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Hyperglycemia-induced degradation of HIF-1α contributes to impaired response of cardiomyocytes to hypoxia
A degradação do HIF-1α induzida pela hiperglicemia contribui para a desregulação na resposta à hipoxia pelos cardiomiócitos
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(A) HL-1 cells were exposed to CoCl<span class="elsevierStyleInf">2</span> (400 μM) for 6 h and then MGO (1 mM or 3 mM) was added in the last 30 min or 3 h. Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control); (B) HL-1 cells were exposed to CoCl<span class="elsevierStyleInf">2</span> (400 μM for 6 h) and MGO (3 mM for the last 3 h of treatment). HIF-1α was immunoprecipitated and immunoprecipitates were probed against HIF-1α and ubiquitin. 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"autores" => array:6 [ 0 => array:3 [ "nombre" => "Ana Rita" "apellidos" => "Ramalho" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 1 => array:3 [ "nombre" => "Adriana" "apellidos" => "Toscano" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 2 => array:3 [ "nombre" => "Paulo" "apellidos" => "Pereira" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 3 => array:3 [ "nombre" => "Henrique" "apellidos" => "Girão" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] ] ] 4 => array:3 [ "nombre" => "Lino" "apellidos" => "Gonçalves" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">a</span>" "identificador" => "aff0005" ] ] ] 5 => array:4 [ "nombre" => "Carla" "apellidos" => "Marques" "email" => array:1 [ 0 => "cmarques@fmed.uc.pt" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">b</span>" "identificador" => "aff0010" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] ] "afiliaciones" => array:2 [ 0 => array:3 [ "entidad" => "Coimbra Hospital and University Center - Cardiology Department, Coimbra, Portugal" "etiqueta" => "a" "identificador" => "aff0005" ] 1 => array:3 [ "entidad" => "CNC.IBILI, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal" "etiqueta" => "b" "identificador" => "aff0010" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "pt" => array:1 [ "titulo" => "A degradação do HIF-1α induzida pela hiperglicemia contribui para a desregulação na resposta à hipoxia pelos cardiomiócitos" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1162 "Ancho" => 2564 "Tamanyo" => 151710 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Cell viability under hypoxic conditions. HL-1 cells were subjected to MGO treatment in the absence (A) or presence of chemical hypoxia (B), and MTT assay was used to assess cell viability. The percentage of cytotoxicity was calculated relative to control (untreated cells). Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate. * p<0.05, significantly different from control cells and MGO 1 mM/30 min; ** p<0.01, significantly different from control cells (one-way analysis of variance with Tukey's multiple comparison test). MGO: methylglyoxal.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Introduction</span><p id="par0060" class="elsevierStylePara elsevierViewall">One of the most important mechanisms involved in the response to oxygen restriction is that regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1), a heterodimer formed of a stable nuclear subunit, HIF-1β, and a labile cytosolic subunit, HIF-1α. When exposed to low oxygen levels, cells mount a complex response that involves a myriad of mechanisms and signaling pathways that help cells to cope with low oxygen tensions. Under normoxia, HIF-1α undergoes hydroxylation of two proline residues (P402 and P564),<a class="elsevierStyleCrossRefs" href="#bib0140"><span class="elsevierStyleSup">1,2</span></a> catalyzed by specific prolyl hydroxylases. Hydroxylated HIF-1α is then recognized by von Hippel-Lindau tumor suppressor protein (pVHL), which is part of an ubiquitin ligase complex that promotes the ubiquitination and subsequent degradation of HIF-1α by the 26S proteasome,<a class="elsevierStyleCrossRefs" href="#bib0150"><span class="elsevierStyleSup">3,4</span></a> a multicatalytic proteolytic complex that recognizes and processes polyubiquitinated proteins through an ATP-dependent process.<a class="elsevierStyleCrossRefs" href="#bib0160"><span class="elsevierStyleSup">5–7</span></a> However, in hypoxic conditions, since HIF-1α is no longer hydroxylated, it accumulates and is translocated into the nucleus, where it dimerizes with the β subunit and initiates a genetic transcription program that includes synthesis of vascular endothelial growth factor (VEGF),<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">8</span></a> which promotes the formation of new blood vessels.</p><p id="par0065" class="elsevierStylePara elsevierViewall">Diabetes is one of the strongest risk factors for heart disease. Indeed, diabetic heart disease, including coronary heart disease, heart failure and diabetic cardiomyopathy, is the leading cause of death in people with diabetes. As with other cells, the cardiomyocyte response to ischemia involves increases in VEGF and angiopoietin 2 (Ang-2) levels that stimulate angiogenesis and myocardial perfusion. However, in diabetic patients, myocardial ischemia results in an increase in Ang-2 levels that is not accompanied by an increase in VEGF levels. In such conditions, Ang-2 exerts an anti-angiogenic effect, leading to programmed cell death of cardiomyocytes.<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">9</span></a> Moreover, in diabetes, an inappropriate cell response to hypoxia has been associated with increased degradation of HIF-1α.<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">10</span></a> Recent studies from our laboratory established that besides the ubiquitin-proteasome pathway (UPP), HIF-1α can also be degraded in the lysosome, through chaperone-mediated autophagy (CMA).<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">11</span></a> However, the molecular mechanisms involved in the destabilization of HIF-1α in diabetes remain elusive. Some studies suggest that methylglyoxal (MGO), a highly reactive byproduct of glycolysis that accumulates in diabetic patients and has been implicated in several complications of the disease,<a class="elsevierStyleCrossRefs" href="#bib0195"><span class="elsevierStyleSup">12,13</span></a> promotes the degradation of HIF-1α with consequent reduction of VEGF expression in hypoxic conditions.<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">2</span></a> These reactive carbonyl compounds form covalent adducts with particular lysine and arginine residues in proteins, altering their life cycle, including stability and function.<a class="elsevierStyleCrossRefs" href="#bib0205"><span class="elsevierStyleSup">14–16</span></a></p><p id="par0070" class="elsevierStylePara elsevierViewall">The main goal of this study was to elucidate the mechanisms whereby diabetes contributes to cardiac dysfunction. Specifically, we aimed to assess how MGO affects the response of cardiomyocytes to hypoxia, particularly HIF-1α levels. We also assessed the role played by UPP in MGO-induced destabilization of HIF-1α.</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Cell culture and treatments</span><p id="par0075" class="elsevierStylePara elsevierViewall">HL-1 adult murine cardiomyocytes (kindly donated by Prof. W. Claycomb, Department of Biochemistry and Molecular Biology, LSUHSC, New Orleans, LA, USA) were cultured in Claycomb Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, and 0.1 mM norepinephrine in an atmosphere of 95% O<span class="elsevierStyleInf">2</span> and 5% CO<span class="elsevierStyleInf">2</span> at 37<span class="elsevierStyleHsp" style=""></span>°C. The HL-1 cells were grown under fibronectin/gelatin, and were treated with CoCl<span class="elsevierStyleInf">2</span>, carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), and MGO (Sigma-Aldrich, St. Louis, MO, USA).</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Cell viability assay</span><p id="par0080" class="elsevierStylePara elsevierViewall">The viability of the HL-1 cells was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, after treatments, cells were incubated with MTT solution (0.5 mg/ml) for two hours at 37<span class="elsevierStyleHsp" style=""></span>°C in a cell culture incubator. Subsequently, supernatants were removed and acidified isopropanol (0.04 N HCl in isopropanol) was added to dissolve the dark blue crystals of formazan produced by metabolically active cells. Formazan was quantified by measuring the absorbance of the samples using an ELISA automated microplate reader (Biotek, Winooski, VT, USA) at 570 nm, with a reference wavelength of 620 nm.</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Western blot</span><p id="par0085" class="elsevierStylePara elsevierViewall">After the treatments, the cells were washed twice with phosphate-buffered saline (PBS) and denatured in a Laemmli buffer, sonicated and boiled at 95<span class="elsevierStyleHsp" style=""></span>°C for 5 min. The denatured proteins were separated by polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with 5% m/v of nonfat milk at room temperature and for 1 h with HIF-1α (1:500; SICGEN, Cantanhede, Portugal), β-actin (1:1000, Sigma Aldrich, St. Louis, MO, USA), GADPH (1:5000; SICGEN, Cantanhede, Portugal) and ubiquitin (1:1000, BioLegend, San Diego, CA, USA) primary antibodies at 4<span class="elsevierStyleHsp" style=""></span>°C overnight, followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. Specific protein bands were detected using an electrochemiluminescence detection system (Bio-Rad, USA). β-actin and GAPDH were used as loading control.</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Chymotrypsin-like activity</span><p id="par0090" class="elsevierStylePara elsevierViewall">The HL-1 cells were washed with PBS, lysed with 50 mM Tris, pH 7.6, supplemented with 1 mM DTT and sonicated. After centrifugation (16<span class="elsevierStyleHsp" style=""></span>000 <span class="elsevierStyleItalic">g</span> for 10 minutes at 4<span class="elsevierStyleHsp" style=""></span>°C), the protein concentration was determined and 40 μg of protein was incubated with 100 μM Suc-LLVY-MCA (Biomol-Enzo Life Sciences, Farmingdale, NY, USA) in a 96-well plate. Chymotrypsin-like activity was monitored for 1 h at 37<span class="elsevierStyleHsp" style=""></span>°C in 5 min periods (excitation wavelength 380 nm; emission wavelength 460 nm). Absorbance was measured on a Biotek Synergy HT spectrophotometer (Biotek, Winooski, VT, USA).</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Immunoprecipitation</span><p id="par0095" class="elsevierStylePara elsevierViewall">Cell lysates were prepared by solubilization in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, pH 7.6), containing protease and phosphatase inhibitors. The samples were centrifuged at 3200 rpm for 5 min, and the supernatants used for immunoprecipitation. Briefly, the supernatants were incubated with antibodies against HIF-1α. Non-specific (anti-GFP) antibodies were used for negative controls. Incubations proceeded overnight at 4<span class="elsevierStyleHsp" style=""></span>°C followed by incubation with protein G-sepharose (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 4<span class="elsevierStyleHsp" style=""></span>°C. The protein G-sepharose sediments were washed in RIPA buffer, eluted in Laemmli buffer, denatured at 95<span class="elsevierStyleHsp" style=""></span>°C for 5 min, and analyzed by western blot.</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Statistical analysis</span><p id="par0100" class="elsevierStylePara elsevierViewall">The results are expressed as mean ± standard error of the mean of at least three independent experiments. The data were analyzed using one-way analysis of variance followed by the Tukey post-hoc test, using GraphPad Prism 5.0 software (San Diego, CA, USA). Differences between means were considered significant for values of p<0.05.</p></span></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Results</span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Methylglyoxal reduces cell viability in HL-1 cells</span><p id="par0105" class="elsevierStylePara elsevierViewall">Hyperglycemia was previously shown to be involved in the loss of cell response to hypoxia. In this study, a cardiac cell line (HL-1) was used to investigate the role of MGO in the regulation of HIF-1α, a transcription factor involved in the cell response to low oxygen tension. The results presented in <a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a>A show that MGO induces a reduction in cell viability in a dose-dependent manner. We then assessed the effect of MGO in cells under hypoxia. To subject the cells to conditions that partially mimic hypoxia, HL-1 cells were incubated with CoCl<span class="elsevierStyleInf">2</span>. HL-1 cells treated with CoCl<span class="elsevierStyleInf">2</span> for 6 h in the presence of 3 mM of MGO added to the culture in the final 3 h present a significant reduction in cell viability compared to cells exposed to hypoxia in the absence of MGO (<a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a>B). Importantly, incubation with CoCl<span class="elsevierStyleInf">2</span> alone did not affect cell viability.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Methylglyoxal induces dose-dependent destabilization of HIF-1α under hypoxia in HL-1 cells</span><p id="par0110" class="elsevierStylePara elsevierViewall">Previous data from our group demonstrated that MGO induces the degradation of HIF-1α in retinal epithelial cells under hypoxia.<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">2</span></a> However, the impact of MGO on HIF-1α in cardiac cells has never been addressed. We first investigated the effects of various MGO concentrations on HIF-1α levels in HL-1 cells under hypoxic conditions. After reaching confluence, the cells were exposed to CoCl<span class="elsevierStyleInf">2</span> to induce chemical hypoxia for 6 h and MGO (1 mM or 3 mM) was added in the last 30 min or 3 h of hypoxia. After the treatments, the cells were harvested and HIF-1α levels were determined by western blotting. As expected, under normoxic conditions HIF-1α was undetectable (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A, lane 1), but after 6 h of incubation with CoCl<span class="elsevierStyleInf">2</span> the amount of HIF-1α increased dramatically (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A, lane 2). However, when cells were simultaneously incubated with CoCl<span class="elsevierStyleInf">2</span> and MGO for 3 h, MGO impaired hypoxia-dependent accumulation of HIF-1α (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A; compare the 110 kDa band in lanes 4 and 6 with lane 2), suggesting that MGO induces degradation of HIF-1α accumulated in the course of hypoxia.</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Methylglyoxal induces ubiquitination of HIF-1α in HL-1 cells</span><p id="par0115" class="elsevierStylePara elsevierViewall">Interestingly, besides the presence of a 110 kDa band, under hypoxia HIF-1α accumulated as a slower migrating smear that most likely corresponded to ubiquitinated forms (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A). Strikingly, when HL-1 cells were simultaneously incubated with CoCl<span class="elsevierStyleInf">2</span> and MGO for 30 min, the intensity of the slower migrating bands was significantly increased, suggesting that ubiquitination was being enhanced (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A). However, for longer periods of incubation with MGO, reduction of both 110 kDa and high molecular weight bands of HIF-1α was observed (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A, lane 4 and lane 6 compared with lane 3 and lane 5), strongly suggesting that HIF-1α undergoes degradation in these conditions. To address whether MGO reduces the ubiquitination of HIF-1α, the protein was immunoprecipitated and probed with anti-ubiquitin antibodies. The results obtained show that levels of ubiquitinated HIF-1α increase in cells treated with MGO (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>B, compare lane 1 with lane 2 of IP). We also assessed the effects on endogenous ubiquitin conjugates by western blot using polyclonal anti-ubiquitin antibodies (<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>B, Input). The results showed an increase in the endogenous conjugates of high molecular weight ubiquitin in cells subjected to hypoxia and in the presence of MGO.</p><p id="par0120" class="elsevierStylePara elsevierViewall">Having established that MGO promotes degradation of HIF-1α under hypoxia, we proceeded to assess the involvement of UPP in the destabilization of HIF-1α. For this, HL-1 cells subjected to hypoxia in the presence of MGO were incubated with a proteasome inhibitor (MG132), after which HIF-1α levels were assessed. In these conditions an accumulation of HIF-1α was observed (<a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>A, compare lane 3 with lane 5), suggesting that MGO induces HIF-1α degradation by the proteasome under hypoxia. Moreover, as expected, under normoxic conditions HIF-1α was undetectable. However, the results show an accumulation of HIF-1α when the cells were treated with a proteasome inhibitor in normoxia, confirming that HIF-1α is mainly degraded by the proteasome in normoxic conditions (<a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>B, compare lane 1 with lane 2).</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0125" class="elsevierStylePara elsevierViewall">We also assessed the effect of hypoxia and MGO on chymotrypsin-like activity of the 20S proteasome by degradation of the Suc-LLVY-MCA fluorogenic peptide (<a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>B). The cells were treated with chemical hypoxia for 6 h in the presence or absence of MGO added to the culture medium in the final 3 h of hypoxia. Incubation with MG132 was used as a positive control. The results presented in <a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>B show that proteasome activity decreased by about 50% in cells subjected to hypoxia. Moreover, simultaneous incubation with MGO did not have additional effects on proteasome activity.</p></span></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Discussion</span><p id="par0130" class="elsevierStylePara elsevierViewall">Diabetes is closely associated with cardiovascular morbidity and mortality, but the specific molecular basis linking diabetes with increased vulnerability to cardiovascular injury remains poorly understood. It has been shown that increased production and accumulation of MGO are hallmarks of diabetes.<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">17</span></a> One of the mechanisms whereby MGO contributes to cytotoxicity is through the modification of proteins involved in cell repair following an insult such as hypoxia,<a class="elsevierStyleCrossRefs" href="#bib0145"><span class="elsevierStyleSup">2,18</span></a> and MGO impairs protein quality control mechanisms in retinal epithelial cells.<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">18</span></a> Similarly, in the present study, we demonstrated that in a cardiac cell line MGO induces a significant decrease in cell viability.</p><p id="par0135" class="elsevierStylePara elsevierViewall">Moreover, diabetic complications are associated not only with hyperglycemia but also with hypoxia. The cellular response to hypoxia is a complex process that involves activation of HIF-1α, which eventually leads to increased transcription of genes that help cells to cope with low oxygen levels. It is well established, for various cell types and tissues, that in diabetic conditions the degradation of HIF-1α is increased in hypoxic conditions, thus compromising cells’ ability to adapt and survive.<a class="elsevierStyleCrossRefs" href="#bib0145"><span class="elsevierStyleSup">2,19–23</span></a> However, the effect of MGO on cardiac cells’ response to low oxygen tension is still unclear. In diabetes, the HIF-1α pathway is deregulated, most likely caused by hyperglycemia. For example, the influence of hyperglycemia on HIF-1α expression and activation in hypoxic conditions is well documented in vivo by the comparative study of biopsy material from chronic diabetic foot ulcers and chronic varicose ulcers.<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">24</span></a> HIF-1α has also been shown to play a role in the pathogenesis of renal interstitial fibrosis, and there appears to be a correlation between intensity of HIF-1α expression and severity of kidney disease.<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">25</span></a> Moreover, HIF-1α plays an important role in the pathogenesis of diabetic retinopathy and is implicated in disease progression, particularly in the early stages of this malady.<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">21</span></a> In the heart, experiments in rats demonstrated that hyperglycemia is associated with increased myocardial infarct size and reduced production of the HIF-1α protein.<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">19</span></a> Previous studies have also assessed the impact of hyperglycemia on HIF-1α gene transcription in hypoxic conditions and concluded that there are no changes in HIF-1α mRNA levels in this situation,<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">26</span></a> ascribing an important role to degradation in the destabilization of HIF-1α in hypoxia. However, the mechanisms underlying this dysfunctional response are far from being clarified. It has been shown that hyperglycemia impairs proteasome function through the action of MGO.<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">27</span></a> We demonstrate that both MGO and hypoxia lead to a decrease in proteasomal chymotrypsin-like activity. Moreover, it has been consistently shown that in diabetic conditions an inappropriate cell response to hypoxia is associated with increased degradation of HIF-1α<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">2</span></a> by both UPP and CMA.<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">11</span></a> Similarly, in this study we show that in cardiomyocytes proteasome inhibitors do not fully prevent MGO-induced HIF degradation, reinforcing the hypothesis that other proteolytic mechanisms account for HIF-1α degradation in cardiomyocytes incubated with MGO.</p><p id="par0140" class="elsevierStylePara elsevierViewall">The present study shows for the first time that MGO promotes the degradation of HIF-1α in cardiomyocytes in hypoxic conditions. However, because proteasome inhibitors do not fully prevent HIF-1α degradation and MGO decreases proteasomal chymotrypsin-like activity, it seems reasonable to suggest that other degradation pathways are likely involved in HIF-1α degradation induced by MGO, possibly by CMA.</p><p id="par0145" class="elsevierStylePara elsevierViewall">Although these results bring new insights into the molecular mechanisms whereby diabetes contributes to diabetic cardiomyopathy, extrapolating them to the heart needs to be confirmed with analysis of samples from animal models and from human diabetic patients.</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Conclusion</span><p id="par0150" class="elsevierStylePara elsevierViewall">Identifying the molecular mechanisms underlying cell responses to oxygen depletion and the defective pathways involved in diabetes is a valuable contribution to a better understanding of the nature of morbid diabetic complications and to the development of new therapeutic strategies directed at the underlying molecular chain of events, in order to reduce the burden of morbidity and mortality that characterizes the disease. Based on this study, we propose that hyperglycemia increases MGO concentrations, which decreases proteasomal activity and induces destabilization of HIF-1α under hypoxic conditions. This destabilization may involve degradation of HIF-1α. However, because proteasome inhibitors do not completely prevent HIF-1α degradation, CMA may be involved in MGO-induced HIF-1α degradation. These results bring new insights into the molecular mechanisms whereby diabetes contributes to diabetic cardiomyopathy.</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Ethical disclosures</span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Protection of human and animal subjects</span><p id="par0155" class="elsevierStylePara elsevierViewall">The authors declare that no experiments were performed on humans or animals for this study.</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Confidentiality of data</span><p id="par0160" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article.</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0155">Right to privacy and informed consent</span><p id="par0165" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appear in this article.</p></span></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0160">Conflicts of interest</span><p id="par0170" class="elsevierStylePara elsevierViewall">The authors have no conflicts of interest to declare.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:13 [ 0 => array:3 [ "identificador" => "xres844008" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction and Objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec839118" "titulo" => "Keywords" ] 2 => array:3 [ "identificador" => "xres844009" "titulo" => "Resumo" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Introdução e objetivos" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusão" ] ] ] 3 => array:2 [ "identificador" => "xpalclavsec839119" "titulo" => "Palavras-chave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 5 => array:3 [ "identificador" => "sec0010" "titulo" => "Methods" "secciones" => array:6 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Cell culture and treatments" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Cell viability assay" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Western blot" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Chymotrypsin-like activity" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "Immunoprecipitation" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "Statistical analysis" ] ] ] 6 => array:3 [ "identificador" => "sec0045" "titulo" => "Results" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0050" "titulo" => "Methylglyoxal reduces cell viability in HL-1 cells" ] 1 => array:2 [ "identificador" => "sec0055" "titulo" => "Methylglyoxal induces dose-dependent destabilization of HIF-1α under hypoxia in HL-1 cells" ] 2 => array:2 [ "identificador" => "sec0060" "titulo" => "Methylglyoxal induces ubiquitination of HIF-1α in HL-1 cells" ] ] ] 7 => array:2 [ "identificador" => "sec0065" "titulo" => "Discussion" ] 8 => array:2 [ "identificador" => "sec0070" "titulo" => "Conclusion" ] 9 => array:3 [ "identificador" => "sec0075" "titulo" => "Ethical disclosures" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0080" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0085" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0090" "titulo" => "Right to privacy and informed consent" ] ] ] 10 => array:2 [ "identificador" => "sec0095" "titulo" => "Conflicts of interest" ] 11 => array:2 [ "identificador" => "xack283535" "titulo" => "Acknowledgments" ] 12 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2016-06-14" "fechaAceptado" => "2016-09-12" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec839118" "palabras" => array:4 [ 0 => "Diabetic heart disease" 1 => "Hypoxia" 2 => "Hypoxia-inducible factor-1α" 3 => "Cardiomyocytes" ] ] ] "pt" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palavras-chave" "identificador" => "xpalclavsec839119" "palabras" => array:4 [ 0 => "Doenças cardiovasculares e diabetes" 1 => "Hipoxia" 2 => "HIF-1α" 3 => "Cardiomiócitos" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Introduction and Objectives</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Cardiovascular disease is the leading cause of mortality and morbidity associated with diabetes. Although impairment of the cell response to hypoxia due to destabilization of the transcription factor hypoxia-inducible factor-1α (HIF-1α), which regulates the expression of genes that help cells to cope with low oxygen tension, has been implicated in diabetes-associated disease, the molecular mechanisms involved remain elusive. It is known that hyperglycemia leads to the enhanced production of methylglyoxal (MGO). Therefore, the main objective of this study was to establish whether MGO leads to the degradation of HIF-1α in cardiomyocytes subjected to hypoxia.</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">The mouse atrial cardiomyocyte cell line, HL-1, was exposed to chemical hypoxia with CoCl<span class="elsevierStyleInf">2</span> in the absence or presence of MGO. Cell viability was assessed by MTT assay, and levels of HIF-1α and endogenous ubiquitin conjugates were determined by western blotting. Proteasome activity was analyzed using a specific chymotrypsin-like fluorogenic substrate.</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">The results obtained indicate that MGO induces time- and dose-dependent degradation of HIF-1α accumulated under hypoxia. Additionally, we show that accumulation of endogenous ubiquitin conjugates in the presence of MGO is associated with decreased proteasome activity.</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Taken together, the results obtained in this study suggest that MGO compromises the ability of cells to adapt to low oxygen tensions, by stimulating the degradation of HIF-1α, likely contributing to the development of diabetes-associated cardiac dysfunction.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction and Objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Methods" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abst0020" "titulo" => "Conclusion" ] ] ] "pt" => array:3 [ "titulo" => "Resumo" "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Introdução e objetivos</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">As complicações cardiovasculares constituem a principal causa de morbimortalidade em doentes diabéticos. A destabilização do fator de transcrição induzido pela hipoxia (HIF-1α), que regula a expressão de genes de adaptação celular a condições de baixos níveis de oxigénio, parece comprometer a resposta celular de diversos tecidos à hipoxia, em condições de hiperglicemia. No entanto, os mecanismos moleculares subjacentes à destabilização do HIF-1α estão ainda por estabelecer. Está bem estabelecido que em situações de hiperglicemia ocorre o aumento da produção de metilglioxal (MGO). Assim, o objetivo deste trabalho foi estabelecer o impacto do MGO na degradação do HIF-1α em cardiomiócitos sujeitos a condições de hipoxia.</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Métodos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">Uma linha celular de cardiomiócitos da aurícula de ratos, HL-1, foi sujeita a hipoxia química com CoCl<span class="elsevierStyleInf">2</span>, na ausência e na presença de MGO. De seguida, avaliou-se a viabilidade celular pelo ensaio de MTT, e determinaram-se os níveis de HIF-1α e de conjugados endógenos de ubiquitina por <span class="elsevierStyleItalic">western blotting</span>. A atividade do proteosoma foi avaliada utilizando um substrato fluorogénico específico do tipo quimotripsina.</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Os resultados obtidos mostram que o MGO induz degradação do HIF-1α em condições de hipoxia de uma forma dependente da dose de MGO utilizada, assim como do tempo de tratamento. Por outro lado, a acumulação de conjugados endógenos de ubiquitina na presença de MGO associa-se a um decréscimo na atividade do proteosoma.</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclusão</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">No conjunto, os resultados sugerem que o MGO compromete a capacidade de adaptação celular a condições de hipoxia através da degradação do HIF-1α, o que pode contribuir para o desenvolvimento da disfunção cardíaca associada à diabetes.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abst0025" "titulo" => "Introdução e objetivos" ] 1 => array:2 [ "identificador" => "abst0030" "titulo" => "Métodos" ] 2 => array:2 [ "identificador" => "abst0035" "titulo" => "Resultados" ] 3 => array:2 [ "identificador" => "abst0040" "titulo" => "Conclusão" ] ] ] ] "nomenclatura" => array:1 [ 0 => array:3 [ "identificador" => "nom0005" "titulo" => "<span class="elsevierStyleSectionTitle" id="sect0065">List of abbreviations</span>" "listaDefinicion" => array:1 [ 0 => array:1 [ "definicion" => array:11 [ 0 => array:2 [ "termino" => "Ang-2" "descripcion" => "<p id="par0005" class="elsevierStylePara elsevierViewall">angiopoietin 2</p>" ] 1 => array:2 [ "termino" => "CMA" "descripcion" => "<p id="par0010" class="elsevierStylePara elsevierViewall">chaperone-mediated autophagy</p>" ] 2 => array:2 [ "termino" => "CoCl<span class="elsevierStyleInf">2</span>" "descripcion" => "<p id="par0015" class="elsevierStylePara elsevierViewall">cobalt chloride</p>" ] 3 => array:2 [ "termino" => "HIF" "descripcion" => "<p id="par0020" class="elsevierStylePara elsevierViewall">hypoxia-inducible factor</p>" ] 4 => array:2 [ "termino" => "MGO" "descripcion" => "<p id="par0025" class="elsevierStylePara elsevierViewall">methylglyoxal</p>" ] 5 => array:2 [ "termino" => "MG132" "descripcion" => "<p id="par0030" class="elsevierStylePara elsevierViewall">carbobenzoxy-L-leucyl-L-leucyl-L-leucinal</p>" ] 6 => array:2 [ "termino" => "MTT" "descripcion" => "<p id="par0035" class="elsevierStylePara elsevierViewall">3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium</p>" ] 7 => array:2 [ "termino" => "PBS" "descripcion" => "<p id="par0040" class="elsevierStylePara elsevierViewall">phosphate-buffered saline</p>" ] 8 => array:2 [ "termino" => "VHL" "descripcion" => "<p id="par0045" class="elsevierStylePara elsevierViewall">Von Hippel-Lindau tumor suppressor protein</p>" ] 9 => array:2 [ "termino" => "UPP" "descripcion" => "<p id="par0050" class="elsevierStylePara elsevierViewall">ubiquitin-proteasome pathway</p>" ] 10 => array:2 [ "termino" => "VEGF" "descripcion" => "<p id="par0055" class="elsevierStylePara elsevierViewall">vascular endothelial growth factor</p>" ] ] ] ] ] ] "multimedia" => array:3 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1162 "Ancho" => 2564 "Tamanyo" => 151710 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Cell viability under hypoxic conditions. HL-1 cells were subjected to MGO treatment in the absence (A) or presence of chemical hypoxia (B), and MTT assay was used to assess cell viability. The percentage of cytotoxicity was calculated relative to control (untreated cells). Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate. * p<0.05, significantly different from control cells and MGO 1 mM/30 min; ** p<0.01, significantly different from control cells (one-way analysis of variance with Tukey's multiple comparison test). MGO: methylglyoxal.</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1535 "Ancho" => 2695 "Tamanyo" => 140685 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Effect of MGO on HIF-1α levels under hypoxia conditions. (A) HL-1 cells were exposed to CoCl<span class="elsevierStyleInf">2</span> (400 μM) for 6 h and then MGO (1 mM or 3 mM) was added in the last 30 min or 3 h. Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control); (B) HL-1 cells were exposed to CoCl<span class="elsevierStyleInf">2</span> (400 μM for 6 h) and MGO (3 mM for the last 3 h of treatment). HIF-1α was immunoprecipitated and immunoprecipitates were probed against HIF-1α and ubiquitin. MGO: methylglyoxal; WB: western blotting.</p>" ] ] 2 => array:7 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1075 "Ancho" => 2562 "Tamanyo" => 105843 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Effect of MGO on UPP activity. (A) HL-1 cells were treated with CoCl<span class="elsevierStyleInf">2</span> (400 μM; 6 h) with MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). The cell extracts were used to measure chymotrypsin-like activity using the fluorogenic substrate Suc-LLVY-AMC. The degree of change in chymotrypsin-like activity was calculated relative to control (untreated) cells. The values in the graph correspond to measurements after 30 min of activity. Data are mean ± standard error of the mean of three independent experiments performed in quadruplicate; (B) HL-1 cells were exposed to CoCl<span class="elsevierStyleInf">2</span> (400 μM; 6 h), MGO (3 mM; 3 h) and MG132 (20 μM; 4 h). Whole-cell extracts were prepared and analyzed by western blotting using anti-HIF-1α or anti-β-actin antibodies (loading control). 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| Ano/Mês | Html | Total | |
|---|---|---|---|
| 2024 Novembro | 10 | 7 | 17 |
| 2024 Outubro | 38 | 46 | 84 |
| 2024 Setembro | 47 | 28 | 75 |
| 2024 Agosto | 34 | 24 | 58 |
| 2024 Julho | 36 | 29 | 65 |
| 2024 Junho | 40 | 20 | 60 |
| 2024 Maio | 34 | 27 | 61 |
| 2024 Abril | 39 | 19 | 58 |
| 2024 Maro | 35 | 22 | 57 |
| 2024 Fevereiro | 34 | 35 | 69 |
| 2024 Janeiro | 28 | 42 | 70 |
| 2023 Dezembro | 26 | 21 | 47 |
| 2023 Novembro | 24 | 23 | 47 |
| 2023 Outubro | 30 | 18 | 48 |
| 2023 Setembro | 34 | 18 | 52 |
| 2023 Agosto | 31 | 15 | 46 |
| 2023 Julho | 26 | 10 | 36 |
| 2023 Junho | 31 | 9 | 40 |
| 2023 Maio | 49 | 29 | 78 |
| 2023 Abril | 27 | 5 | 32 |
| 2023 Maro | 45 | 20 | 65 |
| 2023 Fevereiro | 32 | 21 | 53 |
| 2023 Janeiro | 16 | 9 | 25 |
| 2022 Dezembro | 44 | 26 | 70 |
| 2022 Novembro | 56 | 18 | 74 |
| 2022 Outubro | 38 | 20 | 58 |
| 2022 Setembro | 21 | 23 | 44 |
| 2022 Agosto | 39 | 17 | 56 |
| 2022 Julho | 40 | 34 | 74 |
| 2022 Junho | 37 | 21 | 58 |
| 2022 Maio | 37 | 26 | 63 |
| 2022 Abril | 42 | 33 | 75 |
| 2022 Maro | 33 | 56 | 89 |
| 2022 Fevereiro | 33 | 45 | 78 |
| 2022 Janeiro | 32 | 35 | 67 |
| 2021 Dezembro | 20 | 21 | 41 |
| 2021 Novembro | 29 | 42 | 71 |
| 2021 Outubro | 39 | 40 | 79 |
| 2021 Setembro | 45 | 29 | 74 |
| 2021 Agosto | 40 | 32 | 72 |
| 2021 Julho | 25 | 21 | 46 |
| 2021 Junho | 41 | 32 | 73 |
| 2021 Maio | 48 | 37 | 85 |
| 2021 Abril | 59 | 55 | 114 |
| 2021 Maro | 64 | 25 | 89 |
| 2021 Fevereiro | 50 | 9 | 59 |
| 2021 Janeiro | 29 | 16 | 45 |
| 2020 Dezembro | 33 | 10 | 43 |
| 2020 Novembro | 33 | 19 | 52 |
| 2020 Outubro | 22 | 8 | 30 |
| 2020 Setembro | 46 | 20 | 66 |
| 2020 Agosto | 20 | 8 | 28 |
| 2020 Julho | 42 | 13 | 55 |
| 2020 Junho | 36 | 14 | 50 |
| 2020 Maio | 25 | 8 | 33 |
| 2020 Abril | 34 | 15 | 49 |
| 2020 Maro | 33 | 14 | 47 |
| 2020 Fevereiro | 79 | 47 | 126 |
| 2020 Janeiro | 25 | 8 | 33 |
| 2019 Dezembro | 52 | 9 | 61 |
| 2019 Novembro | 47 | 12 | 59 |
| 2019 Outubro | 48 | 4 | 52 |
| 2019 Setembro | 33 | 6 | 39 |
| 2019 Agosto | 35 | 12 | 47 |
| 2019 Julho | 38 | 9 | 47 |
| 2019 Junho | 39 | 11 | 50 |
| 2019 Maio | 45 | 9 | 54 |
| 2019 Abril | 19 | 16 | 35 |
| 2019 Maro | 67 | 9 | 76 |
| 2019 Fevereiro | 51 | 9 | 60 |
| 2019 Janeiro | 17 | 6 | 23 |
| 2018 Dezembro | 53 | 12 | 65 |
| 2018 Novembro | 45 | 12 | 57 |
| 2018 Outubro | 171 | 23 | 194 |
| 2018 Setembro | 75 | 15 | 90 |
| 2018 Agosto | 72 | 10 | 82 |
| 2018 Julho | 67 | 8 | 75 |
| 2018 Junho | 108 | 10 | 118 |
| 2018 Maio | 76 | 8 | 84 |
| 2018 Abril | 115 | 4 | 119 |
| 2018 Maro | 55 | 10 | 65 |
| 2018 Fevereiro | 27 | 9 | 36 |
| 2018 Janeiro | 33 | 11 | 44 |
| 2017 Dezembro | 60 | 11 | 71 |
| 2017 Novembro | 56 | 11 | 67 |
| 2017 Outubro | 48 | 11 | 59 |
| 2017 Setembro | 46 | 17 | 63 |
| 2017 Agosto | 58 | 20 | 78 |
| 2017 Julho | 55 | 14 | 69 |
| 2017 Junho | 71 | 39 | 110 |
| 2017 Maio | 54 | 60 | 114 |
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