title: CaMKII-dependent phosphorylation of cardiac ryanodine receptors regulates cell death in cardiac ischemia/reperfusion injury.
abstract: Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Mariano N
authors.initials: MN
authors.lastname: Di Carlo
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Matilde
authors.initials: M
authors.lastname: Said
authors.affiliation: Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0636, USA.
authors.forename: Haiyun
authors.initials: H
authors.lastname: Ling
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Valverde
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Verónica C
authors.initials: VC
authors.lastname: De Giusti
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Leandro
authors.initials: L
authors.lastname: Sommese
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Julieta
authors.initials: J
authors.lastname: Palomeque
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Ernesto A
authors.initials: EA
authors.lastname: Aiello
authors.affiliation: Cardiovascular Research Institute, Department of Molecular Physiology and Biophysics, Department of Medicine (in Cardiology), Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Darlene G
authors.initials: DG
authors.lastname: Skapura
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Rinaldi
authors.affiliation: Cardiovascular Research Institute, Department of Molecular Physiology and Biophysics, Department of Medicine (in Cardiology), Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Respress
authors.affiliation: Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0636, USA.
authors.forename: Joan Heller
authors.initials: JH
authors.lastname: Brown
authors.affiliation: Cardiovascular Research Institute, Department of Molecular Physiology and Biophysics, Department of Medicine (in Cardiology), Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Xander H T
authors.initials: XH
authors.lastname: Wehrens
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina. Electronic address: msalas@med.unlp.edu.ar.
authors.forename: Margarita A
authors.initials: MA
authors.lastname: Salas
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT, La Plata, Facultad de Ciencias Médicas, 60 y 120, 1900 La Plata, Argentina. Electronic address: aliciamattiazzi@gmail.com.
authors.forename: Alicia
authors.initials: A
authors.lastname: Mattiazzi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics* / Q000378:metabolism; D016923:Cell Death; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D015428:Myocardial Reperfusion Injury / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009924:Organ Culture Techniques; D010766:Phosphorylation; D061251:Primary Cell Culture; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D019837:Ryanodine Receptor Calcium Release Channel; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: Apoptosis; CaMKII; Ischemia/reperfusion injury; Myocardium; Necrosis; RyR2 PLN