Cardiac Myocyte Nuclei Research Articles

ErbB4 localization to cardiac myocyte nuclei, and its role in myocyte DNA damage response

The intracellular domain of ErbB4 receptor tyrosine kinase is known to translocate to the nucleus of cells where it can regulate p53 transcriptional activity. The purpose of this study was to examine whether ErbB4 can localize to the nucleus of adult rat ventricular myocytes (ARVM), and regulate p53 in these cells. We demonstrate that ErbB4 does locate to the nucleus of cardiac myocytes as a full-length protein, although nuclear location occurs as a full-length protein that does not require Protein Kinase C or γ-secretase activity. Consistent with this we found that only the non-cleavable JM-b isoform of ErbB4 is expressed in ARVM. Doxorubicin was used to examine ErbB4 role in regulation of a DNA damage response in ARVM. Doxorubicin induced p53 and p21 was suppressed by treatment with AG1478, an EGFR and ErbB4 kinase inhibitor, or suppression of ErbB4 expression with small interfering RNA. Thus ErbB4 localizes to the nucleus as a full-length protein, and plays a role in the DNA damage response induced by doxorubicin in cardiac myocytes.

Abstract P193: Nuclear Localization of the α1B-Adrenergic Receptor Subtype Is Required for Hypertrophic Signaling in Cardiac Myocytes

We previously demonstrated that α1-adrenergic receptors (α1-ARs) in the heart are required for physiologic hypertrophy during development and prevent a maladaptive response to pathologic stress. We have also shown that the major subtypes, α1A and α1B, both localize to the nucleus in adult cardiac myocytes. Importantly, we have defined a nuclear α1A-ERK survival and an α1A-PKCδ-cTnI inotropic signaling pathway that both originate at the nucleus and are transduced to cytosolic targets, suggesting that the α1A is required for cardiac myocyte survival and contractility. However, less is known about the molecular function of the α1B. In the current study, we examined the role of the α1B-subtype in hypertrophic signaling. First, we identified a bi-partite nuclear localization sequence (NLS) in the carboxy-terminal tail of the receptor. Mutation of the NLS (α1B-NLSmut) disrupted its localization to the nucleus when expressed in adult cardiac myocytes. We then compared hypertrophic signaling of the wild-type α1B- to the mutated receptor by reconstitution in cardiac myocytes lacking endogenous α1B (α1BKO) receptors. Activation of the wild-type receptor by the α1-agonist phenylephrine in α1BKO myocytes restored hypertrophic signaling, as we observed increased phosphorylation of protein kinase C (PKC) isoforms δ and ε, and histone deacetylases (HDAC) 4 and 5. We also observed increased expression of the hypertrophic gene marker, atrial natriuretic factor (ANF). Expression of the α1B-NLSmut failed to activate hypertrophic signaling in α1BKO cardiac myocytes despite phenylephrine stimulation. Together, our data show that nuclear localization of the α1B-subtype is required for hypertrophic signaling and overall further suggest that α1-adrenergic receptors are functional only at the nucleus in cardiac myocytes.

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.

Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells

Nuclear actin — which is immunologically distinct from cytoplasmic actin — has been documented in a number of differentiated cell types, and cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) have been detected in association with nuclei of adult human cardiac myocytes. It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. We first tested the hypothesis that nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. We also tested the hypothesis that of these five proteins, only actin is present in nuclei of multipotent, bone marrow-derived mesenchymal stem cells (BM-MSCs) from adult rats in culture, but that cTnC, cTnI, cTnT and cTm appear early and uniquely following cardiomyogenic differentiation. Here we show that nuclear actin is present within nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs. We furthermore show that cTnC, cTnI, cTnT and cTm are not only present in myofilaments of ventricular cardiomyocytes in culture but are also within their nuclei; significantly, these four proteins appear between days 3 and 5 in both myofilaments and nuclei of BM-MSCs treated to differentiate into cardiomyogenic cells. These observations indicate that cardiac troponin and tropomyosin could have important cellular function(s) beyond Ca2+-regulation of contraction. While the roles of nuclear-associated actin, troponin subunits and tropomyosin in cardiomyocytes are not known, we anticipate that the BM-MSC culture system described here will be useful for elucidating their function(s), which likely involve cardiac-specific, Ca2+-dependent signaling in the nucleus.

Unresponsive ventricular tachycardia associated with aluminum phosphide poisoning

Inhalation or ingestion of aluminum phosphide (AP) generates phosphine gas on exposure to moisture, which, in turn, produces widespread organ toxicity primarily involving the lungs, heart, liver, and kidneys. Cardiac manifestations of AP poisoning include toxic myocarditis, refractory heart failure, bradyarrhythmias, and tachyarrhythmias including ventricular tachycardia (VT). A 19-year-old depressed male farm worker ingested ten 500-mg tablets of Celphos in a suicide attempt. Each Celphos tablet contains 56% AP. Over the course of 10 hours, the patient developed heart failure and respiratory failure associated with a rise in serum troponin level to 12.7 ng/mL. Serum electrolytes (including magnesium) and serum creatinine levels were normal throughout. His course was further complicated by acidemia and hypotension. These hemodynamic and metabolic abnormalities were initially corrected by assisted ventilation and continuous veno-venous hemofiltration. However, he developed hemodynamically stable sustained monomorphic VT, which proved unresponsive to treatment with intravenous magnesium sulfate and intravenous amiodarone therapy. After a decline in blood pressure, 6 attempts at electrocardioversion failed to restore sinus rhythm, and he died. Postmortem histologic examination of myocardium showed contraction band necrosis, early coagulation necrosis, edema, hemorrhage, and pyknosis of cardiac myocyte nuclei. Ventricular tachycardia associated with AP poisoning has been successfully treated with magnesium sulfate, amiodarone, and electrocardioversion. This case report documents failure of all 3 of these therapeutic modalities.

Mechanisms of Toll-Like Receptor 4 (TLR4)-Mediated Inflammation After Cold Ischemia/Reperfusion in the Heart

Toll-Like Receptor 4 (TLR4) signaling mediates early inflammation after cold ischemia-reperfusion (I/R). We hypothesized that the TLR4 coreceptor CD14, the intracellular adaptor proteins myeloid differentiation factor 88 (MyD88) and TIR domain-containing-adaptor inducing IFNbeta (TRIF) would be required for cold I/R induced inflammation. High mobility group box 1 (HMGB1) is a putative endogenous activator of TLR4. Therefore, we also assessed the contribution of HMGB1 in cold I/R induced inflammation. Syngeneic heart transplants were performed in mice deficient in CD14, MyD88, TRIF, or wild-type mice. In other experiments, anti-HMGB1 neutralizing antibody or control IgG was administered at reperfusion. Donor hearts were subjected to 2 hr of cold ischemia and retrieved after 3 hr of reperfusion. After cold I/R, grafts revealed striking translocation of HMGB1 out of the nucleus in cardiac myocytes. Administration of an anti-HMGB1 neutralizing antibody resulted in reduced systemic interleukin (IL)-6, tumor necrosis factor alpha (TNFalpha), and intercellular adhesion molecule-1 (ICAM-1) messenger RNA (mRNA) levels (P< or =0.05). Compared with controls, CD14 knock-out (KO) mice exhibited significantly lower (P< or =0.05) systemic IL-6 and JE/monocyte chemotractant protein-1 levels after cold I/R. Intragraft TNFalpha and IL-1beta mRNA levels were also significantly lower (P< or =0.05) in CD14 KO grafts. MyD88 KO mice exhibited significantly lower (P< or=0.05) systemic IL-6 levels compared with control mice after cold I/R. Intragraft TNFalpha, IL-6, and ICAM-1 mRNA levels were also significantly lower (P< or =0.05) in MyD88 KO grafts. Significantly lower levels (P< or =0.05) of serum IL-6, monocyte chemotractant protein-1 as well as intragraft TNFalpha, IL-6, IL-1beta, and ICAM-1 were observed after cold I/R in TRIF deficient animals compared with controls. CD14, MyD88, TRIF, and HMGB1 contribute to the inflammatory response that occurs after cold I/R. These results provide insight into the mechanisms of TLR4-mediated inflammation after cold I/R.

A novel heart failure therapy that targets nuclear signaling pathway in cardiaomyocytes

Signals activated by increased hemodynamic overload to the heart finally reach nuclei of cardiac myocytes, change patterns of gene expression and cause their maladaptive hypertrophy. Nuclear acetylation controlled by an intrinsic histone acetyltransferase, p300, is a critical event during this process. However, a pharmacological heart failure therapy that targets this nuclear pathway has yet to be established. A zinc finger protein GATA4 is one of the hypertrophy-responsive transcription factors and forms a complex with an intrinsic histone acetyltransferase, p300.

Nuclear α1-Adrenergic Receptors Signal Activated ERK Localization to Caveolae in Adult Cardiac Myocytes

We previously identified an alpha1-AR-ERK (alpha1A-adrenergic receptor-extracellular signal-regulated kinase) survival signaling pathway in adult cardiac myocytes. Here, we investigated localization of alpha1-AR subtypes (alpha1A and alpha1B) and how their localization influences alpha1-AR signaling in cardiac myocytes. Using binding assays on myocyte subcellular fractions or a fluorescent alpha1-AR antagonist, we localized endogenous alpha1-ARs to the nucleus in wild-type adult cardiac myocytes. To clarify alpha1 subtype localization, we reconstituted alpha1 signaling in cultured alpha1A- and alpha1B-AR double knockout cardiac myocytes using alpha1-AR-green fluorescent protein (GFP) fusion proteins. Similar to endogenous alpha1-ARs and alpha1A- and alpha1B-GFP colocalized with LAP2 at the nuclear membrane. alpha1-AR nuclear localization was confirmed in vivo using alpha1-AR-GFP transgenic mice. The alpha1-signaling partners Galphaq and phospholipase Cbeta1 also colocalized with alpha1-ARs only at the nuclear membrane. Furthermore, we observed rapid catecholamine uptake mediated by norepinephrine-uptake-2 and found that alpha1-mediated activation of ERK was not inhibited by a membrane impermeant alpha1-blocker, suggesting alpha1 signaling is initiated at the nucleus. Contrary to prior studies, we did not observe alpha1-AR localization to caveolae, but we found that alpha1-AR signaling initiated at the nucleus led to activated ERK localized to caveolae. In summary, our results show that nuclear alpha1-ARs transduce signals to caveolae at the plasma membrane in cardiac myocytes.

Phosphorylation and nuclear translocation of serine 722 and serine 910 of focal adhesion kinase in hypertrophic cardiac myocytes of left ventricle of spontaneously hypertensive rats

To investigate the role of focal adhesion kinase (FAK) in cardiac hypertrophy induced by hypertension. Using immunofluorescent labeling, confocal microscopy and Western blot, the expression and subcellular location of FAK-pSer722 and FAK-pSer910 were determined in cardiac myocytes of the left ventricles from 2, 6, 12, and 18 month-old spontaneously hypertensive heart failure (SHHF) rats and age-matched Wistar-Kyoto (WKY) control rats, respectively. There was no obvious difference in FAK-pSer722 and FAK-pSer910 expression between 2 month-old SHHF and WKY rats. In contrast with the control groups, the expression of FAK-pSer722 and FAK-pSer910 significantly increased in cardiac myocytes of the left ventricle, from 6, 12 and 18 month-old SHHF rats. Both FAK-pSer722 and FAK-pSer910 were translocated and acummulated in nuclei of cardiac myocytes from 6, 12, and 18 month-old SHHF rats. Phosphorylation and translocation of serine 722 and serine 910 of phosphorylated FAK play an important role in the de-compensatory cardiac hypertrophy.

Protein Kinase A, Ca2+/Calmodulin-Dependent Kinase II, and Calcineurin Regulate the Intracellular Trafficking of Myopodin between the Z-Disc and the Nucleus of Cardiac Myocytes

Spatial and temporal resolution of intracellular signaling can be achieved by compartmentalizing transduction units. Myopodin is a dual-compartment, actin-bundling protein that shuttles between the nucleus and the Z-disc of myocytes in a differentiation- and stress-dependent fashion. Importin alpha binding and nuclear import of myopodin are regulated by serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. Here we show that in the heart myopodin forms a Z-disc signaling complex with alpha-actinin, calcineurin, Ca2+/calmodulin-dependent kinase II (CaMKII), muscle-specific A-kinase anchoring protein, and myomegalin. Phosphorylation of myopodin by protein kinase A (PKA) or CaMKII mediates 14-3-3 binding and nuclear import in myoblasts. Dephosphorylation of myopodin by calcineurin abrogates 14-3-3beta binding. Activation of PKA or inhibition of calcineurin in adult cardiac myocytes releases myopodin from the Z-disc and induces its nuclear import. The identification of myopodin as a direct target of PKA, CaMKII, and calcineurin defines a novel intracellular signaling pathway whereby changes in Z-disc dynamics may translate into compartmentalized signal transduction in the heart.

Transcriptome analysis of the response to chronic constant hypoxia in zebrafish hearts

Insufficient blood supply during acute infarction and chronic ischemia leads to tissue hypoxia which can significantly alter gene expression patterns in the heart. In contrast to most mammals, some teleost fishes are able to adapt to extremely low oxygen levels. We describe here that chronic constant hypoxia (CCH) leads to a smaller ventricular outflow tract, reduced lacunae within the central ventricular cavity and around the trabeculae and an increase in the number of cardiac myocyte nuclei per area in the hearts of two teleost species, zebrafish (Danio rerio) and cichlids (Haplochromis piceatus). In order to identify the molecular basis for the adaptations to CCH, we profiled the gene expression changes in the hearts of adult zebrafish. We have analyzed over 15,000 different transcripts and found 376 differentially regulated genes, of which 260 genes showed increased and 116 genes decreased expression levels. Two notch receptors (notch-2 and notch-3) as well as regulatory genes linked to cell proliferation were transcriptionally upregulated in hypoxic hearts. We observed a simultaneous increase in expression of IGF-2 and IGFbp1 and upregulation of several genes important for the protection against reactive oxygen species (ROS). We have identified here many novel genes involved in the response to CCH in the heart, which may have potential clinical implications in the future.Electronic supplementary materialThe online version of this article (doi:10.1007/s00360-007-0201-4) contains supplementary material, which is available to authorized users.

Myocarditis Associated with Foot-and-Mouth Disease Virus Type O in Lambs

The present study describes the pathogenetic mechanisms of myocarditis in 9 lambs that died in a foot-and-mouth disease outbreak in Samsun, Turkey. In all the heart samples tested, ELISA and sequencing for phylogenetic analyses showed that the virus, namely O/TUR/Samsun/05, was associated with the PanAsia pandemic strain of foot-and-mouth disease virus (FMDV) type O. The lambs had myocardial lesions but no typical vesicular lesions. In situ reverse transcription showed that many cardiomyocytes and some interstitial cells were positive for FMDV type O. Inflammatory infiltration, hyaline degeneration, and necrosis of sheets of myocytes were observed. The cellular infiltrates were mononuclear cells, including many lymphocytes, macrophages, a few plasma cells, and neutrophils. Major histocompatibility complex Class II+ dendritic and mononuclear cells, gammadelta T cells, CD172A+ and CD14+ macrophages and monocytes, and IgM+ B cells were detected mainly in the infected hearts. Inducible nitric oxide synthetase (iNOS) was seen mostly in areas of inflammation infiltrated by large numbers of cells. Of the 2 alpha-subunits of integrin known to be used as receptors by FMDV in epithelial tissues, CD49e (integrin alpha5) was detected in the membranes of cardiac myocytes with intercalated discs, but CD51 (integrin alphaV) was not detected in cardiac myocytes from infected or normal lambs. Interstitial and inflammatory cells were positive for both integrin subunits. The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL)-positive signal was detected in the nuclei of both cardiac myocytes and interstitial cells from infected lambs. These findings suggest that the iNOS expressed by inflammatory cells in lesions may have a deleterious effect on cardiac myocytes in these lesions.

Analysis of the Signals and Mechanisms Mediating Nuclear Trafficking of GATA-4: LOSS OF DNA BINDING IS ASSOCIATED WITH LOCALIZATION IN INTRANUCLEAR SPECKLES

Nucleocytoplasmic transport of GATA-4 is important in maintaining and regulating normal cardiogenesis and heart function. This report investigates the detailed mechanisms of GATA-4 nuclear transport. We characterized a nonclassical nuclear localization signal between amino acids 270 and 324 that actively transports GATA-4 into the nucleus of both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial arginine residues within this region that mediate active transport predominantly through the nonclassical pathway via interaction with importin beta. These four residues were also essential for the DNA binding activity of GATA-4 and transcriptional activation of cardiac-specific genes. Interestingly, mutation of these residues not only inhibited DNA binding and gene transcription but also resulted in a preferential accumulation of the GATA-4 protein in distinct subnuclear speckles. A cardiac myocyte-specific, chromosome maintenance region 1-dependent nuclear export signal consisting of three essential leucine residues was also identified. The current study provides detailed information on the nuclear shuttling pathways of GATA-4 that represents an additional mechanism of gene regulation.

PPAR Agonist, Pioglitazone Inhibits Interleukin-18 (IL-18)-induced Gene Expression and Apoptosis in Cardiac Myocytes

Interleukin-18 (IL-18) is a proinflammatory cytokine with multiple functions. We and others have reported that increased levels of circulating IL-18 are thought to be one of risk factors for heart failure. Nuclear factor kappa-B (NF-kB) enhances expression of several cytokines in heart failure. We have demonstrated that IL-18 induced c-fos SRE (Serum Response Element), NF-kB activities and apoptosis through translocation of Rho-kinase (Rho-K) into nucleus in cardiac myocytes. Pioglitazone, a PPAR (peroxisome proliferators-activated receptor) agonist has shown anti-inflammatory activities. In this study, we examined the role of Pioglitazone on IL-18-induced gene expression and apoptosis in cardiac myocytes. Pioglitazone inhibited IL-18-induced and RhoA- or Rho-K-induced c-fos SRE and NF-kB activities. Pioglytazone inhibited IL-18-induced Rho-K-fused-GFP (Green Fluorescence Protein) (GFP-Rho-K) translocation into nucleus. However, Pioglitazone did not inhibit RhoA-mediated Rho-K-GFP translocation into nucleus. From these results, Pioglitazone thought to inhibit at least two different sites from IL-18 receptor to SRE and NF-kB. One site is downstream of Rho-K and the other is upstream of RhoA. Meanwhile, IL-18 induced apoptosis. Pioglitazone also inhibited IL-18-induced apoptosis. PPAR might be one of important molecular targets for IL-18-induced cardiovascular events. Inhibition of IL-18-induced translocation of Rho-K into nucleus by Pioglitazone might be one of novel mechanisms for pleiotropic effects of PPAR.

Nuclear Ca 2+/calmodulin-dependent protein kinase II in the murine heart

Ca 2+ signaling through CaMKII is critical in regulating myocyte function with regard to excitation–contraction–relaxation cycles and excitation–transcription coupling. To investigate the role of nuclear CaMKII in cardiac function, transgenic mice were designed and generated to target the expression of a CaMKII inhibitory peptide, AIP (KKALRRQEAVDAL), to the nucleus. The transgenic construct consists of the murine α-myosin heavy chain promoter followed by the expression unit containing nucleotides encoding a four repeat concatemer of AIP (AIP 4) and a nuclear localization signal (NLS). Western blot and immunohistochemical analyses demonstrate that AIP 4 is expressed only in the nucleus of cardiac myocytes of the transgenic mice (NLS-AIP 4). The function of cytoplasmic CaMKII is not affected by the expression of AIP 4 in the nucleus. Inhibition of nuclear CaMKII activity resulted in reduced translocation of HDAC5 from nucleus to cytoplasm in NLS-AIP 4 mouse hearts. Loss of nuclear CaMKII activity causes NLS-AIP 4 mice to have smaller hearts than their nontransgenic littermates. Transcription factors including CREB and NFκB are not regulated by cardiac nuclear CaMKII. With physiological stresses such as pregnancy or aging (8 months), NLS-AIP 4 mice develop hypertrophy symptoms including enlarged atria, systemic edema, sedentariness, and morbidity. RT-PCR analyses revealed that the hypertrophic marker genes, such as ANF and β-myosin heavy chain, were upregulated in pregnancy stressed mice. Our results suggest that absence of adequate Ca 2+ signaling through nuclear CaMKII regulated pathways leads to development of cardiac disease.

Transcriptional Genomics Associates FOX Transcription Factors With Human Heart Failure

Specific transcription factors (TFs) modulate cardiac gene expression in murine models of heart failure, but their relevance in human subjects remains untested. We developed and applied a computational approach called transcriptional genomics to test the hypothesis that a discrete set of cardiac TFs is associated with human heart failure. RNA isolates from failing (n=196) and nonfailing (n=16) human hearts were hybridized with Affymetrix HU133A arrays, and differentially expressed heart failure genes were determined. TF binding sites overrepresented in the -5-kb promoter sequences of these heart failure genes were then determined with the use of public genome sequence databases. Binding sites for TFs identified in murine heart failure models (MEF2, NKX, NF-AT, and GATA) were significantly overrepresented in promoters of human heart failure genes (P<0.002; false discovery rate 2% to 4%). In addition, binding sites for FOX TFs showed substantial overrepresentation in both advanced human and early murine heart failure (P<0.002 and false discovery rate <4% for each). A role for FOX TFs was supported further by expression of FOXC1, C2, P1, P4, and O1A in failing human cardiac myocytes at levels similar to established hypertrophic TFs and by abundant FOXP1 protein in failing human cardiac myocyte nuclei. Our results provide the first evidence that specific TFs identified in murine models (MEF2, NKX, NFAT, and GATA) are associated with human heart failure. Moreover, these data implicate specific members of the FOX family of TFs (FOXC1, C2, P1, P4, and O1A) not previously suggested in heart failure pathogenesis. These findings provide a crucial link between animal models and human disease and suggest a specific role for FOX signaling in modulating the hypertrophic response of the heart to stress in humans.

Nuclear compartmentalization of FAK and FRNK in cardiac myocytes

Focal adhesion kinase (FAK) and FAK-related non-kinase (FRNK) accumulate in the nucleus of cardiac myocytes during hypertensive hypertrophy. Nuclear FAK and FRNK are phosphorylated on different serines and form distinct bright spots. The subnuclear distribution of serine-phosphorylated FAK and FRNK was examined in this study by double labeling with fibrillarin, a component of nucleoli, and Sam68, a constituent of Sam68 nuclear bodies. We also investigated the role of protein kinase C (PKC)-mediated phosphorylation of FAK and FRNK on nuclear translocation. PKC activation by 12-O-tetradecanoylphorbol 13-acetate treatment increased serine phosphorylation of FAK and FRNK. Specifically, FAK was phosphorylated on serine 722 but not serine 910. On the other hand, FRNK was phosphorylated on serine 217, the equivalent site of FAK serine 910, but not serine 30, the homologous site of FAK serine 722. Serine-phosphorylated FAK and FRNK redistributed into the nucleus and formed distinct patterns. FAK with phosphorylation on serine 722 colocalized with Sam68 but not fibrillarin. On the contrary, FRNK phosphorylated on 217 coexisted with fibrillarin but not Sam68. Immunoprecipitation also confirmed that FAK associated with Sam68 and FRNK interacted with fibrillarin, respectively. These results suggest that FAK and FRNK target different nuclear subdomains by their association with distinct nuclear proteins.

Elevated levels of DNA damage due to oxidative stress in serum and myocardium of patients with heart failure

Background: We hypothesized that oxidative damage to DNA might be associated with progression of heart failure. We investigated whether the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, were elevated in serum and myocardium of patients with cardiomyopathies. Methods and Results: Serum levels of 8-OHdG were measured by enzyme immunoassay in 58 patients with dilated cardiomyopathy (DCM) and 23 patients with hypertrophic cardiomyopathy (HCM) and in 20 control subjects. DCM patients had significantly elevated serum levels of 8-OHdG compared with those in control subjects and HCM patients (DCM: 5.2 ± 2.9 ng/mL vs. control: 3.0 ± 1.5 ng/mL, P = 0.0004; DCM vs. HCM: 2.6 ± 0.8 ng/mL, P < 0.0001). The serum levels were significantly correlated with left ventricular end-diastolic diameter determined by echocardiography (P = 0.0009). The levels were significantly inversely correlated with left ventricular ejection fraction determined by left ventriculography (P = 0.0002). Endomyocardial biopsy samples obtained from 12 DCM patients and 5 control subjects with normal cardiac function were studied immunohistochemically for the expression of 8-OHdG. Positive 8-OHdG staining was found in the nuclei of cardiac myocytes of samples from DCM patients but not in those from control subjects. After treatment with carvedilol, the serum levels of 8-OHdG in DCM patients decreased by 19% (P < 0.05) along with amelioration of heart failure. Conclusion: These results indicate that DNA damage due to oxidative stress is implicated in progression of heart failure.

Matrix metalloproteinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro.

Matrix metalloproteinases (MMPs) are traditionally known for their role in extracellular matrix remodeling. Increasing evidence reveals several alternative substrates and novel biological roles for these proteases. Recent evidence showed the intracellular localization of MMP-2 within cardiac myocytes, colocalized with troponin I within myofilaments. Here we investigated the presence of MMP-2 in the nucleus of cardiac myocytes using both immunogold electron microscopy and biochemical assays with nuclear extracts. The gelatinase activity found in both human heart and rat liver nuclear extracts was blocked with MMP inhibitors. In addition, the ability of MMP-2 to cleave poly (ADP-ribose) polymerase (PARP) as a substrate was examined as a possible role for MMP-2 in the nucleus. PARP is a nuclear matrix enzyme involved in the repair of DNA strand breaks, which is known to be inactivated by proteolytic cleavage. PARP was susceptible to cleavage by MMP-2 in vitro in a concentration-dependent manner, yielding novel degradation products of ~66 and <45 kDa. The cleavage of PARP by MMP-2 was also blocked by MMP inhibitors. This is the first characterization of MMP-2 within the nucleus and we hereby suggest its possible role in PARP degradation.

C-Jun is regulated by combination of enhanced expression and phosphorylation in acute-overloaded rat heart

The transient increase in the expression of transcription factors encoded by immediate-early genes has been considered to play a critical role in the coordination of early gene expression during the hypertrophic growth of cardiac myocytes. Here, we investigated the regulation of c-Jun and its upstream activators JNKs in the myocardium of rats subjected to acute pressure overload induced by transverse aortic constriction. Western blotting and immunohistochemistry analysis demonstrated that both JNK1 and JNK2 were transiently activated by pressure overload, but only JNK1 was activated at the nuclei of cardiac myocytes. JNK1 activation was paralleled by phosphorylation of c-Jun at serine-63 in the myocardial nuclear fraction and by an increase in c-Jun expression in cardiac myocytes. A consistent increase in DNA binding of activator protein-1 (AP-1) complex was observed after 10 and 30 min of pressure overload and Supershift assays confirmed that c-Jun was a major component of activated AP-1 complex. Moreover, experiments performed with the specific JNK inhibitor SP-600125 abolished c-Jun phosphorylation and markedly attenuated its expression as well as the expression of the fetal gene beta-myosin heavy chain. Overall, these findings demonstrate a molecular basis for load-induced activation of c-Jun in cardiac myocytes and its connection with the regulation of fetal gene, characteristic of the acute response to pressure overload.