Naoki Nozaki

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.

CD14-Deficient Mice Are Protected Against Lipopolysaccharide-Induced Cardiac Inflammation and Left Ventricular Dysfunction

Background—The molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. CD14 mediates the inflammatory response to lipopolysaccharide (LPS) in various organs including the heart. In this study we investigated the role of CD14 in LPS-induced myocardial dysfunction in vivo. Methods and Results—Wild-type and CD14-deficient (CD14-D) mice were challenged with Escherichia coli LPS. Myocardial tumor necrosis factor, interleukin-1&bgr; (IL-1&bgr;), and NOS2 induction was measured before and 6 hours after LPS challenge. Echocardiographic parameters of left ventricular function were measured before and 6 hours after LPS administration. LPS challenge induced a significant increase in myocardial tumor necrosis factor and IL-1&bgr; mRNA and protein expression in wild-type mice. In contrast, mRNA and protein levels for TNF and IL-1&bgr; were significantly blunted in CD14-D mice. An increase in NOS2 protein was noted within 6 hours of LPS provocation only in the hearts of wild-type mice. This was associated with an increase in ventricular cGMP levels. Activation of nuclear factor-&kgr;B was observed within 30 minutes of LPS in the hearts of wild-type mice but not in CD14-D mice. In wild-type mice, LPS significantly decreased left ventricular fractional shortening, velocity of circumferential shortening, and dP/dtmax. LPS-treated CD14-D mice maintained normal cardiac function. Conclusions—These results suggest that CD14 is important in mediating the proinflammatory response induced by LPS in the heart and that CD14 is necessary for the development of left ventricular dysfunction during LPS-induced shock in vivo.

Reactive oxygen species regulate FLICE inhibitory protein (FLIP) and susceptibility to Fas-mediated apoptosis in cardiac myocytes

OBJECTIVE Fas ligand (FasL) is a key cytokine which initiates apoptosis when FasL binds to its receptor, Fas. Cardiac myocytes are generally resistant to Fas-induced apoptosis. However, sublethal dose of doxorubicin (Dox) can sensitize cardiac myocytes to Fas-induced apoptosis. We investigated the molecular mechanism by which Dox sensitizes cardiac myocytes to Fas-induced apoptosis. FLICE inhibitory protein (FLIP) is a key molecule for blocking Fas-induced apoptosis by functioning as a caspase-8 dominant negative. METHODS AND RESULTS FLIP was constitutively expressed in cultured neonatal rat cardiac myocytes. FLIP protein levels were markedly down-regulated by Dox in a time-dependent and dose-dependent manner. Next, we examined the relation of reactive oxygen species (ROS) by Dox to the expression of FLIP. Both of N-acetylcysteine (NAC) and the combination of superoxide dismutase and catalase restored the decreased FLIP in Dox-treated cardiac myocytes to the basal level. NAC also restored the increased formation of thiobarbituric acid-reactive substance after Dox-treatment. Concurrently, the susceptibility to Fas-mediated apoptosis disappeared with the treatments of the antioxidant agents. Hydrogen peroxide down-regulated FLIP in a dose-dependent fashion and also sensitized cardiac myocytes to Fas-induced apoptosis. CONCLUSIONS FLIP, an inhibitor of apoptosis induced by cytokines of TNF family, contributes at least partly to Dox-induced sensitization to Fas-mediated apoptosis in cardiac myocytes. The expression of FLIP in cardiac myocytes is regulated by ROS.

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.

Serum Levels of Soluble Form of Fas Molecule in Patients with Congestive Heart Failure

Compared with soluble Fas molecule (sFas, an inhibitor of Fas-mediated apoptosis) in normal volunteers, the serum level of sFas significantly increased by 41% in New York Heart Association (NYHA) class III (p <0.05) and by 97% in NYHA class IV patients with congestive heart failure (p <0.001). Furthermore, sFas showed correlations with soluble forms of TNF receptor-p55 (RI) and -p75 (RII) (r = 0.68 and r = 0.56) which inhibit activities of TNF alpha.

Angiotensin-converting enzyme inhibition improves cardiac fatty acid metabolism in patients with congestive heart failure.

Summary This study aimed to examine whether angiotensin‐converting enzyme (ACE) inhibition improved cardiac fatty acid metabolism in patients with congestive heart failure (CHF). Myocardial 123I‐&bgr;‐methyl‐iodophenylpentadecanoic acid (123I‐BMIPP) imaging was performed in 25 patients with CHF and in 10 control subjects. Myocardial 123I‐BMIPP images were obtained 30 min and 4 h after tracer injection. The heart‐to‐mediastinum (H/M) ratio of 123I‐BMIPP uptake and the washout rate of 123I‐BMIPP from the myocardium were calculated. Patients were given enalapril for 6 months, and 123I‐BMIPP imaging was repeated. H/M ratios on early and delayed images were lower in CHF patients than in normal controls (P<0.01). The washout rate of 123I‐BMIPP from the myocardium was faster in CHF patients than in controls (P<0.01). As the severity of the New York Heart Association (NYHA) functional class increased, the H/M ratio decreased and the washout rate increased. The washout rate of 123I‐BMIPP was inversely correlated with left ventricular fractional shortening (R = ‐0.62, P < 0.01). ACE inhibition with enalapril increased the H/M ratio on delayed images (P<0.05) and reduced the washout rate of 123I‐BMIPP (P<0.05) in CHF patients. These data suggest that: (1) angiotensin II‐mediated intracellular signalling activation may be a possible mechanism for the decreased myocardial uptake and enhanced washout of 123I‐BMIPP in heart failure patients; and (2) the improvement in fatty acid metabolism by ACE inhibition may represent a new mechanism for the beneficial effect of this therapy in heart failure. (© 2003 Lippincott Williams & Wilkins)

Ongoing myocardial damage relates to cardiac sympathetic nervous disintegrity in patients with heart failure

Iodine-123-metaiodobenzylguanidine (123I-MIBG) has been used to assess the integrity and function of the cardiac sympathetic nervous system in patients with heart failure. Heart-type fatty acid binding protein (H-FABP) is released into the circulation when the myocardium is injured, and H-FABP has been recently used as a novel marker for the diagnosis of ongoing myocardial damage.ObjectiveThe aim of the present study was to compare cardiac sympathetic nervous activity assessed by123I-MIBG imaging with serum levels of H-FABP in patients with heart failure.MethodsFifty patients with chronic heart failure were studied.123I-MIBG imaging was carried out at 30 min (early) and 240 min (delayed) after the tracer injection. We measured serum levels of H-FABP using a sandwich enzyme linked immunosorbent assay.ResultsHeart to mediastinum (H/M) ratios of123I-MIBG decreased and washout rate increased with higher New York Heart Association (NYHA) functional class. H-FABP, norepinephrine and brain natriuretic peptide (BNP) levels increased as the severity of NYHA class advanced. Delayed H/M ratio was significantly correlated with H-FABP (r = -0.296, p = 0.029) and BNP (r = -0.335, p = 0.0213). Myocardial washout rate of123I-MIBG was also correlated with H-FABP (r = 0.469, p < 0.001), norepinephrine (r = 0.433, p = 0.005), and BNP (r = 0.465, p = 0.001).ConclusionsThese data suggest that cardiac sympathetic nervous activation was associated with ongoing cardiomyocyte damage characterized by an elevated serum level of H-FABP in patients with heart failure.123I-MIBG imaging is an appropriate approach to evaluate non-invasively not only cardiac sympathetic nervous activity, but also latent ongoing myocardial damage in the failing heart.

Dynamic123I-MIBG SPECT reflects sympathetic nervous integrity and predicts clinical outcome in patients with chronic heart failure

Abstract123I-metaiodobenzylguanidine (123I-MIBG) is useful for assessment of the severity and prognosis of patients with chronic heart failure (CHF). To examine123I-MIBG kinetics in the early phase soon after tracer injection, we performed dynamic single photon emission computed tomography (SPECT) in 76 patients with CHF and 17 control subjects. The consecutive 15 images of 2 min-dynamic SPECT were acquired for 30 min after injection. From 0 to 4 min, a significant amount of radioactivity existed in the blood pool, thus we calculated washout rate of123I-MIBG from 4 to 30 min (%WR-E). Patients were followed up with an end-point of cardiac death or re-hospitalization for 16 months (6–30 months). As the NYHA functional class advanced, %WR-E increased (control, NYHA class I, II, and III: 9 ± 4%, 10 ± 5%, 12 ± 5%, and 17 ± 5%*, respectively, *p < 0.01 vs. all other groups). Significant correlation was found between %WR-E and conventional WR from 30 min to 240 min (r = 0.606, p < 0.0001). %WR-E was positively correlated with left ventricular end-diastolic dimension (r = 0.372, p < 0.01 ) and was inversely correlated with left ventricular fractional shortening (r = -0.316, p < 0.02). The normal upper limit of %WR-E was defined as mean + 2SD value of 17 control subjects (17.1%). Patients with abnormally rapid %WR-E levels had a higher cardiac event rate than those with normal %WR-E levels (57% vs. 12%, p < 0.0001). These data suggest that washout rate of123I-MIBG in the early phase from 4 min to 30 min (%WR-E) reflects cardiac sympathetic nervous integrity and is useful to evaluate the severity and prognosis of patients with CHF. The present results indicate a potential role of dynamic SPECT in shortening the123I-MIBG imaging protocol.