Supplementary Material for: To Be Or Not to Be: the “Smoker’s Paradox” – An in-Vitro Study

<b><i>Background/Aims:</i></b> Clinical studies have reported a better outcome of smokers after myocardial infarction compared to non-smokers. The data are controversial, as some clinical studies did not observe this effect. The cell biological processes involved, which might account for a ‘Smoker’s Paradox’, have not been investigated yet. Therefore, the aim was to elucidate the effect of cigarette smoke on the viability of cardiomyocytes in the context of hypoxia and reperfusion. <b><i>Methods:</i></b> HL-1 cells were incubated with different concentrations of cigarette smoke extract (CSE) and subjected to hypoxia/reperfusion to further evaluate influence of CSE on viability of HL-1 cells using flow cytometry analyses, Western Blot and immunofluorescence staining. <b><i>Results:</i></b> Incubation with CSE led to a concentration-dependent reduction in HL-1 viability. Adding hypoxia as a stressor enhanced cell death. Caspase-independent apoptosis was the observed type of cell death partly induced by P53 and apoptosis-inducing-factor. Yet a significant increase in LDH release in cardiomyocytes incubated with 4%, 8% and 16% CSE suggests necrosis with rapid DNA depletion. Interestingly, after hypoxia a decreased LDH release under lower CSE concentrations was observed. Moreover, a concentration-dependent increase in proliferation and a trend for increased ATP availability under hypoxic conditions was shown. <b><i>Conclusions:</i></b> The trend for less LDH release in hypoxia after low-level CSE incubation might represent a switch from necrosis to apoptosis, which in combination with the increase in metabolic activity and ATP availability might account for the ‘Smoker’s Paradox’. These findings could partly explain inconsistent results of previous clinical studies as the data showed strong evidence for the crucial relevance of the amount of cigarettes smoked. We are in need of future studies distinguishing between different types of smokers to finally verify or falsify the ‘Smoker’s Paradox’.