Alternative Splicing of Ryanodine Receptors Modulates Cardiomyocyte Ca ²⁺ Signaling and Susceptibility to Apoptosis

Abstract

Abstract-Ca2ϩ release via type 2 ryanodine receptors (RyR2) regulates cardiac function. Molecular cloning of human RyR2 identified 2 alternatively spliced variants, comprising 30-and 24-bp sequence insertions; yet their role in shaping cardiomyocyte Ca 2ϩ signaling and cell phenotype is unknown. We profiled the developmental regulation and the tissue and species specificity of these variants and showed that their recombinant expression in HL-1 cardiomyocytes profoundly modulated nuclear and cytoplasmic Ca 2ϩ release. All splice variants localized to the sarcoplasmic reticulum, perinuclear Golgi apparatus, and to finger-like invaginations of the nuclear envelope (nucleoplasmic reticulum). Strikingly, the 24-bp splice insertion that was present at low levels in embryonic and adult hearts was essential for targeting RyR2 to an intranuclear Golgi apparatus and promoted the intracellular segregation of this variant. The amplitude variability of nuclear and cytoplasmic Ca 2ϩ fluxes were reduced in nonstimulated cardiomyocytes expressing both 30-and 24-bp splice variants and were associated with lower basal levels of apoptosis. Expression of RyR2 containing the 24-bp insertion also suppressed intracellular Ca 2ϩ fluxes following prolonged caffeine exposure (1 mmol/L, 16 hours) that protected cells from apoptosis. The antiapoptotic effects of this variant were linked to increased levels of Bcl-2 phosphorylation. In contrast, RyR2 containing the 30-bp insertion, which was abundant in human embryonic heart but was decreased during cardiac development, did not protect cardiomyocytes from caffeine-evoked apoptosis. Thus, we provide the first evidence that RyR2 splice variants exquisitely modulate intracellular Ca 2ϩ signaling and are key determinants of cardiomyocyte apoptotic susceptibility. (Circ Res. 2007;100:874-883.)

RNA-binding protein RBM20 represses splicing to orchestrate cardiac pre-mRNA processing

“…A 24-bp exon in RYR2 is upregulated in Rbm20-deficient rats and human cardiomyopathy patients ( Figure 6A). This exon is usually present at low levels in hearts and shifts RYR2 from the ER to the intranuclear Golgi apparatus, with profound effects on intracellular Ca 2+ signaling (27). Mutations in N-terminal domains of RYR2 have been associated with arrhythmogenic right ventricular tachycardia and sudden death (28).…”

Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the Oriental fruit fly,Bactrocera dorsalis(Hendel)

“…Alternative splicing may affect the protein sequence in two ways: (i) by deleting or inserting a sequence and creating long and short isoforms, or (ii) by substituting one segment of the amino acid sequence for another [54]. It was reported that 12 distinct splice variants had been identified in RyR isoforms cloned from mammals [55]. The expressions of RyR splice variants in mammals are regulated both developmentally and in a tissuespecific manner among RyR isoforms [55][56][57][58], and some splice variants can suppress intracellular Ca 2+ fluxes or exhibit distinct Ca 2+ -releasing profiles [10,59,60].…”

“…It was reported that 12 distinct splice variants had been identified in RyR isoforms cloned from mammals [55]. The expressions of RyR splice variants in mammals are regulated both developmentally and in a tissuespecific manner among RyR isoforms [55][56][57][58], and some splice variants can suppress intracellular Ca 2+ fluxes or exhibit distinct Ca 2+ -releasing profiles [10,59,60]. In contrast to mammals, only one RyR gene was found in insects, which showed only a 44 to 46% amino acid identity with the three mammalian RyRs [20].…”

CCDI: a new ligand that modulates mammalian type 1 ryanodine receptor (RyR1)

“…Three major isoforms (RyR1, RyR2 and RyR3) and several spliced variants of RyR are present in mammalian cells (Takeshima et al, 1989;Otsu et al, 1990;Zorzato et al, 1990;Hakamata et al, 1992;Futatsugi et al, 1995;Giannini et al, 1995;Conti et al, 1996;Leeb and Brenig, 1998;Jiang et al, 2003;George et al, 2007). Some organs contain a predominance of one RyR isoform, for example skeletal muscles contain primarily RyR1 (Takeshima et al, 1989;Zorzato et al, 1990) and the heart contains mainly RyR2 (Otsu et al, 1990).…”