title: Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis.
abstract: OBJECTIVE
  Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca loss that mediate altered Ca handling and contractile dysfunction associated with sepsis.
  
  
  METHODS
  Randomized controlled trial.
  
  
  METHODS
  Research laboratorySUBJECTS:: Male wild type and transgenic miceINTERVENTIONS:: We induced sepsis in mice using the colon ascendens stent peritonitis model.
  
  
  RESULTS
  Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca transient amplitude and sarcoplasmic reticulum Ca content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 µM of KN93 prevented the decrease in cell shortening, Ca transient amplitude, and sarcoplasmic reticulum Ca content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23).
  
  
  CONCLUSIONS
  Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca content, Ca transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.
authors.affiliation: 1Centro de Investigaciones Cardiovasculares, Conicet La Plata, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina.2Servicio de Cardiología, Conicet La Plata, Facultad de Veterinaria, Universidad Nacional de La Plata, La Plata, Argentina.3Laboratório de Cardiologia Celular e Molecular - IBCCF - Centro de Ciencias da Saúde, Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil.4Centro Nacional de Biologia Estrutural e Bioimagem - CENABIO-UFRJ, Rio de Janeiro, Brazil.
authors.forename: Marisa
authors.initials: M
authors.lastname: Sepúlveda
authors.forename: Luis A
authors.initials: LA
authors.lastname: Gonano
authors.forename: Manuel
authors.initials: M
authors.lastname: Viotti
authors.forename: Malena
authors.initials: M
authors.lastname: Morell
authors.forename: Paula
authors.initials: P
authors.lastname: Blanco
authors.forename: Micaela
authors.initials: M
authors.lastname: López Alarcón
authors.forename: Isalira
authors.initials: I
authors.lastname: Peroba Ramos
authors.forename: Adriana
authors.initials: A
authors.lastname: Bastos Carvalho
authors.forename: Emiliano
authors.initials: E
authors.lastname: Medei
authors.forename: Martín
authors.initials: M
authors.lastname: Vila Petroff
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009200:Myocardial Contraction / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010084:Oxidation-Reduction / Q000187:drug effects; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011897:Random Allocation; D017382:Reactive Oxygen Species / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D018805:Sepsis / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology*; D013113:Spin Labels; D013318:Stroke Volume / Q000187:drug effects / Q000235:genetics
chemical_list: C527819:AC3-I peptide; D000975:Antioxidants; D003497:Cyclic N-Oxides; D010455:Peptides; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D019837:Ryanodine Receptor Calcium Release Channel; D013113:Spin Labels; C000592748:ryanodine receptor 2. mouse; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium; C001803:tempol