Huei-Ping Tzeng

The Cytoprotective Effects of Tumor Necrosis Factor Are Conveyed Through Tumor Necrosis Factor Receptor–Associated Factor 2 in the Heart

Background—Activation of both type 1 and type 2 tumor necrosis factor (TNF) receptors (TNFR1 and TNFR2) confers cytoprotection in cardiac myocytes. Noting that the scaffolding protein TNF receptor–associated factor 2 (TRAF2) is common to both TNF receptors, we hypothesized that the cytoprotective responses of TNF were mediated through TRAF2. Methods and Results—Mice with cardiac-restricted overexpression of low levels of TNF (MHCsTNF3) and TRAF2 (MHC-TRAF2LC) and mice lacking TNFR1, TNFR2, and TNFR1/TNFR2 were subjected to ischemia (30 minutes) reperfusion (30 minutes) injury ex vivo using a Langendorff apparatus. MHCsTNF3 mice were protected against ischemia-reperfusion injury as shown by a significant ≈30% greater left ventricular developed pressure, ≈80% lower creatine kinase release, and Evans blue dye uptake compared with littermates. The extent of ischemia-reperfusion induced injury was similar in wild-type, TNFR1, and TNFR2 deficient mice; however, mice lacking TNFR1/TNFR2 had a significant ≈40% lower left ventricular developed pressure, a ≈65% greater creatine kinase release, and ≈40% greater Evans blue dye uptake compared with littermates. Interestingly, MHC-TRAF2LC mice had a significant ≈50% lower left ventricular developed pressure, a ≈70% lower creatine kinase release, and ≈80% lower Evans blue dye uptake compared with littermate controls after ischemia-reperfusion injury. Biochemical analysis of the MHC-TRAF2LC hearts showed that there was activation of nuclear factor-kappaB but not c-Jun N-terminal kinase activation. Conclusion—Taken together, these results suggest that TNF confers cytoprotection in the heart through TRAF2-mediated activation of nuclear factor-&kgr;B.

Negative inotropic effects of high-mobility group box 1 protein in isolated contracting cardiac myocytes

High-mobility group box 1 (HMGB1) released from necrotic cells or macrophages functions as a late inflammatory mediator and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients by using fluo-3, and L-type calcium currents by using whole cell perforate configuration of the patch-clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca2+ transient within 5 min (P < 0.01). The immediate negative inotropic effects of HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward l-type calcium currents was also documented by the patch-clamp technique. HMGB1 induced the PKC-epsilon translocation, and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury.

The Development of Myocardial Fibrosis in Transgenic Mice With Targeted Overexpression of Tumor Necrosis Factor Requires Mast Cell–Fibroblast Interactions

Background— Transgenic mice with cardiac-restricted overexpression of tumor necrosis factor (MHCsTNF mice) develop progressive myocardial fibrosis, diastolic dysfunction, and adverse cardiac remodeling. Insofar as tumor necrosis factor (TNF) does not directly stimulate fibroblast collagen synthesis, we asked whether TNF-induced fibrosis was mediated indirectly through interactions between mast cells and cardiac fibroblasts. Methods and Results— Cardiac mast cell number increased 2 to 3 fold (P<0.001) in MHCsTNF mice compared with littermate controls. Outcrossing MHCsTNF mice with mast cell–deficient (c-kit−/−) mice showed that the 11-fold increase (P<0.001) in collagen volume fraction in MHCsTNF/c-kit+/− mice was abrogated in MHCsTNF/c-kit−/− mice, and that the leftward shifted left ventricular pressure–volume curve in the MHCsTNF/c-kit+/− mice was normalized in the MHCsTNF/c-kit−/− hearts. Furthermore, the increase in transforming growth factor &bgr;1 and type I transforming growth factor &bgr; receptor messenger RNA levels was significantly (P=0.03, P=0.01, respectively) attenuated in MHCsTNF/c-kit−/− when compared with MHCsTNF/c-kit+/− mice. Coculture of fibroblasts with mast cells resulted in enhanced &agr;-smooth muscle actin expression, increased proliferation and collagen messenger RNA expression, and increased contraction of 3-dimensional collagen gels in MHCsTNF fibroblasts compared with littermate fibroblasts. The effects of mast cells were abrogated by type I transforming growth factor &bgr; receptor antagonist NP-40208. Conclusions— These results suggest that increased mast cell density with resultant mast cell–cardiac fibroblast cross-talk is required for the development of myocardial fibrosis in inflammatory cardiomyopathy. Cardiac fibroblasts exposed to sustained inflammatory signaling exhibit an increased repertoire of profibrotic phenotypic responses in response to mast cell mediators.

Innate immunity mediates myocardial preconditioning through Toll-like receptor 2 and TIRAP-dependent signaling pathways

Recent studies have implicated Toll-like receptor 2 (TLR2) and TLR4 signaling in delimiting liver and brain injury following ischemia-reperfusion (I/R). To determine whether TLR2 and TLR4 conferred cytoprotection in the heart, we subjected hearts of wild-type (WT) mice and mice deficient in TLR2 (TLR2D), TLR4 (TLR4D), and TIR domain-containing adapter protein (TIRAP-D) to ischemic preconditioning (IPC). Langendorff-perfused hearts were subjected to 30 min ischemia and 60 min reperfusion with or without IPC. IPC resulted in a significant increase (P < 0.05) in the percent recovery of left ventricular developed pressure (%LVDP) in WT mouse hearts (54.4 +/- 2.7% of baseline), whereas there was no significant increase in %LVDP (P > 0.05) in TIRAP-D mouse hearts (43.8 +/- 1.9%) after I/R injury. IPC also resulted in a significant (P < 0.05) decrease in I/R-induced creatine kinase release and Evans blue dye uptake in WT but not TIRAP-D hearts. Interestingly, IPC resulted in a significant (P < 0.05) increase in %LVDP in TLR4-deficient hearts (52.7 +/- 3%) but not in TLR2D hearts (39.3 +/- 1.5%). Pretreatment with a specific TLR2 ligand (Pam3CSK) protected WT hearts against I/R-induced left ventricular dysfunction. The loss of IPC-induced cardioprotection in TIRAP-D mouse hearts was accompanied by a decreased translocation of protein kinase C-epsilon and decreased phosphorylation of GSK-3beta. Taken together, these data suggest that the cardioprotective effect of IPC is mediated, at least in part, through a TLR2-TIRAP-dependent pathway, suggesting that the modulation of this pathway represents a viable target for reducing I/R injury.

Tumor Necrosis Factor Receptor Associated Factor 2 Signaling Provokes Adverse Cardiac Remodeling in the Adult Mammalian Heart

Background—Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal through a common scaffolding protein termed tumor necrosis factor receptor–associated factor 2 (TRAF2); however, virtually nothing is known about TRAF2 signaling in the adult mammalian heart. Methods and Results—We generated multiple founder lines of mice with cardiac-restricted overexpression of TRAF2 and characterized the phenotype of mice with higher expression levels of TRAF2 (myosin heavy chain [MHC]-TRAF2HC). MHC-TRAF2HC transgenic mice developed a time-dependent increase in cardiac hypertrophy, left ventricular dilation, and adverse left ventricular remodeling, and a significant decrease in LV+dP/dt and LV−dP/dt when compared with littermate controls (P<0.05 compared with littermate). During the early phases of left ventricular remodeling, there was a significant increase in total matrix metalloproteinase activity that corresponded with a decrease in total myocardial fibrillar collagen content. As the MHC-TRAF2HC mice aged, there was a significant decrease in total matrix metalloproteinase activity accompanied by an increase in total fibrillar collagen content and an increase in myocardial tissue inhibitor of metalloproteinase-1 levels. There was a significant increase in nuclear factor–&kgr;B activation at 4 to 12 weeks and jun N-terminal kinases activation at 4 weeks in the MHC-TRAF2HC mice. Transciptional profiling revealed that >95% of the hypertrophic/dilated cardiomyopathy–related genes that were significantly upregulated genes in the MHC-TRAF2HC hearts contained &kgr;B elements in their promoters. Conclusions—These results show for the first time that targeted overexpression of TRAF2 is sufficient to mediate adverse cardiac remodeling in the heart.

Dysferlin Mediates the Cytoprotective Effects of TRAF2 Following Myocardial Ischemia Reperfusion Injury

Background We have demonstrated that tumor necrosis factor (TNF) receptor‐associated factor 2 (TRAF2), a scaffolding protein common to TNF receptors 1 and 2, confers cytoprotection in the heart. However, the mechanisms for the cytoprotective effects of TRAF2 are not known. Methods/Results Mice with cardiac‐restricted overexpression of low levels of TRAF2 (MHC‐TRAF2LC) and a dominant negative TRAF2 (MHC‐TRAF2DN) were subjected to ischemia (30‐minute) reperfusion (60‐minute) injury (I/R), using a Langendorff apparatus. MHC‐TRAF2LC mice were protected against I/R injury as shown by a significant ≈27% greater left ventricular (LV) developed pressure after I/R, whereas mice with impaired TRAF2 signaling had a significantly ≈38% lower LV developed pressure, a ≈41% greater creatine kinase (CK) release, and ≈52% greater Evans blue dye uptake after I/R, compared to LM. Transcriptional profiling of MHC‐TRAF2LC and MHC‐TRAF2DN mice identified a calcium‐triggered exocytotic membrane repair protein, dysferlin, as a potential cytoprotective gene responsible for the cytoprotective effects of TRAF2. Mice lacking dysferlin had a significant ≈39% lower LV developed pressure, a ≈20% greater CK release, and ≈29% greater Evans blue dye uptake after I/R, compared to wild‐type mice, thus phenocopying the response to tissue injury in the MHC‐TRAF2DN mice. Moreover, breeding MHC‐TRAF2LC onto a dysferlin‐null background significantly attenuated the cytoprotective effects of TRAF2 after I/R injury. Conclusion The study shows that dysferlin, a calcium‐triggered exocytotic membrane repair protein, is required for the cytoprotective effects of TRAF2‐mediated signaling after I/R injury.

TNF receptor–activated factor 2 mediates cardiac protection through noncanonical NF- κ B signaling

To elucidate the mechanisms responsible for cytoprotective effects of TNF receptor-activated factor 2 (TRAF2) in the heart, we employed genetic gain- and loss-of-function studies ex vivo and in vivo in mice with cardiac-restricted overexpression of TRAF2 (Myh6-TRAF2LC). Crossing Myh6-TRAF2LC mice with mice lacking canonical signaling (Myh6-TRAF2LC/Myh6-IκBαΔN) abrogated the cytoprotective effects of TRAF2 ex vivo. In contrast, inhibiting the JAK/STAT pathway did not abrogate the cytoprotective effects of TRAF2. Transcriptional profiling of WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts suggested that the noncanonical NF-κB signaling pathway was upregulated in the Myh6-TRAF2LC mouse hearts. Western blotting and ELISA for the NF-κB family proteins p50, p65, p52, and RelB on nuclear and cytoplasmic extracts from naive 12-week-old WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts showed increased expression levels and increased DNA binding of p52 and RelB, whereas there was no increase in expression or DNA binding of the p50 and p65 subunits. Crossing Myh6-TRAF2LC mice with RelB-/+ mice (Myh6-TRAF2LC/RelB-/+) attenuated the cytoprotective effects of TRAF2 ex vivo and in vivo. Viewed together, these results suggest that crosstalk between the canonical and noncanonical NF-κB signaling pathways is required for mediating the cytoprotective effects of TRAF2.