The Role And Mechanisms Of Peli1 Involved In Diabetic Cardiomyopathy

Background:Cardiovascular implications are the leading course of death in diabetic patients.Besides micro/macro-vascular changes, cardiomyocytes apoptosis, hypertrophy and interstitial fibrosis are also distinguished. Diabetic cardiomyopathy (DCM) refers to ventricular dysfunction in patients with diabetes in the absence of coronary atherosclerosis and hypertension. Diastolic dysfunction, especially decreased E/A ratio, is recognized as the earliest manifestation of diabetes mellitus induced cardiac dysfunction. Many risk factors such as high glucose, insulin resisitance, lipids metabolic disorder, chronic inflammation, are proved to play an important role in DCM. Although the pathophysiological mechanisms leading to diabetic cardiomyopathy are certainly multifactoriai, decreased mitochondrial abnormalities may play an important role. Notably, recently, the interactions between TLRs and mitochondria are proved to be involved a series of physiological and pathological processes, especially, TLR4 signaling could augment ROS production from mitochondria accompanied with impaired mitochondrial complex I. But in DCM, whether TLRs signaling pathway is involved in mitochondria dysfunction is still unclear. Toll-like receptors (TLRs) are evolutionary conserved pattern recognition receptors and very important for the activation of innate immune system. It has been well documented that TLRs signaling pathway are involved in heart failure. In diabetic mellitus, which has been reported, eleveated FFAs can promote the activation of TLR4, and notably, TLR4 deletion has a protective effect to diabetic cardiomyopathy, but the mechanisms are still unclear. Pellino family (Pelil/2/3) is a group of new defined E3 liagse in Toll-like receptors (TLRs) signaling pathway. Pelil is involved in both myeloid differentiation factor 88(Myd88) depended and non-depended TLRs signaling pathway by interacting with interleukin-1 receptor associated kinase/TNFR-associated factor 6 (IRAK/TRAF6) or receptor interacting proteinl/Toll/IL-1R domain-containing adaptor inducing IFN-β3 (RIP1/TRIF3), and its E3 ligase activity is indispensable in those processes. But the role of Pelil in DCM is still unclear.Aim:to investagate the role of Pelil in DCM; to investagate the role and mechanisms of Pelil in mitochondrial dysfunction in DCM.Results:Part I:1) Pelil expression and its E3 ligase activity increased in diabetic mice hearts. The protein level of Pelil increased about 2 times during the course of the 8 weeks study. And the E3 ligase activity of Pelil was also obviously promoted in diabetic group, Pelil mRNA increased too compared to the control group.2) Pelil conditional knockout ameliorates cardiac diastolic dysfunction in diabetic mice. STZ administrator increased the fasting blood glucose level to 20mM, both in the PelilFiox/Fiox and peincKO group.At 8 weeks endpOint of the study, diabetic groups did not represent significant difference in HW/BW compared with other groups. Meanwhile, the systolic function index EF% and FS% also did not show any change among the groups. But the E/A ratio of diabetic PelilFlox/Flox group decreased from normal 1.74 to 1.26, and Pelil conditional knockout significantly ameliorated that. All of those observations based on no change of mean blood pressure among groups. Unexpectingly, we did not find interstitial fibrosis either diabetic or normal groups. TUNEL stain presented that the apoptosis cells were significantly reduced in diabetic PelicKO group compared to PelilFlox/Flox group.Part Ⅱ:Pelil invovled in FFA induced primary cardiomyocytes cell apoptosis:1) Free fatty acids (FFAs), as expectly, also elevated to about 200μM in diabetic mice blood plasma. Subsequently, we found both the mRNA and protein levels of Pelil were elevated on a time-dependent model in the presence of FFA (200μM Palmatic acid). And the E3 ligase activity of Pelil was also up-regulated even under treatment of FFA only for 30min which there was no change in Pelil protein level. 2) In the presence of FFA, the cleaved-Caspas3,cleaved-Caspas9 and apoptosis factor Bax were greatly up-regulated, and the anti-apoptosis factor Bcl-xl was obviously down-regulated. Reversely, abating Pelil expression by siRNA or Knockout could inverse that effect of FFA. Flow Cytometry analysis presented FFA promoted the MEFs apoptosis from normal 2.9% to 12.8%, but Pelil knockout reduced to 9.5%.Part Ⅲ:1) Pelil suppression improved FFA induced mitochondrial dysfunction. We detected the functional change of mitochondria in the presence of FFA in vitro. Indeed, mitochondria isolated from FFA treated primary cardiomyocytes represented a 61% decrease in OCR compared to BSA control group when supplied with mitochondria complex I substrate malate/glumate and ATP, but not complex II substrate succinate, and siPelil improved the OCR near one times. In intact myocytes, FFA treatment inhibited the OCR both at the basal (14.3%) and the maximum level (37.9%), compared to BSA group, siPelil ameliorated the OCR significantly at the basal (11.1%) and slightly (4.7%) at maximum level. Mitochondria membrane potentials is critical for normal metabolism, JC-1 stain showed that yellow emission fluorescence for normal membrane potentials got down and green emission fluorescence for low membrane potentials got up, and siPelil reversed that phenomenon but not siNC.2) Pelil involved in ROS generation and ATP deficiency in diabetic cardiomyopathy:DHE stain of fresh frozen section presented that ROS level elevated in diabetic PelilFlox/Flox group, but attenuated in PelilcK0 group. In vitro, in primary cardiomyocytes, FFA treatment obviously increased the ROS level in the both indicator, by contrast, Pelil siRN A knockdown attenuated the ROS generation. Similarly, in the presence of FFA, the ROS generation was up-regulated in WT MEFs, but not in Pelil knockout MEFs. Further, we also observed siPelil increased the ATP level in cells which decreased in FFA treatment, these findings was confirmed in Pelil kockout MEFs.3) Pelil located on the outer membrane of mitochondria in the presence of FFA. Confocal laser scanning microscope analysis presented that Pelil located on the outer-membrane of mitochondria in the presence of FFA in primary cardiomyocytes. And more Pelil and TRAF6 were detected in FFA treated mitochondrial lysates than in BSA control. Similarly, Pelil and TRAF6 did not appear in Protease K treated groups, Tom20 an outer membrane protein of mitochondria was also undetectable, but VDAC which locate on both outer and inner membrane, was still detectable.4) Pelil promotes the protein interaction between TRAF6 and ECSIT. Precisely, the ECSIT and binding with TRAF6 was significantly up-regulated under the stimulation of FFA, but Pelil knockout abolished that in MEFs. It was confirmed in primary cardiomyocytes by Pelil siRNA knockdown. And we observed same change in diabetic heart mitochondrial lysates.Conclusions:1. Peli1 and its E3 ligase activity are eleveated in DCM, PelilcKO exhibited an protect effect in diastolic dysfunction at early stage of DCM. Peli1 and its E3 ligase activity plays an important role in DCM.2. FFAs are eleveated in diabetic blood plasma; and Peli1 is involved in FFA induced cardiomyocytes cell apoptosis.3. Peli1 is involved in mitochondrial dysfunction both in DCM and in FFA treated cardiomyocytes. Peli1 promotes TRAF6 interacting with ECSIT on the outer membrane of mitochondria through its E3 ligase activity. The shift of ECSIT from inner-membranes to outer-membranes impairs the mitochondrial complex assembly, then decreases the ATP production and increases the mitochondrial ROS production.