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Characterization of the cytoprotective effect of 6-aminonicotinamide against oxidative stress in cardiac cells

Johannes P. Hofgaard 

Summary

Myocardial infarct is a major cause of mortality in the Western world and there is an urgent need for further development of the existing therapeutic strategies in order to improve the post infarct survival. One general feature of the pathophysiology of ischemia-reperfusion is the rapid production of reactive oxygen species (ROS) upon the restoration of the blood flow. The produced ROS can lead to severe oxidative damage and culminate in myocardial cell death.
In the present work the cytoprotective potential of 6-aminonicotinamide (6AN), an inhibitor of the oxidative pentose phosphate pathway (oxPPP), against simulated ROS-induced reperfusion injury, is studied in a model using H2O2 stress in H9C2 rat cardiac myoblasts and in beating primary neonatal cardiomyocytes.
Pretreatments with 6AN over a range of concentrations 25 - 300 μM, at durations of 6 – 23 hrs, strongly increased cell survival following a 1 hr exposure to H2O2 as measured 2 hrs post-H2O2 stress. The protection induced by 6AN seems to differ from the well-established protective timeframes of the ischemic preconditioning indicating novel mechanisms generating this protective effect. 
The 6AN-induced protection was not triggered or mediated by an opening of the mitochondrial ATPsensitive potassium channel, nor by an opening of the mitochondria high-conductance calciumactivated potassium channel, and was not dependent on protein synthesis or early ROS production, as seen in ischemic preconditioning. The 6AN-induced protective effect was associated with a decrease of total cell reduced glutathione down to 56% of control and with a modest increase (up to 55%) of the cytosolic free Ca2+. The 6ANinduced protection was abolished by ryanodine in the concentration range known to be inhibitory at the skeletal and cardiac types of ryanodine receptors. Although certain 6AN treatment conditions elicited Unfolded Protein Response (a well characterized protective S/ER stress response) in H9C2 cells, this response was not a prerequisite for the induction of cytoprotection. 
Non-selective inhibition of members of the PKC family by chelerythrine, but not a selective inhibition of the Ca2+ -activated isoforms by Gö 6976, abolished the 6AN-mediated protection. This pharmacological inhibition profile suggests a possible partial similarity between the role of the PKC family enzymes in the mechanism of 6AN-induced protection against the oxidative stress, and that of the well-recognized Ca2+-induced preconditioning against ischemia-reperfusion injury. The available data concerning the timeframe of the Ca2+-preconditioning are limited to short term protection studies (a few minutes to max. 30 minutes from treatment to stress). This study indicates that a Ca2+-induced protective state may be maintained for at least 18 hrs. 

In conclusion, the present work presents the first characterization of the 6AN-induced antioxidative cytoprotection in vitro. The findings suggest that 6AN represents a suitable tool to manipulate the cardiomyocyte redox status through interference with the oxPPP on a time scale of hours, and to induce alterations in ryanodine receptor conductance properties sufficient to trigger a Ca2+-mediated, PKC-dependent enhancement of the resistance against oxidative stress. The exact nature of this cytoprotection, including its apparent relationship to the earlier recognized phenomenon of Ca2+ preconditioning, as well as the usefulness of this manipulation at the level of the working heart prior to an imminent ischemia/reperfusion insult, remain to be investigated.