Produção Nacional
Revisado por pares
Deletion of inducible nitric oxide synthase delays the onset of cardiomyocyte electrical remodeling in experimental Chagas disease
2020; Elsevier BV; Volume: 1866; Issue: 12 Linguagem: Inglês
10.1016/j.bbadis.2020.165949
ISSN1879-260X
AutoresDanilo Roman‐Campos, Policarpo A. Sales-Junior, Artur Santos‐Miranda, Julliane V. Joviano‐Santos, Catherine Ropert, Jáder Santos Cruz,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoChagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, has become a global health problem due to the massive migration flow from Latin America to other parts of the globe, including Europe [1,2].The disease is the most common infective cause of cardiomyopathy and it is observed in 11-19% of patients in Europe [3].The time course of the disease is complex and 20-30% of patients with 10-30 years after infection will develop heart failure and/or arrhythmias.Despite its importance, there is scarce information about the molecular physiopathology of the heart in CD.It is known that inducible nitric oxide synthase (iNOS) plays an important role in the macrophage-driven control of parasitic infection, through the formation of peroxynitrite [4][5][6].However, nitric oxide (NO) is an important modulator of cell machinery and its unbalanced production aggravates electrical disturbances during experimental CD [7][8][9][10].We hypothesized that activation of iNOS plays an important role on the electrical remodeling of cardiomyocytes in the acute phase of experimental CD.Thus, we evaluated if iNOS takes part in electrical remodeling of isolated cardiomyocyte in the time course of the acute phase of experimental CD.Eight-week-old male/female C57BL/6 and iNOS -/-mice were infected intraperitoneally with 50 bloodstream trypomastigote form of colombian strain of T. cruzi.Mouse Inflammation Kit from BDTM was used to detect serum cytokine levels.The animals were kindly provided by Professor Dr. Ricardo T. Gazzinelli.Left ventricular cardiomyocytes were obtained using an enzymatic dissociation method with collagenase type II, as previously described [11][12][13].Only calcium-tolerant, quiescent, rod-shaped myocytes showing clear cross striations were studied.Isolated cardiomyocytes from non-infected and infected mice were studied at 15 and 30 days post-infection (dpi).Patch-clamp recordings were obtained using an EPC-9.2 patchclamp amplifier.To measure action potentials (AP) and outward K + currents (I K ) the pipette solution had (in mM): 130 K-aspartate, 20 KCl, 10 HEPES, 2 MgCl 2 , 5 NaCl, 5 EGTA, pH set to 7.2 with KOH.We used Tyrode's as bath solution (in mM) 140 NaCl, 5.4 KCl, 1 MgCl 2 , 1.8 CaCl 2 , 10 HEPES, 10 glucose (pH set at 7.4).To record total outward I K cardiac myocytes were bathed with recording solution containing 100 μM of Cd 2+ to block L-type Ca 2+ currents.Total I K was measured by depolarization steps from -40 to 50 mV (3 s duration) from a holding potential of -80 mV every 15 s.For measurements of L type Ca 2+ current (I CaL ), recording pipettes were filled with internal solution containing (in mM): 120 CsCl, 20 TEACl, 5 NaCl, 10 HEPES, 5 EGTA, pH set to 7.2 with CsOH.I CaL was measured using 1.8 mM of Ca 2+ in extracellular solution.Membrane potential was first stepped from a holding potential of -80 mV to -40 mV for 50 ms and then stepped to different membrane voltages from -40 to 50 mV (300 ms duration).Junction potential was not corrected.The complete patch-clamp methodology is described in [12].Data are expressed as mean ± standard error (SE) and the number of samples is shown as n.Statistical significance of parametric data between multiple groups was determined by one-way or two-way ANOVA, followed by Tukey's post-test.For patch-clamp experiments cardiomyocytes were obtained from at least three animals for each measurement.Survival rate was analyzed using Kaplan-Meier plot.Comparisons were considered to be statistically significant when p < 0.05.During the acute phase of experimental CD excessive NO production contributes to the impairment of electrical properties in cardiomyocytes [12].Thus, we hypothesized that the deletion of iNOS, which is highly activated through IFN-γ activation pathway during the course of the disease, would attenuate the electrical remodeling of cardiomyocytes.Parasitemia were similar for both groups at 15 dpi.However, it was higher in iNOS -/-mice at 30 dpi (Fig. 1A).Higher parasitism was associated with increased, yet not significant, mortality rate in knockout (KO) mice (Fig. 1B) between 20 and 40 dpi, however,
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