Cardiovascular diseases (CVDs), many of which are influenced by exposure to environmental xenobiotics, lack physiologically relevant in vitro models for cardiotoxicity assessment. Although some pollutants have established associations with CVD, the effects of a wide range of potential toxicants remains unknown. Here, we developed a three-dimensional recellularized humanized engineered heart tissue (rHHT) platform by integrating decellularized human left ventricular extracellular matrix with human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), yielding spontaneously contracting tissues that recapitulate key features of native ventricular myocardium. We also generated a hiPSC line stably expressing the calcium indicator GCaMP6f, enabling real-time and longitudinal monitoring of calcium transients. Using ethanol and rotenone as examples, we demonstrate that the rHHT platform provides a sensitive system for evaluating cardiotoxicity and is more stringent than conventional monolayer approaches. This study presents a scalable platform for xenobiotic cardiotoxicity assessment, with potential applicability to high-throughput screening, mechanistic studies, and future personalized medicine applications.

Ibuprofen, a common over-the-counter drug, is taken worldwide to alleviate pain and reduce inflammation. While the recommended dosage is generally regarded as safe for consumption, complications arise when the drug is taken chronically, even in the prescribed quantities. Previous research suggests that ibuprofen alters the productivity of various signal transduction pathways throughout the body and stimulates the production of reactive oxygen species (ROS) in liver tissue. Our research aimed to determine if proteins involved in proteolysis and energy producing pathways were altered in cardiac tissue as a result of ibuprofen usage. The study involved eight nine-week-old female mice divided into two treatment groups: control and ibuprofen. Female control mice received pure water while female ibuprofen mice received 100 mg/kg of ibuprofen in water daily for one week before euthanization. The ibuprofen used corresponds to less than 500 mg ibuprofen/day in humans. Heart samples removed from euthanized mice were homogenized, labeled with Tandem Mass Tags (TMT), and then analyzed by liquid chromatography-tandem Mass Spectrometry (MS). Spectral analysis yielded 120 proteins that were altered in a statistically significant manner (p ≤ 0.0028 according to the Benjamini-Hochberg procedure). Pathway analysis conducted on statistically significantly altered proteins suggests moderate ibuprofen consumption results in changes in aerobic respiration, oxidative stress, the proteasome proteolytic system, the nucleotide metabolic process, and muscle cell development pathways. We conclude that ibuprofen consumption at recommended dosages significantly alters many pathways in cardiac tissue, which may be associated with the clinically observed increased risk of cardiovascular disease for those taking ibuprofen. This research is funded by the NIEHS/Superfund Research Program (P42 ES004699). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

Oxylipins are a group of bioactive fatty acid metabolites generated via enzymatic oxygenation. They are notably involved in inflammation, pain, vascular tone, hemostasis, thrombosis, immunity, and coagulation. Oxylipins have become the focus of therapeutic intervention since they are implicated in many conditions, such as nonalcoholic fatty liver disease, cardiovascular disease, and aging. The liver plays a crucial role in lipid metabolism and distribution throughout the organism. Long-term exposure to pesticides is suspected to contribute to hepatic carcinogenesis via notable disruption of lipid metabolism. Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. The amounts of prometryn documented in the environment, mainly waters, soil and plants used for human and domestic consumption are significantly high. Previous research revealed that prometryn decreased liver development during zebrafish embryogenesis. To understand the mechanisms by which prometryn could induce hepatotoxicity, the effect of prometryn (185 mg/kg every 48 h for seven days) was investigated on hepatic and plasma oxylipin levels in mice. Using an unbiased LC-MS/MS-based lipidomics approach, prometryn was found to alter oxylipins metabolites that are mainly derived from cytochrome P450 (CYP) and lipoxygenase (LOX) in both mice liver and plasma. Lipidomic analysis revealed that the hepatotoxic effects of prometryn are associated with increased epoxide hydrolase (EH) products, increased sEH and mEH enzymatic activities, and induction of oxidative stress. Furthermore, 9-HODE and 13-HODE levels were significantly increased in prometryn treated mice liver, suggesting increased levels of oxidation products. Together, these results support that sEH may be an important component of pesticide-induced liver toxicity.

Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. Significant traces of prometryn are documented in the environment, mainly in waters, soil, and plants used for human and domestic consumption. Previous studies have shown that triazine herbicides have carcinogenic potential in humans. However, there is limited information about the effects of prometryn on the cardiac system in the literature, or the mechanisms and signaling pathways underlying any potential cytotoxic effects are not known. It is important to understand the possible effects of exogenous compounds such as prometryn on the heart. To determine the mechanisms and signaling pathways affected by prometryn (185 mg/kg every 48 h for seven days), we performed proteomic profiling of male mice heart with quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. The data suggest that several major pathways, including energy metabolism, protein degradation, fatty acid metabolism, calcium signaling, and antioxidant defense system were altered in the hearts of prometryn-treated mice. Proteasome and immunoproteasome activity assays and expression levels showed proteasome dysfunction in the hearts of prometryn-treated mice. The results suggest that prometryn induced changes in mitochondrial function and various signaling pathways within the heart, particularly affecting stress-related responses.

Western blotting (immunoblotting) is a powerful and commonly used technique that is capable of detecting or semiquantifying an individual protein from complex mixtures of proteins extracted from cells or tissues. The history surrounding the origin of western blotting, the theory behind the western blotting technique, a comprehensive protocol and the uses of western blotting are presented. Lesser known and significant problems in the western blotting field and troubleshooting of common problems are highlighted and discussed. This work is a comprehensive primer and guide for new western blotting researchers and those interested in a better understanding of the technique or getting better results.

Ibuprofen is one of the most commonly used compounds in the treatment of pain, fever, and inflammation. Despite its beneficial effects, the long-term use of ibuprofen has been previously associated with increased risks for cardiovascular diseases and all-cause mortality. Relatively little is known about the signaling pathways or mechanism(s) involved in ibuprofen’s ability to increase the risk of cardiovascular diseases. Very little is also known about sex-related differences with respect to ibuprofen. We investigated the effects of ibuprofen (100 mg/kg/day, equivalent to about a 500mg tablet/day for humans) on mouse hearts using proteomics. Proteomic results suggest that female mice hearts exposed to ibuprofen for 7 days had tenfold more changes than male mice hearts exposed to ibuprofen for 7 days. Both the expression and activity of β5i immunoproteasome was significantly increased in female mice heart relative to ibuprofen-treated male heart mice. Ibuprofen treatment altered mitochondrial function in both male and female mice. Ibuprofen also induced ROS generation and decreased mitochondrial membrane potential in both male and female mice. However, ibuprofen attenuated mitochondrial complex I activity only in female hearts. Several other signaling pathways were also altered only in female mouse hearts. This study found major ibuprofen-mediated selective effects on male and female mice hearts, with specific sex differences in mitochondrial and proteasome function in mice hearts. NIEHS/Superfund P42 ES004699 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cancer-associated cachexia (CAC) is a critical contributor to pancreatic ductal adenocarcinoma (PDAC) mortality. Thus, there is an urgent need for new strategies to mitigate PDAC-associated cachexia; and the exploration of dietary interventions is a critical component. We previously observed that a ketogenic diet (KD) combined with gemcitabine enhances overall survival in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. In this study, we investigated the effect and cellular mechanisms of a KD in combination with gemcitabine on the maintenance of skeletal muscle mass in KPC mice. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD), a KD, a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. We observed that a KD or a KG-mitigated muscle strength declined over time and presented higher gastrocnemius weights compared CD-fed mice. Mechanistically, we observed sex-dependent effects of KG treatment, including the inhibition of autophagy, and increased phosphorylation levels of eIF2α in KG-treated KPC mice when compared to CG-treated mice. Our data suggest that a KG results in preservation of skeletal muscle mass. Additional research is warranted to explore whether this diet-treatment combination can be clinically effective in combating CAC in PDAC patients.

Acetaminophen (APAP) is a common over-the-counter medication used to treat pain and fever. It can be taken orally, topically, or intravenously and is considered a safe and effective drug when taken at therapeutic doses. However, recent research suggests that taking APAP regularly can result in increased blood pressure and cardiovascular risk. Previous publications found that Non-Steroidal-Anti-Inflammatory-Drugs (NSAIDs) increased Reactive Oxygen Species (ROS) in cardiomyocytes, resulting in cardiac dysfunction. Although APAP is not a NSAID, APAP likely has similar effects. We hypothesized that regular APAP usage resulted in increased ROS levels, leading to potential dysfunction in hearts and other organs. We used the embryonic rat heart cell line H9C2 to test our hypothesis. We conducted a ROS Assay using H9C2 cells treated with dichlorodihydrofluorescein diacetate (DFCDA). The cells were then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM hydrogen peroxide for 1.5 hours before measuring the ROS levels of the cells. We also measured the cell viability of H9C2 cells. Cells were treated with 10μM Alamar Blue, then treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 100μM or 200μM hydrogen peroxide for 48 hours before detecting the percentage of viable cells. The mitochondrial membrane potential (MMP) of cells treated with APAP was also measured. Cells were treated with vehicle, various concentrations of APAP (25μM, 50μM, 100μM, and 200μM), or 250μM p-triflouromethoxyphenylhydrazone (FCCP) for 24 hours before being incubated with JC-10 for one hour. The cells were measured using a fluorescent reader at excitation/emission 490/525 nm and 540/590 nm. For the ROS assay, the cells treated with 100μM APAP, 200μM APAP, and 100μM Hydrogen Peroxide significantly increased total ROS compared to the control. The cell viability assay resulted in a significant decrease in percent viability in cells treated with 200μM APAP, 100μM hydrogen peroxide, and 200μM hydrogen peroxide compared to the control. The MMP Assay resulted in a significant decrease in MMP at all concentrations of APAP and 250μM FCCP compared to the control. These results suggest that treatment of H9C2 cardiac cells with physiological concentrations of APAP causes an increase in intracellular ROS levels, decreased cell viability, and decreased MMP. Overall, APAP causes mitochondrial dysfunction, which is likely to cause cardiomyocyte dysfunction. This research is funded by the NIEHS/Superfund Research Program (P42 ES004699). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The tumor suppressor p53 is thought to play a key role in the maintenance of cell size and homeostasis, but relatively little is known about its role in skeletal muscle. Based on its ability to suppress cell growth, we hypothesized that inhibiting the function of wild-type p53 through the overexpression of a dominant-negative p53 mutant (DDp53) could result in muscle fiber hypertrophy. To test this hypothesis, we electroporated adult rat tibialis anterior muscles with DDp53 and collected the tissue three weeks later. We confirmed successful overexpression of DDp53 on a histological and biochemical level and found pronounced changes to muscle architecture, metabolism, and molecular signaling. Muscle mass, fiber cross-sectional area, and fiber diameter significantly decreased with DDp53 overexpression. We found histopathological changes in DDp53 transfected muscle which were accompanied by increased levels of proteins that are associated with membrane damage and repair. In addition, DDp53 decreased oxidative phosphorylation complex I and V protein levels, and despite its negative effects on muscle mass and fiber size, caused an increase in muscle protein synthesis as assessed via the SUnSET technique. Interestingly, the increase in muscle protein synthesis was concomitant with a decrease in phospho-S6K1 (Thr389). Furthermore, the muscle wasting in the DDp53 electroporated leg was accompanied by a decrease in global protein ubiquitination and an increase in proteasome activity. In conclusion, overexpression of a dominant-negative p53 mutant in skeletal muscle results in decreased muscle mass, myofiber size, histological muscle damage, a metabolic phenotype, and perturbed homeostasis between muscle protein synthesis and degradation.

Pesticides are important chemicals or biological agents that deter or kill pests. The use of pesticides has continued to increase as it is still considered the most effective method to reduce pests and increase crop growth. However, pesticides have other consequences, including potential toxicity to humans and wildlife. Pesticides have been associated with increased risk of cardiovascular disease, cancer, and birth defects. Labels on pesticides also suggest limiting exposure to these hazardous chemicals. Based on experimental evidence, various types of pesticides all seem to have a common effect, the induction of oxidative stress in different cell types and animal models. Pesticide-induced oxidative stress is caused by both reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are associated with several diseases including cancer, inflammation, and cardiovascular and neurodegenerative diseases. ROS and RNS can activate at least five independent signaling pathways including mitochondrial-induced apoptosis. Limited in vitro studies also suggest that exogenous antioxidants can reduce or prevent the deleterious effects of pesticides.

Western Blotting, which is probably better referred to as immunoblotting, is one of the most commonly used biological methods worldwide. This technique is capable of detecting an individual protein from a complex mixture of proteins extracted from cells or tissues. The major steps in the Western Blotting workflow are 1) The separation of proteins based on size, 2) The transfer of separated protein to a suitable stable support, 3) Interaction between the target protein and appropriate primary antibodies. In many cases, a secondary antibody that interacts with the primary antibody is used, and 4) Visualization of the target protein using enhanced chemiluminescence (ECL), fluorescence, or colorimetric methods. A detailed protocol to help users get the most out of their Western Blots is presented. Goal: To help Western Blotting users learn what prevents them from having perfect Western Blots The most common result of your experiment: A good Western blot. Another possible result: No signal detected or weak signal Most likely reasons: 1. Antibody was not suitable (poor quality antibody). 2. Insufficent protein loaded on the gel for the amount of antibody used (or antibody too dluted). 3. Transfer efficiency from gel to membrane was poor. 2. ECL reagent was expired or contaminated. 5. Incorrect secondary antibody used. 6. Blocking agent concentration was too high. For more reasons read WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE

These results indicate that H9c2 and HepG2 cells are sensitive to prometryn in-vitro. However, prometryn effects on the heart and liver tissue in male mice is significantly different. Overall, our data supports the deduction that prometryn affected mitochondrial function, induced oxidative stress in cells, but also alters the ubiquitin-proteasome system and increases oxidative stress in mice liver. These results help towards elucidation of the mechanism by which prometryn could cause diseases.

Background and ObjectiveCardiovascular disease (CVD) is the leading cause of death and a major cause of disability worldwide. Previous studies have shown that the long‐term and chronic use of nonsteroidal anti‐inflammatory drugs (NSAIDs) increases the risk of developing stroke and cardiovascular diseases. Naproxen is an FDA‐approved NSAID that is currently used to treat several medical conditions such as pain, osteoarthritis, rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, tendonitis, etc. Acetaminophen, the active ingredient in Tylenol, is not an NSAID but it has been associated with increased risk of liver toxicity. Similarly, the long‐term exposures to pesticides such as Prometryn amongst others have been established to be dangerous to human lives and capable to disrupt the physiology of different organs in the body, including the cardiovascular system. Prometryn, a diamino‐1,3,5‐triazine, is used as a selective pre‐ and post‐emergence herbicide. It is used to control annual grasses and broadleaf weeds in cotton and celery. This study aimed to investigate the effects of Naproxen, Acetaminophen, and Prometryn in cardiomyocytes and the underlying mechanisms by which it causes cardiovascular dysfunction.MethodsThe embryonic cardiomyocyte cell line H9c2 is commonly used in numerous in vitro studies because morphological parameters of their cells resemble immature embryonic cardiomyocytes. It has been previously shown that H9c2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro. H9c2 cardiomyocytes were plated in a 96 well plate at a concentration of 4000 cells/well. The cells were then treated with different concentrations of Naproxen, Acetaminophen, and Prometryn for 24h at 37°C and 5% CO2. The cell viability, reactive oxygen species (ROS) formation, and mitochondrial membrane potential (ΔΨm) were examined.ResultsH9c2 cells treated with 100μM Acetaminophen and 20μM Prometryn showed significant decreases (> 30%) in cell viability. Hydrogen peroxide (H2O2), which was used as a positive control, also resulted in significant decreases in cell viability at 100μM. ROS formation was significantly increased in the presence of 200–400μM Naproxen. Acetaminophen showed significant amount of ROS formation at a higher concentration of 200μM while Prometryn also showed a significant increase in ROS formation at a higher concentration of 30μM. The effect of varying concentrations of Naproxen showed that ΔΨm was decreased by ~30% at 100μM. Prometryn decreased ΔΨm by ~20% at 10–20μM while Acetaminophen also decreased ΔΨm by ~20% at 100–200μM.ConclusionThese results suggest that both concentration and exposure time are important for NSAID‐induced cardiotoxicity. Acetaminophen, which is not an NSAID, increased ROS generation and decreased ΔΨm at higher concentration. This experimental data suggests that Naproxen, Acetaminophen, and Prometryn causes cardiomyocyte dysfunction at varying concentrations.

Nonsteroidal anti-inflammatory drugs like Advil (Ibuprofen) works via the inhibition of prostaglandin G/H synthases, commonly known as cyclooxygenases (COX-1 and COX-2). The long-term and chronic use of NSAIDs such as Ibuprofen has been shown to increase the risk of developing stroke and cardiovascular diseases (CVDs). Ibuprofen are derived from isobutylphenylpropanoic acid and they are generally used to relieve pain, headaches, muscle aches, fever, and inflammation. It can also be used to treat juvenile idiopathic arthritis, rheumatoid arthritis, patent ductus arteriosus, and pericarditis. The NSAID-mediated precise molecular mechanism that leads to thrombotic risk which eventually results in stroke and CVDs remains largely unknown. In our present study, we investigated the effects of Ibuprofen on protein homeostasis in liver using both male and female mice. We observed that Ibuprofen reduces proteasome activity in the Beta-1 (caspase-like) and Beta-2 (trypsin-like) subunits and enhances the aggregation of ubiquitinated abnormal proteins. We observed that Ibuprofen causes proteasome dysfunction in liver cells by increasing the production of reactive oxygen species (ROS). The experimental data also suggests that proteasome dysfunction in livers from Ibuprofen treated mice differs in male and female mice. Since Vitamin C (ascorbic acid) has been shown to aid in detoxification of free radicals such as ROS, we thereby postulated that Ibuprofen treated mice that were cotreated with Vitamin C should restore proteasome function. We observed an increase in the proteasome activity in the Beta-5 subunit (chymotrypsin-like) with both male and female mice that were cotreated with Ibuprofen and Vitamin C, suggesting that Vitamin C partly restores proteasome function.

Introduction and objectivesPrevious clinical studies have suggested that nonsteroidal anti‐inflammatory drugs (NSAIDs) are associated with increased risk of stroke and cardiovascular disease. Acetaminophen, the active ingredient in Tylenol, is not an NSAID and is associated with increased risk of liver toxicity. The common use of NSAIDs for pain and fever relief is a concern but it is unclear if NSAIDs is also causing adverse effects on other tissues such as the liver, kidney and brain. Previous research in our laboratory has suggested that NSAIDs cause proteasome dysfunction in cardiac tissue. To investigate the possibility that NSAIDs also influence liver function, ibuprofen treated mice were used to determine if this NSAID affects the ubiquitin proteasome system (UPS). The UPS is critical for cell function as it is responsible for degradation of 60–80% of intracellular proteins.Method/ResultsEight‐week‐old male and female C57BL/6J mice were randomly assigned to one of 4 groups: (A) control (B) Group treated with ibuprofen (C) Group treated with ibuprofen + vitamin C (D) Group treated with vitamin C alone. Mice were treated with water or ibuprofen (100 mg/kg) and vitamin C (150 mg/kg) dissolved in drinking water for 6 weeks. Liver from 6 week treated animals were homogenized and cytosol fractions obtained. The three independent proteolytic proteasome activities, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like activity were measured in the different cytosolic liver fractions. The proteasome inhibitor bortezomib was utilized to ensure the activity measured was due to the proteasome.Semi‐quantitative western blotting suggests that the 20S proteasome amount was similar in all 4 groups but the immunoproteasome subunit β5i was increased in both male and female ibuprofen treated and ibuprofen with vitamin C groups. The β1 and β2 proteasome activities were significantly decreased in both male and female mice treated with ibuprofen. Although inclusion of vitamin C with the ibuprofen did not fully restore proteasome activity, it resulted in an improvement in the β1 proteasome activity compared to ibuprofen treated mice. The β5 proteasome activity was also significantly decreased in both male and female mice treated with ibuprofen. However, this decrease was significantly more pronounced in female mice than in male mice suggesting gender‐specific effects of ibuprofen on proteasome function.ConclusionThese results suggest that ibuprofen significantly disrupts the UPS and causes proteasome dysfunction in liver tissue. The experimental data also suggests that proteasome dysfunction in livers from ibuprofen treated mice differs in female and male mice.Support or Funding InformationAmerican Heart Association Award #16GRNT31350040This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.