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

Abstract

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.

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

“…H9c2 cardiomyoblast cells (ATCC, Cat# CRL-1446) were cultured in DMEM/F-12 (Gibco) supplemented with 10% FBS, 1% P/S (Thermo Fisher Scientific) and incubated at 37 °C in a humidified chamber containing 5% CO 2 . For experiments cells were seeded in complete medium and incubated for 48 h at 37 °C, 5% CO 2 , as previously reported [ 21 , 22 , 36 ]. AC16 human cardiomyocytes (Millipore-Sigma Aldrich, Cat.…”

“…Morphological changes were assessed by May-Grunwald Giemsa (MGG) staining, as previously reported [ 36 , 41 ]. H9c2 cells were seeded in 60 mm culture dishes, treated with vehicle (saline, NaCl 0.9%), isoproterenol (ISO) (100 µM) [ 42 ], ISO + PSELT (5 nM) or PSELT alone [ 22 ], while another set of H9c2 cells were treated with vehicle, ISO, ISO + I-PSELT (5 nM) or I-PSELT alone, and incubated in a humidified atmosphere at 37 °C for 48 h. After MGG staining, H9c2 cardiomyocytes were visualized by using Olympus BX41 microscope, and the images were taken with CSV1.14 software, using a CAM XC-30 for image acquisition. Cell-surface area was analyzed by using Image J 1.6 (NIH).…”

“…Gene silencing for SELENOT was performed in H9c2 cardiomyocytes, as previously described [ 21 , 22 ]. Briefly, cells were seeded in 60 mm cell culture dishes and incubated for 48 h at 37 °C, 5% CO 2 .…”

“…H9c2 cells treated with vehicle, ISO (100 μM), ISO + PSELT (5 nM) and PSELT alone were processed as reported previously [ 22 , 36 , 41 ]. After establishing protein concentration in the supernatant by Bradford assay, 50 μg of proteins were loaded on SDS-PAGE gel, then electroblotted onto a nitrocellulose membrane and probed with the primary specific antibody against SELENOT.…”

“…AC16 human cardiomyocytes exposed to vehicle (CTRL), isoproterenol (ISO) (100 µM), ISO + PSELT (5 nM), PSELT for 48 h, were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer overnight at 4 °C. Post-fixation proceeded in buffered osmium tetroxide, followed by dehydration in a graded acetone series, as previously described [ 22 ]. Ultrathin Sects.…”