Background: Laboratory male mice are often housed individually due to aggressive behaviour or experimental requirements. Recent studies have linked singularization with decreased NO-bioavailability and endothelial dysfunction. We sought to determine whether singularization of adolescence healthy mice affects cardiovascular function and outcome of acute myocardial infarction (AMI).

Methods: Male C57Bl/6 mice living in groups of five in cages, where they were running, climbing, and fighting during their active cycle, were randomly assigned to stay there or to live alone for six weeks in small cages where they were predominantly resting. To assess the reversibility of changes induced by singularization, an  additional set of mice was subjected to 4 weeks of voluntary running after 6 weeks of single-housing. Food intake, anatomical and hematological parameters, peripheral blood immune cell numbers, citrate synthase activity, cardiac function, flow-mediated vasodilation and eNOS protein expression were determined. Infarct size per area at risk and post-AMI left ventricular function were measured in all groups after 45 min of left descending artery ligation and 24 h of reperfusion by 2,3,5-triphenyltetrazolium chloride staining.

Results: Food intake and body weight gain were not different between single- and group-housed mice. In contrast, exercise-training stimulated food intake, but prevented body weight gain. Singularization reduced soleus weight/body weight, soleus weight/tibial length, heart weight/body weight ratios and diminished citrate synthase activity in the heart and soleus muscle of single-housed mice as compared to group-housed and exercised animals suggesting a lower level of physical activity. Hematological parameters and immune cell levels measured in peripheral blood were not different between the groups. Singularization induced endothelial dysfunction and reduction of vascular eNOS expression as compared to the group-housed mice. Likewise, singularized mice showed increased infarct size as compared to their group-housed littermates. Moderate voluntary training of singularized mice greatly improved endothelial function, as measured by increased maximal FMD and more sustained vasodilation to shear, upregulated eNOS and reduced infarct size.

Conclusions: In adolescent mice singularization induces endothelial dysfunction, impairs NO-bioavailability and worsens outcome after AMI. These changes were completely reversible by a short period of moderate exercise training. Our findings emphasized that effects of the housing systems on cardiovascular function must be considered in the design of animal experimental studies.