Cytokine mRNA Degradation in Cardiomyocytes Restrains Sterile Inflammation in Pressure-Overloaded Hearts.

BACKGROUND: Proinflammatory cytokines play an important role in the pathogenesis of heart failure. The mechanisms responsible for maintaining sterile inflammation within failing hearts remain poorly defined. Although transcriptional control is important for proinflammatory cytokine gene expression, the stability of mRNA also contributes to the kinetics of immune responses. Regnase-1 is an RNase involved in the degradation of a set of proinflammatory cytokine mRNAs in immune cells. The role of Regnase-1 in nonimmune cells such as cardiomyocytes remains to be elucidated. METHODS: To examine the role of proinflammatory cytokine degradation by Regnase-1 in cardiomyocytes, cardiomyocyte-specific Regnase-1-deficient mice were generated. The mice were subjected to pressure overload by means of transverse aortic constriction to induce heart failure. Cardiac remodeling was assessed by echocardiography as well as histological and molecular analyses 4 weeks after operation. Inflammatory cell infiltration was examined by immunostaining. Interleukin-6 signaling was inhibited by administration with its receptor antibody. Overexpression of Regnase-1 in the heart was performed by adeno-associated viral vector-mediated gene transfer. RESULTS: Cardiomyocyte-specific Regnase-1-deficient mice showed no cardiac phenotypes under baseline conditions, but exhibited severe inflammation and dilated cardiomyopathy after 4 weeks of pressure overload compared with control littermates. Four weeks after transverse aortic constriction, the Il6 mRNA level was upregulated, but not other cytokine mRNAs, including tumor necrosis factor-α, in Regnase-1-deficient hearts. Although the Il6 mRNA level increased 1 week after operation in both Regnase-1-deficient and control hearts, it showed no increase in control hearts 4 weeks after operation. Administration of anti-interleukin-6 receptor antibody attenuated the development of inflammation and cardiomyopathy in cardiomyocyte-specific Regnase-1-deficient mice. In severe pressure overloaded wild-type mouse hearts, sustained induction of Il6 mRNA was observed, even though the protein level of Regnase-1 increased. Adeno-associated virus 9-mediated cardiomyocyte-targeted gene delivery of Regnase-1 or administration of anti-interleukin-6 receptor antibody attenuated the development of cardiomyopathy induced by severe pressure overload in wild-type mice. CONCLUSIONS: The degradation of cytokine mRNA by Regnase-1 in cardiomyocytes plays an important role in restraining sterile inflammation in failing hearts and the Regnase-1-mediated pathway might be a therapeutic target to treat patients with heart failure.

FKBP8 protects the heart from hemodynamic stress by preventing the accumulation of misfolded proteins and endoplasmic reticulum-associated apoptosis in mice.

Protein quality control in cardiomyocytes is crucial to maintain cellular homeostasis. The accumulation of damaged organelles, such as mitochondria and misfolded proteins in the heart is associated with heart failure. During the process to identify novel mitochondria-specific autophagy (mitophagy) receptors, we found FK506-binding protein 8 (FKBP8), also known as FKBP38, shares similar structural characteristics with a yeast mitophagy receptor, autophagy-related 32 protein. However, knockdown of FKBP8 had no effect on mitophagy in HEK293 cells or H9c2 myocytes. Since the role of FKBP8 in the heart has not been fully elucidated, the aim of this study is to determine the functional role of FKBP8 in the heart. Cardiac-specific FKBP8-deficient (Fkbp8-/-) mice were generated. Fkbp8-/- mice showed no cardiac phenotypes under baseline conditions. The Fkbp8-/- and control wild type littermates (Fkbp8+/+) mice were subjected to pressure overload by means of transverse aortic constriction (TAC). Fkbp8-/- mice showed left ventricular dysfunction and chamber dilatation with lung congestion 1week after TAC. The number of apoptotic cardiomyocytes was dramatically elevated in TAC-operated Fkbp8-/- hearts, accompanied with an increase in protein levels of cleaved caspase-12 and endoplasmic reticulum (ER) stress markers. Caspase-12 inhibition resulted in the attenuation of hydrogen peroxide-induced apoptotic cell death in FKBP8 knockdown H9c2 myocytes. Immunocytological and immunoprecipitation analyses indicate that FKBP8 is localized to the ER and mitochondria in the isolated cardiomyocytes, interacting with heat shock protein 90. Furthermore, there was accumulation of misfolded protein aggregates in FKBP8 knockdown H9c2 myocytes and electron dense deposits in perinuclear region in TAC-operated Fkbp8-/- hearts. The data suggest that FKBP8 plays a protective role against hemodynamic stress in the heart mediated via inhibition of the accumulation of misfolded proteins and ER-associated apoptosis.

Toll-like receptor 9 prevents cardiac rupture after myocardial infarction in mice independently of inflammation.

We have reported that the Toll-like receptor 9 (TLR9) signaling pathway plays an important role in the development of pressure overload-induced inflammatory responses and heart failure. However, its role in cardiac remodeling after myocardial infarction has not been elucidated. TLR9-deficient and control C57Bl/6 wild-type mice were subjected to left coronary artery ligation. The survival rate 14 days postoperation was significantly lower in TLR9-deficient mice than that in wild-type mice with evidence of cardiac rupture in all dead mice. Cardiac magnetic resonance imaging showed no difference in infarct size and left ventricular wall thickness and function between TLR9-deficient and wild-type mice. There were no differences in the number of infiltrating inflammatory cells and the levels of inflammatory cytokine mRNA in infarct hearts between TLR9-deficient and wild-type mice. The number of α-smooth muscle actin (αSMA)-positive myofibroblasts and αSMA/Ki67-double-positive proliferative myofibroblasts was increased in the infarct and border areas in infarct hearts compared with those in sham-operated hearts in wild-type mice, but not in TLR9-deficient mice. The class B CpG oligonucleotide increased the phosphorylation level of NF-κB and the number of αSMA-positive and αSMA/Ki67-double-positive cells and these increases were attenuated by BAY1-7082, an NF-κB inhibitor, in cardiac fibroblasts isolated from wild-type hearts. The CpG oligonucleotide showed no effect on NF-κB activation or the number of αSMA-positive and αSMA/Ki67-double-positive cells in cardiac fibroblasts from TLR9-deficient hearts. Although the TLR9 signaling pathway is not involved in the acute inflammatory response in infarct hearts, it ameliorates cardiac rupture possibly by promoting proliferation and differentiation of cardiac fibroblasts.

Glucocorticoids Induce Cardiac Fibrosis via Mineralocorticoid Receptor in Oxidative Stress: Contribution of Elongation Factor Eleven-Nineteen Lysine-Rich Leukemia (ELL).

BACKGROUND: Cardiac fibrosis is considered to be a crucial factor in the development of heart failure. Blockade of the mineralocorticoid receptor (MR) attenuated cardiac fibrosis and improved the prognosis of patients with chronic heart failure but the ligand for MR and the regulatory mechanism of MR pathway in the diseased heart are unclear. Here, we investigated whether glucocorticoids can promote cardiac fibrosis through MR in oxidative stress and the involvement of elongation factor eleven-nineteen lysine-rich leukemia (ELL), a co-activator of MR, in this pathway. METHODS AND RESULTS: The MR antagonist eplerenone attenuated corticosterone-induced collagen synthesis assessed by [(3)H]proline incorporation in rat neonatal cultured cardiac fibroblasts in the presence of H2O2, as an oxidative stress but not in the absence of H2O2. H2O2 increased the ELL expression levels and MR-bound ELL. ELL expression levels and MR-bound ELL were also increased in the left ventricle of heart failure model rats with significant fibrosis and enhanced oxidative stress. Eplerenone did not attenuate corticosterone-induced increase of [(3)H]proline incorporation in the presence of H2O2 after knockdown of ELL expression using small interfering RNA in cardiac fibroblasts. CONCLUSION: Glucocorticoids can promote cardiac fibrosis via MR in oxidative stress, and oxidative stress modulates MR response to glucocorticoids through the interaction with ELL. Preventing cardiac fibrosis by modulating glucocorticoid-MR-ELL pathway may become a new therapeutic strategy for heart failure.

Endovascular aortic repair increases vascular stiffness and alters cardiac structure and function.

BACKGROUND: Endovascular aortic repair (EVAR) is performed in patients with thoracic or abdominal aortic aneurysm because it is less invasive than conventional open repair. However, the effects of EVAR on vascular and cardiac function remain to be clarified. METHODS AND RESULTS: We studied the effects of EVAR on several outcome variables in 40 consecutive patients undergoing EVAR for abdominal and/or thoracic aneurysm with preserved ejection fraction. Echocardiography and brachial-ankle pulse wave velocity (baPWV) data were collected before, 1 week, and 1 year after EVAR. Although no changes in blood pressure were found, baPWV, left ventricular mass index (LVMI), and left atrial volume index were significantly elevated at both post-op time periods after EVAR compared with baseline data. The changes in LVMI correlated with those in baPWV (R=0.32, P<0.05). Among the 22 patients who were successfully followed up, 13 showed deterioration in exercise tolerance 1 year after EVAR. Diastolic wall strain, an index for LV distensibility, was lower at baseline in patients with worsening exercise tolerance than in those with unchanged tolerance. CONCLUSIONS: EVAR increased vascular stiffness and induced LV hypertrophy and diastolic dysfunction without a corresponding elevation of blood pressure in the acute and chronic phases. In addition, low LV distensibility at baseline was associated with the impairment of exercise tolerance. EVAR-induced stiffness of arteries leads to limited clinical symptoms.

A novel heart failure mice model of hypertensive heart disease by angiotensin II infusion, nephrectomy, and salt loading.

Although the mouse heart failure (HF) model of hypertensive heart disease (HHD) is useful to investigate the pathophysiology and new therapeutic targets for HHD, the model using simple experimental procedures and stable phenotypes has not been established. This study aimed to develop a novel mouse HF model of HHD by combining salt loading and uninephrectomy with ANG II infusion. Eight-week-old C57BL/6 male mice were treated with ANG II infusion (AT), ANG II infusion and uninephrectomy (AN), ANG II infusion and salt loading (AS), or ANG II infusion, uninephrectomy, and salt loading (ANS). Systolic blood pressure was significantly elevated and left ventricular (LV) hypertrophy was found in AT, AN, AS, and ANS mice, and there were no significant differences in those parameters between the four groups. At 6 wk after the procedures, only ANS mice showed significant decreases in LV fractional shortening and increases in lung weight with a high incidence. This phenotype was reproducible, and there were few perioperative or early deaths in the experimental procedures. Severe LV fibrosis was found in ANS mice. Oxidative stress was enhanced and small GTPase Rac1 activity was upregulated in the hearts of ANS mice. After the addition of salt loading and uninephrectomy to the ANG II infusion mouse model, cardiac function was significantly impaired, and mice developed HF. This might be a novel and useful mouse HF model to study the transition from compensated LV hypertrophy to HF in HHD.

Interleukin-16 promotes cardiac fibrosis and myocardial stiffening in heart failure with preserved ejection fraction.

BACKGROUND: Chronic heart failure (CHF) with preserved left ventricular (LV) ejection fraction (HFpEF) is observed in half of all patients with CHF and carries the same poor prognosis as CHF with reduced LV ejection fraction (HFrEF). In contrast to HFrEF, there is no established therapy for HFpEF. Chronic inflammation contributes to cardiac fibrosis, a crucial factor in HFpEF; however, inflammatory mechanisms and mediators involved in the development of HFpEF remain unclear. Therefore, we sought to identify novel inflammatory mediators involved in this process. METHODS AND RESULTS: An analysis by multiplex-bead array assay revealed that serum interleukin-16 (IL-16) levels were specifically elevated in patients with HFpEF compared with HFrEF and controls. This was confirmed by enzyme-linked immunosorbent assay in HFpEF patients and controls, and serum IL-16 levels showed a significant association with indices of LV diastolic dysfunction. Serum IL-16 levels were also elevated in a rat model of HFpEF and positively correlated with LV end-diastolic pressure, lung weight and LV myocardial stiffness constant. The cardiac expression of IL-16 was upregulated in the HFpEF rat model. Enhanced cardiac expression of IL-16 in transgenic mice induced cardiac fibrosis and LV myocardial stiffening accompanied by increased macrophage infiltration. Treatment with anti-IL-16 neutralizing antibody ameliorated cardiac fibrosis in the mouse model of angiotensin II-induced hypertension. CONCLUSION: Our data indicate that IL-16 is a mediator of LV myocardial fibrosis and stiffening in HFpEF, and that the blockade of IL-16 could be a possible therapeutic option for HFpEF.

L-Carnitine prevents the development of ventricular fibrosis and heart failure with preserved ejection fraction in hypertensive heart disease.

OBJECTIVES: Prognosis of heart failure with preserved ejection fraction (HFpEF) remains poor because of unknown pathophysiology and unestablished therapeutic strategy. This study aimed to identify a potential therapeutic intervention for HFpEF through metabolomics-based analysis. METHODS AND RESULTS: Metabolomics with capillary electrophoresis time-of-flight mass spectrometry was performed using plasma of Dahl salt-sensitive rats fed high-salt diet, a model of hypertensive HFpEF, and showed decreased free-carnitine levels. Reassessment with enzymatic cycling method revealed the decreased plasma and left-ventricular free-carnitine levels in the HFpEF model. Urinary free-carnitine excretion was increased, and the expression of organic cation/carnitine transporter 2, which transports free-carnitine into cells, was down-regulated in the left ventricle (LV) and kidney in the HFpEF model. L-Carnitine was administered to the hypertensive HFpEF model. L-Carnitine treatment restored left-ventricular free-carnitine levels, attenuated left-ventricular fibrosis and stiffening, prevented pulmonary congestion, and improved survival in the HFpEF model independent of the antihypertensive effects, accompanied with increased expression of fatty acid desaturase (FADS) 1/2, rate-limiting enzymes in forming arachidonic acid, and enhanced production of arachidonic acid, a precursor of prostacyclin, and prostacyclin in the LV. In cultured cardiac fibroblasts, L-carnitine attenuated the angiotensin II-induced collagen production with increased FADS1/2 expression and enhanced production of arachidonic acid and prostacyclin. L-Carnitine-induced increase of arachidonic acid was canceled by knock-down of FADS1 or FADS2 in cultured cardiac fibroblasts. Serum free-carnitine levels were decreased in HFpEF patients. CONCLUSIONS: L-carnitine supplementation attenuates cardiac fibrosis by increasing prostacyclin production through arachidonic acid pathway, and may be a promising therapeutic option for HFpEF.

Ca2+ entry mode of Na+/Ca2+ exchanger as a new therapeutic target for heart failure with preserved ejection fraction.

AIMS: Left ventricular (LV) fibrosis and stiffening play crucial roles in the development of heart failure with preserved ejection fraction (HFPEF). Plasma level of digitalis-like factors (DLFs) is increased in patients with hypertension, a principal underlying cardiovascular disease of HFPEF. Digitalis-like factors inhibit ion-pumping function of Na(+)/K(+)-ATPase and activate the Ca(2+) entry mode of Na(+)/Ca(2+) exchanger (NCX). Digitalis-like factors are known to promote collagen production in fibroblasts. The aim of this study was to explore whether the pharmacological inhibition of the NCX entry mode is effective in the prevention of LV fibrosis and in the development of HFPEF. METHODS AND RESULTS: (i) Dahl salt-sensitive rats fed 8% NaCl diet from age 6 weeks served as hypertensive HFPEF model. In this model, 24 h urine excretion of DLFs was greater than that in the age-matched control at compensatory hypertrophic and heart failure stages. (ii) Continuous administration of ouabain for 14 weeks developed LV fibrosis without affecting blood pressure in Sprague-Dawley rats. (iii) Ouabain elevated intracellular Ca(2+) concentration through the entry of extracellular Ca(2+), increased the phosphorylation level of p42/44 mitogen-activated protein kinases, and enhanced (3)H-proline incorporation in cardiac fibroblast; and SEA0400, the inhibitor of the NCX entry mode, suppressed these effects. (iv) In the HFPEF model, administration of SEA0400 at subdepressor dose improved the survival rate in association with the attenuation of LV fibrosis and stiffening. CONCLUSION: Digitalis-like factors and the subsequently activated NCX entry mode may play an important role in the development of hypertensive HFPEF, and the blockade of the NCX entry mode may be a new therapeutic strategy for this phenotype of heart failure.

Diabetic retinopathy is associated with impaired left ventricular relaxation.

BACKGROUND: Diabetic retinopathy (DR) is an independent predictor of heart failure (HF) in patients with diabetes mellitus (DM). However, it is unclear how DR is related to the development of HF. We hypothesized that DR is associated with left ventricular (LV) diastolic dysfunction, which is well recognized to subsequently result in HF. METHODS AND RESULTS: Data were collected in 63 consecutive patients with DM and LV ejection fraction (EF) ≥50%. Patients were excluded if they had HF diagnosed according to the modified Framingham criteria. Doppler echocardiographic indices including peak early-diastolic mitral annular movement velocity (E') were obtained in each patient.We also assessed the diastolic index of echocardiographic color kinesis (CK-DI), which proportionally decreases with LV relaxation abnormality independently of LV filling pressure, as recently published. The DM patients were divided into groups without (DM-N; n = 30) and with (DM-DR; n = 33) DR. Age, gender, LV end-diastolic dimension, EF, E/A ratio of the transmitral flow velocity curves, E', and E/E' were not different between DM-N and DM-DR. However, CK-DI was significantly lower in DM-DR than DM-N. CONCLUSIONS: DR is associated with LV diastolic dysfunction, and this may at least in part explain the increased incidence of HF in DM patients with DR.

Patients with more coronary yellow plaques have higher risk of stenosis progression within 7 months.

BACKGROUND: Disruption of vulnerable plaques causes acute coronary syndrome and stenosis progression. Yellow plaques are regarded as vulnerable and the number of yellow plaques per vessel (NYP) has been reported as a marker of vulnerable patients. Therefore, we examined if patients with more yellow plaques would have higher risk of stenosis progression. METHODS AND RESULTS: A series of patients (n = 70) who received percutaneous coronary intervention (PCI) and angioscopy was included. Patients were divided into 2 groups according to NYP: group 1 (NYP <4, n = 32) and group 2 (NYP ≥ 4, n = 38). Coronary artery stenosis progression in any segment excluding target lesion of PCI was examined by angiography at 7 months. Maximum yellow color grade of yellow plaques (2.7 ± 0.7 vs. 1.7 ± 1.2, p < 0.0001) and the number of non-target disrupted yellow plaques was larger in group 2 than in group 1 (1.1 ± 1.5 vs. 0.2 ± 0.6, p=0.0017). Progression of coronary stenosis was detected more frequently in group 2 than in group 1 (29% vs. 9%, p = 0.041). The number of sites with stenosis progression was larger in group 2 than in group 1 (0.47 ± 0.98 vs. 0.09 ± 0.30 sites/patient, p = 0.036). CONCLUSIONS: Vulnerable patients with more yellow plaques had higher incidence of stenosis progression. Approximately 30% of vulnerable patients with NYP ≥ 4 had stenosis progression within 7 months.

Competing risks of heart failure with preserved ejection fraction in diabetic patients.

AIMS: The prevalence of heart failure with preserved ejection fraction (HFpEF) has increased in the past two decades, and diabetes mellitus (DM) is frequently associated with HFpEF. Although it has been demonstrated that left ventricular (LV) diastolic and vascular functional abnormalities are generally observed in HFpEF, it remains to be clinically elucidated how an asymptomatic stage progresses to symptomatic HFpEF in DM patients. We aimed to identify risk factors associated with incident HFpEF in DM patients and to evaluate the contribution of LV relaxation and compliance to the development of HFpEF. METHODS AND RESULTS: The study included 544 consecutive Japanese DM patients with ejection fraction ≥50%. Patients with coronary artery disease or serum creatinine ≥2.0 mg/dL were excluded. Multiple logistic regression analysis revealed that obesity, female gender, anaemia, and impaired LV compliance were independently associated with the prevalence of HFpEF, and that age, LV mass index, an index of LV relaxation, estimated glomerular filtration rate, and history of hypertension were not. There was no difference in haemoglobin A1c or brachial-ankle pulse wave velocity between the DM patients with and without HFpEF. CONCLUSIONS: This study suggests that exacerbation of LV compliance impairment, rather than of relaxation abnormality or vascular stiffening, plays a crucial role in the induction of HFpEF in DM patients regardless of the severity of DM and renal dysfunction. Anaemia and obesity may also contribute to the transition from asymptomatic stage to symptomatic HFpEF even without further progression of LV diastolic dysfunction.

Transition from asymptomatic diastolic dysfunction to heart failure with preserved ejection fraction: roles of systolic function and ventricular distensibility.

BACKGROUND: Systolic abnormality, as well as diastolic dysfunction, is observed in patients with heart failure with preserved ejection fraction (HFPEF). However, the role of these 2 conditions in the transition from asymptomatic diastolic dysfunction to symptomatic heart failure remains unclear. We recently demonstrated that diastolic wall strain (DWS) inversely correlates to the myocardial stiffness constant. METHODS AND RESULTS: This study consisted of 127 subjects: 52 consecutive HFPEF patients (HFPEF group), 50 asymptomatic hypertensive patients with ejection fraction ≥50% whose age, gender and left ventricular (LV) mass index matched those of the HFPEF group (HT group) and 25 normal volunteers (Normal group). The tissue Doppler-derived peak systolic and early diastolic velocities of the mitral annulus were significantly decreased in groups HFPEF and HT than in group Normal, but were not significantly different between groups HFPEF and HT. DWS was significantly lower in group HFPEF than in group HT. CONCLUSIONS: The transition from asymptomatic diastolic dysfunction stage to HFPEF stage is not attributed to progression of systolic abnormality, and exacerbation of LV distensibility rather than relaxation plays a crucial role in the development of HFPEF.

Detecting of coronary dissection by coronary magnetic resonance.

A 75-year-old man, who had a history of coronary dissection after percutaneous coronary intervention in left anterior descending coronary artery, underwent coronary magnetic resonance. Magnetic resonance demonstrated coronary dissection in the distal portion of the left anterior descending artery. Both the true lumen with thick vessel wall and the false lumen with thin vessel wall were demonstrated in the cross-sectional images using T1-weighed black blood technique and T2-weighed black blood technique. Soft plaque was located at the twelve o'clock in the true lumen. Invasive coronary angiogram showed long coronary dissection from middle to distal portion of left anterior descending coronary artery. Magnetic resonance was thought to be useful to detect and follow up the coronary dissection noninvasively.