Alternative splicing of ryanodine receptors modulates cardiomyocyte Ca2+ signaling and susceptibility to apoptosis.
Ca(2+) 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(2+) 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(2+) 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(2+) 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(2+) 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(2+) signaling and are key determinants of cardiomyocyte apoptotic susceptibility.
George
Christopher H
CH
Department of Cardiology, Wales Heart Research Institute, School of Medicine, Cardiff University, Heath Park, Cardiff, UK. georgech@cf.ac.uk
Rogers
Sarah A
SA
Bertrand
Benedicte M A
BM
Tunwell
Richard E A
RE
Thomas
N Lowri
NL
Steele
Derek S
DS
Cox
Eryl V
EV
Pepper
Christopher
C
Hazeel
Carolyn J
CJ
Claycomb
William C
WC
Lai
F Anthony
FA
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D002110:Caffeine / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism*; D013045:Species Specificity
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine