CXCR4 Cardiac Specific Knockout Mice Develop a Progressive Cardiomyopathy.

Activation of multiple pathways is associated with cardiac hypertrophy and heart failure. We previously published that CXCR4 negatively regulates ß-adrenergic receptor (ß-AR) signaling and ultimately limits ß-adrenergic diastolic (Ca2+) accumulation in cardiac myocytes. In isolated adult rat cardiac myocytes; CXCL12 treatment prevented isoproterenol-induced hypertrophy and interrupted the calcineurin/NFAT pathway. Moreover; cardiac specific CXCR4 knockout mice show significant hypertrophy and develop cardiac dysfunction in response to chronic catecholamine exposure in an isoproterenol-induced (ISO) heart failure model. We set this study to determine the structural and functional consequences of CXCR4 myocardial knockout in the absence of exogenous stress. Cardiac phenotype and function were examined using (1) gated cardiac magnetic resonance imaging (MRI); (2) terminal cardiac catheterization with in vivo hemodynamics; (3) histological analysis of left ventricular (LV) cardiomyocyte dimension; fibrosis; and; (4) transition electron microscopy at 2-; 6- and 12-months of age to determine the regulatory role of CXCR4 in cardiomyopathy. Cardiomyocyte specific-CXCR4 knockout (CXCR4 cKO) mice demonstrate a progressive cardiac dysfunction leading to cardiac failure by 12-months of age. Histological assessments of CXCR4 cKO at 6-months of age revealed significant tissue fibrosis in knockout mice versus wild-type. The expression of atrial naturietic factor (ANF); a marker of cardiac hypertrophy; was also increased with a subsequent increase in gross heart weights. Furthermore, there were derangements in both the number and the size of the mitochondria within CXCR4 cKO hearts. Moreover, CXCR4 cKO mice were more sensitive to catocholamines, their response to ß-AR agonist challenge via acute isoproterenol (ISO) infusion demonstrated a greater increase in ejection fraction, dp/dtmax, and contractility index. Interestingly, prior to ISO infusion, there were significant differences in baseline hemodynamics between the CXCR4 cKO compared to littermate controls. However, upon administering ISO, the CXCR4 cKO responded in a robust manner overcoming the baseline hemodynamic deficits reaching WT values supporting our previous data that CXCR4 negatively regulates ß-AR signaling. This further supports that, in the absence of the physiologic negative modulation, there is an overactivation of down-stream pathways, which contribute to the development and progression of contractile dysfunction. Our results demonstrated that CXCR4 plays a non-developmental role in regulating cardiac function and that CXCR4 cKO mice develop a progressive cardiomyopathy leading to clinical heart failure.

Alcohol-Mediated Organ Damages: Heart and Brain.

Alcohol is one of the most commonly abused substances in the United States. Chronic consumption of ethanol has been responsible for numerous chronic diseases and conditions globally. The underlying mechanism of liver injury has been studied in depth, however, far fewer studies have examined other organs especially the heart and the central nervous system (CNS). The authors conducted a narrative review on the relationship of alcohol with heart disease and dementia. With that in mind, a complex relationship between inflammation and cardiovascular disease and dementia has been long proposed but inflammatory biomarkers have gained more attention lately. In this review we examine some of the consequences of the altered cytokine regulation that occurs in alcoholics in organs other than the liver. The article reviews the potential role of inflammatory markers such as TNF-α in predicting dementia and/or cardiovascular disease. It was found that TNF-α could promote and accelerate local inflammation and damage through autocrine/paracrine mechanisms. Unraveling the mechanisms linking chronic alcohol consumption with proinflammatory cytokine production and subsequent inflammatory signaling pathways activation in the heart and CNS, is essential to improve our understanding of the disease and hopefully facilitate the development of new remedies.

SDF-1 induces TNF-mediated apoptosis in cardiac myocytes.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation. One such chemokine, Stromal cell-derived factor-1 (SDF-1) also known as CXCL12, and its receptor, CXCR4, are expressed and functional in cardiac myocytes. SDF-1 both stimulates and enhances the cellular signal which attracts potentially beneficial stem cells for tissue repair within the ischemic heart. Paradoxically however, this chemokine is known to act in concert with the inflammatory cytokines of the innate immune response which contributes to cellular injury through the recruitment of inflammatory cells during ischemia. In the present study, we have demonstrated that SDF-1 has dose dependent effects on freshly isolated cardiomyocytes. Using Tunnel and caspase 3-activation assays, we have demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations (pathological concentrations) induced apoptosis. Furthermore, ELISA data demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations upregulated TNF-α protein expression which directly correlated with subsequent apoptosis. There was a significant reduction in SDF-1 mediated apoptosis when TNF-α expression was neutralized which suggests that SDF-1 mediated apoptosis is TNF-α-dependent. The fact that certain stimuli are capable of driving cardiomyocytes into apoptosis indicates that these cells are susceptible to clinically relevant apoptotic triggers. Our findings suggest that the elevated SDF-1 levels seen in a variety of clinical conditions, including ischemic myocardial infarction, may either directly or indirectly contribute to cardiac cell death via a TNF-α mediated pathway. This highlights the importance of this receptor/ligand in regulating the cardiomyocyte response to stress conditions.

CXCR4 and CXCR7 play distinct roles in cardiac lineage specification and pharmacologic ß-adrenergic response.

CXCR4 and CXCR7 are prominent G protein-coupled receptors (GPCRs) for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). This study demonstrates that CXCR4 and CXCR7 induce differential effects during cardiac lineage differentiation and ß-adrenergic response in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using lentiviral vectors to ablate CXCR4 and/or CXCR7 expression, hiPSC-CMs were tested for phenotypic and functional properties due to gene knockdown. Gene expression and flow cytometry confirmed the pluripotent and cardiomyocyte phenotype of undifferentiated and differentiated hiPSCs, respectively. Although reduction of CXCR4 and CXCR7 expression resulted in a delayed cardiac phenotype, only knockdown of CXCR4 delayed the spontaneous beating of hiPSC-CMs. Knockdown of CXCR4 and CXCR7 differentially altered calcium transients and ß-adrenergic response in hiPSC-CMs. In engineered cardiac tissues, depletion of CXCR4 or CXCR7 had opposing effects on developed force and chronotropic response to ß-agonists. This work demonstrates distinct roles for the SDF-1/CXCR4 or CXCR7 network in hiPSC-derived ventricular cardiomyocyte specification, maturation and function.

The duality of chemokines in heart failure.

The failing human heart is a bustling network of intra- and inter-cellular signals and related processes attempting to coordinate a repair mechanism for the injured or diseased myocardium. While our understanding of signaling by mode of cytokines is well understood on a systemic level, we are only now coming to elucidate the role of cytokines in cardiac self-regulation. An increasing number of studies are showing now that cardiomyocytes themselves have not only the ability but also the mandate to produce signals, and play direct roles in how these signals are interpreted. One of the families of cytokines employed by distressed cardiac tissue are chemokines. By regulating the movement of pro-inflammatory cell types to sites of injury, we see now how the myocardium responds to stress. Herein we review the participation of these inflammatory mediators and explore the delicate balance between their protective roles and damaging functions.

Synergistic role of protein phosphatase inhibitor 1 and sarco/endoplasmic reticulum Ca2+ -ATPase in the acquisition of the contractile phenotype of arterial smooth muscle cells.

BACKGROUND: Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. METHODS AND RESULTS: In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. CONCLUSIONS: I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.

CXCR4 gene transfer prevents pressure overload induced heart failure.

Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, ß2-adrenoceptor, and CXCR4 (Cav1.2/ß2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-ß2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest that AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure.

Chemokines and inflammation in heart disease: adaptive or maladaptive?

Heart disease is not only the leading cause of death, disability, and healthcare expense in the US, but also the leading cause of death worldwide. Therefore, treatments to lessen ischemia-related cardiac damage could affect a broad swath of the population and have significant health and fiscal impacts. Cardiac dysfunction has been associated with elevated circulating chemokine levels, both in animals and humans. Most studies in this area have focused on chemokine expression as a prominent feature of the post-infarction inflammatory response. Such studies have investigated the role of chemokines in inflammatory leukocyte recruitment. Other work on this topic has focused on stem-cell therapy or factors e.g. chemokines mobilizing bone marrow progenitor cells as possible avenues for improving contractile dysfunction. Findings from numerous preclinical studies and several initial clinical trials support the feasibility of promoting the recruitment of bone marrow-derived cells to the infarcted heart and increased homing following injury, supporting the notion that cell therapy might have therapeutic potential. They have not, however, addressed the possibility of an autocrine/paracrine effect wherein the chemokine receptors, present on the cardiac myocyte surface, modulate functional responses to stress in which can be adaptive or maladaptive in nature.

ß2-Adrenergic receptor signaling in the cardiac myocyte is modulated by interactions with CXCR4.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α and its G-protein coupled receptor, CXCR4. Stromal cell-derived factor-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective ß-adrenergic receptor (ßAR) agonist, isoproterenol. As a result of the importance of ß-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of ßAR signaling. Our studies demonstrate activation of CXCR4 by stromal cell-derived factor-1 leads to a decrease in ßAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of ßAR downstream targets is ß2AR-dependent. We demonstrated a physical interaction between CXCR4 and ß2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-ß2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-ß2AR complex could represent a hitherto unknown target for therapeutic intervention.

Effects of CXCR4 gene transfer on cardiac function after ischemia-reperfusion injury.

Acute coronary occlusion is the leading cause of death in the Western world. There is an unmet need for the development of treatments to limit the extent of myocardial infarction (MI) during the acute phase of occlusion. Recently, investigators have focused on the use of a chemokine, CXCL12, the only identified ligand for CXCR4, as a new therapeutic modality to recruit stem cells to individuals suffering from MI. Here, we examined the effects of overexpression of CXCR4 by gene transfer on MI. Adenoviruses carrying the CXCR4 gene were injected into the rat heart one week before ligation of the left anterior descending coronary artery followed by 24 hours reperfusion. Cardiac function was assessed by echocardiography couple with 2,3,5-Triphenyltetrazolium chloride staining to measure MI size. In comparison with control groups, rats receiving Ad-CXCR4 displayed an increase in infarct area (13.5% +/- 4.1%) and decreased fractional shortening (38% +/- 5%). Histological analysis revealed a significant increase in CXCL12 and tumor necrosis factor-alpha expression in ischemic area of CXCR4 overexpressed hearts. CXCR4 overexpression was associated with increased influx of inflammatory cells and enhanced cardiomyocyte apoptosis in the infarcted heart. These data suggest that in our model overexpressing CXCR4 appears to enhance ischemia/reperfusion injury possibly due to enhanced recruitment of inflammatory cells, increased tumor necrosis factor-alpha production, and activation of cell death/apoptotic pathways.

beta-Adrenergic receptor activation induces internalization of cardiac Cav1.2 channel complexes through a beta-arrestin 1-mediated pathway.

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of beta-adrenergic receptors (beta-ARs), which leads to an increase in calcium current (I(Ca-L)) density through cardiac Ca(v)1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I(Ca-L) density and kinetics in heart failure often occur in the absence of changes in Ca(v)1 channel expression, arguing for the importance of post-translational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained beta-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and beta-arrestin 1 (beta-Arr1) into clathrin-coated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G(i/o) mediates this response. A peptide that selectively disrupts the interaction between Ca(V)1.2 and beta-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G(i) of the beta-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure.

MCP-1/CCL2 protects cardiac myocytes from hypoxia-induced apoptosis by a G(alphai)-independent pathway.

Chemokines, in addition to their chemotactic properties, act upon resident cells within a tissue and mediate other cellular functions. In a previous study, we demonstrated that CCL2 protects cultured mouse neonatal cardiac myocytes from hypoxia-induced cell death. Leukocyte chemotaxis has been shown to contribute to ischemic injury. While the chemoattractant properties of CCL2 have been established, the protective effects of this chemokine suggest a novel role for CCL2 in myocardial ischemia/reperfusion injury. The present study examined the cellular signaling pathways that promote this protection. Treatment of cardiac myocyte cultures with CCL2 protected them from hypoxia-induced apoptosis. This protection was not mediated through the activation of G(alphai) signaling that mediates monocyte chemotaxis. Inhibition of the ERK1/2 signaling pathway abrogated CCL2 protection. Caspase 3 activation and JNK/SAPK phosphorylation were decreased in hypoxic myocytes co-treated with CCL2 as compared to hypoxia only-treated cultures. Expression of the Bcl-2 family proteins, Bcl-xL and Bag-1, was increased in CCL2-treated myocytes subjected to hypoxia. There was also downregulation of Bax protein levels as a result of CCL2 co-treatment. These data suggest that CCL2 cytoprotection and chemotaxis may occur through distinct signaling mechanisms.

Staurosporine promotes endothelial cell assembly and FAK phosphorylation during in vitro angiogenesis.

In the present study, we report that staurosporine, a known PKC inhibitor, enhanced in vitro angiogenesis. Endothelial cells plated in a three-dimensional matrix formed cords and enclosed structures within 4-6 hours. The cells in cord structures became elongated during the subsequent incubation. Tube formation was confirmed by confocal microscopy. Addition of VEGF enhanced the early responses of endothelial cells, leading to enhanced formation of cords. Staurosporine unexpectedly also enhanced the early endothelial responses, leading to faster alignment of cells and assembly into tube-like structures. At concentrations inhibitory to endothelial cell PKC activity, staurosporine produced 91% and 203% increases in the number of cords and the enclosed structures, respectively, as compared to the controls. Other selective inhibitors of PKC did not stimulate in vitro angiogenesis in the absence or presence of VEGF. Further investigation showed that inhibition of PI-3 kinase and Raf-1 significantly reduced the effects of staurosporine. Staurosporine-induced in vitro angiogenesis required integrins alpha2 and alphavbeta3 and was associated with significantly enhanced FAK phosphorylation. These data indicate that staurosporine enhances in vitro angiogenesis by a means unrelated to its PKC inhibition. The data suggest that enhancement of in vitro angiogenesis by staurosporine involves integrin-mediated signaling, including the stimulation of FAK phosphorylation.

Pharmacological revascularisation in coronary and peripheral vascular disease.

Therapeutic angiogenesis is a novel approach to the treatment of ischaemic or occlusive coronary and peripheral vascular disease. The therapeutic concept is based on the restoration of distal blood flow by the enlargement of existing vessels and tissue perfusion by the induction of new capillaries. Initial studies have focused on the direct application of endothelial growth factors, vascular endothelial growth factor and fibroblast growth factor, or the delivery of genes using either a plasmid or adenoviral vector. Recently, new angiogenic agents such as hypoxia inducible factor-1alpha, fibroblast growth factor-4, Del-1 and hepatocyte growth factor have entered clinical testing. Moreover, stem-cell therapy or factors mobilising bone marrow progenitor cells have provided evidence for a new avenue for therapeutic angiogenesis. Numerous preclinical studies and several initial clinical trials have provided encouraging data in support of the feasibility of promoting biological revascularisation by the administration of angiogenic factors or cells.

Opposing effects mediated by the chemokine receptor CXCR2 on myocardial ischemia-reperfusion injury: recruitment of potentially damaging neutrophils and direct myocardial protection.

BACKGROUND: The timely reperfusion of ischemic myocardium limits infarction, but components of reperfusion, such as inflammation, may be injurious. The chemokine receptor CXCR2 mediates neutrophil chemotaxis. CXCR2 activation also inhibits hypoxia-induced death of isolated cardiac myocytes. This study assesses whether CXCR2 mediates protection in the intact heart and, if so, the magnitude of this protection relative to CXCR2-mediated chemotaxis of potentially damaging inflammatory cells. METHODS AND RESULTS: After ischemia-reperfusion in vivo, CXCR2-/- mice exhibited infarcts that were 50.5% smaller (P<0.05) with 44.3% fewer inflammatory cells (P<0.05) than wild type mice. These data suggest that in this model, CXCR2-mediated chemotaxis may be important in myocardial cell death. To isolate the role of CXCR2 specifically on blood cells, adoptive transfer experiments were performed. After ischemia-reperfusion, infarcts were 53.4% smaller (P<0.05) and contained 65.0% fewer inflammatory cells (P<0.05) in lethally irradiated wild type mice reconstituted with CXCR2-/- compared with wild type bone marrow. Thus, CXCR2 on blood cells is important in myocardial damage, most likely because of CXCR2-mediated chemotaxis. To unmask whether CXCR2 mediates direct myocardial protection in the intact heart, wild type and CXCR2-/- hearts were studied in the absence of blood using Langendorff preparations. In this case, infarcts were 19.7% larger in CXCR2-/- than wild type hearts (P<0.05), revealing a novel CXCR2-mediated cardioprotective effect. CONCLUSIONS: CXCR2 exerts opposing effects on myocardial viability during ischemia-reperfusion with recruitment of damaging inflammatory cells predominant over direct tissue protection.

Chemokine expression in myocardial ischemia: MIP-2 dependent MCP-1 expression protects cardiomyocytes from cell death.

Chemokines are small molecular weight proteins that play important roles in inflammation. Originally described as chemotactic cytokines, chemokines stimulate the influx of leukocytes into specific tissue compartments. These molecules also modulate gene expression in both infiltrating and resident cells to mediate a vast array of cellular functions, and their importance in disease processes has been well documented. This study examined the expression of chemokines during myocardial ischemia and established a pathway by which two, MIP-2 and JE/MCP-1, modulate cardiac myocyte viability during this process. To focus on the direct effects of chemokines on these cells, a mouse model of ischemia without reperfusion was used. The expression of chemokines and chemokine receptors was induced in the left ventricular free wall as early as 1 h post-ischemia, with the most significant increases in MIP-2 (CXCL2) and JE/MCP-1 (CCL2). Expression of their respective receptors, CXCR2 and CCR2, was also induced. Similar changes in gene expression occurred at the mRNA and protein levels in isolated neonatal mouse cardiac myocytes stimulated by hypoxia. Antibody to MIP-2 inhibited hypoxia-induced JE/MCP-1 expression, demonstrating that MIP-2 is critical for this event. Moreover, in vivo intramyocardial injection of either an adenovirus expressing MIP-2 or the recombinant protein itself was sufficient to upregulate JE/MCP-1 production even in the absence of ischemia. Thus, MIP-2 regulates JE/MCP-1 expression both in cell culture and in vivo. Furthermore, JE/MCP-1 markedly decreased hypoxia-induced cell death in cultured cardiac myocytes. Thus, JE/MCP-1 appears to mediate an unanticipated survival pathway in target cardiac myocytes themselves. These findings indicate an important role for MIP-2 and JE/MCP-1 in regulating the response of cardiac myocytes to myocardial ischemia.