Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase

Abstract

G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and β-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and β-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in β-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction. The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore, G protein-coupled receptor kinase isoform 2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.

Guidelines for measuring cardiac physiology in mice

“…To evaluate the current reporting of echocardiographic results, we accessed PubMed on July 21, 2017, and searched for “mouse and American Journal of Physiology and (echocardiography or echocardiographic or ultrasound).” This search resulted in 437 articles, of which 52 articles were published between January 1, 2016, and present as well as 14 articles that were excluded as false positives (two kidney, one liver, one lung, one bone, one RV, two rat not mouse, one fetal not adult, and five with no cardiac echocardiography results). The remaining 38 articles were analyzed both for completeness of information provided and for quality assessment of the echocardiography values reported ( 11 , 13 , 22 , 27 , 30 , 36 , 38 , 39 , 42 , 45 , 52 , 54 , 57 , 59 , 65 , 70 , 75 , 76 , 78 , 85 , 102 , 114 , 117 – 119 , 132 , 147 , 148 , 158 , 160 , 165 , 170 , 172 , 182 , 187 , 193 , 201 , 206 ).…”

Guidelines for authors and reviewers on antibody use in physiology studies

“…The search did not include the use of antibodies for flow cytometry, ELISAs, or blocking studies. From the search results, we evaluated all articles published in 2017, for a total of 28 articles ( 2 , 8 – 11 , 13 , 15 , 16 , 19 , 21 , 22 , 26 – 28 , 30 , 32 , 34 , 41 , 42 , 44 , 45 , 47 , 48 , 50 – 52 , 54 , 58 ). Of these, 79% (22 of 28 articles) used immunoblot analysis, and 39% (11 of 28 articles) used immunohistochemistry (5 articles of the 28 evaluated used both techniques, 18%).…”

Factors affecting the pathophysiology of sepsis, an inflammatory disorder: Key roles of oxidative and nitrosative stress

“…44 Cardiomyocytes show higher expression of NOS2 with increased amounts of released NO during sepsis. 81 During S. Typhimurium peritonitis-induced sepsis, the lack of NOS2 leads to a significant drop in percent of ejection fraction and fractional shortening of heart functions, which was improved upon exogenous NO supplementation. 14 Possibly, the targeted increase in the availability of NO during the onset of sepsis has beneficial effects on microvascular dysfunction.…”