Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning

Abstract

Life-threatening cardiomyopathy is a severe, but common, complication associated with severe trauma or sepsis. Several signaling pathways involved in apoptosis and necroptosis are linked to trauma- or sepsis-associated cardiomyopathy. However, the underling causative factors are still debatable. Heparan sulfate (HS) fragments belong to the class of danger/damage-associated molecular patterns liberated from endothelial-bound proteoglycans by heparanase during tissue injury associated with trauma or sepsis. We hypothesized that HS induces apoptosis or necroptosis in murine cardiomyocytes. By using a novel Medical-In silico approach that combines conventional cell culture experiments with machine learning algorithms, we aimed to reduce a significant part of the expensive and time-consuming cell culture experiments and data generation by using computational intelligence (refinement and replacement). Cardiomyocytes exposed to HS showed an activation of the intrinsic apoptosis signal pathway via cytochrome C and the activation of caspase 3 (both p < 0.001). Notably, the exposure of HS resulted in the induction of necroptosis by tumor necrosis factor α and receptor interaction protein 3 (p < 0.05; p < 0.01) and, hence, an increased level of necrotic cardiomyocytes. In conclusion, using this novel Medical-In silico approach, our data suggest (i) that HS induces necroptosis in cardiomyocytes by phosphorylation (activation) of receptor-interacting protein 3, (ii) that HS is a therapeutic target in trauma- or sepsis-associated cardiomyopathy, and (iii) indicate that this proof-of-concept is a first step toward simulating the extent of activated components in the pro-apoptotic pathway induced by HS with only a small data set gained from the in vitro experiments by using machine learning algorithms.

Ribonuclease 1 attenuates septic cardiomyopathy and cardiac apoptosis in a murine model of polymicrobial sepsis

“…HL-1 cells, a mouse atrial cell line, were provided by William C. Claycomb (Departments of Biochemistry and Molecular Biology and Cell Biology and Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana, USA) (41). Briefly, HL-1 cells were cultured on gelatin/fibronec-tin-coated plates and maintained in Claycomb-Medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (Biochrom), 0.1 mM norepinephrine (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 1% penicillin/streptomycin (Invitrogen) under an atmosphere of 5% CO 2 at 37°C (42)(43)(44)(45). Cardiomyocytes were exposed to 1 × 10 5 NC per milliliter of medium or to 5% septic patient serum in the presence or absence of 2.8 U/mL RNase 1 (Invitrogen) for 16 hours.…”

“…RNA extraction of cardiomyocytes. HL-1 cells were cultivated as described before and harvested by scraping (45). After 3 washing cycles with PBS the RNA was isolated using the NucleoSpin RNA Kit (Macherey-Nagel).…”

“…The amount of cleaved caspase-3 was analyzed using immunofluorescence. As described previously (45), HL-1 cells were grown on coverslips and exposed to necrotic cardiomyocytes or serum of septic patients in presence or absence of 2.8 U/mL RNase 1 for 16 hours. Afterward, cells were fixed with 4% formaldehyde (Sigma-Aldrich) for 15 minutes at room temperature.…”

“…TUNEL labeling. As described previously (45), HL-1 cells were grown in 12-well plates on coverslips coated with gelatin/fibronectin. HL-1 cells were fixed with 4% formaldehyde for 1 hour at room temperature after exposure with necrotic cells in the presence or absence of RNase 1 for 16 hours.…”

“…After washing, cells were permeabilized with 0.1% Triton X-100 for 2 minutes on ice. Next, cells were labeled using the In Situ Cell Death Detection Kit TMR red (Roche) as described (45). Coverslips were covered with 15 μL of ProLong Gold.…”