The decrease of expression of ryanodine receptor sub-type 2 is reversed by gentamycin sulphate in vascular myocytes from mdx mice.

The mdx mouse, a model of the human Duchenne muscular dystrophy, displays impaired contractile function in skeletal, cardiac and smooth muscles. We explored the possibility that ryanodine receptor (RYR) expression could be altered in vascular muscle. The three RYR sub-types were expressed in portal vein myocytes. As observed through mRNA and protein levels, RYR2 expression was strongly decreased in mdx myocytes, whereas RYR3 and RYR1 expression were unaltered. The use of antisense oligonucleotide directed against RYR sub-types indicated that caffeine-induced Ca(2+) response and Ca(2+) spark frequency depended on RYR2 and RYR1. In mdx mice, caffeine-induced Ca(2+) responses were decreased in both amplitude and maximal rate of rise, and the frequency of Ca(2+) sparks was also strongly decreased. The gentamycin treatment was able to increase both the expression of RYR2 and the caffeine-induced Ca(2+) response to the same level as that observed in wild-type mice. Taken together, these results confirm that both RYR1 and RYR2 are required for vascular Ca(2+) signalling and indicate that inhibition of RYR2 expression may account for the decreased Ca(2+) release from the SR in mdx vascular myocytes. Finally, we suggest that gentamycin can restore the Ca(2+) signalling in smooth muscle from mdx mice by increasing RYR2 and dystrophin expression. These results may help explain the reduced efficacy of contraction in vascular myocytes of mdx mice and Duchenne muscular dystrophy-afflicted patients. Gentamycin treatment could be a good therapeutic tool to restore the vascular function.

Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate.

Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation-contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through Gbeta-sensitive PI3K in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II-induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3K transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3K in Ang II-induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.

The voltage-independent B-type Ca2+ channel modulates apoptosis of cardiac myocytes.

In cardiac myocytes, Ca2+ is the second messenger of various physiological processes, including the excitation-contraction coupling phenomenon. Here, we examined how Ca2+ regulates cardiac myocyte apoptosis. Using an adult rat ventricular myocyte culture model, we found that ceramide (Cer) induces an apoptotic process that is highly sensitive to calcium: it is inhibited by the Ca2+buffer BAPTA-AM and is potentiated by the Ca2+ ionophore ionomycin. Cer-treated myocytes maintained their Ca2+ homeostasis and normal activity of their voltage-dependent sarcolemmal Ca2+ entry pathways but showed enhanced activity of B-type Ca2+ channels that were stimulated by Cer in the inside-out patch clamp configuration. Pharmacological modulations of B-type Ca2+ channels modulated Cer-induced apoptosis. Mitochondria were involved in Cer-induced apoptosis, as indicated by the inhibitory effect of cyclosporine A and caspase-9 inhibitor. Both the loss of mitochondrial membrane potential and the caspase-9 activation in Cer-treated myocytes were attenuated by blocking B-type Ca2+ channels. The mitochondrial Ca2+ content was evaluated as changes in [Ca2+]i after a short application of a mitochondrial uncoupler, FCCP, was increased in Cer-treated cells. Together, these results show that Ca2+ modulates Cer-induced cardiac myocyte apoptosis through the coupling of B-type Ca2+ channels with mitochondria.

Comparison between gentamycin and exon skipping treatments to restore ryanodine receptor subtype 2 functions in mdx mouse duodenum myocytes.

In Duchenne muscular dystrophy, a stop-codon mutation in the dystrophin gene induces an impairment of skeletal and smooth muscles contraction. In duodenum from mdx mouse, the disease model, the decrease of contractility was linked with the decrease of calcium signals encoded by ryanodine receptor subtype 2. Aminoglycoside and antisense oligonucleotide strategies were investigated to restore calcium signalling in the mdx mouse. Mdx mice were treated by intraperitoneal injection of gentamycin or 2-O-methyl antisense ribonucleotide directed against exon 23 of dystrophin for 2 weeks. The efficiency of both therapeutic strategies was determined by the level of dystrophin protein expression. The physiological effects of both treatments on ryanodine receptor expression and function were followed by RT-PCR, western blot and calcium measurements. Fourteen days after injection of gentamycin or anti-dystrophin antisense, the expression of dystrophin was recovered in skeletal muscle from treated mdx mice. In duodenum cells, RT-PCR and western blot indicated that the expression of ryanodine receptor subtype 2 was similar in treated mice than in control mice in association with the recovery of caffeine-induced Ca(2+) response. No significant difference was observed in the ryanodine subtype 3-dependent spontaneous Ca(2+) oscillations in untreated and treated mice. Conclusions - these results may help to explain the efficiency of aminoglycoside and anti-dystrophin antisense treatments in smooth muscle. Both treatments could be an interesting therapeutic option to restore smooth muscle contraction in patients with Duchenne muscular dystrophy.

Ryanodine receptors and BK channels act as a presynaptic depressor of neurotransmission in cochlear inner hair cells.

Ryanodine receptors (RyRs) are known to contribute to the regulation of free cytosolic calcium concentration. This family of intracellular calcium channels plays a significant role in calcium-induced-calcium-release (CICR), and have been implicated in calcium-dependent processes requiring exquisite spatio-temporal regulation. In order to characterize the importance of these intracellular calcium channels in cochlear physiology, we perfused the guinea pig cochlea with antagonistic concentrations of ryanodine. The distortion products of the cochlear microphonic and the compound action potential of the auditory nerve were reversibly inhibited by ryanodine (IC(50)=27.3 microm, Hill coefficient=1.9), indicating an action at the cochlear amplifier. Single auditory nerve fibre recordings showed that ryanodine slightly increased spontaneous firing rates by 22%, suggesting an excitatory effect of ryanodine. This paradoxical effect could be explained by an inhibitory action of ryanodine on presynaptic BK channels of inner hair cells (IHC). Indeed, perfusing iberiotoxin also increased the spontaneous firing activity of the auditory nerve fibres. Furthermore, whole-cell patch-clamp recordings demonstrated that ryanodine inhibits BK currents at the IHC level. Conversely, immunohistochemistry demonstrated a strong expression of RyR in IHCs and, more particularly, below the cuticular plate where membranous BK channels are highly expressed. Overall, the study demonstrated a key role for RyR and CICR in signal transduction at the IHCs. We therefore propose that coupled RyR--BK channels act to suppress the fast neurotransmission in IHCs.