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Insulin and cellular stressors both activate p38 MAPK. Insulin protects cardiac tissue in a p38 MAPK-dependent manner. Paradoxically, inhibiting p38 MAPK is also protective. Hsp27 phosphorylation is regulated by p38 MAPK. Insulin was tested in H9c2 cardiomyocytes subjected to media exchange, 6 hours of oxygen-glucose deprivation, and reoxygenation. Insulin suppressed stress-induced phosphorylation of Hsp27 due to media-exchange or oxygen-glucose deprivation. Surprisingly, insulin increased Hsp27 phosphorylation during reoxygenation. Insulin also reduced total p38 MAPK levels. Insulin before oxygen-glucose deprivation prevented both localization of Hsp27 to the nucleus and localization of phospho-p38 MAPK to the cytoplasm. Insulin during oxygen-glucose deprivation caused the localization of phospho-p38 MAPK in the cytoplasm, but did not increase Hsp27 phosphorylation until reoxygenation. In conclusion, insulin may protect before oxygen-glucose deprivation by redirecting phospho-p38 MAPK to the nucleus away from damaging pathways in the cytoplasm and protects during oxygen-glucose deprivation by priming phospho-p38 MAPK to phosphorylate Hsp27. / Insulin was used on a model on H9c2 myotubes to determine the effect of oxygen-glucose deprivation and reoxygenation on the localization and phosphorylation of Hsp27 and p38 MAPK

Most in vitro systems employ the standard cell culture maintenance conditions of 95 % air with 5 % CO2 to balance medium pH, which translates to culture oxygen tensions of approximately 20 % - above the typical ≤ 6 % found in most tissues. The current investigation, therefore, aims to characterize the effect of maintenance and toxicant exposure with a particular focus on the α,β-unsaturated aldehyde, acrolein, in the presence of physiologically relevant oxygen tension using a differentiated H9c2 cardiomyoblast subclone. H9c2 cells were maintained separately in 20.1 and 5 % oxygen, after which cells were differentiated for five days, and then exposed to acrolein in media containing varying concentrations of tricarboxylic acid and glycolytic substrates. Cells were then assessed for viability and metabolism via the MTT conversion assay. H9c2 cells were assessed for mechanistic elucidation to characterize contributors to cellular death, including mitochondrial membrane potential (ΔΨm) reductions (JC-1), intracellular calcium influx (Fluo-4), and PARP activation. Exposure to acrolein in differing oxygen tensions revealed that standard culture cells are particularly sensitive to acrolein, but cells cultured in 5 % oxygen, depending on the medium pyruvate concentration, can be rescued significantly. Further, reductions in ΔΨm were reversed by co-exposure of 5-10 mM EGTA for both culture conditions, while intracellular calcium transients were noted only for standard cultures. The results demonstrate significant metabolic reprogramming which desensitizes differentiated H9c2 to acrolein-induced cytotoxicity. Further, PARP and extracellular calcium contribute to the fate of these cells exposed to acrolein, though clotrimazole-associated TRPM2 channels may not be significantly involved. Conclusively, significant alteration of toxicogenic response was noted in this cell line when cultured under physiologically relevant conditions, and may have a substantial impact on the reliability and predictive power and interpretive application of in vitro-based toxicity models cultured under standard culture conditions, depending on the parent tissue.

Acrolein is an electrophilic α, β-unsaturated aldehyde. Additionally, acrolein is a metabolite of the antineoplastic alkylating agent cyclophosphamide and is implicated in off-target effects, including to bladder hemorrhagic cystitis and cyclophosphamide-induced cardiotoxicity, both of which have led to serious secondary iatrogenic injury during and following chemotherapy. At low concentrations acrolein inhibits cell proliferation without inducing apoptosis, while at high concentrations may result in secondary apoptosis promotion. This investigation assessed the role of the enzyme poly (ADP-ribose) polymerase (PARP) in acrolein induced toxicity using the established toxicological H9c2 (2-1) cardiomyoblast in vitro model. H9c2 (2-1) cells were plated in 24-well plates at 75,000 cells per well three days prior to testing, followed by acrolein dosing at concentrations between 10 µM and 1000µM for either 30 or 55 minutes. PARP activity was quantitatively measured in total cell lysates using a biotin-avidin-conjugated horseradish peroxidase-TMB reporter system in a 96-well microplate formate. The lowest effective dose of toxicity at 30 minute dosing was found at 25 μM (PARP Activity 1.65-fold control) which returned to baseline at 100 μM; concentrations at or above 250 μM results in significant PARP activity reductions (≤ 0.46-fold control). Biomarkers were further characterized for cytotoxicity (AST presence), and viability (MTT reduction) in order to facilitate mechanistic characterization of PARP-mediated acrolein cardiotoxicity. Investigation of a PARP inhibitor was assessed to explore the intervention for acrolein induced cardiac tissue damage.

Methylmercury (MeHg) is known predominantly as a neurotoxicant, however emerging experimental and epidemiological evidence has shown associations between MeHg exposure and the potential for increased risks of cardiovascular diseases. This thesis investigated the in vitro cytotoxic effects of MeHg in two cardiomyocyte cell lines, H9C2 rat neonatal cell line and AC16 adult human cell line. We observed significant increases in cell death at concentrations from 1 – 10 µM. ROS production and intracellular calcium concentrations increased dose-dependantly with MeHgCl exposure. Furthermore, while assessing mitochondrial function, a decline in maximal respiration at 1 µM was seen. However, these observations may in turn be a direct consequence of decreased cell numbers following exposures. Additionally, this study highlighted the differences in cellular bioenergetics which may impact how certain cells respond to contaminant stressors. The distribution of MeHg and total Hg in rat heart tissues was also examined and we observed increasing concentrations of MeHg in high and low dosed rat groups as compared to the vehicle controls. No difference was observed in Hg levels between the normal and high fat and sugar diet groups. The urinary isoprostane levels, which are indicative of systemic oxidative stress, showed significant increases in lean rats exposed to the high dose treatment. It was also observed that a high fat and sugar diet in lean and obese rats can contribute to increasing oxidative stress regardless of the level of contaminants they were dosed with. This thesis demonstrated several in vitro effects of MeHg on heart cells as well as determine the distribution of Hg levels in heart tissues and oxidative stress markers from an in vivo study.

It has been reported that opioid receptor activation mimics ischemic preconditioning, which may protect the heart from the development of infarction. Toll-like receptor 4 (TLR-4) during infarction stimulates cytokine production leading to inflammation and injury of the heart tissue. Our aim was to study the effect of morphine in vitro on the viability and oxidative state of H9c2 cells (rat cardiomyoblasts) and the role of TLR-4 during oxidative stress. Our experiments showed that pretreatment with morphine before tert-butylhydroperoxide (t-BHP)-, 2,2'-bipyridyl (BP)- and lipopolysaccharide (LPS)-induced oxidative stess had protective effect on the viability of H9c2 cells and markedly reduced the production of reactive oxygen species (ROS). The protective effect of morphine was diminished after naloxone treatment, which confirms the role of opioid receptors in preconditioning. TLR-4 inhibition by TAK-242 pretreatment and silencing TLR-4 by RNA interference resulted in a partial increase in cell viability but significant attenuation of ROS production after t-BHP and BP treatment. The action of LPS was reduced in response to TLR-4 silencing. Interestingly, naloxone pretreatment and suppression of TLR-4 markedly alleviated oxidative stress and resulted in a significant improvement of cell viability. We...

Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressant effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic receptor that translocates to the nuclei upon ligand binding and alters gene transcription. Among a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). Although GC induced GILZ expression has been well established in lymphocytes, little is known whether cardiomyocytes respond to GCs by inducing GILZ. Unlike lymphocytes, in which GCs induce apoptosis and GILZ mediates GC induced apoptosis, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression pattern in cardiomyocytes in vivo and in vitro. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs and point to H9C2 cell line as a valid model for studying the biological function of GILZ in cardiomyocytes. I have also determined GILZ functions as GC induced cytoprotective protein against the known cardiac toxicant Doxorubicin. Finally I have determined GILZ stabilizes Bcl-xL pro-survival protein, providing a possible mechanism of cytoprotection in cardiomyocytes.

RNA Binding Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact.