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Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis

Sepúlveda, Marisa PhD1; Gonano, Luis A. MD, PhD1; Viotti, Manuel MD1; Morell, Malena BSc1; Blanco, Paula BVetM2; López Alarcón, Micaela BSc3; Peroba Ramos, Isalira BSc3,4; Bastos Carvalho, Adriana MD3; Medei, Emiliano MD, PhD3,4; Vila Petroff, Martín PhD1

doi: 10.1097/CCM.0000000000002101
Online Laboratory Investigations
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Objectives: Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca2+ handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca2+ loss that mediate altered Ca2+ handling and contractile dysfunction associated with sepsis.

Design: Randomized controlled trial.

Setting: Research laboratory

Subjects: Male wild type and transgenic mice

Interventions: We induced sepsis in mice using the colon ascendens stent peritonitis model.

Measurements and Main 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, Ca2+ transient amplitude and sarcoplasmic reticulum Ca2+ 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, Ca2+ transient amplitude, and sarcoplasmic reticulum Ca2+ 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 Ca2+ leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca2+ content, Ca2+ 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.

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.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ccmjournal).

Supported, in part, by grant PICT 1678 from FONCYT to Dr. Vila Petroff.

Dr. Gonano disclosed off-label product use (calcium and calmodulin-dependent protein kinase II inhibition). Dr. Medei disclosed government work. The remaining authors have disclosed that they do not have any potential conflicts of interest.

Address requests for reprints to: Martín Vila Petroff, PhD, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, 60 y 120, La Plata 1900, Argentina. E-mail: mvila@ciclaplata.org.ar

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