Exogenous HGF Prevents Cardiomyocytes from Apoptosis after Hypoxia via Up-Regulating Cell Autophagy

Background: Hepatocyte growth factor (HGF) is widely known as a protective factor in ischemic myocardium, however HGF sensitive cellular mechanism remained ill-defined. Autophagy at early stage of hypoxia has been demonstrated to play a role in protecting myocardium both in vivo and vitro. We performed this study to investigate the association between the protective effect of HGF and autophagy. Methods: Ventricular myocytes were isolated from neonatal rat heart (NRVMs). We evaluated cardiomyocytes apoptosis by Hoechst staining and flow cytometry. Autophagy was assessed by transmission electron microscope and mRFP-GFP-LC3 adenovirus infection. Mitochondrial membrane potential was estimated by JC-1 staining. Western blotting and ELISA assay were used to quantify protein concentrations. Results: We found that autophagy in NRVMs increased at early stage after hypoxia and HGF release was consistent with the change of autophagy. Exogenous HGF enhanced autophagy and decreased apoptosis, while neutralizing HGF yielded opposite effects. Besides, inhibition of autophagy increased apoptosis of myocytes. Furthermore, exogenous HGF induced Parkin, the marker of mitochondrial autophagy, indicating increased clearance of injured mitochondria. Conclusions: Our results revealed a potential mechanism in which exogenous HGF prevented NRVMs from apoptosis after hypoxia. Upregulation of Parkin through administration of exogenous HGF may be a potential therapeutic strategy ptotecting myocytes during ischemia.

Development of a Vascularized Heterotopic Neonatal Rat Heart Transplantation Model

Background/Purpose: Rodent adult-to-adult heterotopic heart transplantation is a well-established animal model, and the detailed surgical technique with several modifications has been previously described. In immature donor organ transplantation, however, the surgical technique needs to be revised given the smaller size and fragility of the donor graft. Here, we report our surgical technique for heterotopic abdominal (AHTx) and femoral (FHTx) neonatal rat heart transplantation based on an experience of over 300 cases. Methods: Heterotopic heart transplantation was conducted in syngeneic Lewis rats. Neonatal rats (postnatal day 2-4) served as donors. AHTx was performed by utilizing the conventional adult-to-adult transplant method with specific modifications for optimal aortotomy and venous anastomosis. In the FHTx, the donor heart was vascularized by connecting the donor's aorta and pulmonary artery to the recipient's right femoral artery and vein, respectively, in an end-to-end manner. A specifically fashioned butterfly-shaped rubber sheet was used to align the target vessels properly. The transplanted graft was visually assessed for its viability and was accepted as a technical success when the viability met specific criteria. Successfully transplanted grafts were subject to further postoperative evaluation. Forty cases (AHTx and FHTx; n = 20 each) were compared regarding perioperative parameters and outcomes. Results: Both models were technically feasible (success rate: AHTx 75% vs. FHTx 70%) by refining the conventional heterotopic transplant technique. Injury to the fragile donor aorta and congestion of the graft due to suboptimal venous connection were predominant causes of failure, leading to refractory bleeding and poor graft viability. Although the FHTx required significantly longer operation time and graft ischemic time, the in situ graft viabilities were comparable. The FHTx provided better postoperative monitoring as it enabled daily graft palpation and better echocardiographic visualization. Conclusions: We describe detailed surgical techniques for AHTx and FHTx while addressing neonatal donor-specific issues. Following our recommendations potentially reduces the learning curve to achieve reliable and reproducible results with these challenging animal models.

Antibody Reactivity to α-Enolase in Mothers of Children with Congenital Heart Block

Objective.To evaluate the frequency of anti-α-enolase antibodies in the sera of mothers whose children have congenital heart block (CHB), given provocative results in which α-enolase, a membrane protein, was recognized by monoclonal antibodies reactive with the peptide p200 of 52 kDa Ro/SSA in a neonatal rat heart library.Methods.An ELISA using a recombinant α-enolase protein was developed. Sera from 100 anti-Ro52+ CHB mothers in the Research Registry for Neonatal Lupus, 50 patients with systemic lupus erythematosus (SLE; 7 anti-Ro52+), and 48 healthy controls were tested for anti-α-enolase reactivity.Results.There were no significant differences in the median values obtained from CHB mothers, patients with SLE, or controls at each of the dilutions tested. Only 7 (7%) at 1:100 dilution and 2 (2%) at 1:1000 dilution of 100 CHB sera were 3 standard deviations above the mean value obtained for controls. Preincubation with recombinant Ro52 did not inhibit anti-α-enolase reactivity.Conclusion.The low frequency of anti-α-enolase antibodies in the sera of CHB mothers and the absence of apparent cross-reactivity with Ro52 suggest that antibodies to Ro52 are not likely to mediate CHB via binding to α-enolase.