Dorothea Rosenberger

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.

Homocysteine enriched diet leads to prolonged QT interval and reduced left ventricular performance in telemetric monitored mice

BACKGROUND AND AIMS Homocysteine (Hcy) is a sulfur-containing, non-protein amino acid produced in the metabolic pathway of methionine. Hyperhomocysteinemia is associated with cerebro- and cardiovascular disease in industrialized countries, mostly resulting from protein rich diet and sedentary life style. Matrix metalloproteinases are involved in cardiac remodeling, leading to degradation of intercellular junctions, cardiac connexins and basement membranes. The study was designed to investigate the relationship between Hcy, cardiac remodeling, cardiac performance, and rhythm disturbances in an animal model of hyperhomocysteinemia. We tested the hypothesis that induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 leads to connexin 40, connexin 43, connexin 45 expression changes contributing to decreased cardiac performance and disturbed atrioventricular conduction. METHODS AND RESULTS Hcy was added to drinking water of male C57/BL6J mice to achieve moderate Hcy blood levels. ECG was monitored in conscious mice with a telemetric ECG device; echocardiography was used for assessment of left ventricular function. Immunoblotting was used to evaluate matrix metalloproteinase-2, matrix metalloproteinase-9, connexin 40, connexin 43, and connexin 45 expression in cardiac tissue. Animals fed Hcy showed significant prolongation of QRS, QTc, and PR intervals along with reduced left ventricular function. Western blotting showed increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and decreased expression of connexin 40, 43, and 45. CONCLUSION Hcy has been identified as a nutritional factor contributing to cardiovascular disease. Cardiac remodeling induced by matrix metalloproteinase-2 and matrix metalloproteinase-9 and decreased expression of connexin 40, 43, and 45 appears to play a role in the pathomechanism of atrioventricular conduction delay and ventricular dilatation in hyperhomocysteinemia.

Differential expression of γ-aminobutyric acid receptor A (GABAA) and effects of homocysteine

Abstract Background: γ-Aminobutyric acid (GABA) is a known inhibitory neurotransmitter in the mammalian central nervous system, and homocysteine (Hcy) behaves as an antagonist for GABAA receptor. Although the properties and functions of GABAA receptors are well studied in mouse neural tissue, its presence and significance in non-neural tissue remains obscure. The aim of the present study was to examine the expression of GABAA receptor and its subunits in non-neural tissue. Methods: The mice were analyzed. The presence of GABAA receptor and its subunits was evaluated using Western blot and reverse transcription polymerase chain reaction. Results: We report that GABAA receptor protein is abundant in the renal medulla, cortex, heart, left ventricle, aorta and pancreas. Low levels of GABAA receptor protein were detected in the atria of the heart, right ventricle, lung and stomach. The mRNA protein expression of GABAA receptor subunit shows that α1, β1, β3 and γ1 subunits are present only in brain. The mRNA protein expression levels of GABAA receptor α2, α6, β2 and γ3 subunits were highly expressed in brain compared to other tested tissue, while GABAA receptor γ2 subunit was expressed only in brain and kidney. Treatment of microvascular endothelial cells with Hcy decreased GABAA receptor protein level, which was restored to its baseline level in the presence of GABAA receptor agonist, muscimol. The distribution of GABAA and GABAB receptors in wild type mice was determined and tissue-specific expression patterns were found showing that several receptor subtypes were also expressed in the central nervous system. Conclusions: Hcy, a GABAA agonist, was found to decrease GABAA expression levels. These data enlarge knowledge on distribution of GABA receptors and give novel ideas of the effects of Hcy on different organs. Clin Chem Lab Med 2007;45:1777–84.

Arrhythmia and neuronal/endothelial myocyte uncoupling in hyperhomocysteinemia.

Abstract Elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with arrhythmogenesis and sudden cardiac death (SCD). Hcy decreases constitutive neuronal and endothelial nitric oxide (NO), and cardiac diastolic relaxation. Hcy increases the iNOS/NO, peroxynitrite, mitochondrial NADPH oxidase, and suppresses superoxide dismutase (SOD) and redoxins. Hcy activates matrix metalloproteinase (MMP), disrupts connexin-43 and increases collagen/elastin ratio. The disruption of connexin-43 and accumulation of collagen (fibrosis) disrupt the normal pattern of cardiac conduction and attenuate NO transport from endothelium to myocyte (E-M) causing E-M uncoupling, leading to a pro-arrhythmic environment. The goal of this review is to elaborate the mechanism of Hcy-mediated iNOS/NO in E-M uncoupling and SCD. It is known that Hcy creates arrhythmogenic substrates (i.e. increase in collagen/elastin ratio and disruption in connexin-43) and exacerbates heart failure during chronic volume overload. Also, Hcy behaves as an agonist to N-methyl-D-aspartate (NMDA, an excitatory neurotransmitter) receptor-1, and blockade of NMDA-R1 reduces the increase in heart rate-evoked by NMDA-analog and reduces SCD. This review suggest that Hcy increases iNOS/NO, superoxide, metalloproteinase activity, and disrupts connexin-43, exacerbates endothelial-myocyte uncoupling and cardiac failure secondary to inducing NMDA-R1.

Hyperhomocysteinemia and Sudden Cardiac Death: Potential Arrhythmogenic Mechanisms

Elevated levels of serum homocysteine (Hcy) resulting in hyperhomocysteinemia (HHcy) have been implicated in cardiac pathological conditions including: coronary heart disease (CHD), acute myocardial infarction, arrhythmogenesis and sudden cardiac death (SCD). The mechanisms by which HHcy leads to arrhythmogenesis and SCD are unknown. Novel findings indicate that Hcy is an agonist of the N-methyl-D-aspartate receptor (NMDA-R), known to be present in cardiac tissue, and when activated, increases intracellular calcium leading to increased cell excitability. Also, HHcy induces oxidative stress in cardiac cells and activates matrix metalloproteinases (MMPs) that degrade cell membranes and proteins. Here we review the literature relevant to HHcy-induced oxidative stress leading to cardiac tissue remodelling that may adversely affect cell-to-cell impulse conduction, in particular on the hearts specialized conduction system, and may provide substrate for arrhythmogenesis and SCD. Efficacy of B vitamin supplementation in patient populations with HHcy and CHD is also reviewed.

Cardiac Gαs and Gαi Modulate Sympathetic Versus Parasympathetic Mechanisms in Hyperhomocysteinemia

The increase in sympathetic activity is a compensatory mechanism that provides inotropic support to the heart and peripheral vasoconstriction. Increased sympathetic nerve activity is associated with chronic heart failure (CHF). Increased sympathetic nervous activity activates NMDA receptors and GPCRs, which results in the physiological effect of cardiac arrhythmias. It is clear that GPCR activation feeds into the NMDA receptor signal transduction axis, and heightens the physiological effect of cardiac arrhythmias. Effectors that modulate GPCR activation also physically modify the NMDA receptor via intracellular tyrosine kinases. However, it is unknown whether homocysteine-induced NMDAR activation can backtalk to GPCRs and modulate G-proteins: Gαs, Gαi. This bidirectional crosstalk to GPCRs may be mediated by a number of possible events; perhaps calpain or members of the MAPK cascade that includes ERK 1/2 and MMP-9, or even reactive oxygen species may feed back to GPCRs and potentiate G-proteins. Or, perhaps the influx of calcium from NMDAR activation may somehow activate the GPCR signal transduction axis, including G-proteins. Our specific interest begins in knowing exactly whether homocysteine-induced activation of NMDAR can backtalk to GPCRs and modulate G-proteins-Gαs, Gαi-before beginning to elucidate the players that modulate this kind of backtalk. This review addresses the following: pathophysiology of CHF and sudden cardiac death (SCD), causes of cardiac arrhythmias (electrical and structural remodeling), the relationship between homocysteine and CHF, homocysteine and NMDAR activation, the NMDAR signal cascade, GPCR signal cascade, NMDA-to-GPCR crosstalk, and proposal for homocysteine-induced NMDAR and GPCR bidirectional crosstalk that modulates G-proteins.