5000 Characterization and Growth of Human Cardiac Stem Cells
Claudia Bearzi, Stefano Cascapera, Angelo Nascimbene, Claudia Casarsa, Raffaella Rastaldo, Toru Hosoda, Antonella De Angelis, Marcello Rota, Federico Quaini, Konrad Urbanek, Annarosa Leri, Piero Anversa, New York Medical College Cardiovascular Research Inst, Valhalla, NY; Roberto Bolli, University of Louisville Division of Cardiology, Louisville, KY; Jan Kajstura, New York Medical College Cardiovascular Research Inst, Valhalla, NY
The human heart contains a subpopulation of replicating myocytes that is enhanced in pathologic states characterized by acute and chronic cardiac failure. On the assumption that these dividing cells represent amplifying myocytes originated from a pool of undifferentiated cells, effort was made to establish the conditions for the isolation and expansion of potential progenitor cells from surgical specimens of human myocardium. Because of the small size of the samples, we have cultured slices of human atrial and ventricular myocardium by the primary explant technique. The presence of stem cell surface antigens in the outgrowing cardiac cells was determined by immuno-cytochemistry and FACS analysis; c-kitPOS, MDR1POS and Sca-1-likePOS cells were found in 1.8±1.7%, 0.5±0.7% and 1.3±1.9% of the unsorted myocardial cell population, respectively. The fraction of c-kitPOS cells included lineage negative, 52±12%, and early committed cells, 48±12%. After plating, c-kitPOS cells attached rapidly and continued to grow up to P8 undergoing ∼25 population doublings. Ki67 labeling showed that the number of cycling cells remained constant from P1 to P8, 48±10%. For clonal assay, single c-kitPOS cells were deposited in individual wells of a Terasaki plate. After 2–3 weeks, clones were generated and the number of cells in the clones varied from 500 to 1,500. Doubling time was ∼28 hours and ∼90% of cells were labeled by BrdU after 5 days of exposure. In differentiation medium, clonogenic cells gave rise to myocytes, endothelial cells and vascular smooth muscle cells. When human c-kitPOS cardiac progenitor cells were locally injected in the infarcted myocardium of immunodeficient rats and mice, they regenerated myocytes and coronary vessels of human origin. This repair process resulted in an improvement in cardiac function. Importantly, the formation of parenchymal cells, arterioles and capillaries occurred without cell fusion. In conclusion, we have identified a human c-kit positive cardiac stem cell that is self-renewing, clonogenic and multipotent and can reconstitute the dead myocardium after infarction.
5001 Electrophysiology of Human and Porcine Adult Cardiac Stem Cells Isolated from Endomyocardial Biopsies
Rachel Ruckdeschel Smith, M. Roselle Abraham, Johns Hopkins University, Baltimore, MD; Elisa Messina, University La Sapienza, Rome, Italy; Hee Cheol Cho, Johns Hopkins University, Baltimore, MD; Alessandro Giacomello, University La Sapienza, Rome, Italy; Eduardo Marbán, Johns Hopkins University, Baltimore, MD
Cardiac stem cells (CSCs) show great promise for regenerative therapy. We have isolated CSCs from adult human (n = 23) and porcine (n = 60) endomyocardial biopsy specimens, differentiated them in vitro and characterized their electrical properties. When minced and maintained in primary culture for 2–7 days, ventricular or atrial biopsy specimens (∼15 mg) produce small, round cells budding off from the explant. Such cells can be expanded as monolayers with a doubling time of less than one day, or, if grown in suspension culture, form multicellular clusters dubbed cardiospheres (CSps). CSps are partially differentiated toward the cardiac lineage, expressing MHC, TnI, connexin-43, and ANP by immunostaining. Patch clamp reveals that small CSps (<20 cells) manifest only small outward currents and a resting membrane potential of -28+/-9 mV (n = 5). Larger CSps (∼100 cells) variably (n = 2 of 5) express an inward rectifier current like that of adult ventricular myocytes and maintain a resting membrane potential of -82+/-6 mV. CSps co-cultured with neonatal rat ventricular myocytes (NRVMs) become flattened, stellate and contractile after 6 or 7 days. Co-cultured CSp-derived cells are fully excitable: they exhibit spontaneous action potentials (APs) with a maximum diastolic potential of -50mV and an AP morphology akin to nodal cells. CSp-derived cells also cycle calcium and exhibit functional coupling with NRVMs in co-culture, as evidenced by Rhod-2-sensitive intracellular calcium transients synchronous with those of neighboring heart cells by confocal imaging. Thus, CSCs and CSps can be isolated and expanded from routine biopsy specimens; they readily become excitable and contractile in synchrony with cardiomyocytes in vitro. Taken together, these observations provide both a simple method and a solid rationale for the use of CSCs and CSps for autologous cardiac regeneration therapy.
5002 Nodal-Like Cardiac Myocytes Derived From Murine Embryonic Stem Cells Can Be Passaged While Retaining Their Phenotype
Steven M. White, William C. Claycomb, Louisiana State University Health Sciences Center, New Orleans, LA
Little is known about the specification and differentiation of nodal cardiac myocytes due to the lack of appropriate model systems and molecular markers. The proximal 1.5 kilobase region of the chicken GATA6 enhancer is expressed in pre-cardiac mesoderm and in discrete regions of the adult murine heart that contain the sinoatrial and atrioventricular nodes. We have previously demonstrated that the GATA6 enhancer marks pacemaking cells in cultures of differentiating murine embryonic stem (ES) cells. We tested the hypothesis that this GATA6 enhancer can be used to isolate a population of nodal cardiac myocytes from differentiating ES cells. Undifferentiated J1 ES cells were transfected with an expression vector in which the chicken GATA6 enhancer controls expression of the neomycin resistance gene. These cells were differentiated in suspension as aggregates called embryoid bodies. On day 8 of differentiation, embryoid bodies were dissociated into single cells and cultured in the presence of 300 μg/mL G418 for 7 days. Using RT-PCR to analyze the expression of 35 genes, GATA6-positive cells display a unique cardiac gene expression pattern. These cells express high levels of the transcription factors msx2, nkx2.5, gata4, gata6, and tbx3. Additionally, they express high levels of desmin, α-skeletal actin, α-cardiac actin, α- and βMHC, connexins 43 and 45, T- and L-type calcium channel subunit, and the acetylcholine-gated potassium channel with barely detectable levels of ANF, connexin 40, and the inward rectifier potassium channel kir2.1. Electrophysiological (patch-clamp) analyses reveal a heterogeneous population of myocytes that express voltage-gated calcium and potassium currents, as well as an inward, hyperpolarization-activated current (If) and no inward rectifier potassium current (Kir). These GATA6-positive cells have currently been passaged 24 times, frozen, and thawed and retain the same gene expression pattern as the freshly isolated cells. In conclusion, we document the isolation and characterization of a population of nodal-like cardiac myocytes that can be passaged in vitro while retaining their phenotype. This cell population will provide a model system that can be used to study the molecular phenotype of nodal cardiac myocytes.
5003 Bone Marrow-Derived Oct-3/4+ Stem Cells Differentiate Into Cardiac Myocytes In Vitro
Benedetta Pallante, Weill Medical College of Cornell Univ, New York, NY, Jay Edelberg, Cornell University, New York, NY
Background: Bone marrow cells (BMCs) have the ability to differentiate into functional heart tissue, but mechanisms of bone marrow cell plasticity are still largely unknown and have been mostly attributed to fusion with or transdifferentiation. Methods and Results: Our aim was to use a mouse model to identify BMCs that gave rise to cardiac myocytes in vitro and to define mechanisms of this differentiation. Whole BMCs were cultured for 14 days, in IMDM supplemented with 10%FBS, 5 ng/ml FGF-2 and 10 ng/ml VEGF, to induce cardiac differentiation. Cultures were monitored daily. Under these conditions cell aggregates with cardiomyogenic potential appeared at Day-5 and by Day-7 (10.7±1.9 aggregates/animal) clusters of Troponin-T+ cardiac myocytes with spontaneous chronotropic activity started forming at the periphery of 32.9±10.6% of the aggregates. By Day-14 clusters of Troponin-T+ cardiomyocytes detached and became visible in suspension. Molecular analysis by RT-PCR, Q-PCR, and immunostaining of BMC aggregates collected by microdissection (20–30 aggregtaes/6 animals/3 replicates) confirmed expression of the pluripotency-associated gene Oct-3/4 and stem cell markers AFP and alkaline phosphatase, similar to what observed in the cardiomyogenic ES-cell-derived embryoid bodies. Indeed, the expression of phenotypic stem cell genes (Oct-3/4, Dppa3 and Dppa4) was inversely correlated with the induction of cardiac myocyte-specific genes (β and α myosin heavy chains). Conclusions:Overall, the in vitro generation of the BMC-derived cardiac myocytes from Oct-3/4+ EB-like cell clusters provides important mechanistic insights into the cardiomyogenic potential of adult BMCs. The generation of cardiac myocytes in the absence of pre-existing heart cells demonstrates that this process is not mediated by fusion. Moreover, the induction of differentiated cells from a rare sub-population of Oct-3/4+ cells suggests that the cardiac myocytes do not arise from the transdifferentation of other mature cell types. Our results support the potential of autologous bone marrow cells for the regeneration of functional cardiac muscle.
5004 Neovascularization by Skeletal Muscle Stem Cells After Transplantation Into Injured Heart
Bryan Piras, Yasuhiro Nakamura, Xiaohong Wang, University of Minnesota, Minneapolis, MN; Michael A. Rudnicki, Ottawa Health Research Institute, Ottawa, Canada; Jianyi Zhang, Atsushi Asakura; University of Minnesota, Minneapolis, MN
Skeletal muscle stem cells called satellite cells have demonstrated their clinical benefits to autologous cell transplantation for heart repair after injury. Adult skeletal muscle contains a novel stem cell population purified as a side population (SP) by FACS on the basis of Hoechst dye exclusion. The muscle SP cells express stem cell antigen-1 (Sca-1) and contain stem cell populations that give rise to skeletal muscle and hematopoietic cell lineages following transplantation in mice. Here we demonstrate that Sca-1+CD31−CD45−(CD31 is an endothelial cell marker and CD45 is a hematopoietic cell marker) cells in the SP fraction purified from mouse skeletal muscle possess endothelial cell differentiation ability. Following culture on Matrigel, muscle Sca-1+CD31−CD45−SP cells formed capillary-like structures that expressed endothelial cell markers CD31, Flt-1, calveolin and vWF, and displayed an up-take of acetylated low-density lipoprotein, indicating the in vitro endothelial cell differentiation potential of muscle Sca-1+CD31−CD45−SP cells. Following intramuscular injection, muscle Sca-1+CD31−CD45−SP cells differentiated into cells expressing endothelial cell markers and were integrated into vasculatures in regenerating mouse muscle. Therefore, this fraction contains muscle endothelial progenitor cells (mEPCs). We hypothesized that mEPCs are potentially useful stem cells for promoting heart repair by providing vascular endothelial cells in injured heart following cell transplantation. To examine this hypothesis, infarction was induced by coronary arterial ligation in mouse heart and mEPCs were directly injected into the left ventricular of the infarcted heart. As a result, transplanted mEPCs migrated into the injured area, differentiated into cells expressing endothelial cell markers such as Flt-1, CD31, calveolin and vWF, and were integrated into newly formed vasculature structures. These results suggest the contribution of mEPCs to neovascularization in injured heart. Importantly, mEPCs could be expanded ex vivo more than 10 fold while retaining the endothelial cell differentiation ability. In conclusion, mEPCs are potentially useful for therapeutic stem cell transplantation for ischemic heart diseases.
5005 Patterning of Myocardial Growth Is Regulated by the Novel Endocardial Genes Heart of Glass, Santa, and Valentine
John D. Mably, Massachusetts General Hospital, Charlestown, MA; Jau-Nian Chen, UCLA, Los Angeles, CA; Manzoor-Ali P Mohideen, The Pennsylvania State University College of Medicine, Hershey, PA; Mark C. Fishman, The Novartis Institutes for Biomedical Research Inc, Cambridge, MA
The signals required for the normal patterning of the myocardial layer of the vertebrate heart emanate from various sources, including the adjacent endocardium. Disruption of endocardial signals, such as neuregulin, can disrupt heart growth and consequently inhibit normal cardiac function. To dissect and define the mechanisms that regulate myocardial growth, we characterized and positionally cloned three autosomal recessive zebrafish mutants that are distinguished by a dilated and thin-walled heart. These three mutants, heart of glass (heg), santa (san), and valentine (vtn), possess a normal number of cells within the myocardium, but the cells are spread circumferentially along the surface of the endocardium. As a result, the atrium and ventricle develop as two huge, dysfunctional chambers, consisting of a single-cell layered myocardium. All three mutants exhibit a failure to form endocardial cushions, consistent with a disruption of normal endocardial-myocardial signaling pathways. Positional cloning of the genes associated with these three mutants has revealed a putative pathway conserved in vertebrates consisting of a transmembrane molecule (heg), and two intracellular signaling proteins (san and vtn). In situ analysis confirms that heg is expressed in the endocardium of the zebrafish heart by 24 hpf, the stage at which beating chambers are first observed. Human homologs of all three genes exist, and the human san and vtn genes, CCM1 and CCM2 respectively, are associated with the autosomal dominant condition cerebral cavernous malformation (CCM). The zebrafish mutants similarly exhibit defects in vessel patterning, suggestive of a pathogenesis related to the human endothelial vascular lesions. In addition, experiments in zebrafish to inhibit expression of combinations of these three genes results in an increased reproduction of the mutant phenotype, suggesting that the three genes function in a common pathway. In conclusion, we describe three endocardial genes that are vital for patterning the concentric growth of the heart. These data further accentuate the importance of the endocardium as a source of signals essential for coordinating formation of the myocardium and the overall architecture of the vertebrate heart.
5006 Improvement of Heart Function in Chronic Coronary Heart Disease With Chronic Myocardial Infarction: Controlled Study With Intracoronary Autologous Mononuclear Bone Marrow Cell Transplantation (IACT-Study)
Michael Brehm, Matthias Köstering, Tobias Zeus, Thomas Bartsch, Gökmen Turan, Christine Antke, Rüdiger Sorg, Gesine Kögler, Peter Wernet, Hans-Wilhelm Müller, Bodo E. Strauer; Univ of Dusseldorf, Dusseldorf, Germany
Acute myocardial infarction, bone marrow-derived cells improve cardiac function. It is conceivable, but not yet demonstrated, that stem cell therapy may also be useful in chronic infarction. Methods and Results: We treated 18 consecutive patients (48±13 years) with chronic myocardial infarction (5 months to 8 ½ years old) by intracoronary transplantation of autologous bone marrow mononuclear cells (BMCs) and compared them with a consecutive enrolled representative control group (n=18, 52±10 years). After 3 months in the transplantation group, infarct size was reduced by 30%, and global left ventricular ejection fraction (+15%) as well as infarction wall movement velocity (+57%) increased significantly (p<0.001), while in the randomized control group no significant changes were observed during 3 months follow-up in infarct size (27±9 to 26±9%), in left ventricular ejection fraction (51±10 to 52±10%) and in wall movement velocity of infarcted area (1.88±0.76 to 1.91±0.79cm/s). PTCA alone had no effect on left ventricular function in this chronic coronary patients. After bone marrow cell transplantation, there was an improvement of maximum oxygen uptake (V02max, +11%) and of regional 18F-Fluor-Desoxy-Glucose (FDG) uptake (PET) into infarcted tissue (+15%). Conclusions: These results demonstrate that regeneration of infarcted and chronically avital tissue can be realized in humans by bone marrow mononuclear cell transplantation.
5007 Cardiac Ischemia Mobilizes Marrow Progenitors by an MMP-9 Dependent Pathway
Shafie Fazel, Liwen Chen, Richard D. Weisel, Denis Angoulvant, Phillip Cheung, Amir Fazel, Paul W. Fedak, Subodh Verma, Terrence M. Yau, Ren-Ke Li, University of Toronto, Toronto, Canada
Background: Endothelial progenitor cells (EPC) have been purported to impart functional benefit in ischemic myocardium, but clinical data of progenitor cell (c-kit+) mobilization by pro-inflammatory cytokine G-CSF appeared to worsen outcome in unstable coronary plaques. To identify mechanisms for isolated EPC mobilization we examined the pathway involved in EPC trafficking. We hypothesized that matrix metalloproteinase (MMP)-9 is necessary for EPC mobilization to ischemic myocardium. Methods: Cardiac ischemia was induced in wild-type and MMP-9 null mice (n=106) using staged closed-chest ischemia/reperfusion (IR) to segregate cytokine fluctuations due to surgical trauma from those due to IR. On D0, 1, 3, and 7, bone marrow, spleen, blood, and heart samples were collected. Immunoblotting, ELISA, and FACS for protein levels; semi-quantitative RT-PCR for mRNA, zymography for MMP activity, in vitro fibronectin adhesion assay for EPC number; and methylcellulose colony formation assay for marrow progenitor quantification were used. Results: IR↑bone marrow MMP-9 but not MMP-2 or the endogenous MMP-9 inhibitors TIMP-1 and -3. ↑MMP-9 activity was correlated with ↓membrane stem cell factor (mSCF) (D1/D0: 64±5.5%, P<0.05) and with ↑soluable SCF (sSCF) in the bone marrow (D1/D0: 260±98%, P=0.08) and the plasma (D1/D0: 134±15%, P<0.05), without alteration in SCF mRNA levels, suggesting MMP-9 mediated cleavage of mSCF to sSCF. The ↑sSCF caused phosphorylation of its receptor, c-kit. These events corresponded temporally to ↑peripheral EPC (D1/D0: 243±59.8% P<0.05), ↑ heart c-kit+ cells (D3/D0: 255±48%, P<0.05) and ↑heart progenitor colony forming units (D0 to D3: 0±0 to 15.3±0.8 CFU/IRarea) only in injured myocardium. In MMP-9 null IR mice, sSCF did not increase, c-kit was not phosphorylated, and EPCs were not mobilized (D1/D0: 65±7.4%, P=NS) or traffic to the heart (0±0 CFU/IRarea) for up to 7 days. Conclusion: MMP-9 mediated cleavage of SCF and c-kit phosphorylation in the bone marrow is necessary for EPC mobilization. Signaling through c-kit on EPC may allow EPC mobilization without concomitant mobilization of c-kit(-) mature inflammatory cells, potentially permitting restoration of post ischemic cardiac function without adverse vascular events.
5008 Optimized Therapeutic Angiogenesis for Peripheral Ischemia Using Stem Cells Transduced With an Ischemia-Regulated Conditionally Silenced AAV
Keith A. Webster, Matthew J. Gounis, Maria-Grazia Spiga, Regina M. Graham, Shannon Haliko, Amber Wilson, Baruch B. Lieber, Nanette H. Bishopric, Ajay K. Wakhloo, University Miami, Miami, FL
Therapeutic angiogenesis is being tested as a treatment for peripheral and coronary artery diseases. Encouraging results from preclinical studies have not been well reproduced in the more than 1000 patient trials that have been completed. These trials have focused on different isoforms of VEGF or FGF delivered by direct injection of proteins or genes. The latter include plasmid and adenoviral vectors in which expression of the cloned gene is directed by the strong cytomegaloviral promoter. This approach has been questioned because of the transient nature of the therapy and the absence of directional cues for new vessel growth. To address these problems we created AAV vectors where expression of human VEGF165 is directed by a glycolytic enzyme promoter containing a combination of transcriptional silencers and hypoxia-response elements such that expression is restricted to ischemic tissue. The differential of expression in ischemic versus non-ischemic tissue is >200-fold (n=6). Therapeutic angiogenesis was evaluated in the rabbit ischemic hindlimb model comparing the impact of Ad-VEGF, AAV-VEGF, and autologous stem cells transduced with AAV-VEGF. Vessel growth was measured angiographically and by immunohistology. Limbs injected with Ad-VEGF displayed rapid proliferation of endothelial cells evidenced by strong Ki67 staining 2–3 days after therapy (n=4). Angiography showed an intense blush indicative of numerous disorganized capillaries at 1-week and this was supported by anti-CD31 immunostaining. VEGF expression was restricted to the first week of therapy, apoptosis accompanied angiogenesis and all parameters returned to baseline within 3-weeks (n=6). AAVs expressing unregulated or regulated VEGF (AAV-rVEGF) were delivered by vascular infusion and mediated dramatically different results compared with Ad-VEGF. Serum VEGF was low to undetectable but new vessels were produced without significant blush and with an organized directional character (n=3). Injection of autologous stem cells transduced with AAV-VEGF or AAV-rVEGF stimulated angiogenesis and arteriogenesis again without blush (n=3). The results indicate that rAAV alone or transduced into stem cells provides an optimized therapy that may provide a treatment for limb ischemia.
5009 Activation of a Cell Survival Program During Post-Ischemic Heart Failure in Monkeys
You-Tang Shen, Lin Yan, Anthanasios Peppas, Franco Rossi, Dorothy E. Vatner, Junichi Sadoshima, Stephen F. Vatner, Christophe Depre; UMDNJ—NJ Medical School, Newark, NJ
Heart failure (HF) following myocardial infarction (MI) is characterized by increased apoptosis and cell death. We hypothesized that the failing heart activates cytoprotective and anti-apoptotic pathways to limit the extent of apoptosis and necrosis. Specifically, we investigated both by genomics (DNA micro-array) and proteomics (two-dimensional gel electrophoresis followed by mass spectrometry) the regulation of two major pro-survival mechanisms, i.e., anti-apoptosis and autophagy, during post-ischemic HF in a novel non-human primate model, where the changes in genes and proteins should resemble more closely those observed in human HF. MI was induced in 7 monkeys (M. Fascicularis) by ligation of the left anterior descending coronary artery. Eight weeks after MI, the monkeys underwent a three-week protocol of pacing-induced (270 bpm) HF. Four sham monkeys were used as controls (ctrl). Left ventricular dP/dt, an index of cardiac contractility, was significantly reduced in HF versus ctrl (1,750±79 vs 4,251±336 mmHg/sec; P<0.05). Compared to ctrl, the HF group was characterized by a significant (P<0.05) 2- to 4-fold upregulation of anti-apoptotic and cytoprotective molecules (Bcl2, BclXL,inhibitor of apoptosis XIAP, defender against cell death DAD-1, heat-shock proteins HSP70, HSP40, agp-2 and H11 kinase). In addition, the HF group showed a significant (P<0.05) 2- to 4-fold increase of proteins stimulating cellular autophagy (Beclin 1, cathepsin B, Hsc73), another mechanism which promotes cell survival by mediating protein turn-over. Therefore, in contrast to the predicted increase in markers of apoptosis in HF, in a non-human primate model of post-ischemic HF, genes and proteins stimulating apoptosis and cell death were not upregulated. Actually, a diametrically opposite pattern was observed, i.e., activation of pathways participating in anti-apoptosis, cytoprotection and autophagy. Activation of these cytoprotective pathways could act as an important internal defense mechanism in HF, protecting the heart by limiting the extent of cell death.
5010 β2-Adrenergic Receptors Selectively Prevent In Vivo Murine Heart Failure
Anthony Lemaire, Antonio Curcio, Takahisa Noma, Matthew J. Wolf, Duke University Medical Center, Durham, NC; Brian K. Kobilka, Stanford University, Stanford, CA; Howard A. Rockman, Duke University Medical Center, Durham, NC
β-Adrenergic receptors (βARs) are essential in the regulation of the cardiovascular system. Stimulation of β1AR and β2AR subtypes has been shown to activate differential signaling pathways in cardiac myocytes. We tested whether the different properties of the βAR subtypes would lead to a functional difference in vivo for the development of heart failure. Myocardial infarction was induced by coronary ligation in 12-week old wild type (WT) mice and βAR knockout (KO) mice of each subtype: β1ARKO, β2ARKO, and β1AR/β2ARKO (double KO). Cardiac function was measured by serial echocardiography prior to, and then 4, and 8 weeks post-infarction. At 8 weeks, mice were terminated and their hearts removed for histologic and biochemical analysis. Infarct size was similar for all groups, WT (31%±8), β1ARKO (34%±3), β2ARKO (31%±3), and β1AR/β2ARKO (34%±3). At 8 weeks post-MI, β1ARKO mice showed only a 4% decrease in fractional shortening (FS) compared to either the β2ARKO (-40%) or WT (-38%) mice. Only mild LV dilatation at 8 weeks post-MI occurred in the β1ARKO mice (9% increase), whereas marked LV enlargement was seen in the β2ARKO (32%), double KO (21%), and WT (22%) mice. Interestingly, the double KO mice showed a similar decline in %FS as the β2ARKO and WT mice, suggesting that signaling via the β1AR subtype is not the primary pathologic stimulus for cardiac failure. Histologic studies showed significant apoptosis in the β2ARKO mice compared to WT mice. These data demonstrate that β2AR stimulated pathways are protective and preserve cardiac function after MI. Understanding the specific signaling pathways activated by the βAR subtypes may provide insight into novel therapeutic approaches.⇓
5011 Magnetic Targeting and Localization of Endothelial Cells on Implanted Stents
Gurpreet S. Sandhu, Rajiv Gulati, Sorin V. Pislaru, Nicole P. Sandhu, Tyra A. Witt, Robert D. Simari, Mayo Clinic College of Medicine, Rochester, MN
An inability to retain cells at desired vascular sites has limited the development of cell based vascular therapies. We hypothesized that a magnetized stent would be able to capture and retain cells labeled with paramagnetic particles. Outgrowth endothelial cells cultured from rabbit blood were allowed to endocytose green fluorescent labeled superparamagnetic iron oxide (SPIO) particles (Bangs Labs) for 16 hours. Cell membranes were labeled with CM-DiI. SPIO labeled cells remained viable and proliferative. A 0.008” steel wire stent was magnetized with a Neodymium supermagnet. Labeled cells (200,000/ml) were added to the stent in vitro and observed with light microscopy. The magnetized stent rapidly attracted cells up to 5 mm away. Confocal microscopy confirmed surface coverage with labeled cells. The cells did not attach to a control degaussed stent. To address the effect of shear forces of circulating blood, we implanted magnetic stents in rabbit carotid arteries. These stents were delivered via an arteriotomy, and labeled cells were instilled locally while flow was occluded 10 minutes. Pulsatile flow and patency were then confirmed and the vessels were harvested at 3 hours. Confocal microscopy of the explanted struts revealed a layer of double labeled cells attached to the lumen face of the struts (Fig 1). Conclusions: Magnetic force can be used to directly apply paramagnetically labeled cells to metallic surfaces. This biophysical force is capable of capturing and retaining cells within arterial segments. Applications of this technique may include rapid endothelialization of implantable vascular devices, including stents, grafts and ventricular assist devices.
5012 S-Nitrosylation of the L-Type Calcium Channel Alpha 1 Subunit Contributes to Male-Female Differences in Ischemia-Reperfusion Injury Under Adrenergic Stimulation
Junhui Sun, National Institute of Environmental Health Sciences, Research Triangle Park, NC; Charles Steenbergen, Department of Pathology, Duke University Medical Center, Durham, NC; Elizabeth Murphy, National Institute of Environmental Health Sciences, Research Triangle Park, NC
Gender differences in cardioprotection have been observed and a role for nitric oxide (NO) signaling has been suggested based on pharmacological inhibitors, but the mechanism is unclear. In this study, we report evidence that S-nitrosylation of the L-type Ca2+ channel may be an important component of female cardioprotection. We find that female wild-type (WT) C57BL/6J mouse hearts have significantly better recovery of left ventricular developed pressures (LVDP) than males following addition of isoproterenol just prior to 20 minutes of ischemia and 40 minutes of reperfusion. Female hearts from mice lacking eNOS (eNOS−/-) or nNOS (nNOS−/-) did not show improved LVDP recovery of postischemic function, suggesting a role for both NOS isoforms in cardioprotection in females. Following ischemia-reperfusion under adrenergic stimulation, WT females have significantly higher S-nitrosylation of the L-type Ca2+ channel α1 subunit than WT males. Consistent with the lack of a male-female difference in ischemia-reperfusion injury in eNOS−/- and nNOS−/- hearts, there is no male-female differences in S-nitrosylation of the L-type Ca2+ channel in either eNOS−/- or nNOS−/- hearts. We find also that cardiomyocyte-specific caveolin-3-associated eNOS was higher in females than males. We further show that ischemia and reperfusion following isoproterenol results in increased association of nNOS and caveolin-3, suggesting a translocation of nNOS to the plasma membrane, and this association was significantly higher in females than males. These are the first data to directly demonstrate S-nitrosylation of the L-type Ca2+ channel and to show a male-female difference in S-nitrosylation of the L-type Ca2+ channel. Thus, S-nitrosylation of the L-type Ca2+ channel by both eNOS and nNOS may play a critical role in cardioprotection in females.
5013 The Transcriptional Coactivator PGC-1α Regulates Cardiac Mitochondrial Function and Dictates the Supply of ATP Available for Contractile Reserve
Zolt P. Arany, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; Huamei He, Harvard Medical School, Boston, MA; Jiandie Lin, Dana-Farber Cancer Institute, Boston, MA; Kirsten Hoyer, Frederick J. Schoen, Joanne Ingwall, Harvard Medical School, Boston, MA; Bruce Spiegelman, Dana-Farber Cancer Institute, Boston, MA
The beating heart depends on the constant generation of ATP in mitochondria. PGC-1α is a transcriptional coactivator that powerfully regulates mitochondrial biogenesis and function in many organs, but the need for PGC-1α in the heart remains unclear. We hypothesized that PGC-1α is critical in vivo for mitochondrial gene expression and function. We further hypothesized that, if so, PGC-1α is also critical for normal contractile activity. To address these hypotheses, we examined PGC-1α “knockout” (KO) mice generated by homologous recombination. At 3 months, hearts appeared grossly normal by weight and standard histology. Quantitative electron microscopy revealed a normal mitochondrial architecture and volume fraction in cardiac myocytes. Hence, PGC-1α is not required for cardiac mitochondrial biogenesis in vivo. Despite this, however, the expression of genes of mitochondrial biology was almost uniformly depressed by 30 to 50% in KO vs WT hearts, as assayed by PCR and gene profiling. The activities of key mitochondrial enzymes, cytochrome c oxidase and citrate synthase, were also depressed (by 30%, p=.047, and 37%, p=.008, respectively). Hence, PGC-1α is needed for normal mitochondrial gene expression and function. Furthermore, KO hearts had an 18% reduction in ATP levels, as measured by NMR spectroscopy on beating, perfused (Langendorff) hearts. Such a reduction is comparable to that seen in failing hearts. Hence, PGC-1α is required for a normal energy state in the heart. We next measured cardiac function using Langendorff hearts. KO hearts had slightly reduced contractility at baseline, but this was markedly accentuated when challenged with dobutamine (e.g., 20% decrease in +dP/dt, p=.004). At peak challenge, KO hearts recruited only half the increase in contractile reserve as did WT hearts (p=.0003). Hence, PGC-1α is vital for cardiac contractile reserve. Finally, genes typically induced in heart failure were also induced in KO hearts, and serum concentrations of proANP were elevated in KO mice (2-fold, p=.016), consistent with significant cardiac dysfunction in vivo. We conclude that the transcriptional coactivator PGC-1α regulates cardiac mitochondrial function in vivo, and thereby dictates the supply of ATP available for contractile reserve.
5015 Presenilin 2 Directly Interacts with Cardiac Ryanodine Receptor and Regulates the Systolic Function of Heart Muscle
Toshihiro Takeda, Kinya Otsu, Michio Asahi, Osamu Yamaguchi, Michihiko Tada, Masatsugu Hori, Osaka University Graduate School of Medicine, Suita, Japan
In brain, mutations in presenilin 2 (PS2) have been shown to cause familial Alzheimer’s disease. Since PS2 has been shown to interact with sorcin, a penta-EF-hand Ca2+-binding protein, which serves as a modulator of cardiac ryanodine receptor (RyR2), we tested whether PS2 interacts with RyR2 and have a role in excitation-contraction coupling in the heart. Co-immmunoprecipitaion analysis of PS2 with sorcin or RyR2, co-expressed in HEK293 cells, showed that PS2, sorcin and RyR2 interacted each other. The interaction was also observed in mouse heart as well as brain. Immunohistochemistry of heart muscle indicated that PS2 co-localized with RyR2 and sorcin at the Z-lines. The further co-immunoprecipitation study using deletion mutants of PS2 indicated that both C-terminal and N-terminal fragments of PS2 interact with RyR2. Elevated Ca2+ attenuated the association of RyR2 with PS2, whereas the association of sorcin with PS2 was enhanced. PS2KO mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. Invasive hemodynamic analysis revealed that cardiac contractility was increased in PS2KO mice compared to littermate control. The study of isolated papillary muscle showed that peak amplitudes of Ca2+ transients and peak tension were significantly higher in PS2KO mice compared to those in littermate controls. The enhanced Ca2+ transients and contractility in PS2KO mice were observed at low Ca2+, but not at high Ca2+. These results suggest that the activity of RyR2 might be regulated through the Ca2+-dependent dynamic changes of the PS2/sorcin/RyR2 interaction. In conclusion, PS2 interacts with RyR2 in the heart and play an important role in excitation-contraction coupling in the heart.
5016 Endoplasmic Reticulum-Initiated Apoptosis via CHOP/GADD153-Dependent Pathway Contributes to the Development of Heart Failure
Tetsuo Minamino, Ken-Ichiro Okada, Osamu Tsukamoto, Masatsugu Hori, Osaka University Graduate School of Medicine, Suita, Japan; Masafumi Kitkaze, National Cardiovascular Center, Suita, Japan
Endoplasmic reticulum (ER) is recognized as an organelle that participates in protein folding. ER adaptively responds to stresses such as ischemia, oxidative stress, or increased protein synthesis by up-regulating ER chaperones and blocking protein synthesis. However, prolonged and/or excess overload to ER lead to the ER-initiated apoptotic cell death. Since oxidative stress and/or increased protein synthesis, both of which potentially cause ER dysfunction, are observed in hypertrophic and failing hearts, we assessed the hypothesis that the ER-initiated apoptosis due to prolonged ER stress contributes to the development of heart failure. First, we performed immunohistological analysis to clarify whether ER stress was induced in failing human hearts. The proliferation of the ER was prominently observed in the perinuclear area in failing, but not control, human hearts. Furthermore, immunohistochemical analysis revealed that GRP78, an ER stress marker, and CHOP/GADD153, a pro-apoptotic transcription factor that specifically mediates ER-initiated apoptosis, were induced in heart samples of patients with dilated cardiomyopathy, but not of control subject. Then, we assessed the role of prolonged ER stress in the development of heart failure in the experimental model. Mice were subjected to transverse aortic constriction (TAC) or sham operation. Echocardiographic analysis demonstrated that mice 1 and 4 weeks after TAC had cardiac hypertrophy and failure, respectively. GRP78 expression and spliced form of XBP1, another ER stress marker, in hearts continuously increased 1 week after TAC, indicating that pressure overload induced prolonged ER stress. Along with the appearance of cardiac apoptosis in failing mice hearts, CHOP/GADD153 was induced in failing, but not hypertrophic, mice hearts. Pharmacological ER stress inducers up-regulated ER chaperones and induced apoptosis in cultured adult cardiac myocytes, which was prevented by the posttranslational gene silencing of CHOP/GADD153 by RNA interference method. In conclusion, ER stress was observed in failing human hearts and the ER-initiated apoptosis may contribute to the progression from cardiac hypertrophy to failure via CHOP/GADD153-dependent pathway.
5017 Blockade of Brain Renin-Angiotensin-Aldosterone System Inhibits Cardiac Aldosterone and Remodeling Post-Myocardial Infarction
Avtar Lal, Univ Of Ottawa Heart Inst, Ottawa, Canada; John P. Veinot, Ottawa Hospital, Ottawa, Canada; Detlev Ganten, Max-Delbruck Centre for Molecular Medicine, Berlin-Buch, Germany; Frans Leenen, Univ Of Ottawa Heart Inst, Ottawa, Canada
Introduction: Following myocardial infarction (MI), aldosterone and interstitial fibrosis in the left ventricle increase. Transgenic rats (TGR) deficient in brain angiotensinogen and Wistar rats with central infusion of the aldosterone antagonist, spironolactone show marked attenuation of cardiac fibrosis and remodeling post-MI. Hypothesis: The improvements in cardiac remodeling post-MI by blockade of brain renin-angiotensin-aldosterone system may be due-in part-to prevention of the increase in cardiac aldosterone. Methods: MI was induced by acute coronary artery ligation in TGR and control Sprague-Dawley rats (SDR) and in Wistar rats. The effects of MI on cardiac aldosterone and on parameters of cardiac remodeling and dysfunction were evaluated at 8 weeks in TGR and SDR and at 6 weeks post-MI in Wistar rats which were administered vehicle or spironolactone by icv infusion (100ng/hr). Results:Post-MI, LVEDP, aldosterone and interstitial fibrosis in the non-infarcted part of left ventricle showed clear increases in SDR and Wistar rats. The increase in LVEDP was markedly attenuated and the increases in cardiac aldosterone and interstitial fibrosis were prevented in TGR and by icv spironolactone in Wistar rats. Conclusions: We conclude that post-MI the brain renin-angiotensin-aldosterone system activates a cascade of events, among others an increase in cardiac aldosterone, which plays a major role in cardiac remodeling and progressive left ventricular dysfunction. Table: LVEDP (mm Hg) and aldosterone (pg/mg), interstitial fibrosis (septum, %) in left ventricle.
5018 The Beneficial Effects of Postinfarct Cytokine Therapy Are Sustained During Long-term Follow-up
Adam B. Stein, Buddhadeb Dawn, Sumit Tiwari, Yiru Guo, Greg Hunt, Yiming Huang, Arash Rezazadeh, Suzanne T. Ildstad, Roberto Bolli, University of Louisville, Louisville, KY
The efficacy of cytokine therapy in improving left ventricular (LV) function and remodeling after myocardial infarction remains controversial, and the long-term results of such regenerative therapy remain unknown. Accordingly, we sought to determine the effect of postinfarct cytokine therapy on LV function over a longer duration of follow-up. Three groups of mice underwent a 30-min coronary occlusion followed by reperfusion. Mice in groups II (n=9) and III (n=10) received daily s.c. injections of granulocyte-colony stimulating factor (G-CSF)+Flt-3 ligand (FL) or G-CSF+stem cell factor (SCF) starting 4 h after reperfusion. Group I (control, n=8) received equivalent doses of vehicle. LV function and anatomy were assessed by serial echocardiography before and at 48 h, 4 wk, 8 wk, and 16 wk after MI. LV fractional shortening (FS), ejection fraction (EF), and the fractional area change (FAC) were similar before MI and significantly and similarly reduced in all groups at 48 h after MI. At 4 wk, 8 wk, and 16 wk after MI, mice in group I showed progressive deterioration in LV function (Figure) and LV dilatation. In contrast, mice in group II exhibited significantly (P<0.05) higher FS, EF, and FAC at 4 wk, 8 wk, and 16 wk after MI (Figure). Mice in group II demonstrated less dilatation of LV. Mice in group III showed a trend toward improved LV function as compared with group I (Figure). We conclude that postinfarct cytokine therapy, particularly with G-CSF+FL, results in improved LV function that is sustained for at least 4 months after myocardial infarction. These findings support potential future clinical application of cytokine therapy following MI in humans.
5019 Gene Transfer of Extracellular Superoxide Dismutase Protects Against Vascular Dysfunction with Aging
Kathryn A. Brown, Yi Chu, University of Iowa, Iowa City, IA; Donald D. Lund, Veterans Affairs Medical Center, Iowa City, IA; Donald D. Heistad, Frank M. Faraci, University of Iowa, Iowa City, IA
Aging is an independent risk factor for cardiovascular disease, although the exact mechanisms leading to vascular dysfunction have not been fully elucidated. Recent studies suggest that oxidative stress, including superoxide levels, may increase in blood vessels during aging. Levels of superoxide are influenced by the activity of endogenous superoxide dismutases. The goal of this study was to examine the role of extracellular superoxide dismutase (ECSOD) on vascular function and superoxide levels in an animal model of aging. Aortas from young (4–8 months; n=16) and old (29–31 months; n=14) Fischer 344 rats were examined in vitro. Relaxation of aorta to the endothelium-dependent dilator acetylcholine (ACh) was significantly impaired in old rats compared to young rats (e.g. 3 μM ACh produced 57±4% and 84±2% relaxation in old and young rats, respectively; p<0.0001). Three days after intravenous injection of an adenoviral vector expressing human ECSOD (AdECSOD), the response to ACh was improved in old rats (e.g. 3 μM ACh produced 75±3%; n=14; p=0.005) when compared to untreated old rats. There was no difference in relaxation to the endothelium-independent dilator sodium nitroprusside between young, old, and AdECSOD-treated old rats. Superoxide levels, as measured using lucigenin (5 μM) enhanced chemiluminescence, were significantly increased in old rats when compared to young rats; after treatment of old rats with AdECSOD, superoxide levels in aorta were similar in old and young rats. Injection of AdCMVECSODΔHBD, an ECSOD with the heparin-binding domain deleted, had no effect on vascular function or superoxide levels in old rats. These results suggest the following: 1) vascular dysfunction associated with aging is mediated in part by increased levels of superoxide 2) gene transfer of extracellular superoxide dismutase reduces vascular superoxide and dysfunction in old rats, and 3) beneficial effects of ECSOD in old rats require the heparin-binding domain of ECSOD.
5020 Cardiomyocyte-Specific Expression of the Coxsackievirus and Adenovirus Receptor Is Essential for Normal Left Ventricular Development
H. Scott Baldwin, Vanderbilt University, Nashville, TN; Jin-Wen Chen, Children’s Hospital of Philadelphia, Philadelphia, PA; Bin Zhou, Vanderbilt University, Nashville, TN; Jason Shin, Children’s Hospital of Philadelphia, Philadelphia, PA; Michael D Schneider, Baylor College of Medicine, Houston, TX; Jeffrey M Bergelson, Children’s Hospital of Philadelphia, Philadelphia, PA
The coxsackievirus and adenovirus receptor (CAR) is a 46kD integral membrane protein that serves as a receptor for the two viruses most commonly associated with myocarditis. CAR is a transmembrane component of specialized intercellular junctions, including the myocardial intercalated disc; it mediates homotypic cell recognition, and in tumor cells it has been implicated in cell cycle regulation. We used conditional gene targeting to investigate CAR’s possible function during embryonic development. Mice were generated in which CAR exon 2, encoding the molecule’s leader sequence and N-terminus, was flanked by loxP sites (“floxed”). CAR-floxed mice were bred to protamine-Cre transgenics, resulting in germline deletion of exon 2. Homozygous germline deletion of CAR exon 2 (CAR-null) resulted in death by embryonic day 12.5 (E12). Gross examination of embryos at E10 and E11 showed selective bulging of the left ventricle, and engorgement of the cardinal veins. Histologic examination revealed marked hyperplasia of trabecular cardiomyocytes within the left ventricular wall, consistent with accentuated expression of mCAR in the primitive LV as determined by in situ hybridization. In CAR-null animals, 87% (s.d. 9%) of left ventricular cardiomyocytes expressed Ki67 nuclear antigen, a marker of cell proliferation; in contrast, in CAR+/+ littermates, Ki67 was expressed by only 42% (s.d. 6%) of ventricular cardiomyocytes. Interestingly, no defects were observed in the primitive right ventricle, right ventricular outflow tract, or other derivatives of the secondary heart field. Early cardiomyocyte-specific deletion of CAR using transgenic expression of Cre under control of the cardiac troponin T-promoter resulted in a phenotype identical to that seen in CAR-null animals. However, later deletion of mCAR mediated by alpha-MHC Cre did not adversely affect embryonic viability, suggesting an early developmental requirement for CAR-mediated signaling. These results indicate that CAR expression is essential for normal chamber-specific development of the embryonic heart, and suggest that CAR-mediated intercellular interactions regulate cardiomyocyte proliferation during embryonic development.
5021 Structural-Functional Correlation of Two Distinct RYR2 Gene Mutations Linked With Two Phenotipical Inherited Idiopathic Dilated Cardiomyopathy
Guanggen Cui, Luyi Sen, UCLA Medical Ctr Sch Med, Los Angeles, CA
Previously we reported 9 missense point mutations in cardiac ryanodine receptor (RYR2) that have been linked to familial idiopathic dilated cardiomypathy (FIDM). We found that each distinct point missese mutation is responsible for a phenotypical dilated cardiomyopathy. However, the structural-functional relation of these mutations still remains to be revealed. Here we report two of these missense point mutations that are responsible for the distinct abnormalities of the sarcoplasmic reticulum (SR) Ca++ release channel (CRC) properties in two FIDM phenotypes. Both mutations occurred in a functional important that has many CRC regulating protein binding sites. One of them (A2799L) was found co-segregates with the same type of idiopathic dilated cardiomyopathy in 5 families, that is manifested by a predominant impairment in ventricular systolic function, with or with moderate diastolic dysfunction, onset in various age range, without severe arrhythmias. This mutation has a high degree of penetrance. Another missense mutation (L2537S) in this region occurred in 2 families, that shows a relatively low degree of penetrance, and associated with a phenotype of late onset and slowly progressed dilated cardiomyopathy. The full length of wild type or mutant human cardiac CRC cDNAs was functionally expressed in the xenopus oocyte,then fused into the planner lipid bilayer to characterized the single channel properties. The mutant CRC with A2799L mutation mainly caused the frequent transition to subconductance and increase in long-closure time of CRC. The channel open properbility at the full opening state was reduced (47%) compared with that in the wild type CRC (p<0.05). This abnormality was the same as that observed in SR CRC isolated from two probands. The mutant CRC with L2537S mutation has no significant change in the single channel properties per se. Instead, the channel responsiveness to Ca++ and ryanodine was significantly retarded. The dose-response curves for Ca++ and ryanodine were significantly shifted to the right (p<0.05). Our results indicate that the different point mutation is responsible for the distinct functional abnomality of CRC channel and phenotypical dilated cardiomypathy.
5022 RNA Interference Reveals Inhibitory Role of Endogenous RGS2 Protein in Gq/11-mediated Signaling and Hypertrophy in Cardiac Myocytes
Wei Zhang, Jialin Su, Jianming Hao, Xiaomei Xu, Ulrike Mende, Cardiovascular Division, Brigham and Women’s Hospital & Harvard Medical School, Boston, MA
Enhanced Gq/11-mediated signaling in the heart can lead to development and ultimate decompensation of hypertrophy. Regulators of G protein Signaling (RGS) that negatively regulate cardiac Gq/11 signaling could thus be critical modulators of hypertrophy. We recently showed that adenovirally expressed RGS2 potently inhibits Gq/11-mediated phospholipase Cβ (PLCβ) activation and hypertrophy in neonatal and adult ventricular myocytes (NVM and AVM). To date, RGS function has mainly been assessed with recombinant proteins and by heterologous overexpression. The goal of the present study was to determine the functional role of endogenous RGS2, which is of particular importance in light of its low abundance and the presence of other cardiac RGS proteins with potentially overlapping functions. We generated two effective RNA interference (RNAi) constructs targeting adjacent sequences within the RGS2 core domain. Western blots and immunostaining showed that these two constructs almost completely inhibited the expression of co-transfected RGS2 in HEK293, whereas GAPDH and negative RNAi controls (Ctr) had no effect. Importantly, expression of closely related RGS proteins (RGS3, RGS4, RGS5) was unaffected. After optimizing lipid-based transfection conditions, we achieved efficient (>95% of cells) and homogenous uptake of fluorescently labeled siRNA and effective silencing of endogenous RGS2 in NVM and AVM with both RNAi constructs, as shown by RT-PCR. Despite unimpeded presence of RGS3-RGS5, PLCβ activation with phenylephrine and endothelin-1 was enhanced in NVM by 22±5% and 87±12% of Ctr and in AVM by 22±1% and 72±7% of Ctr, resp. Stimulation with either Gq/11-coupled receptor agonist was accompanied by increased protein synthesis (1.6 to 1.9-fold) and cell size in NVM. These data show that endogenous RGS2 negatively regulates Gq/11 signaling and hypertrophy in cardiomyocytes. They suggest that a marked RGS2 down-regulation, which we previously described in hearts with enhanced Gq/11signaling (such as in pressure overload), likely further exacerbates Gq/11signaling and may thereby play a critical role in hypertrophy development in vivo. Furthermore, this study demonstrates the feasibility of RNAi for RGS proteins and in NVM and AVM.
5024 NFkB Represses Hypoxia-Induced Mitochondrial Defects and Cell Death of Ventricular Myocytes
Kelly Regula, Delphine Baetz, Floribeth Aguliar, Lorrie Kirshenbaum, St. Boniface General Hospital Research Centre, Winnipeg, Canada
In this report we provide evidence for the operation of the cellular factor NFκB as a key regulator of the mitochondrial function and the cell death of ventricular myocytes during hypoxia. In contrast to normoxic control cells, ventricular myocytes subjected to hypoxia displayed a 9.1 fold increase (p<0.05) in apoptosis as determined by Hoechst 33258 nuclear staining and vital dyes. Mitochondrial defects consistent with permeability transition pore (PTP)opening, loss of mitochondrial Δψm, and Smac release were observed in cells subjected to hypoxia. This was accompanied by a concomitant increase in the post-mitochondrial caspase 9 and caspase 3 activity in hypoxic myocytes. Adenovirus mediated delivery of wild type IKKb (IKKbwt) resulted in a significant increase in NFkB dependent DAN and gene transcription in ventricular myocytes. Interestingly, cells rendered defective for NFkB activation with a kinase defective IKKbK-M (IKKbmt) or a non-phosphorylatable form of IkBa were sensitized cells to mitochondrial perturbations and hypoxic injury. Hypoxia-induced, mitochondrial defect and cell death were suppressed in cells expressing IKKbwt but in not cells expressing the kinase defective IKKb. To our knowledge, the data provide the first direct evidence that IKKb-mediated NFkB activation suppresses hypoxia-induced cell death of ventricular myocytes through a mechanism that impinges upon the mitochondrial death pathway.
5025 Loss of Fine-Tuning of Gq/11 Signaling: a Potential Novel Mechanism for Cardiac Hypertrophy
Jianming Hao, Wei Zhang, Christina Michalek, Xiaomei Xu, Ulrike Mende, Brigham and Women’s Hospital, Boston, MA
Signal transduction via heterotrimeric G proteins is crucial for normal cardiac function, and altered G protein signaling can lead to the development of cardiac hypertrophy and failure. RGS proteins are negative Regulators of G protein Signaling that likely play a central role in fine-tuning cardiac signaling. Several RGS proteins are expressed in the heart, but little is known about their functional role. We compared the effectiveness of the main cardiac RGS proteins (RGS2, RGS3, RGS4, RGS5, RGS16) in regulating G protein signaling in both neonatal and adult rat ventricular cardiac myocytes (NVM and AVM) using adenoviral gene transfer. Each RGS protein tested exerted a marked inhibitory effect on Gq/11-mediated phospholipase C β activation, a pathway known to lead to cardiac hypertrophy. Accordingly, the hypertrophic response of NVM to Gq/11-coupled receptor stimulation with phenylephrine was blunted by each RGS by over 90%. While RGS3, RGS4, RGS5 and RGS16 also blunted carbachol-induced, Gi/o-mediated inhibition of prestimulated cAMP accumulation by over 80%, RGS2 had no effect. None of the RGS proteins had an effect on isoproterenol-induced, Gs-mediated cAMP accumulation and did not alter adenylyl cyclase directly, as shown by a normal forskolin response. Together, these data demonstrate that RGS2 is a selective inhibitor of Gq/11 signaling in cardiomyocytes, whereas the other cardiac RGS proteins regulate both Gq/11 and Gi/o signaling. We previously showed a selective down-regulation of RGS2 protein in hearts with enhanced Gq/11 signaling (such as in pressure overload) prior to the onset of hypertrophy. In AVM, RGS2 expression was enhanced at 36 hrs, but reduced at 72 hrs in response to enhanced Gq/11 signaling (i.e., phenylephrine or expression of constitutively active Gαq) or activation of protein kinase C by phorbolester. This biphasic response suggests desensitization of RGS2 regulation to continuously elevated Gq/11 signaling. Our results suggest that cardiac RGS proteins play different roles in the heart with RGS2 specifically targeting Gq/11 signaling. A blunted RGS2 inhibitory effect on Gq/11 signaling likely participates in the pathogenesis of cardiac hypertrophy, suggesting a potential novel pharmacological target.