Neonatal Cardiac Myocytes Research Articles

Abstract 191: Phosphodiesterase 9a Modulates Cardiomyocyte Natriuretic Peptide-Stimulated cGMP Pool and Cardiac Hypertrophy

Cardiac hypertrophy in response to pathologic stress is a major contributor to heart disease. Many studies have revealed that compartmentalized cyclic guanosine monophosphate (cGMP) regulation by phosphodiesterases (PDEs) can potently modify acute and chronic cardiac stress responses. PDE5a normally regulates cGMP generated from nitric oxide (NO)-stimulated soluble guanylate cyclase (sGC) but not natriuretic peptide (NP)-stimulated cGMP. Its inhibition blunts maladaptive hypertrophy and remodeling. Regulators of NP-cGMP remain uncertain. Here we reveal that PDE9a, a highly cGMP-specific PDE (×100 affinity vs PDE5a) that is expressed predominantly in the brain modulates myocyte cGMP as well, targeting NP-stimulated pools. PDE9a gene expression is observed in rat neonatal cardiac myocytes (RNCM) and adult mouse myocytes. Protein expression was identified in myocytes by immunohistochemistry, using gene silencing models as a negative control. PDE9a expression is upregulated by various hypertrophic stimuli (phenylephrine (PE) or endothelin-1 (ET-1), and observed in myocytes from pressure-overloaded whole myocardium. PDE9a inhibition with a selective antagonist (PF-9613) or gene silencing significantly abrogated PE- or ET-1-dependent upregulation of pathological-hypertrophy fetal genes and NFAT activity in RNCM, and PDE9a inhibition attenuated ET-1-induced hypertrophy in adult mouse myocytes. PF-9613 had no impact in cells lacking PDE9a (from knockout mice) or after gene silencing. Biochemical and cGMP-sensitive fluorescent probe studies shows PDE9a inhibition augments ANP- but not NO-dependent cGMP stimuli in RNCM and adult myocytes. PDE9a knockout (KO) mice were protected against hypertrophy/remodeling after pressure-overload, accompanied by a rise in myocardial cGMP. In PDE9a KO mice also exhibited less fibrosis and reduced expression of fibrotsis-related genes over wild-type mice. Together, these data identify PDE9a as a novel regulator of myocyte cGMP and hypertrophy that impacts a different compartmentalized cGMP pool to that by PDE5a. Our observations may provide a novel therapeutic approach in the treatment of heart failure.

Physical interaction between calcineurin and Cav3.2 T-type Ca2+ channel modulates their functions

Cav3.2 T-type Ca2+ channel is required for the activation of calcineurin/NFAT signaling in cardiac hypertrophy. We aimed to investigate how Cav3.2 and calcineurin interact. We found that Ca2+ and calmodulin modulate the Cav3.2/calcineurin interaction. Calcineurin binding to Cav3.2 decreases the enzyme’s phosphatase activity and diminishes the channel’s current density. Phenylephrine-induced hypertrophy in neonatal cardiac myocytes is reduced by a cell-permeable peptide with the calcineurin binding site sequence. These data suggest that Cav3.2 regulates calcineurin/NFAT pathway through both the Ca2+ influx and calcineurin binding. Our findings unveiled a reciprocal regulation of Ca2+ signaling which contributes to our understanding of cardiac hypertrophy.

42. Investigation into cardiomyocyte response to cryoablation

Atrial and ventricular tachyarrhythmias represent major cardiovascular diseases that may be treated utilizing thermal therapies. Given this there has been a palpable increase in technologies and approaches of therapeutic intervention, including cryoablation. While increased utilization of cryoablation continues, our fundamental understanding of the response of cardiac cells to the freezing process remains limited. As such we investigated the cellular and molecular response of cardiomyocytes to a range of subfreezing temperatures in an effort to provide further insight to help guide the application of cryoablation for the treatment of cardiac arrhythmias. Primary neonatal rat ventricular cardiac myocytes (NRVCM) and human cardiac myocytes (HCM) were cultured in vitro to study the cellular and molecular effects of thermal excursions. Cells were exposed to a range of temperatures from 37 °C to −40 °C for up to 1 min. Thermal profiles were generated to examine the cooling rates at each target temperature. Samples were then returned to culture and cell survival was measured via metabolic activity, membrane integrity and contractile function. Microfluidic flow cytometry was also conducted to investigate apoptotic involvement and confirm viability assessments of the various populations in a time-course manner. Exposure of NRVCM to temperatures ranging from 37 °C to 0 °C resulted in a minimal reduction in cell viability. Exposure to −5 °C yielded 50% cell death. Temperatures ranging from −10 °C to −20 °C yielded less than 20% survival, whereas exposure to −40 °C resulted in complete cell death. Regardless of the level of cell survival, exposure to temperatures below −5 °C resulted in a loss of spontaneous contraction in surviving cells. HCM demonstrated moderate sensitivity (<30% loss) to temperatures above −10 °C, however a steep decline in cell survival was observed from −12 °C to −25 °C, the point at which all cells were metabolically inactive. Flow cytometric analysis revealed the activation of apoptosis in cardiomyocytes in response to the freezing insult. The level and timing was found to vary with temperature, but not duration of the freeze. Although the utilization of cryoablation for the treatment of arrhythmias is established clinically, there remains a void in our knowledge base of the cellular and molecular responses of cardiac systems to thermal stress. This study, as well as others, are now providing mechanistic answers to the how cryoablation works at the cellular level as well as providing critical characterization data to help guide application and future development of therapeutic application.

The Serum- and Glucocorticoid-inducible Kinases SGK1 and SGK3 Regulate hERG Channel Expression via Ubiquitin Ligase Nedd4-2 and GTPase Rab11

The hERG (human ether-a-go-go-related gene) encodes the α subunit of the rapidly activating delayed rectifier potassium channel (IKr). Dysfunction of hERG channels due to mutations or certain medications causes long QT syndrome, which can lead to fatal ventricular arrhythmias or sudden death. Although the abundance of hERG in the plasma membrane is a key determinant of hERG functionality, the mechanisms underlying its regulation are not well understood. In the present study, we demonstrated that overexpression of the stress-responsive serum- and glucocorticoid-inducible kinase (SGK) isoforms SGK1 and SGK3 increased the current and expression level of the membrane-localized mature proteins of hERG channels stably expressed in HEK 293 (hERG-HEK) cells. Furthermore, the synthetic glucocorticoid, dexamethasone, increased the current and abundance of mature ERG proteins in both hERG-HEK cells and neonatal cardiac myocytes through the enhancement of SGK1 but not SGK3 expression. We have previously shown that mature hERG channels are degraded by ubiquitin ligase Nedd4-2 via enhanced channel ubiquitination. Here, we showed that SGK1 or SGK3 overexpression increased Nedd4-2 phosphorylation, which is known to inhibit Nedd4-2 activity. Nonetheless, disruption of the Nedd4-2 binding site in hERG channels did not eliminate the SGK-induced increase in hERG expression. Additional disruption of Rab11 proteins led to a complete elimination of SGK-mediated increase in hERG expression. These results show that SGK enhances the expression level of mature hERG channels by inhibiting Nedd4-2 as well as by promoting Rab11-mediated hERG recycling.

Role of AKAP79 in â1‐Adrenergic receptor resensitization and recycling

AKAPs (A‐kinase anchoring proteins) have been shown to participate in macromolecular signaling complexes that include protein kinases, phosphodiesterases, ion channels, and also GPCRs. In this study, we examine the role of AKAP79 (also known as AKAP79/150) in the trafficking of β1‐Adrenergic Receptor in vitro in HEK‐293 cells and in neonatal cardiac myocytes derived from AKAP150 homozygous knock out mice.Selective down regulation of AKAP79 in HEK‐293 cells and in rat neonatal cardiac myocytes, inhibited the recycling of the WT β1‐AR in HEK‐293 cells and the reintroduction of siRNA‐resistant AKAP79 rescued the recycling of the agonist‐internalized β1‐AR. Furthermore, in neonatal ventricular myocytes derived from AKAP150−/− mice or from AKAP150+/+ the β1‐AR was internalized, but did not recycle in myocytes from AKAP150−/− mice. The recycling of the β1‐AR was rescued by AKAP79, indicating that AKAP79 was required for the recycling of the β1‐AR to occur and shows that these studies across a spectrum of different cells yielded the same result. In addition we tested the effect of three different truncated mutants of AKAP79, namely AKAP79 [1–360] (ΔPKA‐binding site), AKAP79 [115–412] (Δmembrane and PKC binding domains] and AKAP79 [ΔPIX] (Δcalcineurin‐binding domain] their ability to recapitulate the effect of full‐length AKAP79 on recycling of the β1‐AR in AKAP79‐knock down cells. Immunopricipitation data showed that the interaction between the mutants and β1‐AR remained unchanged. However, none of these AKAP79 mutants was capable of rescuing the recycling of the β1‐AR in HEK‐cells, indicating the entire molecule of AKAP79/150 was required for this effect. Funded by NIH HL‐085848.

Hexokinase Isoforms and Glucose Metabolism in Adult and Neonatal Cardiac Myocytes

Localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria controls their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we compared the effects of HKI and HKII in isolated adult (ARVM) and neonatal rat ventricular myocytes (NRVM) using a set of novel genetically-encoded optical imaging tools to track, in real-time, the subcellular distributions of HKI and HKII, as well as the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically distributes between the mitochondria and cytoplasm. Removal of extracellular glucose displaces HKII from mitochondria in ARVM, but not in NRVM, whereas iodoacetate (IAA) displaces HKII in both. HKI, the predominant isoform in NRVM, always remains bound to mitochondria when overexpressed in either AVRM and NRVM, and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In NVRM, knock-down of HKI, but not HKII, decreased glycolytic activity. Swapping the N terminus between HKI and HKII, as well as specific mutations, revealed that the N terminus of HKI is necessary, but not sufficient, for high affinity mitochondrial binding to promote glycolysis. In conclusion, HKI and HKII play major roles in defining the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.

Abstract 19390: Sirtuin 1 Modulates Cardiac Sodium Channels in vivo

The rapid upstroke in phase 0 of the cardiac action potential is the result of the inward sodium current (I Na ) carried by the cardiac voltage-gated sodium channel Na v 1.5 (SCN5A). We recently reported that the overexpression of Silent Information Regulator 1 (Sirtuin 1, SIRT1), an NAD + dependent protein deacetylase, deacetylated SCN5A, increased its cell surface expression, and increased I Na in HEK-293 cells and rat neonatal cardiac myocytes. Overexpression of a dominant negative SIRT1 construct decreased SCN5A cell surface expression and I Na . The role of acetylation of SCN5A and its regulation by SIRT1 has not been shown in vivo, however. We engineered cardiac specific Sirt1 knockout (cSirt1KO) mice by breeding Sirt1 flox/flox with α-MHC-cre mice on a C57BL-6 background. cSirt1KO compared to littermate Sirt1 flox/flox mice showed much less expression of Sirt1 mRNA (12±6%, n=3) and protein in the heart but not in the kidney, and Scn5a immunoprecipitated from cardiac membrane fractions of the cSirt1KO mice showed an ∼2-fold increase in acetylation (Figure A). At 3 months of age, EKGs from anesthetized cSirt1KO compared to littermate Sirt1 flox/flox mice showed PR prolongation (43±3 ms, n=4, vs. 37±2 ms, n=5; p&lt;0.02) and telemetry from ambulatory cSirt1KO mice showed high degree heart block not seen in the controls (Figure B). Thus, our results show that Scn5a is acetylated in vivo in the mouse, that the degree of acetylation is regulated by cardiac-specific expression of Sirt1, and that decreases in Sirt1 lead to conduction disease similar to those seen in Scn5a +/- mice. These findings raise the possibility that pharmacological manipulation of Sirt1 may provide a means to modulate cardiac I Na and arrhythmias.

D2 dopamine receptor antagonist raclopride induces non‐canonical autophagy in cardiac myocytes

Cell death by autophagy is an important means of maintaining cellular homeostasis in adult cardiac myocytes. Autophagy was previously shown to exert a cardioprotective effect, suggesting that modulation of autophagy pathways is a potential therapeutic strategy in the treatment of heart disease. Although dopamine is known to induce autophagy in neuroblastoma cells, the underlying mechanism and the types of dopamine receptors involved in this process remain unclear. In this study, we used various dopamine receptor antagonists and agonists to identify the specific dopamine receptor that mediates induction of autophagy. We evaluated autophagy induction by the expression of autophagy markers in neonatal rat ventricular cardiac myocytes. We evaluated intracellular calcium levels using Fluo-3/AM and demonstrated autophagy-induced morphological changes in cardiac myocytes using electron microscopy. We also examined the pathway for dopamine-induced autophagy using RNAi-mediated gene knockdown. Raclopride, the well-documented D2 receptor antagonist, significantly upregulated autophagy in cardiac myocytes via an mTOR-independent pathway. There was no difference in intracellular calcium levels between raclopride-treated cells and untreated cells. siRNA-mediated knockdown of Rab9 resulted in decreased expression of autophagy markers in raclopride-treated cells. Interestingly, siRNA-mediated knockdown of Atg7 resulted in a significant increase in Rab9 levels in raclopride-treated cells, suggesting that blocking the classical autophagy pathway results in activation of an alternative pathway. Our study suggests that (1) the D2 dopamine receptor plays a role in autophagy and (2) raclopride mediated a non-canonical autophagy pathway in cardiac myocytes via Rab9.

Sorcin modulates mitochondrial Ca2+ handling and reduces apoptosis in neonatal rat cardiac myocytes

Sorcin localizes in cellular membranes and has been demonstrated to modulate cytosolic Ca(2+) handling in cardiac myocytes. Sorcin also localizes in mitochondria; however, the effect of sorcin on mitochondrial Ca(2+) handling is unknown. Using mitochondrial pericam, we measured mitochondrial Ca(2+) concentration and fluxes in intact neonatal cardiac myocytes overexpressing sorcin. Our results showed that sorcin increases basal and caffeine-stimulated mitochondrial Ca(2+) concentration. This effect was associated with faster Ca(2+) uptake and release. The effect of sorcin was specific for mitochondria, since similar results were obtained with digitonin-permeabilized cells, where cytosolic Ca(2+) flux was disrupted. Furthermore, mitochondria of cardiac myocytes in which sorcin was overexpressed were more Ca(2+)-tolerant. Experiments analyzing apoptotic signaling demonstrated that sorcin prevented 2-deoxyglucose-induced cytochrome c release. Furthermore, sorcin prevented hyperglycemia-induced cytochrome c release and caspase activation. In contrast, antisense sorcin induced caspase-3 activation. Thus, sorcin antiapoptotic properties may be due to modulation of mitochondrial Ca(2+) handling in cardiac myocytes.

Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning

Na(+),K(+)-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na(+),K(+)-ATPase α(1)- and α(3)-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α(1)-cell surface abundance was significantly decreased. Na(+),K(+)-ATPase-activity in crude homogenates and (86)Rb(+) transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na(+),K(+)-ATPase activity and surface expression. This model also revealed that Na(+),K(+)-ATPase-mediated (86)Rb(+) uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na(+),K(+)-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na(+),K(+)-ATPase α(1) known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na(+),K(+)-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na(+),K(+)-ATPase may be independent of ion transport.

Abstract 108: Glycolysis, Cardioprotection and Their Modulation by Differential Interactions of Hexokinase I and II with Mitochondria

Rationale: Neonatal heart is more glycolytically active and less susceptible to anoxia/reoxygenation injury than adult heart. Hexokinase (HK) is the first step in glycolysis and its binding to mitochondria is known to be cardioprotective. Since neonatal cardiac myocytes mainly express the HKI isoform, whereas HKII predominates in adults, we explored the functional consequences of such difference. Objective: To investigate whether differential interactions of HKI and HKII with mitochondria in neonatal (NRVM) and adult rat ventricular myocytes (ARVM) account for their different metabolic profiles and susceptibility to anoxia/reoxygenation injury. Methods and Results: We performed real-time imaging in isolated NRVM and ARVM expressing the genetically-encoded biosensors FLIPglu-600µM to track intracellular glucose metabolism and YFP-tagged HK constructs to track subcellular HK localization, together with a propidium iodide-based cell death assay. We show that HKI remains bound to mitochondria in both NRVM and ARVM, while HKII distributes between the mitochondria and cytoplasm. Extracellular glucose removal displaced HKII from mitochondria in ARVM, but not in NRVM, whereas iodoacetate (IAA) displaces HKII in both. These differences were associated with a 35-fold greater glycolytic activity and markedly enhanced resistance to anoxia/reoxygenation injury in NVRM. NRVM became comparably susceptible to anoxia/reoxygenation injury only when treated with IAA to suppress HK interaction with mitochondria (whereas glucose removal alone had no effect). Conclusions: HKs exhibit stronger interaction with mitochondria in NRVM, accounting for their enhanced glycolytic activity and increased resistance to anoxia/reoxygenation injury. HKI may be superior to HKII in protecting the heart because of its stronger interaction with mitochondria.

Abstract 36: Disrupting the Delta Protein Kinase C Interaction with the “d” Subunit of F1Fo ATP Synthase Protects Against Ischemia-Reperfusion Injury in Perfused Rat Hearts

Cardiac ischemia / reperfusion (IR) injury is associated with severe energy deprivation and is the number one cause of death world-wide. Mitochondrial F1Fo ATP synthase produces &gt;90% of cardiac energy in mammals, yet few studies have targeted its role in IR injury. Previously, we identified a hypoxia-induced interaction of delta protein kinase C (dPKC) with the “d” subunit of F1Fo ATP synthase (dF1Fo) in neonatal cardiac myocytes, which inhibits F1Fo function. In the present work we investigated the hypothesis that a novel peptide inhibitor of the dPKC-dF1Fo interaction would preserve ATP and reduce infarct-size in isolated rat hearts subjected to IR injury. This peptide [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] contains HIV-Tat protein transduction and mitochondrial targeting domains, the dPKC-dF1Fo inhibitor sequence, and a FLAG epitope. In hearts exposed to global ischemia, or IR, dPKC co-immuno-precipitated with dF1Fo. Pretreatment with the dPKC-dF1Fo inhibitor exacerbated cardiac ATP loss by 1.9-fold (n=5, p&lt;0.03) following 10 min of global ischemia. However; following a pro-longed IR exposure ATP levels were enhanced by 2.1-fold (p&lt;0.02, n=5). These opposing effects of the[[Unable to Display Character: ]]dPKC-dF1Fo inhibitor on ATP levels are likely due to relief of dPKC inhibition of the different modes of the F1Fo complex during ischemia (ATPase) and oxygenated reperfusion (ATP synthase). We next used 2,3,5, tetrazolium chloride staining techniques to determine if the dPKC-dF1Fo inhibitor had infarct-sparing effects following prolonged IR. In hearts exposed to 30 min of global ischemia and 150 min of reperfusion the dPKC-dF1Fo inhibitor reduced infarct size, (expressed as the percentage of total LV area) from 45 + 3 % (n=6) to 22 + 3 % (n=6, p &lt; 0.01). Delivery and stability of the dPKC-dF1Fo inhibitor in hearts was confirmed by FLAG immunoreactivity in western blots conducted on mitochondria isolated the left ventricle. This is the first demonstration that perfusion with the dPKC-dF1Fo inhibitor prior to IR improves ATP recovery and reduces infarction in intact mammalian hearts. Our results support the potential for this peptide as a first-in-class translational agent for combating cardiac IR injury.

Mitochondrial Ca 2+ uptake contributes to buffering cytoplasmic Ca 2+ peaks in cardiomyocytes

Mitochondrial ability of shaping Ca(2+) signals has been demonstrated in a large number of cell types, but it is still debated in heart cells. Here, we take advantage of the molecular identification of the mitochondrial Ca(2+) uniporter (MCU) and of unique targeted Ca(2+) probes to directly address this issue. We demonstrate that, during spontaneous Ca(2+) pacing, Ca(2+) peaks on the outer mitochondrial membrane (OMM) are much greater than in the cytoplasm because of a large number of Ca(2+) hot spots generated on the OMM surface. Cytoplasmic Ca(2+) peaks are reduced or enhanced by MCU overexpression and siRNA silencing, respectively; the opposite occurs within the mitochondrial matrix. Accordingly, the extent of contraction is reduced by overexpression of MCU and augmented by its down-regulation. Modulation of MCU levels does not affect the ATP content of the cardiomyocytes. Thus, in neonatal cardiac myocytes, mitochondria significantly contribute to buffering the amplitude of systolic Ca(2+) rises.

Improving Cardiac Conduction With a Skeletal Muscle Sodium Channel by Gene and Cell Therapy

The voltage-gated Na+ channel is a critical determinant of the action potential (AP) upstroke. Increasing Na+ conductance may speed AP propagation. In this study, we propose use of the skeletal muscle Na+ channel SkM1 as a more favorable gene than the cardiac isoform SCN5A to enhance conduction velocity in depolarized cardiac tissue. We used cells that electrically coupled with cardiac myocytes as a delivery platform to introduce the Na+ channels. Human embryonic kidney 293 cells were stably transfected with SkM1 or SCN5A. SkM1 had a more depolarized (18 mV shift) inactivation curve than SCN5A. We also found that SkM1 recovered faster from inactivation than SCN5A. When coupled with SkM1 expressing cells, cultured myocytes showed an increase in the dV/dtmax of the AP. Expression of SCN5A had no such effect. In an in vitro cardiac syncytium, coculture of neonatal cardiac myocytes with SkM1 expressing but not SCN5A expressing cells significantly increased the conduction velocity under both normal and depolarized conditions. In an in vitro reentry model induced by high-frequency stimulation, expression of SkM1 also enhanced angular velocity of the induced reentry. These results suggest that cells carrying a Na+ channel with a more depolarized inactivation curve can improve cardiac excitability and conduction in depolarized tissues.

Exposing cardiomyocytes to subclinical concentrations of doxorubicin rapidly reduces their creatine transport

Doxorubicin is commonly used to treat leukemia, lymphomas, and solid tumors, such as soft tissue sarcomas or breast cancer. A major side effect of doxorubicin therapy is dose-dependent cardiotoxicity. Doxorubicin's effects on cardiac energy metabolism are emerging as key elements mediating its toxicity. We evaluated the effect of doxorubicin on [(14)C]creatine uptake in rat neonatal cardiac myocytes and HL-1 murine cardiac cells expressing the human creatine transporter protein. A significant and irreversible decrease in creatine transport was detected after an incubation with 50-100 nmol/l doxorubicin. These concentrations are well below peak plasma levels (5 μmol/l) and within the ranges (25-250 nmol/l) for steady-state plasma concentrations reported after the administration of 15-90 mg/m(2) doxorubicin for chemotherapy. The decrease in creatine transport was not solely because of increased cell death due to doxorubicin's cytotoxic effects. Kinetic analysis showed that doxorubicin decreased V(max), K(m), and creatine transporter protein content. Cell surface biotinylation experiments confirmed that the amount of creatine transporter protein present at the cell surface was reduced. Cardiomyocytes rely on uptake by a dedicated creatine transporter to meet their intracellular creatine needs. Our findings show that the cardiomyocellular transport capacity for creatine is substantially decreased by doxorubicin administration and suggest that this effect may be an important early event in the pathogenesis of doxorubicin-mediated cardiotoxicity.

Down-Regulation of Replication Factor C-40 (RFC40) Causes Chromosomal Missegregation in Neonatal and Hypertrophic Adult Rat Cardiac Myocytes

BackgroundAdult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied.MethodsWe performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12.ResultsRFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers.ConclusionOur novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

Antagonists of organic anion transporters 1 and 3 ameliorate adverse cardiac remodelling induced by uremic toxin indoxyl sulfate

Circulating toxins in patients with chronic kidney disease may attenuate cardiac function [1,2]. Specifically, indoxyl sulfate, one such non-dialysable uremic toxin, has direct pro-hypertrophic and pro-fibrotic effects on cardiac myocytes and fibroblasts [3]. This toxin significantly increases neonatal cardiac myocyte hypertrophy and fibroblast collagen synthesis within a clinically relevant concentration range [3]. We sought to determine potential approaches to blockade of indoxyl sulfate-induced cardiac remodelling.

Reduction of Na/K-ATPase Potentiates Marinobufagenin-induced Cardiac Dysfunction and Myocyte Apoptosis

Decreases in cardiac Na/K-ATPase have been documented in patients with heart failure. Reduction of Na/K-ATPase α1 also contributes to the deficiency in cardiac contractility in animal models. Our previous studies demonstrate that reduction of cellular Na/K-ATPase causes cell growth inhibition and cell death in renal proximal tubule cells. To test whether reduction of Na/K-ATPase in combination with increased cardiotonic steroids causes cardiac myocyte death and cardiac dysfunction, we examined heart function in Na/K-ATPase α1 heterozygote knock-out mice (α1(+/-)) in comparison to wild type (WT) littermates after infusion of marinobufagenin (MBG). Adult cardiac myocytes were also isolated from both WT and α1(+/-) mice for in vitro experiments. The results demonstrated that MBG infusion increased myocyte apoptosis and induced significant left ventricle dilation in α1(+/-) mice but not in their WT littermates. Mechanistically, it was found that in WT myocytes MBG activated the Src/Akt/mTOR signaling pathway, which further increased phosphorylation of ribosome S6 kinase (S6K) and BAD (Bcl-2-associated death promoter) and protected cells from apoptosis. In α1(+/-) myocytes, the basal level of phospho-BAD is higher compared with WT myocytes, but MBG failed to induce further activation of the mTOR pathway. Reduction of Na/K-ATPase also caused the activation of caspase 9 but not caspase 8 in these cells. Using cultures of neonatal cardiac myocytes, we demonstrated that inhibition of the mTOR pathway by rapamycin also enabled MBG to activate caspase 9 and induce myocyte apoptosis.

P300‐dependent acetylation of Histone H3 at lysine 56 is required for cardioprotection and DNA damage response

Backgroundp300 is important for transcription‐dependent DNA damage repair. Acetylation of Histone H3 at Lysine 56 (AcH3K56) by p300 has been suggested to be important for initiation of DNA damage response.HypothesisAcetyltransferase p300 loss is causally related to reduced DNA damage response during aging and genotoxic stress.Methodsp300 was knocked down in neonatal cardiac myocytes using siRNA and compared to a non‐targeting sequence. Doxorubicin (Dox) was used to model genotoxic stress. Cycloheximide (CY)(5μg/ml) was used to assess p300 stability.ResultsIn cardiac myocytes, DOX caused a p300‐HAT activity dependent rapid and sustained stabilization of p300 protein (3.7±1.9 fold vs. control at 8h; p&lt;0.05). AcH3K56 co localized with p300 after DOX treatment. p300 knockdown increased DOX‐induced myocyte death (26.9±13.5%; p&lt;0.05) and caspase3 cleavage, decreased AcH3K56, blunted phosphorylation of H2AX, ATM, and BRCA1, and reduced phosphorylation and acetylation of p53. Converse effects were seen with activation of p300 by HDAC inhibitors TSA and SAHA (HDACi). Further, p300KD completely abolished HDACi mediated cardioprotection and increase in AcH3K56. This could also explain accelerated aging phenotype of mice with heterozygous p300 loss.ConclusionGenotoxic stress causes a post‐transcriptional stabilization of p300 that is required for the DNA damage response and cardioprotection.The work was supported by NIH R01‐ HL7109, the Florida Heart Research Institute and AHA Pre‐doctoral fellowship to Sumit Jain.

Lipophilic And Hydrophilic Statins Differentially Modulate Endoplasmic Reticulum Stress Response In Cardiac Myocytes

Statins inhibit HMG‐CoA reductase, target mevalonic acid synthesis, and limit cholesterol biosynthesis. HMG‐CoA reductase is expressed in the membrane of the endoplasmic reticulum (ER). Statins are prescribed to prevent cardiovascular events.In cultured neonatal mouse cardiac myocytes the lipophilic statin atorvastatin and the hydrophilic statin pravastatin both up‐regulated PDI, indicating unfolded protein response (UPR) meant to relieve ER stress. Only atorvastatin increased ER stress, growth arrest, and induced apoptosis via induction of CHOP, Puma, active Caspase‐3 and PARP. Dose‐dependent release of LDH was only observed in atorvastatin treated cells (1–10 μM). Hearts of mice treated with atorvastatin (5mg/kg/day for 7 months) showed protein aggresomes and autophagosomes when compared to vehicle treated controls. While atorvastatin changed mitochondrial ultrastructure, no differences in cardiac function, exercise ability or creatine kinase levels were found.We show differential activation of ER stress by atorvastatin and pravastatin in cardiac myocytes. Our results provide a novel mechanism through which specific statins therapeutically modulate the balance of UPR/ER stress responses thereby possibly influencing cardiac remodeling.