Midkine Deteriorates Cardiac Remodeling via Epidermal Growth Factor Receptor Signaling in Chronic Kidney Disease.

In chronic kidney disease, activation of the epidermal growth factor receptor (EGFR) leads to cardiac hypertrophy, which affects morbidity and mortality. In patients with renal insufficiency and heart failure, the expression of midkine, a heparin-binding growth factor, is increased. Therefore, we investigated the association between midkine and EGFR in the induction of cardiac hypertrophy and dysfunction in chronic kidney disease. We performed subtotal nephrectomies in midkine-knockout mice and wild-type mice. We found that subtotal nephrectomy-induced cardiac hypertrophy and phosphorylation of extracellular signal-regulated kinase 1/2 and AKT were attenuated in midkine-knockout mice compared with wild-type mice. An antiphosphotyrosine receptor antibody array was used to demonstrate that EGFR phosphorylation in the heart was also lower in midkine-knockout mice than in wild-type mice. Midkine induced EGFR, extracellular signal-regulated kinase 1/2, and AKT phosphorylation and led to hypertrophy in neonatal rat cardiomyocytes. Pretreatment with EGFR inhibitors or EGFR silencing suppressed midkine-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, induction of fetal cardiac gene expression, and hypertrophy in cardiomyocytes. To confirm the association between midkine and EGFR in vivo, mice subjected to subtotal nephrectomy were treated with the EGFR inhibitor gefitinib. Gefitinib treatment attenuated subtotal nephrectomy-induced cardiac hypertrophy. These results indicate that midkine might be a key mediator of cardiorenal interactions through EGFR activation, which plays a crucial role in cardiac hypertrophy in chronic kidney disease.

HECT-Type Ubiquitin E3 Ligase ITCH Interacts With Thioredoxin-Interacting Protein and Ameliorates Reactive Oxygen Species-Induced Cardiotoxicity.

BACKGROUND: The homologous to the E6-AP carboxyl terminus (HECT)-type ubiquitin E3 ligase ITCH is an enzyme that plays a pivotal role in posttranslational modification by ubiquitin proteasomal protein degradation. Thioredoxin-interacting protein (TXNIP) is a negative regulator of the thioredoxin system and an endogenous reactive oxygen species scavenger. In the present study, we focused on the functional role of ubiquitin E3 ligase ITCH and its interaction with TXNIP to elucidate the mechanism of cardiotoxicity induced by reactive oxygen species, such as doxorubicin and hydrogen peroxide. METHODS AND RESULTS: Protein interaction between TXNIP and ITCH in cardiomyocyte was confirmed by immunoprecipitation assays. Overexpression of ITCH increased proteasomal TXNIP degradation and augmented thioredoxin activity, leading to inhibition of reactive oxygen species generation, p38 MAPK, p53, and subsequent intrinsic pathway cardiomyocyte apoptosis in reactive oxygen species-induced cardiotoxicity. Conversely, knockdown of ITCH using small interfering RNA inhibited TXNIP degradation and resulted in a subsequent increase in cardiomyocyte apoptosis. Next, we generated a transgenic mouse with cardiac-specific overexpression of ITCH, called the ITCH-Tg mouse. The expression level of TXNIP in the myocardium in ITCH-Tg mice was significantly lower than WT littermates. In ITCH-Tg mice, cardiac dysfunction and remodeling were restored compared with WT littermates after doxorubicin injection and myocardial infarction surgery. Kaplan-Meier analysis revealed that ITCH-Tg mice had a higher survival rate than WT littermates after doxorubicin injection and myocardial infarction surgery. CONCLUSION: We demonstrated, for the first time, that ITCH targets TXNIP for ubiquitin-proteasome degradation in cardiomyocytes and ameliorates reactive oxygen species-induced cardiotoxicity through the thioredoxin system.

The Clinical Value of Nongated Dual-Source Computed Tomography in Atrial Fibrillation Catheter Ablation.

INTRODUCTION: Using a high-pitch dual-source CT (DSCT), we aimed to quantify the amounts of contrast media, radiation doses, and image qualities in patients undergoing pulmonary vein (PV) isolation. METHODS AND RESULTS: The study enrolled 60 patients who were randomly assigned in a 1: 1: 1 ratio to undergo ECG-gated 64-slice multidetector computed tomography (MDCT; group I, n = 20), ECG-gated 128-DSCT (group II, n = 20), and nongated 128-DSCT (group III, n = 20). The total amount of contrast media was lower in groups II and III compared with group I (I: 54.7 ± 5.6, II: 26.6 ± 2.7, and III: 28.7 ± 6.9 mL, P < 0.001). The CT dose index was lower in groups II and III compared with group I (I: 73.1 ± 5.2, II: 3.5 ± 0.1, and III: 3.7 ± 0.1 mGy, P < 0.001). The dose length product was lower in groups II and III compared with group I (I: 1154.8 ± 82.8, II: 75.4 ± 2.3, and III: 77.2 ± 1.9 mGy × cm, P < 0.001). The total CT effective radiation dose was lower in groups II and III compared with group I (I: 16.2 ± 1.2, II: 1.1 ± 0.1, and III: 1.1 ± 0.1 mSv, P < 0.001). The total CT scan duration was shorter in group III compared with groups I and II (I: 30.8 ± 2.2, II: 23.4 ± 3.6, and III: 16.0 ± 2.4 minutes, P < 0.001). There were no significant differences in quality for integrated electroanatomical mapping (EAM) and parameters associated with PV isolation among the 3 groups. CONCLUSION: Nongated 128-DSCT provides sufficient image quality to allow integrated EAM while exposing the patient to less contrast media, lower radiation doses, and shorter CT scan durations.

Deficiency of Long Pentraxin PTX3 Promoted Neointimal Hyperplasia after Vascular Injury.

AIM: Pentraxin 3 (PTX3) is a novel marker for the primary local activation of innate immunity and inflammatory responses. Although clinical and experimental evidence suggests that PTX3 is associated with atherosclerosis, the relationship between PTX3 and vascular remodeling after wall injury remains to be determined. We investigated the effects of PTX3 on neointimal hyperplasia following wire vascular injury. METHODS: PTX3 systemic knockout (PTX3-KO) mice and wild-type littermate (WT) mice were subjected to wire-mediated endovascular injury. At four weeks after wire-mediated injury, the areas of neointimal and medial hyperplasia were evaluated. RESULTS: The PTX3-KO mice exhibited higher hyperplasia/media ratios than the WT mice after wire injury, and the degree of Mac-3-positive macrophage accumulation was significantly higher in the PTX3-KO mice than in the WT mice. Furthermore, the PTX3-KO mice showed a much greater increase in the number of PCNA-stained cells in the vascular wall than that observed in the WT mice. CONCLUSIONS: A deficiency of PTX3 results in deteriorated neointimal hyperplasia after vascular injury via the effects of macrophage accumulation and vascular smooth muscle cell proliferation and migration.

Diacylglycerol kinase α exacerbates cardiac injury after ischemia/reperfusion.

Early coronary reperfusion of the ischemic myocardium is a desired therapeutic goal for the preservation of myocardial function. However, reperfusion itself causes additional myocardium injuries. Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade has been implicated in the cardioprotective effects occurring after ischemia/reperfusion (I/R). DAG kinase (DGK) controls cellular DAG levels by converting DAG to phosphatidic acid, and may act as an endogenous regulator of DAG-PKC signaling. In the present study, we examined the functional role of DGKα in cardiac injury after I/R in in vivo mouse hearts. We generated transgenic mice with cardiac-specific overexpression of DGKα (DGKα-TG). The left anterior descending coronary artery was transiently occluded for 20 min and reperfused for 24 h in DGKα-TG mice and wild-type littermate (WT) mice. The levels of phosphorylation activity of PKCε, extracellular-signal regulated kinase (ERK) 1/2, and p70 ribosomal S6 kinase (p70S6K) were increased after I/R in WT mouse hearts. However, in DGKα-TG mice, activation of PKCε, ERK1/2, and p70S6K was attenuated compared to WT mice. After 24 h, Evans blue/triphenyltetrazolium chloride double staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that DGKα-TG mice had significantly larger myocardial infarctions and larger numbers of TUNEL-positive cardiomyocytes than WT mice. Echocardiography and cardiac catheterization revealed that left ventricular systolic function was more severely depressed in DGKα-TG mice than in WT mice after I/R. These findings suggest that DGKα exacerbates I/R injury by inhibiting the cardioprotective effects of PKCε, ERK1/2, and p70S6K activation.

A novel mouse model of aortic valve stenosis induced by direct wire injury.

OBJECTIVE: The response-to-tissue-injury theory is currently the favorite paradigm to investigate valve pathology. To the best of our knowledge, there are currently no in vivo valve injury models. There are few calcific aortic valve stenosis (AVS) models that develop hemodynamically significant stenosis. Here, we investigated the effect of direct mechanical injury on aortic valves in vivo and developed a novel mouse model of calcific AVS. APPROACH AND RESULTS: Aortic valve injury was created by inserting and moving a spring guidewire under echocardiographic guidance into the left ventricle of male C57/BL6 mice via right common carotid artery. Serial echocardiographic measurements revealed that aortic velocity was increased 1 week after injury and persistently increased until 16 weeks after injury. AVS mice showed a higher heart weight/body weight ratio and decreased left ventricular fractioning shortening 4 weeks after injury, compared with sham mice. We found remarkable proliferation of valve leaflets 4 weeks after injury. Proliferative valves showed increased production of reactive oxygen species and expression of inflammatory cytokines and osteochondrogenic factors. Alizarin red staining showed valvular calcification 12 weeks after injury. CONCLUSIONS: We report a novel calcific AVS model to support the response-to-tissue-injury theory. This model may be a valuable tool for analyzing the mechanism of AVS and assessing therapeutic options.

Midkine exacerbates pressure overload-induced cardiac remodeling.

Midkine is a multifunctional growth factor, and its serum levels are increased with the functional severity of heart failure. This study aimed to examine the role of midkine in heart failure pathogenesis. Midkine expression levels were increased in the kidney and lung after transverse aortic constriction (TAC) surgery, but not sufficiently increased in the heart. After TAC, phosphorylation of extracellular signal-regulated kinase1/2 and AKT, and the expression levels of foetal genes in the heart were considerably increased in transgenic mice with cardiac-specific overexpression of midkine (MK-Tg) compared with wild-type (WT) mice. MK-Tg mice showed more severe cardiac hypertrophy and dysfunction, and showed lower survival rate after TAC than WT mice. We conclude that midkine plays a critical role in cardiac hypertrophy and remodelling.

Atrial endothelial impairment through Toll-like receptor 4 signaling causes atrial thrombogenesis.

Low-grade inflammation associated with heart failure (HF) is known to deteriorate cardioembolic stroke in patients with atrial fibrillation (AF). Little is known about the relationship between atrial endothelial impairment induced by innate immunity and thrombus formation. We examined whether atrial endothelial impairment through Toll-like receptor (TLR) 4 signaling causes atrial thrombogenesis. TLR4, heat shock protein 60, and vascular cell adhesion molecule (VCAM)-1 expression were higher in the atrium of AF patients who underwent valve replacement surgery with HF compared with those without it (p < 0.05). We created thoracic transverse aortic constriction (TAC) in TLR4 knock-out (KO) and wild-type (WT) mice. Atrial thrombosis was observed less frequently in TLR4 KO mice (4/15) than in WT mice (16/20) 4 weeks after TAC despite similar severity of heart failure. The decrease in endothelial nitric oxide synthase (eNOS) phosphorylation and increase in VCAM-1 and plasminogen activator inhibitor (PAI)-1 expression, observed in the atrium of WT mice following TAC, were significantly attenuated in TLR4 KO mice (p < 0.05). Nuclear factor-κB (NF-κB) activation after TAC was attenuated in TLR4 KO mice compared with WT mice. Activation of mitogen-activated protein kinase p38 (p38) after TAC was also attenuated in TLR4 KO mice (p < 0.05). Thus, increased VCAM-1 and PAI-1, and decreased eNOS phosphorylation through the TLR4/NFκB/p38 pathway, may be associated with atrial thrombogenesis in the heart failure mice model. Atrial endothelial impairment through the TLR4 signaling may play a role in atrial thrombogenesis in AF patients with HF.

The impact of renal tubular damage, as assessed by urinary ß2-microglobulin-creatinine ratio, on cardiac prognosis in patients with chronic heart failure.

BACKGROUND: Renal dysfunction was reported to be closely associated with clinical outcomes in patients with chronic heart failure (CHF). Renal tubulointerstitial damage has been shown to be an important factor in the development of renal dysfunction as well as glomerular damage. However, the impact of renal tubular damage on clinical outcomes in patients with CHF remains to be determined. METHODS AND RESULTS: Urinary ß2-microglobulin-creatinine ratio was measured in 315 patients with CHF. Renal tubular damage was defined as a urinary ß2-microglobulin-creatinine ratio ≥ 300 µg/g, as previously reported. Patients were prospectively followed up for a median period of 1097 days. There were 91 cardiac events, including 16 cardiac deaths and 75 rehospitalizations for worsening heart failure. Log10 urinary ß2-microglobulin-creatinine ratio was increased with worsening New York Heart Association functional class. Multivariate analysis revealed that renal tubular damage was an independent predictor of cardiac events. Kaplan-Meier analysis demonstrated that the rate of cardiac events was higher in patients with renal tubular damage compared with those without it. Patients were divided into 4 groups according to the presence of chronic kidney disease and renal tubular damage. The Cox proportional hazard analysis revealed that comorbidity of chronic kidney disease and renal tubular damage was associated with the highest risk for cardiac events compared with other groups. CONCLUSIONS: Renal tubular damage was related to the severity of heart failure and was associated with poor outcomes in patients with CHF. Renal tubular damage could add clinical information to chronic kidney disease in patients with CHF.

Cardiac nuclear high mobility group box 1 prevents the development of cardiac hypertrophy and heart failure.

AIMS: High mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription, and repair. However, little is known about the effects of nuclear HMGB1 on cardiac hypertrophy and heart failure. The aim of this study was to examine whether nuclear HMGB1 plays a role in the development of cardiac hypertrophy induced by pressure overload. METHODS AND RESULTS: Analysis of human biopsy samples by immunohistochemistry showed decreased nuclear HMGB1 expression in failing hearts compared with normal hearts. Nuclear HMGB1 decreased in response to both endothelin-1 (ET-1) and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes, where nuclear HMGB1 was acetylated and translocated to the cytoplasm. Overexpression of nuclear HMGB1 attenuated ET-1 induced cardiomyocyte hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) and wild-type (WT) mice. Cardiac hypertrophy after TAC was attenuated in HMGB1-Tg mice and the survival rate after TAC was higher in HMGB1-Tg mice than in WT mice. Induction of foetal cardiac genes was decreased in HMGB1-Tg mice compared with WT mice. Nuclear HMGB1 expression was preserved in HMGB1-Tg mice compared with WT mice and significantly attenuated DNA damage after TAC was attenuated in HMGB1-TG mice. CONCLUSION: These results suggest that the maintenance of stable nuclear HMGB1 levels prevents hypertrophy and heart failure by inhibiting DNA damage.

Long pentraxin PTX3 exacerbates pressure overload-induced left ventricular dysfunction.

BACKGROUND: Left ventricular hypertrophy is enhanced by an inflammatory state and stimulation of various cytokines. Pentraxin 3 (PTX3) is rapidly produced in response to inflammatory signals, and high plasma PTX3 levels are seen in patients with heart failure. This study aimed to examine the influence of PTX3 on cardiac hypertrophy and left ventricular dysfunction with respect to pressure overload. METHODS AND RESULTS: PTX3 systemic knockout (PTX3-KO) mice, transgenic mice with cardiac-specific overexpression of PTX3 (PTX3-TG), and the respective wild-type (WT) littermate mice were subjected to transverse aortic constriction (TAC) or a sham operation. Cardiac PTX3 expression increased after TAC in WT mice. In vitro, hydrogen peroxide induced the expression of PTX3 in both cardiac myocytes and cardiac fibroblasts. Recombinant PTX3 phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) in cardiac fibroblasts. Phosphorylation of cardiac ERK1/2 and nuclear factor kappa-B after TAC was attenuated in the PTX3-KO mice but was enhanced in the PTX3-TG mice compared with WT mice. Interleukin-6 and connective tissue growth factor production was lower in the PTX3-KO mice than in the WT mice, but this was augmented in the PTX3-TG mice than in the WT mice. Echocardiography revealed that adverse remodeling with left ventricular dysfunction, as well as with increased interstitial fibrosis, was enhanced in PTX3-TG mice, while these responses were suppressed in PTX3-KO mice. CONCLUSION: The local inflammatory mediator PTX3 directly modulates the hypertrophic response and ventricular dysfunction following an increased afterload.

Acidic urine is associated with poor prognosis in patients with chronic heart failure.

Renal dysfunction is reported to be associated with poor outcomes in patients with chronic heart failure (CHF). A recent study showed that acidic urine is related to chronic kidney disease, which is a risk factor for the development of CHF. However, it remains to be determined whether acidic urine is associated with poor outcomes in patients with CHF. We measured urine pH using dipsticks in 537 patients with CHF. Acidic urine was defined as urine pH ≤5.5. Patients were prospectively followed during a median follow-up period of 556 days. There were 145 cardiac events. Prevalence of acidic urine was increased with advancing stage of chronic kidney disease. Patients with acidic urine had a more severe New York Heart Association functional class compared with those with normal urine. In the multivariate Cox proportional hazard analysis, acidic urine was independently associated with poor outcomes in patients with CHF after adjustment of confounding factors. A Kaplan-Meier analysis demonstrated that the rate of cardiac events was higher in patients with acidic urine than in those with normal urine. The presence of acidic urine can reliably identify patients at high risk of future cardiac events in patients with CHF.

Renal tubulointerstitial damage is associated with short-term cardiovascular events in patients with myocardial infarction.

BACKGROUND: Urinary ß2 microglobulin (U-ß2MG) is a more sensitive and accurate marker of tubulointerstitial damage. The etiology of glomerular damage is related to the occurrence of major adverse cardiovascular events (MACE) in patients with myocardial infarction (MI); however, the prognostic importance of tubulointerstitial damage in patients with ST-segment elevation MI (STEMI) has not been established. The aim of this study was to elucidate whether renal tubulointerstitial damage is associated with the occurrence of MACE in patients after STEMI undergoing percutaneous coronary intervention. METHODS AND RESULTS: The degree of renal tubulointerstitial damage was evaluated by measuring the U-ß2MG level in 89 consecutive STEMI patients. There were 22 MACEs during the follow-up period. Patients with MACE had higher U-ß2MG levels than those without MACE, and the U-ß2MG level was an independent predictor for MACE. A Kaplan-Meier analysis revealed that the group with higher U-ß2MG levels corrected for urinary creatinine was associated with a greater risk for MACE. CONCLUSIONS: An elevated U-ß2MG level was associated with the occurrence of MACE in STEMI patients who underwent PCI. Renal tubulointerstitial damage is therefore considered to be associated with the occurrence of MACE.

Serum pregnancy-associated plasma protein a in patients with heart failure.

BACKGROUND: Pregnancy-associated plasma protein A (PAPP-A) proteolyzes insulin-like growth factor (IGF)-binding proteins and thus increases IGF-1 bioactivity. PAPP-A has been reported to be involved in various pathophysiologic abnormalities; however, the clinical significance of PAPP-A has not been examined in cases of heart failure (HF). We hypothesized that PAPP-A levels might be correlated with the severity of HF. METHODS AND RESULTS: PAPP-A and B-type natriuretic peptide (BNP) levels were measured in 262 subjects (182 HF patients and 80 control subjects). PAPP-A levels were higher in patients with HF than in control subjects and increased with advancing New York Heart Association functional class. There were 53 cardiac events during a mean follow-up period of 796 days. PAPP-A levels were higher in patients with cardiac events than in event-free patients. Patients were divided into 3 groups on the basis of their PAPP-A and BNP levels. Kaplan-Meier analysis demonstrated that the group with both high BNP with high PAPP-A had a significantly higher cardiac event rate than other groups. CONCLUSIONS: Serum PAPP-A levels were related to the severity of HF and associated with a high risk for adverse cardiac events in HF patients, suggesting that PAPP-A might be involved in the pathogenesis of HF.

Elevated cystatin C levels predict the incidence of vasospastic angina.

BACKGROUND: Cystatin C, a marker for early stage chronic kidney disease, has been shown to be involved in cardiovascular disease. The relationship between serum cystatin C levels and coronary vasospastic angina (VSA), however, remains to be elucidated. The aim of the present study was to investigate whether elevated cystatin C levels predict the incidence of VSA. METHODS AND RESULTS: One hundred and ten patients were referred to hospital due to suspected VSA. VSA was evoked in 59 patients by a vasospasm provocation test with administration of acetylcholine into the coronary arteries. The patients with VSA had lower levels of high-density lipoprotein cholesterol and a higher history of cigarette smoking, higher levels of triglyceride, high-sensitivity C-reactive protein, and higher cystatin C levels compared with those without VSA. There were no differences in serum creatinine or estimated glomerular filtration rate between patients with and without VSA. Multivariate logistic regression indicated that history of smoking (odds ratio, 2.956 P<0.05) and cystatin C levels (odds ratio, 2.285; P<0.01) were independently associated with the incidence of VSA. CONCLUSIONS: Elevated cystatin C levels were associated with higher incidence of VSA, suggesting that mild renal dysfunction may be implicated in the pathogenesis of coronary artery spasm.

Iodine-123-metaiodobenzylguanidine imaging can predict future cardiac events in heart failure patients with preserved ejection fraction.

OBJECTIVE: Iodine-123-metaiodobenzylguanidine ((123)I-MIBG) has been used to assess the function of the cardiac sympathetic nervous system in patients with chronic heart failure (HF). The usefulness of (123)I-MIBG imaging for evaluating patients with heart failure with preserved ejection fraction (HFPEF) has not been established. METHODS: We performed (123)I-MIBG scintigraphy and echocardiography and measured the plasma brain natriuretic peptide (BNP) levels of 117 consecutive HF patients (64 men, mean age 66 ± 14 years) with a left ventricular ejection fraction (LVEF) of ≥50% who were admitted to our hospital. Patients were divided into 2 groups according to the New York Heart Association (NYHA) functional class. RESULTS: The (123)I-MIBG delayed heart-to-mediastinum (H/M) ratio was significantly lower, and the washout rate (WR) was higher in patients with HFPEF with advanced NYHA functional class (NYHA functional class I and II vs. III: 1.90 ± 0.34 vs. 1.49 ± 0.32, p < 0.0001; 25.9 ± 13.4 vs. 46.9 ± 16.3%, p < 0.0001, respectively). On the other hand, the (123)I-MIBG WR was not correlated with LVEF and had a weak correlation with plasma BNP levels (R = 0.207, p = 0.0346). Moreover, patients with a high (123)I-MIBG WR showed a poor clinical outcome (p = 0.0033). CONCLUSIONS: (123)I-MIBG imaging provides independent prognostic information in patients with HFPEF.