Supplemental Material for
Genetic deletion of the adaptor protein p66Shc increases susceptibility to shortterm ischemic myocardial injury via intracellular salvage pathways
Alexander Akhmedov*1,2 PhD; Fabrizio Montecucco*3,4, MD, PhD; Vincent
Braunersreuther3, PhD; Giovanni G. Camici1,2, PhD; Philipp Jacob1,2, PhD; Martin F.
Reiner1,2, MD; Martina Glanzmann1,2, MSc; Fabienne Burger3,4, MSc; Francesco
Paneni1,2,5, MD; Katia Galan3; Graziano Pelli3; Nicolas Vuilleumier6,7, MD;
Alexandre Belin8; Jean-Paul Vallée8, MD; Francois Mach3#, MD; Thomas F.
Lüscher1,2#, MD, PhD
1
Supplementary Figures
2
Supplementary figure 1. Basal arteriole density in heart and brain. A.
Quantifications of smooth muscle actin staining content in frozen heart sections (p=0.4). B.
Representative images of SMA stained middle heart sections are shown. C. Quantifications of
SMA staining content in frozen brain sections (p=0.4). D. Representative images of SMA stained
middle brain sections are shown. Data are expressed as mean±SEM (n=3 per group).
3
4
Supplementary figure 2. Basal vessel density in heart and brain. A. Quantifications
of CD31 staining content in frozen heart sections (p=0.7). B. Representative images of CD31
stained middle heart sections are shown. C. Quantifications of CD31 staining content in frozen
brain sections (p=0.7). D. Representative images of CD31 stained middle brain sections are
shown. Data are expressed as mean±SEM (n=3 per group).
5
6
Supplementary figure 3. Vessel density in the infarcted hearts from WT and p66/-
mice. A. Quantifications of CD31 staining content in frozen heart sections (p=0.8465). B.
Representative images of CD31 stained middle heart sections are shown. Lower panel
represents higher magnification of the area within the rectangle. C. Quantifications of SMA
staining content in frozen heart sections (p=0.0762). D. Representative images of SMA stained
middle heart sections are shown. Lower panel represents higher magnification of the area
within the rectangle. Data are expressed as mean±SEM (n=3 per group).
7
8
Supplementary figure 4. Increasing cardiac levels of p66Shc protein at different
time points after the reperfusion. After 30 min ischemia followed by either 12 or 24 h of
reperfusion, WT mouse hearts were lysed and the total levels of p66Shc was assessed by
Western blot (n=3 in each group). Basal expression of p66Shc was determined in hearts of the
age-matched WT mice without myocardial infarction. Data are shown as the mean±SEM.
*P<0.05 vs basal (one-tailed, Mann-Whitney nonparametric test).
9
10
Supplementary figure 5. Vessel density in the infarcted hearts from p66Shc and
scramble siRNA injected WT mice. A. Quantifications of CD31 staining content in frozen
heart sections (p=0.6905). B. Representative images of CD31 stained middle heart sections are
shown. Lower panel represents higher magnification of the area within the rectangle. C.
Quantifications of SMA staining content in frozen heart sections (p=1). D. Representative
images of SMA stained middle heart sections are shown. Lower panel represents higher
magnification of the area within the rectangle. Data are expressed as mean±SEM (n=3 per
group).
11
12
Supplementary figure 6. Genetic deletion of p66Shc does not impact postinfarction leukocyte recruitment and ROS release within mouse hearts. WT and
p66-/- mice were submitted to 30 minutes of ischemia followed by 24 hours of reperfusion.
Data are expressed as mean±SEM (n=5-8 per group) Differences between groups were
statistically not significant for all stainings. A. Quantification of infiltrated neutrophils (Ly-6G+
cells) per area in frozen sections of infarcted hearts (p=0.4908). Left panel, representative
images of neutrophil infiltration are shown. B. Quantification of macrophages (CD68+ cells) per
area in frozen sections of infarcted hearts (p=0.8518). Left panel, representative images of
macrophage infiltration are shown. C. Quantifications of 4-HNE staining content in frozen
sections of infarcted hearts (p=0.4136). Left panel, representative images of 4-HNE stained
middle heart sections are shown. D. Quantifications of Di bromo tyrosine (DiBrY) content in
frozen sections of infarcted hearts (p=0.2824). Left panel, representative images of DiBrY
stained middle heart sections are shown. E. Quantifications of superoxide (DHE staining)
content in frozen sections of infarcted hearts (p=0.4136). Left panel, representative images of
representative images of DHE stained middle heart sections and correspondent images of the
same middle heart sections stained with 4',6-diamidino-2-phenylindole (DAPI), visualizing
nuclear DNA are shown.
13
14
Supplementary figure 7. In vivo knockdown of p66Shc does not affect postinfarction leukocyte recruitment and ROS release within mouse hearts. WT mice
were pre-injected with scrambled (scr siRNA) or p66Shc siRNA were submitted to 30 minutes of
ischemia followed by 24 hours of reperfusion. Data are expressed as mean±SEM (n=4-6 per
group). Differences between groups were statistically not significant for all stainings. A.
Quantification of infiltrated neutrophils (Ly-6G+ cells) per area in frozen sections of infarcted
hearts (p=1). Left panel, representative images of neutrophil infiltration are shown. B.
Quantification of macrophages (CD68+ cells) per area in frozen sections of infarcted hearts
(p=0.9048). Left panel, representative images of macrophage infiltration are shown. C.
Quantifications of 4-HNE staining content in frozen sections of infarcted hearts (p=0.4286). Left
panel, representative images of 4-HNE stained middle heart sections are shown. D.
Quantifications of Di bromo tyrosine (DiBrY) content in frozen sections of infarcted hearts
(p=0.2468). Left panel, representative images of DiBrY stained middle heart sections are shown.
E. Quantifications of superoxide (DHE staining) content in frozen sections of infarcted hearts
(p=0.5368). Left panel, representative images of representative images of DHE stained middle
heart sections and correspondent images of the same middle heart sections stained with 4',6diamidino-2-phenylindole (DAPI), visualizing nuclear DNA are shown.
15
16
Supplementary figure 8. Basal levels of ROS in heart and brain. A. Quantifications
of 4-HNE staining content in frozen heart sections (p=1). Right panel, representative images of
4-HNE stained middle heart sections are shown. B. Quantifications of 4-HNE staining content in
frozen brain sections (p=0.7). Right panel, representative images of 4-HNE stained middle brain
sections are shown. Data are expressed as mean±SEM (n=3 per group).
17
18
Supplementary figure 9. Basal levels of ROS in heart and brain. A. Quantifications
of Di bromo tyrosine (DiBrY) content in frozen heart sections (p=0.4). Right panel,
representative images of DiBrY stained middle heart sections are shown. B. Quantifications of
Di bromo tyrosine (DiBrY) content in frozen brain sections (p=1). Right panel, representative
images of DiBrY stained middle brain sections are shown. Data are expressed as mean±SEM
(n=3 per group).
19
Supplementary figure 10. Genetic deletion of p66Shc is not associated with
modulation of extracellular signal-regulated mitogen-activated protein kinase
(ERK 1/2) within infarcted hearts. After 30 min ischemia followed by few minutes (5-15
minutes) of reperfusion, mouse hearts were lysed and the phosphorylation of MAP kinase
ERK1/2 was assessed by Western blot (n=5-7 in each group, p=0.0823 for 5 min and p=1 for 15
min). Data are shown as the mean±SEM.
20