Faculty Bibliography

Coronary heart disease remains the principle cause of mortality in the United States. During aging, the efficiency of the cardiovascular system is decreased and the aged heart is less tolerant to ischemic injury. ATP-sensitive K(+) (K(ATP) ) channels protect the myocardium against ischemic damage. We investigated how aging affects cardiac K(ATP) channels in the Fischer 344 rat model. Expression of K(ATP) channel subunit mRNA and protein levels was unchanged in hearts from 26-month-old vs. 4-month-old rats. Interestingly, the mRNA expression of several other ion channels (> 80) was also largely unchanged, suggesting that posttranscriptional regulatory mechanisms occur during aging. The whole-cell K(ATP) channel current density was strongly diminished in ventricular myocytes from aged male rat hearts (also observed in aged C57BL/6 mouse myocytes). Experiments with isolated patches (inside-out configuration) demonstrated that the K(ATP) channel unitary conductance was unchanged, but that the inhibitory effect of cytosolic ATP on channel activity was enhanced in the aged heart. The mean patch current was diminished, consistent with the whole-cell data. We incorporated these findings into an empirical model of the K(ATP) channel and numerically simulated the effects of decreased cytosolic ATP levels on the human action potential. This analysis predicts lesser activation of K(ATP) channels by metabolic impairment in the aged heart and a diminished action potential shortening. This study provides insights into the changes in K(ATP) channels during aging and suggests that the protective role of these channels during ischemia is significantly compromised in the aged individual.

The endogenous phospholipid lysophosphatidic acid (LPA) regulates fundamental cellular processes such as proliferation, survival, motility, and invasion implicated in homeostatic and pathological conditions. Hence, delineation of the full range of molecular mechanisms by which LPA exerts its broad effects is essential. We report avid binding of LPA to the receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily, and mapping of the LPA binding site on this receptor. In vitro, RAGE was required for LPA-mediated signal transduction in vascular smooth muscle cells and C6 glioma cells, as well as proliferation and migration. In vivo, the administration of soluble RAGE or genetic deletion of RAGE mitigated LPA-stimulated vascular Akt signaling, autotaxin/LPA-driven phosphorylation of Akt and cyclin D1 in the mammary tissue of transgenic mice vulnerable to carcinogenesis, and ovarian tumor implantation and development. These findings identify novel roles for RAGE as a conduit for LPA signaling and suggest targeting LPA-RAGE interaction as a therapeutic strategy to modify the pathological actions of LPA.

Aldose reductase (AR), an enzyme mediating the first step in the polyol pathway of glucose metabolism, is associated with complications of diabetes mellitus and increased cardiac ischemic injury. We investigated whether deleterious effects of AR are due to its actions specifically in cardiomyocytes. We created mice with cardiac specific expression of human AR (hAR) using the alpha-myosin heavy chain (MHC) promoter and studied these animals during aging and with reduced fatty acid (FA) oxidation. hAR transgenic expression did not alter cardiac function or glucose and FA oxidation gene expression in young mice. However, cardiac overexpression of hAR caused cardiac dysfunction in older mice. We then assessed whether hAR altered heart function during ischemia reperfusion. hAR transgenic mice had greater infarct area and reduced functional recovery than non-transgenic littermates. When the hAR transgene was crossed onto the PPAR alpha knockout background, another example of greater heart glucose oxidation, hAR expressing mice had increased heart fructose content, cardiac fibrosis, ROS, and apoptosis. In conclusion, overexpression of hAR in cardiomyocytes leads to cardiac dysfunction with aging and in the setting of reduced FA and increased glucose metabolism. These results suggest that pharmacological inhibition of AR will be beneficial during ischemia and in some forms of heart failure.

The multi-ligand receptor RAGE was discovered on account of its ability to bind and transduce the cell stress-provoking signals of advanced glycation endproducts (AGEs). The finding that RAGE also bound pro-inflammatory molecules set the stage for linking RAGE and inflammation to the pathogenesis of diabetic macro- and microvascular complications. In this review, we focus on the roles of RAGE and its ligands in diabetes complications. We recount the findings from mice, rats, swine and human subjects suggesting that RAGE action potently contributes to vascular, inflammatory and end-organ stress and damage in types 1 and 2 diabetes. We detail the efforts to track ligands and RAGE in human subjects with diabetes to address if this axis may be a biomarker reflective of the state of the diabetic complications. Lastly, we suggest specific strategies to tackle AGE-ligand-RAGE interactions as potential therapeutic targets for diabetes and its complications.

Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3beta (p-GSK3beta). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3beta inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3beta was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3beta was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3beta and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3beta inhibitors improved p-GSK3beta expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3beta was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) alpha/beta inhibitor displayed significant increases in p-Akt and p-GSK3beta expression, and resulted in decreased LDH release. In summary, AR mediates changes in p-GSK3beta, in part, via PKCalpha/beta and Akt during I/R.

Insulin resistance is a characteristic feature of obesity and type 2 diabetes mellitus and impacts the heart in various ways. Impaired insulin-mediated glucose uptake is a uniformly observed characteristic of the heart in these states, although changes in upstream kinase signaling are variable and dependent on the severity and duration of the associated obesity or diabetes mellitus. The understanding of the physiological and pathophysiological role of insulin resistance in the heart is evolving. To maintain its high energy demands, the heart is capable of using many metabolic substrates. Although insulin signaling may directly regulate cardiac metabolism, its main role is likely the regulation of substrate delivery from the periphery to the heart. In addition to promoting glucose uptake, insulin regulates long-chain fatty acid uptake, protein synthesis, and vascular function in the normal cardiovascular system. Recent advances in understanding the role of metabolic, signaling, and inflammatory pathways in obesity have provided opportunities to better understand the pathophysiology of insulin resistance in the heart. This review will summarize our current understanding of metabolic mechanisms for and consequences of insulin resistance in the heart and will discuss potential new areas for investigating novel mechanisms that contribute to insulin resistance in the heart.

Cardiovascular disease represents the major cause of morbidity and mortality in patients with diabetes mellitus. Studies by us and others have implicated increased flux via aldose reductase (AR) as a key player in mediating diabetic complications, including cardiovascular complications. Data suggest that increased flux via AR in diabetics perpetuates increased injury after myocardial infarction, accelerates atherosclerotic lesion formation, and promotes restenosis via multiple mechanisms. Most importantly, studies have shown that increased generation of reactive oxygen species due to flux via AR has been a common feature in animal models of diabetic cardiovascular disease. Taken together, these considerations place AR in the center of biochemical and molecular stresses that characterize the cardiovascular complications of diabetes. Stopping AR-dependent signaling may hold the key to interrupting cycles of cellular perturbation and tissue damage in diabetic cardiovascular complications.

Background: RAGE and its ligands have been implicated in the pathogenesis of I/R injury through pathways of inflammation and apoptosis. We hypothesized that RAGE-directed quantitative imaging of myocardial uptake of 99mTc-anti-RAGE F(ab')2 in a mouse model of I/R can detect RAGE expression in the myocardium and show quantitative differences between early (18-20 h) and later times (48 h) after reperfusion. Methods: Twenty-four wild-type (WT) mice underwent left anterior descending coronary artery (LAD) occlusion for 30 min followed by reperfusion for 18-20 h (n = 8) or 48 h (n = 12) and 3 WT mice underwent sham operation without coronary intervention. Twenty-seven mice (including shams) were injected with 19.98 +/- 1.78 MBq 99mTc anti-RAGE F(ab')2 and 5 h later, mice were injected with 6.14 +/- 2.0 MBq 201Tl and immediately underwent dual isotope SPECT/CT imaging. Four WT mice were injected with control nonspecific F(ab')2 and similarly imaged at 18-20 h. At the completion of imaging, hearts were removed, radioactivity counted, and sectioned for histology. Results: The uptake of 99mTc-anti-RAGE F(ab')2 in the ischemic zone from the scans as mean percentage injected dose (%ID) was significantly greater at 18-20 h (5.7 +/- 2.1 x 10-3%) compared to 48 h (1.4 +/- 1.1 x 10-3%; p < 0.001) following reperfusion. Disease and antibody controls showed no focal uptake in the infarct. Gamma well counting of the myocardium from experiments performed at 18-20 h and 48 h post reperfusion supported the quantitative scan data. Immunohistochemical staining of the myocardium showed greater caspase-3 and RAGE staining at 18-20 h vs. 48 h (p = 0.04 and p = 0.01, respectively). On dual immunofluorescence, RAGE colocalized mainly with injured cardiomyocytes undergoing apoptosis. Conclusion: RAGE expression in reperfusion injury can be imaged in-vivo using a novel 99mTc-anti-RAGE F(ab')2. This imaging approach may be useful in evaluating contribution of RAGE to myocardial ischemic injury in diseases with increased expression such as diabetes

Rationale: The mammalian diaphanous-related formin (mDia1), governs microtubule and microfilament dynamics while functioning as an effector for Rho small GTP-binding proteins during key cellular processes such as adhesion, cytokinesis, cell polarity, and morphogenesis. The cytoplasmic domain of the receptor for advanced glycation endproducts binds to the formin homology 1 domain of mDia1; mDia1 is required for receptor for advanced glycation endproducts ligand-induced cellular migration in transformed cells. Objective: Because a key mechanism in vascular remodeling is the induction of smooth muscle cell migration, we tested the role of mDia1 in this process. Methods and Results: We report that endothelial denudation injury to the murine femoral artery significantly upregulates mDia1 mRNA transcripts and protein in the injured vessel, particularly in vascular smooth muscle cells within the expanding neointima. Loss of mDia1 expression significantly reduces pathological neointimal expansion consequent to injury. In primary murine aortic smooth muscle cells, mDia1 is required for receptor for advanced glycation endproducts ligand-induced membrane translocation of c-Src, which leads to Rac1 activation, redox phosphorylation of AKT/glycogen synthase kinase 3beta, and consequent smooth muscle cell migration. Conclusions: We conclude that mDia1 integrates oxidative and signal transduction pathways triggered, at least in part, by receptor for advanced glycation endproducts ligands, thereby regulating pathological neointimal expansion.

The receptor for advanced glycation end products (RAGE) is expressed in the heart in cardiomyocytes, vascular cells, fibroblasts, and in infiltrating inflammatory cells. Experiments in murine, rat, and swine models of injury suggest that RAGE and the ligands of RAGE are upregulated in key injuries to the heart, including ischemia/reperfusion injury, diabetes, and inflammation. Pharmacological antagonism of RAGE or genetic deletion of the receptor in mice is strikingly protective in models of these stresses. Data emerging from human studies suggest that measurement of levels of RAGE ligands or soluble RAGEs in plasma or serum may correlate with the degree of heart failure. Taken together, the ligand-RAGE axis is implicated in heart failure and we predict that therapeutic antagonism of RAGE might be a unique target for therapeutic intervention in this disorder.