AJP: Heart and Circulatory Physiology recent issues

Cardiovascular physiology, emotions, and clinical applications: are we ready for prime time?

Pagani, M., Lucini, D. — 2008-07-14

The proverbial chicken or the egg? Dissection of the role of cell-free hemoglobin versus reactive oxygen species in sickle cell pathophysiology

Krajewski, M. L., Hsu, L. L., Gladwin, M. T. — 2008-07-14

The complexity of genotype-phenotype relations associated with loss-of-function sodium channel mutations and the role of in silico studies

Wilde, A. A. M., Coronel, R. — 2008-07-14

Sex differences in circulating and renal angiotensins of hypertensive mRen(2).Lewis but not normotensive Lewis rats

2008-07-14

Sex differences in blood pressure are evident in experimental models and human subjects, yet the mechanisms underlying this disparity remain equivocal. The current study sought to define the extent of male-female differences in the circulating and tissue renin-angiotensin aldosterone systems (RAASs) of congenic mRen(2).Lewis and control Lewis rats. Male congenics exhibited higher systolic blood pressure than females [200 ± 4 vs. 146 ± 7 mmHg, P < 0.01] or Lewis males and females [113 ± 2 vs. 112 ± 2 mmHg, P > 0.05]. Plasma ANG II levels were twofold higher in male congenics [47 ± 3 vs. 19 ± 3 pM, P < 0.01] and fivefold higher than in male or female Lewis rats [6 ± 1 vs. 6 ± 1 pM]. ANG I levels were also highest in the males; however, plasma ANG-(1-7) was higher in female congenics. Male congenics exhibited greater circulating renin and angiotensin-converting enzyme (ACE) activities, as well as angiotensinogen, than female littermates. Renal cortical and medullary ANG II levels were also higher in the male congenics versus all the other groups; ANG I was lower in the males. Cortical ACE2 activity was higher in male congenics, yet neprilysin activity and protein were greater in the females, which may contribute to reduced renal levels of ANG II. These data reveal that sex differences in both the circulating and renal RAAS are apparent primarily in the hypertensive group. The enhanced activity of the RAAS in male congenics may contribute to the higher pressure and tissue injury evident in the strain.

ATP/UTP activate cation-permeable channels with TRPC3/7 properties in rat cardiomyocytes

2008-07-14

Extracellular purines and pyrimidines have major effects on cardiac rhythm and contraction. ATP/UTP are released during various physiopathological conditions, such as ischemia, and despite degradation by ectonucleotidases, their interstitial concentrations can markedly increase, a fact that is clearly associated with arrhythmia. In the present whole cell patch-clamp analysis on ventricular cardiomyocytes isolated from various mammalian species, ATP and UTP elicited a sustained, nonselective cationic current, I_ATP. UDP was ineffective, whereas 2'(3')-O-(4-benzoylbenzoyl)-ATP was active, suggesting that P2Y₂ receptors are involved. I_ATP resulted from the binding of ATP^4– to P2Y₂ purinoceptors. I_ATP was maintained after ATP removal in the presence of guanosine 5'-[-thio]triphosphate and was inhibited by U-73122, a PLC inhibitor. Single-channel openings are rather infrequent under basal conditions. ATP markedly increased opening probability, an effect prevented by U-73122. Two main conductance levels of 14 and 23 pS were easily distinguished. Similarly, in fura-2-loaded cardiomyocytes, Mn²⁺ quenching and Ba²⁺ influx were significant only in the presence of ATP or UTP. Adult rat ventricular cardiomyocytes expressed transient receptor potential channel TRPC1, -3, -4, and -7 mRNA and the TRPC3 and TRPC7 proteins that coimmunoprecipitated. Finally, the anti-TRPC3 antibody added to the patch pipette solution inhibited I_ATP. In conclusion, activation of P2Y₂ receptors, via a G protein and stimulation of PLCβ, induces the opening of heteromeric TRPC3/7 channels, leading to a sustained, nonspecific cationic current. Such a depolarizing current could induce cell automaticity and trigger the arrhythmic events during an early infarct when ATP/UTP release occurs. These results emphasize a new, potentially deleterious role of TRPC channel activation.

Persistent alterations in heart rate variability, baroreflex sensitivity, and anxiety-like behaviors during development of heart failure in the rat

Henze, M., Hart, D., Samarel, A., Barakat, J., Eckert, L., Scrogin, K. — 2008-07-14

Depressed heart rate variability and mood are associated with increased mortality in patients with congestive heart failure (CHF). Here autonomic indexes were assessed 3 and 7 wk after left coronary artery ligation in telemetered rats, after which anxiety-like behaviors were assessed in an elevated plus maze. Low frequency (LF) and high frequency (HF) heart rate variability were reduced in CHF rats 3 wk after infarction (LF, 1.60 ± 0.52 vs. 6.97 ± 0.79 ms²; and HF, 1.53 ± 0.39 vs. 6.20 ± 1.01 ms²; P < 0.01). The number of sequences of interbeat intervals that correlated with arterial pressure was decreased in CHF rats at 3 and 7 wk (week 3, 26.60 ± 10.85 vs. 59.75 ± 11.4 sequences, P < 0.05; and week 7, 20.80 ± 8.97 vs. 65.38 ± 5.89 sequences, P < 0.01). Sequence gain was attenuated in CHF rats by 7 wk (1.34 ± 0.06 vs. 2.70 ± 0.29 ms/mmHg, P < 0.01). Coherence between interbeat interval and mean arterial blood pressure variability in the LF domain was reduced in CHF rats at 3 (0.12 ± 0.03 vs. 0.26 ± 0.05 k², P < 0.05) and 7 (0.16 ± 0.02 vs. 0.31 ± 0.05 k², P < 0.05) wk. CHF rats invariably entered the open arm of the elevated plus maze first and spent more time in the open arms (36.0 ± 15% vs. 4.6 ± 1.9%, P < 0.05). CHF rats also showed a tendency to jump head first off the apparatus, whereas controls did not. Together the data indicate that severe autonomic dysfunction is accompanied by escape-seeking behaviors in rats with verified CHF.

Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

Kaul, D. K., Zhang, X., Dasgupta, T., Fabry, M. E. — 2008-07-14

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human - and β^S-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E₂ (PGE₂) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.

Subepicardial phase 0 block and discontinuous transmural conduction underlie right precordial ST-segment elevation by a SCN5A loss-of-function mutation

2008-07-14

Two mechanisms are generally proposed to explain right precordial ST-segment elevation in Brugada syndrome: 1) right ventricular (RV) subepicardial action potential shortening and/or loss of dome causing transmural dispersion of repolarization; and 2) RV conduction delay. Here we report novel mechanistic insights into ST-segment elevation associated with a Na⁺ current (I_Na) loss-of-function mutation from studies in a Dutch kindred with the COOH-terminal SCN5A variant p.Phe2004Leu. The proband, a man, experienced syncope at age 22 yr and had coved-type ST-segment elevations in ECG leads V1 and V2 and negative T waves in V2. Peak and persistent mutant I_Na were significantly decreased. I_Na closed-state inactivation was increased, slow inactivation accelerated, and recovery from inactivation delayed. Computer-simulated I_Na-dependent excitation was decremental from endo- to epicardium at cycle length 1,000 ms, not at cycle length 300 ms. Propagation was discontinuous across the midmyocardial to epicardial transition region, exhibiting a long local delay due to phase 0 block. Beyond this region, axial excitatory current was provided by phase 2 (dome) of the M-cell action potentials and depended on L-type Ca²⁺ current ("phase 2 conduction"). These results explain right precordial ST-segment elevation on the basis of RV transmural gradients of membrane potentials during early repolarization caused by discontinuous conduction. The late slow-upstroke action potentials at the subepicardium produce T-wave inversion in the computed ECG waveform, in line with the clinical ECG.

The genetic contribution to heart rate and heart rate variability in quiescent mice

2008-07-14

Recent studies have suggested a genetic component to heart rate (HR) and HR variability (HRV). However, a systematic examination of the genetic contribution to the variation in HR and HRV has not been performed. This study investigated the genetic contribution to HR and HRV using a wide range of inbred and recombinant inbred (RI) mouse strains. Electrocardiogram data were recorded from 30 strains of inbred mice and 29 RI strains. Significant differences in mean HR and total power (TP) HRV were identified between inbred strains and RI strains. Multiple significant differences within the strain sets in mean low-frequency (LF) and high-frequency (HF) power were also found. No statistically significant concordance was found between strain distribution patterns for HR and HRV phenotypes. Genomewide interval mapping identified a significant quantitative trait locus (QTL) for HR [LOD (likelihood of the odds) score = 3.763] on chromosome 6 [peak at 53.69 megabases (Mb); designated HR 1 (Hr1)]. Suggestive QTLs for TP were found on chromosomes 2, 4, 5, 6, and 14. A suggestive QTL for LF was found on chromosome 16; for HF, we found one significant QTL on chromosome 5 (LOD score = 3.107) [peak at 53.56 Mb; designated HRV-high-frequency 1 (Hrvhf1)] and three suggestive QTLs on chromosomes 2, 11 and 15. In conclusion, the results demonstrate a strong genetic component in the regulation of resting HR and HRV evidenced by the significant differences between strains. A lack of correlation between HR and HRV phenotypes in some inbred strains suggests that different sets of genes control the phenotypes. Furthermore, QTLs were found that will provide important insight to the genetic regulation of HR and HRV at rest.

Microvessel vascular smooth muscle cells contribute to collagen type I deposition through ERK1/2 MAP kinase, {alpha}v{beta}3-integrin, and TGF-{beta}1 in response to ANG II and high glucose

Belmadani, S., Zerfaoui, M., Boulares, H. A., Palen, D. I., Matrougui, K. — 2008-07-14

This study determines that vascular smooth muscle cell (VSMC) signaling through extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein (MAP) kinase, vβ₃-integrin, and transforming growth factor (TGF)-β1 dictates collagen type I network induction in mesenteric resistance arteries (MRA) from Type 1 diabetic (streptozotocin) or hypertensive (HT; ANG II) mice. Isolated MRA were subjected to a pressure-passive-diameter relationship. To delineate cell types and mechanisms, cultured VSMC were prepared from MRA and stimulated with ANG II (100 nM) and high glucose (HG, 22 mM). Pressure-passive-diameter relationship reduction was associated with increased collagen type I deposition in MRA from HT and diabetic mice compared with control. Treatment of HT and diabetic mice with neutralizing TGF-β1 antibody reduced MRA stiffness and collagen type I deposition. Cultured VSMC stimulated with HG or ANG II for 5 min increased ERK1/2-MAP kinase phosphorylation, whereas a 48-h stimulation induced latent TGF-β1, vβ₃-integrin, and collagen type 1 release in the conditioned media. TGF-β1 bioactivity and Smad2 phosphorylation were vβ₃-integrin-dependent, since β₃-integrin antibody and vβ₃-integrin inhibitor (SB-223245, 10 µM) significantly prevented TGF-β1 bioactivity and Smad2 phosphorylation. Pretreatment of VSMC with ERK1/2-MAP kinase inhibitor (U-0126, 1 µM) reduced vβ₃-integrin, TGF-β1, and collagen type 1 content. Additionally, vβ₃-integrin antibody, SB-223245, TGF-β1-small-intefering RNA (siRNA), and Smad2-siRNA (40 nM) prevented collagen type I network formation in response to ANG II and HG. Together, these data provide evidence that resistance artery fibrosis in Type 1 diabetes and hypertension is a consequence of abnormal collagen type I release by VSMC and involves ERK1/2, vβ₃-integrin, and TGF-β1 signaling. This pathway could be a potential target for overcoming small artery complications in diabetes and hypertension.

A novel signaling pathway of ADP-ribosyl cyclase activation by angiotensin II in adult rat cardiomyocytes

Gul, R., Kim, S.-Y., Park, K.-H., Kim, B.-J., Kim, S.-J., Im, M.-J., Kim, U.-H. — 2008-07-14

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca²⁺-mobilizing second messenger, cADP-ribose (cADPR), from NAD⁺. In this study, we investigated the molecular basis of ADPR-cyclase activation in the ANG II signaling pathway and cellular responses in adult rat cardiomyocytes. The results showed that ANG II generated biphasic intracellular Ca²⁺ concentration increases that include a rapid transient Ca²⁺ elevation via inositol trisphosphate (IP₃) receptor and sustained Ca²⁺ rise via the activation of L-type Ca²⁺ channel and opening of ryanodine receptor. ANG II-induced sustained Ca²⁺ rise was blocked by a cADPR antagonistic analog, 8-bromo-cADPR, indicating that sustained Ca²⁺ rise is mediated by cADPR. Supporting the notion, ADPR-cyclase activity and cADPR production by ANG II were increased in a time-dependent manner. Application of pharmacological inhibitors and immunological analyses revealed that cADPR formation was activated by sequential activation of Src, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), phospholipase C (PLC)-1, and IP₃-mediated Ca²⁺ signal. Inhibitors of these signaling molecules not only completely abolished the ANG II-induced Ca²⁺ signals but also inhibited cADPR formation. Application of the cADPR antagonist and inhibitors of upstream signaling molecules of ADPR-cyclase inhibited ANG II-stimulated hypertrophic responses, which include nuclear translocation of Ca²⁺/calcineurin-dependent nuclear factor of activated T cells 3, protein expression of transforming growth factor-β1, and incorporation of [³H]leucine in cardiomyocytes. Taken together, these findings suggest that activation of ADPR-cyclase by ANG II entails a novel signaling pathway involving sequential activation of Src, PI 3-kinase/Akt, and PLC-1/IP₃ and that the activation of ADPR-cyclase can lead to cardiac hypertrophy.

15-Lipoxygenase metabolites contribute to age-related reduction in acetylcholine-induced hypotension in rabbits

Aggarwal, N. T., Gauthier, K. M., Campbell, W. B. — 2008-07-14

Arachidonic acid (AA) metabolites from the 15-lipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium-derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis, and nitric oxide and prostaglandin-independent relaxations to acetylcholine (ACh) and AA decreased with age (neonates to 16-wk-old). We characterized age-dependent ACh-hypotensive responses in vivo in 1-, 4-, 8-, and 16-wk-old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACh (4–4,000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACh responses decreased with age (P > 0.001). In the absence or presence of N-nitro-l-arginine methyl ester (l-NAME) and indomethacin (Indo), maximum responses in 1 (–54.7 ± 7.4 and –37.9 ± 3.9%)- and 4 (–48.8 ± 2.4 and –35.5 ± 7.8%)-wk-old rabbits were higher than 8 (–30.0 ± 2.8 and –26.6 ± 4.4%)- and 16 (–36.7 ± 3.5 and –27.3 ± 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and N-nitro-l-arginine, ACh relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4-wk-old rabbits treated with l-NAME and Indo, the maximum ACh hypotension was reduced by the potassium channel inhibitors apamin and charybdotoxin to –6.9 ± 0.9%, by apamin alone to –19.5 ± 1.4%, and by BW755C to –18.8 ± 3.5%. The present study indicates that the age-related decrease in ACh-induced hypotension is mediated by the decreased synthesis of the 15-LO-1 metabolites THETAs and HEETAs.

Calcium-activated chloride current contributes to action potential alternations in left ventricular hypertrophy rabbit

2008-07-14

T-wave alternans, characterized by a beat-to-beat change in T-wave morphology, amplitude, and/or polarity on the ECG, often heralds the development of lethal ventricular arrhythmias in patients with left ventricular hypertrophy (LVH). The aim of our study was to examine the ionic basis for a beat-to-beat change in ventricular repolarization in the setting of LVH. Transmembrane action potentials (APs) from epicardium and endocardium were recorded simultaneously, together with transmural ECG and contraction force, in arterially perfused rabbit left ventricular wedge preparation. APs and Ca²⁺-activated chloride current (I_Cl,Ca) were recorded from left ventricular myocytes isolated from normal rabbits and those with renovascular LVH using the standard microelectrode and whole cell patch-clamping techniques, respectively. In the LVH rabbits, a significant beat-to-beat change in endocardial AP duration (APD) created beat-to-beat alteration in transmural voltage gradient that manifested as T-wave alternans on the ECG. Interestingly, contraction force alternated in an opposite phase ("out of phase") with APD. In the single myocytes of LVH rabbits, a significant beat-to-beat change in APD was also observed in both left ventricular endocardial and epicardial myocytes at various pacing rates. APD alternans was suppressed by adding 1 µM ryanodine, 100 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 100 µM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The density of the Ca²⁺-activated chloride currents (I_Cl,Ca) in left ventricular myocytes was significantly greater in the LVH rabbits than in the normal group. Our data indicate that abnormal intracellular Ca²⁺ fluctuation may exert a strong feedback on the membrane I_Cl,Ca, leading to a beat-to-beat change in the net repolarizing current that manifests as T-wave alternans on the ECG.

Different effects of palmitoyl-L-carnitine and palmitoyl-CoA on mitochondrial function in rat ventricular myocytes

2008-07-14

Although mitochondrial oxidative catabolism of fatty acid (FA) is a major energy source for the adult mammalian heart, cardiac lipotoxity resulting from elevated serum FA and enhanced FA use has been implicated in the pathogenesis of heart failure. To investigate the effects of intermediates of FA metabolism [palmitoyl-l-carnitine (Pal-car) and palmitoyl-CoA (Pal-CoA)] on mitochondrial function, we measured membrane potential (_m), opening of the mitochondrial permeability transition pore (mPTP), and the production of ROS in saponin-treated rat ventricular myocytes with a laser scanning confocal microscope. Our results revealed that 1) lower concentrations of Pal-car (1 and 5 µM) caused a slight hyperpolarization of _m [tetramethylrhodamine ethyl ester (TMRE) intensity increased to 115.5 ± 5.4% and 110.7 ± 1.6% of baseline, respectively, P < 0.05] but did not open the mPTP, 2) a higher concentration of Pal-car (10 µM) depolarized _m (TMRE intensity decreased to 61.9 ± 12.2% of baseline, P < 0.01) and opened the mPTP (calcein intensity decreased to 70.7 ± 2.8% of baseline, P < 0.01), 3) Pal-CoA depolarized _m without opening the mPTP, and 4) only the higher concentration of Pal-car (10 µM) increased ROS generation (2',7'-dichlorofluorescein diacetate intensity increased to 3.4 ± 0.3-fold of baseline). We concluded that excessive exogenous intermediates of long-chain saturated FA may disturb mitochondrial function in different ways between Pal-car and Pal-CoA. The distinct mechanisms of the deteriorating effects of long-chain FA on mitochondrial function are important for our understanding of the development of cardiac diseases in systemic metabolic disorders.

Catestatin (chromogranin A344-364) is a novel cardiosuppressive agent: inhibition of isoproterenol and endothelin signaling in the frog heart

2008-07-14

The catecholamine release-inhibitory catestatin [Cts; human chromogranin (Cg) A_352-372, bovine CgA_344-364] is a vasoreactive and anti-hypertensive peptide derived from CgA. Using the isolated avascular frog heart as a bioassay, in which the interactions between the endocardial endothelium and the subjacent myocardium can be studied without the confounding effects of the vascular endothelium, we tested the direct cardiotropic effects of bovine Cts and its interaction with β-adrenergic (isoproterenol, ISO) and endothelin-1 (ET-1) signaling. Cts dose-dependently decreased stroke volume and stroke work, with a threshold concentration of 11 nM, approaching the in vivo level of the peptide. Cts reduced contractility by inhibiting phosphorylation of phospholamban (PLN). Furthermore, the Cts effect was abolished by pretreatment with either nitric oxide synthase (N^G-monomethyl-l-arginine) or guanylate cyclase (ODQ) inhibitors, or an ET_B receptor (ET_BR) antagonist (BQ-788). Cts also noncompetitively inhibited the positive inotropic action of ISO. In addition, Cts inhibited the positive inotropic effect of ET-1, mediated by ET_A receptors, and did not alter the negative inotropic ET-1 influence mediated by ET_BR. Cts action through ET_BR was further suggested when, in the presence of BQ-788, Cts failed to inhibit the positive inotropism of both ISO and ET-1 stimulation and PLN phosphorylation. We concluded that the cardiotropic actions of Cts, including the β-adrenergic and ET-1 antagonistic effects, support a novel role of this peptide as an autocrine-paracrine modulator of cardiac function, particularly when the stressed heart becomes a preferential target of both adrenergic and ET-1 stimuli.

Endothelial nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo

Kellogg, D. L., Zhao, J. L., Wu, Y. — 2008-07-14

Nitric oxide (NO) participates in locally mediated vasodilation induced by increased local skin temperature (T_loc) and in sympathetically mediated vasodilation during whole body heat stress. We hypothesized that endothelial NOS (eNOS) participates in the former, but not the latter, response. We tested this hypothesis by examining the effects of the eNOS antagonist N^G-amino-l-arginine (l-NAA) on skin blood flow (SkBF) responses to increased T_loc and whole body heat stress. Microdialysis probes were inserted into forearm skin for drug delivery. One microdialysis site was perfused with l-NAA in Ringer solution and a second site with Ringer solution alone. SkBF [laser-Doppler flowmetry (LDF)] and blood pressure [mean arterial pressure (MAP)] were monitored, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF ÷ MAP). In protocol 1, T_loc was controlled with LDF/local heating units. T_loc initially was held at 34°C and then increased to 41.5°C. In protocol 2, after a normothermic period, whole body heat stress was induced (water-perfused suits). At the end of both protocols, 58 mM sodium nitroprusside was perfused at both microdialysis sites to cause maximal vasodilation for data normalization. In protocol 1, CVC at 34°C T_loc did not differ between l-NAA-treated and untreated sites (P > 0.05). Local skin warming to 41.5°C T_loc increased CVC at both sites. This response was attenuated at l-NAA-treated sites (P < 0.05). In protocol 2, during normothermia, CVC did not differ between l-NAA-treated and untreated sites (P > 0.05). During heat stress, CVC rose to similar levels at l-NAA-treated and untreated sites (P > 0.05). We conclude that eNOS is predominantly responsible for NO generation in skin during responses to increased T_loc, but not during reflex responses to whole body heat stress.

Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway

Kim, S.-J., Abdellatif, M., Koul, S., Crystal, G. J. — 2008-07-14

Chronic treatment with insulin-like growth factor I (IGF-I) improves contractile function in congestive heart failure and ischemic cardiomyopathy. The present study investigated the effect of chronic treatment with IGF-I on intrinsic myocyte function and the role of the phosphatidylinositol (PI)3-kinase-Akt-sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2a signaling cascade in these responses. Myocytes were isolated from 23 adult rats and cultured with and without IGF-I (10^–6 M). After 48 h of treatment, myocyte function was evaluated. IGF-I increased contractile function (percent contraction, 7.7 ± 0.3% vs. 4.5 ± 0.3%; P < 0.01) and accelerated relaxation time (time for 70% relengthening, 81 ± 4 vs. 106 ± 5 ms; P < 0.05) compared with untreated myocytes [control (Con)]. The enhanced function was associated with an increase in Ca²⁺ transients assessed by fura-2 (340/380 nm; IGF-I, 0.42 ± 0.02 vs. Con, 0.25 ± 0.01; P < 0.01). The PI3-kinase inhibitor LY-249002 (10^–9 M) abolished the enhanced function caused by IGF-I. IGF-I increased both Akt and SERCA2a protein levels 2.5- and 4.8-fold, respectively, compared with those of Con (P < 0.01); neither phospholamban nor calsequestrin was affected. To evaluate whether the SERCA2a protein was directly mediated by Akt-SERCA2a signaling, IGF-I-induced changes in the SERCA2a protein were compared in myocytes transfected with adenovirus harboring either constitutively active Akt [multiplicity of infection (MOI), 15] or dominant negative Akt (dnAkt; MOI, 15). The ability of IGF-I to upregulate the SERCA2a protein in myocytes transected with active Akt was absent in dnAkt myocytes. Taken together, our findings indicate that chronic treatment with IGF-I enhances intrinsic myocyte function and that this effect is due to an enhancement in intracellular Ca²⁺ handling, secondary to the activation of the PI3-kinase-Akt-SERCA2a signaling cascade.

The benefit of medium-chain triglyceride therapy on the cardiac function of SHRs is associated with a reversal of metabolic and signaling alterations

2008-07-14

The spontaneously hypertensive rat (SHR) is a model of cardiomyopathy that displays a genetic defect in cardiac fatty acid (FA) translocase/CD36, a plasma membrane long-chain FA transporter. Therapy with medium-chain FAs, which do not require CD36-facilitated transport, has been shown to improve cardiac function and hypertrophy in SHRs despite persistent hypertension. However, little is known about the underlying molecular mechanisms. The aim of this study was to document the impact of medium-chain triglyceride (MCT) therapy in SHRs on the expression level and activity of metabolic enzymes and signaling pathways. Four-week-old male SHRs were administered MCT (SHR-MCT) or long-chain triglyceride (SHR-LCT) for 16 wk. We used Wistar-Kyoto (WKY) rats as controls (WKY-MCT and WKY-LCT). The SHR-MCT group displayed improved cardiac dysfunction [as assessed by left ventricular (LV) end-diastolic pressure and the positive and negative first derivatives of LV pressure/P value], a shift in the β-myosin heavy chain (MHC)-to--MHC ratio, and cardiac hypertrophy compared with the SHR-LCT group without an effect on blood pressure. Administration of MCT of SHRs reversed the LCT-induced reduction in the cardiac FA metabolic enzymatic activities of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and medium-chain acyl-CoA dehydrogenase (MCAD). In the SHR-MCT group, the protein expression and transcriptional regulation of myocardial peroxisome proliferator-activated receptor-, which regulates the transcription of LCHAD and MCAD genes, corresponded to the changes seen in those enzymatic activities. Furthermore, MCT intake caused an inhibition of JNK activation in SHR hearts. Collectively, the observed changes in the myocardial activity of metabolic enzymes and signaling pathways may contribute to the improved cardiac dysfunction and hypertrophy in SHRs following MCT therapy.

The sex-specific impact of systolic hypertension and systolic blood pressure on arterial-ventricular coupling at rest and during exercise

2008-07-14

In healthy subjects the arterial system and the left ventricle (LV) are tightly coupled at rest to optimize cardiac performance. Systolic hypertension (SH) is a major risk factor for heart failure and is associated with structural and functional alterations in the arteries and the LV. The effects of SH and resting systolic blood pressure (SBP) on arterial-ventricular coupling (E_aI/E_LVI) at rest, at peak exercise, and during recovery are not well described. We noninvasively characterized E_aI/E_LVI as end-systolic volume index/stroke volume index in subjects who were normotensive (NT, n = 203) or had SH (brachial SBP ≥140 mmHg, n = 79). Cardiac volumes were measured at rest and throughout exhaustive upright cycle exercise with gated blood pool scans. E_aI/E_LVI reserve was calculated by subtracting peak from resting E_aI/E_LVI. At rest, E_aI/E_LVI did not differ between SH and NT men but was 23% (P = 0.001) lower in SH vs. NT women. E_aI/E_LVI did not differ between SH and NT men or women at peak exercise or during recovery. Nevertheless, E_aI/E_LVI reserve was 61% (P < 0.001) lower in SH vs. NT women. Similarly, resting SBP (as a continuous variable) was not associated with E_aI/E_LVI in men (β = –0.12, P = 0.17) but was inversely associated with E_aI/E_LVI in women (β = –0.47, P < 0.001). SH and a higher resting brachial SBP are associated with a lower E_aI/E_LVI at rest in women but not in men, and SH women have an attenuated E_aI/E_LVI reserve. Whether a smaller E_aI/E_LVI reserve leads to functional limitations warrants further examination.

Functionally distinct sodium channels in ventricular epicardial and endocardial cells contribute to a greater sensitivity of the epicardium to electrical depression

2008-07-14

A greater depression of the action potential (AP) of the ventricular epicardium (Epi) versus endocardium (Endo) is readily observed in experimental models of acute ischemia and Brugada syndrome. Endo and Epi differences in transient outward K⁺ current and/or ATP-sensitive K⁺ channel current are believed to contribute to the differential response. The present study tested the hypothesis that the greater sensitivity of Epi is due in part to its functionally distinct early fast Na⁺ current (I_Na). APs were recorded from isolated Epi and Endo tissue slices and coronary-perfused wedge preparations before and after exposures to elevated extracellular K⁺ concentration ([K⁺]_o; 6–12 mM). I_Na was recorded from Epi and Endo myocytes using whole cell patch-clamp techniques. In tissue slices, increasing [K⁺]_o to 12 mM reduced V_max to 51.1 ± 5.3% and 26.8 ± 9.6% of control in Endo (n = 9) and Epi (n = 14), respectively (P < 0.05). In wedge preparations (n = 12), the increase in [K⁺]_o caused selective depression of Epi APs and transmural conduction slowing and block. I_Na density was not significantly different between Epi (n = 14) and Endo (n = 15) cells, but Epi cells displayed a more negative half-inactivation voltage [–83.6 ± 0.1 and –75.5 ± 0.3 mV for Epi (n = 16) and Endo (n = 16), respectively, P < 0.05]. Our data suggest that reduced I_Na availability in ventricular Epi may contribute to its greater sensitivity to electrical depression and thus may contribute to the R-ST segment changes observed under a variety of clinical conditions including acute myocardial ischemia, severe hyperkalemia, and Brugada syndrome.

Chronic insulin treatment amplifies PDGF-induced motility in differentiated aortic smooth muscle cells by suppressing the expression and function of PTP1B

Zhuang, D., Pu, Q., Ceacareanu, B., Chang, Y., Dixit, M., Hassid, A. — 2008-07-14

Hyperinsulinemia plays a major role in the pathogenesis of vascular disease. Restenosis occurs at an accelerated rate in hyperinsulinemia and is dependent on increased vascular smooth muscle cell movement from media to neointima. PDGF plays a critical role in mediating neointima formation in models of vascular injury. We have reported that PDGF increases the levels of protein tyrosine phosphatase PTP1B and that PTP1B suppresses PDGF-induced motility in cultured cells and that it attenuates neointima formation in injured carotid arteries. Others have reported that insulin enhances the mitogenic and motogenic effects of PDGF in cultured smooth muscle cells and that hyperinsulinemia promotes vascular remodeling. In the present study, we tested the hypothesis that insulin amplifies PDGF-induced cell motility by suppressing the expression and function of PTP1B. We found that chronic but not acute treatment of cells with insulin enhances PDGF-induced motility in differentiated cultured primary rat aortic smooth muscle cells and that it suppresses PDGF-induced upregulation of PTP1B protein. Moreover, insulin suppresses PDGF-induced upregulation of PTP1B mRNA levels, PTP1B enzyme activity, and binding of PTP1B to the PDGF receptor-β, and it enhances PDGF-induced PDGF receptor phosphotyrosylation. Treatment with insulin induces time-dependent upregulation of phosphatidylinositol 3-kinase (PI3-kinase)- and activation of Akt, an enzyme downstream of PI3-kinase. Finally, inhibition of PI3-kinase activity, or its function, by pharmacological or genetic means rescues PTP1B activity in insulin-treated cells. These observations uncover novel mechanisms that explain how insulin amplifies the motogenic capacity of the pivotal growth factor PDGF.

Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

Radek, K. A., Kovacs, E. J., Gallo, R. L., DiPietro, L. A. — 2008-07-14

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1 protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.

The transcriptional coactivator PGC-1{alpha} is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis

2008-07-14

High-capacity mitochondrial ATP production is essential for normal function of the adult heart, and evidence is emerging that mitochondrial derangements occur in common myocardial diseases. Previous overexpression studies have shown that the inducible transcriptional coactivator peroxisome proliferator-activated receptor- coactivator (PGC)-1 is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1 (PGC-1^–/– mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1 is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1^–/– mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1^–/– mice compared with PGC-1^+/+ controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1^–/– mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1^–/– fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1^–/– mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. ¹³C NMR revealed that hearts from PGC-1^–/– mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during β-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1^–/– mice relative to PGC-1^+/+ following a fast. Overall, these results demonstrate that PGC-1 is essential for the maintenance of maximal, efficient cardiac mitochondrial fatty acid oxidation, ATP synthesis, and myocardial lipid homeostasis.

Structure and torsion of the normal and situs inversus totalis cardiac left ventricle. I. Experimental data in humans

Delhaas, T., Kroon, W., Decaluwe, W., Rubbens, M., Bovendeerd, P., Arts, T. — 2008-07-14

In 1926, the famous American pediatric cardiologist, Dr. Helen B. Taussig, observed that in situs inversus totalis (SIT) main gross anatomical structures and the deep muscle bundles of the ventricles were a mirror image of the normal structure, while the direction of the superficial muscle bundles remained unchanged (H. B. Taussig, Bull Johns Hopkins Hosp 39: 199–202, 1926). She and we wondered about the implication of this observation for left ventricular (LV) deformation in SIT. We used magnetic resonance tagging to obtain information on LV deformation, rotation, and torsion from a series of tagged images in five evenly distributed, parallel, short-axis sections of the heart of nine controls and eight persons with SIT without other structural (cardiac) defect. In the controls, during ejection, the apex rotated counterclockwise with respect to the base, when looking from the apex. Furthermore, the base-to-apex gradient in rotation (torsion) was negative and similar at all longitudinal levels of the LV. In SIT hearts, torsion was positive near the base, indicating mirrored myofiber orientations compared with the normal LV. Contrary to expectations, torsion in the apical regions of SIT LVs was as in normal ones, reflecting a normal internal myocardial architecture. The transition zone with zero torsion, found between the apex and base, suggests that the heart structure in SIT is essentially different from that in the normal heart. This provides a unique possibility to study regulatory mechanisms for myocardial fiber orientation and mechanical load, which has been dealt with in the companion paper by Kroon et al.

Structure and torsion in the normal and situs inversus totalis cardiac left ventricle. II. Modeling cardiac adaptation to mechanical load

Kroon, W., Delhaas, T., Bovendeerd, P., Arts, T. — 2008-07-14

Mathematical models provide a suitable platform to test hypotheses on the relation between local mechanical stimuli and responses to cardiac structure and geometry. In the present model study, we tested hypothesized mechanical stimuli and responses in cardiac adaptation to mechanical load on their ability to estimate a realistic myocardial structure of the normal and situs inversus totalis (SIT) left ventricle (LV). In a cylindrical model of the LV, 1) mass was adapted in response to myofiber strain at the beginning of ejection and to global contractility (average systolic pressure), 2) cavity volume was adapted in response to fiber strain during ejection, and 3) myofiber orientations were adapted in response to myofiber strain during ejection and local misalignment between neighboring tissue parts. The model was able to generate a realistic normal LV geometry and structure. In addition, the model was also able to simulate the instigating situation in the rare SIT LV with opposite torsion and transmural courses in myofiber direction between the apex and base [Delhaas et al. (6)]. These results substantiate the importance of mechanical load in the formation and maintenance of cardiac structure and geometry. Furthermore, in the model, adapted myocardial architecture was found to be insensitive to fiber misalignment in the transmural direction, i.e., myofiber strain during ejection was sufficient to generate a realistic transmural variation in myofiber orientation. In addition, the model estimates that, despite differences in structure, global pump work and the mass of the normal and SIT LV are similar.

Effects of isoproterenol treatment for 7 days on inflammatory mediators in the rat aorta

2008-07-14

The aim of the present study was to evaluate the effect of overstimulation of β-adrenoceptors on vascular inflammatory mediators. Wistar rats were treated with the β-adrenoceptor agonist isoproterenol (0.3 mg·kg^–1·day^–1 sc) or vehicle (control) for 7 days. At the end of treatment, the right carotid artery was catheterized for arterial and left ventricular (LV) hemodynamic evaluation. Isoproterenol treatment increased LV weight but did not change hemodynamic parameters. Aortic mRNA and protein expression were quantified by real-time RT-PCR and Western blot analysis, respectively. Isoproterenol enhanced aortic mRNA and protein expression of IL-1β (124% and 125%) and IL-6 (231% and 40%) compared with controls but did not change TNF- expression. The nuclear-to-cytoplasmatic protein expression ration of the NF-B p65 subunit was increased by isoproterenol treatment (51%); in addition, it reduced the cytoplasmatic expression of IB- (52%) in aortas. An electrophoretic mobility shift assay was performed using the aorta, and increased NF-B DNA binding (31%) was observed in isoproterenol-treated rats compared with controls (P < 0.05). Isoproterenol treatment increased phenylephrine-induced contraction in aortic rigs (P < 0.05), which was significantly reduced by superoxide dismutase (150 U/ml) and sodium salicylate (5 mM). Cotreatment with thalidomide (150 mg·kg^–1·day^–1 for 7 days) also reduced hyperreactivity to phenylephrine induced by isoproterenol. In conclusion, overstimulation of β-adrenoceptors increased proinflammatory cytokines and upregulated NF-B in the rat aorta. Moreover, local oxidative stress and the proinflammatory state seem to play key roles in the altered vascular reactivity of the rat aorta induced by chronic β-adrenergic stimulation.

Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats

Koyanagi, T., Wong, L. Y., Inagaki, K., Petrauskene, O. V., Mochly-Rosen, D. — 2008-07-14

Hypertension induced by high-salt diet in Dahl salt-sensitive rats leads to compensatory cardiac hypertrophy by ~11 wk, cardiac dysfunction at ~17 wk, and death from cardiac dysfunction at ~21 wk. It is unclear what molecular hallmarks distinguish the compensatory hypertrophy from the decompensated cardiac dysfunction phase. Here we compared the gene expression in rat cardiac tissue from the compensatory hypertrophic phase (11 wk, n = 6) with the cardiac dysfunction phase (17 wk, n = 6) and with age-matched normotensive controls. Messenger RNA levels of 93 genes, selected based on predicted association with cardiac dysfunction, were measured by quantitative real-time PCR. In the hypertrophic phase, the expression of three genes, atrial natriuretic peptide (ANP; P = 0.0089), brain natriuretic peptide (P = 0.0012), and endothelin-1 precursor (P = 0.028), significantly increased, whereas there was decreased expression of 24 other genes including SOD2 (P = 0.0148), sarco(endo)plasmic reticulum Ca²⁺-ATPase 2a (P = 0.0002), and ryanodine receptor 2 (P = 0.0319). In the subsequent heart cardiac dysfunction phase, the expression of an additional 20 genes including inducible nitric oxide synthase (NOS; P = 0.0135), angiotensin I-converting enzyme (P = 0.0082), and IL-1β (P < 0.0001) increased, whereas the expression of seven genes decreased compared with those of age-matched controls. Furthermore, the expression of 22 genes, including prepro-endothelin-1, ANP, angiotensin I-converting enzyme, β₁-adrenergic receptor, SOD2, and endothelial NOS, significantly changed in the cardiac dysfunction phase compared with the compensatory hypertrophic phase. Finally, principal component analysis successfully segregated animals with decompensatory cardiac dysfunction from controls, as well as from animals at the compensated hypertrophy phase, suggesting that we have identified molecular markers for each stage of the disease.

Inhibition of brain proinflammatory cytokine synthesis reduces hypothalamic excitation in rats with ischemia-induced heart failure

Kang, Y.-M., Zhang, Z.-H., Xue, B., Weiss, R. M., Felder, R. B. — 2008-07-14

The expression of proinflammatory cytokines increases in the hypothalamus of rats with heart failure (HF). The pathophysiological significance of this observation is unknown. We hypothesized that hypothalamic proinflammatory cytokines upregulate the activity of central neural systems that contribute to increased sympathetic nerve activity in HF, specifically, the brain renin-angiotensin system (RAS) and the hypothalamic-pituitary-adrenal (HPA) axis. Rats with HF induced by coronary ligation and sham-operated controls (SHAM) were treated for 4 wk with a continuous intracerebroventricular infusion of the cytokine synthesis inhibitor pentoxifylline (PTX, 10 µg/h) or artificial cerebrospinal fluid (VEH). In VEH-treated HF rats, compared with VEH-treated SHAM rats, the hypothalamic expression of proinflammatory cytokines was increased, along with key components of the brain RAS (renin, angiotensin-converting enzyme, angiotensin type 1 receptor) and corticotropin-releasing hormone, the central indicator of HPA axis activation, in the paraventricular nucleus (PVN) of the hypothalamus. The expression of other inflammatory/excitatory mediators (superoxide, prostaglandin E₂) was also increased, along with evidence of chronic neuronal excitation in PVN. VEH-treated HF rats had higher plasma levels of norepinephrine, ANG II, interleukin (IL)-1β, and adrenocorticotropic hormone, increased left ventricular end-diastolic pressure, and increased wet lung-to-body weight ratio. With the exception of plasma IL-1β, an indicator of peripheral proinflammatory cytokine activity, all measures of neurohumoral excitation were significantly lower in HF rats treated with intracerebroventricular PTX. These findings suggest that the increase in brain proinflammatory cytokines observed in rats with ischemia-induced HF is functionally significant, contributing to neurohumoral excitation by activating brain RAS and the HPA axis.

Triglyceride-rich lipoprotein lipolysis increases aggregation of endothelial cell membrane microdomains and produces reactive oxygen species

Wang, L., Sapuri-Butti, A. R., Aung, H. H., Parikh, A. N., Rutledge, J. C. — 2008-07-14

Triglyceride-rich lipoprotein (TGRL) lipolysis may provide a proinflammatory stimulus to endothelium. Detergent-resistant plasma membrane microdomains (lipid rafts) have a number of functions in endothelial cell inflammation. The mechanisms of TGRL lipolysis-induced endothelial cell injury were investigated by examining endothelial cell lipid rafts and production of reactive oxygen species (ROS). Lipid raft microdomains in human aortic endothelial cells were visualized by confocal microscopy with fluorescein isothiocyanate-labeled cholera toxin B as a lipid raft marker. Incubation of Atto565-labeled TGRL with lipid raft-labeled endothelial cells showed that TGRL colocalized with the lipid rafts, TGRL lipolysis caused clustering and aggregation of lipid rafts, and colocalization of TGRL remnant particles on the endothelial cells aggregated lipid rafts. Furthermore, TGRL lipolysis caused translocation of low-density lipoprotein receptor-related protein, endothelial nitric oxide synthase, and caveolin-1 from raft regions to nonraft regions of the membrane 3 h after treatment with TGRL lipolysis. TGRL lipolysis significantly increased the production of ROS in endothelial cells, and both NADPH oxidase and cytochrome P-450 inhibitors reduced production of ROS. Our studies suggest that alteration of lipid raft morphology and composition and ROS production could contribute to TGRL lipolysis-mediated endothelial cell injury.

Diacylglycerol kinase-{varepsilon} restores cardiac dysfunction under chronic pressure overload: a new specific regulator of G{alpha}q signaling cascade

2008-07-14

G_q protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGK acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGK prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGK (DGK-TG) using an -myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGK-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGK-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGK-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGK-TG mice, and the survival rate after TAC was higher in DGK-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGK-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGK-TG mice. In conclusion, these results demonstrate the first evidence that DGK restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGK may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.

Reduced heart size and increased myocardial fuel substrate oxidation in ACC2 mutant mice

2008-07-14

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC2) is a key regulator of mitochondrial fatty acid (FA) uptake via carnitine palmitoyltransferase 1 (CPT1). To test the hypothesis that oxidative metabolism is upregulated in hearts from animals lacking ACC2 (employing a transgenic Acc2-mutant mouse), we assessed cardiac function in vivo and determined rates of myocardial substrate oxidation ex vivo. When examined by echocardiography, there was no difference in systolic function, but left ventricular mass of the Acc2-mutant (MUT) mouse was significantly reduced (~25%) compared with wild-types (WT). Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts. Exogenous oxidation rates of oleate were increased ~22%, and, unexpectedly, exogenous glucose oxidation rates were also increased in MUT hearts. Using a hyperinsulinemic-euglycemic clamp, we found that glucose uptake in MUT hearts was increased by ~83%. Myocardial triglyceride levels were significantly reduced in MUT vs. WT while glycogen content was the same. In parallel, transcript levels of PPAR and its target genes, pyruvate dehydrogenase kinase-4 (PDK-4), malonyl-CoA decarboxylase (MCD), and mCPT1, were downregulated in MUT mice. In summary, we report that 1) Acc2-mutant hearts exhibit a marked preference for the oxidation of both glucose and FAs coupled with greater utilization of endogenous fuel substrates (triglycerides), 2) attenuated mTOR signaling may result in reduced heart sizes observed in Acc2-mutant mice, and 3) Acc2-mutant hearts displayed normal functional parameters despite a significant decrease in size.

Cyclic AMP acts through Rap1 and JNK signaling to increase expression of cutaneous smooth muscle {alpha}2C-adrenoceptors

Eid, A. H., Chotani, M. A., Mitra, S., Miller, T. J., Flavahan, N. A. — 2008-07-14

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of _2C-adrenoceptors (_2C-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of _2C-ARs from the transGolgi to the cell surface. The expression of _2C-ARs in human cutaneous VSMCs is under dual regulation by cyclic AMP: gene transcription is inhibited by cyclic AMP acting through protein kinase A but is increased by cyclic AMP acting through the exchange protein directly activated by cyclic AMP (EPAC) and the GTP-binding protein Rap1. Experiments were performed to further characterize the Rap1 signaling pathway. Forskolin (10 µM), the selective EPAC activator, 8-pCPT-2'-O-Me-cyclic AMP (CMC; 100 µM), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun NH₂-terminal kinase (JNK) in human cutaneous VSMCs. This was associated with the increased phosphorylation of c-Jun and activation of an activator protein (AP)-1 reporter construct, which were inhibited by the JNK inhibitor SP600125 (3 µM). Rap1CA increased the activity of an _2C-AR promoter-reporter construct, which was inhibited by SP600125 (3 µM) or by the mutation of an AP-1 binding site in the _2C-AR promoter. Furthermore, forskolin (10 µM) or CMC (100 µM) increased the expression of the _2C-AR protein, and these effects were inhibited by SP600125 (3 µM). Therefore, cyclic AMP increases the expression of _2C-ARs in cutaneous VSMCs by activating a novel Rap1 signaling pathway, mediated by the activation of JNK, AP-1, and the subsequent transcriptional activation of the _2C-AR gene. By increasing the expression of cold-responsive _2C-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon.

ACTH-induced hypertension is dependent on the ouabain-binding site of the {alpha}2-Na+-K+-ATPase subunit

2008-07-14

ACTH-induced-hypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabain-resistant ₂-Na⁺-K⁺-ATPase subunits (2^R/R mice). Since tail-cuff measurements involve restraint stress, the present study used telemetry to distinguish between an effect of ACTH on resting BP vs. an ACTH-enhanced stress response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant 2^R/R mice vs. wild-type mice (ouabain-sensitive ₂-Na⁺-K⁺-ATPase, 2^S/S mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in 2^S/S (104.0 ± 2.6 to 117.7 ± 3.0 mmHg) but not in 2^R/R mice (108.2 ± 3.2 to 111.5 ± 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of ₂-Na⁺-K⁺-ATPase on vascular smooth muscle by endogenous cardiotonic steroids, we measured BP and regional blood flow. Results suggest a differential sensitivity of renal, mesenteric, and cerebral circulations to ACTH and that the response depends on the ouabain sensitivity of the ₂-Na⁺-K⁺-ATPase. Baseline cardiac performance was elevated in 2^S/S but not 2^R/R mice. Overall, the data establish that the ₂-Na⁺-K⁺-ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations, presumably caused by elevated levels of circulating cardiotonic steroids.

Xanthine oxidase and mitochondria contribute to vascular superoxide anion generation in DOCA-salt hypertensive rats

Viel, E. C., Benkirane, K., Javeshghani, D., Touyz, R. M., Schiffrin, E. L. — 2008-07-14

Vascular superoxide anion (O₂^•–) levels are increased in DOCA-salt hypertensive rats. We hypothesized that the endothelin (ET)-1-induced generation of ROS in the aorta and resistance arteries of DOCA-salt rats originates partly from xanthine oxidase (XO) and mitochondria. Accordingly, we blocked XO and the mitochondrial oxidative phosphorylation chain to investigate their contribution to ROS production in mesenteric resistance arteries and the aorta from DOCA-salt rats. Systolic blood pressure rose in DOCA-salt rats and was reduced after 3 wk by apocynin [NAD(P)H oxidase inhibitor and/or radical scavenger], allopurinol (XO inhibitor), bosentan (ET_A/B receptor antagonist), BMS-182874 (BMS; ET_A receptor antagonist), and hydralazine. Plasma uric acid levels in DOCA-salt rats were similar to control unilaterally nephrectomized (UniNx) rats, reduced with allopurinol and bosentan, and increased with BMS. Levels of thiobarbituric acid-reacting substances were increased in DOCA-salt rats versus UniNx rats, and BMS, bosentan, and hydralazine prevented their increase. Dihydroethidium staining showed reduced O₂^•– production in mesenteric arteries and the aorta from BMS- and bosentan-treated DOCA-salt rats compared with untreated DOCA-salt rats. Increased O₂^•– derived from XO was reduced or prevented by all treatments in mesenteric arteries, whereas bosentan and BMS had no effect on aortas from DOCA-salt rats. O₂^•– generation decreased with in situ treatment by tenoyltrifluoroacetone and CCCP, inhibitors of mitochondrial electron transport complexes II and IV, respectively, whereas rotenone (mitochondrial complex I inhibitor) had no effect. Our findings demonstrate the involvement of ET_A receptor-modulated O₂^•– derived from XO and from mitochondrial oxidative enzymes in arteries from DOCA-salt rats.

Vitamin D derivatives acutely reduce endothelium-dependent contractions in the aorta of the spontaneously hypertensive rat

Wong, M. S. K., Delansorne, R., Man, R. Y. K., Vanhoutte, P. M. — 2008-07-14

The available evidence suggests that vitamin D has cardiovascular effects besides regulating calcium homeostasis. To examine the effect of 1,25-dihydroxyvitamin D₃, the major metabolite of vitamin D, on endothelium-dependent contractions, aortic rings of spontaneously hypertensive rats (SHR) were suspended in organ chambers for isometric force measurements. Rings were incubated with N-nitro-l-arginine methyl ester (l-NAME) and then exposed to increasing concentrations of acetylcholine, ATP, or the calcium ionophore to trigger contractions. This was done in the absence or presence of 1,25-dihydroxyvitamin D₃. The release of prostacyclin after acetylcholine or A-23187 stimulation was also measured. The cytosolic-free calcium concentration was measured by confocal microscopy after incubation with the fluorescent dyes fluo-4 and fura red. The presence of vitamin D receptors was confirmed using immunohistochemistry. Acetylcholine- and ATP-induced endothelium-dependent contractions were significantly reduced compared with those obtained in the absence of the drug. This effect was not present if A-23187 was used as an agonist. The acetylcholine- but not the A-23187-induced release of prostacyclin was reduced by the acute administration of 1,25-dihydroxyvitamin D₃. Exposure to 1,25-dihydroxyvitamin D₃ reduced the increase in cytosolic-free calcium concentration caused by acetylcholine but not by A-23187 in cells. Vitamin D receptors were densely distributed in the endothelium. Inecalcitol (19-nor-14-epi-23-yne-1,25-dihydroxyvitamin D₃), a synthetic analog of vitamin D, caused a comparable depression of endothelium-dependent contractions as 1,25-dihydroxyvitamin D₃. These results demonstrate that vitamin D₃ modulates vascular tone by reducing calcium influx into the endothelial cells and hence decreasing the production of endothelium-derived contracting factors.

Intracellular calcium dynamics at the core of endocardial stationary spiral waves in Langendorff-perfused rabbit hearts

2008-07-14

In vitro models of sustained monomorphic ventricular tachycardia (MVT) are rare and do not usually show spiral reentry on the epicardium. We hypothesized that MVT is associated with the spiral wave in the endocardium and that this stable reentrant propagation is supported by a persistently elevated intracellular calcium (Ca_i) transient at the core of the spiral wave. We performed dual optical mapping of transmembrane potential (V_m) and Ca_i dynamics of the right ventricular (RV) endocardium in Langendorff-perfused rabbit hearts (n = 12). Among 64 induced arrhythmias, 55% were sustained MVT (>10 min). Eighty percent of MVT showed stationary spiral waves (>10 cycles, cycle length: 128 ± 14.6 ms) in the endocardial mapped region, anchoring to the anatomic discontinuities. No reentry activity was observed in the epicardium. During reentry, the amplitudes of V_m and Ca_i signals were higher in the periphery and gradually decreased toward the core. At the core, maximal V_m and Ca_i amplitudes were 42.95 ± 5.89% and 43.95 ± 9.46%, respectively, of the control (P < 0.001). However, the trough of the V_m and Ca_i signals at the core were higher than those in the periphery, indicating persistent V_m and Ca_i elevations during reentry. BAPTA-AM, a calcium chelator, significantly reduced the maximal Ca_i transient amplitude and prevented sustained MVT and spiral wave formation in the mapped region. These findings indicate that endocardial spiral waves often anchor to anatomic discontinuities causing stable MVT in normal rabbit ventricles. The spiral core is characterized by diminished V_m and Ca_i amplitudes and persistent V_m and Ca_i elevations during reentry.

Modeling flow in collecting lymphatic vessels: one-dimensional flow through a series of contractile elements

Macdonald, A. J., Arkill, K. P., Tabor, G. R., McHale, N. G., Winlove, C. P. — 2008-07-14

The lymphatic system comprises a series of elements, lymphangions, separated by valves and possessed of active, contractile walls to pump interstitial fluid from its collection in the terminal lymphatics back to the main circulation. Despite its importance, there is a dearth of information on the fluid dynamics of the lymphatic system. In this article, we describe linked experimental and computational work aimed at elucidating the biomechanical properties of the individual lymphangions. We measure the static and dynamic mechanical properties of excised bovine collecting lymphatics and develop a one-dimensional computational model of the coupled fluid flow/wall motion. The computational model is able to reproduce the pumping behavior of the real vessel using a simple contraction function producing fast contraction pulses traveling in the retrograde direction to the flow.

In vivo administration of calpeptin attenuates calpain activation and cardiomyocyte loss in pressure-overloaded feline myocardium

2008-07-14

Calpain activation is linked to the cleavage of several cytoskeletal proteins and could be an important contributor to the loss of cardiomyocytes and contractile dysfunction during cardiac pressure overload (PO). Using a feline right ventricular (RV) PO model, we analyzed calpain activation during the early compensatory period of cardiac hypertrophy. Calpain enrichment and its increased activity with a reduced calpastatin level were observed in 24- to 48-h-PO myocardium, and these changes returned to basal level by 1 wk of PO. Histochemical studies in 24-h-PO myocardium revealed the presence of TdT-mediated dUTP nick-end label (TUNEL)-positive cardiomyocytes, which exhibited enrichment of calpain and gelsolin. Biochemical studies showed an increase in histone H2B phosphorylation and cytoskeletal binding and cleavage of gelsolin, which indicate programmed cardiomyocyte cell death. To test whether calpain inhibition could prevent these changes, we administered calpeptin (0.6 mg/kg iv) by bolus injections twice, 15 min before and 6 h after induction of 24-h PO. Calpeptin blocked the following PO-induced changes: calpain enrichment and activation, decreased calpastatin level, caspase-3 activation, enrichment and cleavage of gelsolin, TUNEL staining, and histone H2B phosphorylation. Although similar administration of a caspase inhibitor, N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VD-fmk), blocked caspase-3 activation, it did not alleviate other aforementioned changes. These results indicate that biochemical markers of cardiomyocyte cell death, such as sarcomeric disarray, gelsolin cleavage, and TUNEL-positive nuclei, are mediated, at least in part, by calpain and that calpeptin may serve as a potential therapeutic agent to prevent cardiomyocyte loss and preserve myocardial structure and function during cardiac hypertrophy.

Intradermal angiotensin II administration attenuates the local cutaneous vasodilator heating response

Stewart, J. M., Taneja, I., Raghunath, N., Clarke, D., Medow, M. S. — 2008-07-14

The vasodilation response to local cutaneous heating is nitric oxide (NO) dependent and blunted in postural tachycardia but reversed by angiotensin II (ANG II) type 1 receptor (AT₁R) blockade. We tested the hypothesis that a localized infusion of ANG II attenuates vasodilation to local heating in healthy volunteers. We heated the skin of a calf to 42°C and measured local blood flow to assess the percentage of maximum cutaneous vascular conductance (%CVC_max) in eight healthy volunteers aged 19.5–25.5 years. Initially, two experiments were performed; in one, Ringer solution was perfused in three catheters, the response to heating was measured, 2 µg/l losartan, 10 mM nitro-l-arginine (NLA), or NLA + losartan was added to perfusate, and the heat response was remeasured; in another, 10 µM ANG II was given, the heat response was measured, losartan, NLA, or NLA + losartan was added to ANG II, and the heat response was reassessed. The heat response decreased with ANG II, particularly the plateau phase (47 ± 5 vs. 84 ± 3 %CVC_max). Losartan increased baseline conductance in both experiments (from 8 ± 1 to 20 ± 2 and 12 ± 1 to 24 ± 3). Losartan increased the ANG II response (83 ± 4 vs. 91 ± 6 in Ringer). NLA decreased both angiotensin and Ringer responses (31 ± 4 vs. 43 ± 3). NLA + losartan blunted the Ringer response (48 ± 2), but the ANG II response (74 ± 5) increased. In a second set of experiments, we used dose responses to ANG II (0.1 nM to 10 µM) with and without NLA + losartan to confirm graded responses. Sodium ascorbate (10 mM) restored the ANG II-blunted heating plateau. NO synthase and AT₁R inhibition cause an NO-independent angiotensin-mediated vasodilation with local heating. ANG II mediates the AT₁R blunting of local heating, which is not exclusively NO dependent, and is improved by antioxidant supplementation.

Increased protein O-GlcNAc modification inhibits inflammatory and neointimal responses to acute endoluminal arterial injury

2008-07-14

Inflammation plays a major role in vascular disease. We have shown that leukocyte infiltration and inflammatory mediator expression contribute to vascular remodeling after endoluminal injury. This study tested whether increasing protein O-linked-N-acetylglucosamine (O-GlcNAc) levels with glucosamine (GlcN) and O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) inhibits acute inflammatory and neointimal responses to endoluminal arterial injury. Ovariectomized rats were treated with a single injection of GlcN (0.3 mg/g ip), PUGNAc (7 nmol/g ip) or vehicle (V) 2 h before balloon injury of the right carotid artery. O-GlcNAc-modified protein levels decreased markedly in injured arteries of V-treated rats at 30 min, 2 h, and 24 h after injury but returned to control (contralateral uninjured) levels after 14 days. Both GlcN and PUGNAc increased O-GlcNAc-modified protein levels in injured arteries compared with V controls at 30 min postinjury; the GlcN-mediated increase persisted at 24 h but was not evident at 14 days. Proinflammatory mediator expression increased markedly after injury and was reduced significantly (30–50%) by GlcN and PUGNAc. GlcN and PUGNAc also inhibited infiltration of neutrophils and monocytes in injured arteries. Chronic (14 days) treatment with GlcN reduced neointima formation in injured arteries by 50% compared with V controls. Acute GlcN and PUGNAc treatment increases O-GlcNAc-modified protein levels and inhibits acute inflammatory responses in balloon-injured rat carotid arteries; 14 day GlcN treatment inhibits neointima formation in these vessels. Augmenting O-GlcNAc modification of proteins in the vasculature may represent a novel anti-inflammatory and vasoprotective mechanism.

The role of eNOS, iNOS, and NF-{kappa}B in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin

2008-07-14

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS^–/– and iNOS^–/– mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-B dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS^–/– and iNOS^–/– mice received ATV (10 mg·kg^–1·day^–1; ATV⁺) or water alone (ATV^–) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-B in WT mice. It also increased myocardial COX2 activity. In eNOS^–/– mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-B was not activated by ATV in the eNOS^–/– mice. In the iNOS^–/– mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-B was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-B, dependent. Activation of COX2 is dependent on iNOS.

Chronic doxycycline exposure accelerates left ventricular hypertrophy and progression to heart failure in mice after thoracic aorta constriction

2008-07-14

Tetracycline is a powerful tool for controlling the expression of specific transgenes (TGs) in various tissues, including heart. In these mouse systems, TG expression is repressed/enhanced by adding doxycycline (Dox) to the diet. However, Dox has been shown to attenuate matrix metalloproteinase (MMP) expression and activity in various tissues, and MMP inactivation mitigates left ventricular (LV) remodeling in animal models of heart failure. Therefore, we examined the influence of Dox on LV remodeling and MMP expression in mice after transverse aortic constriction (TAC). One month after TAC, cardiac hypertrophy (99% vs. 67%) and the proportion of mice exhibiting congestive heart failure (CHF, 74% vs. 32%) were higher in the TAC + Dox group than in the TAC group (P < 0.05). These differences were no longer seen 2 mo after TAC, although LV was more severely dilated in TAC + Dox mice than in TAC mice (P < 0.05). One month after TAC, the increase in brain natriuretic peptide and β-myosin heavy chain mRNA levels was 1.6 and 1.7 times higher, respectively, in TAC + Dox mice than in TAC mice (P < 0.01). MMP-2 gelatin zymographic activity increased 1.9- and 2.4-fold in TAC and TAC + Dox mice, respectively (P < 0.01 and P < 0.05 relative to respective sham-operated animals), but the difference between TAC + Dox and TAC mice did not reach statistical significance. Dox did not significantly alter TAC-associated perivascular and interstitial myocardial fibrosis. These findings demonstrate that Dox accelerates the onset of cardiac hypertrophy and the progression to CHF following TAC in mice. Accordingly, care should be taken when designing and interpreting studies based on TG mouse models of LV hypertrophy using the tetracycline-regulated (tet)-on/tet-off system.