca²⁺-imaging showed a reduction in Ca²⁺ baseline for both D-cis-diltiazem and L-cis-diltiazem. However, D-cis-diltiazem did not affect the light induced Ca²⁺ response in cones, while L-cis-diltiazem strongly suppressed this response.

Conclusions : Our preliminary data suggests that pharmacological block of both VGCC and CNGC can decrease photoreceptor Ca²⁺ levels and the activity of calpains. The differential effect of L-cis- and D-cis diltiazem on light-induced responses indicates that only CNGC inhibition can reduce Ca²⁺-levels in photoreceptor outer segments.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.