Cardiac Lysates Research Articles

Rearrangement of the Protein Phosphatase 1 Interactome During Heart Failure Progression.

Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors. Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%-40%), and severe HF (ejection fraction <30%). Cardiac lysates were subjected to affinity purification with anti-PP1c antibodies followed by high-resolution mass spectrometry. PP1 regulatory subunit 7 (Ppp1r7) was knocked down in mouse cardiomyocytes and HeLa cells with adeno-associated virus serotype 9 and siRNA, respectively. Calcium imaging was performed on isolated ventricular myocytes. Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum. PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular "sponge" of PP1c.

Abstract 528: Cell Specific Signaling Regulated by PI3Kγ Modulates Myofibroblast Differentiation

Phosphoinositide 3 Kinase γ (PI3Kγ) regulates anti-apoptotic Akt signaling. Previous studies have established a role for PI3Kγ in cardiac fibrosis, a key underlying cause of heart failure. However, less is known about the mechanism by which PI3Kγ regulates cardiac myofibroblast differentiation, hallmark of tissue fibrosis, characterized by smooth muscle α-actin (αSMA) overexpression. Measurement of αSMA abundance in cardiac lysates from PI3Kγ null mice (PI3Kγ -/- ) showed significant baseline and pressure overload induced upregulation compared to wildtype (WT), indicating that loss of PI3Kγ predisposes the hearts towards fibrosis. Furthermore, isolated cardiac fibroblasts (CF) from PI3Kγ -/- exhibited a myofibroblast phenotype with αSMA in stress fibers. Correspondingly, immunoblotting showed significantly higher αSMA in PI3Kγ -/- CF than WT. It has been previously shown that fibroblast growth factor mediated activation of the signaling pathway downregulates αSMA and that this inhibition of αSMA expression is through negative regulation by extracellular regulated kinase (ERK). To understand whether PI3Kγ regulates ERK signaling in a cell specific manner and thereby possibly αSMA, the phosphorylation of ERK by insulin stimulation were compared in CF isolated from WT and PI3Kγ -/- . Intriguingly, there was significant loss of ERK phosphorylation in CF from PI3Kγ -/- when compared to CF from WT. However, ERK phosphorylation was not altered in cardiomyocytes (CM) due to the absence of PI3Kγ. Confirming this differential regulation of ERK in CM and CF, there was no change in the association of ERK and protein phosphatase 2A (PP2A) in CM of WT and PI3Kγ -/- . However, there was increased association of PP2A with ERK in CF of PI3Kγ -/- when compared to CF from WT. This is because in the CM, ERK is regulated by Dual Specificity Phosphatase (DUSP8). However, in the CF, PP2A is the major regulator of ERK and thus PI3Kγ plays a major role in ERK signaling in CF. Consistent with previous observations, we found that association of DUSP8 with ERK was not observed in CF. Moreover, ERK-DUSP8 interaction was not dependent on the presence or absence of PI3Kγ. These data indicate that PI3Kγ regulates signaling pathways in a cell specific manner with respect to cardiac remodeling.

Abstract 109: PI3Kγ Regulates Release of Myocyte-derived Factors Responsible for Myofibroblast Differentiation and Cardiac Fibrosis

Phosphoinositide 3 Kinase γ (PI3Kγ) is a lipid kinase that regulates downstream anti-apoptotic Akt signaling. Thus, pressure overload in PI3Kγ null (PI3Kγ -/- ) mice leads to significant cardiac fibrosis, a key underlying cause of fatal heart failure. Classical hallmark of tissue fibrosis is differentiation of fibroblasts to myofibroblasts characterized by smooth muscle α-actin (αSMA) overexpression. However, less is known about the role of PI3Kγ in cardiac myofibroblast differentiation. Assessment of αSMA expression in cardiac lysates from WT and PI3Kγ -/- showed significant baseline upregulation in PI3Kγ -/- showing that loss of PI3Kγ predisposes the hearts towards fibrosis. To directly confirm that PI3Kγ -/- cardiac fibroblasts (CF) exhibit a myofibroblast phenotype, CF were isolated from hearts of WT and PI3Kγ -/- and assessed by immunostaining for αSMA in stress fibers. Greater number of CF from PI3Kγ -/- exhibited αSMA in stress fibers than CF from WT. Correspondingly, immunoblotting showed significantly higher expression of αSMA in PI3Kγ -/- CF compared to WT showing enhanced myofibroblast differentiation by PI3Kγ -/- fibroblasts. Surprisingly, abundance of αSMA protein is significantly reduced in the hearts of mice with cardiomyocyte-specific expression of kinase-dead PI3Kγ (PI3Kγ inact ) in the PI3Kγ -/- (PI3Kγ inact /PI3Kγ -/- ) suggesting that myocytes derived factors responsible for myofibroblast differentiation are regulated by kinase-independent function of PI3Kγ. To directly evaluate the PI3Kγ-dependent cardiomyocyte derived factors responsible for myofibroblast differentiation; fibroblasts were treated with conditioned media derived from primary adult cardiomyocytes from WT, PI3Kγ -/- and PI3Kγ inact /PI3Kγ -/- mice. Conditioned media derived from PI3Kγ -/- showed pro-fibrotic effects, while that from PI3Kγ inact /PI3Kγ -/- showed fibrosis protective biological activity compared to WT. These findings reveal that kinase-independent function of PI3Kγ is a key regulator of the myocyte-initiated pathway that ultimately drives myofibroblast conversion. Proteomic analysis of conditioned media identified several pro-fibrotic factors that are regulated by PI3Kγ, the results of which will be discussed.

Abstract 284: Elevation of Cardiac NAD+ Levels Improves Mitochondrial Function in Mice Lacking Sirtuin 3

The mitochondria rely heavily on the ratio between NAD+ and its reduced form NADH to maintain proper function and generate 95% of cardiac cellular energy. Sirtuin 3 (SIRT3) is the major mitochondrial deacetylase and its activity level has been linked to the NAD+/NADH ratio. Numerous studies have shown that deletion of SIRT3 results in hyperacetylation and impairment of mitochondrial enzymes involved in fatty acid metabolism and in the mitochondrial respiratory chain. Because impairments in these processes have shown to be associated with decreases in the NAD+/NADH ratio and contribute to deficits in energy production, we hypothesized that the SIRT3 knockout mice (SIRT3 -/- ) would have a decreased NAD+/NADH ratio and that normalizing the ratio would improve mitochondrial respiratory function and inner membrane potential. In the present study, we observed increases in both NAD+ (1.1-fold, n=3, p&lt;0.05) and NADH (1.5-fold) in cardiac tissue from 14-week old SIRT3 -/- mice compared to wild-type (WT) controls, resulting in a 26±2.2% reduction in the NAD+/NADH ratio. These changes correlated with decreased Complex I ADP stimulated respiration (173±16 vs 118±14 nmolO 2 /min/mg for WT and SIRT3 -/- , respectively, n=3, p&lt;0.05) and suppressed mitochondrial membrane potential. Intraperitoneal injection (I.P.) of Nicotinamide Riboside (NR) increased NAD+ levels in cardiac tissue lysates (WT 1.5-fold and SIRT3 -/- 1.4-fold, n=5, p&lt;0.05) and in mitochondria isolated (WT 1.9-fold and SIRT3 -/- 1.7-fold) from the mice with no significant changes in NADH levels. Therefore, the NR I.P. injections normalized the NAD+/NADH ratio, partially restored the Complex I supported mitochondrial respiration (123±11 vs 157±8 nmolO 2 /min/mg for vehicle and NR treated SIRT3 -/- mice, respectively, n=3, p&lt;0.05), and improved mitochondrial membrane potential in the SIRT3 -/- mice. These results suggest that increasing cardiac NAD+ levels can rescue mitochondrial dysfunction independent of SIRT3 protein deacetylation and warrants further investigation. The next step will be to test whether increasing cardiac NAD+ levels can improve mitochondrial function and reduce injury in SIRT3 -/- mice subjected to chronic heart stress induced by transverse aortic constriction surgery.

Abstract 471: Kinase-independent Function of PI3Kγ Enables ERK Activation

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including cell survival. PI3Kγ, a member of the PI3K family, is activated in response to G-protein coupled receptor (GPCR) stimulation leading to extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, less is known about the underlying mechanisms of PI3Kγ-directed ERK activation. Knockdown of PI3Kγ showed that PI3Kγ not only regulates ERK phosphorylation in response to GPCR stimulation but also to receptor tyrosine kinase activation in HEK 293 cells. The key role of PI3Kγ in ERK activation was further validated by loss of insulin-stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs demonstrating a kinase-independent role of PI3Kγ in regulating ERK function. Mechanistic studies showed that PI3Kγ regulates ERK activation by inhibiting ERK dephosphorylation following stimulation thereby, sustaining ERK phosphorylation and activation. Critically, PI3Kγ regulates ERK dephosphorylating phosphatase PP2A by interacting and sequestering PP2A from ERK maintaining ERK phosphorylation, which is evidenced by increased PP2A association with ERK in KO MEFs. Consistently, ERK activation was completely abolished in KO MEFs following carvedilol or insulin suggesting an essential role for PI3Kγ in ERK activation pathway. Correspondingly, primary cardiac fibroblasts isolated from KO mice showed complete loss of insulin-stimulated ERK phosphorylation compared to WT mice. This is intriguing given that GSK3 phosphorylation and not ERK phosphorylation is regulated by inhibition of PP2A through kinase-independent mechanism of PI3Kγ in the total cardiac lysates. Even though GSK3 and ERK are substrates for PP2A, our findings that ERK is regulated by kinase-independent function PI3Kγ suggest the existence of this unique regulation in fibroblasts and not in cardiomyocytes. Thus, kinase activity of PI3Kγ may contribute to cardiac-pathology while kinase-independent function could be beneficial and will be discussed in presentation.

Infarcted Myocardium-Primed Dendritic Cells Improve Remodeling and Cardiac Function After Myocardial Infarction by Modulating the Regulatory T Cell and Macrophage Polarization.

Inflammatory responses play a critical role in left ventricular remodeling after myocardial infarction (MI). Tolerogenic dendritic cells (tDCs) can modulate immune responses, inducing regulatory T cells in a number of inflammatory diseases. We generated tDCs by treating bone marrow-derived dendritic cells with tumor necrosis factor-α and cardiac lysate from MI mice. We injected MI mice, induced by a ligation of the left anterior descending coronary artery in C57BL/6 mice, twice with tDCs within 24 hours and at 7 days after the ligation. In vivo cardiac magnetic resonance imaging and ex vivo histology confirmed the beneficial effect on postinfarct left ventricular remodeling in MI mice treated with tDCs. Subcutaneously administered infarct lysate-primed tDCs near the inguinal lymph node migrated to the regional lymph node and induced infarct tissue-specific regulatory T-cell populations in the inguinal and mediastinal lymph nodes, spleen, and infarcted myocardium, indicating that a local injection of tDCs induces a systemic activation of MI-specific regulatory T cells. These events elicited an inflammatory-to-reparative macrophage shift. The altered immune environment in the infarcted heart resulted in a better wound remodeling, preserved left ventricular systolic function after myocardial tissue damage, and improved survival. This study showed that tDC therapy in a preclinical model of MI was potentially translatable into an antiremodeling therapy for ischemic tissue repair.

Abstract 423: Kinase Independent Signaling of PI3Kγ Mediates Cardiac Fibrosis

Phosphoinositide 3 Kinase γ (PI3Kγ) belongs to a family of lipid kinases genetic deletion of which leads to pressure overload induced cardiac fibrosis in mice. However, the mechanism by which PI3Kγ mediates cardiac fibrosis is unknown. Cardiac fibrosis is a key underlying cause of fatal heart failure. A well-known fibrogenic mechanism is the generation of myofibroblasts, which are characterized by overexpression of smooth muscle α-actin (αSMA). Myofibroblast is a fibrosis-effector cell that produces pro-fibrotic cytokines and exuberant extracellular matrix that leads to cardiac fibrosis. To evaluate the role of PI3Kγ in fibrotic phenotype, cardiac tissue lysates from 3 months old WT and PI3Kγ null (PI3Kγ -/- ) mice were assessed for the expression of αSMA. Interestingly, there is significant up-regulation of αSMA in PI3Kγ -/- in comparison to littermate controls (WT) even at baseline suggesting that loss of PI3Kγ predisposes the hearts towards fibrosis. To directly confirm that PI3Kγ -/- cardiac fibroblasts (CF) exhibit a myofibroblast phenotype even at baseline, CF were isolated from hearts of WT and PI3Kγ -/- mice and assessed for myofibroblast phenotype by immunostaining for αSMA in stress fibers. Fluorescence microscopy on the CF from PI3Kγ -/- mice showed intense immunostaining for αSMA with greater number of cells exhibiting αSMA in stress fibers when compared to CF from WT mice. Consistently, immunoblotting showed significantly higher αSMA protein levels in PI3Kγ -/- CF compared to WT CF suggesting that PI3Kγ -/- fibroblasts are “primed” to undergo myofibroblast differentiation. To determine the role of kinase-independent function of PI3Kγ in vivo, we generated unique mice lines with cardiomyocyte-specific expression of either kinase-dead PI3Kγ (PI3Kγ inact ) or constitutively active PI3Kγ ( Myr PI3Kγ) in the global PI3Kγ -/- (PI3Kγ inact /PI3Kγ -/- or Myr PI3Kγ/PI3Kγ -/- ) and measured αSMA. Surprisingly, abundance of αSMA protein is significantly reduced in PI3Kγ inact /PI3Kγ -/- when compared to WT and PI3Kγ -/- mice. These data reveal that kinase-independent function of PI3Kγ is a key component in the myocyte-initiated pathway that ultimately drives CF to become myofibroblasts uncovering a novel mechanism of regulating pro-fibrotic signals.

Abstract 478: FGF23 Expression in Cardiac Fibroblasts Is Augmented by S100/Calgranulins-Mediated Inflammation and Associated With Cardiac Hypertrophy, but Not in Angiotensin II-Induced Cardiac Hypertrophy

Background: Serum S100A12 and fibroblast growth factor (FGF) 23 are biomarkers for cardiovascular mortality in patients with chronic kidney disease (CKD) and are associated with left ventricular hypertrophy (LVH). FGF23 is induced in cultured cardiac fibroblasts in response to cytokines including IL-6, TNF-a, LPS and S100/calgranulins. Moreover, hBAC-S100 transgenic mice with CKD had increased FGF23 in valvular interstitial cells and exhibited LVH. The present study was designed to examine cardiac FGF23 expression in other murine models of LVH in the absence of CKD. Methods: Hearts from five groups of male mice were studied: (i) C57BL6/J with transgenic expression a bacterial artificial chromosome of the human S100/calgranulins (S1008/9 and S100A12, hBAC-S100), (ii) wild type littermates, (iii) LDLR-/- infused with saline (29 days, 0.9%), (iv) LDLR-/- infused with angiotensin (Ang) II (29 days, 1000 ng/kg/min), and (v) fibroblast specific depletion of angiotensin II type 1a receptor (AT1aR) (S100A4-Cre x AT1aR-/- x LDLR-/-) infused with AngII. Results: hBAC-S100, but not wild type littermate mice, developed significant LVH at 10 months by heart weight/body weight (5.9 ±1.1 mg/g vs. 4.2 ±0.8, p&lt;0.04), decreased E/A ratio, and increased LVPW thickness, and associated with increased expression of FGF23 mRNA and protein in cardiac tissue lysates (2-4 fold increase). Similarly, Ang II induced significant LVH compared to saline infused LDLR-/- mice (6.1±1.3 vs. 3.6 ±0.9 mg/g, p&lt;0.01), and associated with increased mRNA for hypertrophic genes (ANP, BNP, b-MHC, CTGF and Col1a1). However, there was no significant difference in FGF23 mRNA and protein between Ang II and saline infused mice. Cardiac hypertrophy was attenuated in AngII-infused mice with deficiency of AT1aR (S100A4-Cre+/-xAT1aRxLDLR-/-). In vitro, Ang II (100nM) did not induce FGF23 in valvular interstitial fibroblasts or myocytes. Summary: Transgenic expression of S100/calgranulins is sufficient to induce LVH in aged mice with normal renal function, and this is associated with FGF23 expression in cardiac interstitial fibroblasts. Future studies are needed to determine whether cardiac FGF23 promotes LVH in a paracrine manner. However, FGF23 does not play a role in Ang II-induced LVH.

0116 : Phosphodiesterases regulate cAMP level in the mitochondria of adult cardiomyocytes

The heart is highly energy-dependent with most of its energy provided by mitochondria. Mitochondria also play a role in many essential cellular processes including metabolite synthesis, redox balance, calcium homeostasis and cell death. Therefore, maintaining a functional population of mitochondria is critical for cardiac function and identification of novel regulatory mechanisms. Recently, a soluble adenylyl cyclase (sAC) has been revealed to serve as a local source of the second messenger cAMP in the mitochondrial matrix in response to bicarbonate and calcium. PDE2A, a cGMP-stimulated cAMP-hydrolyzing phosphodiesterase (PDE), was also shown to be expressed in the mitochondrial matrix of liver and brain. To gain new insights into the control of mitochondrial pool of cAMP, we investigated the role of various isoforms of PDEs in isolated cardiac mitochondria. Western blot analysis of PDE protein expression revealed positive bands for PDE2A, 3A, 4A and 4B subtypes in cardiac mitochondria. Basal cAMP-degrading enzymatic activities were determined by radioenzymatic assay in cardiac mitochondrial lysates with 1μm cAMP as substrate. PDE2 represented the largest mitochondrial cAMP PDE activity (35% of total) and its activity was enhanced ~2-fold by 5μM cGMP and was inhibited by the PDE2 inhibitor, Bay 60-7550 (100nm). PDE3 and PDE4 activities represented, respectively, 30% and 25% of total, and they were inhibited by their respective inhibitors, cilostamide (1μm) and Ro 20- 1724 (10 μM). Accordingly, measurements by ELISA of mitochondrial cAMP levels confirmed that inhibition of PDE2, 3 and 4 families leads to an increase in cAMP level. In conclusion, at least three PDE families and four PDE subtypes are located in the cardiac mitochondrial matrix, participating to a local signaling pathway with sAC to control cAMP level. Our findings unravel a cAMP signaling cascade in cardiac mitochondria which may have implications for the metabolic control of cardiac function.

Alterations in the Interactome of Serine/Threonine Protein Phosphatase Type-1 in Atrial Fibrillation Patients

BackgroundAtrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown. ObjectivesThe purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits. MethodsCardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry–based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit–PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation. ResultsA total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone. ConclusionsSubcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c–R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca2+ remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF.

Functional Cardiac Lipolysis in Mice Critically Depends on Comparative Gene Identification-58

Efficient catabolism of cellular triacylglycerol (TG) stores requires the TG hydrolytic activity of adipose triglyceride lipase (ATGL). The presence of comparative gene identification-58 (CGI-58) strongly increased ATGL-mediated TG catabolism in cell culture experiments. Mutations in the genes coding for ATGL or CGI-58 in humans cause neutral lipid storage disease characterized by TG accumulation in multiple tissues. ATGL gene mutations cause a severe phenotype especially in cardiac muscle leading to cardiomyopathy that can be lethal. In contrast, CGI-58 gene mutations provoke severe ichthyosis and hepatosteatosis in humans and mice, whereas the role of CGI-58 in muscle energy metabolism is less understood. Here we show that mice lacking CGI-58 exclusively in muscle (CGI-58KOM) developed severe cardiac steatosis and cardiomyopathy linked to impaired TG catabolism and mitochondrial fatty acid oxidation. The marked increase in ATGL protein levels in cardiac muscle of CGI-58KOM mice was unable to compensate the lack of CGI-58. The addition of recombinant CGI-58 to cardiac lysates of CGI-58KOM mice completely reconstituted TG hydrolytic activities. In skeletal muscle, the lack of CGI-58 similarly provoked TG accumulation. The addition of recombinant CGI-58 increased TG hydrolytic activities in control and CGI-58KOM tissue lysates, elucidating the limiting role of CGI-58 in skeletal muscle TG catabolism. Finally, muscle CGI-58 deficiency affected whole body energy homeostasis, which is caused by impaired muscle TG catabolism and increased cardiac glucose uptake. In summary, this study demonstrates that functional muscle lipolysis depends on both CGI-58 and ATGL.

Enhanced differentiation potential of human amniotic mesenchymal stromal cells by using three-dimensional culturing

The therapeutic potential of human amniotic mesenchymal stromal cells (hAMSCs) remains limited because of their differentiation towards mesenchymal stem cells (MSCs) following adherence. The aim of this study was to develop a three-dimensional (3-D) culture system that would permit hAMSCs to differentiate into cardiomyocyte-like cells. hAMSCs were isolated from human amnions of full-term births collected after Cesarean section. Immunocytochemistry, immunofluorescence and flow cytometry analyses were undertaken to examine hAMSC marker expression for differentiation status after adherence. Membrane currents were determined by patch clamp analysis of hAMSCs grown with or without cardiac lysates. Freshly isolated hAMSCs were positive for human embryonic stem-cell-related markers but their marker profile significantly shifted towards that of MSCs following adherence. hAMSCs cultured in the 3-D culture system in the presence of cardiac lysate expressed cardiomyocyte-specific markers, in contrast to those maintained in standard adherent cultures or those in 3-D cultures without cardiac lysate. hAMSCs cultured in 3-D with cardiac lysate displayed a cardiomyocyte-like phenotype as observed by membrane currents, including a calcium-activated potassium current, a delayed rectifier potassium current and a Ca(2+)-resistant transient outward K(+) current. Thus, although adherence limits the potential of hAMSCs to differentiate into cardiomyocyte-like cells, the 3-D culture of hAMSCs represents a more effective method of their culture for use in regenerative medicine.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Rare incidence of congestive heart failure in gastrointestinal stromal tumor and other sarcoma patients receiving imatinib mesylate

The authors sought to determine the incidence and severity of cardiovascular toxicity caused by imatinib mesylate in gastrointestinal stromal tumor (GIST) and other sarcoma patients, and to explore cardiotoxicity caused by imatinib mesylate using cell culture and in vitro models. To determine the incidence and significance of serious cardiac adverse events in GIST and other sarcoma patients receiving imatinib mesylate, the authors performed a retrospective analysis of 219 consecutive patients treated with imatinib mesylate. In vitro studies of imatinib mesylate on cultured cardiomyocytes and biochemical studies of cardiac lysates from mice treated with imatinib mesylate were performed to define the potential cardiotoxic effects of imatinib mesylate. Grade 3 or 4 potentially cardiotoxic adverse events (mostly edema or effusions) occurred in 8.2% of patients, were manageable with medical therapy, and infrequently required dose reduction or discontinuation of imatinib mesylate. Arrhythmias, acute coronary syndromes, or heart failure were uncommon, occurring in <1% of treated patients. However, administration of imatinib in a mouse model system resulted in inhibition of activation of protein kinases that are known to be important in the cardiac stress response. The authors concluded that imatinib is an uncommon cause of cardiotoxicity, and that the cardiovascular adverse events that occur are manageable when recognized and treated. Nevertheless, our preclinical findings suggest that imatinib remains a potential cardiotoxin. Furthermore, the cardiac consequences of long-term imatinib therapy remain unknown. We therefore recommend treatment of risk factors for cardiovascular disease in imatinib-treated patients in accord with the American Heart Association guidelines for the prevention and treatment of heart failure.

NCS‐1 modulates expression of calcium handling proteins

We have previously shown that neuronal‐calcium sensor‐1 (NCS‐1) was present in rat heart and that overexpression of this small binding protein in isolated ventricular myocytes lead to an increase in contractile function. To further elucidate the physiological role of NCS‐1 in the heart, we examine cellular mechanisms by which overexpression of NCS‐1 could augment contractile function. Isolated adult rat ventricular myocytes were infected with adenovirus constructs (MOI: 100) expressing either NCS‐1 or β‐gal (control) for 48 hr. Cells were then washed and collected. Total cardiac lysate proteins were separated on SDS‐PAGE gels and expression levels of major E‐C coupling‐associated proteins were determined. Preliminary results suggest that expression of SERCA‐2a was decreased in the NCS‐1‐infected cells when compared to control cells. Expression of phospholamban (PLB), a negative regulator of SERCA‐2a activity, was unchanged while levels of phosphorylated PLB were significantly increased in NCS‐1‐infected cells, which is consistent with an increase in cardiac contractility. Finally, expression of NCX‐1 was not altered in the NCS‐1‐infected cells. Taken together, our data suggest that in addition to directly interacting with cardiac myofilament proteins, NCS‐1 may also regulate contractile function by modulating expression of key calcium handling proteins. Supported by UAMS Department of Medicine Seed Money Award (ST).

Abstract 1330: Divergent Control of the Mitochondrial Death Gene BNIP3 by Opposing Actions of the Cellular Factors Nuclear Factor kappaB and E2F-1 in Ventricular Myocytes.

The hypoxia-inducible death factor Bnip3 is known to provoke mitochondrial perturbations and cell death of ventricular myocytes. The transcriptional control processes that govern Bnip3 gene expression under basal and inducible conditions remain cryptic. Sequence analysis of the Bnip3 promoter revealed the presence of distinct but overlapping DNA binding elements for the cell cycle factor E2F-1 and cellular factor NF-κB. Previously, we reported a survival role for NF-κB in ventricular myocytes. As a step toward elucidating the regulation of Bnip3 gene expression in ventricular myocytes, we tested the impact of E2F-1 and NF-κB on basal and inducible expression of Bnip3. A 2.0 fold increase in Bnip3 gene transcription was observed in cells expression wild type E2F-1 but not in cells expressing an E2F-1 mutant defective for DNA binding. Interestingly, basal Bnip3 gene transcription was increased by 2.5 fold in myocytes rendered defective for NF-κB activation with a non-phosphorylatable form of IκBα. Importantly, genetic ablation of E2F-1 inhibited basal and inducible Bnip3 transcription in NF-κB defective cells. Expression of the p65 subunit of NF-κB in NF-κB defective cells inhibited E2F-1 mediated Bnip3 transcription. Western blot analysis of cardiac cell lysate revealed that p65 NF-κB immunoprecipitated with E2F-1. ChIP analysis of the Bnip3 promoter indicated that the p65 NF-κB bound DNA under normoxic conditions. During hypoxia E2F-1 activity increased where as p65 NF-κB protein levels were decreased. ChIP analysis revealed increased binding of E2F-1 to the Bnip3 promoter during hypoxia which coincided with a 3.5 fold increase in Bnip3 gene transcription. IKKβ mediated activation of NF-κB activation abrogated hypoxia-induced E2F-1 binding to the Bnip3 promoter and Bnip3 gene transcription. To our knowledge our data provide the first direct evidence that a novel relationship exists between p65 NF-κB and E2F-1 for basal and hypoxia-inducible regulation of the Bnip3 promoter. Furthermore, our data highlight a novel survival pathway by which NF-κB averts hypoxia - induced cell death by antagonizing the E2F-1 dependent transcription of Bnip3 in ventricular myocytes.