The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

Discovery of a novel 1,3,4-oxadiazol-2-one-based NLRP3 inhibitor as a pharmacological agent to mitigate cardiac and metabolic complications in an experimental model of diet-induced metaflammation.

Inspired by the recent advancements in understanding the binding mode of sulfonylurea-based NLRP3 inhibitors to the NLRP3 sensor protein, we developed new NLRP3 inhibitors by replacing the central sulfonylurea moiety with different heterocycles. Computational studies evidenced that some of the designed compounds were able to maintain important interaction within the NACHT domain of the target protein similarly to the most active sulfonylurea-based NLRP3 inhibitors. Among the studied compounds, the 1,3,4-oxadiazol-2-one derivative 5 (INF200) showed the most promising results being able to prevent NLRP3-dependent pyroptosis triggered by LPS/ATP and LPS/MSU by 66.3 ± 6.6% and 61.6 ± 11.5% and to reduce IL-1ß release (35.5 ± 8.8% µM) at 10 µM in human macrophages. The selected compound INF200 (20 mg/kg/day) was then tested in an in vivo rat model of high-fat diet (HFD)-induced metaflammation to evaluate its beneficial cardiometabolic effects. INF200 significantly counteracted HFD-dependent "anthropometric" changes, improved glucose and lipid profiles, and attenuated systemic inflammation and biomarkers of cardiac dysfunction (particularly BNP). Hemodynamic evaluation on Langendorff model indicate that INF200 limited myocardial damage-dependent ischemia/reperfusion injury (IRI) by improving post-ischemic systolic recovery and attenuating cardiac contracture, infarct size, and LDH release, thus reversing the exacerbation of obesity-associated damage. Mechanistically, in post-ischemic hearts, IFN200 reduced IRI-dependent NLRP3 activation, inflammation, and oxidative stress. These results highlight the potential of the novel NLRP3 inhibitor, INF200, and its ability to reverse the unfavorable cardio-metabolic dysfunction associated with obesity.

Palmitate-Induced Cardiac Lipotoxicity Is Relieved by the Redox-Active Motif of SELENOT through Improving Mitochondrial Function and Regulating Metabolic State.

Cardiac lipotoxicity is an important contributor to cardiovascular complications during obesity. Given the fundamental role of the endoplasmic reticulum (ER)-resident Selenoprotein T (SELENOT) for cardiomyocyte differentiation and protection and for the regulation of glucose metabolism, we took advantage of a small peptide (PSELT), derived from the SELENOT redox-active motif, to uncover the mechanisms through which PSELT could protect cardiomyocytes against lipotoxicity. To this aim, we modeled cardiac lipotoxicity by exposing H9c2 cardiomyocytes to palmitate (PA). The results showed that PSELT counteracted PA-induced cell death, lactate dehydrogenase release, and the accumulation of intracellular lipid droplets, while an inert form of the peptide (I-PSELT) lacking selenocysteine was not active against PA-induced cardiomyocyte death. Mechanistically, PSELT counteracted PA-induced cytosolic and mitochondrial oxidative stress and rescued SELENOT expression that was downregulated by PA through FAT/CD36 (cluster of differentiation 36/fatty acid translocase), the main transporter of fatty acids in the heart. Immunofluorescence analysis indicated that PSELT also relieved the PA-dependent increase in CD36 expression, while in SELENOT-deficient cardiomyocytes, PA exacerbated cell death, which was not mitigated by exogenous PSELT. On the other hand, PSELT improved mitochondrial respiration during PA treatment and regulated mitochondrial biogenesis and dynamics, preventing the PA-provoked decrease in PGC1-α and increase in DRP-1 and OPA-1. These findings were corroborated by transmission electron microscopy (TEM), revealing that PSELT improved the cardiomyocyte and mitochondrial ultrastructures and restored the ER network. Spectroscopic characterization indicated that PSELT significantly attenuated infrared spectral-related macromolecular changes (i.e., content of lipids, proteins, nucleic acids, and carbohydrates) and also prevented the decrease in membrane fluidity induced by PA. Our findings further delineate the biological significance of SELENOT in cardiomyocytes and indicate the potential of its mimetic PSELT as a protective agent for counteracting cardiac lipotoxicity.

Quercetin and Its Derivative Counteract Palmitate-Dependent Lipotoxicity by Inhibiting Oxidative Stress and Inflammation in Cardiomyocytes.

Cardiac lipotoxicity plays an important role in the pathogenesis of obesity-related cardiovascular disease. The flavonoid quercetin (QUE), a nutraceutical compound that is abundant in the "Mediterranean diet", has been shown to be a potential therapeutic agent in cardiac and metabolic diseases. Here, we investigated the beneficial role of QUE and its derivative Q2, which demonstrates improved bioavailability and chemical stability, in cardiac lipotoxicity. To this end, H9c2 cardiomyocytes were pre-treated with QUE or Q2 and then exposed to palmitate (PA) to recapitulate the cardiac lipotoxicity occurring in obesity. Our results showed that both QUE and Q2 significantly attenuated PA-dependent cell death, although QUE was effective at a lower concentration (50 nM) when compared with Q2 (250 nM). QUE decreased the release of lactate dehydrogenase (LDH), an important indicator of cytotoxicity, and the accumulation of intracellular lipid droplets triggered by PA. On the other hand, QUE protected cardiomyocytes from PA-induced oxidative stress by counteracting the formation of malondialdehyde (MDA) and protein carbonyl groups (which are indicators of lipid peroxidation and protein oxidation, respectively) and intracellular ROS generation, and by improving the enzymatic activities of catalase and superoxide dismutase (SOD). Pre-treatment with QUE also significantly attenuated the inflammatory response induced by PA by reducing the release of key proinflammatory cytokines (IL-1ß and TNF-α). Similar to QUE, Q2 (250 nM) also significantly counteracted the PA-provoked increase in intracellular lipid droplets, LDH, and MDA, improving SOD activity and decreasing the release of IL-1ß and TNF-α. These results suggest that QUE and Q2 could be considered potential therapeutics for the treatment of the cardiac lipotoxicity that occurs in obesity and metabolic diseases.