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Accelerated Autophagy of Cecal Ligation and Puncture-Induced Myocardial Dysfunction and Its Correlation with Mammalian Target of Rapamycin Pathway in Rats

Background: Recent studies have indicated that autophagy is involved in sepsis-induced myocardial dysfunction. This study aimed to investigate the change of autophagy in cecal ligation and puncture (CLP)-induced myocardium dysfunction and its relationship with mammalian target of rapamycin (mTOR) pathway. Methods: Totally, 12 rats were randomly divided into CLP group or sham-operated (SHAM) group. Cardiac tissues were harvested 18 h after CLP or sham operation. Pathology was detected by hematoxylin and eosin staining, cardiac functions by echocardiography, distribution of microtubule-associated protein light chain 3 type II (LC3II) by immunohistochemical staining, and autophagic vacuoles by transmission electron microscopy. Moreover, phosphorylation of mTOR (p-mTOR), phosphorylation of S6 kinase-1 (PS6K1), and LC3II and p62 expression were measured by western blotting. Pearson's correlation coefficient was used to analyze the correlation of two parameters. Results: The results by pathology and echocardiography revealed that there was obvious myocardial injury in CLP rats (left ventricle ejection fraction: SHAM 0.76 ± 0.06 vs. CLP 0.59 ± 0.11, P < 0.01; fractional shortening: SHAM 0.51 ± 0.09 vs. CLP 0.37 ± 0.06, P < 0.05). We also found that the autophagy process was elevated by CLP, the ratio of LC3II/LC3I was increased (P < 0.05) while the expression of p62 was decreased (P < 0.05) in the CLP rats, and there were also more autophagosomes and autolysosomes in the CLP rats. Furthermore, the mTOR pathway in CLP myocardium was inhibited when compared with the sham-operated rats; p-mTOR (P < 0.01) and PS6K1 (P < 0.05) were both significantly suppressed following CLP challenge. Interestingly, we found that the mTOR pathway was closely correlated with the autophagy processes. In our study, while p-mTOR in the myocardium was significantly correlated with p62 (r = 0.66, P = 0.02), PS6K1 was significantly positively correlated with p62 (r = 0.70, P = 0.01) and negatively correlated with LC3II (r = −0.71, P = 0.01). Conclusions: The autophagy process in the myocardium was accelerated in CLP rats, which was closely correlated with the inhibition of the mTOR pathway.

while p62 has an adaptor function to recognize ubiquitinated proteins that need to be removed from the cytoplasm during autophagy, and its amount is generally considered to inversely correlate with autophagic activity. [8][9][10] As a housekeeping process, autophagy is vital for the normal structure and functions of the heart. [11] It also plays an important role ischemia-reperfusion (I/R) injury. [12] Evidence suggests that autophagy protects against I/R injury. Recent studies have found that autophagy also plays an important role in septic myocardial depression. There is evidence showing that autophagic activity in cardiomyocytes changes during sepsis, but the results are still unclear. [13,14] Mammalian target of rapamycin (mTOR) is a master sensor of energy status and will be inhibited in the state of energy depletion. [15] Myocardial energy depletion plays a major role in myocardial dysfunction during sepsis. [16,17] There are little data describing the role of the mTOR pathway during sepsis in the heart. [18] Furthermore, among various pathways known to regulate autophagy in mammalian cells, mTOR complex 1 (mTORC1)/S6 kinase-1 (S6K1) signaling pathway is one of the best-understood pathways. [19] Accumulating evidence has indicated that the mTORC1/S6K1 pathway negatively regulates autophagy, so the relationship between the mTOR pathway and the change of autophagy process in cecal ligation and puncture (CLP)-induced myocardial dysfunction was investigated in this study.

Animal model preparation
All procedures used in this study were in accordance with our institutional guidelines that complied with the international ethics and humane standards for animal use. Every effort was made to minimize the number of animals and reduce their suffering.
Healthy Wistar rats, male, aged 7-8 weeks, and weight of 220 ± 20 g were obtained from the Animal Facility Center, Peking Union Medical College Hospital. All animals were housed in a pathogen-free facility and used according to the protocols approved by the IACUC of Peking Union Medical College Hospital. Totally, 12 rats were randomly divided into sham-operated (SHAM) group or CLP group. CLP was performed as previously described to establish a mid-grade sepsis model. [20] In brief, the cecum was ligated at half the distance between the distal pole and the base of the cecum. Two cecal punctures were made with a 22G needle, and a droplet of feces was forced out from both the mesenteric and antimesenteric penetration holes. Sham-operated rats were subjected to the same laparotomy without CLP. After surgery, all animals immediately received a subcutaneous injection of sterile saline (0.9% NaCl, 5 ml per 100 g body weight) for resuscitation.

Hematoxylin and eosin staining
The left ventricle (LV) hearts of rats were transversely cut at a 2 mm-thickness, immediately fixed in 4% paraformaldehyde, and embedded in paraffin. Sections of 3-μm thickness were affixed to slides, deparaffinized, and stained with hematoxylin and eosin (H and E) to evaluate the morphological changes of the heart.

Transmission electron microscopy analysis
Transmission electron microscopy was performed as described previously. [21] The samples after pretreatment were cut into thin sections (90 nm) that were viewed at 120 kV with a H7650 transmission electron microscope (HITACHI, Tokyo, Japan). Micrographs were obtained using a Philips CM12 (10-15 per sample) by random sampling.

Immunohistochemistry
Formalin-fixed, paraffin-embedded sections of the rat hearts were subjected to immunohistochemical analysis as described previously. [22] The heart sections were immune with anti-LC3 antibody (ab48394, Abcam, 1:1000 dilution). All slides were viewed using a DP70 Olympus digital microscope camera at ×200 and ×400. Images were captured with Olympus DP-Soft823 version 3.2 acquisition software (Olympus Corporation, Tokyo, Japan).

Echocardiography examination
An Ultrasonic Machine (M-Turbo SonoSite, USA) equipped with a 15-MHz transducer was used for noninvasive transthoracic echocardiography. Rats were anaesthetized intraperitoneally with pentobarbital (70-80 mg/kg) at 18 h after CLP and situated in the supine position on a warming pad. LV end diastolic and systolic dimensions were measured. The LV ejection fraction (LVEF) and fractional shortening (FS) were also calculated from M-mode echocardiograms. Data from three consecutive selected cardiac cycles were analyzed and averaged.

Statistical analysis
Data were analyzed by SPSS 18.0 software (SPSS Inc., IBM Corp., Armonk, NY, USA). All data for continuous variables in this study had normal distributions and were shown as mean ± standard deviation. Differences were assessed using analysis of independent-samples t-test. Pearson's correlation coefficient was used to analyze the correlation of two parameters. A P < 0.05 was considered statistically significant.

Cardiomyopathy and cardiac dysfunction in cecal ligation and puncture rats
H and E staining revealed that there were pathological changes in CLP rats. The myocardium of SHAM rats had a normal architecture and clear myocyte boundaries, whereas CLP rats after 18 h of sepsis showed marked myocardial injury with myocardial necrosis and interstitial edema adjacent to localized extravasation of red blood cells. These results indicated cardiomyopathy of the sepsis model established by CLP [ Figure 1a]. As shown in Figure 1b, LVEF and FS were measured at 18 h after treatment. Compared with the sham-operated rats, the LVEF and FS were both significantly reduced in the CLP group (LVEF: SHAM 0.76 ± 0.06 vs. CLP 0.59 ± 0.11, P < 0.01; FS: SHAM 0.51 ± 0.09 vs. CLP 0.37 ± 0.06, P < 0.05).

Increased expression of light chain 3 type II and decreased expression of p62 in cecal ligation and puncture rats
Autophagy is under the control of the gene of LC3. LC3II is a main mediator in autophagy and functions at an early stage of phagophore expansion. We found that LC3II/LC3I in the LV was significantly increased at 18 h after CLP compared with sham-operated rats [P < 0.05, Figure 2a]. We also evaluated LC3II in the LV by immunohistochemical staining. In immunohistochemical staining experiments, autophagosome was evaluated by examining the punctate form (type II) of LC3. As shown in Figure 2b, LV sections from CLP rats showed an increase in LC3II expression compared with sham-operated rats; we even observed LC3B globular structures in some sections from CLP rats. Since p62 has an adaptor function to recognize the ubiquitinylated proteins and to be removed from the cytoplasm during autophagy, its amount is generally considered to inversely correlate with autophagic activity. As shown in Figure 2c, the expression of p62 in the heart from CLP rats was lower than that from sham-operated rats (P < 0.05).

More autophagosomes and autolysosomes in cecal ligation and puncture rats
As shown in Figure 3, LV tissues from the sham-operated rats showed normal structure with proper mitochondria distribution, while CLP rats revealed cellular disorganization and myofibrillar disarray. Autophagic vacuoles were also observed through transmission electron microscopy; we found that there were more larger autophagosomes and more autolysosomes compared with sham-operated rats.

Inhibited mammalian target of rapamycin pathway in myocardium of cecal ligation and puncture rats
To investigate the role of mTOR pathway in sepsis-induced myocardial dysfunction, we detected   the level of p-mTOR (phosphorylation at Ser2448) and PS6K1 (phosphorylation at Thr389), and we found that the level of p-mTOR and PS6K1 was both decreased in the myocardium of CLP rats (P < 0.01; P < 0.05, Figure 4].

dIscussIon
The autophagy process has been indicated correlated with sepsis-induced myocardial dysfunction; however, the role of autophagy in the heart has been controversial, and its relationship with mTOR pathway is not well known. In this study, we found that compared with sham-operated rats, the autophagy process in myocardium was accelerated in CLP rats, and mTOR pathway was inhibited and closely correlated with the change of autophagic protein, indicating that the change of autophagy process in CLP rats may be mediated by mTOR pathway.
Autophagy is a dynamic process in which damaged organelles and long-lived proteins are delivered to the lysosome for degradation and recycling. [7] During this dynamic process, the outer membrane of the autophagosomes (a double-membrane vesicle) fuses with the lysosomal membrane forming an autolysosome. Within the lysosome, the inner autophagosomal membrane and autophagosomal compartment are degraded and the resulting molecules are recycled. As a housekeeping process, autophagy is vital for the normal structure and functions of the heart. [11] In addition, autophagy plays a critical role in the maintenance of cardiac functions by removing damaged proteins and subcellular organelles under stress conditions. [23] It also plays an important role in the modulation of I/R injury. [12] However, there was little evidence showing the change of entire process of autophagy in cardiomyocytes during sepsis. In our study, we found that 18 h after CLP, cardiomyocytes show a considerably higher LC3II/LC3I ratio (a marker of autophagy induction), accompanied by an increased number of autophagosomes. The autolysosomal (the product of an autophagosome fusing with a lysosome) numbers were also increased accompanied by a decrease in the expression of SQSTM1/p62 receptor proteins. These results indicate that the autophagy process was complete and accelerated in CLP rats. However, in a study by Hsieh et al., [13] they found that the process of autophagy in myocardial cells was not complete at 24 h after CLP. They found that localization of LC3 and LAMP1 (a lysosome marker) was decreased 24 h after CLP. They also observed that there were few autolysosomes in CLP mice. The reason why we got different results may be that we obtained myocardial tissues at different stages of sepsis. A recent study demonstrated that autophagy was activated initially in sepsis, followed by a subsequent phase of incompletion in the liver, [24] which may be similar in the heart. Even though the timing chosen was almost same, we ligated the cecum at half the distance between the distal pole and base of the cecum to establish the mid-grade sepsis model, whereas they ligated the cecum just below the ileocecal junction for high-grade sepsis. As a result, the results of our study may indicate the process of autophagy process in the heart during early stage of sepsis, which is a critical stage to detect the  b a myocardial dysfunction and repair timely. Furthermore, in this study, we investigated the formation and degradation of autophagosomes during autophagic process in the LV in CLP-induced septic mice. We found that the formation and the degradation of autophagosomes were both increased in the LV at 18 h after CLP, which confirmed the above dynamic change of autophagy process in myocardium of CLP-induced septic mice.
Furthermore, we found accompanied with the accelerated process of autophagy that the mTOR pathway was inhibited in CLP rats. p-mTOR and PS6K1, downstream targets of mTORC1, were significantly suppressed following CLP challenge. mTOR is a master sensor of energy status and will be inhibited in the state of energy depletion. Under nutrient-rich growth conditions, mTOR supports cellular growth and suppresses autophagy. On the contrary, in response to nutrient starvation, mTOR pathway is inhibited and autophagy is induced to provide energy source. Recent studies showed that in myocardial ischemia, the mTOR signaling pathway is inhibited by AMPK. [25] Energy depletion also plays a major role in septic cardiac dysfunction induced by mitochondrial dysfunction and reduced cardiac fatty acid oxidation. [26] Our previous studies have found that the mTOR pathway in CD8(+) T-cell was changed during the sepsis. [27] However, there is little evidence to prove the relevance of mTOR in sepsis-induced cardiac dysfunction, and nobody has confirmed the relationship between the mTOR pathway and the change of autophagy process in CLP-induced myocardial dysfunction.
Most importantly, we found that p-mTOR was significantly positively correlated with p62, PS6K1 was significantly positively correlated with p62 and negatively correlated with LC3B, which means that the activity of mTOR pathway was closely correlated with the change of autophagic protein, indicating that the change of autophagy process in CLP rats may be mediated by mTOR pathway. However, there was a limitation in our inference. To reveal mTOR pathway mediated the change of autophagy process in sepsis-induced myocardial dysfunction, it is necessary for us to inhibit or accelerate the mTOR pathway using the methods of pharmacology or gene knockout. In consideration of this, we will use mTOR myocardial-specific knockout mice to confirm our inference in our next step experiment.
In conclusion, the autophagy process in myocardium was accelerated in CLP rats, which was correlated with the inhibition of the mTOR pathway. Investigation the dynamic process of autophagy could help us clear out the controversial role of autophagy in CLP-induced myocardial dysfunction. Whether mTOR pathway could be used as a target of protection of sepsis-induced myocardial dysfunction in the future requires further study.

Financial support and sponsorship
The work was supported by a grant of the National Natural Science Foundation of China (No. 81601657).

Conflicts of interest
There are no conflicts of interest.