Circulation Research current issue

[Editorials] Ten Good Years

Marban, E. — 2009-07-01

[Corrections] Correction

2009-07-01

[Editorials] Calcineurin Finds a New Partner in the L-Type Ca2+ Channel

Pitt, G. S. — 2009-07-01

[Editorials] Limits to Growth of Native Collateral Vessels: Just One Mouse CLIC Away From Unlimited Collateral Perfusion?

Waltenberger, J. — 2009-07-01

[Reports] Avoidance of Transient Cardiomyopathy in Cardiomyocyte-Targeted Tamoxifen-Induced MerCreMer Gene Deletion Models

2009-07-01

Cardiac myocyte targeted MerCreMer transgenic mice expressing tamoxifen-inducible Cre driven by the -myosin heavy chain promoter are increasingly used to control gene expression in the adult heart. Here, we show tamoxifen-mediated MerCreMer (MCM) nuclear translocation can induce severe transient dilated cardiomyopathy in mice with or without loxP transgenes. The cardiomyopathy is accompanied by marked reduction of energy/metabolism and calcium-handling gene expression (eg, PGC1-, peroxisome proliferator-activated , SERCA2A), all fully normalized with recovery. MCM-negative/flox-positive controls display no dysfunction with tamoxifen. Nuclear Cre translocation and equally effective gene knockdown without cardiomyopathy is achievable with raloxifene, suggesting toxicity is not simply from Cre. Careful attention to controls, reduced tamoxifen dosing and/or use of raloxifene is advised with this model.

[Molecular Medicine] Peroxisome Proliferator-Activated Receptor {delta} Regulates Extracellular Matrix and Apoptosis of Vascular Smooth Muscle Cells Through the Activation of Transforming Growth Factor-{beta}1/Smad3

2009-07-01

Homeostasis of the extracellular matrix and apoptosis of vascular smooth muscle cells (VSMCs) are key components in the regulation of the stability of atherosclerotic plaques. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) regulates extracellular matrix synthesis and degradation through transforming growth factor-β1 and its effector, Smad3. Activation of PPAR strongly amplified the expression of types I and III collagen, fibronectin, elastin, and TIMP-3 (tissue inhibitor of metalloproteinases 3), but not of TIMP-1, matrix metalloproteinase-2 or -9. The effect of PPAR on the expression of type III collagen was dually regulated by the direct binding of PPAR and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, in the type III collagen gene promoter. The activation of PPAR attenuated apoptotic cell death in VSMCs induced by oxidized low-density lipoprotein, and similar antiapoptotic effects were observed on treatment of cells with exogenous type I and/or III collagen. Administration of a PPAR ligand GW501516 to mice also suppressed elastase-induced cell death of aortic VSMCs. These results suggest that PPAR-induced upregulation of extracellular matrix proteins exerts an antiapoptotic effect, thereby maintaining the stability of atherosclerotic plaques. Specific ligands of PPAR may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.

[Molecular Medicine] Activating Transcription Factor 3 Constitutes a Negative Feedback Mechanism That Attenuates Saturated Fatty Acid/Toll-Like Receptor 4 Signaling and Macrophage Activation in Obese Adipose Tissue

2009-07-01

Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid-stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor- production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor- promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKA^y mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation.

[Molecular Medicine] Nitric Oxide-Independent Vasodilator Rescues Heme-Oxidized Soluble Guanylate Cyclase From Proteasomal Degradation

2009-07-01

Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the β heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both and β subunits. Collectively, our data establish oxidation–ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.

[Cellular Biology] Angiotensin II-Mediated Adaptive and Maladaptive Remodeling of Cardiomyocyte Excitation-Contraction Coupling

2009-07-01

Cardiac hypertrophy is associated with alterations in cardiomyocyte excitation–contraction coupling (ECC) and Ca²⁺ handling. Chronic elevation of plasma angiotensin II (Ang II) is a major determinant in the pathogenesis of cardiac hypertrophy and congestive heart failure. However, the molecular mechanisms by which the direct actions of Ang II on cardiomyocytes contribute to ECC remodeling are not precisely known. This question was addressed using cardiac myocytes isolated from transgenic (TG1306/1R [TG]) mice exhibiting cardiac specific overexpression of angiotensinogen, which develop Ang II–mediated cardiac hypertrophy in the absence of hemodynamic overload. Electrophysiological techniques, photolysis of caged Ca²⁺ and confocal Ca²⁺ imaging were used to examine ECC remodeling at early (20 weeks of age) and late (60 weeks of age) time points during the development of cardiac dysfunction. In young TG mice, increased cardiac Ang II levels induced a hypertrophic response in cardiomyocyte, which was accompanied by an adaptive change of Ca²⁺ signaling, specifically an upregulation of the Na⁺/Ca²⁺ exchanger–mediated Ca²⁺ transport. In contrast, maladaptation was evident in older TG mice, as suggested by reduced sarcoplasmic reticulum Ca²⁺ content resulting from a shift in the ratio of plasmalemmal Ca²⁺ removal and sarcoplasmic reticulum Ca²⁺ uptake. This was associated with a conserved ECC gain, consistent with a state of hypersensitivity in Ca²⁺-induced Ca²⁺ release. Together, our data suggest that chronic elevation of cardiac Ang II levels significantly alters cardiomyocyte ECC in the long term, and thereby contractility, independently of hemodynamic overload and arterial hypertension.

[Cellular Biology] Physical and Functional Interaction Between Calcineurin and the Cardiac L-Type Ca2+ Channel

2009-07-01

The L-type Ca²⁺ channel (LTCC) is the major mediator of Ca²⁺ influx in cardiomyocytes, leading to both mechanical contraction and activation of signaling cascades. Among these Ca²⁺-activated cascades is calcineurin, a protein phosphatase that promotes hypertrophic growth of the heart. Coimmunoprecipitations from heart extracts and pulldowns using heterologously expressed proteins provided evidence for direct binding of calcineurin at both the N and C termini of ₁1.2. At the C terminus, calcineurin bound specifically at amino acids 1943 to 1971, adjacent to a well-characterized protein kinase (PK)A/PKC/PKG phospho-acceptor site Ser1928. In vitro assays demonstrated that calcineurin can dephosphorylate ₁1.2. Channel function was increased in voltage-clamp recordings of I_Ca,L from cultured cardiomyocytes expressing constitutively active calcineurin, consistent with previous observations in cardiac hypertrophy in vivo. Conversely, acute suppression of calcineurin pharmacologically or with specific peptides decreased I_Ca,L. These data reveal direct physical interaction between the LTCC and calcineurin in heart. Furthermore, they demonstrate that calcineurin induces robust increases in I_Ca,L and highlight calcineurin as a key modulator of pathological electrical remodeling in cardiac hypertrophy.

[Cellular Biology] Gene Expression Profiling of the Forming Atrioventricular Node Using a Novel Tbx3-Based Node-Specific Transgenic Reporter

2009-07-01

The atrioventricular (AV) node is a recurrent source of potentially life-threatening arrhythmias. Nevertheless, limited data are available on its developmental control or molecular phenotype. We used a novel AV nodal myocardium–specific reporter mouse to gain insight into the gene programs determining the formation and phenotype of the developing AV node. In this reporter, green fluorescent protein (GFP) expression was driven by a 160-kbp bacterial artificial chromosome with Tbx3 and flanking sequences. GFP was selectively active in the AV canal of embryos and AV node of adults, whereas the Tbx3-positive AV bundle and sinus node were devoid of GFP, demonstrating that distinct regulatory sequences and pathways control expression in the components of the conduction system. Fluorescent AV nodal and complementary Nppa-positive chamber myocardial cell populations of embryonic day 10.5 embryos and of embryonic day 17.5 fetuses were purified using fluorescence-activated cell sorting, and their expression profiles were assessed by genome-wide microarray analysis, providing valuable information concerning their molecular identities. We constructed a comprehensive list of sodium, calcium, and potassium channel genes specific for developing nodal or chamber myocardium. Furthermore, the data revealed that the AV node and the chamber (working) myocardium phenotypes diverge during development but that the functional gene classes characterizing both subtypes are maintained. One of the repertoires identified in the AV node–specific gene profiles consists of multiple neurotrophic factors and semaphorins, not yet appreciated to play a role in nodal development, revealing shared characteristics between nodal and nervous system development.

[Integrative Physiology] The I{kappa}B Kinase {beta}/Nuclear Factor {kappa}B Signaling Pathway Protects the Heart From Hemodynamic Stress Mediated by the Regulation of Manganese Superoxide Dismutase Expression

2009-07-01

Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-B signaling pathway regulates cell death, however, the effect of NF-B pathway on cell death can vary in different cells or stimuli. The purpose of the present study was to clarify the in vivo role of the NF-B pathway in response to pressure overload. First, we subjected C57Bl6/J mice to pressure overload by means of transverse aortic constriction (TAC) and examined the activity of the NF-B pathway in response to pressure overload. IB kinase (IKK) and NF-B were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKβ-deficient mice (CKO). CKO displayed normal global cardiac structure and function compared with control littermates. We subjected CKO and control mice to pressure overload. One week after TAC, CKO showed cardiac dilation, dysfunction, and lung congestion, which are characteristics of heart failure. The number of apoptotic cells in the hearts of CKO mice increased significantly after TAC. The levels of manganese superoxide dismutase mRNA and protein expression in CKO after TAC were significantly attenuated compared with control mice. The levels of oxidative stress and c-Jun N-terminal kinase (JNK) activation in CKO after TAC were significantly greater than those in control mice. Isoproterenol-induced cell death of isolated adult CKO cardiomyocytes was inhibited by treatment with either a manganese superoxide dismutase mimetic or a JNK inhibitor. Thus, the IKKβ/NF-B signaling pathway plays a protective role in cardiomyocytes because of the attenuation of oxidative stress and JNK activation in a setting of acute pressure overload.

[Integrative Physiology] Cardiac Muscle Ring Finger-1 Increases Susceptibility to Heart Failure In Vivo

2009-07-01

Muscle ring finger (MuRF)1 is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates and heterodimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal, whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy, indicating cooperative control of muscle mass by MuRF1 and MuRF2. To identify the unique role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg⁺) hearts exhibited a nonprogressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by transaortic constriction (TAC) induced rapid failure of MuRF1 Tg⁺ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg⁺ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg⁺ mice did not differ from wild-type mice despite the depressed contractility following TAC. In comparing the level and activity of creatine kinase (CK) between wild-type and MuRF1 Tg⁺ hearts, we found that mCK and CK-M/B protein levels were unaffected in MuRF1 Tg⁺ hearts; however, total CK activity was significantly inhibited. We conclude that increased expression of cardiac MuRF1 results in a broad disruption of primary metabolic functions, including alterations in CK activity that leads to increased susceptibility to heart failure following TAC. This study demonstrates for the first time a role for MuRF1 in the regulation of cardiac energetics in vivo.

[Integrative Physiology] Chloride Intracellular Channel-4 Is a Determinant of Native Collateral Formation in Skeletal Muscle and Brain

Chalothorn, D., Zhang, H., Smith, J. E., Edwards, J. C., Faber, J. E. — 2009-07-01

The capacity of the collateral circulation to lessen injury in occlusive vascular disease depends on the density and caliber of native (preexisting) collaterals, as well as their ability to outwardly remodel in ischemia. Native collateral conductance varies widely among healthy individuals, yet little is known about what specifies collateral formation. Chloride intracellular channel (CLIC)4 protein is required for endothelial cell hollowing, a process necessary for vessel formation during embryogenesis and ischemia. Whether CLIC4 has other physiological roles in vascular biology is uncertain. We studied collateral formation and remodeling in mice deficient in CLIC1 and CLIC4. Vascular responses to femoral artery ligation were similar in Clic1^–/– and wild-type mice. In contrast, immediately after ligation perfusion dropped more in Clic4^–/– than wild-type mice, suggesting fewer preexisting collaterals, a finding confirmed by angiography, greater ischemia, and worse recovery of perfusion; however, collateral remodeling was unaffected. Likewise, native cerebral collateral density in Clic4^–/– (but not Clic1^–/–) mice was reduced, resulting in severe infarctions. This was associated with impaired perinatal formation and stabilization of nascent collaterals. Clic4 hemizygous mice had intermediate deficits in the above parameters, suggesting a gene-dose effect. Ischemia augmented CLIC1 and CLIC4 expression similarly in wild-type mice. However, CLIC1 increased 3-fold more in Clic4^–/– mice, suggesting compensation. Despite greater ischemia in Clic4^–/– mice, hypoxia-inducible factor-1, vascular endothelial growth factor (VEGF) and angiopoietin-2 increased less compared to wild-type, suggesting CLIC4 exerts influences upstream of hypoxia-inducible factor-1-VEGF signaling. Hence, CLIC4 represents the second gene that, along with VEGF shown by us previously, specifies native collateral formation.

[Integrative Physiology] MIF Deficiency Reduces Chronic Inflammation in White Adipose Tissue and Impairs the Development of Insulin Resistance, Glucose Intolerance, and Associated Atherosclerotic Disease

2009-07-01

Chronic inflammation in white adipose tissue (WAT) is positively associated with obesity, insulin resistance (IR) and the development of type 2 diabetes. The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is an essential, upstream component of the inflammatory cascade. This study examines whether MIF is required for the development of obesity, IR, glucose intolerance, and atherosclerosis in the LDL receptor-deficient (Ldlr^–/–) mouse model of disease. Ldlr^–/– mice develop IR and glucose intolerance within 15 weeks, whereas Mif^–/–Ldlr^–/– littermates are protected. MIF deficiency does not affect obesity and lipid risk factors but specifically reduces inflammation in WAT and liver, as reflected by lower plasma serum amyloid A and fibrinogen levels at baseline and under inflammatory conditions. Conversely, MIF stimulates the in vivo expression of human C-reactive protein, an inflammation marker and risk factor of IR and cardiovascular disease. In WAT, MIF deficiency reduces nuclear c-Jun levels and improves insulin sensitivity; MIF deficiency also reduces macrophage accumulation in WAT and blunts the expression of two proteins that regulate macrophage infiltration (intercellular adhesion molecule-1, CD44). Mechanistic parallels to WAT were observed in aorta, where the absence of MIF reduces monocyte adhesion, macrophage lesion content, and atherosclerotic lesion size. These data highlight the physiological importance of chronic inflammation in development of IR and atherosclerosis and suggest that MIF is a potential therapeutic target for reducing the inflammatory component of metabolic and cardiovascular disorders.