title: Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance.
abstract: Spontaneous sarcoplasmic reticulum (SR) Ca2+release events increased in fructose-rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca2+load. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2-S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD-WT mice, which was prevented by co-treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD-SR-autocamide inhibitory peptide (AIP) mice, expressing the SR-targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD-SR-AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD-WT mice but not in FRD-S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition.
  
  
  
  The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+/calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+spontaneous events in the absence of SR Ca2+load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favour Ca2+transit between the two organelles.
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs. Médicas, UNLP, La Plata, Argentina.
authors.forename: Marilen
authors.initials: M
authors.lastname: Federico
authors.affiliation: Laboratorio de Análisis de Imágenes, Facultad de Cs. Veterinarias, UNLP, La Plata, Argentina.
authors.forename: Enrique L
authors.initials: EL
authors.lastname: Portiansky
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs. Médicas, UNLP, La Plata, Argentina.
authors.forename: Leandro
authors.initials: L
authors.lastname: Sommese
authors.affiliation: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Alvarado
authors.affiliation: Servicio de Ecocardiografía, Facultad de Veterinaria, UNLP, La Plata, Argentina.
authors.forename: Paula G
authors.initials: PG
authors.lastname: Blanco
authors.affiliation: Laboratorio de Análisis de Imágenes, Facultad de Cs. Veterinarias, UNLP, La Plata, Argentina.
authors.forename: Carolina N
authors.initials: CN
authors.lastname: Zanuzzi
authors.affiliation: Department of Genome Science, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors.forename: John
authors.initials: J
authors.lastname: Dedman
authors.affiliation: Department of Genome Science, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors.forename: Marcia
authors.initials: M
authors.lastname: Kaetzel
authors.affiliation: Cardiovascular Research Institute, Departments of Molecular Physiology and Biophysics, Medicine (in Cardiology), Pediatrics; and Center for Space Medicine, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Xander H T
authors.identifier: 0000-0001-5044-672X
authors.initials: XHT
authors.lastname: Wehrens
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs. Médicas, UNLP, La Plata, Argentina.
authors.forename: Alicia
authors.initials: A
authors.lastname: Mattiazzi
authors.affiliation: Centro de Investigaciones Cardiovasculares, CCT-La Plata-CONICET, Facultad de Cs. Médicas, UNLP, La Plata, Argentina.
authors.forename: Julieta
authors.identifier: 0000-0002-4407-818X
authors.initials: J
authors.lastname: Palomeque
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001145:Arrhythmias, Cardiac / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D009202:Cardiomyopathies / Q000378:metabolism; D002460:Cell Line; D018149:Glucose Intolerance / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium
keywords: CaMKII; apoptosis; diabetes; mitochondria; sarcoplasmic reticulum