Walter E. Rodriguez

Dietary copper supplementation reverses hypertrophic cardiomyopathy induced by chronic pressure overload in mice

Sustained pressure overload causes cardiac hypertrophy and the transition to heart failure. We show here that dietary supplementation with physiologically relevant levels of copper (Cu) reverses preestablished hypertrophic cardiomyopathy caused by pressure overload induced by ascending aortic constriction in a mouse model. The reversal occurs in the continued presence of pressure overload. Sustained pressure overload leads to decreases in cardiac Cu and vascular endothelial growth factor (VEGF) levels along with suppression of myocardial angiogenesis. Cu supplementation replenishes cardiac Cu, increases VEGF, and promotes angiogenesis. Systemic administration of anti-VEGF antibody blunts Cu regression of hypertrophic cardiomyopathy. In cultured human cardiomyocytes, Cu chelation blocks insulin-like growth factor (IGF)-1– or Cu-stimulated VEGF expression, which is relieved by addition of excess Cu. Both IGF-1 and Cu activate hypoxia-inducible factor (HIF)-1α and HIF-1α gene silencing blocks IGF-1– or Cu-stimulated VEGF expression. HIF-1α coimmunoprecipitates with a Cu chaperone for superoxide dismutase-1 (CCS), and gene silencing of CCS, but not superoxide dismutase-1, prevents IGF-1– or Cu-induced HIF-1α activation and VEGF expression. Therefore, dietary Cu supplementation improves the condition of hypertrophic cardiomyopathy at least in part through CCS-mediated HIF-1α activation of VEGF expression and angiogenesis.

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.

Mitochondrial mechanism of oxidative stress and systemic hypertension in hyperhomocysteinemia

Formation of homocysteine (Hcy) is the constitutive process of gene methylation. Hcy is primarily synthesized by de‐methylation of methionine, in which s‐adenosyl‐methionine (SAM) is converted to s‐adenosyl‐homocysteine (SAH) by methyltransferase (MT). SAH is then hydrolyzed to Hcy and adenosine by SAH‐hydrolase (SAHH). The accumulation of Hcy leads to increased cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased. In this process, Ca2+‐dependent mitochondrial nitric oxide synthase (mtNOS) and calpain are induced which lead to cytoskeletal de‐arrangement and cellular remodeling. This process generates peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction. Chronic exposure to Hcy instigates endothelial and vascular dysfunction and increases vascular resistance causing systemic hypertension. To compensate, the heart increases its load which creates adverse cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia. J. Cell. Biochem.

Cytochrome P450 (CYP) 2J2 gene transfection attenuates MMP-9 via inhibition of NF-κβ in hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase‐9 (MMP‐9). Cytochrome P450 (CYP) epoxygenase‐2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF‐κβ. However, the underlying molecular mechanisms for MMP‐9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)‐induced MMP‐9 activation. CYP2J2 was over‐expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF‐κβ‐mediated MMP‐9 regulation were evaluated using Western blot, in‐gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K‐dependent AKT signal. Hcy induced the nuclear translocation of NF‐κβ via downregulation of IKβα (endogenous cytoplasmic inhibitor of NF‐κβ). Hcy induced MMP‐9 activation by increasing NF‐κβ–DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9‐EET phosphorylated the AKT and attenuated Hcy‐induced MMP‐9 activation. This occurred, in part, by the inhibition of NF‐κβ nuclear translocation, NF‐κβ–DNA binding and activation of IKβα. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP‐9 signal. J. Cell. Physiol. 215: 771–781, 2008.

Hyperhomocysteinemic Diabetic Cardiomyopathy: Oxidative Stress, Remodeling, and Endothelial-Myocyte Uncoupling

Accumulation of oxidized-matrix (fibrosis) between the endothelium (the endothelial cells embedded among the myocytes) and cardiomyocytes is a hallmark of diabetes mellitus and causes diastolic impairment. In diabetes mellitus, elevated levels of homocysteine activate matrix metalloproteinase and disconnect the endothelium from myocytes. Extracellular matrix functionally links the endothelium to the cardiomyocyte and is important for their synchronization. However, in diabetes mellitus, a disconnection is caused by activated metalloproteinase, with subsequent accumulation of oxidized matrix between the endothelium and myocyte. This contributes to endothelial-myocyte uncoupling and leads to impaired diastolic relaxation of the heart in diabetes mellitus. Elevated levels of homocysteine in diabetes are attributed to impaired homocysteine metabolism by glucose and insulin and decreased renal clearance. Homocysteine induces oxidative stress and is inversely related to the expression of peroxisome proliferators activated receptor (PPAR). Several lines of evidence suggest that ablation of the matrix metalloproteinase (MMP-9) gene ameliorates the endothelial-myocyte uncoupling in diabetes mellitus. Homocysteine competes for, and decreases the PPARγ activity. In diabetes mellitus, endothelial-myocyte uncoupling is associated with matrix metalloproteinase activation and decreased PPARγ activity. The purpose of this review is to discuss the role of endothelial-myocyte uncoupling in diabetes mellitus and increased levels of homocysteine, causing activation of latent metalloproteinases, decreased levels of thioredoxin and peroxiredoxin, and cardiac tissue inhibitor of metalloproteinase (CIMP) in response to antagonizing PPARγ.

Mechanisms of endothelial dysfunction with development of type 1 diabetes mellitus: Role of insulin and C‐peptide

Complications associated with insulin‐dependent diabetes mellitus (type‐1diabetes) primarily represent vascular dysfunction that has its origin in the endothelium. While many of the vascular changes are more accountable in the late stages of type‐1diabetes, changes that occur in the early or initial functional stages of this disease may precipitate these later complications. The early stages of type‐1diabetes are characterized by a diminished production of both insulin and C‐peptide with a significant hyperglycemia. During the last decade numerous speculations and theories have been developed to try to explain the mechanisms responsible for the selective changes in vascular reactivity and/or tone and the vascular permeability changes that characterize the development of type‐1diabetes. Much of this research has suggested that hyperglycemia and/or the lack of insulin may mediate the observed functional changes in both endothelial cells and vascular smooth muscle. Recent studies suggest several possible mechanisms that might be involved in the observed decreases in vascular nitric oxide (NO) availability with the development of type‐1 diabetes. In addition more recent studies have indicated a direct role for both endogenous insulin and C‐peptide in the amelioration of the observed endothelial dysfunction. These results suggest a synergistic action between insulin and C‐peptide that facilitates increase NO availability and may suggest new clinical treatment modalities for type‐1 diabetes mellitus.

Ciglitazone, a PPARγ agonist, ameliorates diabetic nephropathy in part through homocysteine clearance

Diabetes and hyperhomocysteinemia (HHcy) are two independent risk factors for glomeruloslerosis and renal insufficiency. Although PPARgamma agonists such as ciglitazone (CZ) are known to modulate diabetic nephropathy, the role of CZ in diabetes-associated HHcy and renopathy is incompletely defined. We tested the hypothesis that induction of PPARgamma by CZ decreases tissue Hcy level; this provides a protective role against diabetic nephropathy. C57BL/6J mice were administered alloxan to create diabetes. Mice were grouped to 0, 1, 10, 12, and 16 wk of treatment; only 12- and 16-wk animals received CZ in drinking water after a 10-wk alloxan treatment. In diabetes, PPARgamma cDNA, mRNA, and protein expression were repressed, whereas an increase in plasma and glomerular Hcy levels was observed. CZ normalized PPARgamma mRNA and protein expression and glomerular level of Hcy, whereas plasma level of Hcy remained unchanged. GFR was dramatically increased at 1-wk diabetic induction, followed by hypofiltration at 10 wk, and was normalized by CZ treatment. This result corroborated with glomerular and preglomerular arteriole histology. A steady-state increase of RVR in diabetic mice became normal with CZ treatment. CZ ameliorated decrease bioavailability of NO in the diabetic animal. Glomerular MMP-2 and MMP-9 activities as well as TIMP-1 expression were increased robustly in diabetic mice and normalized with CZ treatment. Interestingly, TIMP-4 expression was opposite to that of TIMP-1 in diabetic and CZ-treated groups. These results suggested that diabetic nephropathy exacerbated glomerular tissue level of Hcy, and this caused further deterioration of glomerulus. CZ, however, protected diabetic nephropathy in part by activating PPARgamma and clearing glomerular tissue Hcy.

Restoration of contractility in hyperhomocysteinemia by cardiac-specific deletion of NMDA-R1

Homocysteine (HCY) activated mitochondrial matrix metalloproteinase-9 and led to cardiomyocyte dysfunction, in part, by inducing mitochondrial permeability (MPT). Treatment with MK-801 [N-methyl-d-aspartate (NMDA) receptor antagonist] ameliorated the HCY-induced decrease in myocyte contractility. However, the role of cardiomyocyte NMDA-receptor 1 (R1) activation in hyperhomocysteinemia (HHCY) leading to myocyte dysfunction was not well understood. We tested the hypothesis that the cardiac-specific deletion of NMDA-R1 mitigated the HCY-induced decrease in myocyte contraction, in part, by decreasing nitric oxide (NO). Cardiomyocyte-specific knockout of NMDA-R1 was generated using cre/lox technology. NMDA-R1 expression was detected by Western blot and confocal microscopy. MPT was determined using a spectrophotometer. Myocyte contractility and calcium transients were studied using the IonOptix video-edge detection system and fura 2-AM loading. We observed that HHCY induced NO production by agonizing NMDA-R1. HHCY induced the MPT by agonizing NMDA-R1. HHCY caused a decrease in myocyte contractile performance, maximal rate of contraction and relaxation, and prolonged the time to 90% peak shortening and 90% relaxation by agonizing NMDA-R1. HHCY decreased contraction amplitude with the increase in calcium concentration. The recovery of calcium transient was prolonged in HHCY mouse myocyte by agonizing NMDA-R1. It was suggested that HHCY increased mitochondrial NO levels and induced MPT, leading to the decline in myocyte mechanical function by agonizing NMDA-R1.

Nitrotyrosinylation, remodeling and endothelial‐myocyte uncoupling in iNOS, cystathionine beta synthase (CBS) knockouts and iNOS/CBS double knockout mice

Increased levels of homocysteine (Hcy), recognized as hyperhomocysteinemia (HHcy), were associated with cardiovascular diseases. There was controversy regarding the detrimental versus cardio protective role of inducible nitric oxide synthase (iNOS) in ischemic heart disease. The aim of this study was to test the hypothesis that the Hcy generated nitrotyrosine by inducing the endothelial nitric oxide synthase, causing endothelial‐myocyte (E‐M) coupling. To differentiate the role of iNOS versus constitutive nitric oxide synthase (eNOS and nNOS) in Hcy‐mediated nitrotyrosine generation and matrix remodeling in cardiac dysfunction, left ventricular (LV) tissue was analyzed from cystathionine beta synthase (CBS) heterozygote knockout, iNOS homozygote knockout, CBS−/+/iNOS−/− double knockout, and wild‐type (WT) mice. The levels of nitrotyrosine, MMP‐2 and ‐9 (zymographic analysis), and fibrosis (by trichrome stain) were measured. The endothelial‐myocyte function was determined in cardiac rings. In CBS−/+ mice, homocysteine was elevated and in iNOS−/− mice, nitric oxide was significantly reduced. The nitrotyrosine and matrix metalloproteinase‐9 (MMP‐9) levels were elevated in double knockout and CBS−/+ as compared to WT mice. Although MMP‐2 levels were similar in CBS−/+, iNOS−/−, and CBS−/+/iNOS−/−, the levels were three‐ to fourfold higher than WT. The levels of collagen were similar in CBS−/+ and iNOS−/−, but they were threefold higher than WT. Interesting, the levels of collagen increased sixfold in double knockouts, compared to WT, suggesting synergism between high Hcy and lack of iNOS. Left ventricular hypertrophy was exaggerated in the iNOS−/− and double knockout, and mildly increased in the CBS−/+, compared to WT mice. The endothelial‐dependent relaxation was attenuated to the same extent in the CBS−/+ and iNOS−/−, compared to WT, but it was robustly blunted in double knockouts. The results concluded that homocysteine generated nitrotyrosine in the vicinity of endothelium, caused MMP activation and endothelium‐myocyte uncoupling. The generation of nitrotyrosine was independent of iNOS. J. Cell. Biochem. 106: 119–126, 2009.

Congenic expression of tissue inhibitor of metalloproteinase in Dahl-salt sensitive hypertensive rats is associated with reduced LV hypertrophy

Although congenic translocation of a segment from chromosome 10 from Lewis rat, containing an extracellular proteinase inhibitor gene, decreased blood pressure in Dahl-salt sensitive (DSS) rats, the relationship between the levels of matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP), and cardiac function was unclear. In this study we investigated the cardiac effects of congenic translocation of a segment from chromosome 10 from Lewis rat, containing an extracellular proteinase inhibitor gene, in Dahl-salt sensitive rats. To test the hypothesis that left ventricular (LV) hypertrophy in DSS rats was due to high MMP and low TIMP levels and the decrease in blood pressure in congenic rats was associated with increase in proteinase inhibitor expression, cardiac function and levels of MMP and TIMP were determined in 16 weeks male DSS (D), Lewis (L) and congenic (CL-10) rats. Cardiac function was assessed by electrocardiography, echocardiography and a Millar catheter in LV cavity. LV MMP and TIMP levels were measured by Q-RT-PCR and Western blot analyses. In L, D and CL-10 rats, heart weight/body weight (g/g) were 3.73 ± 0.06, 4.45 ± 0.04 and 3.35 ± 0.05 × 10−3, respectively, suggesting significant (p < 0.05) LV hypertrophy (LVH) in D group. The ST duration was longer in D group compared with L group, suggesting coronary vasospasm, but normalized in CL-10 rats. The fractional shortening and ejection fraction were decreased in D group as compared with L group, but normalized in CL-10 groups. LV diameter was increased in D group as compared to L group, but normalized in CL-10 groups. The levels of MMP-9 were higher and TIMP were lower in D as compared to L groups, but normalized in CL-10 rats. Compared with control non-congenic Dahl rats, congenic rats exhibited lower blood pressure, amelioration of LV remodelling and dysfunction, as well as coronary abnormalities. In addition, congenic animals exhibited reduced myocardial expression of MMP-9, but increased expression of MMP-2 and TIMP-4 compared to non congenic animals. We concluded that the congenic transfer of TIMP ameliorated LV hypertrophy and cardiac dysfunction.