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The organization of muscle is the product of functional adaptation over several length scales spanning from the sarcomere to the muscle bundle. One possible strategy for solving this multiscale coupling problem is to physically constrain the muscle cells in microenvironments that potentiate the organization of their intracellular space. We hypothesized that boundary conditions in the extracellular space potentiate the organization of cytoskeletal scaffolds for directed sarcomeregenesis. We developed a quantitative model of how the cytoskeleton of neonatal rat ventricular myocytes organizes with respect to geometric cues in the extracellular matrix. Numerical results and in vitro assays to control myocyte shape indicated that distinct cytoskeletal architectures arise from two temporally-ordered, organizational processes: the interaction between actin fibers, premyofibrils and focal adhesions, as well as cooperative alignment and parallel bundling of nascent myofibrils. Our results suggest that a hierarchy of mechanisms regulate the self-organization of the contractile cytoskeleton and that a positive feedback loop is responsible for initiating the break in symmetry, potentiated by extracellular boundary conditions, is required to polarize the contractile cytoskeleton., Author Summary How muscle is organized impacts its function. However, understanding how muscle organizes is challenging, as the process occurs over several length scales. We approach this multiscale coupling problem by constraining the overall shapes of muscle cells to indirectly control the organization of their intracellular space. We hypothesized the cellular boundary conditions direct the organization of cytoskeletal scaffolds. We developed a model of how the cytoskeleton of cardiomyocytes organizes with respect to boundary cues. Our computational and experimental results to control myocyte shape indicated that distinct muscle architectures arise from two main organizational mechanisms: the interaction between actin fibers, premyofibrils and focal adhesions, as well as cooperative alignment and parallel bundling of more mature myofibrils. We show that a hierarchy of processes regulate the self-organization of cardiomyocytes. Our results suggest that a symmetry break, due to the boundary conditions imposed on the cell, is responsible for polarization of the contractile cytoskeletal organization.

BACKGROUND: Low frequency (4-12 cpm) spontaneous fluctuations of the cerebrovascular tone (vasomotion) and oscillations of the cerebral blood flow (CBF) have been reported in diseases associated with endothelial dysfunction. Since endothelium-derived nitric oxide (NO) suppresses constitutively the release and vascular effects of thromboxane A(2) (TXA(2)), NO-deficiency is often associated with activation of thromboxane receptors (TP). In the present study we hypothesized that in the absence of NO, overactivation of the TP-receptor mediated cerebrovascular signaling pathway contributes to the development of vasomotion and CBF oscillations. METHODOLOGY/PRINCIPAL FINDINGS: Effects of pharmacological modulation of TP-receptor activation and its downstream signaling pathway have been investigated on CBF oscillations (measured by laser-Doppler flowmetry in anesthetized rats) and vasomotion (measured by isometric tension recording in isolated rat middle cerebral arteries, MCAs) both under physiological conditions and after acute inhibition of NO synthesis. Administration of the TP-receptor agonist U-46619 (1 µg/kg i.v.) to control animals failed to induce any changes of the systemic or cerebral circulatory parameters. Inhibition of the NO synthesis by nitro-L-arginine methyl ester (L-NAME, 100 mg/kg i.v.) resulted in increased mean arterial blood pressure and a decreased CBF accompanied by appearance of CBF-oscillations with a dominant frequency of 148±2 mHz. U-46619 significantly augmented the CBF-oscillations induced by L-NAME while inhibition of endogenous TXA(2) synthesis by ozagrel (10 mg/kg i.v.) attenuated it. In isolated MCAs U-46619 in a concentration of 100 nM, which induced weak and stable contraction under physiological conditions, evoked sustained vasomotion in the absence of NO, which effect could be completely reversed by inhibition of Rho-kinase by 10 µM Y-27632. CONCLUSION/SIGNIFICANCE: These results suggest that hypersensitivity of the TP-receptor-Rho-kinase signaling pathway contributes to the development of low frequency cerebral vasomotion which may propagate to vasospasm in pathophysiological states associated with NO-deficiency.

Author(s): Bishop, Joseph R; Passos-Bueno, Maria Rita; Fong, Loren; Stanford, Kristin I; Gonzales, Jon C; Yeh, Erika; Young, Stephen G; Bensadoun, Andre; Witztum, Joseph L; Esko, Jeffrey D; Moulton, Karen S | Abstract: BackgroundLipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism.Methods and findingsWe examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia.ConclusionsThis is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

Background Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines. Methodology and Principal Findings Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines. Conclusions and Significance The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

The t-tubules of mammalian ventricular myocytes are invaginations of the cell membrane that occur at each Z-line. These invaginations branch within the cell to form a complex network that allows rapid propagation of the electrical signal, and hence synchronous rise of intracellular calcium (Ca2+). To investigate how the t-tubule microanatomy and the distribution of membrane Ca2+ flux affect cardiac excitation-contraction coupling we developed a 3-D continuum model of Ca2+ signaling, buffering and diffusion in rat ventricular myocytes. The transverse-axial t-tubule geometry was derived from light microscopy structural data. To solve the nonlinear reaction-diffusion system we extended SMOL software tool (http://mccammon.ucsd.edu/smol/). The analysis suggests that the quantitative understanding of the Ca2+ signaling requires more accurate knowledge of the t-tubule ultra-structure and Ca2+ flux distribution along the sarcolemma. The results reveal the important role for mobile and stationary Ca2+ buffers, including the Ca2+ indicator dye. In agreement with experiment, in the presence of fluorescence dye and inhibited sarcoplasmic reticulum, the lack of detectible differences in the depolarization-evoked Ca2+ transients was found when the Ca2+ flux was heterogeneously distributed along the sarcolemma. In the absence of fluorescence dye, strongly non-uniform Ca2+ signals are predicted. Even at modest elevation of Ca2+, reached during Ca2+ influx, large and steep Ca2+ gradients are found in the narrow sub-sarcolemmal space. The model predicts that the branched t-tubule structure and changes in the normal Ca2+ flux density along the cell membrane support initiation and propagation of Ca2+ waves in rat myocytes., Author Summary In cardiac muscle cells, calcium (Ca2+) is best known for its role in contraction activation. A remarkable amount of quantitative data on cardiac cell structure, ion-transporting protein distributions and intracellular Ca2+ dynamics has been accumulated. Various alterations in the protein distributions or cell ultra-structure are now recognized to be the primary mechanisms of cardiac dysfunction in a diverse range of common pathologies including cardiac arrhythmias and hypertrophy. Using a 3-D computational model, incorporating more realistic transverse-axial t-tubule geometry and considering geometric irregularities and inhomogeneities in the distribution of ion-transporting proteins, we analyze several important spatial and temporal features of Ca2+ signaling in rat ventricular myocytes. This study demonstrates that the computational models could serve as powerful tools for prediction and analyses of how the Ca2+ dynamics and cardiac excitation-contraction coupling are regulated under normal conditions or certain pathologies. The use of computational and mathematical approaches will help also to better understand aspects of cell functions that are not currently amenable to experimental investigation.

Background The assessment of baroreflex sensitivity (BRS) has emerged as prognostic tool in cardiology. Although available computer-assisted methods, measuring spontaneous fluctuations of heart rate and blood pressure in the time and frequency domain are easily applicable, they do not allow for quantification of BRS during cardiovascular adaption processes. This, however, seems an essential criterion for clinical application. We evaluated a novel algorithm based on trigonometric regression regarding its ability to map dynamic changes in BRS and autonomic tone during cardiovascular provocation in relation to gender and age. Methodology/Principal Findings We continuously recorded systemic arterial pressure, electrocardiogram and respiration in 23 young subjects (25±2 years) and 22 middle-aged subjects (56±4 years) during cardiovascular autonomic testing (metronomic breathing, Valsalva manoeuvre, head-up tilt). Baroreflex- and spectral analysis was performed using the algorithm of trigonometric regressive spectral analysis. There was an age-related decline in spontaneous BRS and high frequency oscillations of RR intervals. Changes in autonomic tone evoked by cardiovascular provocation were observed as shifts in the ratio of low to high frequency oscillations of RR intervals and blood pressure. Respiration at 0.1 Hz elicited an increase in BRS while head-up tilt and Valsalva manoeuvre resulted in a downregulation of BRS. The extent of autonomic adaption was in general more pronounced in young individuals and declined stronger with age in women than in men. Conclusions/Significance The trigonometric regressive spectral analysis reliably maps age- and gender-related differences in baroreflex- and autonomic function and is able to describe adaption processes of baroreceptor circuit during cardiovascular stimulation. Hence, this novel algorithm may be a useful screening tool to detect abnormalities in cardiovascular adaption processes even when resting values appear to be normal.

Background During vascular injury, vascular smooth muscle cells (SMCs) and fibroblasts/myofibroblasts (FBs/MFBs) are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms. Methodology/principal findings Exposure to 8 dyn/cm(2) laminar flow shear stress (2-dimensional, 2-D) for 15 h significantly reduced expression of alpha-smooth muscle actin (alpha-SMA), smooth muscle protein 22 (SM22), SM myosin heavy chain (SM-MHC), smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH(2)O, approximately 0.05 dyn/cm(2), 3-D), and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of alpha-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor) and heparinase III (an enzyme to cleave heparan sulfate) significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of alpha-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs). Conclusions/significance The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation.

Restenosis remains the main complication of balloon angioplasty and/or stent implantation. Preclinical testing of new pharmacologic agents preventing restenosis largely rely on porcine models, where restenosis is assessed after endothelial abrasion of the arterial wall or stent implantation. We combined endothelial cell denudation and implantation of stents to develop a new clinically relevant porcine model of restenosis, and used this model to determine the effects of an α4 integrin inhibitor, ELN 457946, on restenosis. Balloon-angioplasty endothelial cell denudation and subsequent implantation of bare metal stents in the left anterior descending coronary, iliac, and left common carotid arteries was performed in domestic pigs, treated with vehicle or ELN 457946, once weekly via subcutaneous injections, for four weeks. After 1 month, histopathology and morphometric analyses of the arteries showed complete healing and robust, consistent restenotic response in stented arteries. Treatment with ELN 457946 resulted in a reduction in the neointimal response, with decreases in area percent stenosis between 12% in coronary arteries and 30% in peripheral vessels. This is the first description of a successful pig model combining endothelial cell denudation and bare metal stent implantation. This new double injury model may prove particularly useful to assess pharmacological effects of drug candidates on restenosis, in coronary and/or peripheral arteries. Furthermore, the ELN 457946 α4 integrin inhibitor, administered subcutaneously, reduced inflammation and restenosis in stented coronary and peripheral arteries in pigs, therefore representing a promising systemic therapeutic approach in reducing restenosis in patients undergoing angioplasty and/or stent implantation.

Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca2+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca2+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca2+ dynamics: 1) the biphasic increment during the upstroke of the Ca2+ transient resulting from the delay between the peripheral and central SR Ca2+ release, and 2) the relative contribution of SL Ca2+ current and SR Ca2+ release to the Ca2+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca2+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca2+ release sites define the interface between Ca2+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca2+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca2+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca2+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes., Author Summary In the human heart, the contraction of atrial and ventricular muscle cells is based largely on common mechanisms. There is, however, a fundamental difference in the cellular calcium dynamics that underlie the contractile function. Here, we have developed a computational model of the human atrial cell that convincingly reproduces the experimentally observed characteristics of the electrical activity and the cyclic fluctuations of the intracellular calcium concentration. With the model, we evaluate the relative roles of the most important cellular calcium transport mechanisms and their impact on the electrical behavior of the cell. Our simulations predict that the amount of calcium released from the cellular stores during each electrical cycle crucially regulates the excitability of the human atrial cell. Furthermore, the results indicate that the cellular sodium accumulation related to faster heart rates is one of the main mechanisms driving the adaptation of cardiac electrical activity. Finally, we conclude that the presented model also provides a useful framework for future studies of human atrial cells.

The transcriptome, as the pool of all transcribed elements in a given cell, is regulated by the interaction between different molecular levels, involving epigenetic, transcriptional, and post-transcriptional mechanisms. However, many previous studies investigated each of these levels individually, and little is known about their interdependency. We present a systems biology study integrating mRNA profiles with DNA–binding events of key cardiac transcription factors (Gata4, Mef2a, Nkx2.5, and Srf), activating histone modifications (H3ac, H4ac, H3K4me2, and H3K4me3), and microRNA profiles obtained in wild-type and RNAi–mediated knockdown. Finally, we confirmed conclusions primarily obtained in cardiomyocyte cell culture in a time-course of cardiac maturation in mouse around birth. We provide insights into the combinatorial regulation by cardiac transcription factors and show that they can partially compensate each other's function. Genes regulated by multiple transcription factors are less likely differentially expressed in RNAi knockdown of one respective factor. In addition to the analysis of the individual transcription factors, we found that histone 3 acetylation correlates with Srf- and Gata4-dependent gene expression and is complementarily reduced in cardiac Srf knockdown. Further, we found that altered microRNA expression in Srf knockdown potentially explains up to 45% of indirect mRNA targets. Considering all three levels of regulation, we present an Srf-centered transcription network providing on a single-gene level insights into the regulatory circuits establishing respective mRNA profiles. In summary, we show the combinatorial contribution of four DNA–binding transcription factors in regulating the cardiac transcriptome and provide evidence that histone modifications and microRNAs modulate their functional consequence. This opens a new perspective to understand heart development and the complexity cardiovascular disorders., Author Summary An evolutionary conserved orchestra of transcription factors controls cardiac development and function. More recently the contributions of epigenetic and post-transcriptional mechanisms like histone modifications and microRNAs have been identified. The interplay between these regulatory mechanisms is still an open question. However, perturbations of the cardiac transcriptome, triggered by all three levels of regulation, are underlying cardiovascular disease such as congenital heart malformations. Here, we show the impact of the interdependencies of four key transcription factors (Gata4, Mef2a, Nkx2.5, and Srf) and the contribution of activating histone modifications and microRNAs on the cardiac transcriptome. We found that even these non-paralogous transcription factors can partially compensate each other's function. Our data show that histone 3 acetylation correlates with Srf- and Gata4- dependent gene activation. Moreover, we predict a large proportion of indirect Srf targets to be regulated by Srf-dependent microRNAs, which thus might represent an important intermediate layer of regulation. Taken together, we suggest that the different levels regulating cardiac mRNA profiles have a high degree of interdependency and the potential to buffer each other, which presents a starting point to understand the phenotypic variability typically seen in complex cardiovascular disorders.

Background Nitric oxide (NO) has long been recognized to affect muscle contraction [1], both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins. Principal Findings Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo. Significance These data show that nitrosylation signaling acts as a molecular “gear shift” for myosin—an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.

Background To identify molecular mechanisms underlying SCN5A-related sick sinus syndrome (SSS), a rare type of SSS, in parallel experiments we elucidated the electrophysiological properties and the cell surface localization of thirteen human Nav1.5 (hNav1.5) mutant channels previously linked to this disease. Methodology/Principal Findings Mutant hNav1.5 channels expressed by HEK293 cells and Xenopus oocytes were investigated by whole-cell patch clamp and two-microelectrode voltage clamp, respectively. HEK293 cell surface biotinylation experiments quantified the fraction of correctly targeted channel proteins. Our data suggested three distinct mutant channel subtypes: Group 1 mutants (L212P, P1298L, DelF1617, R1632H) gave peak current densities and cell surface targeting indistinguishable from wild-type hNav1.5. Loss-of-function of these mutants resulted from altered channel kinetics, including a negative shift of steady-state inactivation and a reduced voltage dependency of open-state inactivation. Group 2 mutants (E161K, T220I, D1275N) gave significantly reduced whole-cell currents due to impaired cell surface localization (D1275N), altered channel properties at unchanged cell surface localization (T220I), or a combination of both (E161K). Group 3 mutant channels were non-functional, due to an almost complete lack of protein at the plasma membrane (T187I, W1421X, K1578fs/52, R1623X) or a probable gating/permeation defect with normal surface localisation (R878C, G1408R). Conclusions/Significance This study indicates that multiple molecular mechanisms, including gating abnormalities, trafficking defects, or a combination of both, are responsible for SCN5A-related familial SSS.

Background Hemolymph circulation in mosquitoes is primarily controlled by the contractile action of a dorsal vessel that runs underneath the dorsal midline and is subdivided into a thoracic aorta and an abdominal heart. Wave-like peristaltic contractions of the heart alternate in propelling hemolymph in anterograde and retrograde directions, where it empties into the hemocoel at the terminal ends of the insect. During our analyses of hemolymph propulsion in Anopheles gambiae, we observed periodic ventral abdominal contractions and hypothesized that they promote extracardiac hemolymph circulation in the abdominal hemocoel. Methodology/Principal Findings We devised methods to simultaneously analyze both heart and abdominal contractions, as well as to measure hemolymph flow in the abdominal hemocoel. Qualitative and quantitative analyses revealed that ventral abdominal contractions occur as series of bursts that propagate in the retrograde direction. Periods of ventral abdominal contraction begin only during periods of anterograde heart contraction and end immediately following a heartbeat directional reversal, suggesting that ventral abdominal contractions function to propel extracardiac hemolymph in the retrograde direction. To test this functional role, fluorescent microspheres were intrathoracically injected and their trajectory tracked throughout the hemocoel. Quantitative measurements of microsphere movement in extracardiac regions of the abdominal cavity showed that during periods of abdominal contractions hemolymph flows in dorsal and retrograde directions at a higher velocity and with greater acceleration than during periods of abdominal rest. Histochemical staining of the abdominal musculature then revealed that ventral abdominal contractions result from the contraction of intrasegmental lateral muscle fibers, intersegmental ventral muscle bands, and the ventral transverse muscles that form the ventral diaphragm. Conclusions/Significance These data show that abdominal contractions potentiate extracardiac retrograde hemolymph propulsion in the abdominal hemocoel during periods of anterograde heart flow.

Background: Within the general population, levels of C-reactive protein (CRP) are positively associated with atherosclerotic cardiovascular disease (CVD). Whether CRP is causally implicated in atherogenesis or is the results of atherosclerosis is disputed. A role of CRP to protect endothelium-derived nitric oxide (EDNO) has been suggested. We examined the association of CRP with EDNO-dependent vasomotor function and subclinical measures of atherosclerosis and arteriosclerosis in patients with raised CRP resulting from rheumatoid arthritis (RA).Methodology/Principal Findings: Patients with RA (n = 59) and healthy control subjects (n = 123), underwent measures of high sensitivity CRP, flow-mediated dilation (FMD, dependent on EDNO), intima-media thickness (IMT, a measure of subclinical atherosclerosis) and aortic pulse wave velocity (PWV, a measure of arteriosclerosis). IMT and PWV were elevated in patients with RA compared to controls but FMD was similar in the two groups. In patients with RA, IMT and PWV were not correlated with CRP but FMD was positively independently correlated with CRP (P

Background Besides its well-established role in nerve cell survival and adaptive plasticity, brain-derived neurotrophic factor (BDNF) is also involved in energy homeostasis and cardiovascular regulation. Although BDNF is present in the systemic circulation, it is unknown whether plasma BDNF correlates with circulating markers of dysregulated metabolism and an adverse cardiovascular profile. Methodology/Principal Findings To determine whether circulating BDNF correlates with indices of metabolic and cardiovascular health, we measured plasma BDNF levels in 496 middle-age and elderly subjects (mean age ∼70), in the Baltimore Longitudinal Study of Aging. Linear regression analysis revealed that plasma BDNF is associated with risk factors for cardiovascular disease and metabolic syndrome, regardless of age. In females, BDNF was positively correlated with BMI, fat mass, diastolic blood pressure, total cholesterol, and LDL-cholesterol, and inversely correlated with folate. In males, BDNF was positively correlated with diastolic blood pressure, triglycerides, free thiiodo-thyronine (FT3), and bioavailable testosterone, and inversely correlated with sex-hormone binding globulin, and adiponectin. Conclusion/Significance Plasma BDNF significantly correlates with multiple risk factors for metabolic syndrome and cardiovascular dysfunction. Whether BDNF contributes to the pathogenesis of these disorders or functions in adaptive responses to cellular stress (as occurs in the brain) remains to be determined.

Background: Diurnal variations in the incidence of events such as heart attack and stroke suggest a role for circadian rhythms in the etiology of cardiovascular disease. The aim of this study was to assess the influence of the suprachiasmatic nucleus (SCN) circadian clock on cardiovascular function.Methodology/Principal Findings: Heart rate (HR), blood pressure (BP) and locomotor activity (LA) were measured in circadian mutant (Vipr2(-/-)) mice and wild type littermates, using implanted radio-telemetry devices. Sleep and wakefulness were studied in similar mice implanted with electroencephalograph (EEG) electrodes. There was less diurnal variation in the frequency and duration of bouts of rest/activity and sleep/wake in Vipr2(-/-) mice than in wild type (WT) and short "ultradian" episodes of arousal were more prominent, especially in constant conditions (DD). Activity was an important determinant of circadian variation in BP and HR in animals of both genotypes; altered timing of episodes of activity and rest (as well as sleep and wakefulness) across the day accounted for most of the difference between Vipr2(-/-) mice and WT. However, there was also a modest circadian rhythm of resting HR and BP that was independent of LA.Conclusions/Significance: If appropriate methods of analysis are used that take into account sleep and locomotor activity level, mice are a good model for understanding the contribution of circadian timing to cardiovascular function. Future studies of the influence of sleep and wakefulness on cardiovascular physiology may help to explain accumulating evidence linking disrupted sleep with cardiovascular disease in man.

Pharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lipid absorption using the zebrafish system. A pilot screen of an unbiased chemical library identified novel compounds that inhibited processing of fluorescent lipid analogues in live zebrafish larvae. Secondary assays identified those compounds suitable for testing in mammals and provided insight into mechanism of action, which for several compounds could be distinguished from ezetimibe, a drug used to inhibit cholesterol absorption in humans that broadly inhibited lipid absorption in zebrafish larvae. These findings support the utility of zebrafish screening assays to identify novel compounds that target complex physiological processes.

Background The changes in the actomyosin crossbridge cycle underlying altered contractility of the heart are not well described, despite their importance to devising rational treatment approaches. Methodology/Principal Findings A rat ischemia–reperfusion model was used to determine the transitions of the crossbridge cycle impacted during ischemia. Compared to perfused hearts, the maximum force per cross-sectional area and Ca2+ sensitivity of fibers from ischemic hearts were both reduced. Muscle activation by photolytic release of Ca2+ and ATP suggested that the altered contractility was best described as a reduction in the rate of activation of noncycling actomyosin crossbridges to activated, cycling states. More specifically, the apparent forward rate constant of the transition between the nonforce bearing A-M.ADP.Pi state and the bound, force bearing AM*.ADP.Pi state was reduced in ischemic fibers, suggesting that this transition is commensurate with initial crossbridge activation. These results suggested an alteration in the relationship between the activation of thin filament regulatory units and initial crossbridge attachment, prompting an examination of the post-translational state of troponin (Tn) T and I. These analyses indicated a reduction in the diphosphorylated form of TnT during ischemia, along with lower Ser23/24 phosphorylation of TnI. Treatment of perfused fibers by 8-Br-cAMP increased Ser23/24 phosphorylation of TnI, altering the reverse rate constant of the Pi isomerization in a manner consistent with the lusitropic effect of β-adrenergic stimulation. However, similar treatment of ischemic fibers did not change TnI phosphorylation or the kinetics of the Pi isomerization. Conclusions Ischemia reduces the isomerization from A-M.ADP.Pi to AM*.ADP.Pi, altering the kinetics of crossbridge activation through a mechanism that may be mediated by altered TnT and TnI phosphorylation.

BackgroundThe pulmonary microcirculation is the chief regulatory site for resistance in the pulmonary circuit. Despite pulmonary microvascular dysfunction being implicated in the pathogenesis of several pulmonary vascular conditions, there are currently no techniques for the specific assessment of pulmonary microvascular integrity in humans. Peak hyperemic flow assessment using thermodilution-derived mean transit-time (T(mn)) facilitate accurate coronary microcirculatory evaluation, but remain unvalidated in the lung circulation. Using a high primate model, we aimed to explore the use of T(mn) as a surrogate of pulmonary blood flow for the purpose of measuring the novel indices Pulmonary Flow Reserve [PFR = (maximum hyperemic)/(basal flow)] and Pulmonary Index of Microcirculatory Resistance [PIMR = (maximum hyperemic distal pulmonary artery pressure)x(maximum hyperemic T(mn))]. Ultimately, we aimed to investigate the effect of progressive pulmonary microvascular obstruction on PFR and PIMR.Methods and resultsTemperature- and pressure-sensor guidewires (TPSG) were placed in segmental pulmonary arteries (SPA) of 13 baboons and intravascular temperature measured. T(mn) and hemodynamics were recorded at rest and following intra-SPA administration of the vasodilator agents adenosine (10-400 microg/kg/min) and papaverine (3-24 mg). Temperature did not vary with intra-SPA sensor position (0.010+/-0.009 v 0.010+/-0.009 degrees C; distal v proximal; p = 0.1), supporting T(mn) use in lung for the purpose of hemodynamic indices derivation. Adenosine (to 200 microg/kg/min) & papaverine (to 24 mg) induced dose-dependent flow augmentations (40+/-7% & 35+/-13% T(mn) reductions v baseline, respectively; pConclusionsThermodilution-derived mean transit time can be accurately and reproducibly measured in the pulmonary circulation using TPSG. Mean transit time-derived PFR and PIMR can be assessed using a TPSG and adenosine or papaverine as hyperemic agents. These novel indices detect progressive pulmonary microvascular obstruction and thus have with a potential role for pulmonary microcirculatory assessment in humans.

BackgroundSudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated.Methodology/principal findingsLeft ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [(3)H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled B(max) value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls).ConclusionsIn the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism.

Background Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies. Methodology/Principal Findings Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner. Conclusions/Significance Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.

Background Hypoxic states of the cardiovacular system are undoubtedly associated with the most frequent diseases of modern time. Therefore, understanding hypoxic resistance encountered after physiological adaptation such as chronic hypoxia, is crucial to better deal with hypoxic insult. In this study, we examine the role of energetic modifications induced by chronic hypoxia (CH) in the higher tolerance to oxygen deprivation. Methodology/Principal Findings Swiss mice were exposed to a simulated altitude of 5500 m in a barochamber for 21 days. Isolated perfused hearts were used to study the effects of a decreased oxygen concentration in the perfusate on contractile performance (RPP) and phosphocreatine (PCr) concentration (assessed by 31P-NMR), and to describe the integrated changes in cardiac energetics regulation by using Modular Control Analysis (MoCA). Oxygen reduction induced a concomitant decrease in RPP (−46%) and in [PCr] (−23%) in Control hearts while CH hearts energetics was unchanged. MoCA demonstrated that this adaptation to hypoxia is the direct consequence of the higher responsiveness (elasticity) of ATP production of CH hearts compared with Controls (−1.88±0.38 vs −0.89±0.41, p

Inherited cardiomyopathy (CM) represents a diverse group of cardiac muscle diseases that present with a broad spectrum of symptoms ranging from benign to highly malignant. Contributing to this genetic complexity and clinical heterogeneity is the emergence of a cohort of patients that are double or compound heterozygotes who have inherited two different CM mutant alleles in the same or different sarcomeric gene. These patients typically have early disease onset with worse clinical outcomes. Little experimental attention has been directed towards elucidating the physiologic basis of double CM mutations at the cellular-molecular level. Here, dual gene transfer to isolated adult rat cardiac myocytes was used to determine the primary effects of co-expressing two different CM-linked mutant proteins on intact cardiac myocyte contractile physiology. Dual expression of two CM mutants, that alone moderately increase myofilament activation, tropomyosin mutant A63V and cardiac troponin mutant R146G, were shown to additively slow myocyte relaxation beyond either mutant studied in isolation. These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles alphaTmA63V and cTnI R193H, which approached a functional threshold. Interestingly, a combination of a CM myofilament deactivating mutant, troponin C G159D, together with an activating mutant, cTnIR193H, produced a hybrid phenotype that blunted the strong activating phenotype of cTnIR193H alone. This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination. Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients.

BACKGROUND: The sequence of events leading to the development of insulin resistance (IR) as well as the underlying pathophysiological mechanisms are incompletely understood. As reductionist approaches have been largely unsuccessful in providing an understanding of the pathogenesis of IR, there is a need for an integrative, time-resolved approach to elucidate the development of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Male ApoE3Leiden transgenic mice exhibiting a humanized lipid metabolism were fed a high-fat diet (HFD) for 0, 1, 6, 9, or 12 weeks. Development of IR was monitored in individual mice over time by performing glucose tolerance tests and measuring specific biomarkers in plasma, and hyperinsulinemic-euglycemic clamp analysis to assess IR in a tissue-specific manner. To elucidate the dynamics and tissue-specificity of metabolic and inflammatory processes key to IR development, a time-resolved systems analysis of gene expression and metabolite levels in liver, white adipose tissue (WAT), and muscle was performed. During HFD feeding, the mice became increasingly obese and showed a gradual increase in glucose intolerance. IR became first manifest in liver (week 6) and then in WAT (week 12), while skeletal muscle remained insulin-sensitive. Microarray analysis showed rapid upregulation of carbohydrate (only liver) and lipid metabolism genes (liver, WAT). Metabolomics revealed significant changes in the ratio of saturated to polyunsaturated fatty acids (liver, WAT, plasma) and in the concentrations of glucose, gluconeogenesis and Krebs cycle metabolites, and branched amino acids (liver). HFD evoked an early hepatic inflammatory response which then gradually declined to near baseline. By contrast, inflammation in WAT increased over time, reaching highest values in week 12. In skeletal muscle, carbohydrate metabolism, lipid metabolism, and inflammation was gradually suppressed with HFD. CONCLUSIONS/SIGNIFICANCE: HFD-induced IR is a time- and tissue-dependent process that starts in liver and proceeds in WAT. IR development is paralleled by tissue-specific gene expression changes, metabolic adjustments, changes in lipid composition, and inflammatory responses in liver and WAT involving p65-NFkB and SOCS3. The alterations in skeletal muscle are largely opposite to those in liver and WAT.

BACKGROUND: Evidence from animal studies indicates the importance of an interaction between the sympathetic nervous system and the endothelium for cardiovascular regulation. However the interaction between these two systems remains largely unexplored in humans. The aim of this study was to investigate whether directly recorded sympathetic vasoconstrictor outflow is related to a surrogate marker of endothelial function in healthy individuals. METHODS AND RESULTS: In 10 healthy normotensive subjects (3 f/7 m), (age 37+/-11 yrs), (BMI 24+/-3 kg/m(2)) direct recordings of sympathetic action potentials to the muscle vascular bed (MSNA) were performed and endothelial function estimated with the Reactive Hyperaemia- Peripheral Arterial Tonometry (RH-PAT) technique. Blood samples were taken and time spent on leisure-time physical activities was estimated. In all subjects the rate between resting flow and the maximum flow, the Reactive Hyperemic index (RH-PAT index), was within the normal range (1.9-3.3) and MSNA was as expected for age and gender (13-44 burst/minute). RH-PAT index was inversely related to MSNA (r = -0.8, p = 0.005). RH-PAT index and MSNA were reciprocally related to time (h/week) spent on physical activity (p = 0.005 and p = 0.006 respectively) and platelet concentration (PLT) (p = 0.02 and p = 0.004 respectively). CONCLUSIONS: Our results show that sympathetic nerve activity is related to a surrogate marker of endothelial function in healthy normotensive individuals, indicating that sympathetic outflow may be modulated by changes in endothelial function. In this study time spent on physical activity is identified as a predictor of sympathetic nerve activity and endothelial function in a group of healthy individuals. The results are of importance in understanding mechanisms underlying sympathetic activation in conditions associated with endothelial dysfunction and emphasise the importance of a daily exercise routine for maintenance of cardiovascular health.

Background The repolarizing cardiac rapid delayed rectifier current, I(Kr), is composed of ERG1 channels. It has been suggested that two isoforms of the ERG1 protein, ERG1a and ERG1b, both contribute to I(Kr). Marked heterogeneity in the kinetic properties of native I(Kr) has been described. We hypothesized that the heterogeneity of native I(Kr) can be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the functional consequences of differential expression of ERG1 isoforms were explored as a potential mechanism underlying native heterogeneity of action potential duration (APD) and restitution. Methodology/principal findings The results show that the heterogeneity of native I(Kr) can be reproduced in heterologous expression systems by differential expression of ERG1a and ERG1b isoforms. Characterization of the macroscopic kinetics of ERG1 currents demonstrated that these were dependent on the relative abundance of ERG1a and ERG1b. Furthermore, we used a computational model of the ventricular cardiomyocyte to show that both APD and the slope of the restitution curve may be modulated by varying the relative abundance of ERG1a and ERG1b. As the relative abundance of ERG1b was increased, APD was gradually shortened and the slope of the restitution curve was decreased. Conclusions/significance Our results show that differential expression of ERG1 isoforms may explain regional heterogeneity of I(Kr) kinetics. The data demonstrate that subunit dependent changes in channel kinetics are important for the functional properties of ERG1 currents and hence I(Kr). Importantly, our results suggest that regional differences in the relative abundance of ERG1 isoforms may represent a potential mechanism underlying the heterogeneity of both APD and APD restitution observed in mammalian hearts.

The identification of NOX4 as a major source of oxidative stress in stroke and demonstration of dramatic protection after stroke in mice by NOX4 deletion or NOX inhibition, opens up new avenues for treatment., Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4 −/−) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4 −/− mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy., Author Summary Stroke is the second leading cause of death worldwide. Today, only one approved therapy exists—a drug that breaks down blood clots—the effectiveness of which is moderate, and it can only be used in about 10% of patients because of contraindications. New therapeutic strategies that are translatable to humans and more rigid thresholds of relevance in pre-clinical stroke models are needed. One candidate mechanism is oxidative stress, which is the damage caused by reactive oxygen species (ROS). Antioxidant approaches that specifically target ROS have thus far failed in clinical trials. For a more effective approach, we focus here on targeting ROS at its source by investigating an enzyme involved in generating ROS, known as NADPH oxidase type 4, or NOX4. We found that NOX4 causes oxidative stress and death of nerve cells after a stroke. Deletion of the NOX4-coding gene in mice, as well as inhibiting the ROS-generating activity of NOX with a pharmacological inhibitor, reduces brain damage and improves neurological function, even when given hours after a stroke. Importantly, neuroprotection was preserved in old male and female Nox4 −/− mice as well as in Nox4 −/− mice subjected to permanent ischemia. NOX4 thus represents a most promising new therapeutic target for reducing oxidative stress in general, and in brain injury due to stroke in particular.

BACKGROUND: Meprin metalloproteases are thought to be involved in basic physiological functions such as cell proliferation and tissue differentiation. However, the specific functions of these enzymes are still ambiguous, although a variety of growth factors and structural proteins have been identified as meprin substrates. The discovery of meprins alpha(1), alpha(2) and beta in teleost fish provided the basis for uncovering their physiological functions by gene silencing in vivo. METHODOLOGY/PRINCIPAL FINDINGS: A Morpholino knockdown in zebrafish embryos targeting meprin alpha(1) and beta mRNA caused defects in general tissue differentiation. But meprin alpha(2) morphants were affected more specifically and showed severe failures in the formation of the vascular system provoking the hypothesis of a pro-angiogenic effect. The blood circulation was largely diminished resulting in erythrocyte accumulation. These phenotypes mimic a previously described VEGF-A morphant, revealing a possible role of meprin alpha in VEGF-A activation. Indeed, human recombinant meprin alpha processed the vascular endothelial growth factor-A (VEGF-A) specifically, revealing the same cleavage products detectable for VEGF from zebrafish whole lysate. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that meprin metalloproteases are important for cell differentiation and proliferation already during embryogenesis, predominantly by the activation of growth factors. Thus, we conclude that meprins play a significant role in VEGF-A processing, subsequently regulating angiogenesis. Therefore, meprin alpha might be a new therapeutic target in cardiovascular diseases or in tumor growth inhibition.

BACKGROUND: Loss-of-function mutations in SCN5A, the gene encoding Na(v)1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a(+/-) mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. METHODOLOGY/PRINCIPAL FINDINGS: Based on ECG, 10-week-old Scn5a(+/-) mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS< or = 18 ms; QRS in wild-type littermates: 10-18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a(+/-) mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a(+/-) mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A-mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a(+/-) mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a(+/-) mice had similar Na(v)1.5 mRNA but higher Na(v)1.5 protein expression, and moderately larger I(Na) current than severely affected Scn5a(+/-) mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a(+/-) mice than in mildly affected ones. CONCLUSIONS: Scn5a(+/-) mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a(+/-) mice, phenotype severity correlates with wild-type Na(v)1.5 protein expression.

UNLABELLED: Ingestion of glucosinolates has previously been reported to improve endothelial function in spontaneously hypertensive rats, possibly because of an increase in NO availability in the endothelium due to an attenuation of oxidative stress; in our study we tried to see if this also would be the case in humans suffering from essential hypertension.METHODS: 40 hypertensive individuals without diabetes and with normal levels of cholesterol were examined. The participants were randomized either to ingest 10 g dried broccoli sprouts, a natural donor of glucosinolates with high in vitro antioxidative potential, for a 4 week period or to continue their ordinary diet and act as controls. Blood pressure, endothelial function measured by flow mediated dilation (FMD) and blood samples were obtained from the participants every other week and the content of glucosinolates was measured before and after the study. Measurements were blinded to treatment allocation.RESULTS: In the interventional group overall FMD increased from 4% to 5.8% in the interventional group whereas in the control group FMD was stable (4% at baseline and 3.9% at the end of the study). The change in FMD in the interventional group was mainly due to a marked change in FMD in two participants while the other participants did not have marked changes in FMD. The observed differences were not statistically significant. Likewise significant changes in blood pressure or blood samples were not detected between or within groups. Diastolic blood pressure stayed essentially unchanged in both groups, while the systolic blood pressure showed a small non significant decrease (9 mm Hg) in the interventional group from a value of 153 mm Hg at start.CONCLUSION: Daily ingestion of 10 g dried broccoli sprouts does not improve endothelial function in the presence of hypertension in humans.TRIAL REGISTRATION: Clinicaltrials.gov NCT00252018.

International audience; BACKGROUND: A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERalpha) could be involved in the transduction of the vascular benefits of polyphenols. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used ERalpha deficient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, Provinols, or delphinidin, an anthocyanin that possesses similar pharmacological profile, is mediated by ERalpha. Indeed, Provinols, delphinidin and ERalpha agonists, 17-beta-estradiol and PPT, are able to induce endothelial vasodilatation in aorta from ERalpha Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing the effects of ERalpha completely prevented the effects of Provinols and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between delphinidin and ERalpha activator site is demonstrated using both binding assay and docking. Most interestingly, the ability of short term oral administration of Provinols to decrease response to serotonin and to enhance sensitivity of the endothelium-dependent relaxation to acetylcholine, associated with concomitant increased NO production and decreased superoxide anions, was completely blunted in ERalpha deficient mice. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benefits via ERalpha activation. It is a major breakthrough bringing new insights of the potential therapeutic of polyphenols against cardiovascular pathologies.

Acute effects of sex steroid hormones likely contribute to the observation that post-pubescent males have shorter QT intervals than females. However, the specific role for hormones in modulating cardiac electrophysiological parameters and arrhythmia vulnerability is unclear. Here we use a computational modeling approach to incorporate experimentally measured effects of physiological concentrations of testosterone, estrogen and progesterone on cardiac ion channel targets. We then study the hormone effects on ventricular cell and tissue dynamics comprised of Faber-Rudy computational models. The “female” model predicts changes in action potential duration (APD) at different stages of the menstrual cycle that are consistent with clinically observed QT interval fluctuations. The “male” model predicts shortening of APD and QT interval at physiological testosterone concentrations. The model suggests increased susceptibility to drug-induced arrhythmia when estradiol levels are high, while testosterone and progesterone are apparently protective. Simulations predict the effects of sex steroid hormones on clinically observed QT intervals and reveal mechanisms of estrogen-mediated susceptibility to prolongation of QT interval. The simulations also indicate that acute effects of estrogen are not alone sufficient to cause arrhythmia triggers and explain the increased risk of females to Torsades de Pointes. Our results suggest that acute effects of sex steroid hormones on cardiac ion channels are sufficient to account for some aspects of gender specific susceptibility to long-QT linked arrhythmias., Author Summary It is well known that female gender is an independent risk factor for some types of cardiac arrhythmias. However, it has been difficult to determine how much of a role physiological concentrations of circulating sex steroid hormones play in gender linked arrhythmia susceptibility because the cardiac system is so extraordinarily complex. Here we employ a computational strategy, based on experimental measurements, to tease out the individual contributions of estrogen, progesterone and testosterone on cardiac electrical behavior and then make predictions about their effects in combination and in the presence of drugs. The computational models convincingly reproduce observed fluctuations of QT intervals (as recorded on the ECG (electrocardiogram), the QT interval reflects the time period between ventricular excitation and relaxation) through the menstrual cycle in females and effects of testosterone on ECG parameters. Our simulations also predict that testosterone and progesterone are protective against drug-induced arrhythmias, while estrogen likely exacerbates the breakdown of normal cardiac electrical activity in the presence of QT-prolonging drugs.

BACKGROUND: Cardiac cell transplantation is compromised by low cell retention and poor graft viability. Here, the effects of co-injecting adipose tissue-derived stem cells (ASCs) with biopolymers on cell cardiac retention, ventricular morphometry and performance were evaluated in a rat model of myocardial infarction (MI). METHODOLOGY/PRINCIPAL FINDINGS: 99mTc-labeled ASCs (1x10(6) cells) isolated from isogenic Lewis rats were injected 24 hours post-MI using fibrin a, collagen (ASC/C), or culture medium (ASC/M) as vehicle, and cell body distribution was assessed 24 hours later by gamma-emission counting of harvested organs. ASC/F and ASC/C groups retained significantly more cells in the myocardium than ASC/M (13.8+/-2.0 and 26.8+/-2.4% vs. 4.8+/-0.7%, respectively). Then, morphometric and direct cardiac functional parameters were evaluated 4 weeks post-MI cell injection. Left ventricle (LV) perimeter and percentage of interstitial collagen in the spare myocardium were significantly attenuated in all ASC-treated groups compared to the non-treated (NT) and control groups (culture medium, fibrin, or collagen alone). Direct hemodynamic assessment under pharmacological stress showed that stroke volume (SV) and left ventricle end-diastolic pressure were preserved in ASC-treated groups regardless of the vehicle used to deliver ASCs. Stroke work (SW), a global index of cardiac function, improved in ASC/M while it normalized when biopolymers were co-injected with ASCs. A positive correlation was observed between cardiac ASCs retention and preservation of SV and improvement in SW post-MI under hemodynamic stress. CONCLUSIONS: We provided direct evidence that intramyocardial injection of ASCs mitigates the negative cardiac remodeling and preserves ventricular function post-MI in rats and these beneficial effects can be further enhanced by administering co-injection of ASCs with biopolymers.

Background The most well known reproductive consequence of residence at high altitude (HA >2700 m) is reduction in fetal growth. Reduced fetoplacental oxygenation is an underlying cause of pregnancy pathologies, including intrauterine growth restriction and preeclampsia, which are more common at HA. Therefore, altitude is a natural experimental model to study the etiology of pregnancy pathophysiologies. We have shown that the proximate cause of decreased fetal growth is not reduced oxygen availability, delivery, or consumption. We therefore asked whether glucose, the primary substrate for fetal growth, might be decreased and/or whether altered fetoplacental glucose metabolism might account for reduced fetal growth at HA. Methods Doppler and ultrasound were used to measure maternal uterine and fetal umbilical blood flows in 69 and 58 residents of 400 vs 3600 m. Arterial and venous blood samples from mother and fetus were collected at elective cesarean delivery and analyzed for glucose, lactate and insulin. Maternal delivery and fetal uptakes for oxygen and glucose were calculated. Principal Findings The maternal arterial – venous glucose concentration difference was greater at HA. However, umbilical venous and arterial glucose concentrations were markedly decreased, resulting in lower glucose delivery at 3600 m. Fetal glucose consumption was reduced by >28%, but strongly correlated with glucose delivery, highlighting the relevance of glucose concentration to fetal uptake. At altitude, fetal lactate levels were increased, insulin concentrations decreased, and the expression of GLUT1 glucose transporter protein in the placental basal membrane was reduced. Conclusion/Significance Our results support that preferential anaerobic consumption of glucose by the placenta at high altitude spares oxygen for fetal use, but limits glucose availability for fetal growth. Thus reduced fetal growth at high altitude is associated with fetal hypoglycemia, hypoinsulinemia and a trend towards lactacidemia. Our data support that placentally-mediated reduction in glucose transport is an initiating factor for reduced fetal growth under conditions of chronic hypoxemia.

Mechanotransduction modulates cellular functions as diverse as migration, proliferation, differentiation, and apoptosis. It is crucial for organ development and homeostasis and leads to pathologies when defective. However, despite considerable efforts made in the past, the molecular basis of mechanotransduction remains poorly understood. Here, we have investigated the genetic basis of mechanotransduction in Drosophila. We show that the fly heart senses and responds to mechanical forces by regulating cardiac activity. In particular, pauses in heart activity are observed under acute mechanical constraints in vivo. We further confirm by a variety of in situ tests that these cardiac arrests constitute the biological force-induced response. In order to identify molecular components of the mechanotransduction pathway, we carried out a genetic screen based on the dependence of cardiac activity upon mechanical constraints and identified Painless, a TRPA channel. We observe a clear absence of in vivo cardiac arrest following inactivation of painless and further demonstrate that painless is autonomously required in the heart to mediate the response to mechanical stress. Furthermore, direct activation of Painless is sufficient to produce pauses in heartbeat, mimicking the pressure-induced response. Painless thus constitutes part of a mechanosensitive pathway that adjusts cardiac muscle activity to mechanical constraints. This constitutes the first in vivo demonstration that a TRPA channel can mediate cardiac mechanotransduction. Furthermore, by establishing a high-throughput system to identify the molecular players involved in mechanotransduction in the cardiovascular system, our study paves the way for understanding the mechanisms underlying a mechanotransduction pathway., Author Summary Cells sense mechanical forces and design an appropriate response crucial for cell and organ shape and differentiation during development, as well as for physiological adaptation. In particular, cardiac muscle continuously adapts to the mechanical constraints generated by its own rhythmic contractile activity. Consequently, defects in mechanosensation lead to severe pathologies, including cardiomyopathies and atherosclerosis. However, despite their well recognized functional importance, the molecular mechanisms of mechanotransduction are poorly understood. Here we study the Drosophila heart to investigate the genetic basis of mechanotransduction. We show that the heart responds to mechanical constraints by diastolic heart arrests, and we demonstrate that this phenotype can be used to identify genes controlling this particular mechanotransduction pathway. We show that the cation channel, Painless, first identified in the pain response pathway, also plays an essential function in the mechanotransduction pathway. The model system we have developed allows, for the first time, analysis of gene function in a mechanotransduction process in vivo, in the presence of endogenous mechanical constraints. These results establish the basis for an in-depth characterization of mechanotransduction pathways.

BACKGROUND Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca(2+)] ([Ca(2+)](i)) and subsequent activation of K(Ca) 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration. METHODOLOGY/PRINCIPAL FINDINGS We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+)- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM), dipyridamole (100 microM), DIDS (100 microM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca(2+)](i), in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity. CONCLUSIONS/SIGNIFICANCE The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+)-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+) for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

BackgroundMechanical overload leads to cardiac hypertrophy and mechanical unloading to cardiac atrophy. Both conditions produce similar transcriptional changes including a re-expression of fetal genes, despite obvious differences in phenotype. MicroRNAs (miRNAs) are discussed as superordinate regulators of global gene networks acting mainly at the translational level. Here, we hypothesized that defined sets of miRNAs may determine the direction of cardiomyocyte plasticity responses.Methodology/principal findingsWe employed ascending aortic stenosis (AS) and heterotopic heart transplantation (HTX) in syngenic Lewis rats to induce mechanical overloading and unloading, respectively. Heart weight was 26±3% higher in AS (n = 7) and 33±2% lower in HTX (n = 7) as compared to sham-operated (n = 6) and healthy controls (n = 7). Small RNAs were enriched from the left ventricles and subjected to quantitative stem-loop specific RT-PCR targeting a panel of 351 miRNAs. In total, 153 miRNAs could be unambiguously detected. Out of 72 miRNAs previously implicated in the cardiovascular system, 40 miRNAs were regulated in AS and/or HTX. Overall, HTX displayed a slightly broader activation pattern for moderately regulated miRNAs. Surprisingly, however, the regulation of individual miRNA expression was strikingly similar in direction and amplitude in AS and HTX with no miRNA being regulated in opposite direction. In contrast, fetal hearts from Lewis rats at embryonic day 18 exhibited an entirely different miRNA expression pattern.ConclusionsTaken together, our findings demonstrate that opposite changes in cardiac workload induce a common miRNA expression pattern which is markedly different from the fetal miRNA expression pattern. The direction of postnatal adaptive cardiac growth does, therefore, not appear to be determined at the level of single miRNAs or a specific set of miRNAs. Moreover, miRNAs themselves are not reprogrammed to a fetal program in response to changes in hemodynamic load.

Background Individuals of African descent in the United States suffer disproportionately from diseases with a metabolic etiology (obesity, metabolic syndrome, and diabetes), and from the pathological consequences of these disorders (hypertension and cardiovascular disease). Methodology/Principal Findings Using a combination of genetic/genomic and bioinformatics approaches, we identified a large number of genes that were both differentially expressed between American subjects self-identified to be of either African or European ancestry and that also contained single nucleotide polymorphisms that distinguish distantly related ancestral populations. Several of these genes control the metabolism of simple carbohydrates and are direct targets for the SREBP1, a metabolic transcription factor also differentially expressed between our study populations. Conclusions/Significance These data support the concept of stable patterns of gene transcription unique to a geographic ancestral lineage. Differences in expression of several carbohydrate metabolism genes suggest both genetic and transcriptional mechanisms contribute to these patterns and may play a role in exacerbating the disproportionate levels of obesity, diabetes, and cardiovascular disease observed in Americans with African ancestry.

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxigenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes.

Rapid arterial O2 desaturation during apnea in the preterm infant has obvious clinical implications but to date no adequate explanation for why it exists. Understanding the factors influencing the rate of arterial O2 desaturation during apnea () is complicated by the non-linear O2 dissociation curve, falling pulmonary O2 uptake, and by the fact that O2 desaturation is biphasic, exhibiting a rapid phase (stage 1) followed by a slower phase when severe desaturation develops (stage 2). Using a mathematical model incorporating pulmonary uptake dynamics, we found that elevated metabolic O2 consumption accelerates throughout the entire desaturation process. By contrast, the remaining factors have a restricted temporal influence: low pre-apneic alveolar causes an early onset of desaturation, but thereafter has little impact; reduced lung volume, hemoglobin content or cardiac output, accelerates during stage 1, and finally, total blood O2 capacity (blood volume and hemoglobin content) alone determines during stage 2. Preterm infants with elevated metabolic rate, respiratory depression, low lung volume, impaired cardiac reserve, anemia, or hypovolemia, are at risk for rapid and profound apneic hypoxemia. Our insights provide a basic physiological framework that may guide clinical interpretation and design of interventions for preventing sudden apneic hypoxemia., Author Summary When breathing stops, the flow of O2 into and the flow of CO2 out of the body cease. Such an event, termed an apnea, can be especially dangerous in preterm infants in whom it can lead to a rapid decline in arterial O2 saturation, reaching rates of 3–8% per second, rapidly reducing O2 to a level that could lead to neurological damage. Despite extensive experimental research, we have a poor mechanistic understanding of the causes of rapidly developing hypoxemia. We describe a new mathematical model that allows examination of the importance of the major cardiorespiratory factors that are likely to influence the speed at which arterial hypoxemia worsens during apnea. We found that high metabolic rate as well as reduced pre-apneic ventilation, lung volume, cardiac output, hemoglobin content, blood O2 affinity, and blood volume accelerate the development of hypoxemia during apnea. Importantly, the cardiorespiratory factors that contribute to rapid hypoxemia are all pertinent to the preterm infant during early postnatal development. Thus the newborn is highly susceptible to rapid and severe desaturation, potentially explaining the propensity of preterm infants, particularly those with apnea, to neurological impairment.

Background: Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts. Methods and Findings: A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca2+-activated K+ current (BKCa) in most (88%) human cardiac fibroblasts, a delayed rectifier K+ current (IKDR) and a transient outward K+ current (Ito) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K+ current (IKir) in 24% of cells, and a chloride current (ICl) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na+ currents (INa) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (INa.TTX, IC50 = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (INa.TTXR, IC50 = 1.8 μM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BKCa), Kv1.5, Kv1.6 (responsible for IKDR), Kv4.2, Kv4.3 (responsible for Ito), Kir2.1, Kir2.3 (for IKir), Clnc3 (for ICl), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX), and NaV1.5 (for INa.TTXR). Conclusions: These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling. © 2009 Li et al., published_or_final_version

Metabolic substrate utilization of the human failing heart is an area of controversy. The purpose of this study is to directly quantify myocardial substrate utilization in moderately severe heart failure, type 2 diabetes and healthy controls using simultaneous coronary sinus and arterial blood sampling. Patients with heart failure (n = 9, mean NYHA 2.7+/-0.5), with type 2 diabetes (n = 5) and with normal heart function (n = 10) were studied after an overnight fast in connection with electrophysiological investigations/treatments.A systemic infusion of [(2)H(2)]palmitate allowed for the calculation of absolute palmitate extraction across the heart. Blood samples were analysed for non-esterified fatty acids, triacylglycerol, glycerol, glucose, pyruvate, lactate, 3-hydroxybutyrate, and blood gases after simultaneous sampling of arterial and coronary sinus blood. Arterio-coronary sinus metabolite concentration differences and fractional extractions for all substrates were similar between the groups. The absolute NEFA uptakes assessed by [(2)H(2)]palmitate extraction were also similar between the groups. Using direct measurements of metabolic substrate uptake by arterio-venous difference technique, the compensated human failing heart does not appear to have reduced myocardial fatty acid uptake.

International audience; BackgroundBone marrow-derived endothelial progenitor cells (EPCs) are critical for neovascularization. We hypothesized that microparticles (MPs), small fragments generated from the plasma membrane, can activate angiogenic programming of EPCs.Methodology/Principal FindingsWe studied the effects of MPs obtained from wild type (MPsPPARα+/+) and knock-out (MPsPPARα−/−) mice on EPC differentiation and angiogenesis. Bone marrow-derived cells were isolated from WT or KO mice and were cultured in the presence of MPsPPARα+/+ or MPsPPARα−/− obtained from blood of mice. Only MPsPPARα+/+ harboring PPARα significantly increased EPC, but not monocytic, differentiation. Bone marrow-derived cells treated with MPsPPARα+/+ displayed increased expression of pro-angiogenic genes and increased in vivo angiogenesis. MPsPPARα+/+ increased capillary-like tube formation of endothelial cells that was associated with enhanced expressions of endothelial cell-specific markers. Finally, the effects of MPsPPARα+/+ were mediated by NF-κB-dependent mechanisms.Conclusions/SignificanceOur results underscore the obligatory role of PPARα carried by MPs for EPC differentiation and angiogenesis. PPARα-NF-κB-Akt pathways may play a pivotal stimulatory role for neovascularization, which may, at least in part, be mediated by bone marrow-derived EPCs. Improvement of EPC differentiation may represent a useful strategy during reparative neovascularization.

Background Treatment of arterial bifurcation lesions using drug-eluting stents (DES) is now common clinical practice and yet the mechanisms governing drug distribution in these complex morphologies are incompletely understood. It is still not evident how to efficiently determine the efficacy of local drug delivery and quantify zones of excessive drug that are harbingers of vascular toxicity and thrombosis, and areas of depletion that are associated with tissue overgrowth and luminal re-narrowing. Methods and Results We constructed two-phase computational models of stent-deployed arterial bifurcations simulating blood flow and drug transport to investigate the factors modulating drug distribution when the main-branch (MB) was treated using a DES. Simulations predicted extensive flow-mediated drug delivery in bifurcated vascular beds where the drug distribution patterns are heterogeneous and sensitive to relative stent position and luminal flow. A single DES in the MB coupled with large retrograde luminal flow on the lateral wall of the side-branch (SB) can provide drug deposition on the SB lumen-wall interface, except when the MB stent is downstream of the SB flow divider. In an even more dramatic fashion, the presence of the SB affects drug distribution in the stented MB. Here fluid mechanic effects play an even greater role than in the SB especially when the DES is across and downstream to the flow divider and in a manner dependent upon the Reynolds number. Conclusions The flow effects on drug deposition and subsequent uptake from endovascular DES are amplified in bifurcation lesions. When only one branch is stented, a complex interplay occurs – drug deposition in the stented MB is altered by the flow divider imposed by the SB and in the SB by the presence of a DES in the MB. The use of DES in arterial bifurcations requires a complex calculus that balances vascular and stent geometry as well as luminal flow.

Loss of mitochondrial function is a fundamental determinant of cell injury and death. In heart cells under metabolic stress, we have previously described how the abrupt collapse or oscillation of the mitochondrial energy state is synchronized across the mitochondrial network by local interactions dependent upon reactive oxygen species (ROS). Here, we develop a mathematical model of ROS-induced ROS release (RIRR) based on reaction-diffusion (RD-RIRR) in one- and two-dimensional mitochondrial networks. The nodes of the RD-RIRR network are comprised of models of individual mitochondria that include a mechanism of ROS-dependent oscillation based on the interplay between ROS production, transport, and scavenging; and incorporating the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and Ca2+ handling. Local mitochondrial interaction is mediated by superoxide (O2 .−) diffusion and the O2 .−-dependent activation of an inner membrane anion channel (IMAC). In a 2D network composed of 500 mitochondria, model simulations reveal ΔΨm depolarization waves similar to those observed when isolated guinea pig cardiomyocytes are subjected to a localized laser-flash or antioxidant depletion. The sensitivity of the propagation rate of the depolarization wave to O2.− diffusion, production, and scavenging in the reaction-diffusion model is similar to that observed experimentally. In addition, we present novel experimental evidence, obtained in permeabilized cardiomyocytes, confirming that ΔΨm depolarization is mediated specifically by O2 .−. The present work demonstrates that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR. Moreover, the findings have uncovered a novel aspect of the synchronization mechanism, which is that clusters of mitochondria that are oscillating can entrain mitochondria that would otherwise display stable dynamics. The work identifies the fundamental mechanisms leading from the failure of individual organelles to the whole cell, thus it has important implications for understanding cell death during the progression of heart disease., Author Summary Cardiac cell injury and death is a key component of cardiac diseases such as heart failure or myocardial infarction, thus it is important to understand the earliest steps leading up to irreversible cell damage. Mitochondria are the organelles responsible for generating the energy required to keep the cell running, yet they are particularly vulnerable to damage by toxic byproducts of metabolism, which include reactive oxygen species (ROS). ROS wreak havoc on cellular functions by attacking proteins, lipids and DNA, so the cardiac cell has evolved sophisticated defenses to remove them. The work we present in this paper using a computer model of the mitochondrial network describes how ROS generated inside the cell can spread from one mitochondrion to the next in a positive feedback process known as ROS-induced ROS release. Understanding this process will help us to find ways to intervene in this catastrophic mechanism to prevent loss of cell function and the associated cardiac arrhythmias and contractile problems leading to sudden cardiac death.

BACKGROUND: Cardiomyocytes derived from murine embryonic stem (ES) cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP) in regulation of membrane potentials and Ca(2+) currents has not been investigated in developmental cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of ANP in regulating L-type Ca(2+) channel current (I(CaL)) in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs) in early developmental stage (EDS) cardiomyocytes, embryonic bodies (EB) as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I(CaL) in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS) cells. However, after stimulation of I(CaL) by isoproterenol (ISO) in LDS cells, ANP inhibited the response in about 70% cells. The depression of I(CaL) induced by ANP was not affected by either Nomega, Nitro-L-Arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG) selective inhibitor, in either EDS and LDS cells; whereas depression of I(CaL) by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl) adenine (EHNA), a selective inhibitor of type 2 phosphodiesterase(PDE2) in most cells tested. CONCLUSION/SIGNIFICANCES: Taken together, these results indicate that ANP induced depression of action potentials and I(CaL) is due to activation of particulate guanylyl cyclase (GC), cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3', 5'-cyclic monophophate (cAMP)-cAMP-dependent protein kinase (PKA) in early cardiomyogenesis.

We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2)>0.7) and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

Platt's 1964 essay on strong inference [1] illuminates a rational approach to scientific inquiry that integrates seamlessly with current investigations on the operation of complex biological systems. Yet in re-examining the 1964 essay in light of current trends, it is apparent that the groundbreaking approach has failed to become universal. Here it is argued that both the opportunity and the need to follow Platt's advice are now greater than ever. A revised method of strong inference for systems biology is presented and applied to analyze longstanding questions in cardiac energy metabolism. It is shown how this logical framework combined with computational-based hypothesis testing illuminates unresolved questions regarding how the energetic state of the heart is maintained in response to changes in the rate of ATP hydrolysis.

BACKGROUND:Spontaneous circulation returns to less than half of adult cardiac arrest victims who received in-hospital resuscitation. One clue for this disheartening outcome arises from the prognosis that asystole invariably takes place, after a time lag, on diagnosis of brain stem death. The designation of brain stem death as the point of no return further suggests that permanent impairment of the brain stem cardiovascular regulatory machinery precedes death. It follows that a crucial determinant for successful revival of an arrested heart is that spontaneous circulation must resume before brain stem death commences. Here, we evaluated the hypothesis that maintained functional integrity of the rostral ventrolateral medulla (RVLM), a neural substrate that is intimately related to brain stem death and central circulatory regulation, holds the key to the vital time-window between cardiac arrest and resumption of spontaneous circulation. METHODOLOGY/PRINCIPAL FINDINGS:An animal model of brain stem death employing the pesticide mevinphos as the experimental insult in Sprague-Dawley rats was used. Intravenous administration of lethal doses of mevinphos elicited an abrupt cardiac arrest, accompanied by elevated systemic arterial pressure and anoxia, augmented neuronal excitability and enhanced microvascular perfusion in RVLM. This period represents the vital time-window between cardiac arrest and resumption of spontaneous circulation in our experimental model. Animals with restored spontaneous circulation exhibited maintained neuronal functionality in RVLM beyond this critical time-window, alongside resumption of baseline tissue oxygen and enhancement of local blood flow. Intriguingly, animals that subsequently died manifested sustained anoxia, diminished local blood flow, depressed mitochondrial electron transport activities and reduced ATP production, leading to necrotic cell death in RVLM. That amelioration of mitochondrial dysfunction and bioenergetic failure in RVLM by coenzyme Q10, the mobile electron carrier in mitochondrial respiratory chain, or oxygenation restored spontaneous circulation further established a causal relationship between functionality of RVLM and resumed spontaneous circulation after cardiac arrest. CONCLUSIONS/SIGNIFICANCE:We conclude that whereas necrotic cell death because of bioenergetic failure triggered by anoxia in RVLM, which precipitates brain stem death, negates resuscitation of an arrested heart, maintained functional integrity of this neural substrate holds the key to resumption of spontaneous circulation after cardiac arrest in rats.

Oxygen metabolism is a strong predictor of the general health and fitness of an organism. In this study, we hypothesized that a divergence in intrinsic aerobic fitness would co-segregate with susceptibility for cardiovascular dysfunction. To test this hypothesis, cardiac function was assessed in rats specifically selected over nineteen generations for their low (LCR) and high (HCR) intrinsic aerobic running capacity. As an integrative marker of native aerobic capacity, run time to exhaustion between LCR and HCR rats had markedly diverged by 436% at generation nineteen of artificial selection. In vivo assessment of baseline cardiac function by echocardiography and catheter-based conductance micromanometry showed no marked difference in cardiac performance. However, when challenged by exposure to acute hypoxia, cardiac pump failure occurred significantly earlier in LCR rats compared to HCR animals. Acute cardiac decompensation in LCR rats was exclusively due to the development of intractable irregular ventricular contractions. Analysis of isolated cardiac myocytes showed significantly slower sarcomeric relaxation and delayed kinetics of calcium cycling in LCR myocytes compared to HCR myocytes. This study also revealed that artificial selection for low native aerobic capacity is a novel pathologic stimulus that results in myosin heavy chain isoform switching in the heart as shown by increased levels of beta-MHC in LCR rats. Together, these results provide evidence that alterations in sub-cellular calcium handling and MHC isoform composition are associated with susceptibility to compensatory cardiac remodeling and hypoxia induced pump failure in animals with low intrinsic aerobic capacity.