Circulation Research Online First

[Article] Protein Kinase A-Mediated Phosphorylation of cMyBP-C Increases Proximity of Myosin Heads to Actin in Resting Myocardium

2008-07-03

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein-C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I₁₁/I₁₀, in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C^-/-) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase (approximately 50%) in I₁₁/I₁₀ (0.22±0.03 versus 0.33±0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I₁₁/I₁₀ between untreated and PKA-treated cMyBP-C^-/- myocardium (0.40±0.06 versus 0.42±0.05). Although lattice spacing did not change after treatment in wild-type (45.68±0.84 nm versus 45.64±0.64 nm), treatment of cMyBP-C^-/- myocardium increased lattice spacing (46.80±0.92 nm versus 49.61±0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross-bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross-bridges to actin and increasing the probability of interaction with actin on contraction.

[Article] Regulation of Endothelial Nitric Oxide Synthase and Postnatal Angiogenesis by Rac1

Sawada, N., Salomone, S., Kim, H.-H., Kwiatkowski, D. J., Liao, J. K. — 2008-07-03

Diminished bioavailability of nitric oxide is a hallmark of endothelial dysfunction and is associated with a broad spectrum of vascular disorders such as impaired angiogenesis. Because Rac1, a Rho family member, mediates cellular motility and generation of reactive oxygen species, it could be involved in the regulation of endothelial nitric oxide production. However, the pathophysiological consequences of postnatal endothelial Rac1 deletion on endothelial function have not been determined. We generated endothelial-specific Rac1 haploinsufficient mice (EC-Rac1^+/-) using Cre-loxP technology. The EC-Rac1^+/- mice have decreased expression and activity of endothelial nitric oxide synthase (eNOS), impaired endothelium-dependent vasorelaxation, and mild hypertension compared with control (Rac1^+/flox) or wild-type mice. Hind limb ischemia model and aortic capillary sprouting assay showed that eNOS activity and angiogenesis was impaired in EC-Rac1^+/- mice. Indeed, Rac1 promotes eNOS gene transcription through p21-activated kinase but not NADPH oxidase, increases eNOS mRNA stability, and enhances eNOS activity by promoting endothelial uptake of L-arginine. These findings indicate that endothelial Rac1 is essential for endothelium-dependent vasomotor response and ischemia-induced angiogenesis. These effects of Rac1 on endothelial function are largely due to the upregulation of eNOS through multiple mechanisms that are mediated, in part, by p21-activated kinase. Therapeutic strategies to enhance Rac1 function, therefore, may be important for preventing endothelial dysfunction.

[Article] Enhancing Mitochondrial Ca2+ Uptake in Myocytes From Failing Hearts Restores Energy Supply and Demand Matching

Liu, T., O'Rourke, B. — 2008-07-03

Mitochondrial ATP production is continually adjusted to energy demand through coordinated increases in oxidative phosphorylation and NADH production mediated by mitochondrial Ca²⁺([Ca²⁺]_m). Elevated cytosolic Na⁺ impairs [Ca²⁺]_m accumulation during rapid pacing of myocytes, resulting in a decrease in NADH/NAD⁺ redox potential. Here, we determined 1) if accentuating [Ca²⁺]_m accumulation prevents the impaired NADH response at high [Na⁺]_i; 2) if [Ca²⁺]_m handling and NADH/NAD⁺ balance during stimulation is impaired with heart failure (induced by aortic constriction); and 3) if inhibiting [Ca²⁺]_m efflux improves NADH/NAD⁺ balance in heart failure. [Ca²⁺]_m and NADH were recorded in cells at rest and during voltage clamp stimulation (4Hz) with either 5 or 15 mmol/L [Na⁺]_i. Fast [Ca²⁺]_m transients and a rise in diastolic [Ca²⁺]_m were observed during electric stimulation. [Ca²⁺]_m accumulation was [Na⁺]_i-dependent; less [Ca²⁺]_m accumulated in cells with 15 Na⁺ versus 5 mmol/L Na⁺ and NADH oxidation was evident at 15 mmol/L Na⁺, but not at 5 mmol/L Na⁺. Treatment with either the mitochondrial Na⁺/Ca²⁺ exchange inhibitor CGP-37157 (1 µmol/L) or raising cytosolic P_i (2 mmol/L) enhanced [Ca²⁺]_m accumulation and prevented the NADH oxidation at 15 mmol/L [Na⁺]_i. In heart failure myocytes, resting [Na⁺]_i increased from 5.2±1.4 to 16.8±3.1mmol/L and net NADH oxidation was observed during pacing, whereas NADH was well matched in controls. Treatment with CGP-37157 or lowering [Na⁺]_i prevented the impaired NADH response in heart failure. We conclude that high [Na⁺]_i (at levels observed in heart failure) has detrimental effects on mitochondrial bioenergetics, and this impairment can be prevented by inhibiting the mitochondrial Na⁺/Ca²⁺ exchanger.

[Article] Involvement of Heat Shock Factor 1 in Statin-Induced Transcriptional Upregulation of Endothelial Thrombomodulin

Fu, Q., Wang, J., Boerma, M., Berbee, M., Qiu, X., Fink, L. M., Hauer-Jensen, M. — 2008-07-03

Statins upregulate endothelial thrombomodulin (TM) by mechanisms that involve members of the Kruppel-like factor family. Although Kruppel-like factors are unequivocally implicated in this process, experimental evidence points to additional mechanisms. Deletion/mutation analysis of reporter constructs was used to demonstrate that mutation of the SP1/Kruppel-like factor element in the TM promoter only partially abolishes statin-induced TM upregulation, whereas simultaneous mutation of relevant heat shock elements and SP1/Kruppel-like factor element completely prevents statin-induced TM upregulation, thus demonstrating a role for heat shock factors (HSFs). We further identified the pathway by which statins increase binding of HSF1 to heat shock elements in the TM promoter. Specifically, statins caused NO-dependent dissociation of HSF1 from heat shock protein 90, nuclear translocation of HSF1, and binding to heat shock elements in the TM promoter. Statins also decreased nuclear content of the HSF1 chaperone 14 to 3–3β. In addition to reducing TM upregulation, inhibition of HSF1 reduced statin-induced upregulation of tissue plasminogen activator, whereas downregulation of thrombomospondin, plasminogen activator inhibitor 1, or connective tissue growth factor was unaffected. Knockdown of 14 to 3–3β or inhibition of HSF1 phosphorylation enhanced the effect of statins on TM and tissue plasminogen activator, but did not influence thrombomospondin, plasminogen activator inhibitor 1, or connective tissue growth factor. These data demonstrate that HSF1 is involved in statin-induced regulation of TM. They also suggest that analogous mechanisms may apply to genes that are upregulated by statins, but not to downregulated genes. These results may have broad implications and suggest the use of heat shock protein modulators to selectively regulate pleiotropic statin effects.

[Article] Lidocaine-Induced Brugada Syndrome Phenotype Linked to a Novel Double Mutation in the Cardiac Sodium Channel

2008-07-03

Brugada syndrome has been linked to mutations in SCN5A. Agents that dissociate slowly from the sodium channel such as flecainide and ajmaline unmask the Brugada syndrome electrocardiogram and precipitate ventricular tachycardia/fibrillation. Lidocaine, an agent with rapid dissociation kinetics, has previously been shown to exert no effect in patients with Brugada syndrome. We characterized a novel double mutation of SCN5A (V232I in DI-S4+L1308F in DIII-S4) identified in a rare case of lidocaine (1 mg/kg)-induced Brugada syndrome. We studied lidocaine blockade of I_Na generated by wild-type and V232I+L1308F mutant cardiac sodium channels expressed in mammalian TSA201 cells using patch clamp techniques. Despite no significant difference in steady-state gating parameters between V232I+L1308F and wild-type sodium currents at baseline, use-dependent inhibition of I_Na by lidocaine was more pronounced in V232I+L1308F versus wild-type (73.0±0.1% versus 18.23±0.04% at 10 µmol/L measured at 10 Hz, respectively). A dose of 10 µmol/L lidocaine also caused a more negative shift of steady-state inactivation in V232I+L1308F versus wild-type (-14.1±0.3 mV and -4.8±0.3 mV, respectively). The individual mutations produced a much less accentuated effect. We report the first case of lidocaine-induced Brugada electrocardiogram phenotype. The double mutation in SCN5A, V232I, and L1308F alters the affinity of the cardiac sodium channel for lidocaine such that the drug assumes Class IC characteristics with potent use-dependent block of the sodium channel. Our results demonstrate an additive effect of the 2 missense mutations to sensitize the sodium channel to lidocaine. These findings suggest caution when treating patients carrying such genetic variations with Class I antiarrhythmic drugs.

[Article] Connexin40 Imparts Conduction Heterogeneity to Atrial Tissue

2008-07-03

Impulse propagation in cardiac tissue is a complex process in which intercellular coupling through gap junction channels is a critical component. Connexin40 (Cx40) is an abundant gap junction protein that is expressed in atrial myocytes. Alterations in the expression of Cx40 have been implicated in atrial arrhythmogenesis. The purpose of the current study was to assess the role of Cx40 in atrial impulse propagation. High-resolution optical mapping was used to study conduction in the right and left atrial appendages of isolated Langendorff-perfused murine hearts. Wild-type (Cx40^+/+), heterozygous (Cx40^±), and knockout (Cx40^-/-) mice, both adult and embryonic, were studied to assess the effects of reduced Cx40 expression on sinus node function and conduction velocity at different pacing cycle lengths (100 and 60 ms). In both adult and late-stage embryonic Cx40^+/+ mice, heterogeneity in CV was found between the right and left atrial appendages. Either partial (Cx40^±) or complete (Cx40^-/-) deletion of Cx40 was associated with the loss of conduction heterogeneity in both adult and embryonic mice. Additionally, sinus node impulse initiation was found to be ectopic in Cx40^-/- mice at 15.5 days postcoitus, whereas Cx40^+/+ mice showed normal activation occurring near the crista terminalis. Our findings suggest that Cx40 plays an essential role in establishing interatrial conduction velocity heterogeneity in the murine model. Additionally, we describe for the first time a developmental requirement for Cx40 in normal sinus node impulse initiation at 15.5 days postcoitus.

[Article] Reactive Oxygen Species-Induced Activation of p90 Ribosomal S6 Kinase Prolongs Cardiac Repolarization Through Inhibiting Outward K+ Channel Activity

2008-07-03

p90 ribosomal S6 kinase (p90RSK) is activated in cardiomyopathies caused by conditions such as ischemia/reperfusion injury and diabetes mellitus in which prolongation of cardiac repolarization and frequent arrhythmias are common. Molecular mechanisms underlying the electric remodeling in cardiac diseases are largely unknown. In the present study, we determined the role of p90RSK activation in the modulation of voltage-gated K⁺ channel activity determining cardiac repolarization. Mice with increased cardiac p90RSK activity due to transgenic expression of p90RSK (p90RSK-Tg) had prolongation of QT intervals and of ventricular myocyte action potential durations. Fast transient outward K⁺ current (I_to,f), slow delayed outward K⁺ current (I_K,slow), and steady-state K⁺ current (I_SS) were significantly decreased in p90RSK-Tg mouse ventricular myocytes. mRNA levels of Kv4.3, Kv4.2, Kv1.5, Kv2.1, and KChIP2 from ventricles between p90RSK-Tg and nontransgenic littermate control mice were similar, as assessed by quantitative reverse transcriptase–polymerase chain reaction, indicating that p90RSK regulates voltage-gated K⁺ channels through posttranslational modification. Kv4.3- and Kv1.5- rather than Kv4.2- and Kv2.1-encoded channels in HEK 293 cells were inhibited by p90RSK. In vitro phosphorylation analysis showed that Kv4.3 was phosphorylated by p90RSK at 2 conserved sites, Ser516 and Ser550. p90RSK expression significantly inhibited Kv4.3- and Kv4.3 and KChIP2-encoded channel activities in HEK 293 cells, whereas p90RSK's effects were blocked by amino acid mutation(s) at phosphorylation site(s) in Kv4.3. Hydrogen peroxide, a mediator of induced cardiac p90RSK activation in ischemia/reperfusion and diabetes mellitus, had effects similar to those of p90RSK on Kv4.3- or Kv4.3- and KChIP2-encoded channels. Fluoromethylketone, a specific p90RSK inhibitor, abolished hydrogen peroxide effects. These findings indicate that p90RSK activation is critical for reactive oxygen species-mediated inhibition of voltage-gated K⁺ channel activity and leads to prolongation of cardiac repolarization.

[Article] Role of Nox2-Based NADPH Oxidase in Bone Marrow and Progenitor Cell Function Involved in Neovascularization Induced by Hindlimb Ischemia

2008-07-01

Bone marrow (BM) is the major reservoir for endothelial progenitor cells (EPCs). Postnatal neovascularization depends on not only angiogenesis but also vasculogenesis, which is mediated through mobilization of EPCs from BM and their recruitment to the ischemic sites. Reactive oxygen species (ROS) derived from Nox2-based NADPH oxidase play an important role in postnatal neovascularization; however, their role in BM and EPC function is unknown. Here we show that hindlimb ischemia of mice significantly increases Nox2 expression and ROS production in BM-mononuclear cells (BMCs), which is associated with an increase in circulating EPC-like cells. Mice lacking Nox2 show reduction of ischemia-induced flow recovery, ROS levels in BMCs, as well as EPC mobilization from BM. Transplantation of wild-type (WT)-BM into Nox2-deficient mice rescues the defective neovascularization, whereas WT mice transplanted with Nox2-deficient BM show reduced flow recovery and capillary density compared to WT-BM transplanted control. Intravenous infusion of WT- and Nox2-deficient BMCs into WT mice reveals that neovascularization and homing capacity are impaired in Nox2-deficient BMCs in vivo. In vitro, Nox2-deficient c-kit⁺Lin^- BM stem/progenitor cells show impaired chemotaxis and invasion as well as polarization of actins in response to stromal derived factor (SDF), which is associated with blunted SDF-1–mediated phosphorylation of Akt. In conclusion, Nox2-derived ROS in BM play a critical role in mobilization, homing, and angiogenic capacity of EPCs and BM stem/progenitor cells, thereby promoting revascularization of ischemic tissue. Thus, NADPH oxidase in BM and EPCs is potential therapeutic targets for promoting neovascularization in ischemic cardiovascular diseases.

[Article] Endothelial Cells Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine TGF-{beta} Signaling Pathway

2008-06-26

Mechanical forces are potent modulators of the growth and hypertrophy of vascular cells. We examined the molecular mechanisms through which mechanical force and hypertension modulate endothelial cell regulation of vascular homeostasis. Exposure to mechanical strain increased the paracrine inhibition of vascular smooth muscle cells (VSMCs) by endothelial cells. Mechanical strain stimulated the production of perlecan and heparan sulfate glycosaminoglycans by endothelial cells. By inhibiting the expression of perlecan with an antisense vector we demonstrated that perlecan was essential to the strain-mediated effects on endothelial cell growth control. Mechanical regulation of perlecan expression in endothelial cells was governed by a mechanotransduction pathway requiring autocrine transforming growth factor β (TGF-β) signaling and intracellular signaling through the ERK pathway. Immunohistochemical staining of the aortae of spontaneously hypertensive rats demonstrated strong correlations between endothelial TGF-β, phosphorylated signaling intermediates, and arterial thickening. Further, studies on ex vivo arteries exposed to varying levels of pressure demonstrated that ERK and TGF-β signaling were required for pressure-induced upregulation of endothelial HSPG. Our findings suggest a novel feedback control mechanism in which net arterial remodeling to hemodynamic forces is controlled by a dynamic interplay between growth stimulatory signals from VSMCs and growth inhibitory signals from endothelial cells.

[Article] Protein Kinase D Is a Key Regulator of Cardiomyocyte Lipoprotein Lipase Secretion After Diabetes

2008-06-26

The diabetic heart switches to exclusively using fatty acid (FA) for energy supply and does so by multiple mechanisms including hydrolysis of lipoproteins by lipoprotein lipase (LPL) positioned at the vascular lumen. We determined the mechanism that leads to an increase in LPL after diabetes. Diazoxide (DZ), an agent that decreases insulin secretion and causes hyperglycemia, induced a substantial increase in LPL activity at the vascular lumen. This increase in LPL paralleled a robust phosphorylation of Hsp25, decreasing its association with PKC, allowing this protein kinase to phosphorylate and activate protein kinase D (PKD), an important kinase that regulates fission of vesicles from the golgi membrane. Rottlerin, a PKC inhibitor, prevented PKD phosphorylation and the subsequent increase in LPL. Incubating control myocytes with high glucose and palmitic acid (Glu+PA) also increased the phosphorylation of Hsp25, PKC, and PKD in a pattern similar to that seen with diabetes, in addition to augmenting LPL activity. In myocytes in which PKD was silenced or a mutant form of PKC was expressed, high Glu+PA were incapable of increasing LPL. Moreover, silencing of cardiomyocyte Hsp25 allowed phorbol 12-myristate 13-acetate to elicit a significant phosphorylation of PKC, an appreciable association between PKC and PKD, and a vigorous activation of PKD. As these cells also demonstrated an additional increase in LPL, our data imply that after diabetes, PKD control of LPL requires dissociation of Hsp25 from PKC, association between PKC and PKD, and vesicle fission. Results from this study could help in restricting cardiac LPL translocation, leading to strategies that overcome contractile dysfunction after diabetes.

[Article] Localized {alpha}4 Integrin Phosphorylation Directs Shear Stress-Induced Endothelial Cell Alignment

2008-06-26

Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized 4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress–induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial 4 integrins became phosphorylated on Ser⁹⁸⁸. In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced 4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable 4(S⁹⁸⁸A) mutation failed to align in response to shear stress, thus establishing 4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent 4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress.

[Article] Vascular Endothelial Growth Factor Receptor-1 Regulates Postnatal Angiogenesis Through Inhibition of the Excessive Activation of Akt

2008-06-26

Vascular endothelial growth factor (VEGF) binds both VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2). Activation of VEGFR-2 is thought to play a major role in the regulation of endothelial function by VEGF. Recently, specific ligands for VEGFR-1 have been reported to have beneficial effects when used to treat ischemic diseases. However, the role of VEGFR-1 in angiogenesis is not fully understood. In this study, we showed that VEGFR-1 performs "fine tuning" of VEGF signaling to induce neovascularization. We examined the effects of retroviral vectors expressing a small interference RNA that targeted either the VEGFR-1 gene or the VEGFR-2 gene. Deletion of either VEGFR-1 or VEGFR-2 reduced the ability of endothelial cells to form capillaries. Deletion of VEGFR-1 markedly reduced endothelial cell proliferation and induced premature senescence of endothelial cells. In contrast, deletion of VEGFR-2 significantly impaired endothelial cell survival. When VEGFR-1 expression was blocked, VEGF constitutively activated Akt signals and thus induced endothelial cell senescence via a p53-dependent pathway. VEGFR-1^+/- mice exhibited an increase of endothelial Akt activity and showed an impaired neovascularization in response to ischemia, and this impairment was ameliorated in VEGFR-1^+/- Akt1^+/- mice. These results suggest that VEGFR-1 plays a critical role in the maintenance of endothelial integrity by modulating the VEGF/Akt signaling pathway.

[Article] Role of Sphingosine-1-Phosphate Phosphohydrolase 1 in the Regulation of Resistance Artery Tone

2008-06-26

Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S1P-degrading function, we hypothesized that S1P-phosphohydrolase 1 (SPP1) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP1. Overexpression of SPP1 (via transfection of a SPP1^wt) reduced resting tone, Ca²⁺ sensitivity, and myogenic vasoconstriction, whereas reduced SPP1 expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP1 (SPP1^H208A) had no effect on any parameter tested, suggesting that catalytic activity of SPP1 is critical. The enhanced myogenic tone that follows overexpression of S1P-generating enzyme sphingosine kinase 1 (Sk1^wt) was functionally antagonized by coexpression with SPP1^wt but not SPP1^H208A. SPP1 modulated vasoconstriction in response to 1 to 100 nmol/L exogenous S1P, a concentration range that was characterized as S1P₂-dependent, based on the effect of S1P₂ inhibition by antisense oligonucleotides and 1 µmol/L JTE013. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (1) restored S1P responses that were attenuated by SPP1^wt overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP1 is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk1^wt and S1P₂ receptor activation. SPP1 accesses extracellular S1P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP1 and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure.

[Article] The del22q11.2 Candidate Gene Tbx1 Controls Regional Outflow Tract Identity and Coronary Artery Patterning

2008-06-26

TBX1, encoding a T-box containing transcription factor, is the major candidate gene for del22q11.2 or DiGeorge syndrome, characterized by craniofacial and cardiovascular defects including tetralogy of Fallot and common arterial trunk. Mice lacking Tbx1 have severe defects in the development of pharyngeal derivatives including cardiac progenitor cells of the second heart field that contribute to the arterial pole of the heart. The outflow tract of Tbx1 mutant embryos is short and narrow resulting in common arterial trunk. Here we show by a series of genetic crosses using transgene markers of second heart field derived myocardium and coronary endothelial cells that a subdomain of myocardium normally observed at the base of the pulmonary trunk is reduced and malpositioned in Tbx1 mutant hearts. This defect is associated with anomalous coronary artery patterning. Both right and left coronary ostia form predominantly at the right/ventral sinus in mutant hearts, proximal coronary arteries coursing across the normally coronary free ventral region of the heart. We have identified Semaphorin3c as a Tbx1-dependent gene expressed in subpulmonary myocardium. Our results implicate second heart field development in coronary artery patterning and provide new insights into the association between conotruncal defects and coronary artery anomalies.

[Article] Unexpected Structural and Functional Consequences of the R33Q Homozygous Mutation in Cardiac Calsequestrin. A Complex Arrhythmogenic Cascade in a Knock In Mouse Model

2008-06-26

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disorder characterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2^R33Q/R33Q mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2^R33Q/R33Q mice develop bidirectional VT on exposure to environmental stress whereas CASQ2^R33Q/R33Q myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2^R33Q/R33Q hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2^R33Q/R33Q CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) impaired clustering of mutant CASQ2.

[Article] Glycogen Synthase Kinase-3 Inactivation Is Not Required for Ischemic Preconditioning or Postconditioning in the Mouse

2008-06-26

The inactivation of glycogen synthase kinase-3β (GSK-3β) is proposed as the event integrating protective pathways initiated by preconditioning and other interventions. The inactivation of GSK-3 is thought to decrease the probability of opening of the mitochondrial permeability transition pore. The aim of this study was to verify the role of GSK-3 using a targeted mouse line lacking the critical N-terminal serine within GSK-3β (Ser9) and the highly homologous GSK-3 (Ser21), which when phosphorylated results in kinase inactivation. Postconditioning with 10 cycles of 5 seconds of reperfusion/5 seconds of ischemia and preconditioning with 6 cycles of 4 minutes of ischemia/6 minutes of reperfusion, similarly reduced infarction of the isolated perfused mouse heart in response to 30 minutes of global ischemia and 120 minutes of reperfusion. Preconditioning caused noticeable inactivating phosphorylation of GSK-3. However, both preconditioning and postconditioning still protected hearts of homozygous GSK-3 double knockin mice. Moreover, direct pharmacological inhibition of GSK-3 catalytic activity with structurally diverse inhibitors before or after ischemia failed to recapitulate conditioning protection. Nonetheless, cyclosporin A, a direct mitochondrial permeability transition pore inhibitor, reduced infarction in hearts from both wild-type and homozygous GSK-3 double knockin mice. Furthermore, in adult cardiac myocytes from GSK-3 double knockin mice, insulin exposure was still as effective as cyclosporin A in delaying mitochondrial permeability transition pore opening. Our results, which include a novel genetic approach, suggest that the inhibition of GSK-3 is unlikely to be the key determinant of cardioprotective signaling in either preconditioning or postconditioning in the mouse.

[Article] The Role of Oxidant Stress on AT1 Receptor Expression in Neurons of Rabbits With Heart Failure and in Cultured Neurons

Liu, D., Gao, L., Roy, S. K., Cornish, K. G., Zucker, I. H. — 2008-06-19

We have previously reported that the expression of Angiotensin II (Ang II) type 1 receptors (AT1R) was increased in the rostral ventrolateral medulla (RVLM) of rabbits with chronic heart failure (CHF) and in the RVLM of normal rabbits infused with intracerebroventricular (ICV) Ang II. The present study investigated whether oxidant stress plays a role in Ang II–induced AT1R upregulation and its relationship to the transcription factor activator protein 1 (AP1) in CHF rabbits and in the CATHa neuronal cell line. In CATHa cells, Ang II significantly increased AT1R mRNA by 123±11%, P<0.01; c-Jun mRNA by 90±20%, P<0.01; c-fos mRNA by 148±49%, P<0.01; NADPH oxidase activity by 126±43%, P<0.01 versus untreated cells. Tempol and Apocynin reversed the increased expression of AT1R mRNA, c-Jun mRNA, c-fos mRNA, and superoxide production induced by Ang II. We also examined the effect of ICV Tempol on the RVLM of CHF rabbits. Compared to vehicle treated CHF rabbits, Tempol significantly decreased AT1R protein expression (1.6±0.29 versus 0.88±0.16, P<0.05), phosphorylated Jnk protein (0.4±0.05 versus 0.2±0.04, P<0.05), cytosolic phosphorylated c-Jun (0.56±0.1 versus 0.36±0.05, P<0.05), and nuclear phosphorylated c-Jun (0.67±0.1 versus 0.3±0.08, P<0.01). Tempol also significantly decreased the AP-1–DNA binding activity in the RVLM of CHF rabbits compared to the vehicle group (9.14x10³ versus 41.95x10³ gray level P<0.01). These data suggest that Ang II induces AT1R upregulation at the transcriptional level by induction of oxidant stress and activation of AP1 in both cultured neuronal cells and in intact brain of rabbits. Antioxidant agents may be beneficial in CHF and other states where brain Ang II is elevated by decreasing AT1R expression through the Jnk and AP1 pathway.

[Article] Redox-Sensitive Signaling by Angiotensin II Involves Oxidative Inactivation and Blunted Phosphorylation of Protein Tyrosine Phosphatase SHP-2 in Vascular Smooth Muscle Cells From SHR

2008-06-19

Angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) involves reactive oxygen species (ROS) through unknown mechanisms. We propose that Ang II induces phosphorylation of growth signaling kinases by redox-sensitive regulation of protein tyrosine phosphatases (PTP) in VSMCs and that augmented Ang II signaling in spontaneously hypertensive rats (SHRs) involves oxidation/inactivation and blunted phosphorylation of the PTP, SHP-2. PTP oxidation was assessed by the in-gel PTP method. SHP-2 expression and activity were evaluated by immunoblotting and by a PTP activity assay, respectively. SHP-2 and Nox1 were downregulated by siRNA. Ang II induced oxidation of multiple PTPs, including SHP-2. Basal SHP-2 content was lower in SHRs versus WKY. Ang II increased SHP-2 phosphorylation and activity with blunted responses in SHRs. Ang II—induced SHP-2 effects were inhibited by valsartan (AT₁R blocker), apocynin (NAD(P)H oxidase inhibitor), and Nox1 siRNA. Ang II stimulation increased activation of ERK1/2, p38MAPK, and AKT, with enhanced effects in SHR. SHP-2 knockdown resulted in increased AKT phosphorylation, without effect on ERK1/2 or p38MAPK. Nox1 downregulation attenuated Ang II–mediated AKT activation in SHRs. Hence, Ang II regulates PTP/SHP-2 in VSMCs through AT₁R and Nox1-based NAD(P)H oxidase via two mechanisms, oxidation and phosphorylation. In SHR Ang II–stimulated PTP oxidation/inactivation is enhanced, basal SHP-2 expression is reduced, and Ang II–induced PTP/SHP-2 phosphorylation is blunted. These SHP-2 actions are associated with augmented AKT signaling. We identify a novel redox-sensitive SHP-2–dependent pathway for Ang II in VSMCs. SHP-2 dysregulation by increased Nox1-derived ROS in SHR is associated with altered Ang II–AKT signaling.

[Article] Interleukin-10 From Transplanted Bone Marrow Mononuclear Cells Contributes to Cardiac Protection After Myocardial Infarction

2008-06-19

Bone marrow mononuclear cells (BM-MNCs) have successfully been used as a therapy for the improvement of left ventricular (LV) function after myocardial infarction (MI). It has been suggested that paracrine factors from BM-MNCs may be a key mechanism mediating cardiac protection. We previously performed microarray analysis and found that the pleiotropic cytokine interleukin-10 (IL-10) was highly upregulated in human progenitor cells in comparison with adult endothelial cells and CD14⁺ cells. Moreover, BM-MNCs secrete significant amounts of IL-10, and IL-10 could be detected from progenitor cells transplanted in infarcted mouse hearts. Specifically, intramyocardial injection of WT BM-MNCs led to a significant decrease in LV end diastolic pressure (LVEDP) and LV end systolic volume (LVESV) compared to hearts injected with either diluent or IL-10 KO BM-MNCs. Furthermore, intramyocardial injection of WT BM-MNCs led to a significant increase in stroke volume (SV) and rate of the development of pressure over time (+dP/dt) compared to hearts injected with either diluent or IL-10 KO BM-MNCs. The IL-10–dependent improvement provided by transplanted cells was not caused by reduced infarct size, neutrophil infiltration, or capillary density, but rather was associated with decreased T lymphocyte accumulation, reactive hypertrophy, and myocardial collagen deposition. These results suggest that BM-MNCs mediate cardiac protection after myocardial infarction and this is, at least in part, dependent on IL-10.

[Article] Neurotrophin p75 Receptor (p75NTR) Promotes Endothelial Cell Apoptosis and Inhibits Angiogenesis. Implications for Diabetes-Induced Impaired Neovascularization in Ischemic Limb Muscles

2008-06-19

Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75^NTR), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we show that gene transfer-induced p75^NTR expression impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells (EPCs) and inhibits angiogenesis in vitro. Moreover, intramuscular p75^NTR gene delivery impairs neovascularization and blood flow recovery in a mouse model of limb ischemia. These disturbed functions are associated with suppression of signaling mechanisms implicated in EC survival and angiogenesis. In fact, p75^NTR depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels of ITGB1 [beta (1) integrin], BIRC5 (survivin), PTTG1 (securin) and VEZF1. Diabetic mice, which typically show impaired postischemic muscular neovascularization and blood perfusion recovery, have these defects corrected by intramuscular gene transfer of a dominant negative mutant form of p75^NTR. Collectively, our data newly demonstrate the antiangiogenic action of p75^NTR and open new avenues for the therapeutic use of p75^NTR inhibition to combat diabetes-induced microvascular liabilities.

[Article] Extracellular Release of the Atheroprotective Heat Shock Protein 27 Is Mediated by Estrogen and Competitively Inhibits acLDL Binding to Scavenger Receptor-A

2008-06-19

We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ERβ)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27^o/e) mice were crossed to apoE^-/- mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (P≤0.001) in atherosclerotic lesion area in apoE^-/-HSP27^o/e mice compared to apoE^-/- mice, but primarily in females. Serum HSP27 levels were >10-fold higher in female apoE^-/-HSP27^o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r²=0.78, P≤0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)—an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A^-/- mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1β and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.

[Article] Enhanced Cellular Uptake of Remnant High-Density Lipoprotein Particles. A Mechanism for High-Density Lipoprotein Lowering in Insulin Resistance and Hypertriglyceridemia

2008-06-12

A low level of high-density lipoprotein (HDL) cholesterol is characteristic of insulin resistance and hypertriglyceridemia and likely contributes to the increased risk of cardiovascular disease associated with these conditions. One pathway involves enhanced clearance of lipolytically modified HDL particles, but the underlying mechanisms remain poorly understood. Here, we examine the effect of triglyceride enrichment and hepatic lipase hydrolysis on HDL binding, internalization, and degradation in cultured liver and kidney cells. Maximal binding of remnant HDL (HDL enriched with triglycerides followed by hepatic lipase hydrolysis), but not binding affinity, was markedly higher than native and triglyceride-rich HDL in both HepG2 cells and HEK293 cells. Compared with native and triglyceride-rich HDL, remnant HDL was internalized to a greater extent in both cell types and was more readily degraded in HEK293 cells. The increased binding of remnant HDL was not mediated by the low-density lipoprotein receptor or SR-BI (scavenger receptor class B type I), because enhanced remnant HDL binding was observed in low-density lipoprotein receptor–deficient cells with or without SR-BI overexpression. Disruption of cell surface heparan sulfate proteoglycans or blockage of apolipoprotein E–mediated lipoprotein binding also did not abolish the enhanced remnant HDL binding. Our observations indicate that remodeling of triglyceride-enriched HDL by hepatic lipase may result in enhanced binding, internalization, and degradation in tissues involved in HDL catabolism, contributing to rapid clearance and overall lowering of plasma HDL cholesterol in insulin resistance and hypertriglyceridemia.

[Article] Engineering Robust and Functional Vascular Networks In Vivo With Human Adult and Cord Blood-Derived Progenitor Cells

2008-06-12

The success of therapeutic vascularization and tissue engineering will rely on our ability to create vascular networks using human cells that can be obtained readily, can be expanded safely ex vivo, and can produce robust vasculogenic activity in vivo. Here we describe the formation of functional microvascular beds in immunodeficient mice by coimplantation of human endothelial and mesenchymal progenitor cells isolated from blood and bone marrow. Evaluation of implants after 1 week revealed an extensive network of human blood vessels containing erythrocytes, indicating the rapid formation of functional anastomoses within the host vasculature. The implanted endothelial progenitor cells were restricted to the luminal aspect of the vessels; mesenchymal progenitor cells were adjacent to lumens, confirming their role as perivascular cells. Importantly, the engineered vascular networks remained patent at 4 weeks in vivo. This rapid formation of long-lasting microvascular networks by postnatal progenitor cells obtained from noninvasive sources constitutes an important step forward in the development of clinical strategies for tissue vascularization.

[Article] Forced Alignment of Mesenchymal Stem Cells Undergoing Cardiomyogenic Differentiation Affects Functional Integration With Cardiomyocyte Cultures

2008-06-12

Alignment of cardiomyocytes (CMCs) contributes to the anisotropic (direction-related) tissue structure of the heart, thereby facilitating efficient electric and mechanical activation of the ventricles. This study aimed to investigate the effects of forced alignment of stem cells during cardiomyogenic differentiation on their functional integration with CMC cultures. Labeled neonatal rat (nr) mesenchymal stem cells (nrMSCs) were allowed to differentiate into functional heart muscle cells in different cell-alignment patterns during 10 days of coculture with nrCMCs. Development of functional cellular properties was assessed by measuring impulse transmission across these stem cells between 2 adjacent nrCMC fields, cultured onto microelectrode arrays and previously separated by a laser-dissected channel (230±10 µm) for nrMSC transplantation. Coatings in these channels were microabraded in a direction (1) parallel or (2) perpendicular to the channel or were (3) left unabraded to establish different cell patterns. Application of cells onto microabraded coatings resulted in anisotropic cell alignment within the channel. Application on unabraded coatings resulted in isotropic (random) alignment. On coculture, conduction across seeded nrMSCs occurred from day 1 (perpendicular and isotropic) or day 6 (parallel) onward. Conduction velocity across nrMSCs at day 10 was highest in the perpendicular (11±0.9 cm/sec; n=12), intermediate in the isotropic (7.1±1 cm/sec; n=11) and lowest in the parallel configuration (4.9±1 cm/sec; n=11) (P<0.01). nrCMCs and fibroblasts served as positive and negative control, respectively. Also, immunocytochemical analysis showed alignment-dependent increases in connexin 43 expression. In conclusion, forced alignment of nrMSCs undergoing cardiomyogenic differentiation affects the time course and degree of functional integration with surrounding cardiac tissue.

[Article] Heparan Sulfate in Perlecan Promotes Mouse Atherosclerosis. Roles in Lipid Permeability, Lipid Retention, and Smooth Muscle Cell Proliferation

2008-06-05

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2^3/3). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2^3/3 mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2^3/3 smooth muscle cells was reduced. In vivo, at 20 minutes influx of human ¹²⁵I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2^3/3 mice compared to ApoE0 mice. However, at 72 hours accumulation of ¹²⁵I-LDL was similar in ApoE0/Hspg2^3/3 and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2^3/3 mice showed decreased staining for apoB and increased smooth muscle -actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

[Article] Late-Breaking Basic Science Abstracts From the American Heart Association Scientific Sessions 2005. November 13-16, 2005, Dallas Convention Center Dallas, TX

2005-11-09