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Voltage-gated Ca2+ channels formed by subunits (class D Ca2+ channels) tightly regulate neurotransmitter release from cochlear inner hair cells (IHCs) by controlling the majority of depolarisation-induced Ca2+ entry. We have recently shown that the absence of these channels can cause deafness and degeneration of outer hair cells (OHCs) and IHCs in α1D-deficient mice (α1D−/−) (Platzer et al., 2000. Cell 102, 89–97). We investigated the time-dependent patterns of degeneration during postnatal development in the α1D−/− mouse cochlea using light and electron microscopy. At postnatal day 3 (P3), electron microscopy revealed no morphological aberrations in sensory cells, in afferent as well as in efferent nerve endings. But at P7 we observed a beginning degeneration of afferent nerve fibres by electron microscopy. By P15, we found a loss of OHCs in apical turns but electron microscopy revealed no ultrastructural changes in IHCs and efferent axons as compared to C57 black control animals (C57BL). We demonstrated by serial ultrathin sectioning of 15 days old α1D−/− mice that intact efferent nerve fibres formed direct contacts with IHCs as the degeneration of afferent nerve fibres progressed. We also saw a notable degeneration of spiral ganglion cells at P15. By 8 months, nearly all spiral ganglion and sensory cells of the organ of Corti were absent. Random ultrathin sectioning gave the impression that synaptic bodies abundant in wild-type animals were absent in nearly all α1D−/− mice investigated. We conclude that besides presumably reduced synaptic bodies the absence of class D L-type Ca2+ channels does not prevent morphological development of the cochlea until P3 but may cause cochlear degeneration thereafter. The observed pattern of degeneration involves afferent nerve fibres (P7) followed by cell bodies in the spiral ganglion (P15), OHCs (P15) and IHCs (after P15). [Copyright &y& Elsevier]

The L-type calcium channel (LTCC) is one of the major ion channels that are known to be associated with the electrical remodeling of atrial fibrillation (AF). In AF, there is significant downregulation of the LTCC, but the underlying mechanism for such downregulation is not clear. We have previously reported that microRNA-499 (miR-499) is significantly upregulated in patients with permanent AF and that KCNN3, the gene that encodes the small-conductance calcium-activated potassium channel 3 (SK3), is a target of miR-499. We found that CACNB2, an important subunit of the LTCC, is also a target of miR-499. We hypothesize that miR-499 plays an important role in AF electrical remodeling by regulating the expression of CACNB2 and the LTCC. In atrial tissue from patients with permanent AF, CACNB2 was significantly downregulated by 67% ( n  = 4, p  < 0.05) compared to those from patients with no history of AF. Transfection of miR-499 mimic into HL-1 cells, a mouse hyperplastic atrial cardiac myocyte cell-line, resulted in the downregulation of CACNB2 protein expression, while that of miR-499 inhibitor upregulated CACNB2 protein expression. Binding of miR-499 to the 3' untranslated region of CACNB2 was confirmed by luciferase reporter assay and by the increased presence of CACNB2 mRNA in Argonaute pulled-down microRNA-induced silencing complexes after transfection with the miR-499 mimic. In addition, downregulation of CACNB2 resulted in the downregulation of protein levels of the pore-forming α-subunit (CACNA1C). In conclusion, upregulation of atrial miR-499 induces the downregulation of CACNB2 expression and may contribute to the electrical remodeling in AF.