Table 1.
Myocardial hemodynamics during in vivo experiments.
Fig 1.
SPC decreases cardiac infarct size, increases LV contractile function and attenuates histopathologic organ damage following I/R.
(A) Rats were sacrificed at the end of reperfusion and the hearts removed and stained with TTC for the measurement of myocardial infarct area. The infarct size was expressed as a percentage of area at risk. n = 6 /group. (B) Echocardiography was performed at the end of reperfusion and representative M-mode echocardiograms were recorded in all groups. Rats without LAD occlusion served as basal controls (SHAM group). n = 10 /group. (C) LV tissues were retrieved at the end of reperfusion, paraffin section was prepared and subjected to the H&E staining. Representative H&E staining images are shown (magnification, 200×). Scale bar: 50μm. n = 3 /group. The columns and errors bars represent means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. I/R group.
Fig 2.
SPC ameliorates mitochondrial disorder and dysfunction after I/R.
(A) LV tissues were harvested for examination of myocardial ultrastructure by transmission electron microscopy (TEM). Typical TEM images obtained at a magnification (20000×) of cardiac ultrastructure in all groups. Note that myofilaments were absent (*) and damaged mitochondria (^) nearby the autophagosomes (→). Scale bar: 2μm. n = 3 /group. M: mitochondria; S: sarcomeres. (B) SPC prevents depletion of ATP stores in I/R hearts and the ATP content in all groups are shown. n = 6 /group. (C) After 2 h of reperfusion, LV tissues were obtained and analyzed by real-time PCR for the expression levels of Cycs, Cox4il, Ndufa2, Ndufa4, Ndufa8, Cox7a1, Cox7a2 and TFAM. n = 6 /group. (D) LV were collected and prepared for immunoblots. Representative immunoblots and semiquantitative analysis of Nrf1 and PGC-1 in each group of rats after reperfusion. The blots for Tubulin were served as loading controls. n = 4 /group. All data are presented as means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. I/R group.
Fig 3.
SPC inhibits oxidative stress brought about by the I/R injury.
(A) The cardiac levels of ROS in each group are shown by DHE staining. The image was obtained by a confocal microscope. SPC-treatment significantly lowered the increased DHE fluorescent intensity induced by the I/R injury. Scale bar: 20μm. n = 3 /group. (B) The cardiac GSH, GSSG and MDA levels were measured using enzymatic kits, and the GSH/GSSG ratios derived from the GSSG and GSH contents. n = 6 /group. (C) Protein carbonyl content was examined as carbonyl-containing 2,4-DNPH adducts by immunoblotting. Protein carbonylation of SPC group was likewise lower than the I/R group. n = 4 /group. (D) The immunoblot analysis SOD2 and HO-1 expression at the end of reperfusion. The blots for Tubulin were served as loading controls. n = 4 /group. The columns and errors bars represent means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. I/R group.
Fig 4.
SPC narrows the I/R-induced accumulation of protein aggregates.
(A) Cardiac tissues at papillary muscles level were collected and the cryosectioning was prepared. The immunohistochemical analysis of protein aggregates stained with anti-Vimentin is shown. Hoechst 33342 was used to stain cell nuclei. The immunofluorescence was examined by a confocal microscope. Scale bar: 20μm. n = 3 /group (B) Representative immunoblots of nuclear protein from all groups detected with a Vimentin-specific antibody. H3 was utilized to normalize the amount of protein. n = 4 /group. The columns and errors bars represent means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. I/R group.
Fig 5.
SPC plays an important regulatory role in the expression of class I/III PI3K and the interaction among Bcl2, Beclin1 and Vps34.
(A) LV tissues were collected from rats at the end of 2h reperfusion. The immunoblotting for phosphor-Akt (Ser473), Akt, phosphor- mTOR (Ser2448) and mTOR were performed. n = 4 /group. (B) Immunoblotting was used to examine the expression levels of Vps34, Bcl2 and Beclin1. Representative protein images and quantitative analysis were shown. The blots for Tubulin were served as loading controls. n = 4 /group. (C) Effect of SPC on I/R-induced association between beclin1 and Vps34. Upper panel, Cell lysates from LV tissues were immunoprecipitated (IP) with Beclin1 antibody and immunoblotted (IB) for Vps34 and Beclin1; Lower panel, Lysates were immunoprecipitated with Vps34 antibody and immunoblotted for Vps34 and Beclin1. The columns and errors bars represent means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. I/R group.
Fig 6.
SPC can protect against I/R-induced mitochondrial fragmentation and ensuing mitophagy.
(A) The expression levels of LC3 II/I, p62, Atg 5 and Atg 7 are pivotal markers of autophagosome formation and clearance. Representative immunoblot for these proteins in the myocardial tissues from all groups and densitometric quantification. n = 4 /group. (B) The autophagosome clearance related Lamp 2 was found out. Representative protein bands and densitometric quantification were shown. Tubulin was used to an internal control. n = 4 per group. (C) Representative electron micrograph of the heart from the I/R group obtained at a magnification of 100000 times. Asterisk (*) indicates dissolved myofilaments. Scale bar: 400nm. n = 3 /group. (D) Immunoblotting was used to examine the levels of Drp1, Opa1 and Parkin. Representative protein images and quantitative analysis were shown. Expression was normalized to Tubulin for each sample. n = 4 /group. The columns and errors bars represent means ± SD. * P < 0.05 vs. SHAM group; # P < 0.05 vs. I/R group.