Emma Robinson

Nox2 NADPH oxidase promotes pathologic cardiac remodeling associated with Doxorubicin chemotherapy.

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2-/-) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2-/- versus WT mice (fractional shortening: 29.5±1.4 versus 25.7±1.0%; P<0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2-/- versus WT mice (ejection fraction: 52.6±2.5 versus 28.5±2.3%, LVdP/dtmin: -8,379±416 versus -5,198±527 mmHg s(-1); end-diastolic pressure-volume relation: 0.051±0.009 versus 0.114±0.012; P<0.001). Furthermore, in response to doxorubicin, Nox2-/- mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, transforming growth factor-β3, and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy.

Significance of peroxisome proliferator-activated receptors in the cardiovascular system in health and disease.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription factors that belong to the nuclear receptor superfamily. Three isoforms of PPAR have been identified, alpha, delta and gamma, which play distinct roles in the regulation of key metabolic processes, such as glucose and lipid redistribution. PPARalpha is expressed predominantly in the liver, kidney and heart, and is primarily involved in fatty acid oxidation. PPARgamma is mainly associated with adipose tissue, where it controls adipocyte differentiation and insulin sensitivity. PPARdelta is abundantly and ubiquitously expressed, but as yet its function has not been clearly defined. Activators of PPARalpha (fibrates) and gamma (thiazolidinediones) have been used clinically for a number of years in the treatment of hyperlipidaemia and to improve insulin sensitivity in diabetes. More recently, PPAR activation has been found to confer additional benefits on endothelial function, inflammation and thrombosis, suggesting that PPAR agonists may be good candidates for the treatment of cardiovascular disease. In this regard, it has been demonstrated that PPAR activators are capable of reducing blood pressure and attenuating the development of atherosclerosis and cardiac hypertrophy. This review will provide a detailed discussion of the current understanding of basic PPAR physiology, with particular reference to the cardiovascular system. It will also examine the evidence supporting the involvement of the different PPAR isoforms in cardiovascular disease and discuss the current and potential future clinical applications of PPAR activators.

Conformational change of mitochondrial complex I increases ROS sensitivity during ischemia.

AIMS Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS. RESULTS We found that in the heart, t½ of complex I deactivation during ischemia was 10 min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis. INNOVATION A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting. CONCLUSION The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

The gastrointestinal peptide obestatin induces vascular relaxation via specific activation of endothelium‐dependent NO signalling

BACKGROUND AND PURPOSE Obestatin is a recently discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation.

Interaction between anandamide and sphingosine-1-phosphate in mediating vasorelaxation in rat coronary artery

BACKGROUND AND PURPOSE Anandamide and sphingosine‐1‐phosphate (S1P) both regulate vascular tone in a variety of vessels. This study aimed to examine the mechanisms involved in the regulation of coronary vascular tone by anandamide and S1P, and to determine whether any functional interaction occurs between these receptor systems.

Selective targeting of glucagon‐like peptide‐1 signalling as a novel therapeutic approach for cardiovascular disease in diabetes

Glucagon‐like peptide‐1 (GLP‐1) is an incretin hormone whose glucose‐dependent insulinotropic actions have been harnessed as a novel therapy for glycaemic control in type 2 diabetes. Although it has been known for some time that the GLP‐1 receptor is expressed in the CVS where it mediates important physiological actions, it is only recently that specific cardiovascular effects of GLP‐1 in the setting of diabetes have been described. GLP‐1 confers indirect benefits in cardiovascular disease (CVD) under both normal and hyperglycaemic conditions via reducing established risk factors, such as hypertension, dyslipidaemia and obesity, which are markedly increased in diabetes. Emerging evidence indicates that GLP‐1 also exerts direct effects on specific aspects of diabetic CVD, such as endothelial dysfunction, inflammation, angiogenesis and adverse cardiac remodelling. However, the majority of studies have employed experimental models of diabetic CVD and information on the effects of GLP‐1 in the clinical setting is limited, although several large‐scale trials are ongoing. It is clearly important to gain a detailed knowledge of the cardiovascular actions of GLP‐1 in diabetes given the large number of patients currently receiving GLP‐1‐based therapies. This review will therefore discuss current understanding of the effects of GLP‐1 on both cardiovascular risk factors in diabetes and direct actions on the heart and vasculature in this setting and the evidence implicating specific targeting of GLP‐1 as a novel therapy for CVD in diabetes.

Surgical optimization and characterization of a minimally invasive aortic banding procedure to induce cardiac hypertrophy in mice

Left ventricular pressure overload in response to aortic banding is an invaluable model for studying progression of cardiac hypertrophy and transition to heart failure. Traditional aortic banding has recently been superceded by minimally invasive transverse aortic banding (MTAB), which does not require ventilation so is less technically challenging. Although the MTAB approach is superior, few laboratories have documented success, and minimal information on the model is available. The aim of this study was to optimize conditions for MTAB and to characterize the development and progression of cardiac hypertrophy. Isofluorane proved the most suitable anaesthetic for MTAB surgery in mice, and 1 week after surgery the MTAB animals showed significant increases in systolic blood pressure (MTAB 110 ± 6 mmHg versus sham 78 ± 3 mmHg, n= 7, P < 0.0001) and heart weight to body weight ratio (MTAB 6.2 ± 0.2 versus sham 5.1 ± 0.1, n= 12, P < 0.001), together with systolic (e.g. fractional shortening, MTAB 31.7 ± 1%versus sham 36.6 ± 1.4%, P= 0.01) and diastolic dysfunction (e.g. left ventricular end‐diastolic pressure, MTAB 12.7 ± 1.0 mmHg versus sham 6.7 ± 0.8 mmHg, P < 0.001). Leucocyte infiltration to the heart was evident after 1 week in MTAB hearts, signifying an inflammatory response. More pronounced remodelling was observed 4 weeks postsurgery (heart weight to body weight ratio, MTAB 9.1 ± 0.6 versus sham 4.6 ± 0.04, n= 10, P < 0.0001) and fractional shortening was further decreased (MTAB 24.3 ± 2.5%versus sham 43.6 ± 1.7%, n= 10, P= 0.003), together with a significant increase in cardiac fibrosis and further cardiac inflammation. Our findings demonstrate that MTAB is a relevant experimental model for studying development and progression of cardiac hypertrophy, which will be highly valuable for future studies examining potential novel therapeutic interventions in this setting.

Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts

Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts.

GLUCAGON-LIKE PEPTIDE-1 PROTECTS AGAINST CARDIAC DYSFUNCTION AND EXTRACELLULAR MATRIX REMODELLING IN EXPERIMENTAL DIABETES

Glucagon-like peptide-1 (GLP-1) is an insulin-releasing hormone with established cardiovascular actions. Here, we investigated effects of exendin-4, a stable GLP-1 mimetic, on cardiac remodelling in experimental diabetes. Male C57BL/6J mice were injected with streptozotocin (STZ; 50 mg/kg/day for 5 days) or vehicle control prior to starting infusion with exendin-4 (25 nmol/kg/day) at 4 weeks. Continuous treatment with exendin-4 for 8 weeks had no effect on body weight but reduced blood glucose in STZ-treated animals (HbA1c: control 6.6±0.3 vs STZ saline 11.8±1.1, p<0.01; exendin-4 9.4±0.9 vs STZ exendin-4 6.5±0.3%, p=NS; n=4–9). Echocardiography indicated that systolic function, assessed by fractional shortening, was similar between groups. However, diastolic dysfunction observed after STZ treatment was attenuated by exendin-4 (mitral valve E/A: STZ saline 1.17±0.04 vs STZ exendin-4 1.51±0.09, p<0.05; n=3–8). Interestingly, these functional effects were associated with an improved pro-fibrotic gene expression profile, as assessed by real-time RT-PCR. For example, expression of procollagen I mRNA was reduced in STZ animals after exendin-4 treatment (STZ saline 4.32±0.27 vs STZ exendin-4 3.02±0.37 arbitrary units, p<0.05; n=5–8), and similar patterns were observed for procollagen III and fibronectin. Furthermore, differential STZ-induced effects on mRNA expression of matrix metalloproteinase-2 (MMP-2) (control 7.30±0.46 vs STZ saline 9.21±0.49, p<0.05; exendin-4 6.50±0.11 vs STZ exendin-4 7.70±0.45 arbitrary units, p=NS; n=5–7) and MMP-9 (STZ saline 1.70±0.35 vs STZ exendin-4 3.84±0.63 arbitrary units, p<0.05; n=5–8) were inhibited by exendin-4. These data indicate that GLP-1 protects against adverse cardiac remodelling in diabetes via modulation of the extracellular matrix, although the underlying mechanisms remain unclear.

Investigation of mechanisms underlying the interaction between Nox2 NADPH oxidase and PPAR-α in left ventricular hypertrophy.

NADPH oxidases and peroxisome proliferator–activated receptor-α (PPAR-α) play key roles in left ventricular hypertrophy (LVH) with emerging evidence supporting an important interaction. To investigate the nature of this interplay, gene-modified mice lacking PPAR-α (PPAR-α−/−) or Nox2 (Nox2−/−), and wild-type (WT) controls underwent thoracic aortic constriction (TAC) or sham surgery (n>8) before study at 7 days. TAC-induced increases in LV/body weight were abolished in both PPAR-α−/− and Nox2−/− mice (WT: 10.8±2.1, PPAR-α−/−: 1.6±1.8, Nox2−/−: 1.7±3.0%; p<0.05), whereas LV contractile dysfunction (echocardiographic fractional shortening) was accentuated in PPAR-α−/− mice (WT: −15.6±2.1, PPAR-α−/−: −28.0±3.8%; p<0.05), but unaltered in Nox2−/− mice. Interestingly, associated increases in PPAR-α mRNA (real-time RT-PCR) in WT TAC versus sham m ice (1.31±0.08 vs 1.01±0.05 arbitrary units; p<0.05) were reversed in Nox2−/− mice (0.83±0.11 vs 1.11±0.10; p<0.05), whilst parallel reductions in Nox2 mRNA were evident in WT (0.49±0.03 vs 1.03±0.09; p<0.05) but not PPAR-α−/− mice. These data clearly suggest that cross-talk between PPAR-α and Nox2 plays an important role in LVH. To elucidate underlying mechanisms, a combined proteomic/transcriptomic approach using DIGE gel-LC-MS proteomics and Illumina mouse Ref-8 beadchips was employed in LV tissue (n=4/group). Data analysis by DAVID functional annotation tools identified several genes whose TAC-regulated differential expression (proteomics: F.C>1.2, p<0.05; transcriptomics: F.C>1.2, p<0.001) was significantly altered in the absence of PPAR-α and/or Nox2, including integrin-α/-β subunits, desmin, and AP-1 subunits c-Fos and c-Jun. These potential key mediators provide exciting new avenues of investigation which may uncover novel mechanisms underlying important interaction between PPAR-α and Nox2 in LVH.