Long term survival of childhood cancers is now more than 70%. Anthracyclines, including doxorubicin, are some of the most efficacious anticancer drugs available. However, its use as a chemotherapeutic agent is severely hindered by its dose-limiting toxicities. Most notably observed is cardiotoxicity, but other organ systems are also degraded by doxorubicin use. Despite the years of its use and the amount of information written about this drug, an understanding of its cellular mechanisms is not fully appreciated. The mechanisms by which doxorubicin induces cytotoxicity in target cancer cells have given insight about how the drug damages cardiomyocytes. The major mechanisms of doxorubicin actions are thought to be as an oxidant generator and as an inhibitor of topoisomerase 2. However, other signaling pathways are also invoked with significant consequences for the cardiomyocyte. Further the interaction between oxidant generation and topoisomerase function has only recently been appreciated and the consequences of this interaction are still not fully understood. The unfortunate consequences of doxorubicin within cardiomyocytes have promoted the search for new drugs and methods that can prevent or reverse the damage caused to the heart after treatment in cancer patients. Alternative protocols have lessened the impact on newly diagnosed cancer patients. However the years of doxorubicin use have generated a need for monitoring the onset of cardiotoxicity as well as understanding its potential long-term consequences. Although a fairly clear understanding of the short-term pathologic mechanisms of doxorubicin actions has been achieved, the long-term mechanisms of doxorubicin induced heart failure remain to be carefully delineated.

Children surviving cancer and chemotherapy are at risk for adverse health events including heart failure that may be delayed by years. Although the early effects of doxorubicin-induced cardiotoxicity may be attributed to a direct effect on the cardiomyocytes, the mechanisms underlying the delayed or late effects (8–20 yr) are unknown. The goal of this project was to develop a model of late-onset doxorubicin-induced cardiotoxicity to better delineate the underlying pathophysiology responsible. The underlying hypothesis was that doxorubicin-induced “late-onset cardiotoxicity” was the result of mitochondrial dysfunction leading to cell failure and death. Wistar rats, 3–4 wk of age, were randomly assigned to vehicle or doxorubicin injection groups (1–45 mg/kg). Cardiovascular function was unaltered at the lower dosages (1–15 kg/mg), but beginning at 6 mo after injection significant cardiac degradation was observed in the 45 mg/kg group. Doxorubicin significantly increased myocardial mitochondrial DNA (mtDNA) damage. In contrast, in isolated c-kit left ventricular (LV) cells, doxorubicin treatment did not increase mtDNA damage. Biomarkers of senescence within the LV were significantly increased, suggesting accelerated aging of the LV. Doxorubicin also significantly increased LV histamine content suggestive of mast cell activation. With the use of flow cytometry, a significant expansion of the c-kit and stage-specific embryonic antigen 1 cell populations within the LV were concomitant with significant decreases in the circulating peripheral blood population of these cells. These results are consistent with the concept that doxorubicin induced significant damage to the cardiomyocyte population and that although the heart attempted to compensate it eventually succumbed to an inability for self-repair.

Background: Different mechanisms of diabetic-induced NO dysfunction have been proposed and central to most of them are significant changes in eNOS function as the rate-limiting step in NO bioavailability. eNOS exists in both monomeric and dimeric conformations, with the dimeric form catalyzing the synthesis of nitric oxide, while the monomeric form catalyzes the synthesis of superoxide (O₂ ^-). Diabetic-induced shifts to decrease the dimer:monomer ratio is thought to contribute to the degradation of nitric oxide (NO) bioavailability. Exercise has long been useful in the management of diabetes. Although exercise-induced increases expression of eNOS has been reported, it is unclear if exercise may alter the functional coupling of eNOS. Methods: To investigate this question, Goto-Kakizaki rats (a model of type II diabetes) were randomly assigned to a 9-week running program (train) or sedentary (sed) groups. Results: Exercise training significantly (p < .05) increased plantaris muscle cytochrome oxidase, significantly improved glycosylated hemoglobin (sed: 7.33 ± 0.56%; train: 6.1 ± 0.18%), ad improved insulin sensitivity. Exercise increased both total eNOS expression and the dimer:monomer ratio in the left ventricle LV (sed: 11.7 ± 3.2%; train: 41.4 ± 4.7%). Functional analysis of eNOS indicated that exercise induced significant increases in nitric oxide (+28%) production and concomitant decreases in eNOS-dependent superoxide (-12%) production. This effect was observed in the absence of tetrahydrobiopterin (BH₄), but not in the presence of exogenous BH₄. Exercise training also significantly decreased NADPH-dependent O₂ ^- activity. Conclusion: Exercise-induced increased eNOS dimerization resulted in an increased coupling of the enzyme to facilitate production of NO at the expense of ROS generation. This shift that could serve to decrease diabetic-related oxidative stress, which should serve to lessen diabetic-related complications.

Cardiovascular disease is the leading cause of mortality in diabetics, and it has a complex etiology that operates on several levels. Endothelial dysfunction and increased generation of reactive oxygen species are believed to be an underlying cause of vascular dysfunction and coronary artery disease in diabetes. This impairment is likely the result of decreased bioavailability of nitric oxide (NO) within the vasculature. However, it is unclear whether hyperglycemia per se stimulates NADPH oxidase-derived superoxide generation in vascular tissue. This study focused on whether NADPH oxidase-derived superoxide is elevated in vasculature tissue evoking endothelial/smooth muscle dysfunction in the hyperglycemic (169±4 mg%) Goto-Kakizaki (GK) rat. By dihydroethidine fluorescence staining, we determined that aorta superoxide levels were significantly elevated in 9 month-old GK compared with age matched Wistar (GK; 195±6%, Wistar; 100±3.5%). Consistent with these findings, 10⁻⁶ mol/L acetylcholine-induced relaxation of the carotid artery was significantly reduced in GK rats compared with age matched Wistar (GK; 41±7%, Wistar; 100±5%) and measurements in the aorta showed a similar trend (p = .08). In contrast, relaxation to the NO donor SNAP was unaltered in GK compared to Wistar. Endothelial dysfunction was reversed by lowering of superoxide with apocynin, a specific Nox inhibitor. The major findings from this study are that chronic hyperglycemia induces significant vascular dysfunction in both the aorta and small arteries. Hyperglycemic induced increases in NAD(P)H oxidase activity that did not come from an increase in the expression of the NAD(P)H oxidase subunits, but more likely as a result of chronic activation via intracellular signaling pathways.

Mitochondrial dysfunction has a significant role in the development and complications of diabetic cardiomyopathy. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) mutations are also associated with different types of cancer and neurodegenerative diseases. The goal of this study was to determine if chronically elevated glucose increase in mtDNA damage contributed to mitochondrial dysfunction and identify the underlying basis for mtDNA damage. H9c2 myotubes (a cardiac-derived cell line) were studied in the presence of 5.5, 16.5, or 33.0 mM glucose for up to 13 days. Tests of mitochondria function (Complex I and IV activity and ATP generation) were all significantly depressed by elevated media glucose. Intramitochondrial superoxide and intracellular superoxide levels were transiently increased during the experimental period. AnnexinV binding (a marker of apoptosis) was significantly increased after 7 and 13 days of high glucose. Thirteen days of elevated glucose significantly increased mtDNA damage globally and across the region encoding for the three subunits of cytochrome oxidase. Using mitochondria isolated from cells chronically exposed to elevated glucose, we observed significant increases in topoisomerase-linked DNA cleavage. Mitochondria-dependent DNA cleavage was significantly exacerbated by H₂O₂ and that immunoprecipitation of mitochondrial extracts with a mtTOP1 antibody significantly decreased DNA cleavage, indicating that at least part of this activity could be attributed to mtTOP1. We conclude that even mild increases in glucose presentation compromised mitochondrial function as a result of a decline in mtDNA integrity. Separate from a direct impact of oxidative stress on mtDNA, ROS-induced alteration of mitochondrial topoisomerase activity exacerbated and propagated increases in mtDNA damage. These findings are significant in that the activation/inhibition state of the mitochondrial topoisomerases will have important consequences for mitochondrial DNA integrity and the well being of the myocardium.