Cultured Mouse Cardiomyocytes Research Articles

Direct Binding of Synaptopodin 2-Like Protein to Alpha-Actinin Contributes to Actin Bundle Formation in Cardiomyocytes.

Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.

Abstract Mo013: Regulation of Ferroptosis by Wnt5a in the Heart: Implication in Cardio-Oncology

Ferroptosis is an iron and lipid peroxidation dependent form of cell death. It has been implicated in ischemia/reperfusion injury, heart failure, and doxorubicin (Dox)-induced cardiotoxicity (DIC). While Wingless-type family member (Wnt) pathways are crucial for heart development and remodeling, the role of Wnt5a in regulating ferroptosis in cardiac cells and its relevance to DIC remain unclear. In the present study, we investigate the role of Wnt5a signaling in cardiac ferroptosis and its implication in cardio-oncology. We observed an increase in Wnt5a secretion in H9C2 cardiac cells upon treatment with ferroptosis inducers such as ferric ammonium citrate (FAC 2 mM) and RSL3 (20 nM). Live/Dead viability analyses in cultured mouse cardiomyocytes (CMs) and cardiac fibroblasts (CFs) revealed that the Wnt5a antagonist BOX5 (100 µM) attenuated ferroptosis induced by FAC or RSL3 in both CMs and CFs. Similarly, FAC- and RSL3-induced ferroptotic death rates were reduced in CMs from Wnt5a conditional knockout (cKO) mice. Recombinant Wnt5a (rWnt5a) induced CM death in a dose-dependent manner (5, 7, 10 µg/ml) and was partially prevented by Fer-1 (10 µM), a selective ferroptosis inhibitor. Moreover, rWnt5a (10 µg/ml) also effectively induced CF ferroptosis which was inhibited by Fer-1. rWnt5a and its mimicking peptide Foxy5 also modulated the sensitivity to various ferroptosis inducers. Furthermore, FAC-, RSL3- and rWnt5a-induced ferroptosis were attenuated in CMs from frizzled 2 receptor cKO mice. We further delineated the role of Wnt5a upregulation in Dox-induced ferroptosis and the development of DIC. In a chronic DIC mouse model, Wnt5a upregulation in the heart correlated with left ventricular dysfunction which was abrogated in Wnt5a cKO mice. Live/Dead viability analyses showed that Dox induced ferroptosis which was partially prevented by Fer-1 in CMs. Inhibition of the Wnt5a pathway by BOX5 led to a decrease in the rate of Dox-induced ferroptosis, while the sensitivity to ferroptosis was enhanced by rWnt5a. Our study has shown for the first time that the production and secretion of Wnt5a in an autocrine and/or paracrine manner promotes ferroptosis in CMs and CFs. Wnt5a is likely to be a critical mediator of Dox-induced ferroptosis and cardiotoxicity.

The involvement and therapeutic potential of lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway in arsenic trioxide-induced cardiotoxicity

Background/AimsArsenic trioxide (ATO) is the first-line therapeutic drug for acute promyelocytic leukemia. However, the cardiotoxicity of ATO limits its clinical application. This study aims to explore the long noncoding RNA (lncRNA) involved molecular mechanism in ATO-induced cardiotoxicity and to identify available prevention strategies.MethodsATO was administered to mice or primary cultured mouse cardiomyocytes. Small interfering RNA targeting lncRNA Kcnq1ot1 (si-Kcnq1ot1) was used to knockdown lncRNA Kcnq1ot1. MiR-34a-5p mimic and antisense morpholino oligonucleotide targeting miR-34a-5p (AMO-34a-5p) were used to upregulate and downregulate the expression of miR-34a-5p, respectively. TUNEL staining was conducted to detect cell DNA damage. Flow cytometry assay was used to detect cell apoptosis. Western blot was conducted to detect Bcl-2, Bax and Sirt1 protein expression. Real-time PCR was used to detect lncRNA Kcnq1ot1, miR-34a-5p, and Sirt1 mRNA expression. Dual-luciferase reporter assay was performed to validate the predicted binding site.ResultsATO induced apoptosis in cardiomyocytes both in vivo and in vitro. Simultaneously, the expression of lncRNA Kcnq1ot1 and Sirt1 was downregulated, and miR-34a-5p was upregulated. MiR-34a-5p has binding sites with lncRNA Kcnq1ot1 and Sirt1. Knockdown of lncRNA Kcnq1ot1 induced apoptosis of cardiomyocytes, with increased miR-34a-5p and decreased Sirt1 expression. Inhibition of miR-34a-5p attenuated si-Kcnq1ot1-induced apoptosis in cardiomyocytes. Therefore, the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 signaling pathway is involved in ATO-induced cardiotoxicity. Propranolol alleviated ATO-induced apoptosis in cardiomyocytes both in vivo and in vitro, which was related to the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 signaling pathway.ConclusionThe lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway is involved in ATO-induced cardiotoxicity. Propranolol can attenuate ATO-induced cardiotoxicity at least partially through the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway. Combined administration with propranolol may be a new strategy for alleviating the cardiotoxicity of ATO.

Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption

Kinetic analysis of intracellular calcium (Ca2+) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca2+ kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca2+ kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca2+ amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly.

MALAT1-mediated recruitment of the histone methyltransferase EZH2 to the microRNA-22 promoter leads to cardiomyocyte apoptosis in diabetic cardiomyopathy

Diabetic patients often have a heightened risk of cardiomyopathy, even in the absence of traditional risk factors such as hypertension and atherosclerotic coronary artery disease. Diabetic cardiomyopathy is characterized by a typical cardiomyopathy specific to diabetes, the pathogenesis of which has yet to be fully elucidated. As a well-documented oncogenic long noncoding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been implicated in a variety of pathological processes, including diabetic complications. This study aimed to evaluate the functional roles of MALAT1 in the pathogenesis of diabetic cardiomyopathy. Spontaneously diabetic (db/db) C57BL/Ks mice were employed to establish diabetic cardiomyopathy models in vivo and high glucose (HG)-cultured mouse cardiomyocytes for myocardial damage models in vitro. Mouse left ventricular volume and function were evaluated by echocardiography, while the myocyte cross-sectional area was calculated to evaluate the degree of myocardial hypertrophy. TUNEL staining and flow cytometric analysis were performed to evaluate myocardial damage and cardiomyocyte apoptosis. Silencing of MALAT1 was found to attenuate cardiac dysfunction and inhibit cardiomyocyte apoptosis in db/db mice and HG-cultured mouse cardiomyocytes. MALAT1 recruited the histone methyltransferase EZH2 to the miR-22 promoter region and inhibited its expression. EZH2 induced an increased in the expression of ATP-binding cassette transporter A1 (ABCA1), which was identified to be a target gene of miR-22. Silencing of EZH2 was found to improve cardiac function and prevent cardiomyocyte apoptosis in db/db mice and HG-cultured mouse cardiomyocytes in the presence of MALAT1, suggesting that MALAT1 mediated myocardial damage by recruiting EZH2 to the miR-22 promoter. Taken together, this study's findings provide evidence confirming our hypothesis, suggesting the involvement of MALAT1 in the processes of cardiac function and cardiomyocyte apoptosis via the EZH2/miR-22/ABCA1 signaling cascade, which has potential therapeutic implications for the understanding of diabetic cardiomyopathy.

Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity.

We recently reported that doxorubicin decreased the expression of calpain-1/2, while inhibition of calpain activity promoted doxorubicin-induced cardiac injury in mice. In this study, we investigated whether and how elevation of calpain-2 could affect doxorubicin-triggered cardiac injury. Transgenic mice with inducible cardiomyocyte-specific expression of calpain-2 were generated. An acute cardiotoxicity was induced in both transgenic mice and their relevant wild-type littermates by injection of a single dose of doxorubicin (20mg/kg) and cardiac injury was analyzed 5 days after doxorubicin injection. Cardiomyocyte-specific up-regulation of calpain-2 did not induce any adverse cardiac phenotypes under physiological conditions by age 3 months, but significantly reduced myocardial injury and improved myocardial function in doxorubicin-treated mice. Cardiac protection of calpain-2 up-regulation was also observed in a mouse model of chronic doxorubicin cardiotoxicity. Up-regulation of calpain-2 increased the protein levels of mitogen activated protein kinase phosphatase-1 (MKP-1) in cultured mouse cardiomyocytes and heart tissues. Over-expression of MKP-1 prevented, whereas knockdown of MKP-1 augmented doxorubicin-induced apoptosis in cultured cardiomyocytes. Moreover, knockdown of MKP-1 offset calpain-2-elicited protective effects against doxorubicin-induced injury in cultured cardiomyocytes. Mechanistically, up-regulation of calpain-2 reduced the protein levels of phosphatase and tensin homolog and consequently promoted Akt activation, leading to increased MKP-1 protein steady-state levels by inhibiting its degradation. Collectively, this study reveals a new role of calpain-2 in promoting MKP-1 expression via phosphatase and tensin homolog/Akt signaling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury.

Abstract 471: Development of a Cardiomyocyte Targeting Delivery System Utilizing Cardiosphere-Derived Cell Exosomes

Research has shown that treating the heart with stem cell populations, including cardiosphere-derived cells (CDCs), post myocardial infarction stimulates regeneration, angiogenesis, and functional improvement. While this treatment has shown promise in early stage clinical trials, there remains a gap in the ability to efficiently deliver tissue-specific agents directly to the heart while avoiding nonspecific delivery to other organs. Our aim was to develop an efficient delivery system that can target cardiomyocytes and transport drugs, siRNA, and/or miRNA to aid in the treatment of cardiovascular diseases. Methods: Lamp-2b was cloned with cDNA from C2C12 cells and ligated downstream of the CMV promoter in a lentiviral transfer plasmid. Primers designed to encode a cardiomyocyte targeting peptide were used to introduce the targeting ligand between XhoI and BspEI at the N terminus of Lamp-2b forming the final vector (LV-CMP). We subsequently generated CMP lentivirus by triple transfection of LV-CMP with helper plasmids pLP/VSVG and psPAX2 into 293T cells. CDCs were transduced at 1 MOI in the presence of polybrene. Exosomes were isolated from infected and non-infected CDCs and labelled with a nucleic acid dye. Cultured mouse cardiomyocytes were incubated with 1 ug of fluorescent labeled CDC-exosomes or CMP CDC-exosomes for 24 hours then imaged. Results: To achieve exosomal cardiomyocyte targeting, we engineered CDCs to express Lamp-2b, an exosomal membrane protein, fused to a cardiomyocyte specific peptide. Primary cardiomyocytes showed a 3.8-fold increase in uptake of targeted CMP CDC-exosomes in vitro when compared with their non-engineered CDC control, establishing the efficacy of our system and laying the groundwork for further experiments to assess the efficiency of cardiac localization and cardiomyocyte uptake in vivo. Conclusions: Cardiomyocytes show enhanced in vitro uptake of CDC-derived exosomes engineered to express a cardiomyocyte targeting peptide fused with Lamp-2b. Improving exosomal uptake by cardiomyocytes following myocardial infarction would allow for improved delivery of beneficial drug and gene therapies and may result in an increase in viable myocardium and improved functional capacity of the heart.

Abstract 404: Sphingosine-1-phosphate Receptor 1 Inhibits Pathological Cardiac Remodeling Through Histone Deacetylases

Sphingosine-1-phosphate receptor 1 (S1P 1 , encoded by S1pr1 ) is a G protein-coupled receptor that signals in multiple cell types including endothelium and cardiomyocytes. We observed decreased cardiac S1PR1 mRNA in humans with nonischemic cardiomyopathy; these patterns were also observed in mice after cardiac stress. To assess the importance of reduced S1pr1 expression, we exposed S1pr1 heterozygous mice to isoproterenol or angiotensin II. Subsequently, hearts from S1pr1+/- mice displayed significantly higher markers of hypertrophy, inflammation, fibrosis, and oxidative stress than wild-type littermates. Cardiomyocyte-specific deletion of S1pr1 caused pronounced increases in the same markers. In wild-type mice, co-administration of the S1P 1 -specific agonist SEW2871 rescued the pathologic effects of angiotensin II. Because class I histone deacetylases (HDACs) contribute to pathological remodeling, we investigated interactions between S1P 1 and HDAC activities as a potential mechanism. In cultured mouse cardiomyocytes, angiotensin II increased expression of histone deacetylases 1 and 2, as well as other markers of pathological remodeling; co-treatment with SEW2871 inhibited these responses. Moreover, HDAC inhibition in S1pr1+/- mice blunted the pathological response to angiotensin II. These results indicate that S1P 1 protects by modulating HDAC1/2 during cardiac stress and support S1P 1 agonism as a therapeutic strategy for pathological cardiac remodeling in humans.

PCSK9 expression in the ischaemic heart and its relationship to infarct size, cardiac function, and development of autophagy.

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapy to treat hypercholesterolaemia and related cardiovascular diseases. This study determined if PCSK9 can regulate infarct size, cardiac function, and autophagy during ischaemia. Mice hearts were subjected to left coronary artery (LCA) occlusion. There was intense expression of PCSK9 in the zone bordering the infarct area in association with marked cardiac contractile dysfunction in the wild-type mice. This region also revealed intense autophagy. To assess the role of PCSK9 in the evolution of infarct size and function and development of autophagy, we used wild-type mice pre-treated with two different PCSK9 inhibitors (Pep2-8 and EGF-A) or mice lacking PCSK9 gene. Both strategies resulted in smaller infarcts and improved cardiac function following LCA ligation. PCSK9 inhibition also markedly reduced autophagy. Relationship between myocardial ischaemia and PCSK9 expression and autophagy was examined in cultured mouse cardiomyocytes. Exposure of cardiomyocytes to hypoxia resulted in prompt PCSK9 expression and autophagy signals; both were blocked by HIF-1α siRNA. Further, treatment of cardiomyocytes with recombinant PCSK9 during hypoxia induced, and treatment with PCSK9 siRNA reduced, autophagy suggesting a possible role of PCSK9 in the determination of autophagy. Other studies revealed activation of ROS-ATM-LKB1-AMPK axis as a possible mechanism of PCSK-induced autophagy. Hearts of humans with recent infarcts also showed expression of PCSK9 and autophagy in the border zone-similar to that in the infarcted mouse heart. PCSK9 is up-regulated in the ischaemic hearts and determines development of infarct size, cardiac function, and autophagy.

Environmentally Persistent Free Radicals Cause Apoptosis in HL-1 Cardiomyocytes.

Samples of environmental particulate matter contain environmentally persistent free radicals (EPFRs) capable of sustained generation of oxygen radicals. While exposure to EPFRs produces cardiac toxicity and oxidative stress in experimental animals, the underlying mechanisms are largely unknown. To determine whether EPFRs could directly damage cardiomyocytes, cultured mouse cardiomyocytes (HL-1) and primary rat adult left ventricular myocytes (ALVM) were incubated with an EPFR consisting of 1,2-dichlorobenzene chemisorbed to CuO-coated silica beads (DCB230). Treatment with DCB230 killed both HL-1 and ALVM in a dose- and time-dependent manner. The cytotoxic effects of DCB230 were significantly attenuated by treatment with α-tocopherol. One to 2h after exposure to DCB230, there were significant reductions in mitochondrial membrane potential and significant increases in cleaved caspase-9, but no significant increases in DNA damage or cell death. After 8h of treatment, there were significant increases in caspase-3, caspase-9, DNA damage and PARP cleavage associated with significant cell death. Together, these data indicate that DCB230 kills HL-1 myocytes by inducing oxidative stress that initiates apoptosis, with the intrinsic or mitochondrial pathway acting early in the apoptotic signaling process.

MiR-1 mediated suppression of Sorcin regulates myocardial contractility through modulation of Ca2+ signaling

MicroRNAs are negative gene regulators and play important roles in cardiac development and disease. As evident by cardiomyopathy following cardiac-specific Dicer knockdown they also are required for maintaining normal cardiac contractile function but the specific role of miR-1 in the process is poorly understood. To characterize the role of miR-1 in particular and to identify its specific targets we created a tamoxifen-inducible, cardiac-specific Dicer knockdown mouse and demonstrated that Dicer downregulation results in a dramatic and rapid decline in cardiac function concurrent with significantly reduced levels of miR-1. The importance of miR-1 was established by miR-1 antagomir treatment of wild-type mice, which replicated the cardiac-specific Dicer knockdown phenotype. Down-regulation of miR-1 was associated with up-regulation of its predicted target Sorcin, an established modulator of calcium signaling and excitation-contraction coupling, subsequently verified as a miR-1 target with luciferase constructs. siRNA-mediated knockdown of Sorcin effectively rescued the cardiac phenotypes after Dicer or miR-1 knockdown affirming Sorcin as a critical mediator of the acute cardiomyopathy observed. The regulatory relationship between miR-1 and Sorcin was further confirmed in cultured mouse cardiomyocytes where modulation of miR-1 was associated with discordant Sorcin levels and dysregulation of calcium signaling. Pathological relevance of our findings included decreased miR-1 and increased Sorcin expression in end-stage cardiomyopathy. These findings demonstrate the importance of miR-1 in cardiac function and in the pathogenesis of heart failure via Sorcin-dependent calcium homeostasis.

G i -Biased β 2 AR Signaling Links GRK2 Upregulation to Heart Failure

Phosphorylation of β(2)-adrenergic receptor (β(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of β(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to βAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant β(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type β(2)AR (WT-TG) or a mutant β(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased β(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of β(2)AR by GRK and resultant increase in G(i)-biased β(2)AR signaling play an important role in the development of heart failure. Our data show that enhanced β(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased β(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.

Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, but fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils and contribute insight into mechanisms of myofibrillogenesis and disassembly.

Local overexpression of HB-EGF exacerbates remodeling following myocardial infarction by activating noncardiomyocytes

Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are cardiogenic and cardiohypertrophic growth factors. Although the therapeutic effects of IGF and HGF have been well demonstrated in injured hearts, it is uncertain whether natural upregulation of HB-EGF after myocardial infarction (MI) plays a beneficial or pathological role in the process of remodeling. To answer this question, we conducted adenoviral HB-EGF gene transduction in in vitro and in vivo injured heart models, allowing us to highlight and explore the HB-EGF-induced phenotypes. Overexpressed HB-EGF had no cytoprotective or additive death-inducible effect on Fas-induced apoptosis or oxidative stress injury in primary cultured mouse cardiomyocytes, although it significantly induced hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. Locally overexpressed HB-EGF in the MI border area in rabbit hearts did not improve cardiac function or exhibit an angiogenic effect, and instead exacerbated remodeling at the subacute and chronic stages post-MI. Namely, it elevated the levels of apoptosis, fibrosis, and the accumulation of myofibroblasts and macrophages in the MI area, in addition to inducing left ventricular hypertrophy. Thus, upregulated HB-EGF plays a pathophysiological role in injured hearts in contrast to the therapeutic roles of IGF and HGF. These results imply that regulation of HB-EGF may be a therapeutic target for treating cardiac hypertrophy and fibrosis.

Expression of Protein S in the Murine Heart and Cultured Mouse Cardiomyocytes Is Down-regulated by Cytokines

Protein S (PS), a co-factor of activated protein C, is a vitamin K-dependent anticoagulant protein and is known to be produced extrahepatically. In the present study, the concentration of PS mRNA was determined tissue by tissue in the mouse, and it was high in lung, adrenal and heart as well as in liver. We further investigated the effects of lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 (IL-1) on the PS mRNA expression in murine tissues in vivo. Although LPS and TNF-alpha significantly decreased the expression level of PS mRNA in all tissues examined (e. g., lung, liver, heart, and kidney) and the PS antigen level in plasma, the suppressive effect of IL-1 on PS gene expression was limited to heart. More specifically, considerable amounts of PS mRNA and antigen were expressed in a cultured mouse cardiomyocyte cell line, and again, treatment with IL-1 decreased the PS expression in these cells. These observations raise a possibility that the expression of cardiac PS may contribute to the regional anticoagulant potential in heart, and suggest that the decreased PS expression by cytokines may result in an increase in the systemic and/or regional prothrombotic potential under inflammatory conditions.

Coupling function of endogenous alpha(1)- and beta-adrenergic receptors in mouse cardiomyocytes.

Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. beta(1)-ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective beta(2)-AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. beta(2)-AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. alpha(1)-ARs fail to activate phospholipase C, the extracellular signal-regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated by Pasteurella multocida toxin (a G(q) alpha-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via beta-ARs in mouse cardiomyocytes, but beta-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in alpha(1)-AR signaling and a defect in beta(2)-AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.

Inhibition by metallothionein of doxorubicin toxicity in cultured mouse cardiomyocytes: [formula omitted], Department of Medicine, University of Louisville, Louisville, KY 40292

Inhibition by metallothionein of doxorubicin toxicity in cultured mouse cardiomyocytes: [formula omitted], Department of Medicine, University of Louisville, Louisville, KY 40292