Lorena García

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes.

Aggresomes are dynamic structures formed when the ubiquitin-proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with gamma-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.

Increased levels of oxidative stress, subclinical inflammation, and myocardial fibrosis markers in primary aldosteronism patients.

Background Patients with primary aldosteronism experience greater left ventricular hypertrophy and a higher frequency of cardiovascular events than do essential hypertensive patients with comparable blood pressure levels. Aldosterone has been correlated with increased oxidative stress, endothelial inflammation, and fibrosis, particularly in patients with heart disease. Aim To evaluate oxidative stress, subclinical endothelial inflammation, and myocardial fibrosis markers in patients with primary aldosteronism and essential hypertension. Design and individuals We studied 30 primary aldosteronism patients and 70 control essential hypertensive patients, matched by age, sex and median blood pressure. For all patients, we measured the serum levels of aldosterone, plasma renin activity, malondialdehyde (MDA), xanthine oxidase, metalloproteinase-9, ultrasensitive C-reactive protein and amino terminal propeptides of type I (PINP), and type III procollagen. We also evaluated the effect of PA treatment in 19 PA individuals. Results PA patients showed elevated levels of MDA (1.70 ± 0.53 versus 0.94 ± 0.65 μmol/l, P <0.001) and PINP (81.7 ± 50.6 versus 49.7 ± 27 mg/l, P = 0.002) compared with essential hypertensive controls. We found a positive correlation between MDA, PINP, and the serum aldosterone/plasma renin activity ratio in primary aldosteronism patients. Clinically, treating primary aldosteronism patients decreased MDA and PINP levels. Conclusion We detected higher levels of MDA and PINP in primary aldosteronism patients, suggesting increased oxidative stress and myocardial fibrosis in these individuals. Treating primary aldosteronism patients reduced MDA and PINP levels, which may reflect the direct effect of aldosterone greater than endothelial oxidative stress and myocardial fibrosis, possibly mediated by a mineralocorticoid receptor.

Pleiotropic effects of atorvastatin in heart failure: role in oxidative stress, inflammation, endothelial function, and exercise capacity.

BACKGROUND Increased oxidative stress, a common feature in chronic heart failure, has been associated with inflammation, endothelial dysfunction, and extracellular matrix degradation. Statins have known anti-inflammatory and anti-oxidant effects; however, their role in chronic heart failure is still controversial. METHODS This was a prospective study of 38 patients with stable systolic chronic heart failure. Patients received a 4-week placebo course, followed by atorvastatin 20 mg/day for 8 weeks. Oxidative stress, inflammation and remodeling markers, brachial artery flow-mediated vasodilation, and 6-minute walk test were evaluated at baseline, 4, and 8 weeks. RESULTS Mean age was 58 +/- 12. Mean left ventricular ejection fraction was 27% +/- 12%. No significant differences were observed between measurements at baseline and after placebo. Atorvastatin induced a significant decrease of matrix metalloproteinase-9 activity, high-sensitivity C-reactive protein, tumor necrosis factor-alpha, interleukin-6, and malondialdehyde, and a significant increase of endothelial superoxide dismutase activity when compared with placebo. No differences in tissue inhibitor of matrix metalloproteinase and matrix metalloproteinase-2 activities were observed. Atorvastatin use was associated with an improved flow-dependent brachial vasodilation and exercise capacity in the 6-minute walk test. CONCLUSIONS In chronic heart failure patients, atorvastatin therapy is associated with a decrease of inflammation and extracellular matrix remodeling, improving both endothelial function and exercise capacity.

Mitochondria, Myocardial Remodeling, and Cardiovascular Disease

The process of muscle remodeling lies at the core of most cardiovascular diseases. Cardiac adaptation to pressure or volume overload is associated with a complex molecular change in cardiomyocytes which leads to anatomic remodeling of the heart muscle. Although adaptive at its beginnings, the sustained cardiac hypertrophic remodeling almost unavoidably ends in progressive muscle dysfunction, heart failure and ultimately death. One of the features of cardiac remodeling is a progressive impairment in mitochondrial function. The heart has the highest oxygen uptake in the human body and accordingly it has a large number of mitochondria, which form a complex network under constant remodeling in order to sustain the high metabolic rate of cardiac cells and serve as Ca2+ buffers acting together with the endoplasmic reticulum (ER). However, this high dependence on mitochondrial metabolism has its costs: when oxygen supply is threatened, high leak of electrons from the electron transport chain leads to oxidative stress and mitochondrial failure. These three aspects of mitochondrial function (Reactive oxygen species signaling, Ca2+ handling and mitochondrial dynamics) are critical for normal muscle homeostasis. In this article, we will review the latest evidence linking mitochondrial morphology and function with the process of myocardial remodeling and cardiovascular disease.

Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.

Angiotensin-(1-9) reverses experimental hypertension and cardiovascular damage by inhibition of the angiotensin converting enzyme/Ang II axis.

Background: Little is known about the biological effects of angiotensin-(1–9), but available evidence shows that angiotensin-(1–9) has beneficial effects in preventing/ameliorating cardiovascular remodeling. Objective: In this study, we evaluated whether angiotensin-(1–9) decreases hypertension and reverses experimental cardiovascular damage in the rat. Methods and results: Angiotensin-(1–9) (600 ng/kg per min for 2 weeks) reduced already-established hypertension in rats with early high blood pressure induced by angiotensin II infusion or renal artery clipping. Angiotensin-(1–9) also improved cardiac (assessed by echocardiography) and endothelial function in small-diameter mesenteric arteries, cardiac and aortic wall hypertrophy, fibrosis, oxidative stress, collagen and transforming growth factor type &bgr; − 1 protein expression (assessed by western blot). The beneficial effect of angiotensin-(1–9) was blunted by coadministration of the angiotensin type 2(AT2) receptor blocker PD123319 (36 ng/kg per min) but not by coadministration of the Mas receptor blocker A779 (100 ng/kg per min). Angiotensin-(1–9) treatment also decreased circulating levels of Ang II, angiotensin-converting enzyme activity and oxidative stress in aorta and left ventricle. Whereas, Ang-(1–9) increased endothelial nitric oxide synthase mRNA levels in aorta as well as plasma nitrate levels. Conclusion: Angiotensin-(1–9) reduces hypertension, ameliorates structural alterations (hypertrophy and fibrosis), oxidative stress in the heart and aorta and improves cardiac and endothelial function in hypertensive rats. These effects were mediated by the AT2 receptor but not by the angiotensin-(1–7)/Mas receptor axis.

Impaired cardiac autophagy in patients developing postoperative atrial fibrillation

OBJECTIVES Postoperative atrial fibrillation (POAF) is a common complication after on-pump heart surgery. Several histologic abnormalities, such as interstitial fibrosis and vacuolization, have been described in atrial samples from patients developing POAF. This ultrastructural remodeling has been associated with the establishment of a proarrhythmic substrate. We studied whether atrial autophagy is activated in patients who develop POAF. METHODS A total of 170 patients in sinus rhythm who had undergone elective coronary artery bypass grafting were included. Systemic inflammatory markers were measured at baseline and 72 hours after surgery. During the procedure, samples of the right atrial appendages were obtained for evaluation of remodeling by light and electron microscopy. Protein ubiquitination and autophagy-related LC3B processing were assessed by Western blot. RESULTS Of these patients, 22% developed POAF. The level of high-sensitivity C-reactive protein, fibrosis, inflammation, myxoid degeneration, and ubiquitin-aggregates in the atria did not differ between patients with and without POAF. Electron microphotographs of those with POAF showed a significant accumulation of autophagic vesicles and lipofuscin deposits. Total protein ubiquitination was similar in the patients with and without POAF, but LC3B processing was markedly reduced in those with POAF, suggesting a selective impairment in autophagic flow. CONCLUSIONS This study provides novel evidence that ultrastructural atrial remodeling characterized by an impaired cardiac autophagy is present in patients developing POAF after coronary artery bypass surgery.

Iron induces protection and necrosis in cultured cardiomyocytes: Role of reactive oxygen species and nitric oxide.

We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 microM iron (added as FeCl(3)-Na nitrilotriacetate, Fe-NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe-NTA concentrations (100 microM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe-NTA (20-40 microM) or the nitric oxide donor Delta-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe-NTA (20 microM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 microM Fe-NTA also attenuated the necrotic cell death triggered by 100 microM Fe-NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe-NTA (20 microM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis.

Serum uric acid correlates with extracellular superoxide dismutase activity in patients with chronic heart failure.

Increased serum uric acid has been identified as an independent risk factor for cardiovascular disease. However, because of its antioxidant capacity, uric acid may play a beneficial role in endothelial function. This paradoxical relationship between uric acid and endothelial function in chronic heart failure patients remains poorly understood. Thirty‐eight chronic heart failure patients (New York Heart Association functional class II–III, mean age 58±10 years and mean left ventricular ejection fraction 25±8%) and twelve age‐and‐sex‐matched healthy controls were studied. Chronic heart failure patients showed higher uric acid levels (7.3±2.3 mg/dL vs. 6.1±0.2 mg/dL, p<0.05) and lower extracellular superoxide dismutase activity (136±36 U ml−1 min−1 vs. 203±61 U ml−1 min−1, p<0.01) and endothelium‐dependent vasodilatation (4.0±1.6% v. 9.1±3.0%, p<0.01) when compared with control subjects. In chronic heart failure patients, correlations between both uric acid levels and extracellular superoxide dismutase activity (r=0.45; p<0.01), and uric acid and endothelium‐dependent vasodilatation (r=0.35; p=0.03) were detected. These correlations were not observed in healthy individuals, suggesting a positive effect of uric acid on endothelial function partially mediated by modulation of extracellular superoxide dismutase activity in chronic heart failure.

TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways.

Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia-reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.