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OBJECTIVE—A high-protein diet (HPD) is known to promote the reduction of body fat, but the mechanisms underlying this change are unclear. AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) function as majors regulators of cellular metabolism that respond to changes in energy status, and recent data demonstrated that they also play a critical role in systemic energy balance. Here, we sought to determine whether the response of the AMPK and mTOR pathways could contribute to the molecular effects of an HPD. RESEARCH DESIGN AND METHODS—Western blotting, confocal microscopy, chromatography, light microscopy, and RT-PCR assays were combined to explore the anorexigenic effects of an HPD. RESULTS—An HPD reduced food intake and induced weight loss in both normal rats and ob/ob mice. The intracerebroventricular administration of leucine reduced food intake, and the magnitude of weight loss and reduction of food intake in a leucine-supplemented diet are similar to that achieved by HPD in normal rats and in ob/ob mice, suggesting that leucine is a major component of the effects of an HPD. Leucine and HPD decrease AMPK and increase mTOR activity in the hypothalamus, leading to inhibition of neuropeptide Y and stimulation of pro-opiomelanocortin expression. Consistent with a cross-regulation between AMPK and mTOR to control food intake, our data show that the activation of these enzymes occurs in the same specific neuronal subtypes. CONCLUSIONS—These findings provide support for the hypothesis that AMPK and mTOR interact in the hypothalamus to regulate feeding during HPD in a leucine-dependent manner.

Upper arm circumference (UAC) measurement is necessary for the proper sizing of cuffs and is recommended for accurate blood pressure (BP) assessment. The aim of this report is to identify and quantify the relationships between UAC and the usual anthropometric measurements of body fat distribution and cardiac structure in hypertensive subjects. We evaluated 339 patients (202 women and 137 men) by medical history, physical examination, anthropometry, metabolic and inflammatory parameters, and echocardiography. Partial correlation analyses adjusted for age and body mass index revealed that anthropometric variables were significantly associated with echocardiographic parameters exclusively in women. In this regard, UAC correlated with interventricular septum thickness, posterior wall thickness, and relative wall thickness >or=0.45, while waist circumference was related to left cardiac chamber diameter. Multivariate analyses including age, body mass index, systolic BP, homeostasis model assessment index, and use of antihypertensive medications demonstrated that UAC was an independent predictor of left ventricular wall thickness and concentric hypertrophy in women. Further linear regression analyses revealed that waist circumference was an independent predictor of left ventricular end-diastolic and left atrial diameters in this gender. Overall, these findings suggest that UAC determination might serve not only as a routine approach preceding BP evaluation but also as a simple and feasible predictor of adverse LV remodeling in hypertensive women.

Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Focal adhesion kinase (FAK) has attracted particular attention as a mediator of hypertrophy induced by increased load. Here, we demonstrate increased expression and phosphorylation of FAK in the hypertrophic left ventricles (LVs) of aortic-banded mice. We used an RNA interference strategy to examine whether FAK signaling plays a role in the pathophysiology of load-induced LV hypertrophy and failure. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing ( approximately 70%) in both normal and hypertrophic LVs. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of beta-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study provides novel evidence that FAK may be involved in multiple aspects of the pathophysiology of cardiac hypertrophy and failure induced by pressure overload.

Recent characterization of the ability of uncoupling protein 2 (UCP2) to reduce ATP production and inhibit insulin secretion by pancreatic beta-cells has placed this mitochondrial protein as a candidate target for therapeutics in diabetes mellitus. In the present study we evaluate the effects of short-term treatment of two animal models of type 2 diabetes mellitus with an antisense oligonucleotide to UCP2. In both models, Swiss mice (made obese and diabetic by a hyperlipidic diet) and ob/ob mice, the treatment resulted in a significant improvement in the hyperglycemic syndrome. This effect was due not only to an improvement of insulin secretion, but also to improved peripheral insulin action. In isolated pancreatic islets, the partial inhibition of UCP2 increased ATP content, followed by increased glucose-stimulated insulin secretion. This was not accompanied by increased expression of enzymes involved in protection against oxidative stress. The evaluation of insulin action in peripheral tissues revealed that the inhibition of UCP2 expression significantly improved insulin signal transduction in adipose tissue. In conclusion, short-term inhibition of UCP2 expression ameliorates the hyperglycemic syndrome in two distinct animal models of obesity and diabetes. Metabolic improvement is due to a combined effect on insulin-producing pancreatic islets and in at least one peripheral tissue that acts as a target for insulin.

Introduction: Previously(Lassaletta et al,PMC3943541)we showed that Rapamycin treatment of normal pigs subjected to an acute myocardial ischemia-reperfusion injury (IRI) was associated with increase myocardial necrosis and refractory left ventricle (LV) defibrillations. In an effort to better understand rapamycin effects we applied a non-bias genomic analysis to determine if microRNAs were modified by rapamycin treatment in this pre-clinical animal model of IRI. Hypothesis: We hypothesized that microRNA expression were modified by rapamycin-treated and would identify novel and meaningful pharmacological mechanisms related to its detrimental effects on reperfusion injury. Methods: Young male Yorkshire swine (16-19 kg) received either no drug (controls, n=5) or were given 4mg of oral rapamycin daily (n=5). After two weeks of treatment, all animals underwent median sternotomy and the mid-LAD was occluded for 60 minutes followed by 120 minutes of reperfusion. RNA was extracted from LV non-ischemic and ischemic areas from each group and processed (according to the manufacture′s specifications at Brown University Genomic Core facility) for microRNA (miRNA) expression analysis using a Affymetrix GeneChip miRNA3.0 Array containing over 25,000 probe sets of which 257 miRNA probes of domestic pigs. Data were processed using MetaCore version 5.0 (GeneGo, St Joseph, MI) for both statistical (FDR: p Results: miRNA regulated in 5 animals per group were annotated for human orthologs. A total of 23 miRNAs were differentially expressed. Interestingly, all miRNAs were down-regulated in the rapamycin-treated animals. Of those, nine had human orthologs: miR 181c-3p was highly represented followed by miR323a-5p, miR1245b-3p, miR4324, miR331-5p, miR491-5p and miR490-3p. Conclusions: Recent studies show that miRNA 181c regulates myocardial mitochondrial function through cytochrome c oxidase and complex IV gene expressions (PMCID: PMC4014556)and regulates Glucose-regulated protein (GRP78)/BiP [an endoplasmic reticulum stress (ER) chaperone](PMID: 24696166). Hence It is likely that rapamycin effects (detrimental or not) are associated with mitochondrial function and ER stress response.

Nature Communications 5: Article number: 5519 (2014); Published: 16 October 2014; Updated: 23 March 2015 An image in Supplementary Fig. 11a in this Article, representing the negative control, was inadvertently duplicated from the ‘myc-FAK’-transfected group. The correct version of the figure appearsbelow.

Tumor necrosis factor-alpha (TNF-alpha) is known to participate in the wastage syndrome that accompanies cancer and severe infectious diseases. More recently, a role for TNF-alpha in the pathogenesis of type 2 diabetes mellitus and obesity has been shown. Much of the regulatory action exerted by TNF-alpha upon the control of energy stores depends on its action on the hypothalamus. In this study, we show that TNF-alpha activates canonical pro-inflammatory signal transduction pathways in the hypothalamus of rats. These signaling events lead to the transcriptional activation of an early responsive gene and to the induction of expression of cytokines and a cytokine responsive protein such as interleukin-1beta, interleukin-6, interleukin-10 and suppressor of cytokine signalling-3, respectively. In addition, TNF-alpha induces the expression of neurotransmitters involved in the control of feeding and thermogenesis. Thus, TNF-alpha may act directly in the hypothalamus inducing a pro-inflammatory response and the modulation of expression of neurotransmitters involved in energy homeostasis.

Normotensive and hypertensive subjects with and without left ventricular (LV) hypertrophy (LV mass index [LVMI] >51 g/m 2.7 ) were examined by conventional echocardiography and tissue Doppler imaging of mitral annulus motion. The subgroups included nonobese normotensive subjects (n=16; age 51±9 years; 11 female; systolic blood pressure [SBP] 109±11 mm Hg, body mass index [BMI] 24±2.7 kg/m 2 ; LVMI 32±5.5 g/m 2.7 ), nonobese hypertensive subjects without LV hypertrophy (n=16; age 54±12 years; 12 female; SBP 166±15 mm Hg; BMI 25±2.7 kg/m 2 ; LVMI 42±5.5 g/m 2.7 ), and hypertensive subjects with LV hypertrophy (n=22; age 56±10 years; 10 female; SBP 181±19 mm Hg; BMI 26±2.3 kg/m 2 ; LVMI 69±16 g/m 2.7 ). Ejection fraction was comparable among the subgroups, but midwall fractional shortening was reduced in hypertensive subjects with LV hypertrophy (≈26%). Isovolumic relaxation time was increased in subjects with LV hypertrophy, whereas mitral wave A velocity was increased in hypertensive subjects with and without LV hypertrophy. Tissue Doppler imaging mitral annulus systolic (S M ) and diastolic (E M ) velocities were reduced in subjects with and without LV hypertrophy compared with normotensive subjects. There was a positive correlation between S M and E M ( r =0.68; P M , r =−0.41; P =0.002; E M , r =−0.56; P

There are subsets of patients with ischemic cardiomyopathy for whom the optimal treatment strategies are not clear. The objective of this study was to delineate the relationship between clinical outcomes and surgical procedure in patients who were treated either with a coronary artery bypass graft or with a coronary artery bypass graft and additional ventricular restoration.The study population comprised 137 consecutive patients with anterior myocardial infarction. All patients had an ejection fraction50% and left ventricle end-systolic volume index80 ml/m(2). The patients were divided into a viable and a non-viable group according to anterior myocardium viability, which was determined by a thallium-201 test. The viable group underwent a revascularization and was randomized into two groups for additional ventricular reconstruction. Group 1a comprised 35 patients with viable anterior wall who underwent surgical revascularization. Group 1b comprised 39 patients with viable anterior wall who underwent surgical revascularization and ventricular restoration. Group 2 comprised 69 patients with non-viable anterior wall who underwent revascularization and ventricular reconstruction. The preoperative and postoperative ejection fractions, end-systolic volume, mitral regurgitation, mortality, and heart failure symptoms were compared among groups.Complete 2-year follow-up was achieved in 127 (92.7%) patients. Ejection fraction improved in all groups compared with preoperative values and it was greater in group 1b than in group 1a (p0.001) at 2 years. There were no postoperative deaths in group 1a, one in group 1b, and two in group 2. After 2 years, group 1b was significantly smaller than group 1a (p0.01) in relation to mitral regurgitation of grades 1 to 2+. End-systolic volume was significantly smaller in group 1b than in group 1a (p0.001), it was smaller in group 1a than in group 2 (p0.001), and it was smaller in group 1b than in group 2 (p0.001). Heart failure class (NYHA) was reduced in all groups and events were significantly smaller in patients with end-systolic volume lesser than 120 ml/m(2) (p0.05).We have demonstrated that the short-term and mid-term outcomes of coronary artery surgery alone in patients with a large left ventricle are inferior to coronary artery surgery plus ventricular restoration.

Focal adhesion kinase (FAK) has been shown to be activated in cardiac myocytes exposed to mechanical stress. However, details of how mechanical stimuli induce FAK activation are unknown. We investigated whether signaling events mediated by the RhoA/Rho-associated coiled coil-containing kinase (ROCK) pathway are involved in regulation of stretch-induced FAK phosphorylation at Tyr397 in neonatal rat ventricular myocytes (NRVMs). Immunostaining showed that RhoA localized to regions of myofilaments alternated with phalloidin (actin) staining. The results of coimmunoprecipitation assays indicated that FAK and RhoA are associated in nonstretched NRVMs, but cyclic stretch significantly reduced the amount of RhoA recovered from anti-FAK immunoprecipitates. Cyclic stretch induced rapid and sustained (up to 2 h) increases in phosphorylation of FAK at Tyr397 and ERK1/2 at Thr202/Tyr204. Blockade of RhoA/ROCK signaling by pharmacological inhibitors of RhoA ( Clostridium botulinum C3 exoenzyme) or ROCK (Y-27632, 10 μmol/l, 1 h) markedly attenuated stretch-induced FAK and ERK1/2 phosphorylation. Similar effects were observed in cells treated with the inhibitor of actin polymerization cytochalasin D. Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of β-myosin heavy chain mRNA. Similar results were seen in cells transfected with FAK antisense oligonucleotide. These findings demonstrate that RhoA/ROCK signaling plays a crucial role in stretch-induced FAK phosphorylation, presumably by coordinating upstream events operationally linked to the actin cytoskeleton.

This study examined whether focal adhesion kinase (FAK) plays a role in the differentiation of C2C12myoblasts into myotubes. Differentiation of C2C12myoblasts induced by switch to differentiation culture medium was accompanied by a transient reduction of FAK phosphorylation at Tyr-397 (to ∼50%, at 1 and 2 h), followed by an increase thereafter (to 240% up to 5 days), although FAK protein expression remained unchanged. FAK and phosphorylated FAK were found at the edge of lamellipodia in proliferating cells, whereas the later increase in FAK phosphorylation in differentiating cells was accompanied by its preferential location at the tip of well-organized actin stress fibers. Hyperexpression of FAK autophosphorylation site (Tyr-397) mutant (MT-FAK) reduced FAK phosphorylation at Tyr-397 in proliferating cells and was accompanied by reduction of cyclin D1 and increase of myogenin expression. These cells failed to progress to myotubes in differentiation medium. In contrast, hyperexpression of a wild-type FAK construction (WT-FAK) increased baseline and abolished the transient reduction of FAK phosphorylation at Tyr-397 in serum-starved C2C12cells. Cells transfected with WT-FAK failed to reduce cyclin D1 and to increase myogenin expression, as well as to progress to terminal differentiation in differentiation medium. These data indicate that FAK signaling plays a critical role in the control of cell cycle as well as in the progression of C2C12cells to terminal differentiation. Transient inhibition of FAK phosphorylation at Tyr-397 contributes to trigger the myogenic genetic program, but its later activation is also central to terminal differentiation into myotubes.

In isolated rat hearts perfused with HEPES and red blood cell-enriched buffers, we examined changes in left ventricular pressure induced by increases in heart rate or infusion of adenosine to investigate whether the negative force-frequency relation and the positive inotropic effect of adenosine are related to an inadequate oxygen supply provided by crystalloid perfusates. Hearts perfused with HEPES buffer at a constant flow demonstrated a negative force-frequency relation, whereas hearts perfused with red blood cell-enriched buffer exhibited a positive force-frequency relation. In contrast, HEPES buffer-perfused hearts showed a concentration-dependent increase in left ventricular systolic pressure [EC50 = 7.0 ± 1.2 nM, maximal effect (Emax) = 104 ± 2 and 84 ± 2 mmHg at 0.1 μM and baseline, respectively] in response to adenosine, whereas hearts perfused with red blood cell-enriched buffer showed no change in left ventricular pressure. The positive inotropic effect of adenosine correlated with the simultaneous reduction in heart rate ( r = 0.67, P < 0.01; EC50 = 3.8 ± 1.4 nM, baseline 228 ± 21 beats/min to a minimum of 183 ± 22 beats/min at 0.1 μM) and was abolished in isolated hearts paced to suppress the adenosine-induced bradycardia. In conclusion, these results indicate that the negative force-frequency relation and the positive inotropic effect of adenosine in the isolated rat heart are related to myocardial hypoxia, rather than functional peculiarities of the rat heart.

Background Low-renin (volume-dependent) hypertension represents 25-30% of all cases of primary hypertension. Endothelial dysfunction and vascular remodeling are associated with hypertension but their relevance to volume-dependent hypertension (VDH) is not yet known. To evaluate this, flow-mediated dilation (FMD) of the brachial artery and the carotid intima-media thickness in the distal common carotid artery were measured and compared between renin-dependent mild-hypertensive patients (RDH) and controls. Method and Results The study group comprised 40 mild-hypertensive patients and 25 controls. Plasma renin activity (PRA), plasma aldosterone concentration, angiotensin II and nitrite/nitrate plasma levels were measured. According to PRA, subjects were classified as VDH ( 0.6 ng · ml-1 · h -1). Vascular function was evaluated by FMD before and after reactive hyperemia (RH) and glyceryl-trinitrate (GTN) administration. FMD in response to RH and GTN in the VDH group when compared with RDH group was 10.2±2.8% vs 13.3±3.6% (p=0.01); and 16.0±3.5% vs 19.9±4.5% (p=0.01), respectively. Conclusion This study showed impaired FMD and reduced GTN response in mildly hypertensive patients with low-renin plasma levels. (Circ J 2005; 69: 1380 -1385)

Insulin and angiotensin II (AngII) may act through overlapping intracellular pathways to promote cardiac myocyte growth. In this report insulin and AngII signaling, through the phosphatidylinositol 3-kinase (PI 3-kinase) and MAPK pathways, were compared in cardiac tissues of control and obese Zucker rats. AngII induced Janus kinase 2 tyrosine phosphorylation and coimmunoprecipitation with insulin receptor substrate 1 (IRS-1) and IRS-2 as well as an increase in tyrosine phosphorylation of IRS and its association with growth factor receptor-binding protein 2. Simultaneous treatment with both hormones led to marked increases in the associations of IRS-1 and -2 with growth factor receptor-binding protein 2 and in the dual phosphorylation of ERK1/2 compared with the administration of AngII or insulin alone. In contrast, an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity was induced by both hormones. Insulin stimulated the phosphorylation of MAPK equally in lean and obese rats. Conversely, insulin-induced phosphorylation of Akt in heart was decreased in obese rats. Pretreatment with losartan did not change insulin-induced activation of ERK1/2 and attenuated the reduction of Akt phosphorylation in the heart of obese rats. Thus, the imbalance between PI 3-kinase-Akt and MAPK signaling pathways in the heart may play a role in the development of cardiovascular abnormalities observed in insulin-resistant states, such as in obese Zucker rats.

Angiotensin II (Ang II) exerts a potent growth stimulus on the heart and vascular wall. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) intracellular signaling pathway by Ang II mediates at least some of the mitogenic responses to this hormone. In other signaling systems that use the JAK/STAT pathway, proteins of the suppressor of cytokine signaling (SOCS) family participate in signal regulation. In the present study it is demonstrated that SOCS3 is constitutively expressed at a low level in rat heart and neonatal rat ventricular myocytes. Ang II at a physiological concentration enhances the expression of SOCS3 mRNA and protein, mainly via AT1 receptors. After induction, SOCS3 associates with JAK2 and impairs further activation of the JAK2/STAT1 pathway. Pretreatment of rats with a specific phosphorthioate antisense oligonucleotide to SOCS3, reverses the desensitization to angiotensin signaling, as detected by a fall in c-Jun expression after repetitive infusions of the hormone. Thus, SOCS3 is induced by Ang II in rat heart and neonatal rat ventricular myocytes and participates in the modulation of the signal generated by this hormone. (Endocrinology 144: 4586 – 4596, 2003)

We investigated the effects of acute pressure overload on activation of p160ROCK in rat myocardium. Constriction of transverse aorta, controlled to increase peak systolic pressure of ascending aorta by ∼40 mmHg, induced a rapid association of RhoA with Dbl-3 and p160ROCK. The binding of p160ROCK to RhoA was rapidly increased, peaking at 30 min (∼3.5-fold), but reduced to lower levels (∼1.9-fold) by 60 min of pressure overload. The activity of immunoprecipitated p160ROCK toward myosin light chain increased ∼2.5-fold within 10 min but decreased to lower levels (∼1.6-fold) after 60 min of pressure overload. Confocal microscopic analysis indicated that pressure overload induced the formation of aggregates of p160ROCK and RhoA along the longitudinal axis of cardiac myocytes. Immunoelectron microscopic analysis showed that pressure overload induced the association of p160ROCK and RhoA to Z-line, T-tubule, and subsarcolemmal areas. The rapid activation of p160ROCK by pressure overload and its aggregation in subcellular structures involved in transmission of mechanical force suggest a role for this enzyme in the mechanobiochemical transduction in the myocardium.

We compared the myocardial lesions caused by the long-term inhibition of nitric oxide (NO) biosynthesis with those associated with renovascular hypertension (two-kidney, one-clip model [2K-1C]) and superimposed streptozotocin-induced diabetes mellitus (DM).Prospective trial.University laboratory.Male Wistar rats were classified into the following groups: (1) a control group; (2) the L-NAME group (treatment with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester [L-NAME], 75 micro mol per rat per day, orally); (3) the 2K-1C group (renovascular hypertension); (4) the DM group (treatment with streptozotocin, 60 mg/kg via intraperitoneal route); and (5) the 2K-1C plus DM group (renovascular hypertension and streptozotocin-induced DM). Arterial BP was measured by a tail-cuff method for 3 weeks, after which histologic and stereological analysis of the heart was done and cardiac NO synthase type 3 (NOS3) levels were assessed by Western blotting. The circulating levels of nitrates/nitrites and thromboxane B(2) (TXB(2), the stable metabolite of thromboxane A(2)) were also measured.In DM and 2K-1C rats, the myocardial lesions consisted mainly of recent myocardial infarcts, which were more severe in the 2K-1C plus DM group. In L-NAME-treated rats, multiple foci of reparative fibrosis and fresh myocardial necrosis resembled the severe lesions found in the 2K-1C plus DM group. Although NOS3 protein expression increased (19 to 44%; p0.05) in all treated groups, serum nitrate/nitrite levels decreased only in the L-NAME group and the 2K-1C plus DM group. These two groups also showed a more pronounced increase in TXB(2) concentrations.These results indicate that the association of hypertension and DM mimics the alterations induced by L-NAME in rats, which suggests a role for NO in the pathophysiology of hypertensive-diabetic cardiomyopathy.

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here, we show that αB-crystallin confers protection to FAK against calpain-mediated proteolysis under mechanical stress in cardiomyocytes. Biochemical assays, chemical cross-linking coupled to mass spectrometry experiments, mutational analyses and Förster resonance energy transfer (FRET) were combined to investigate the basis of FAK and αB-crystallin interaction. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation by mechanical stress. αB-crystallin silencing results in calpain-dependent FAK depletion and in increased apoptosis of cardiomyocytes. The overexpression of a myc-FAK construct or treatment with a calpain inhibitor (E64) restored the survival of cardiomyocytes depleted of αB-crystallin. The association between FAK and αB-crystallin was also demonstrated to occur in response to pressure overload in rat left ventricle. The myocardial depletion of αB-crystallin by siRNA results in enhanced apoptosis of cardiomyocytes and myocardial fibrosis in overloaded hearts. These alterations were markedly attenuated in the overloaded left ventricles of transgenic mice with cardiac specific FAK expression. These findings define a novel mechanism by which αB-crystallin controls FAK function, with impact on cardiomyocyte survival and cardiac remodelling in response to stress.

Signaling by the calcium-dependent phosphatase calcineurin (Cn) plays key roles in regulating cardiac development, hypertrophy, and pathological remodeling. Cn binds to and is negatively regulated by calsarcins (CS), a family of muscle-specific proteins. However, the molecular mechanisms involved in the inhibition of Cn by CS remain unclear. Understanding the architecture and structure of Cn-CS complex is critical to unravel the regulation of Cn by CS. Here we combined biochemical assays, chemical cross-linking coupled to mass spectrometry experiments (MS/MS), mutational analysis and a modeling strategy for structural characterization of CnA-CS1 assembly. The MS/MS data obtained from the cross-linked peptides of both proteins were used to guide an in silico docking of their polypeptide models. The protein complex models with the smallest estimated binding energy were clustered according to structural similarity and submitted to molecular dynamics simulation. The interacting surface of CnA was mapped in a pocket between the 1st and 3rd α-helixes and surrounding loops, while the corresponding surface of CS1 was mapped to the carboxyterminal loops within the Leu179-Phe185, Phe195-Ser199 and Thr250-Leu264 regions. Notably, the region of CnA that interacts with CS1 was found to be located in close proximity, but not coincident, to the β-sheet 14, the main binding site for the PxIxIT sequence of NFAT. Experiments performed with several CnA (FLAG-CnA) and CS1 (myc-CS1) mutants were used to validate the structural model of the CnA-CS1 assembly. The Lys40 (CnA) and Glu254 (CS1) residues were identified as critical for the complex stability. The model that emerges from this study supports the notion that CS1 interacts with an allosteric site to inhibit the activity of CnA. Alternatively, the close proximity of the CS1 to NFAT interacting site supports an interference of CS1 on the ability of CnA to bind and activate NFAT.

Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.

Abstract —Studies have indicated a complex functional interaction between angiotensin (Ang) II and NO in the heart. The purpose of the present study was to examine the protein expression and tissue distribution of NO synthases 1 (NOS1) and 3 (NOS3) in the myocardium of rats that underwent continuous infusion of Ang II at 2 different rates (10 and 40 ng · kg −1 · min −1 ) for 6 days. Mean arterial pressure increased by ≈15 mm Hg in rats infused with Ang II at 40 ng · kg −1 · min −1 , but it remained close to the values observed in saline-infused rats (≈110 mm Hg) when Ang II was infused at 10 ng · kg −1 · min −1 . The protein expression of a 160-kDa NOS1 and a 135-kDa NOS3 were found to increase (≈200%) in the myocardium of rats infused with both subpressor and pressor doses of Ang II. Immunohistochemistry studies showed that NOS1 and NOS3 are differentially expressed in myocardial cells. NOS1 was detected in cardiac myocytes and in smooth muscle cells of small and large coronary arteries, whereas NOS3 was detected in the endothelium and in perivascular and interstitial tissues, but NOS3 was not detected in cardiac or smooth muscle cells. Ang II infusion enhanced the tissue immunoreactivity of both isoforms in their specific locations but did not change the distribution throughout the myocardium. Myocardium staining with anti–angiotensin type 1 (AT 1 ) receptor antibody indicated that AT 1 receptor is expressed in cardiac myocytes, coronary smooth muscle cells, and interstitial and perivascular tissues. Ang II infusion did not change the protein expression and distribution of AT 1 receptor in the myocardium. These results indicate that long-term increases in the circulating levels of Ang II modulate the protein expression of NOS1 and NOS3 and, consequently, the function of the local myocardial NO system.

Studies were carried out to determine the relative importance of volume and hemodilution on hemodynamic adjustments to acute volume expansion. Systemic and renal hemodynamics were monitored in unanesthetized and unrestrained rats during progressive and equivalent blood volume expansion with saline (Sal; 1, 2, and 4% body wt), 7% BSA solution (0.35, 0.7, and 1.4% body wt), and reconstituted whole blood from donor rats (WBL; 0.35, 0.7, and 1.4% body wt). Mean arterial pressure remained unchanged in Sal and BSA but increased progressively in WBL-expanded rats (from 92 to 106 mmHg after maximal expansion). In Sal and BSA-expanded rats, cardiac output (CO) and renal blood flow (RBF) increased (CO: Sal from 19 to 20, 22, and 25; BSA from 21 to 23, 27, and 31; RBF: Sal from 1.6 to 1.8, 2.2, and 2.5; BSA from 2 to 2.4, 2.7, and 3.1 ml · min−1 · 100 g body wt−1), whereas total peripheral (TPR) and renal vascular (RVR) resistance decreased in parallel with the expansions. After expansion with WBL, CO increased progressively but less extensively than in cell-free expanded rats (21 to 22, 24, and 26 ml · min−1 · 100 g body wt−1), whereas TPR and RVR remained unchanged. Systemic hematocrit (Hct) decreased approximately the same after expansion with Sal or BSA solutions but remained unchanged after expansion with WBL. Isovolemic hemodilution to Hct levels comparable to those seen after maximal expansion with cell-free solutions also reduced SVR and RVR, although less extensively. These findings suggest that in unanesthetized rats hemodilution plays a major role in the systemic and renal hemodynamics during expansion.

Abstract —Mechanical overload elicits functional and structural adaptive mechanisms in cardiac muscle. Signaling pathways linked to integrin/cytoskeleton complexes may have a function in mediation of the effects of mechanical stimulus in myocardial cells. We investigated the tyrosine phosphorylation and the assembly of the multicomponent signaling complex associated with focal adhesion kinase (Fak) and the actin cytoskeleton in the overloaded myocardium of rats. Pressure overload induced a 3-fold increase in Fak tyrosine phosphorylation within 3 minutes after a 60-mm Hg rise in aortic pressure. A pressure stimulus that lasted for 60 minutes was accompanied by a 5-fold increase in the amount of tyrosine-phosphorylated Fak, and a stimulus as low as 10 mm Hg doubled the amount of tyrosine-phosphorylated Fak in the myocardium within 10 minutes. Pressure overload also induced a time-dependent association of actin with Fak and an increase in the amount of Fak detected in the cytoskeletal fraction of the myocardium. These events were paralleled by c-Src activation and binding to Fak and by an association of Grb2 and p85 subunit of phosphatidylinositol 3-kinase with Fak. Erk1/2 and Akt, two possible downstream effectors of Fak via Grb2 and phosphatidylinositol 3-kinase, were also shown to be activated in parallel with Fak. These findings show that pressure overload induced a rapid activation of the Fak multiple signaling complex in the myocardium of rats, which suggests that this mechanism may have a role in mechanotransduction in the myocardium.

Autoregulation of renal blood flow (RBF) was studied in rats that underwent equivalent blood volume expansion with saline (Sal; 5% body wt), 7% BSA solution (1.4% body wt), and reconstituted whole blood from donor rats (WBL; 1.4% body wt). Renal perfusion pressure (RPP) and renal neural reflexes were prevented by clamping RPP and sectioning the vagus, baro/chemoreceptor, and renal nerves. Sal and BSA expansion increased RBF by ∼60%, whereas no effect was observed with WBL. RBF autoregulation was markedly attenuated after expansion with cell-free solutions, but no change occurred in WBL-expanded rats. Correction of the fall in hematocrit in Sal- and BSA-expanded rats restored RBF and its autoregulation to control levels. Expansion with Sal or BSA after inhibition of renal vascular tone with intrarenal infusion of papaverine still increased RBF and further changed the RBF-RPP relationship. These findings suggest that the hemodilution plays a central role in the reduction of renal vascular resistance and in the attenuation of the autoregulatory efficiency of renal circulation that accompany expansion with cell-free solutions.

The present study examined the factors responsible for triggering renal hemodynamic adjustments during acute volume expansion. The renal hemodynamic effects of graded volume expansion with 0.9% saline (Sal; 1, 2, and 4% of body wt), 7% BSA solution (0.35, 0.70, and 1.4% body wt), or whole blood from a donor rat (WBL; 0.35, 0.70, and 1.4% body wt) were compared in rats anesthetized with pentobarbital sodium. Neural influences on the kidney were eliminated by vagus nerves, baro/chemoreceptor afferents, and renal nerves section, and renal perfusion pressure was controlled at constant level (∼120 mmHg) throughout the experiments. In Sal- and BSA-expanded rats, renal blood flow (RBF) increased (Sal: 15, 40, 71%; BSA 17, 49, 107%) and renal vascular resistance (RVR) decreased in parallel with the degree of volume expansion (RVR: Sal 17, 31, 44%; and BSA: 15, 35, 54%). Renal hemodynamics remained unaltered after expansion with WBL. In rats expanded with Sal or BSA, correction of the fall of hematocrit restored RBF and RVR to control levels. Interference with tubuloglomerular feedback by uretheral obstruction had no effect on the decrease in RVR with Sal or BSA. Inhibition of the vascular tone by intrarenal papaverine infusion also did not alter the renal hemodynamic response to volume expansion with Sal or BSA. These findings suggest that the changes in renal hemodynamics after acute expansion are likely mediated by changes in rheologic properties of the blood rather than by changes in active vascular tone.

Studies in our laboratory and others have demonstrated that arginine vasopressin (AVP) exerts potent vasoconstrictor actions on the vessels supplying the renal medulla. The physiological importance of these vascular effects of AVP has been difficult to assess because of high endogenous levels of AVP in anesthetized, surgically prepared animals. We have developed a decerebrated, hypophysectomized, renal-denervated rat model that enables us to study the effects of low levels of AVP on the pressure-diuresis, relationship under acute conditions. These rats maintain normal mean arterial pressure (MAP) and plasma AVP (2.5 pg/ml). Cortical and medullary blood flow (CBF and MBF, respectively) were measured by laser-Doppler flowmetry and total renal blood flow (RBF) by transit time flowmetry. Renal interstitial fluid pressure (RIFP) and urinary sodium excretion (UNaV) responses were determined during controlled increases of MAP produced by aortic occlusion below the renal arteries. From a baseline of 97 +/- 2 mmHg, 30% increases in MAP resulted in a 63% increase in MBF, 35% increase in RIFP, and sixfold increase in UNaV, whereas CBF and RBF remained unchanged. Infusion of AVP (0.50 ng.kg-1.min-1, which increased plasma AVP from normal control levels of 3 pg/ml to 11 pg/ml) produced no change in baseline MAP, RBF, or CBF but lowered MBF by 24%, RIFP by 26%, and UNaV by 71%. The slope of the relationship of AP and UNaV, MBF, and RIP was reduced to nearly zero by these small increases of plasma AVP. We conclude that an increase of plasma AVP in the range that occurs with water restriction decreases MBF selectively and greatly attenuates the arterial pressure-MBF and pressure-natriuretic relationship.

Na hipertensão primária, ou essencial, a elevação da resistência periférica é a principal responsável pelo aumento da pressão arterial. Isso ocorre pela redução do calibre das arteríolas determinada pela combinação, em grau variado, de fatores funcionais e fatores estruturais. A vasoconstrição pode ser causada pela produção excessiva de fatores pressores (angiotensina, vasopressina, endotelina e sistema nervoso simpático etc), ou pela deficiência dos fatores depressores (óxido nítrico, prostaciclina e peptídio natriurético atrial). O componente estrutural é, geralmente, representado pela hipertrofia da camada média que passa a ocupar parte do espaço intravascular. Importante ressaltar que os fatores que, ativamente, contraem ou dilatam os vasos têm, também, efeitos tróficos sobre a camada muscular, estimulando ou inibindo o seu espessamento. A redução da luz das arteríolas pode resultar, igualmente, de “remodelagem”, quando ocorre redução tanto do diâmetro interno como externo, sem modificações da massa. O componente genético é responsável pela produção dos complexos fatores pressores e depressores que regulam a pressão arterial. Também, ele é o responsável pela susceptibilidade individual aos fatores ambientais (sal e estresse, por exemplo), que sobrecarregam o sistema e podem gerar o desequilíbrio causador da hipertensão.

A genome-wide search for multiple loci influencing salt-loaded systolic blood pressure (NaSBP) variation among 188 F2 progeny from a cross between the Brown-Norway and spontaneously hypertensive rat strains was pursued in an effort to gain insight into the polygenic basis of blood pressure regulation. The results suggest that loci within five to six genomic regions collectively explain approximately 43% of the total NaSBP variation exhibited among the 188 F2 progeny. Many of these loci are in regions that previous studies have not implicated in blood pressure regulation. Ultimately, however, this study not only sheds light on the multigenic basis of blood pressure but provides further evidence that the identification of the genetic determinants of polygenic traits in mammals is possible with modern biometrical and molecular genetic tools in controlled settings (i.e., breeding paradigm and model organism).

BACKGROUND Changing from a supine to an orthostatic posture is associated with substantial increments in leg blood pressure (BP) levels, which could ultimately influence the hemodynamic burden imposed on the heart. This study investigated the relationship between brachial and leg BP measurements and the left cardiac chamber's structure and assessed the role of body posture changes in this regard. METHODS One hundred and thirty normotensive, nondiabetic, nonsmoking, normolipemic subjects were evaluated by a clinical history, anthropometry, the analysis of metabolic parameters, echocardiography, and the measurement of BP in the arm and the calf in both supine and orthostatic positions. RESULTS Significant correlation coefficients between the leg BP measurements and the cardiac structure were detected, especially between the orthostatic pulse pressure (PP) and the left ventricular (LV) wall thickness (r = 0.38; P < 0.001), the orthostatic PP and the LV mass (r = 0.37; P < 0.001), and the orthostatic systolic BP (SBP) and the left atrial size (r = 0.35; P < 0.001). Stepwise and standard regression analysis adjusted for brachial BP and anthropometric and metabolic variables confirmed that the leg orthostatic PP was independently related to the LV wall thickness and mass. Moreover, the leg orthostatic SBP was associated with the left atrial dimension even after adding the LV mass to the statistical models. Finally, triglyceride levels and body surface area showed significant relationship with leg orthostatic PP and SBP, whereas brachial orthostatic PP and SBP were only associated with age and anthropometric variables. CONCLUSIONS Orthostatic leg BP is independently associated with the cardiac structure in normotensive subjects.

Myocardial fibrosis is maladaptive, accelerating the evolution of diseased hearts to failure. The pathogenesis of myocardial fibrosis is critically dependent on complex processes of activation (i.e. enhanced proliferation, production and secretion of soluble factors, collagen and matrix metalloproteinases) and terminal differentiation of cardiac fibroblasts into myofibroblasts, resultant from the mobilization of numerous signaling molecules by physical and humoral stimuli. Noting that Focal Adhesion Kinase (FAK) is activated in areas of ongoing myocardial fibrosis, we sought to examine whether it is a critical mediator of fibrogenesis in load-induced hypertrophic hearts. Isolated fibroblasts from hypertrophic hearts of mice subjected to transverse aortic constriction (TAC; 1 to 8 weeks) were highly activated as recognized by markers that indicate enhanced proliferation (nuclear Ki67), production of collagen and matrix metalloproteinase-2 (MMP-2) and differentiation into myofibroblasts (expression of α-smooth muscle actin - α-SMA). In these cells, FAK was upregulated, as also were its dowstream pathways Src/ERK1/2 and PI3K/AKT/mTOR. Depletion of FAK (∼80%) after treatment with small interfering RNA (siRNA-FAK) markedly attenuated cardiac hypertrophy and fibrosis, and significantly reduced the number of activated fibroblasts harvested from overloaded hearts. Restoration of FAK function by overexpressing a full-length FAK construct in these cells, selectively enhanced the activity of the downstream PI3K/AKT/mTOR and rescued the activated phenotype of fibroblasts. Transfection with an inactive FAK mutant (Tyr397 substituted by phenylalanine) did not rescue the activated phenotype of fibroblasts harvested from overloaded hearts depleted of FAK. However, cells harvested from overloaded hearts depleted of FAK and treated with the mTOR activating aminoacid leucine showed typical phenotype of activated fibroblasts. These findings uncover a role for FAK in regulating the signaling cascade PI3K/AKT/mTOR in cardiac fibroblasts, which seems to be critical for the pathogenesis of myocardial fibrosis in hypertrophic hearts. Targeting this pathway may provide a novel strategy for treating hypertrophic heart diseases.

Background Reactive oxygen species have been implicated in the physiopathogenesis of hypertensive end-organ damage. This study investigated the impact of the C242T polymorphism of the p22-phox gene (CYBA) on left ventricular structure in Brazilian hypertensive subjects. Methods We cross-sectionally evaluated 561 patients from 2 independent centers [Campinas (n = 441) and Vitória (n = 120)] by clinical history, physical examination, anthropometry, analysis of metabolic and echocardiography parameters as well as p22-phox C242T polymorphism genotyping. In addition, NADPH-oxidase activity was quantified in peripheral mononuclear cells from a subgroup of Campinas sample. Results Genotype frequencies in both samples were consistent with the Hardy- Weinberg equilibrium. Subjects with the T allele presented higher left ventricular mass/height2.7 than those carrying the CC genotype in Campinas (76.8 ± 1.6 vs 70.9 ± 1.4 g/m2.7; p = 0.009), and in Vitória (45.6 ± 1.9 vs 39.9 ± 1.4 g/m2.7; p = 0.023) samples. These results were confirmed by stepwise regression analyses adjusted for age, gender, blood pressure, metabolic variables and use of anti-hypertensive medications. In addition, increased NADPH-oxidase activity was detected in peripheral mononuclear cells from T allele carriers compared with CC genotype carriers (p = 0.03). Conclusions The T allele of the p22-phox C242T polymorphism is associated with higher left ventricular mass/height2.7 and increased NADPH-oxidase activity in Brazilian hypertensive patients. These data suggest that genetic variation within NADPH-oxidase components may modulate left ventricular remodeling in subjects with systemic hypertension.

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 A(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.

The mechanisms by which dietary sodium modulates cardiovascular risk are not fully understood. This study investigated whether sodium intake is related to carotid structure and hemodynamics and to plasma matrix metalloproteinase (MMP) activity in hypertensive adults. One hundred thirty-four participants were cross-sectionally evaluated by clinical history, anthropometry, carotid ultrasound, and analysis of hemodynamic, inflammatory, and metabolic variables. Daily sodium intake (DSI) was estimated by 24-h recall, discretionary sodium, and a FFQ. In 42 patients, plasma MMP-2 and MMP-9 activities were also analyzed. The mean DSI was 5.52 ± 0.29 g/d. Univariate analysis showed that DSI correlated with common carotid artery systolic and diastolic diameter (r = 0.36 and 0.34; both P0.001), peak and mean circumferential tension (r = 0.44 and 0.39; both P0.001), Young's Elastic Modulus (r = 0.40; P0.001), intima-media thickness (r = 0.19; P0.05), and internal carotid artery resistive index (r = 0.20; P0.05). Multivariate analyses revealed that only artery diameter, circumferential wall tension, and Young's Elastic Modulus were independently associated with DSI. Conversely, plasma MMP-9 activity was associated with DSI (r = 0.53; P0.001) as well as with common carotid systolic diameter (r = 0.33; P0.05) and Young's Elastic Modulus (r = 0.38; P0.01). In conclusion, sodium intake is associated with carotid alterations in hypertensive adults independently of systemic hemodynamic variables. The present findings also suggest that increased MMP-9 activity might play a role in sodium-induced vascular remodeling.

LEOPARD syndrome (LS) is an autosomal dominant "RASopathy" that manifests with congenital heart disease. Nearly all cases of LS are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11) gene that encodes the SH2 domain-containing PTP-2 (SHP2). RASopathies typically affect components of the RAS/MAPK pathway, yet it remains unclear how PTPN11 mutations alter cellular signaling to produce LS phenotypes. We therefore generated knockin mice harboring the Ptpn11 mutation Y279C, one of the most common LS alleles. Ptpn11(Y279C/+) (LS/+) mice recapitulated the human disorder, with short stature, craniofacial dysmorphia, and morphologic, histologic, echocardiographic, and molecular evidence of hypertrophic cardiomyopathy (HCM). Heart and/or cardiomyocyte lysates from LS/+ mice showed enhanced binding of Shp2 to Irs1, decreased Shp2 catalytic activity, and abrogated agonist-evoked Erk/Mapk signaling. LS/+ mice also exhibited increased basal and agonist-induced Akt and mTor activity. The cardiac defects in LS/+ mice were completely reversed by treatment with rapamycin, an inhibitor of mTOR. Our results demonstrate that LS mutations have dominant-negative effects in vivo, identify enhanced mTOR activity as critical for causing LS-associated HCM, and suggest that TOR inhibitors be considered for treatment of HCM in LS patients.

In the present study we analyzed the cardiovascular, respiratory and behavioral responses evoked in rats by KCN. Hypertension, hyperpnea and alerting behavior were characteristic responses in unanesthetized animals. Selective denervation of aortic and carotid bifurcation areas confirmed the carotid body chemoreceptor (CBC) origin of these responses. Sino-aortic denervation, as well as selective carotid sinus denervation abolished the responses, while, after selective aortic denervation, the responses were not different from those of the control rats. The administration of prazosin abolished the hypertensive response but did not change the bradycardic response, whereas the administration of atropine eliminated the bradycardic response and potentiated the hypertensive response to KCN. Increasing doses of KCN (10–80 μg/rat) produced proportionally larger bradycardia, hypertension and hyperventilation. Slight attenuation of bradycardic responses to KCN were observed in rats under chloralose anesthesia, while marked depression of bradycardic responses were observed under pentobarbitone anesthesia. Both anesthetics changed the hypertensive responses to hypotensive responses. In conclusion, the cardiovascular, respiratory and behavioral responses to intravenous KCN are a good functional test to CBC stimulation in unanesthetized rats, producing simultaneously intense bradycardia with hyperventilation and behaviour responses.

BACKGROUND Experimental data demonstrated that inactivation of toll-like receptor (TLR) pathway components attenuated left ventricular (LV) remodeling induced by pressure overload. This study investigated the impact of TLR6 Ser249Pro polymorphism on LV structure in hypertensive subjects. METHODS A sample of 443 patients (266 women and 177 men) was evaluated by clinical history, physical examination, analysis of inflammatory and metabolic parameters, echocardiography, and genotyping of the TLR6 variant. Moreover, the relationship between genotypes and in vitro responsiveness of peripheral blood monocytic cells to TLR agonists was also assessed. RESULTS Homozygous women for the TLR6 249Ser allele had lower LV posterior wall thickness (9.4 + or - 0.4 vs. 10.5 + or - 0.1 mm; P = 0.02), interventricular septum thickness (9.7 + or - 0.3 vs. 10.7 + or - 0.1 mm; P = 0.03), and LV relative wall thickness (0.39 + or - 0.02 vs. 0.44 + or - 0.01; P = 0.02) than women with other genotypes. These results were confirmed by stepwise regression analyses adjusted by potential confounders. Conversely, homozygous men for the 249Ser variant showed no differences in LV structure in comparison to males carrying the 249Pro allele. In addition, monocytes from hypertensive women homozygous for the 249Ser allele showed a lower release of tumor necrosis factor-alpha and interleukin-6 in response to zymosan (TLR6 agonist), but not to lipopolysaccharide (TLR4 agonist). CONCLUSION These data suggest that hypertensive women homozygous for the TLR6 249Ser polymorphism might exhibit lower LV wall thickness and reduced TLR6-mediated inflammatory response than females carrying the major allele.

AIMS: Mitral valve prolapse (MVP) is associated with aortic root (AoR) enlargement in patients with inherited connective tissue disorders. This report evaluated whether MVP is related to AoR dimension in a large population with otherwise normal echocardiographic parameters. METHODS AND RESULTS: We retrospectively analysed echocardiograms performed by a single echocardiographer between 2001 and 2007 for various clinical indications. Six hundred and twenty-seven subjects with isolated MVP were found and then matched by sex, age, and body mass index to 627 individuals without MVP. The whole sample included 454 men and 800 women with an average age of 37.9 +/- 0.3 years and a body mass index of 23.7 +/- 0.1 kg/m(2). MVP subjects had a higher AoR diameter (30.4 +/- 0.1 vs. 29.5 +/- 0.1 cm; P < 0.0001) compared with controls. Furthermore, multivariate analyses demonstrated an independent association between MVP and AoR size (P < 0.0001) in a model that included age, gender, body mass index, body surface area, blood pressure levels, and left ventricular mass index as confounding variables. CONCLUSION: Isolated MVP is an independent predictor of greater AoR size in a large population with otherwise normal echocardiographic parameters.

BACKGROUND:The activation of the members of the myocyte enhancer factor-2 family (MEF2A, B, C and D) of transcription factors promotes cardiac hypertrophy and failure. However, the role of its individual components in the pathogenesis of cardiac hypertrophy remains unclear. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we investigated whether MEF2C plays a role in mediating the left ventricular hypertrophy by pressure overload in mice. The knockdown of myocardial MEF2C induced by specific small interfering RNA (siRNA) has been shown to attenuate hypertrophy, interstitial fibrosis and the rise of ANP levels in aortic banded mice. We detected that the depletion of MEF2C also results in lowered levels of both PGC-1alpha and mitochondrial DNA in the overloaded left ventricle, associated with enhanced AMP:ATP ratio. Additionally, MEF2C depletion was accompanied by defective activation of S6K in response to pressure overload. Treatment with the amino acid leucine stimulated S6K and suppressed the attenuation of left ventricular hypertrophy and fibrosis in the aforementioned aortic banded mice. CONCLUSION/SIGNIFICANCE:These findings represent new evidences that MEF2C depletion attenuates the hypertrophic responses to mechanical stress and highlight the potential of MEF2C to be a target for new therapies to cardiac hypertrophy and failure.

Mechanical stress is a major triggering stimulus for the installation of cardiac hypertrophy as well as for the structural and functional deterioration occurring in the hypertrophy decompensation. The sensing of mechanical forces and their conversion into biochemical signals depend on the integrity of subcellular structures such as the costameres and Z-disks. Signaling molecules concentrated into these structures are thought to be activated by the stress-induced deformation of structural proteins. Evidence also indicates that Ca2+ may be involved in mediating the mechanical forces conversion into biochemical signals and biological responses. Ca2+ channels, transporters and activated proteins are concentrated at the junctions between the T-tubules and the sarcoplasmic reticulum which are in close proximity to the costameres and Z-disks. This provides a structural basis for the influence of mechanical forces on Ca2+ transport and on the events related to signaling molecules clustered in the costameres and the Z-disks. Emerging data reviewed here are providing insight into how Ca2+ and mechanical mediated signaling are coordinated to modulate the functional and trophic responses of cardiac myocytes to mechanical stress.

Introduction Angiotensin-converting enzyme inhibitors (ACEi) have been successfully used for patients with cardiac dysfunction after myocardial infarction. Objective The aim of the present study was to investigate cardiac effects of ACEi in sickle cell disease (SCD) patients, as there are no previous reports regarding these effects. Methods Enalapril was administered to 9 SCD patients with microalbuminuria. Nine SCD patients without microalbuminuria, matched according to age, diagnosis and levels of haemoglobin, haematocrit and foetal haemoglobin did not receive enalapril and were followed up as the control group during the same period of study. Echocardiograms were performed before the study entry and after 36 months of follow-up. Results At 36 months of follow-up, significant increases in left ventricular mass, left ventricular mass index, posterior left ventricular wall thickness in end-diastole, interventricular septal wall thickness in end-diastole, and aortic root diameter values were seen in untreated, but not in enalapril-treated patients. No major changes were seen in left ventricular systolic diameter, diastolic dimension and ejection fraction, and left atrial diameter, in both groups, along the observational period. Conclusion The results of this study suggest that enalapril prevents cardiac remodelling in SCD patients. However, a large trial concerning the response to enalapril in patients with SCD should be carried out to further clarify this issue.

An RP-HPLC method for the analysis of adenosine (ADO) has been developed and validated. In the present study, we report an RP-HPLC-based method with modifications of mobile phase and shorter retention time that substantially improved the efficiency of ADO analysis. The HPLC separation of the ADO was achieved on a C18 column, using a mobile phase consisting of water, containing 7% v/v ACN, at a flow rate of 0.8 mL/min. The column effluent was monitored by UV detection at 260 nm. A linear response was achieved over the concentration range of 0.25-100.00 micromol/L. The analytical method inter- and intra-run accuracy and precision were better than +/- 15%. The LOQ was 0.25 micromol/L, with ADO detection in the range of 6.25 pmol per sample. The method has been applied to the study of adenosine kinase (AK) kinetics.

FAK (focal adhesion kinase) has been shown to mediate the hypertrophic growth of the left ventricle. Experimental results also suggest that FAK may contribute to the structural and functional deterioration of the chronically overloaded left ventricle. In the present study, we postulated that FAK expression and phosphorylation may be altered in the volume-overloaded heart in humans. FAK expression and phosphorylation at Tyr397 were detected by Western blotting and immunohistochemistry in samples from endomyocardial biopsies from patients with MR (mitral regurgitation; n =21) and donor subjects ( n =4). Hearts from patients with MR had degenerated cardiac myocytes and areas of fibrosis. In this group, the myocardial collagen area was increased (18% in MR hearts compared with 3% in donor hearts respectively) and correlated negatively with left ventricular ejection fraction ( r =−0.74; P >0.001). FAK expression and phosphorylation at Tyr397 (a marker of the enzyme activity) were increased in samples from MR hearts compared with those from donor hearts (3.1- and 4.9-fold respectively). In myocardial samples from donor hearts, anti-FAK staining was almost exclusively restricted to cardiac myocytes; however, in myocardial samples from MR hearts, staining with the anti-FAK antibody was found to occur in myocytes and the interstitium. There was a positive correlation between collagen and the interstitial areas stained with the anti-FAK antibody ( r =0.76; P >0.001). Anti-FAK and anti-vimentin staining of the interstitial areas of samples from MR hearts were extensively superimposed, indicating that most of the interstitial FAK was located in fibroblasts. In conclusion, FAK expression and phosphorylation are increased and may contribute to the underlying structural and functional abnormalities in the volume-overloaded heart in humans. Abbreviations: FAK, focal adhesion kinase; LAD, left atrial diameter; LV, left ventricular; LVEDD, LV end-diastolic diameter; LVEF, LV ejection fraction; LVESD, LV end-systolic diameter; LVMI, LV mass index; MR, mitral regurgitation; PASP, pulmonary artery systolic pressure; RAP, right atrial pressure; TRITC, tetramethylrhodamine β-isothiocyanate

The p22-phox subunit is an essential component of NAD(P)H oxidase enzymatic complex, which is considered the major source of oxidative stress products in the cardiovascular system. The -930G allele of p22-phox has been associated with higher promoter activity, increased NAD(P)H oxidase-mediated oxidative stress and hypertension. We recently reported that left ventricular hypertrophy is accompanied by increased myocardial p22-phox expression in aortic-banded rats, suggesting that this protein might be involved in hypertensive cardiac hypertrophy.