Dulce Elena Casarini

Aerobic Exercise Training–Induced Left Ventricular Hypertrophy Involves Regulatory MicroRNAs, Decreased Angiotensin-Converting Enzyme-Angiotensin II, and Synergistic Regulation of Angiotensin-Converting Enzyme 2-Angiotensin (1-7)

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-angiotensin (Ang) system was studied in trained rats assigned to 3 groups: (1) sedentary; (2) swimming trained with protocol 1 (T1, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T1 and T2; however, Ang II type 1 receptor mRNA levels (69% in T1 and 99% in T2) and protein expression (240% in T1 and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T1 and T2 were shown to increase ACE2 activity and protein expression and Ang (1-7) levels in the heart. Exercise increased microRNA-27a and 27b, targeting ACE and decreasing microRNA-143 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type 1 receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (1-7), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-angiotensin system counteracts the classic cardiac renin-angiotensin system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-angiotensin system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart.

Swimming training increases cardiac vagal activity and induces cardiac hypertrophy in rats

The effect of swimming training (ST) on vagal and sympathetic cardiac effects was investigated in sedentary (S, N = 12) and trained (T, N = 12) male Wistar rats (200-220 g). ST consisted of 60-min swimming sessions 5 days/week for 8 weeks, with a 5% body weight load attached to the tail. The effect of the autonomic nervous system in generating training-induced resting bradycardia (RB) was examined indirectly after cardiac muscarinic and adrenergic receptor blockade. Cardiac hypertrophy was evaluated by cardiac weight and myocyte morphometry. Plasma catecholamine concentrations and citrate synthase activity in soleus muscle were also determined in both groups. Resting heart rate was significantly reduced in T rats (355 +/- 16 vs 330 +/- 20 bpm). RB was associated with a significantly increased cardiac vagal effect in T rats (103 +/- 25 vs 158 +/- 40 bpm), since the sympathetic cardiac effect and intrinsic heart rate were similar for the two groups. Likewise, no significant difference was observed for plasma catecholamine concentrations between S and T rats. In T rats, left ventricle weight (13%) and myocyte dimension (21%) were significantly increased, suggesting cardiac hypertrophy. Skeletal muscle citrate synthase activity was significantly increased by 52% in T rats, indicating endurance conditioning. These data suggest that RB induced by ST is mainly mediated parasympathetically and differs from other training modes, like running, that seems to mainly decrease intrinsic heart rate in rats. The increased cardiac vagal activity associated with ST is of clinical relevance, since both are related to increased life expectancy and prevention of cardiac events.

Collecting Duct Renin Is Upregulated in Both Kidneys of 2-Kidney, 1-Clip Goldblatt Hypertensive Rats

Renin in collecting duct cells is upregulated in chronic angiotensin II–infused rats via angiotensin II type 1 receptors. To determine whether stimulation of collecting duct renin is a blood pressure–dependent effect; changes in collecting duct renin and associated parameters were assessed in both kidneys of 2-kidney, 1-clip Goldblatt hypertensive (2K1C) rats. Renal medullary tissues were used to avoid the contribution of renin from juxtaglomerular cells. Systolic blood pressure increased to 184±9 mm Hg in 2K1C rats (n=19) compared with sham rats (121±6 mm Hg; n=12). Although renin immunoreactivity markedly decreased in juxtaglomerular cells of nonclipped kidneys (NCK: 0.2±0.0 versus 1.0±0.0 relative ratio) and was augmented in clipped kidneys (CK: 1.7±1.0 versus 1.0±0.0 relative ratio), its immunoreactivity increased in cortical and medullary collecting ducts of both kidneys of 2K1C rats (CK: 2.8±1.0 cortex; 2.1±1.0 medulla; NCK: 4.6±2.0 cortex, 3.2±1.0 medulla versus 1.0±0.0 in sham kidneys). Renal medullary tissues of 2K1C rats showed greater levels of renin protein (CK: 1.4±0.2; NCK: 1.5±0.3), renin mRNA (CK: 5.8±2.0; NCK: 4.9±2.0), angiotensin I (CK: 120±18 pg/g; NCK: 129±13 pg/g versus sham: 67±6 pg/g), angiotensin II (CK: 150±32 pg/g; NCK: 123±21 pg/g versus sham: 91±12 pg/g; P<0.05), and renin activity (CK: 8.6 &mgr;g of angiotensin I per microgram of protein; NCK: 8.3 &mgr;g of angiotensin I per microgram of protein; sham: 3.4 &mgr;g of angiotensin I per microgram of protein) than sham rats. These data indicate that enhanced collecting duct renin in 2K1C rats occurs independently of blood pressure. Upregulation of distal tubular renin helps to explain how sustained intrarenal angiotensin II formation occurs even during juxtaglomerular renin suppression, thus allowing maintained effects on tubular sodium reabsorption that contribute to the hypertension.

NADPH oxidase and enhanced superoxide generation in intrauterine undernourished rats: involvement of the renin–angiotensin system

OBJECTIVE We previously reported that intrauterine undernutrition increased the oxidative stress by decreasing superoxide dismutase activity. In the present study, we tested whether NADPH oxidase, xanthine oxidase, cyclooxygenase or nitric oxide synthase are responsible for the increased O(2)(-) generation observed in rats submitted to intrauterine undernutrition. In addition, we investigated the effect of angiotensin II (ANG II) on O(2)(-) production via activation of NADPH oxidase. METHODS Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 16 weeks of age, the rats were used for the study of intravital fluorescence microscopy; microvascular reactivity, local ANG II concentration and AT(1), p22(phox) and gp91(phox) gene expression. In this study only the male offspring was used. RESULTS Treatment of mesenteric arterioles with the xanthine oxidase inhibitor oxypurinol, the nitric oxide synthase inhibitor L-NAME or the cyclooxygenase inhibitor diclofenac did not significantly change superoxide production. Thus, these vascular sources of superoxide were not responsible for the increased superoxide concentration. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased superoxide generation and improved vascular function. On the other hand, intrauterine undernutrition did not alter the gene expression for p22(phox) and gp91(phox). The fact that the local ANG II concentration was increased and the attenuation of oxidative stress by blocking AT(1) receptor with losartan, led us to suggest that ANG II induces O(2)(-) generation in intrauterine undernourished rats. CONCLUSION Our study shows that NADPH oxidase inhibition attenuated superoxide anion generation and ameliorated vascular function in rats submitted to intrauterine undernutrition. Although it is not clear which mechanisms are responsible for the increase in NADPH oxidase activity, a role for ANG II-mediated superoxide production via activation of NADPH oxidase is suggested.

Increase in kinins on post-exercise hypotension in normotensive and hypertensive volunteers.

Abstract Post-exercise hypotension is an important event for blood pressure regulation, especially in hypertensive individuals. Although post-exercise hypotension is a well-known phenomenon, the mechanism responsible is still unclear. The kallikrein-kinin system is involved in blood pressure control, but its role in post-exercise hypotension has not yet been investigated. Thus, the purpose of this study was to investigate the involvement of the vasodilators bradykinin and des-Arg9-BK and kallikrein activity in post-exercise hypotension promoted by 35 min of cycle ergometer (CE) or circuit weight-training (CWT) bouts in normotensive and hypertensive individuals. A significant decrease in mean arterial pressure at 45 and 60 min after CE and 45 min after CWT was observed in normotensive individuals. Hypertensive values of mean arterial pressure were significantly reduced at 45 and 60 min after CE and at 60 min after CWT. Before exercise, plasma bradykinin concentrations and kallikrein activity were higher in hypertensive compared to normotensive volunteers. Kinin levels increased in the groups evaluated at the end of the training period and 60 min post-exercise. These data suggest that the kallikrein-kinin system may be involved in post-exercise hypotension in normotensive and hypertensive individuals subjected to CE and CWT bouts.

Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure : role of exercise training

Sympathetic hyperactivity (SH) is a hallmark of heart failure (HF), and several lines of evidence suggest that SH contributes to HF-induced skeletal myopathy. However, little is known about the influence of SH on skeletal muscle morphology and metabolism in a setting of developing HF, taking into consideration muscles with different fiber compositions. The contribution of SH on exercise tolerance and skeletal muscle morphology and biochemistry was investigated in 3- and 7-mo-old mice lacking both alpha(2A)- and alpha(2C)-adrenergic receptor subtypes (alpha(2A)/alpha(2C)ARKO mice) that present SH with evidence of HF by 7 mo. To verify whether exercise training (ET) would prevent skeletal muscle myopathy in advanced-stage HF, alpha(2A)/alpha(2C)ARKO mice were exercised from 5 to 7 mo of age. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF and preserved exercise tolerance and muscular norepinephrine with no changes in soleus morphology. In contrast, plantaris muscle of alpha(2A)/alpha(2C)ARKO mice displayed hypertrophy and fiber type shift (IIA --> IIX) paralleled by capillary rarefaction, increased hexokinase activity, and oxidative stress. At 7 mo, alpha(2A)/alpha(2C)ARKO mice displayed exercise intolerance and increased muscular norepinephrine, muscular atrophy, capillary rarefaction, and increased oxidative stress. ET reestablished alpha(2A)/alpha(2C)ARKO mouse exercise tolerance to 7-mo-old wild-type levels and prevented muscular atrophy and capillary rarefaction associated with reduced oxidative stress. Collectively, these data provide direct evidence that SH is a major factor contributing to skeletal muscle morphological changes in a setting of developing HF. ET prevented skeletal muscle myopathy in alpha(2A)/alpha(2C)ARKO mice, which highlights its importance as a therapeutic tool for HF.

Cardiovascular adaptations in rats submitted to a resistance-training model

1. The present study sought to evaluate cardiovascular adaptations, such as blood pressure (BP), heart rate (HR) and cardiac hypertrophy, to resistance training (RT) in a rat model.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 microM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Exercise Training Reduces Sympathetic Modulation on Cardiovascular System and Cardiac Oxidative Stress in Spontaneously Hypertensive Rats

BACKGROUND Spontaneously hypertensive rats (SHRs) show increased cardiac sympathetic activity, which could stimulate cardiomyocyte hypertrophy, cardiac damage, and apoptosis. Norepinephrine (NE)-induced cardiac oxidative stress seems to be involved in SHR cardiac hypertrophy development. Because exercise training (ET) decreases sympathetic activation and oxidative stress, it may alter cardiac hypertrophy in SHR. The aim of this study was to determine, in vivo, whether ET alters cardiac sympathetic modulation on cardiovascular system and whether a correlation exists between cardiac oxidative stress and hypertrophy. METHODS Male SHRs (15-weeks old) were divided into sedentary hypertensive (SHR, n = 7) and exercise-trained hypertensive rats (SHR-T, n = 7). Moderate ET was performed on a treadmill (5 days/week, 60 min, 10 weeks). After ET, cardiopulmonary reflex responses were assessed by bolus injections of 5-HT. Autoregressive spectral estimation was performed for systolic arterial pressure (SAP) with oscillatory components quantified as low (LF: 0.2-0.75 Hz) and high (HF: 0.75-4.0 Hz) frequency ranges. Cardiac NE concentration, lipid peroxidation, antioxidant enzymes activities, and total nitrates/nitrites were determined. RESULTS ET reduced mean arterial pressure, SAP variability (SAP var), LF of SAP, and cardiac hypertrophy and increased cardiopulmonary reflex responses. Cardiac lipid peroxidation was decreased in trained SHRs and positively correlated with NE concentrations (r = 0.89, P < 0.01) and heart weight/body weight ratio (r = 0.72, P < 0.01), and inversely correlated with total nitrates/nitrites (r = -0.79, P < 0.01). Moreover, in trained SHR, cardiac total nitrates/nitrites were inversely correlated with NE concentrations (r = -0.82, P < 0.01). CONCLUSIONS ET attenuates cardiac sympathetic modulation and cardiac hypertrophy, which were associated with reduced oxidative stress and increased nitric oxide (NO) bioavailability.