Objective: To determine the role of APN in rosiglitazone (RSG) cardioprotection against ischemic heart injury.

Methods and Results: Adult male wild-type (WT) and APN knockdown/knockout (APN^+/- and APN^-/-) mice were treated with vehicle or RSG (20 mg/kg per day), and subjected to coronary artery ligation 3 days after beginning treatment. In WT mice, RSG (7 days) significantly increased adipocyte APN expression, elevated plasma APN levels (2.6-fold), reduced infarct size (17% reduction), decreased apoptosis (0.23±0.02% versus 0.47±0.04% TUNEL-positive in remote nonischemic area), attenuated oxidative stress (48.5% reduction), and improved cardiac function (P<0.01). RSG-induced APN production and cardioprotection were significantly blunted (P<0.05 versus WT) in APN^+/-, and completely lost in APN^-/- (P>0.05 versus vehicle-treated APN^-/- mice). Moreover, treatment with RSG for up to 14 days significantly improved the postischemic survival rate of WT mice (P<0.05 versus vehicle group) but not APN knockdown/knockout mice.

Conclusions: The cardioprotective effects of PPAR- agonists are critically dependent on its APN stimulatory action, suggesting that under pathological conditions where APN expression is impaired (such as advanced type 2 diabetes), the harmful cardiovascular effects of PPAR- agonists may outweigh its cardioprotective benefits.

Objective: The objective of this study was to elucidate the role of Sema3E/plexinD1 in postnatal angiogenesis.

Methods and Results: Sema3E inhibited cell growth and tube formation by suppressing the vascular endothelial growth factor (VEGF) signaling pathway. Expression of Sema3E and plexinD1 was markedly upregulated in ischemic limbs of mice (2.5- and 4.5-fold increase for Sema3E and plexinD1, respectively), and inhibition of this pathway by introduction of the plexinD1-Fc gene or disruption of Sema3E led to a significant increase of blood flow recovery (1.6- and 1.5-fold increase for the plexinD1-Fc gene treatment and Sema3E disruption, respectively). Hypoxia activated the tumor suppressor protein p53, thereby upregulating Sema3E expression. Expression of p53 and Sema3E was enhanced in diabetic mice compared with normal mice (2- and 1.3-fold increase for p53 and Sema3E, respectively). Consequently, neovascularization after VEGF treatment was poor in the ischemic tissues of diabetic mice, whereas treatment with VEGF plus plexinD1-Fc markedly improved neovascularization.

Conclusions: These results indicate that inhibition of Sema3E may be a novel strategy for therapeutic angiogenesis, especially when VEGF is ineffective.

Objective: To elucidate the role of COX-2 in blood pressure homeostasis using COX-1>COX-2 mice, in which the COX-1 expression is controlled by COX-2 regulatory elements.

Methods and Results: COX-1>COX-2 mice developed systolic hypertension relative to wild types (WTs) on a high-salt diet (HSD); this was attenuated by a PGI₂ receptor agonist. HSD increased expression of COX-2 in WT mice and of COX-1 in COX-1>COX-2 mice in the inner renal medulla. The HSD augmented in all strains urinary prostanoid metabolite excretion, with the exception of the major PGI₂ metabolite that was suppressed on regular chow and unaltered by the HSD in both mutants. Furthermore, renal inner renal medulla expression of the receptor for PGI₂, but not for other prostanoids, was depressed by HSD in WT and even more so in both mutant strains. Increasing osmolarity augmented expression of COX-2 in WT renal medullary interstitial cells and again the increase in formation of PGI₂ observed in WTs was suppressed in cells derived from both mutants. Intramedullary infusion of the PGI₂ receptor agonist increased urine volume and sodium excretion in mice.

Conclusions: These studies suggest that dysregulated expression of the COX-2 dependent, PGI₂ biosynthesis/response pathway in the renal inner renal medulla undermines the homeostatic response to a HSD. Inhibition of this pathway may contribute directly to the hypertensive response to NSAIDs.

Objective: Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart.

Methods and Results: In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from simulated ischemia–induced cell death, whereas dominant-negative Derl3 decreased ERAD and increased simulated ischemia–induced cardiomyocyte death.

Conclusions: This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.

Objective: We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro.

Methods and Results: We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance–associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1.

Conclusions: These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

Objective: We tested the hypothesis that postshock arrhythmias occur because of spontaneous sarcoplasmic reticulum Ca release, delayed afterdepolarization (DAD), and triggered activity (TA) from tissues with high sensitivity of resting membrane voltage (V_m) to elevated intracellular calcium (Ca_i) (high diastolic Ca_i–voltage coupling gains).

Methods and Results: We simultaneously mapped Ca_i and V_m on epicardial (n=14) or endocardial (n=14) surfaces of Langendorff-perfused rabbit ventricles. Spontaneous Ca_i elevation (SCaE) was noted after defibrillation in 32% of ventricular tachycardia/ventricular fibrillation at baseline and in 81% during isoproterenol infusion (0.01 to 1 µmol/L). SCaE was reproducibly induced by rapid ventricular pacing and inhibited by 3 µmol/L of ryanodine. The SCaE amplitude and slope increased with increasing pacing rate, duration, and dose of isoproterenol. We found TAs originating from 6 of 14 endocardial surfaces but none from epicardial surfaces, despite similar amplitudes and slopes of SCaEs between epicardial and endocardial surfaces. This was because DADs were larger on endocardial surfaces as a result of higher diastolic Ca_i–voltage coupling gain, compared to those of epicardial surfaces. Purkinje-like potentials preceded TAs in all hearts studied (n=7). I_K1 suppression with CsCl (5 mmol/L, n=3), BaCl₂ (3 µmol/L, n=3), and low extracellular potassium (1 mmol/L, n=2) enhanced diastolic Ca_i–voltage coupling gain and enabled epicardium to also generate TAs.

Conclusions: Higher diastolic Ca_i–voltage coupling gain is essential for genesis of TAs and may underlie postshock arrhythmias arising from Purkinje fibers. I_K1 is a major factor that determines the diastolic Ca_i–voltage coupling gain.

Objective: We sought to define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP.

Methods and Results: Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH₄ levels, and NO production in human endothelial cells. Laminar, but not oscillatory shear stress, caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH₄ levels in vivo.

Conclusions: These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH₄ to be produced in response to laminar shear stress.

Objective: To clarify the role of central ACE2 using a new transgenic mouse model with human (h)ACE2 under the control of a synapsin promoter, allowing neuron-targeted expression in the central nervous system.

Methods and Results: Syn-hACE2 (SA) transgenic mice exhibit high hACE2 protein expression and activity throughout the brain. Baseline hemodynamic parameters (telemetry), autonomic function, and spontaneous baroreflex sensitivity (SBRS) were not significantly different between SA mice and nontransgenic littermates. Brain-targeted ACE2 overexpression attenuated the development of neurogenic hypertension (Ang II infusion: 600 ng/kg per minute for 14 days) and the associated reduction of both SBRS and parasympathetic tone. This prevention of hypertension by ACE2 overexpression was reversed by blockade of the Ang-(1-7) receptor (D-Ala7-Ang-[1-7]; 600 ng/kg per minute). Brain angiotensin II type 2 (AT₂)/AT₁ and Mas/AT₁ receptor ratios were significantly increased in SA mice. They remained higher following Ang II infusion but were dramatically reduced after Ang-(1-7) receptor blockade. ACE2 overexpression resulted in increased NOS and NO levels in the brain, and prevented the Ang II–mediated decrease in NOS expression in regions modulating blood pressure regulation.

Conclusions: ACE2 overexpression attenuates the development of neurogenic hypertension partially by preventing the decrease in both SBRS and parasympathetic tone. These protective effects might be mediated by enhanced NO release in the brain resulting from Mas and AT₂ receptor upregulation. Taken together, our data highlight the compensatory role of central ACE2 and its potential benefits as a therapeutic target for neurogenic hypertension.

Objective: To determine whether hyperlipidemia affects apoptotic cell clearance, and identify the molecular pathways downstream of VSMC apoptosis that may promote inflammation.

Methods and Results: We find that human VSMCs are potent and efficient phagocytes of apoptotic human VSMCs, but phagocytosis is significantly reduced by oxidized low-density lipoprotein in vitro or hyperlipidemia in vivo. Necrotic human aortic VSMCs release IL-1, which induces IL-6 and MCP-1 production from viable human VSMCs in vitro. In contrast, secondary necrotic VSMCs release both IL-1 and caspase-activated IL-1β, augmenting IL-6 and MCP-1 production. Conditionally inducing VSMC apoptosis in situ in hyperlipidemic SM22-hDTR/ApoE^-/- mice to levels seen in human plaques increases serum MCP-1, tumor necrosis factor , and IL-6, which is prevented by blocking IL-1.

Conclusions: We conclude that VSMC necrosis releases IL-1, whereas secondary necrosis of apoptotic VSMCs releases both IL-1 and β. IL-1 from necrotic VSMCs induces the surrounding viable VSMCs to produce proinflammatory cytokines. Thus, failed clearance of apoptotic VSMCs caused by hyperlipidemia in vivo may promote the increased serum cytokines and chronic inflammation associated with atherosclerosis.

Objective: This study aimed to investigate possible role of Rad in cardiac EC coupling and β-adrenergic receptor (βAR) inotropic mechanism.

Methods and Results: Adenoviral overexpression of Rad by 3-fold in rat cardiomyocytes suppressed LCC current (I_Ca), [Ca²⁺]_i transients, and contractility by 60%, 42%, and 38%, respectively, whereas the "gain" function of EC coupling was significantly increased, due perhaps to reduced "redundancy" of LCC in triggering sarcoplasmic reticulum release. Conversely, 70% Rad knockdown by RNA interference increased I_Ca (50%), [Ca²⁺]_i transients (52%) and contractility (58%) without altering EC coupling efficiency; and the dominant negative mutant RadS105N exerted a similar effect on I_Ca. Rad upregulation caused depolarizing shift of LCC activation and hastened time-dependent LCC inactivation; Rad downregulation, however, failed to alter these attributes. The Na⁺/Ca²⁺ exchange activity, sarcoplasmic reticulum Ca²⁺ content, properties of Ca²⁺ sparks and propensity for Ca²⁺ waves all remained unperturbed regardless of Rad manipulation. Rad overexpression, but not knockdown, negated βAR effects on I_Ca and Ca²⁺ transients.

Conclusion: These results establish Rad as a novel endogenous regulator of cardiac EC coupling and βAR signaling and support a parsimonious model in which Rad buffers Ca_vβ to modulate LCC activity, EC coupling, and βAR responsiveness.

Objective: We asked whether the presence of LAP2 was required for normal cardiac function.

Methods and Results: To study the molecular mechanisms of the disease, we analyzed heart structure and function in complete and conditional Lap2^-/- mice as well as Lap2^-/-/Mdx mutants. Unlike conditional deletion of LAP2 in late embryonic striated muscle, its complete knockout caused systolic dysfunction in young mice, accompanied by sporadic fibrosis in old animals, as well as deregulation of major cardiac transcription factors GATA4 and myocyte enhancer factor 2c. Activation of compensatory pathways, including downregulation of β-adrenergic receptor signaling, resulted in reduced responsiveness of the myocardium to chronic β-adrenergic stimulation and stalled the progression of LAP2-deficient hearts from hypertrophy toward cardiac failure. Dystrophin deficiency in an Mdx background resulted in a transient rescue of the Lap2^-/- phenotype.

Conclusions: Our data suggest a novel role of LAP2 in the maintenance of cardiac function under normal and stress conditions.

Objective: In this study, we confirm that perivascular mast cell content correlates with the number of nerve fibers in the adventitia of human coronary atherosclerotic plaque specimen. Because peripheral C-type nerve fibers secrete, among others, substance P, a potent mast cell activator, we set out to study effects of adventitial administration of this neuropeptide on mast cell dependent destabilization of carotid artery plaques in apolipoprotein E–deficient (apoE^-/-) mice.

Methods and Results: Substance P treatment significantly enhanced the number and activation status of adventitial mast cells compared to controls and promoted intraplaque hemorrhages. These phenomena could be prevented by coadministration of the neurokinin-1 receptor antagonist spantide I and did not occur in mast cell deficient apoE^-/- mice, establishing the critical involvement of mast cells in substance P–elicited plaque destabilization.

Conclusions: Our data suggest that neurotransmitters such as substance P are capable of promoting mast cell dependent plaque destabilization and provide a new, direct link between neural factors and vascular inflammation.

Objective: We aimed to characterize the cellular mechanism by which GPCR crosstalk regulates mitogen-activated protein kinase (MAPK) activation.

Methods and Results: Adrenergic receptors on cardiac fibroblasts were manipulated to examine the role of arrestin in the spatiotemporal regulation of extracellular signal-regulated kinase (ERK)1/2 MAPK signaling. We show a general mechanism in which arrestin activation by one GPCR is capable of regulating signaling originating from another GPCR. Activation of Gq coupled–receptor signaling leads to prolonged ERK1/2 MAPK phosphorylation, nuclear accumulation, and cellular proliferation. Interestingly, coactivation of these receptors with the β-adrenergic receptors induced transient ERK signaling localized within the cytosol, which attenuated cell proliferation. Further studies revealed that recruitment of arrestin3 to the β₂-adrenergic receptor orchestrates the sequestration of Gq-coupled receptor–induced ERK to the cytosol through direct binding of ERK to arrestin.

Conclusions: This is the first evidence showing that arrestin3 acts as a coordinator to integrate signals from multiple GPCRs. Our studies not only provide a novel mechanism explaining the integration of mitogenic signaling elicited by different GPCRs, but also underscore the critical role of signaling crosstalk among GPCRs in vivo.

Objective: Here, we determined the potential role of CaMKII-B in regulating cardiomyocyte viability and explored the underlying mechanism.

Methods and Results: In cultured neonatal rat cardiomyocytes, the expression of CaMKII-B and CaMKII-C was inversely regulated in response to H₂O₂-induced oxidative stress with a profound reduction of the former and an increase of the later. Similarly, in vivo ischemia/reperfusion (IR) led to an opposite regulation of these CaMKII isoforms in a rat myocardial IR model. Notably, overexpression of CaMKII-B protected cardiomyocytes against oxidative stress-, hypoxia-, and angiotensin II–induced apoptosis, whereas overexpression of its cytosolic counterpart promoted apoptosis. Using cDNA microarray, real time-PCR and Western blotting, we demonstrated that overexpression of CaMKII-B but not CaMKII-C elevated expression of heat shock protein (HSP)70 family members, including inducible (i)HSP70 and its homolog (Hst70). Moreover, overexpression of CaMKII-B led to phosphorylation and activation of heat shock factor (HSF)1, the primary transcription factor responsible for HSP70 gene regulation. Importantly, gene silencing of iHSP70, but not Hst70, abolished CaMKII-B-mediated protective effect, indicating that only iHSP70 was required for CaMKII-B elicited antiapoptotic signaling.

Conclusions: We conclude that cardiac CaMKII-B and CaMKII-C were inversely regulated in response to oxidative stress and IR injury, and that in contrast to CaMKII-C, CaMKII-B serves as a potent suppressor of cardiomyocyte apoptosis triggered by multiple death-inducing stimuli via phosphorylation of HSF1 and subsequent induction of iHSP70, marking both CaMKII- isoforms as promising therapeutic targets for the treatment of ischemic heart disease.

Objective: Here, we show that cellular prion protein (PrP) serves as an effective surface marker for isolating nascent cardiomyocytes as well as cardiomyogenic progenitors.

Methods and Results: Embryonic stem (or embryo-derived) cells were analyzed using flow cytometry to detect surface expression of PrP and intracellular myosin heavy chain (Myhc) proteins. Sorted cells were then analyzed for their differentiation potential.

Conclusions: PrP⁺ cells from beating embryoid bodies (EBs) frequently included nascent Myhc⁺ cardiomyocytes. Cultured PrP⁺ cells further differentiated, giving rise to cardiac troponin I⁺ definitive cardiomyocytes with either an atrial or a ventricular identity. These cells were electrophysiologically functional and able to survive in vivo after transplantation. Combining PrP with a second marker, platelet-derived growth factor receptor (PDGFR), enabled us to identify an earlier cardiomyogenic population from prebeating EBs, the PrP⁺PDGFR⁺ (PRa) cells. The Myhc^- PRa cells expressed cardiac transcription factors, such as Nkx2.5, T-box transcription factor 5, and Isl1 (islet LIM homeobox 1), although they were not completely committed. In mouse embryos, PRa cells in cardiac crescent at the 1 to 2 somite stage were Myhc⁺, whereas they were Myhc^- at headfold stages. PRa cells clonally expanded in methlycellulose cultures. Furthermore, single Myhc^- PRa cell–derived colonies contained both cardiac and smooth muscle cells. Thus, PrP demarcates a population of bipotential cardiomyogenic progenitor cells that can differentiate into cardiac or smooth muscle cells.

Objective: Here, we examined the hypothesis that phosphorylation of Jak2 upstream of PI3K is impaired in diabetic hearts by an angiotensin II type 1 (AT₁) receptor–mediated mechanism.

Methods and Results: Infarct size (as percentage of risk area) after 20-minute ischemia/2-hour reperfusion was larger in a rat model of type 2 diabetes (Otsuka–Long–Evans–Tokushima fatty [OLETF] rat) than in its control (Long–Evans–Tokushima–Otsuka [LETO] rat) (60.4±1.6% versus 48.4±1.3%). Activation of Jak2-mediated signaling by erythropoietin or DADLE ([D-Ala2, D-Leu5]-enkephalin acetate), a -opioid receptor agonist, limited infarct size in LETO rats (27.7±3.4% and 24.8±5.0%) but not in OLETF rats (53.9±5.3% and 55.0±2.2%). Blockade of the AT₁ receptor by valsartan or losartan for 2 weeks restored the myocardial response of OLETF rats to erythropoietin-induced infarct size limitation (39.4±4.9% and 31.2±7.5). In OLETF rats, erythropoietin failed to phosphorylate both Jak2 and Akt, and calcineurin activity was significantly higher than in LETO rats. Two-week treatment with valsartan normalized calcineurin activity in OLETF rats and restored the response of Jak2 to erythropoietin. This effect of AT₁ receptor blockade was mimicked by inhibition of calcineurin by FK506.

Conclusions: These results suggest that the diabetic heart is refractory to protection by Jak2-activating ligands because of AT₁ receptor–mediated upregulation of calcineurin activity.

Objective: We sought to study effects of CXCL4 on macrophage differentiation with possible relevance for atherogenesis.

Methods and Results: Flow cytometry for expression of surface markers in macrophage colony–stimulating factor (M-CSF)– and CXCL4-induced macrophages demonstrated virtually complete absence of the hemoglobin scavenger receptor CD163 in CXCL4-induced macrophages. mRNA for CD163 was downregulated as early as 2 hours after CXCL4. CD163 protein reached a minimum after 3 days, which was not reversed by treatment of cells with M-CSF. The CXCL4 effect was entirely neutralized by heparin, which bound CXCL4 and prevented CXCL4 surface binding to monocytes. Pretreatment of cells with chlorate, which inhibits glycosaminoglycan synthesis, strongly inhibited CXCL4-dependent downregulation of CD163. Similar to recombinant CXCL4, releasate from human platelets also reduced CD163 expression. CXCL4-differentiated macrophages were unable to upregulate the atheroprotective enzyme heme oxygenase-1 at the RNA and protein level in response to hemoglobin–haptoglobin complexes. Immunofluorescence of human atherosclerotic plaques demonstrated presence of both CD68⁺CD163⁺ and CD68⁺CD163^- macrophages. PF4 and CD163 gene expression within human atherosclerotic lesions were inversely correlated, supporting the in vivo relevance of CXCL4-induced downregulation of CD163.

Conclusions: CXCL4 may promote atherogenesis by suppressing CD163 in macrophages, which are then unable to upregulate the atheroprotective enzyme heme oxygenase-1 in response to hemoglobin.

Objective: Here, we investigated the role of MAO-A in maladaptive hypertrophy and heart failure.

Methods and Results: We report that MAO-A activity is triggered in isolated neonatal and adult myocytes on stimulation with norepinephrine, followed by increase in cell size, reactive oxygen species production, and signs of maladaptive hypertrophy. All of these in vitro changes occur, in part, independently from - and β-adrenergic receptor–operated signaling and are inhibited by the specific MAO-A inhibitor clorgyline. In mice with left ventricular dilation and pump failure attributable to pressure overload, norepinephrine catabolism by MAO-A is increased accompanied by exacerbated oxidative stress. MAO-A inhibition prevents these changes, and also reverses fetal gene reprogramming, metalloproteinase and caspase-3 activation, as well as myocardial apoptosis. The specific role of MAO-A was further tested in mice expressing a dominant-negative MAO-A (MAO-A^neo), which were more protected against pressure overload than their wild-type littermates.

Conclusions: In addition to adrenergic receptor–dependent mechanisms, enhanced MAO-A activity coupled with increased intramyocardial norepinephrine availability results in increased reactive oxygen species generation, contributing to maladaptive remodeling and left ventricular dysfunction in hearts subjected to chronic stress.

Objective: This study was undertaken to investigate the expression and function of S100B and its receptor, the receptor for advanced glycation end products (RAGE) in both postinfarction myocardium and in a rat neonatal myocyte culture model.

Methods and Results: In a rat model of myocardial infarction following coronary artery ligation, we demonstrate in periinfarct myocytes, upregulation of RAGE, induction of S100B, and release into plasma with consequent myocyte apoptosis. Using a coimmunoprecipitation strategy, we demonstrate a direct interaction between S100B and RAGE. In rat neonatal cardiac myocyte cultures, S100B at concentrations ≥50 nmol/L induced myocyte apoptosis, as evidenced by increased terminal DNA fragmentation, TUNEL, cytochrome c release from mitochondria to cytoplasm, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p53, increased expression and activity of proapoptotic caspase-3, and decreased expression of antiapoptotic bcl-2. Transfection of a full-length cDNA of RAGE or a dominant-negative mutant of RAGE resulted in increased or attenuated S100B-induced myocyte apoptosis, respectively. Inhibition of ERK1/2 by U0126/PD-98059 or overexpression of a dominant negative p53 comparably inhibited S100B-induced myocyte apoptosis.

Conclusions: These results suggest that interaction of RAGE and its ligand S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53 signaling. This receptor-mediated mechanism is uniquely amenable to therapeutic intervention.

Objective: This study used a novel multiscale electromechanical model of the rabbit ventricles to investigate the origin of and the substrate for spontaneous arrhythmias arising from ischemia-induced electrophysiological and mechanical changes.

Methods and Results: Two stages of ischemia were simulated. Dynamic mechanoelectrical feedback was modeled as spatially and temporally nonuniform membrane currents through mechanosensitive channels, the conductances of which depended on local strain rate. Our results reveal that both strains and strain rates were significantly larger in the central ischemic zone than in the border zone. However, in both ischemia stages, a VPB originated from the ischemic border in the left ventricular apical endocardium because of mechanically induced suprathreshold depolarizations. It then traveled fully intramurally until emerging from the ischemic border on the anterior epicardium. Reentry was formed only in the advanced ischemia stage as the result of a widened temporal excitable gap. Mechanically induced delayed afterdepolarization-like events contributed to the formation of reentry by further decreasing the already reduced-by-hyperkalemia local excitability, causing extended conduction block lines and slowed conduction in the ischemic region.

Conclusions: Mechanically induced membrane depolarizations in the ischemic region are the mechanism by which mechanical activity contributes to both the origin of and substrate for spontaneous arrhythmias under the conditions of acute regional ischemia.

Objective: Our goal was to determine the role of resident intimal DCs in the initiation of atherosclerosis.

Methods and Results: En face immunostaining of nascent atherosclerotic lesions in low-density lipoprotein receptor–deficient (Ldlr^-/-) mice fed a cholesterol-rich diet for 5 or 10 days demonstrated that foam cells expressed DC markers CD11c, 33D1, and major histocompatibility complex class II. Transmission electron microscopy revealed that the majority of intimal lipid was intracellular. The role of resident intimal DCs in lesion formation was verified by their conditional depletion using transgenic mice expressing the simian diphtheria toxin receptor in CD11c⁺ cells. A single injection of diphtheria toxin depleted intimal CD11c⁺ DCs by >98% within 24 hours, with 25% and 75% recovery at 1 and 3 weeks, respectively. When bred onto the Ldlr^-/- background, intimal DC depletion with diphtheria toxin during 5 days of lesion formation reduced the intimal lipid area by 55% relative to undepleted controls. Transmission electron microscopy revealed few foam cells in DC-depleted mice and abundant accumulation of subendothelial extracellular lipid.

Conclusions: Induction of hypercholesterolemia in mice triggers rapid ingestion of lipid by resident intimal DCs, which initiate nascent foam cell lesion formation.

Objective: We aimed to examine the effect of class II HDACs on post–myocardial infarction remodeling in male and female mice.

Methods and Results: Here we show that the absence of HDAC5 or -9 in female mice protects against maladaptive remodeling following myocardial infarction, which coincides with upregulation of estrogen-responsive genes in the heart. This genetic reprogramming coincides with a pronounced increase in expression of the estrogen receptor (ER) gene, which we show to be a direct MEF2 target gene. ER also directly interacts with class II HDACs. Cardioprotection resulting from the absence of HDAC5 or -9 in female mice can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of the ER target gene vascular endothelial growth factor-a.

Conclusions: Our results reveal a novel gender-specific pathway of cardioprotection mediated by ER and its regulation by MEF2 and class II HDACs.

Objective: We tested the hypothesis that the structural heterogeneity of the ventricular wall modulates globally applied stretches to create heterogeneous strain distributions that lead to the initiation of arrhythmias.

Methods and Results: We applied global stretches to arterially perfused rabbit right ventricular tissue preparations. The distribution of strain (determined by marker tracking) and the transmembrane potential (measured by optical mapping) were simultaneously recorded while accounting for motion artifacts. The 3D structure of the preparations was also examined using a laser displacement meter. To examine whether such observations can be translated to the physiological condition, we performed similar measurements in whole heart preparations while applying volume pulses to the right ventricle. At the tissue level, larger stretches (≥20%) caused synchronous excitation of the entire preparation, whereas medium stretches (10% and 15%) induced focal excitation. We found a significant correlation between the local strain and the local thickness, and the probability for focal excitation was highest for medium stretches. In the whole heart preparations, we observed that such focal excitations developed into reentrant arrhythmias.

Conclusions: Global stretches of intermediate strength, rather than intense stretches, created heterogeneous strain (excitation) distributions in the ventricular wall, which can trigger fatal arrhythmias.

Objective: To prevent the "normal" downregulation of miR-133a induced by an acute hypertrophic stimulus in the adult heart.

Methods and Results: miR-133a is downregulated in transverse aortic constriction (TAC) and isoproterenol-induced hypertrophy, but not in 2 genetic hypertrophy models. Using MYH6 promoter-directed expression of a miR-133a genomic precursor, increased cardiomyocyte miR-133a had no effect on postnatal cardiac development assessed by measures of structure, function, and mRNA profile. However, increased miR-133a levels increased QT intervals in surface electrocardiographic recordings and action potential durations in isolated ventricular myocytes, with a decrease in the fast component of the transient outward K⁺ current, I_to,f, at baseline. Transgenic expression of miR-133a prevented TAC-associated miR-133a downregulation and improved myocardial fibrosis and diastolic function without affecting the extent of hypertrophy. I_to,f downregulation normally observed post-TAC was prevented in miR-133a transgenic mice, although action potential duration and QT intervals did not reflect this benefit. miR-133a transgenic hearts had no significant alterations of basal or post-TAC mRNA expression profiles, although decreased mRNA and protein levels were observed for the I_to,f auxiliary KChIP2 subunit, which is not a predicted target.

Conclusions: These results reveal striking differences between in vitro and in vivo phenotypes of miR expression, and further suggest that mRNA signatures do not reliably predict either direct miR targets or major miR effects.

Objective: We hypothesized that extracellular HSP60 (exHSP60) would mediate apoptosis via TLR-4.

Methods and Results: Adult rat cardiac myocytes were treated with HSP60, either recombinant human or with HSP60 purified from the media of injured rat cardiac myocytes. ExHSP60 induced apoptosis in cardiac myocytes, as detected by increased caspase 3 activity and increased DNA fragmentation. Apoptosis could be reduced by blocking antibodies to TLR-4 and by nuclear factor B binding decoys, but not completely inhibited, even though similar treatment blocked lipopolysaccharide-induced apoptosis. Three distinct controls showed no evidence for involvement of a ligand other than exHSP60 in the mediation of apoptosis.

Conclusions: This is the first report of HSP60-induced apoptosis via the TLRs. HSP60-mediated activation of TLR-4 may be a mechanism of myocyte loss in heart failure, where HSP60 has been detected in the plasma.

Objective: To test the role of S100A12 in vascular inflammation, we generated and analyzed mice expressing human S100A12 in vascular smooth muscle under control of the smooth muscle 22 promoter because S100A12 is not present in mice.

Methods and Results: Transgenic mice displayed pathological vascular remodeling with aberrant thickening of the aortic media, disarray of elastic fibers, and increased collagen deposition, together with increased latent matrix metalloproteinase-2 protein and reduction in smooth muscle stress fibers leading to a progressive dilatation of the aorta. In primary aortic smooth muscle cell cultures, we found that S100A12 mediates increased interleukin-6 production, activation of transforming growth factor β pathways and increased metabolic activity with enhanced oxidative stress. To correlate our findings to human aortic aneurysmal disease, we examined S100A12 expression in aortic tissue from patients with thoracic aortic aneurysm and found increased S100A12 expression in vascular smooth muscle cells.

Conclusions: S100A12 expression is sufficient to activate pathogenic pathways through the modulation of oxidative stress, inflammation and vascular remodeling in vivo.

Objective: To characterize the role of Inpp5f in regulating cardiac hypertrophy.

Methods and Results: We generated homozygous Inpp5f knockout mice and cardiac specific Inpp5f overexpression transgenic mice. We evaluated their hearts for biochemical, structural and functional changes. Inpp5f knockout mice have augmented hypertrophy and reactivation of the fetal gene program in response to stress when compared to wild-type littermates. Furthermore, cardiac overexpression of Inpp5f in transgenic mice reduces hypertrophic responsiveness.

Conclusions: Our results suggest that Inpp5f is a functionally important endogenous modulator of cardiac myocyte size and of the cardiac response to stress.

Objective: To explore the mechanism underlying the roles of 14-3-33 and filamin A in regulating the VWF binding function of GPIb-IX.

Methods and Results: A truncation mutant of GPIb (565) deleting the C-terminal 14-3-33 binding sites retained 14-3-33 binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIb totally abolished 14-3-33 binding, indicating that the residues 551 to 564 of GPIb are important in the interaction between 14-3-33 and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIb suggesting phosphorylation of a population of GPIb molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIb eliminated the association of 14-3-33 with 565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIb with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions.

Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-33 to central filamin A binding site of GPIb, and the dimeric 14-3-33 binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

Objective: We investigated the net effects of acute hypoxia and catecholamines on the cardiac action potential.

Methods and Results: We incorporated all published data on the effects of hypoxia on the late Na⁺ current (I_Na-L), the fast Na⁺ current (I_Na), the basal L-type Ca²⁺ channel current (I_Ca-L), and the slow (I_Ks) and rapid components of the delayed rectifier K⁺-current (I_Kr) in the absence and presence of β-adrenergic receptor (β-AR) stimulation into the Luo–Rudy model of the action potential. Hypoxia alone had little effect on the action potential configuration or action potential duration. However in the presence of β-AR stimulation, hypoxia caused a prolongation of the action potential and early afterdepolarizations (EADs) and spontaneous tachycardia were induced. Experiments performed in guinea pig ventricular myocytes confirmed the modeling results.

Conclusions: EADs occur predominantly because of the increased sensitivity of I_Ca-L to β-AR stimulation during hypoxia. β-AR stimulation is necessary to induce EADs as EADs are never observed during hypoxia in the absence of β-AR stimulation.

Objective: To analyze connexin changes and their potential contribution to the atrial fibrillation (AF) substrate during the development and reversal of CHF.

Methods and Results: Three groups of dogs were studied: CHF induced by 2-week ventricular tachypacing (240 bpm, n=15); CHF dogs allowed a 4-week nonpaced recovery interval after 2-week tachypacing (n=16); and nonpaced sham controls (n=19). Left ventricular (LV) end-diastolic pressure and atrial refractory periods increased with CHF and normalized on CHF recovery. CHF caused abnormalities in atrial conduction indices and increased the duration of burst pacing-induced AF (DAF, from 22±7 seconds in control to 1100±171 seconds, P<0.001). CHF did not significantly alter overall atrial connexin (Cx)40 and Cx43 mRNA and protein expression levels, but produced Cx43 dephosphorylation, increased Cx40/Cx43 protein expression ratio and caused Cx43 redistribution toward transverse cell-boundaries. All of the connexin-alterations reversed on CHF recovery, but CHF-induced conduction abnormalities and increased DAF (884±220 seconds, P<0.001 versus control) remained. The atrial fibrous tissue content increased from 3.6±0.7% in control to 14.7±1.5% and 13.3±2.3% in CHF and CHF recovery, respectively (both P<0.01 versus control), with transversely running zones of fibrosis physically separating longitudinally directed muscle bundles. In an ionically based action potential/tissue model, fibrosis was able to account for conduction abnormalities associated with CHF and recovery.

Conclusions: CHF causes atrial connexin changes, but these are not essential for CHF-related conduction disturbances and AF promotion, which are rather related primarily to fibrotic interruption of muscle bundle continuity.

Objective: Our objective in the present study was to characterize the signal transduction mechanism of phosphatidylinositol 3-kinase (PI3K) involvement in Ang II–induced stimulation of central neuronal activity in cultured neurons and Ang II–induced inhibition of baroreflex in spontaneously hypertensive rats (SHR) versus WKY rats.

Methods and Results: Application of Ang II to neurons produced a 42% greater increase in neuronal firing in cells from the SHR than the WKY rat. Although the Ang II–mediated increase in firing rate was abolished entirely by the protein kinase (PK)C inhibitor GF109230 in the WKY, blockade of both PKC and PI3K activity was necessary in the SHR. This was associated with an increased ability of Ang II to stimulate NADPH oxidase–reactive oxygen species (ROS)–mediated signaling involving phosphorylation of the p47phox subunit of the NADPH oxidase and was dependent on the activation of PI3K in the SHR. Inhibition of PI3K resulted in the reduction of levels of p47phox phosphorylation, NADPH oxidase activity, ROS levels, and ultimately neuronal activity in cells from the SHR but not the WKY rat. In addition, in working heart–brainstem preparations, inhibition of PKC activity in the nucleus of the solitary tract in situ abolished the Ang II–mediated depression of cardiac and sympathetic baroreceptor reflex gain in the WKY. In contrast, PKC inhibition in the nucleus of the solitary tract of SHR only partially reduced the effect of Ang II on the baroreceptor reflex gain.

Conclusions: These observations demonstrate that PI3K in the cardiovascular brainstem regions of the SHR may be selectively involved in Ang II–mediated signaling that includes a reduction in baroreceptor reflex function, presumably via a NADPH-ROS mediated pathway.

Objective: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI_PKC-P) expressing mutant cTnI (S43E, S45E, T144E).

Methods and Results: Two-dimensional gel analysis showed 7.2±0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model–based analysis for integrative interpretation. Compared to wild-type mice, cTnI_PKC-P mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F_max). Additionally, cTnI_PKC-P mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI_PKC-P mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI_PKC-P mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state.

Conclusions: A modest increase in PKC-induced cTnI phosphorylation (7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).

Objective: To investigate in cultured myocytes from adult cat and rat, 2 species in which Ang II has opposite inotropic effects, the signaling cascade involved in Ang II–induced apoptosis.

Methods and Results: Ang II (1 µmol/L) reduced cat/rat myocytes viability by 40%, in part, because of apoptosis (TUNEL/caspase-3 activity). In both species, apoptosis was associated with reactive oxygen species (ROS) production, Ca²⁺/calmodulin–dependent protein kinase (CaMK)II, and p38 mitogen-activated protein kinase (p38MAPK) activation and was prevented by the ROS scavenger MPG (2-mercaptopropionylglycine) or the NADPH oxidase inhibitor DPI (diphenyleneiodonium) by CaMKII inhibitors (KN-93 and AIP [aryl hydrocarbon receptor–interacting protein]) or in transgenic mice expressing a CaMKII inhibitory peptide and by the p38MAPK inhibitor, SB202190. Furthermore, p38MAPK overexpression exacerbated Ang II–induced cell mortality. Moreover, although KN-93 did not affect Ang II–induced ROS production, it prevented p38MAPK activation. Results further show that CaMKII can be activated by Ang II or H₂O₂, even in the presence of the Ca²⁺ chelator BAPTA-AM, in myocytes and in EGTA-Ca²⁺–free solutions in the presence of the calmodulin inhibitor W-7 in in vitro experiments.

Conclusions: (1) The Ang II–induced apoptotic cascade converges in both species, in a common pathway mediated by ROS-dependent CaMKII activation which results in p38MAPK activation and apoptosis. (2) In the presence of Ang II or ROS, CaMKII may be activated at subdiastolic Ca²⁺ concentrations, suggesting a new mechanism by which ROS reset the Ca²⁺ dependence of CaMKII to extremely low Ca²⁺ levels.

Objective: The goal of the present study was therefore to gain insights into the role of NF-B pathway specifically in mouse cardiomyocytes by conditional deletion of the NF-B essential modulator (NEMO).

Methods and Results: Using a Cre/loxP system, we disrupted the Nemo gene in a cardiomyocyte-specific manner in the heart, which simulated gene expression changes underlying human heart failure and caused adult-onset dilated cardiomyopathy accompanied by inflammation and apoptosis. Pressure overload challenges of NEMO-deficient young hearts precociously induced the functional decrements that develop spontaneously in older knockout animals. Moreover, oxidative stress in NEMO-deficient cardiomyocytes is a critical pathological component that can be attenuated with antioxidant diet in vivo.

Conclusions: These results reveal an essential physiological role for NEMO-mediated signaling in the adult heart to maintain cardiac function in response to age-related or mechanical challenges, in part through modulation of oxidative stress.

Objective: Herein, we generated a cardiac-specific overexpression model, carrying nonphosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20^S16A). By subjecting this model to ischemia/reperfusion, we addressed whether: (1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; (2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and (3) the aggregation pattern of Hsp20 is altered by its phosphorylation.

Methods and Results: Our results demonstrated that Hsp20^S16A hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis, compared with non-TG hearts. Interestingly, autophagy was activated in non-TG hearts but significantly inhibited in Hsp20^S16A hearts following ischemia/reperfusion. Accordingly, pretreatment of Hsp20^S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20^S16A hearts. Furthermore, on ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20^S16A hearts.

Conclusions: Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be attributable to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.