[Show abstract][Hide abstract] ABSTRACT: Background. Aortocaval fistula (ACF)-induced volume overload (VO) heart failure (HF) results in progressive left ventricular (LV) dysfunction. Hemodynamic load reversal during pre-HF (4 weeks post-ACF; REV) results in rapid structural but delayed functional recovery. This study investigated myocyte and myofilament function in ACF and REV and tested the hypothesis that a myofilament Ca(2+) sensitizer would improve VO-induced myofilament dysfunction in ACF and REV. Methods and Results. Following the initial Sham or ACF surgery in male Sprague-Dawley rats (200-240g) at Week 0, REV surgery and experiments were performed at Weeks 4 and 8, respectively. In ACF, decreased LV function is accompanied by impaired sarcomeric shortening and force generation and decreased Ca(2+) sensitivity, while in REV, impaired LV function is accompanied by decreased Ca(2+) sensitivity. IV Levo elicited the best inotropic and lusitropic responses and was selected for chronic oral studies. Subsets of ACF and REV rats were given vehicle (Veh; water) or Levo (1 mg/kg) in drinking water from Weeks 4-8. Levo improved systolic (%FS, Ees, PRSW) and diastolic (tau, dP/dtmin) function in ACF and REV. Levo improved Ca(2+) sensitivity without altering the amplitude and kinetics of the intracellular Ca(2+) transient. In ACF-Levo, increased cMyBP-C Ser-273 and Ser-302 and cTnI Ser-23/24 phosphorylation correlated with improved diastolic relaxation, while in REV-Levo, increased cMyBP-C Ser-273 phosphorylation and increased α-to-β-MHC correlated with improved diastolic relaxation. Conclusion. Levo improves LV function, and myofilament composition and regulatory protein phosphorylation likely play a key role in improving function.
[Show abstract][Hide abstract] ABSTRACT: Objectives:
The purpose of this study is to characterize the cytokine response of preterm newborns with surgical necrotizing enterocolitis (NEC) or spontaneous intestinal perforation (SIP) before surgical treatment and to relate these finding to intestinal disease (NEC vs. SIP).
The study was a 14-month prospective, cohort study of neonates undergoing surgery or drainage for NEC or SIP or surgical ligation of patent ductus arteriosus (PDA). Multiplex cytokine detection technology was used to analyze six inflammatory markers: interleukin-2, interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 β (IL-1β), interferon-gamma, and tumor necrosis factor-α (TNF-α).
Patients with NEC had much higher median preoperative levels of IL-6 (NEC: 8,381 pg/mL; SIP: 36 pg/mL; PDA: 25 pg/mL, p < 0.001), IL-8 (NEC: 18,438 pg/mL; SIP: 2,473 pg/mL; PDA: 1,110 pg/mL, p = 0.001), TNF-α (NEC: 161 pg/mL; SIP: 77 pg/mL; PDA: 71 pg/mL, p < 0.001), and IL-1β (NEC: 85 pg/mL; SIP: 31 pg/mL; PDA: 24 pg/mL, p = 0.001). Patients with NEC totalis (NEC-totalis had the highest levels of IL-8 and were significantly different from infants with limited NEC (28,141 vs. 11,429 pg/mL, p = 0.03).
Surgical NEC is a profoundly more proinflammatory disease than SIP. The cytokine profiles of patients with SIP are closer to those of a nonseptic surgical neonate.
No preview · Article · Aug 2013 · American Journal of Perinatology
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES:: Our goal was to evaluate the role of three anesthetic techniques in altering the stress response in children undergoing surgery for repair of congenital heart diseases utilizing cardiopulmonary bypass in the setting of fast tracking or early tracheal extubation. Furthermore, we wanted to evaluate the correlation between blunting the stress response and the perioperative clinical outcomes. DESIGN:: Prospective, randomized, double-blinded study. SETTING:: Single center from December 2008 to May of 2011. PATIENTS:: Forty-eight subjects (low-dose fentanyl plus placebo, n = 16; high-dose fentanyl plus placebo, n = 17; low-dose fentanyl plus dexmedetomidine, n = 15) were studied between ages 30 days to 3 years old who were scheduled to undergo repair for a ventricular septal defect, atrioventricular septal defect, or Tetralogy of Fallot. METHODS:: Children undergoing surgical repair of congenital heart disease were randomized to receive low-dose fentanyl (10 mcg/kg; low-dose fentanyl), high-dose fentanyl (25mcg/kg; high-dose fentanyl), or low-dose fentanyl plus dexmedetomidine (as a 1 mcg/kg loading dose followed by infusion at 0.5mcg/kg/hr until separation from cardiopulmonary bypass. In addition, patients received a volatile anesthetic agent as needed to maintain hemodynamic stability. Blood samples were tested for metabolic, hormonal and cytokine markers at baseline, after sternotomy, after the start of cardiopulmonary bypass, at the end of the procedure and at 24 hours postoperatively. MEASUREMENTS AND MAIN RESULTS:: Forty-eight subjects (low-dose fentanyl plus placebo, n = 16; high-dose fentanyl plus placebo, n = 17; low-dose fentanyl plus dexmedetomidine, n = 15) were studied. Subjects in the low-dose fentanyl plus placebo group had significantly higher levels of adrenocorticotropic hormone, cortisol, glucose, lactate, and epinephrine during the study period. The lowest levels of stress markers were seen in the high-dose fentanyl plus placebo group both over time (adrenocorticotropic hormone, p = 0.01; glucose, p = 0.007) and at individual time points (cortisol and lactate at the end of surgery, epinephrine poststernotomy; p < 0.05). Subjects in the low-dose fentanyl plus dexmedetomidine group had lower lactate levels at the end of surgery compared with the low-dose fentanyl plus placebo group (p < 0.05). Although there were no statistically significant differences in plasma cytokine levels between the three groups, the low-dose fentanyl plus placebo group had significantly higher interleukin-6:interleukin-10 ratio at 24 hours postoperatively (p < 0.0001). In addition, when compared with the low-dose fentanyl plus placebo group, the low-dose fentanyl plus dexmedetomidine group showed lower norepinephrine level from baseline at poststernotomy, after start of cardiopulmonary bypass, and end of surgery (p ≤ 0.05). Subjects in the low-dose fentanyl plus placebo group had more postoperative narcotic requirement (p = 0.004), higher prothrombin time (p ≤ 0.03), and more postoperative chest tube output (p < 0.05). Success of fast tracking was not significantly different between groups (low-dose fentanyl plus placebo 75%, high-dose fentanyl plus placebo 82%, low-dose fentanyl plus dexmedetomidine 93%; p = 0.39). CONCLUSIONS:: The use of low-dose fentanyl was associated with the greatest stress response, most coagulopathy, and highest transfusion requirement among our cohorts. Higher dose fentanyl demonstrated more favorable blunting of the stress response. When compared with low-dose fentanyl alone, the addition of dexmedetomidine improved the blunting of the stress response, while achieving better postoperative pain control.
No preview · Article · May 2013 · Pediatric Critical Care Medicine
[Show abstract][Hide abstract] ABSTRACT: Background:
With advances in cardiac care, patients with congenital heart disease, including single ventricle (SV) physiology, now survive into adulthood. These patients often suffer from congestive heart failure (CHF) with overexpression of serum biomarkers. Strain and strain rate (SR) may better describe the myocardial mechanics of a failing SV. Our objective was to determine the correlation between strain/SR and biomarkers in adult patients with SV and CHF.
Adult patients (age ≥8 years) with a SV were enrolled. Strain/SR in a 16-segment model of a SV was measured using 2D speckle echocardiography. Serum levels of interleukin 6, interleukin 8, matrix metalloproteinase 9, procollagen I C-terminal peptide (PCIP), cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), pro-B-type natriuretic peptide, nitrotryrosine, tissue growth factor beta (TGF-β), tumor necrosis factor alpha, vascular endothelial growth factor, and creatinine (Cr) were measured. Patients underwent a complete 6 Minute Walk Test (MWT). Pearson correlation coefficient was used. P <.05 was considered significant.
Ten patients with SV (LV = 7, RV = 3) were enrolled. Mean age was 35.5 years (25-42 years). Mean single ventricular ejection fraction (SVEF) was 47%. ICTP correlated with the basal, mid, and apical anterolateral SR, as well as apical anterior and inferior SR. PCIP correlated with mid anterolateral, basal anteroseptal, and mid inferolateral SR. TGF-β correlated with apical inferior SR. Cr correlated with mid inferior-septal and apical lateral SR. 6 MWT negatively correlated with the apical anterior septum SR.
ICTP, Cr, and PCIP correlated best with segmental SR values. Our results provide a preliminary platform for future studies to follow the results of treatment modalities using strain/SR and biomarkers for CHF in this population.
Full-text · Article · Oct 2012 · Congenital Heart Disease
[Show abstract][Hide abstract] ABSTRACT: Previous studies from our laboratory showed that coronary arterioles from type 2 diabetic mice undergo inward hypertrophic remodeling and reduced stiffness. The aim of the current study was to determine if coronary resistance microvessels (CRMs) in Ossabaw swine with metabolic syndrome (MetS) undergo remodeling distinct from coronary conduit arteries. Male Ossabaw swine were fed normal (n = 7, Lean) or hypercaloric high-fat (n = 7, MetS) diets for 6 mo, and then CRMs were isolated and mounted on a pressure myograph. CRMs isolated from MetS swine exhibited decreased luminal diameters (126 ± 5 and 105 ± 9 μm in Lean and MetS, respectively, P < 0.05) with thicker walls (18 ± 3 and 31 ± 3 μm in Lean and MetS, respectively, P < 0.05), which doubled the wall-to-lumen ratio (14 ± 2 and 30 ± 2 in Lean and MetS, respectively, P < 0.01). Incremental modulus of elasticity (IME) and beta stiffness index (BSI) were reduced in CRMs isolated from MetS pigs (IME: 3.6 × 10(6) ± 0.7 × 10(6) and 1.1 × 10(6) ± 0.2 × 10(6) dyn/cm(2) in Lean and MetS, respectively, P < 0.001; BSI: 10.3 ± 0.4 and 7.3 ± 1.8 in Lean and MetS, respectively, P < 0.001). BSI in the left anterior descending coronary artery was augmented in pigs with MetS. Structural changes were associated with capillary rarefaction, decreased hyperemic-to-basal coronary flow velocity ratio, and augmented myogenic tone. MetS CRMs showed a reduced collagen-to-elastin ratio, while immunostaining for the receptor for advanced glycation end products was selectively increased in the left anterior descending coronary artery. These data suggest that MetS causes hypertrophic inward remodeling of CRMs and capillary rarefaction, which contribute to decreased coronary flow and myocardial ischemia. Moreover, our data demonstrate novel differential remodeling between coronary micro- and macrovessels in a clinically relevant model of MetS.
[Show abstract][Hide abstract] ABSTRACT: Current surgical management of volume overload-induced heart failure (HF) leads to variable recovery of left ventricular (LV) function despite a return of LV geometry. The mechanisms that prevent restoration of function are unknown but may be related to the timing of intervention and the degree of LV contractile impairment. This study determined whether reduction of aortocaval fistula (ACF)-induced LV volume overload during the compensatory stage of HF results in beneficial LV structural remodeling and restoration of pump function. Rats were subjected to ACF for 4 wk; a subset then received a load-reversal procedure by closing the shunt using a custom-made stent graft approach. Echocardiography or in vivo pressure-volume analysis was used to assess LV morphology and function in sham rats; rats subjected to 4-, 8-, or 15-wk ACF; and rats subjected to 4-wk ACF followed by 4- or 11-wk reversal. Structural and functional changes were correlated to LV collagen content, extracellular matrix (ECM) proteins, and hypertrophic markers. ACF-induced volume overload led to progressive LV chamber dilation and contractile dysfunction. Rats subjected to short-term reversal (4-wk ACF + 4-wk reversal) exhibited improved chamber dimensions (LV diastolic dimension) and LV compliance that were associated with ECM remodeling and normalization of atrial and brain natriuretic peptides. Load-independent parameters indicated LV systolic (preload recruitable stroke work, Ees) and diastolic dysfunction (tau, arterial elastance). These changes were associated with an altered α/β-myosin heavy chain ratio. However, these changes were normalized to sham levels in long-term reversal rats (4-wk ACF + 11-wk reversal). Acute hemodynamic changes following ACF reversal improve LV geometry, but LV dysfunction persists. Gradual restoration of function was related to normalization of eccentric hypertrophy, LV wall stress, and ECM remodeling. These results suggest that mild to moderate LV systolic dysfunction may be an important indicator of the ability of the myocardium to remodel following the reversal of hemodynamic overload.
[Show abstract][Hide abstract] ABSTRACT: Little is known about the impact of type 2 diabetes mellitus (DM) on coronary arteriole remodeling. The aim of this study was to determine the mechanisms that underlie coronary arteriole structural remodeling in type 2 diabetic (db/db) mice. Passive structural properties of septal coronary arterioles isolated from 12- to 16-week-old diabetic db/db and control mice were assessed by pressure myography. Coronary arterioles from 12-week-old db/db mice were structurally similar to age-matched controls. By 16 weeks of age, coronary wall thickness was increased in db/db arterioles (p < 0.01), while luminal diameter was reduced (control: 118 ± 5 μm; db/db: 102 ± 4 μm, p < 0.05), augmenting the wall-to-lumen ratio by 58% (control: 5.9 ± 0.6; db/db: 9.5 ± 0.4, p < 0.001). Inward hypertrophic remodeling was accompanied by a 56% decrease in incremental elastic modulus (p < 0.05, indicating decreased vessel coronary wall stiffness) and a ~30% reduction in coronary flow reserve (CFR) in diabetic mice. Interestingly, aortic pulse wave velocity and femoral artery incremental elastic modulus were increased (p < 0.05) in db/db mice, indicating macrovascular stiffness. Molecular tissue analysis revealed increased elastin-to-collagen ratio in diabetic coronaries when compared to control and a decrease in the same ratio in the diabetic aortas. These data show that coronary arterioles isolated from type 2 diabetic mice undergo inward hypertrophic remodeling associated with decreased stiffness and increased elastin-to-collagen ratio which results in a decreased CFR. This study suggests that coronary microvessels undergo a different pattern of remodeling from macrovessels in type 2 DM.
Full-text · Article · Jul 2011 · Archiv für Kreislaufforschung
[Show abstract][Hide abstract] ABSTRACT: Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G(1)/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D(1) expression, as well as p27(Kip) upregulation. Cell division kinase 2 (cdc2) phosphorylation at G(2)/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.
Preview · Article · Mar 2011 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Receptor-independent G-protein regulators provide diverse mechanisms for signal input to G-protein-based signaling systems, revealing unexpected functional roles for G-proteins. As part of a broader effort to identify disease-specific regulators for heterotrimeric G-proteins, we screened for such proteins in cardiac hypertrophy using a yeast-based functional screen of mammalian cDNAs as a discovery platform. We report the identification of three transcription factors belonging to the same family, transcription factor E3 (TFE3), microphthalmia-associated transcription factor, and transcription factor EB, as novel receptor-independent activators of G-protein signaling selective for Gα(16). TFE3 and Gα(16) were both up-regulated in cardiac hypertrophy initiated by transverse aortic constriction. In protein interaction studies in vitro, TFE3 formed a complex with Gα(16) but not with Gα(i3) or Gα(s). Although increased expression of TFE3 in heterologous systems had no influence on receptor-mediated Gα(16) signaling at the plasma membrane, TFE3 actually translocated Gα(16) to the nucleus, leading to the induction of claudin 14 expression, a key component of membrane structure in cardiomyocytes. The induction of claudin 14 was dependent on both the accumulation and activation of Gα(16) by TFE3 in the nucleus. These findings indicate that TFE3 and Gα(16) are up-regulated under pathologic conditions and are involved in a novel mechanism of transcriptional regulation via the relocalization and activation of Gα(16).
Preview · Article · Mar 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: G-protein coupled receptor (GPCR) signaling represents one of the most conserved and ubiquitous means in mammalian cells for transferring information across the plasma membrane to the intracellular environment. Heterotrimeric G-protein subunits play key roles in transducing these signals, and intracellular regulators influencing the activation state and interaction of these subunits regulate the extent and duration of GPCR signaling. One class of intracellular regulator, the non-receptor activators of G-protein signaling (or AGS proteins), are the major focus of this review. AGS proteins provide a basis for understanding the function of heterotrimeric G-proteins in both GPCR-driven and GPCR independent cellular signaling pathways.
No preview · Article · Jul 2006 · Seminars in Cell and Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: As part of a broader effort to identify postreceptor signal regulators involved in specific diseases or organ adaptation, we used an expression cloning system in Saccharomyces cerevisiae to screen cDNA libraries from rat ischemic myocardium, human heart, and a prostate leiomyosarcoma for entities that activated G protein signaling in the absence of a G protein coupled receptor. We report the characterization of activator of G protein signaling (AGS) 8 (KIAA1866), isolated from a rat heart model of repetitive transient ischemia. AGS8 mRNA was induced in response to ventricular ischemia but not by tachycardia, hypertrophy, or failure. Hypoxia induced AGS8 mRNA in isolated adult ventricular cardiomyocytes but not in rat aortic smooth muscle cells, endothelial cells, or cardiac fibroblasts, suggesting a myocyte-specific adaptation mechanism involving remodeling of G protein signaling pathways. The bioactivity of AGS8 in the yeast-based assay was independent of guanine nucleotide exchange by Galpha, suggesting an impact on subunit interactions. Subsequent studies indicated that AGS8 interacts directly with Gbetagamma and this occurs in a manner that apparently does not alter the regulation of the effector PLC-beta(2) by Gbetagamma. Mechanistically, AGS8 appears to promote G protein signaling by a previously unrecognized mechanism that involves direct interaction with Gbetagamma.
Preview · Article · Feb 2006 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The identification of AGS proteins as receptor-independent activators of G-protein signaling reveals unexpected mechanisms for the regulation of heterotrimeric G-protein activation and has opened up new areas of research related to the role of G proteins as signal transducers. In addition to their obvious interest associated with G-protein-coupled receptor signaling, AGS proteins might provide alternative binding partners for G-protein subunits that enable them to serve unexpected functions related to cell division, differentiation and organelle structure that might operate independently of a GPCR. Thus, these proteins and the concepts advanced with their discovery highlight the diversity associated with G-protein signaling and present new avenues for the development of therapeutics that target G-protein signaling.
No preview · Article · Oct 2005 · Trends in Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: Heterotrimeric G-proteins are key transducers for signal transfer from outside the cell, mediating signals emanating from cell-surface G-protein coupled receptors (GPCR). Many, if not all, subtypes of heterotrimeric G-proteins are also regulated by accessory proteins that influence guanine nucleotide binding, guanosine triphosphate (GTP) hydrolysis, or subunit interactions. One subgroup of such accessory proteins (activators of G-protein signaling; AGS proteins) refer to a functionally defined group of proteins that activate selected G-protein signaring systems in the absence of classical G-protein coupled receptors. AGS and related proteins provide unexpected insights into the regulation of the G-protein activation-deactivation cycle. Different AGS proteins function as guanine nucleotide exchange factors or guanine nucleotide dissociation inhibitors and may also influence subunit interactions by interaction with GBgamma. These proteins play important roles in the generation or positioning of signaling complexes and of the regulation of GPCR signaling, and as alternative binding partners for G-protein subunits. Perhaps of even broader impact is the discovery that AGS proteins provide a foundation for the concept that heterotrimeric G-protein subunits are processing signals within the cell involving intrinsic cues that do not involve the classical signal input from a cell surface GPCR.
No preview · Article · Feb 2005 · Reviews of Physiology, Biochemistry and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: AGS1/RASD1 is a Ras-related protein identified as a dexamethasone-inducible cDNA and as a signal regulator in various functional and protein-interaction screens. As an initial approach to define the role of AGS1/RASD1 as a Ras-family member, we determined its influence on cell growth/survival. In clonogenic assays with NIH-3T3 murine fibroblast cells, the MCF-7 human breast cancer cell line and the human lung adenocarcinoma cell line A549, AGS1/RASD1 markedly diminished the number of G418-resistant colonies, whereas the Ras subgroup member K-Ras was without effect. A549 cell infection with adenovirus engineered to express AGS1/RASD1 (Ad.AGS1) inhibited log phase growth in vitro and increased the percentage of cells undergoing apoptosis. The anti-growth action was also observed in vivo as the expression of AGS1/RASD1 inhibited the subcutaneous tumor growth of A549 cells in athymic nude mice. These data indicate that AGS1/RASD1, a member of the Ras superfamily of small G-proteins that often promotes cell growth and tumor expansion, plays an active role in preventing aberrant cell growth.
[Show abstract][Hide abstract] ABSTRACT: Activators of G-protein signaling 1-3 (AGS1-3) were identified in a functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor. We report the isolation and characterization of an additional AGS protein (AGS4) from a human prostate leiomyosarcoma cDNA library. AGS4 is identical to G18.1b, which is encoded by a gene within the major histocompatibility class III region of chromosome 6. The activity of AGS4 in the yeast-based functional screen was selective for G(i2)/G(i3) and independent of guanine-nucleotide exchange by G(i)alpha. RNA blots indicated enrichment of AGS4/G18.1b mRNA in heart, placenta, lung, and liver. Immunocytochemistry with AGS4/G18.1b-specific antisera indicated a predominant nonhomogeneous, extranuclear distribution within the cell following expression in COS7 or Chinese hamster ovary cells. AGS4/G18.1b contains three G-protein regulatory motifs downstream of an amino terminus domain with multiple prolines. Glutathione S-transferase (GST)-AGS4/G18.1b fusion proteins interacted with purified G(i)alpha, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to purified G(i)alpha(1). AGS4/G18.1b was also complexed with G(i)alpha(3) in COS7 cell lysates following cell transfection. However, AGS4/G18.1b did not alter the generation of inositol phosphates in COS7 cells cotransfected with the Gbetagamma-regulated effector phospholipase C-beta2. These data suggest either that an additional signal is required to position AGS4/G18.1b in the proper cellular location where it can access heterotrimer and promote subunit dissociation or that AGS4 serves as an alternative binding partner for G(i)alpha independent of Gbetagamma participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.
No preview · Article · Jul 2004 · Journal of Biological Chemistry