Hydrocortisone cardioprotection in ischaemia/reperfusion injury involves antioxidant mechanisms.

BACKGROUND: Glucocorticoid (GR) and mineralocorticoid (MR) receptors are highly expressed in cardiac tissue, and both can be activated by corticosteroids. MR activation, in acute myocardial infarction (AMI), worsens cardiac function, and increase NHE activity contributing to the deleterious process. In contrast, effects of GR activation are not fully understood, probably because of the controversial scenario generated by using different doses or potencies of corticosteroids. AIMS: We tested the hypothesis that an acute dose of hydrocortisone (HC), a low-potency glucocorticoid, in a murine model of AMI could be cardioprotective by regulating NHE1 activity, leading to a decrease in oxidative stress. MATERIALS AND METHODS: Isolated hearts from Wistar rats were subjected to regional ischemic protocol. HC (10 nmol/L) was added to the perfusate during early reperfusion. Infarct size and oxidative stress were determined. Isolated papillary muscles from non-infarcted hearts were used to evaluate HC effect on sodium-proton exchanger 1 (NHE1) by analysing intracellular pH recovery from acute transient acidosis. RESULTS: HC treatment decreased infarct size, improved cardiac mechanics, reduced oxidative stress after AMI, while restoring the decreased level of the pro-fusion mitochondrial protein MFN-2. Co-treatment with the GR-blocker Mifepristone avoided these effects. HC reduced NHE1 activity by increasing the NHE1 pro-inhibiting Ser648 phosphorylation site and its upstream kinase AKT. HC restored the decreased AKT phosphorylation and anti-apoptotic BCL-2 protein expression detected after AMI. CONCLUSIONS: Our results provide the first evidence that acute HC treatment during early reperfusion induces cardioprotection against AMI, associated with a non-genomic HC-triggered NHE1 inhibition by AKT and antioxidant action that might involves mitochondrial dynamics improvement.

COVID-19 patients display changes in lymphocyte subsets with a higher frequency of dysfunctional CD8lo T cells associated with disease severity.

This work examines cellular immunity against SARS-CoV-2 in patients from Córdoba, Argentina, during two major waves characterized by different circulating viral variants and different social behavior. Using flow cytometry, we evaluated the main lymphocyte populations of peripheral blood from hospitalized patients with moderate and severe COVID-19 disease. Our results show disturbances in the cellular immune compartment, as previously reported in different cohorts worldwide. We observed an increased frequency of B cells and a significant decrease in the frequency of CD3+ T cells in COVID-19 patients compared to healthy donors (HD). We also found a reduction in Tregs, which was more pronounced in severe patients. During the first wave, the frequency of GZMB, CD107a, CD39, and PD-1-expressing conventional CD4+ T (T conv) cells was significantly higher in moderate and severe patients than in HD. During the second wave, only the GZMB+ T conv cells of moderate and severe patients increased significantly. In addition, these patients showed a decreased frequency in IL-2-producing T conv cells. Interestingly, we identified two subsets of circulating CD8+ T cells with low and high CD8 surface expression in both HD and COVID-19 patients. While the percentages of CD8hi and CD8lo T cells within the CD8+ population in HD are similar, a significant increase was observed in CD8lo T cell frequency in COVID-19 patients. CD8lo T cell populations from HD as well as from SARS-CoV-2 infected patients exhibited lower frequencies of the effector cytokine-producing cells, TNF, IL-2, and IFN-γ, than CD8hi T cells. Interestingly, the frequency of CD8lo T cells increased with disease severity, suggesting that this parameter could be a potential marker for disease progression. Indeed, the CD8hi/CD8lo index helped to significantly improve the patient's clinical stratification and disease outcome prediction. Our data support the addition of, at least, a CD8hi/CD8lo index into the panel of biomarkers commonly used in clinical labs, since its determination may be a useful tool with impact on the therapeutic management of the patients.

Different cytokine and chemokine profiles in hospitalized patients with COVID-19 during the first and second outbreaks from Argentina show no association with clinical comorbidities.

Background: COVID-19 severity has been linked to an increased production of inflammatory mediators called "cytokine storm". Available data is mainly restricted to the first international outbreak and reports highly variable results. This study compares demographic and clinical features of patients with COVID-19 from Córdoba, Argentina, during the first two waves of the pandemic and analyzes association between comorbidities and disease outcome with the "cytokine storm", offering added value to the field. Methods: We investigated serum concentration of thirteen soluble mediators, including cytokines and chemokines, in hospitalized patients with moderate and severe COVID-19, without previous rheumatic and autoimmune diseases, from the central region of Argentina during the first and second infection waves. Samples from healthy controls were also assayed. Clinical and biochemical parameters were collected. Results: Comparison between the two first COVID-19 waves in Argentina highlighted that patients recruited during the second wave were younger and showed less concurrent comorbidities than those from the first outbreak. We also recognized particularities in the signatures of systemic cytokines and chemokines in patients from both infection waves. We determined that concurrent pre-existing comorbidities did not have contribution to serum concentration of systemic cytokines and chemokines in COVID-19 patients. We also identified immunological and biochemical parameters associated to inflammation which can be used as prognostic markers. Thus, IL-6 concentration, C reactive protein level and platelet count allowed to discriminate between death and discharge in patients hospitalized with severe COVID-19 only during the first but not the second wave. Conclusions: Our data provide information that deepens our understanding of COVID-19 pathogenesis linking demographic features of a COVID-19 cohort with cytokines and chemokines systemic concentration, presence of comorbidities and different disease outcomes. Altogether, our findings provide information not only at local level by delineating inflammatory/anti-inflammatory response of patients but also at international level addressing the impact of comorbidities and the infection wave in the variability of cytokine and chemokine production upon SARS-CoV-2 infection.

Myocardial Impact of NHE1 Regulation by Sildenafil.

The cardiac Na+/H+ exchanger (NHE1) is a membrane glycoprotein fundamental for proper cell functioning due its multiple housekeeping tasks, including regulation of intracellular pH, Na+ concentration, and cell volume. In the heart, hyperactivation of NHE1 has been linked to the development of different pathologies. Several studies in animal models that reproduce the deleterious effects of ischemia/reperfusion injury or cardiac hypertrophy have conclusively demonstrated that NHE1 inhibition provides cardioprotection. Unfortunately, NHE1 inhibitors failed to reproduce these effects in the clinical arena. The reasons for those discrepancies are not apparent yet. However, a reasonable clue to consider would be that drugs that completely abolish the exchanger activity, including that its essential housekeeping function may not be the best therapeutic approach. Therefore, interventions tending to specifically reduce its hyperactive state without affecting its basal activity emerge as a novel potential gold standard. In this regard, a promising goal seems to be the modulation of the phosphorylation state of the cytosolic tail of the exchanger. Recent own experiments demonstrated that Sildenafil, a phosphodiesterase 5A inhibitor drug that has been widely used for the treatment of erectile dysfunction is able to decrease NHE1 phosphorylation, and hence reduce its hyperactivity. In connection, growing evidence demonstrates cardioprotective properties of Sildenafil against different cardiac pathologies, with the distinctive characteristic of directly affecting cardiac tissue without altering blood pressure. This mini-review was aimed to focus on the regulation of NHE1 activity by Sildenafil. For this purpose, experimental data reporting Sildenafil effects in different animal models of heart disease will be discussed.

PDE5 inhibition improves cardiac morphology and function in SHR by reducing NHE1 activity: Repurposing Sildenafil for the treatment of hypertensive cardiac hypertrophy.

Previously, we have shown that an increased cGMP-activated protein Kinase (PKG) activity after phosphodiesterase 5 (PDE5) inhibition by Sildenafil (SIL), leads to myocardial Na+/H+ exchanger (NHE1) inhibition preserving its basal homeostatic function. Since NHE1 is hyperactive in the hypertrophied myocardium of spontaneous hypertensive rats (SHR), while its inhibition was shown to prevent and revert this pathology, the current study was aimed to evaluate the potential antihypertrophic effect of SIL on adult SHR myocardium. We initially tested the inhibitory capability of SIL on NHE1 in isolated cardiomyocytes of SHR by comparing H+ efflux during the recovery from an acid load. After confirmed that effect, eight-month-old SHR were chronically treated for one month with SIL through drinking water. Compared to their littermate controls, SIL-treated rats presented a decreased NHE1 activity, which correlated with a reduction in its phosphorylation level assigned to activation of a PKG-p38 MAP kinase-PP2A signaling pathway. Moreover, treated animals showed a decreased oxidative stress that appears to be a consequence of a decreased mitochondrial NHE1 phosphorylation. Treated SHR showed a significant reduction in the pro-hypertrophic phosphatase calcineurin, despite slight tendency to decrease hypertrophy was detected. When SIL treatment was prolonged to three months, a significant decrease in myocardial hypertrophy and interstitial fibrosis that correlated with a lower myocardial stiffness was observed. In conclusion, the current study provides evidence concerning the ability of SIL to revert established cardiac hypertrophy in SHR, a clinically relevant animal model that resembles human essential hypertension.

[Covid-19 disease. Report of the first confirmed case in Córdoba (Argentina) and literatura review]. / Enfermedad covid-19. Reporte del primer caso confirmado en Córdoba (Argentina) y revisión de la literatura.

Introduction: COVID-19 disease is an acute viral pneumonia caused by SARS-CoV-2. Its main means of transmission is from person to person through respiratory droplets. It presents as a febrile condition with respiratory symptoms and high transmissibility rate. There is no vaccine, so the clinical suspicion and immediate isolation of the patient are essential Objective: The purpose of this work is to report the first confirmed case of COVID-19 disease in Córdoba (Argentina), highlighting telemedicine as a novel form of outpatient management. Methodology: We describe the first confirmed case of COVID-19 disease in Córdoba (Argentina), which presented in a patient without comorbidities in good clinical condition, and therefore ambulatory management using telemedicine was decided, which was satisfactory. Results: Outpatient management using telemedicine turned out to be optimal, avoiding the use of beds and the transmission of the virus within the hospital through management at home. Conclusion: Outpatient management of selected patients avoids virus transmission within the hospital and saturation of beds. Telemedicine is an effective and novel strategy for this situation.

Introducción: La enfermedad COVID-19 es una neumonía viral aguda causada por el SARS-CoV-2. Su principal medio de transmisión es de persona a persona a través de gotas respiratorias. Se presenta como un cuadro febril con síntomas respiratorios con una alta tasa de transmisibilidad. No existe vacuna, por lo que la sospecha y aislamiento inmediato del enfermo son primordiales. Objetivo: El propósito de este trabajo es informar el primer caso de enfermedad COVID-19 confirmado en Córdoba (Argentina), destacando a la telemedicina como una novedosa forma de manejo ambulatorio. Metodología: Se describe el primer caso confirmado de enfermedad COVID-19 en Córdoba (Argentina), en un paciente sin comorbilidades, con buen estado clínico, manejándose el mismo mediante telemedicina. Resultados: El manejo ambulatorio mediante telemedicina resultó ser óptimo, evitando la utilización de camas y la transmisión del virus intrahospitalaria mediante el manejo en su domicilio. Conclusión: El manejo ambulatorio de pacientes seleccionados evita la transmisión del virus dentro del hospital y la saturación de camas. La telemedicina es una estrategia efectiva y novedosa para esta situación.

Silencing of epidermal growth factor receptor reduces Na+/H+ exchanger 1 activity and hypertensive cardiac hypertrophy.

Pathological cardiac hypertrophy (PCH) can be triggered by epidermal growth factor receptor (EGFR) transactivation. Progression of PCH can be prevented by inhibition of hyperactive Na+/H+ exchanger isoform 1 (NHE1). We first aimed, to limit PCH of spontaneously hypertensive rats (SHR) by specific and localized silencing of cardiac EGFR, and second to study the connection of its activation pathway with cardiac NHE1 activity. Short hairpin RNA (shRNA) against EGFR was delivered with a lentivirus (l-shEGFR) in the cardiac left ventricle (LV) wall. Protein expression was analyzed by immunoblots, and NHE1 activity was indirectly measured in isolated papillary muscles by rate of pHi recovery from transient acidification. EGFR protein expression in the LV was reduced compared to the group injected with l-shSCR (Scrambled sequence) without changes in ErbB2 or ErbB4. Hypertrophic parameters together with cardiomyocytes cross sectional area were reduced in animals injected with l-shEGFR. Echocardiographic analysis exhibited a reduced fractional shortening in the l-shSCR group 30 days following treatment that was not observed in l-shEGFR group. l-shEGFR treated rats presented a reduced basal production of reactive oxygen species and decreased lipid peroxidation. NHE1 activity was significantly diminished in hearts with a partial EGFR silencing, without modification of its protein expression. We conclude that specifically silencing cardiac EGFR expression prevents progression of PCH through a pathway that involves a decrease in the NHE1 activity. Lentiviral vectors prove to be a valuable tool for long term expression of shRNA, bringing the possibility to extend its use in clinical area.

p38-MAP Kinase Negatively Regulates the Slow Force Response to Stretch in Rat Myocardium through the Up-Regulation of Dual Specificity Phosphatase 6 (DUSP6).

BACKGROUND/AIMS: Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na⁺/H⁺ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. METHODS: Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na⁺-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. RESULTS: The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. CONCLUSION: Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation.

p38 mitogen activated protein kinase mediates cardiac Na+/H+ exchanger inhibition induced by Sildenafil.

Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal-regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.

Epidermal Growth Factor Receptor Silencing Blunts the Slow Force Response to Myocardial Stretch.

BACKGROUND: Myocardial stretch increases force biphasically: the Frank-Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]-EGFR). METHODS AND RESULTS: Silencing capability of p-shEGFR was assessed in EGFR-GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA-EGFR-injected hearts showed ≈60% reduction of EGFR protein expression compared with shRNA-SCR-injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was ≈130% of initial rapid phase in the shRNA-SCR group, while it was blunted in shRNA-EGFR-expressing muscles. Angiotensin II (Ang II)-dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate-free medium, demonstrating an increase in shRNA-SCR-injected myocardium, an effect not observed in the silenced group. Ang II- or EGF-triggered reactive oxygen species production was significantly reduced in shRNA-EGFR-injected hearts compared with that in the shRNA-SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox-sensitive pathway, leading to p90RSK activation in shRNA-SCR-injected myocardium, an effect that was absent in the shRNA-EGFR group. CONCLUSIONS: Our results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox-sensitive kinase activation pathway, leading to SFR development.

Synthetic DAF-12 modulators with potential use in controlling the nematode life cycle.

Dafachronic acids (DAs) are 3-keto cholestenoic acids bearing a carboxylic acid moiety at the end of the steroid side chain. These compounds interact with the DAF-12 receptor, a ligand-dependent transcription factor that acts as a molecular switch mediating the choice between arrest at diapause or progression to reproductive development and adult lifespan in different nematodes. Recently, we reported that the 27-nor-Δ4-DA was able to directly activate DAF-12 in a transactivation cell-based luciferase assay and rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants. In the present paper, to investigate further the relationship between the structure of the steroid side chain and DAF-12 activity, we evaluated the in vitro and in vivo activity of Δ4-DA analogues with modified side chains using transactivation cell-based assays and daf-9(dh6) C. elegans mutants. Our results revealed that introduction of a 24,25-double bond on the cholestenoic acid side chain did not affect DAF-12 activity, whereas shortening the side chain lowered the activity. Most interestingly, the C24 alcohol 24-hydroxy-4-cholen-3-one (6) was an antagonist of the DAF-12 receptor both in vitro and in vivo.

27-Nor-Δ(4)-dafachronic acid is a synthetic ligand of Caenorhabditis elegans DAF-12 receptor.

27-Nor-Δ(4)-dafachronic acid was prepared in nine steps and 14% overall yield by two sequential 2-carbon homologations from 20ß-carboxyaldehyde-4-pregnen-3-one. Its activity was evaluated in vivo, where it rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants and restored their normal resistance to oxidative stress. 27-Nor-Δ(4)-dafachronic acid was also able to directly bind and activate DAF-12 in a transactivation cell-based luciferase reporter assay, although it was less active than the corresponding 25R-and 25S dafachronic acids. The binding mode of the 27-Nor steroid was studied by molecular dynamics using a homology model of the CeDAF-12 receptor.