Angiogenesis. Panel A. Representative semithin sections stained with toluidine-blue from the left ventricle of a control and an IH mouse.

<p>The arrows indicate endothelial cells. The horizontal bar represents 10 µm. Panel B. Representative Western blots reporting the protein expression of VEGF-R2, VEGF55, VEGF42 as well as α-actinin (loading control). Panel C. Data related to angiogenesis as obtained in all available hearts (mean±SEM, n=6/6 control and intermittent hypoxia, respectively), * marks P<0.05 with respect to control, Student’s two-tailed t-test. The capillary count per unit area, as well as the expression of VEGF-R2, VEGF isoforms VEGF42 and VEGF55 as normalized for α-actinin (Western blot) are reported. Panels D and E. Transmission electron microscopy images of left ventricle sections from an IH mouse. In panel C, arrows (→) indicate endothelial junctions. In panel D, arrow heads (►) show a cellular “bridge” that partitions a pre-existing capillary in the process of neo-angiogenesis. Ca, capillary lumen; Ec, erythrocytes; Ej, endothelial junctional complexes; Pc, pericyte. The horizontal bar represents 0.25 µm.</p

Stress proteins.

<p>Panel <b>A</b>. Sample Western blot images illustrating the protein expressions of HO-1, HSP70, GRP94 (referred to α-actin). Panel <b>B</b>. Double immunofluorescence analysis to investigate the nuclear localization of the transcription factor CHOP. The left column shows representative staining using anti-CHOP antibodies (red fluorescence), whereas the middle column shows α−sarcoglycan antibodies (green fluorescence). Nuclei were counterstained with DAPI (blue fluorescence). Arrows indicate cardiomyocyte nuclei positive for CHOP staining, which appears pink when merged with DAPI (right column). The upper and lower rows report a sample control and intermittent hypoxia heart. Bar: 50 µm. Panel <b>C</b>. Quantification of stress proteins from densitometry analysis of Western blots and analysis of Panel B images in all available samples (mean±SEM, n=6/6). *, P<0.05 with respect to control, Student’s two-tailed t-test. This panel also shows the effect of wortmannin on the expression of HO-1 and CHOP (n=5/5).</p

Hemodynamic data.

<p>Panel <b>A</b>. Pressure-volume loops obtained in ten consecutive contractions in two representative mice from either group. Panel <b>B</b>. Some hemodynamic data (mean±SEM) obtained in all the examined mice (n=7/6 control and intermittent hypoxia, respectively), * marks P<0.05 with respect to control, Student’s two-tailed t-test. The diastolic (clear) and systolic (shaded) volumes, systolic (clear) and diastolic (shaded) pressures, +dP/dt_max (shaded) and -dP/dt_max (clear), and the cardiac output (clear) are reported.</p

Signaling. Western blots of Akt, its phosphorylated isoform P-Akt, eNOS, its phosphorylated isoform P-eNOS, HIF-1α and Nrf2 in all available samples (mean±SEM, n=6/6 and 5/5 without and with wortmannin, respectively).

<p>*, P<0.05 with respect to control, Student’s two-tailed t-test.</p

Scheme of the intermittent hypoxia protocol used in this study.

<p>FIO₂: fraction of O₂ in inspired air (%).</p