Tetsuro Shishido

Toll-Like Receptor-2 Modulates Ventricular Remodeling After Myocardial Infarction

Background—Toll-like receptors (TLRs) are members of the interleukin-1 receptor family and transduce similar signals as interleukin-1 receptor in response to exogenous pathogens. Recent studies have demonstrated that TLRs are activated by endogenous signals, such as heat shock proteins and oxidative stress, that may contribute to ventricular remodeling after myocardial infarction. In this study, we determined whether TLR-2 was involved in cardiac remodeling after myocardial infarction. Methods and Results—Myocardial infarction was induced by surgical left anterior descending coronary artery ligation on wild-type (WT) mice and TLR-2–knockout (KO) mice. The survival rate was significantly higher in KO mice than in WT mice 4 weeks after myocardial infarction (65% versus 43%, P <0.03). Infarct size and degree of inflammatory cell infiltration in infarct area were similar between WT and KO mice. However, myocardial fibrosis in the noninfarct area of KO mice was much less than in WT mice (P <0.01) and was accompanied by reduced transforming growth factor-&bgr;1 and collagen type 1 mRNA expressions (P <0.01 and P <0.05, respectively). Left ventricular dimensions at end diastole were smaller in KO mice than in WT mice at 1 week (P <0.05) and 4 weeks (P <0.01) after surgery. Furthermore, fractional shortening was higher (27.7±2.5% versus 21.2±2.6%, P <0.05, at 1 week, and 24.3±2.0% versus 16.6±2.5%, P <0.01, at 4 weeks) in KO mice compared with WT mice. Conclusions—These data suggest that TLR-2 plays an important role in ventricular remodeling after myocardial infarction.

Modulation of Doxorubicin-Induced Cardiac Dysfunction in Toll-Like Receptor-2–Knockout Mice

Background—Toll-like receptors (TLRs) are members of the interleukin-1 receptor family and are involved in the responsiveness to pathogen-associated molecular patterns. Recent studies have demonstrated that TLRs are activated by endogenous signals, such as heat shock proteins and oxidative stress, which may contribute to congestive heart failure. Oxidative stress is one of the major factors in doxorubicin (Dox)-induced cardiac dysfunction. Thus, we hypothesized that TLRs contribute to the pathogenesis of Dox-induced cardiac dysfunction. Methods and Results—Cardiac dysfunction was induced by a single injection of Dox (20 mg/kg IP) into wild-type (WT) mice and TLR-2–knockout (KO) mice. Five days after Dox injection, left ventricular dimension at end-diastole was smaller and fractional shortening was higher in KO mice compared with WT mice (P<0.01). Nuclear factor-&kgr;B activation and production of proinflammatory cytokines after Dox were suppressed in KO mice compared with WT mice (P<0.01). The numbers of TUNEL-positive nuclei and Dox-induced caspase-3 activation were less in KO mice than in WT mice (P<0.01). Survival rate was significantly higher in KO mice than in WT mice 10 days after Dox injection (46% vs 11%, P<0.05). Conclusions—These findings suggest that TLR-2 may play a role in the regulation of inflammatory and apoptotic mediators in the heart after Dox administration.

Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy

Background— Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. Methods and Results— We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1−/− mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. Conclusions— Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Extracellular Signal-Regulated Kinase 5 SUMOylation Antagonizes Shear Stress–Induced Antiinflammatory Response and Endothelial Nitric Oxide Synthase Expression in Endothelial Cells

Shear stress–induced extracellular signal-regulated kinase (ERK)5 activation and the consequent regulation of Kruppel-like factor 2 and endothelial nitric oxide synthase expression represents one of the antiinflammatory and vascular tone regulatory mechanisms maintaining normal endothelial function. Endothelial dysfunction is a major initiator of atherosclerosis, a vascular pathology often associated with diabetes. Small ubiquitin-like modifier (SUMO) covalently attaches to certain residues of specific target transcription factors and could inhibit its activity. We investigated whether H2O2 and AGE (advanced glycation end products), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity and laminar flow–induced endothelial nitric oxide synthase expression through ERK5 SUMOylation. H2O2 and AGE induced endogenous ERK5 SUMOylation. In addition, ERK5 SUMOylation was increased in the aortas from diabetic mice. ERK5 transcriptional activity, but not kinase activity, was inhibited by expression of Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), suggesting the involvement of ERK5 SUMOylation on its transcriptional activity. Point-mutation analyses showed that ERK5 is covalently modified by SUMO at 2 conserved sites, Lys6 and Lys22, and that the SUMOylation defective mutant of ERK5, dominant negative form of Ubc9 (DN-Ubc9), and small interfering RNA PIAS1 reversed H2O2 and AGE–mediated reduction of shear stress–mediated ERK5/myocyte enhancer factor 2 transcriptional activity, as well as promoter activity of Kruppel-like factor 2. Finally, PIAS1 knockdown reversed the inhibitory effect of H2O2 in shear stress–induced Kruppel-like factor 2 and endothelial nitric oxide synthase expression. These data clearly defined SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and suggested this process as among the molecular mechanisms of diabetes-mediated endothelial dysfunction.

ERK5 Activation Inhibits Inflammatory Responses via Peroxisome Proliferator-activated Receptor δ (PPARδ) Stimulation

Peroxisome proliferator-activated receptors (PPAR) decrease the production of cytokine and inducible nitric-oxide synthase (iNOS) expression, which are associated with aging-related inflammation and insulin resistance. Recently, the involvement of the induction of heme oxygenase-1 (HO-1) in regulating inflammation has been suggested, but the exact mechanisms for reducing inflammation by HO-1 remains unclear. We found that overexpression of HO-1 and [Ru(CO)3Cl2]2, a carbon monoxide (CO)-releasing compound, increased not only ERK5 kinase activity, but also its transcriptional activity measured by luciferase assay with the transfection of the Gal4-ERK5 reporter gene. This transcriptional activity is required for coactivation of PPARδ by ERK5 in C2C12 cells. [Ru(CO)3Cl2]2 activated PPARδ transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-κB activity by ERK5 activation was reversed by a dominant negative form of PPARδ suggesting that ERK5/PPARδ activation is required for the anti-inflammatory effects of CO and HO-1. Based on these data, we propose a new mechanism by which CO and HO-1 mediate anti-inflammatory effects via activating ERK5/PPARδ, and ERK5 mediates CO and HO-1-induced PPARδ activation via its interaction with PPARδ.

Inhibiting p90 Ribosomal S6 Kinase Prevents Na+-H+ Exchanger–Mediated Cardiac Ischemia-Reperfusion Injury

Background— Pharmacological and genetic studies indicate that the Na+-H+ exchanger isoform 1 (NHE1) plays a critical role in myocardial ischemia and reperfusion (I/R) injury. We found that p90 ribosomal S6 kinase (RSK) phosphorylated serine 703 of NHE1, stimulating 14–3–3 binding and NHE1 activity. Therefore, we hypothesized that inhibiting RSK in cardiomyocytes would prevent NHE1 activation and decrease I/R-mediated injury. Methods and Results— To examine the role of RSK in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative RSK (DN-RSK-TG). DN-RSK-TG hearts demonstrated normal basal cardiac function and morphology. However, myocardial infarction (left coronary artery occlusion for 45 minutes) in DN-RSK-TG hearts was significantly reduced at 24 hours of reperfusion from 46.9±5.6% area at risk in nontransgenic littermate controls to 26.0±4.2% in DN-RSK-TG (P<0.01). Cardiomyocyte apoptosis was significantly reduced after I/R in DN-RSK (0.9±0.2%) compared with nontransgenic littermate controls (6.2±2.6%). Importantly, activation of RSK and interaction of 14–3–3 with NHE1, necessary for agonist-stimulated NHE1 activity, were increased by I/R and inhibited by 70% in DN-RSK-TG (P<0.01). Next, we transduced rat neonatal cardiomyocytes with adenovirus-expressing DN-RSK (Ad.DN-RSK) and measured NHE1 activity. The baseline rate of pH recovery in acid-loaded cells was equal in cells expressing LacZ or DN-RSK. However, NHE1 activation by 100 &mgr;mol/L H2O2 was significantly inhibited in cells expressing DN-RSK (0.16±0.02 pH units/min) compared with Ad.LacZ (0.49±0.13 pH units/min). Apoptosis induced by 12 hours of anoxia followed by 24 hours’ reoxygenation was significantly reduced in cells expressing Ad.DN-RSK (18.6±2.0%) compared with Ad.LacZ (29.3±5.4%). Conclusions— In summary, RSK is a novel regulator of cardiac NHE1 activity by phosphorylating NHE1 serine 703 and a new pathological mediator of I/R injury in the heart.

The prognostic importance of objective nutritional indexes in patients with chronic heart failure

BACKGROUND Although malnutrition indicates an unfavorable prognosis in some clinical settings, the association between nutritional indexes and outcomes for patients with chronic heart failure (CHF) is unclear. METHODS AND RESULTS All the previously established objective nutritional indexes were evaluated. The controlling nutritional status score (CONUT), prognostic nutritional index (PNI), and geriatric nutritional risk index (GNRI) were determined for 388 consecutive patients with CHF (mean age 69.6±12.3 years). The prevalence of malnutrition in this cohort was 60-69%. Patients were followed prospectively, with the endpoints being death due to a cardiovascular event or re-hospitalization. There were 130 events, including 33 deaths and 97 re-hospitalizations, during a mean follow-up period of 28.4 months. Patients experiencing cardiovascular events showed impaired nutritional status, higher CONUT scores, lower PNI scores, and lower GNRI scores, compared with those who did not experience cardiovascular events. CONUT score [hazard ratio 40.9, 95% confidence interval (CI) 10.8-154.8], PNI score (hazard ratio 6.4, 95% CI 5.4-25.1), and GNRI score (hazard ratio 11.6, 95% CI 3.7-10.0) were independently associated with cardiovascular events. Kaplan-Meier analysis showed that there was a significantly higher incidence of cardiovascular events in patients who were malnourished than in those who were not. CONCLUSION Malnutrition was common in patients with CHF. Evaluation of nutritional status may provide additional prognostic information in patients with CHF.

Cardiac-Specific Overexpression of Diacylglycerol Kinase ζ Prevents Gq Protein-Coupled Receptor Agonist-Induced Cardiac Hypertrophy in Transgenic Mice

Background— Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqα protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. Methods and Results— To test this hypothesis, we generated transgenic (DGK&zgr;-TG) mice with cardiac-specific overexpression of DGK&zgr;. There were no differences in heart size and heart weight between DGK&zgr;-TG and wild-type littermate mice. The left ventricular function was normal in DGK&zgr;-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGK&zgr;-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGK&zgr;-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGK&zgr;-TG mouse hearts, suggesting that DGK&zgr; regulated PKC activity by controlling cellular diacylglycerol levels. Conclusions— These results demonstrated the first evidence that DGK&zgr; negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

Effects of MEK5/ERK5 Association on Small Ubiquitin-Related Modification of ERK5: Implications for Diabetic Ventricular Dysfunction After Myocardial Infarction

Diabetes mellitus (DM) contributes to the exacerbation of left ventricle (LV) dysfunction after myocardial infarction (MI). Activation of ERK5, an atypical mitogen activated protein kinase with transcriptional activity, inhibits apoptosis and LV dysfunction after doxorubicin treatment. SUMOylation has been proposed as a negative regulator of various transcription factors. In the current study, we investigated the role of ERK5-SUMOylation in ERK5 transcriptional activity as well as on DM-mediated exacerbation of LV dysfunction and apoptosis after MI. ERK5 wild-type transcriptional activity was inhibited by Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), but not in the ERK5-SUMOylation-site defective mutant (K6R/K22R). H2O2 and high glucose, 2 well-known mediators of diabetes, induced ERK5-SUMOylation, and the K6R/K22R mutant, dominant negative form of Ubc9, and siRNA-PIAS1 reversed H2O2-mediated reduction of ERK5 transcriptional activity in cardiomyocytes, indicating the presence of SUMOylation-dependent ERK5 transcriptional repression. Constitutively active form of MEK5&agr; (CA-MEK5&agr;) inhibited ERK5-SUMOylation independent of kinase activity, but dependent on MEK5-ERK5 association. To investigate the pathological role of ERK5-SUMOylation in DM mice after MI, we used cardiac specific CA-MEK5&agr; transgenic mice (CA-MEK5&agr;-Tg). MI was induced in streptozotocin (STZ)-injected (DM+MI group) or vehicle-injected mice (MI group) by ligating the left coronary artery. The ERK5-SUMOylation was increased in the DM+MI, but not in the MI group. ERK5-SUMOylation, the exacerbation of LV dysfunction, and the number of TUNEL-positive cells in DM+MI was significantly inhibited in CA-MEK5&agr;-Tg mice. Of note, we could not detect any difference of cardiac function after MI in non-diabetic CA-MEK5&agr;-Tg and non-transgenic littermate control mice. These results demonstrated that ERK5 transcriptional activity is subject to downregulation by diabetes-dependent SUMOylation, which resulted in a proapoptotic condition contributing to poor post-MI LV function.

Activation of distinct signal transduction pathways in hypertrophied hearts by pressure and volume overload

Abstract.ObjectiveTwo types of hemodynamic overload, pressure and volume overload, result in morphologically distinct types of cardiac remodeling. We explored the possibility that distinct hemodynamic overload may differentially activate the signal transduction pathway.MethodsPressure and volume overload were induced by thoracic aortic banding and carotid–jugular shunt formation in rabbits, respectively. Phosphorylation activities of mitogen–activated protein (MAP) kinase families, Akt, and signal transducer and activator of transcription (STAT) 3 in the left ventricular myocardium were determined by Western blotting using phospho–specific antibodies and were compared between hypertrophied hearts by pressure and volume overload.ResultsPressure and volume overload produced concentric and eccentric cardiac hypertrophy in rabbits, respectively. In pressure–overloaded hearts, extracellular signal–regulated kinase (ERK) 1/2, p38 MAP kinase, and STAT3 were transiently activated prior to hypertrophic changes. In contrast, activation of ERK1/2, but not p38 MAP kinase and STAT3, was observed only at 12 weeks after shunt surgery. Pressure overload evoked short and biphasic activation of Akt at 15 min and 1 day after aortic banding. In contrast, volume overload induced sustained activation of Akt from 1 day to 1 week. Concordant phosphorylation of downstream targets of Akt, glycogen synthase kinase–3β (GSK–3β) and p70 ribosomal S6 kinase (p70S6K), in response to Akt activation was observed at 15 min after pressure overload. However in volume–overloaded hearts, phosphorylation of GSK-3β and p70S6K was observed at 6 weeks and at 6 and 12 weeks, respectively, and was not coincident with Akt activation. These findings suggest that phosphorylation of GSK-3β and p70S6K is regulated by an alternative pathway other than Akt in volume–overloaded hearts.ConclusionPressure and volume overload–induced cardiac hypertrophy is associated with distinct patterns of activation of signal transduction pathways. These data may suggest that stimulus–specific heterogeneity in the signaling pathway plays a role in determining the type of cardiac hypertrophy.