Unraveling the Role of 12- and 20- HETE in Cardiac Pathophysiology: G-Protein-Coupled Receptors, Pharmacological Inhibitors, and Transgenic Approaches.

ABSTRACT: Arachidonic acid-derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy, and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs:EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450-derived and lipoxygenase- derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far have employed the use of enzyme inhibitors, HETE antagonists, and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.

Paired Pressure-Volume Loop Analysis and Biaxial Mechanical Testing Characterize Differences in Left Ventricular Tissue Stiffness of Volume Overload and Angiotensin-Induced Pressure Overload Hearts.

Pressure overload (PO) and volume overload (VO) of the heart result in distinctive changes to geometry, due to compensatory structural remodeling. This remodeling potentially leads to changes in tissue mechanical properties. Understanding such changes is important, as tissue modulus has an impact on cardiac performance, disease progression, and influences on cell phenotype. Pressure-volume (PV) loop analysis, a clinically relevant method for measuring left ventricular (LV) chamber stiffness, was performed in vivo on control rat hearts and rats subjected to either chronic PO through Angiotensin-II infusion (4-weeks) or VO (8-weeks). Immediately following PV loops, biaxial testing was performed on LV free wall tissue to directly measure tissue mechanical properties. The ß coefficient, an index of chamber stiffness calculated from the PV loop analysis, increased 98% in PO (n = 4) and decreased 38% in VO (n = 5) compared to control (n = 6). Material constants of LV walls obtained from ex vivo biaxial testing (n = 9-10) were not changed in Angiotensin-II induced PO and decreased by about half in VO compared to control (47% in the circumferential and 57% the longitudinal direction). PV loop analysis showed the expected increase in chamber stiffness of PO and expected decrease in chamber stiffness of VO. Biaxial testing showed a decreased modulus of the myocardium of the VO model, but no changes in the PO model, this suggests the increased chamber stiffness in PO, as shown in the PV loop analysis, may be secondary to changes in tissue mass and/or geometry but not an increase in passive tissue mechanical properties.

Decreased Substrate Stiffness Promotes a Hypofibrotic Phenotype in Cardiac Fibroblasts.

A hypofibrotic phenotype has been observed in cardiac fibroblasts (CFs) isolated from a volume overload heart failure model, aortocaval fistula (ACF). This paradoxical phenotype results in decreased ECM synthesis despite increased TGF-ß presence. Since ACF results in decreased tissue stiffness relative to control (sham) hearts, this study investigates whether the effects of substrate stiffness could account for the observed hypofibrotic phenotype in CFs isolated from ACF. CFs isolated from ACF and sham hearts were plated on polyacrylamide gels of a range of stiffness (2 kPa to 50 kPa). Markers related to cytoskeletal and fibrotic proteins were measured. Aspects of the hypofibrotic phenotype observed in ACF CFs were recapitulated by sham CFs on soft substrates. For instance, sham CFs on the softest gels compared to ACF CFs on the stiffest gels results in similar CTGF (0.80 vs. 0.76) and transgelin (0.44 vs. 0.57) mRNA expression. The changes due to stiffness may be explained by the observed decreased nuclear translocation of transcriptional regulators, MRTF-A and YAP. ACF CFs appear to have a mechanical memory of a softer environment, supported by a hypofibrotic phenotype overall compared to sham with less YAP detected in the nucleus, and less CTGF and transgelin on all stiffnesses.

Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure.

Hemodynamic load regulates cardiac remodeling. In contrast to pressure overload (increased afterload), hearts subjected to volume overload (VO; preload) undergo a distinct pattern of eccentric remodeling, chamber dilation, and decreased extracellular matrix content. Critical profibrotic roles of cardiac fibroblasts (CFs) in postinfarct remodeling and in response to pressure overload have been well established. Little is known about the CF phenotype in response to VO. The present study characterized the phenotype of primary cultures of CFs isolated from hearts subjected to 4 wk of VO induced by an aortocaval fistula. Compared with CFs isolated from sham hearts, VO CFs displayed a "hypofibrotic" phenotype, characterized by a ~50% decrease in the profibrotic phenotypic markers α-smooth muscle actin, connective tissue growth factor, and collagen type I, despite increased levels of profibrotic transforming growth factor-ß1 and an intact canonical transforming growth factor-ß signaling pathway. Actin filament dynamics were characterized, which regulate the CF phenotype in response to biomechanical signals. Actin polymerization was determined by the relative amounts of G-actin monomers versus F-actin. Compared with sham CFs, VO CFs displayed ~78% less F-actin and an increased G-actin-to-F-actin ratio (G/F ratio). In sham CFs, treatment with the Rho kinase inhibitor Y-27632 to increase the G/F ratio resulted in recapitulation of the hypofibrotic CF phenotype observed in VO CFs. Conversely, treatment of VO CFs with jasplakinolide to decrease the G/F ratio restored a more profibrotic response (>2.5-fold increase in α-smooth muscle actin, connective tissue growth factor, and collagen type I). NEW & NOTEWORTHY The present study is the first to describe a "hypofibrotic" phenotype of cardiac fibroblasts isolated from a volume overload model. Our results suggest that biomechanical regulation of actin microfilament stability and assembly is a critical mediator of cardiac fibroblast phenotypic modulation.

Enhanced Steatosis and Fibrosis in Liver of Adult Offspring Exposed to Maternal High-Fat Diet.

Early life exposures can increase the risk of developing chronic diseases including nonalcoholic fatty liver disease. Maternal high-fat diet increases susceptibility to development of steatosis in the offspring. We determined the effect of maternal high-fat diet exposure in utero and during lactation on offspring liver histopathology, particularly fibrosis. Female C57Bl/6J mice were fed a control or high-fat diet (HFD) for 8 weeks and bred with lean males. Nursing dams were continued on the same diet with offspring sacrificed during the perinatal period or maintained on either control or high-fat diet for 12 weeks. Increased hepatocyte proliferation and stellate cell activation were observed in the liver of HFD-exposed pups. Offspring exposed to perinatal high-fat diet and high-fat diet postweaning showed extensive hepatosteatosis compared to offspring on high-fat diet after perinatal control diet. Offspring exposed to perinatal high-fat diet and then placed on control diet for 12 weeks developed steatosis and pericellular fibrosis. Importantly, we found that exposure to perinatal high-fat diet unexpectedly promotes more rapid disease progression of nonalcoholic fatty liver disease, with a sustained fibrotic phenotype, only in adult offspring fed a postweaning control diet.

Congenital heart disease-causing Gata4 mutation displays functional deficits in vivo.

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans.

Myofilament dysfunction as an emerging mechanism of volume overload heart failure.

Two main hemodynamic overload mechanisms [i.e., volume and pressure overload (VO and PO, respectively] result in heart failure (HF), and these two mechanisms have divergent pathologic alterations and different pathophysiological mechanisms. Extensive evidence from animal models and human studies of PO demonstrate a clear association with alterations in Ca(2+) homeostasis. By contrast, emerging evidence from animal models and patients with regurgitant valve disease and dilated cardiomyopathy point toward a more prominent role of myofilament dysfunction. With respect to VO HF, key features of excitation-contraction coupling defects, myofilament dysfunction, and extracellular matrix composition will be discussed.

Effects of a myofilament calcium sensitizer on left ventricular systolic and diastolic function in rats with volume overload heart failure.

Aortocaval fistula (ACF)-induced volume overload (VO) heart failure (HF) results in progressive left ventricular (LV) dysfunction. Hemodynamic load reversal during pre-HF (4 wk post-ACF; REV) results in rapid structural but delayed functional recovery. This study investigated myocyte and myofilament function in ACF and REV and tested the hypothesis that a myofilament Ca(2+) sensitizer would improve VO-induced myofilament dysfunction in ACF and REV. Following the initial sham or ACF surgery in male Sprague-Dawley rats (200-240 g) at week 0, REV surgery and experiments were performed at weeks 4 and 8, respectively. In ACF, decreased LV function is accompanied by impaired sarcomeric shortening and force generation and decreased Ca(2+) sensitivity, whereas, in REV, impaired LV function is accompanied by decreased Ca(2+) sensitivity. Intravenous levosimendan (Levo) elicited the best inotropic and lusitropic responses and was selected for chronic oral studies. Subsets of ACF and REV rats were given vehicle (water) or Levo (1 mg/kg) in drinking water from weeks 4-8. Levo improved systolic (% fractional shortening, end-systolic elastance, and preload-recruitable stroke work) and diastolic (τ, dP/dtmin) function in ACF and REV. Levo improved Ca(2+) sensitivity without altering the amplitude and kinetics of the intracellular Ca(2+) transient. In ACF-Levo, increased cMyBP-C Ser-273 and Ser-302 and cardiac troponin I Ser-23/24 phosphorylation correlated with improved diastolic relaxation, whereas, in REV-Levo, increased cMyBP-C Ser-273 phosphorylation and increased α-to-ß-myosin heavy chain correlated with improved diastolic relaxation. We concluded that Levo improves LV function, and myofilament composition and regulatory protein phosphorylation likely play a key role in improving function.

In vivo and in vitro cardiac responses to beta-adrenergic stimulation in volume-overload heart failure.

Hearts in volume overload (VO) undergo progressive ventricular hypertrophy resulting in chronic heart failure that is unresponsive to ß-adrenergic agonists. This study compared left ventricular (LV) and isolated cardiomyocyte contractility and ß-adrenergic responsiveness in rats with end-stage VO heart failure (HF). Adult male Sprague-Dawley rats were studied 21 weeks after aortocaval fistula (ACF) or sham surgery. Echocardiography revealed decreased fractional shortening accompanied by increased LV chamber diameter and decreased eccentric dilatation index at end-stage ACF compared to sham. Hemodynamic measurements showed a decrease in the slope of end-systolic pressure-volume relationship, indicating systolic dysfunction. Isolated LV myocytes from ACF exhibited decreased peak sarcomere shortening and kinetics. Both Ca2+ transient amplitude and kinetics were increased in ACF myocytes, with no change under the integrated Ca2+ curves relating to contraction and relaxation phases. Increases in ryanodine receptor and phospholamban phosphorylation, along with a decrease in SERCA2 levels, were observed in ACF. These changes were associated with decreased expression of ß-myosin heavy chain, cardiac troponin I and cardiac myosin binding protein-C. In vivo inotropic responses to ß-adrenergic stimulation were attenuated in ACF. Interestingly, ACF myocytes exhibited a similar peak shortening to those of sham in response to a ß-adrenergic agonist. The protein expression of the gap junction protein connexin-43 was decreased, although its phosphorylation at Ser-368 increased. These changes were associated with alterations in Src and ZO-1. In summary, these data suggest that the disconnect in ß-adrenergic responsiveness between in vivo and in vitro conditions may be associated with altered myofilament Ca2+ sensitivity and connexin-43 degradation.

Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease.

The mature aortic valve is composed of a structured trilaminar extracellular matrix that is interspersed with aortic valve interstitial cells (AVICs) and covered by endothelium. Dysfunction of the valvular endothelium initiates calcification of neighboring AVICs leading to calcific aortic valve disease (CAVD). The molecular mechanism by which endothelial cells communicate with AVICs and cause disease is not well understood. Using a co-culture assay, we show that endothelial cells secrete a signal to inhibit calcification of AVICs. Gain or loss of nitric oxide (NO) prevents or accelerates calcification of AVICs, respectively, suggesting that the endothelial cell-derived signal is NO. Overexpression of Notch1, which is genetically linked to human CAVD, retards the calcification of AVICs that occurs with NO inhibition. In AVICs, NO regulates the expression of Hey1, a downstream target of Notch1, and alters nuclear localization of Notch1 intracellular domain. Finally, Notch1 and NOS3 (endothelial NO synthase) display an in vivo genetic interaction critical for proper valve morphogenesis and the development of aortic valve disease. Our data suggests that endothelial cell-derived NO is a regulator of Notch1 signaling in AVICs in the development of the aortic valve and adult aortic valve disease.

Collagen XIV is important for growth and structural integrity of the myocardium.

Collagen XIV is a fibril-associated collagen with an interrupted triple helix (FACIT). Previous studies have shown that this collagen type regulates early stages of fibrillogenesis in connective tissues of high mechanical demand. Mice null for Collagen XIV are viable, however formation of the interstitial collagen network is defective in tendons and skin leading to reduced biomechanical function. The assembly of a tightly regulated collagen network is also required in the heart, not only for structural support but also for controlling cellular processes. Collagen XIV is highly expressed in the embryonic heart, notably within the cardiac interstitium of the developing myocardium, however its role has not been elucidated. To test this, we examined cardiac phenotypes in embryonic and adult mice devoid of Collagen XIV. From as early as E11.5, Col14a1(-/-) mice exhibit significant perturbations in mRNA levels of many other collagen types and remodeling enzymes (MMPs, TIMPs) within the ventricular myocardium. By post natal stages, collagen fibril organization is in disarray and the adult heart displays defects in ventricular morphogenesis. In addition to the extracellular matrix, Col14a1(-/-) mice exhibit increased cardiomyocyte proliferation at post natal, but not E11.5 stages, leading to increased cell number, yet cell size is decreased by 3 months of age. In contrast to myocytes, the number of cardiac fibroblasts is reduced after birth associated with increased apoptosis. As a result of these molecular and cellular changes during embryonic development and post natal maturation, cardiac function is diminished in Col14a1(-/-) mice from 3 months of age; associated with dilation in the absence of hypertrophy, and reduced ejection fraction. Further, Col14a1 deficiency leads to a greater increase in left ventricular wall thickening in response to pathological pressure overload compared to wild type animals. Collectively, these studies identify a new role for type XIV collagen in the formation of the cardiac interstitium during embryonic development, and highlight the importance of the collagen network for myocardial cell survival, and function of the working myocardium after birth.

Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery.

Proximal spinal muscular atrophy (SMA) is a debilitating neurological disease marked by isolated lower motor neuron death and subsequent atrophy of skeletal muscle. Historically, SMA pathology was thought to be limited to lower motor neurons and the skeletal muscles they control, yet there are several reports describing the coincidence of cardiovascular abnormalities in SMA patients. As new therapies for SMA emerge, it is necessary to determine whether these non-neuromuscular systems need to be targeted. Therefore, we have characterized left ventricular (LV) function of SMA mice (SMN2+/+; SMNΔ7+/+; Smn-/-) and compared it with that of their unaffected littermates at 7 and 14 days of age. Anatomical and physiological measurements made by electrocardiogram and echocardiography show that affected mouse pups have a dramatic decrease in cardiac function. At 14 days of age, SMA mice have bradycardia and develop a marked dilated cardiomyopathy with a concomitant decrease in contractility. Signs of decreased cardiac function are also apparent as early as 7 days of age in SMA animals. Delivery of a survival motor neuron-1 transgene using a self-complementary adeno-associated virus serotype 9 abolished the symptom of bradycardia and significantly decreased the severity of the heart defect. We conclude that severe SMA animals have compromised cardiac function resulting at least partially from early bradycardia, which is likely attributable to aberrant autonomic signaling. Further cardiographic studies of human SMA patients are needed to clarify the clinical relevance of these findings from this SMA mouse.

PYK2 signaling is required for PDGF-dependent vascular smooth muscle cell proliferation.

Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G(1)/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D(1) expression, as well as p27(Kip) upregulation. Cell division kinase 2 (cdc2) phosphorylation at G(2)/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.

Coronary arterioles in type 2 diabetic (db/db) mice undergo a distinct pattern of remodeling associated with decreased vessel stiffness.

Little is known about the impact of type 2 diabetes mellitus (DM) on coronary arteriole remodeling. The aim of this study was to determine the mechanisms that underlie coronary arteriole structural remodeling in type 2 diabetic (db/db) mice. Passive structural properties of septal coronary arterioles isolated from 12- to 16-week-old diabetic db/db and control mice were assessed by pressure myography. Coronary arterioles from 12-week-old db/db mice were structurally similar to age-matched controls. By 16 weeks of age, coronary wall thickness was increased in db/db arterioles (p < 0.01), while luminal diameter was reduced (control: 118 ± 5 µm; db/db: 102 ± 4 µm, p < 0.05), augmenting the wall-to-lumen ratio by 58% (control: 5.9 ± 0.6; db/db: 9.5 ± 0.4, p < 0.001). Inward hypertrophic remodeling was accompanied by a 56% decrease in incremental elastic modulus (p < 0.05, indicating decreased vessel coronary wall stiffness) and a ~30% reduction in coronary flow reserve (CFR) in diabetic mice. Interestingly, aortic pulse wave velocity and femoral artery incremental elastic modulus were increased (p < 0.05) in db/db mice, indicating macrovascular stiffness. Molecular tissue analysis revealed increased elastin-to-collagen ratio in diabetic coronaries when compared to control and a decrease in the same ratio in the diabetic aortas. These data show that coronary arterioles isolated from type 2 diabetic mice undergo inward hypertrophic remodeling associated with decreased stiffness and increased elastin-to-collagen ratio which results in a decreased CFR. This study suggests that coronary microvessels undergo a different pattern of remodeling from macrovessels in type 2 DM.

Histopathologic evaluation of patent ductus arteriosus stents after hybrid stage I palliation.

The aim of this study was to determine the histopathology of patent ductus arteriosus (PDA) in-stent stenosis after hybrid stage I palliation. The hybrid approach to palliation of hypoplastic left heart syndrome can be complicated by the development of in-stent stenosis of the PDA. This may obstruct retrograde aortic arch flow, decrease systemic circulation, and lead to interstage interventional procedures. Stented PDA samples removed from eight patients undergoing comprehensive stage II repair were examined by way of radiography and histochemistry (hematoxylin and eosin, Movat pentachrome, α-smooth muscle actin, and proliferating cell nuclear antigen). A retrospective chart review of the patients was also performed. PDA stents were in place in the PDA for a mean period of 169 ± 28 days in patients who had a mean age of 176 ± 30 days at the time of stent removal. Stent deployment caused chronic inflammation, caused fibrin deposition, and induced vascular smooth muscle-cell (VSMC) proliferation in the area immediately surrounding the stent struts. The neointimal region was composed largely of smooth muscle cells that appeared to be fully differentiated by the lack of PCNA staining. Neointimal thickening occurs in the PDA after stent placement for hybrid palliation of HLHS and is the result of inflammation, extracellular matrix deposition, and smooth muscle-cell proliferation in the peristrut region. This finding suggests that proliferating VSMCs in the peristrut region may provide the impetus for inward neointimal formation and therefore the manifestation of in-stent stenosis.

Extracellular matrix remodeling during the progression of volume overload-induced heart failure.

Volume overload-induced heart failure results in progressive left ventricular remodeling characterized by chamber dilation, eccentric cardiac myocyte hypertrophy and changes in extracellular matrix (ECM) remodeling changes. The ECM matrix scaffold is an important determinant of the structural integrity of the myocardium and actively participates in force transmission across the LV wall. In response to this hemodynamic overload, the ECM undergoes a distinct pattern of remodeling that differs from pressure overload. Once thought to be a static entity, the ECM is now regarded to be a highly adaptive structure that is dynamically regulated by mechanical stress, neurohormonal activation, inflammation and oxidative stress, that result in alterations in collagen and other matrix components and a net change in matrix metalloproteinase (MMP) expression and activation. These changes dictate overall ECM turnover during volume overload hear failure progression. This review will discuss the cellular and molecular mechanisms that dictate the temporal patterns of ECM remodeling during heart disease progression.

Right ventricular remodeling in restrictive ventricular septal defect.

Restrictive ventricular septal defect (rVSD) presents with little/no hemodynamic aberrations despite a patent septal defect. Clinically, these patients are observed with the hope that the defect will functionally close over time without the need for surgical repair and development of heart failure. Without evidence supporting a definitive therapeutic strategy, rVSD patients may have increased risk of a poor outcome. We tested the hypothesis that rVSD results in subclinical RV diastolic dysfunction and molecular remodeling. Five pigs underwent surgical rVSD creation. Echocardiography, hemodynamics, myocyte contractility experiments, and proteomics/Western blot were performed 6-weeks post-rVSD and in controls. *p<0.05. LV and RV hemodynamics in rVSD were comparable to controls. The tricuspid valve early/late diastolic inflow velocity ratio (TV E/A ratio) decreased from 1.6+/-0.05 in controls to 1.0+/-0.08* in rVSD, indicating RV diastolic dysfunction. rVSD RV myocytes showed abnormalities in contraction (departure velocity (Vd) -51%*, Vd time +55%*) and relaxation (return velocity (Vr) -50%*, Vr time +62%*). Mitochondrial proteins (fatty acid, TCA cycle) increased 2-fold*, indicating heightened RV work. Desmin protein upregulated 285%* in rVSD RV myocardium, suggesting cytoskeletal remodeling. rVSD causes RV diastolic dysfunction, myocyte functional impairment, and mitochondrial/cytoskeletal protein upregulation in our model. Desmin upregulation may hinder sarcomeric organization/relaxation, representing a key subclinical early marker for future RV dysfunction. TV E/A measurements are a non-invasive modality to assess rVSD patients for diastolic dysfunction. Translational research applications may lead to fundamental changes in the clinical management of rVSD by providing evidence for early repair of the defect.

Role of advanced glycation end products with oxidative stress in resistance artery dysfunction in type 2 diabetic mice.

OBJECTIVE: Type 2 diabetes is associated with increased advanced glycation end product (AGE) formation and vasculopathy. We hypothesized that AGEs contribute to resistance artery dysfunction. METHODS AND RESULTS: Type 2 diabetic db(-)/db(-) (diabetic) and nondiabetic db(-)/db(+) (control) mice were treated with the AGE inhibitor (aminoguanidine: 50 mg/Kg/d) for 3 months. Isolated mesenteric resistance arteries (MRAs) were mounted in an arteriograph. Pressure-induced myogenic tone (MT) was increased in diabetic mice but was unaffected by aminoguanidine treatment. Phenylephrine-induced contraction and nitric oxide donor-induced endothelium-independent relaxation were similar in all groups. In diabetic mice, endothelium-dependent relaxation in response to shear-stress or acetylcholine was altered and was associated with reduced eNOS protein and mRNA expression. Aminoguanidine treatment improved endothelial function and restored eNOS expression. AGE formation and hypoxia markers (plasminogen activator inhibitor 1 and Bnip3) were increased in MRA from diabetic mice and normalized with Aminoguanidine. Primary cultured endothelial cells (ECs) isolated from resistance arteries subjected to high glucose for 48 hours showed decreased eNOS expression and phosphorylation in response to calcium ionophore. High glucose decreased antioxidant protein (MnSOD) and increased prooxidant proteins (gp91phox) expression leading to increased oxidative stress generation, as assessed by DHE staining and endothelial NADH/NADPH oxidase activity. The preincubation of ECs with aminoguanidine restored eNOS-phosphorylation and expression as well as the balance between pro- and antioxidant factors induced by high glucose. CONCLUSIONS: We provide evidence of a link between AGEs, oxidative stress, and resistance artery EC dysfunction in type 2 diabetic mice. Thus, AGEs and oxidative stress may be a potential target for overcoming diabetic microvessels complications.

Ecstasy produces left ventricular dysfunction and oxidative stress in rats.

AIMS: Our aim was to determine whether the repeated, binge administration of 3,4-methylenedioxymethamphetamine (ecstasy; MDMA) produces structural and/or functional changes in the myocardium that are associated with oxidative stress. METHODS AND RESULTS: Echocardiography and pressure-volume conductance catheters were used to assess left ventricular (LV) structure and function in rats subjected to four ecstasy binges (9 mg/kg i.v. for 4 days, separated by a 10 day drug-free period). Hearts from treated and control rats were used for either biochemical and proteomic analysis or the isolation of adult LV myocytes. After the fourth binge, treated hearts showed eccentric LV dilation and diastolic dysfunction. Systolic function was not altered in vivo; however, the magnitude of the contractile responses to electrical stimulation was significantly smaller in myocytes from rats treated in vivo with ecstasy compared with myocytes from control rats. The magnitude of the peak increase in intracellular calcium (measured by Fura-2) was also significantly smaller in myocytes from ecstasy-treated vs. control rats. The relaxation kinetics of the intracellular calcium transients were significantly longer in myocytes from ecstasy-treated rats. Ecstasy significantly increased nitrotyrosine content in the left ventricle. Proteomic analysis revealed increased nitration of contractile proteins (troponin-T, tropomyosin alpha-1 chain, myosin light polypeptide, and myosin regulatory light chain), mitochondrial proteins (Ub-cytochrome-c reductase and ATP synthase), and sarcoplasmic reticulum calcium ATPase. CONCLUSION: The repeated binge administration of ecstasy produces eccentric LV dilation and dysfunction that is accompanied by oxidative stress. These functional responses may result from the redox modification of proteins involved in excitation-contraction coupling and/or mitochondrial energy production. Together, these results indicate that ecstasy has the potential to produce serious cardiac toxicity and ventricular dysfunction.