Soichiro Usui

Inhibition of Endogenous Mst1 Prevents Apoptosis and Cardiac Dysfunction Without Affecting Cardiac Hypertrophy After Myocardial Infarction

Mammalian sterile 20–like kinase-1 (Mst1) plays an important role in mediating cardiac myocyte apoptosis in response to ischemia/reperfusion. Whether or not Mst1 is also involved in the long-term development of heart failure after myocardial infarction (MI) is unknown. We addressed this issue using transgenic mice with cardiac specific overexpression of dominant negative Mst1 (Tg-DN-Mst1). The left coronary artery was permanently ligated, and the size of MI was similar between Tg-DN-Mst1 and nontransgenic controls (NTg). After 4 weeks, Mst1 was significantly activated in the remodeling area in NTg, but not in Tg-DN-Mst1. Although left ventricular (LV) enlargement was significantly attenuated in Tg-DN-Mst1 compared with NTg, neither LV weight/body weight nor myocyte cross sectional area was statistically different between Tg-DN-Mst1 and NTg. LV ejection fraction was significantly greater in Tg-DN-Mst1 than in NTg (53 versus 38%, P<0.01), whereas LV end-diastolic pressure (6 versus 12 mm Hg, P<0.05) and lung weight/body weight (9.8 versus 12.2 P<0.05) were significantly smaller in Tg-DN-Mst1 than in NTg. The number of TUNEL-positive myocytes (0.17 versus 0.28%, P<0.05) and amount of interstitial fibrosis (5.0 versus 7.1%, P<0.05) in the remodeling area were significantly less in Tg-DN-Mst1 than in NTg. Upregulation of matrix metalloproteinase 2 and proinflammatory cytokines was significantly attenuated in Tg-DN-Mst1. These results indicate that endogenous Mst1 plays an important role in mediating cardiac dilation, apoptosis, fibrosis, and cardiac dysfunction, but not cardiac hypertrophy, after MI. Inhibition of Mst1 improves cardiac function without attenuating cardiac hypertrophy. Thus, Mst1 may be an important target of heart failure treatment.

S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species

AIMS Sphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype. METHODS AND RESULTS SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice. CONCLUSION These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects.

Blood Lipid Mediator Sphingosine 1-Phosphate Potently Stimulates Platelet-derived Growth Factor-A and -B Chain Expression through S1P1-Gi-Ras-MAPK-dependent Induction of Krüppel-like Factor 5

Platelet-derived growth factors (PDGFs), potent mitogens and chemoattractants for mesenchymal cell types, play essential roles in development of several organs including blood vessels, kidney, and lung, and are also implicated in the pathogenesis of atherosclerosis and malignancies. Blood lipid mediator sphingosine 1-phosphate (S1P) regulates migration, proliferation, and apoptosis in a variety of cell types through multiple G protein-coupled receptors of the Edg family, and is necessary for vascular formation at the developmental stage. We found in the present study that S1P induced severalfold increases in the mRNA and protein levels of PDGF-A and -B chains in vascular smooth muscle cells and neointimal cells. S1P stimulation of PDGF mRNA and protein expression was abolished by the small interfering RNA duplexes targeting S1P1/Edg1 receptor subtype. S1P stimulated the small GTPase Ras in a Gi-dependent manner, and activated ERK and p38 MAPK in Gi- and Ras-dependent manners. Pertussis toxin pretreatment, adenovirus-mediated Asn17Ras expression, the MEK inhibitor PD98059, or the p38 MAPK inhibitor SB203580 markedly suppressed PDGF mRNA and protein up-regulation, indicating the involvement of Gi-Ras-ERK/p38 MAPK in S1P stimulation of PDGF expression. S1P stimulated expression of the transcription factor KLF5 in manners dependent on Gi, Ras, and ERK/p38 MAPK. Down-regulation of KLF5 by small interfering RNA duplexes abolished S1P-induced PDGF-A and -B chain expression. On the other hand, overexpression of KLF5 stimulated basal and S1P-induced PDGF expression. Either S1P stimulation or KLF5 overexpression increased the PDGF-B promoter activity in a cis-element-dependent manner. These results reveal the S1P1-triggered, Gi-Ras-ERK/p38 MAPK-KLF5-dependent, stimulatory regulation of PDGF gene transcription in vascular smooth muscle cells.

Endogenous Muscle Atrophy F-Box Mediates Pressure Overload–Induced Cardiac Hypertrophy Through Regulation of Nuclear Factor-κB

Rationale: Overexpression of muscle atrophy F-box (MAFbx/atrogin-1), an E3 ubiquitin ligase, induces proteasomal degradation in cardiomyocytes. The role of endogenous MAFbx in regulating cardiac hypertrophy and failure remains unclear. Objective: We investigated the role of MAFbx in regulating cardiac hypertrophy and function in response to pressure overload. Transverse aortic constriction (TAC) was applied to MAFbx knockout (KO) and wild-type (WT) mice. Methods and Results: Expression of MAFbx in WT mice was significantly increased by TAC. TAC-induced increases in cardiac hypertrophy were significantly smaller in MAFbx KO than in WT mice. There was significantly less lung congestion and interstitial fibrosis in MAFbx KO than in WT mice. MAFbx KO also inhibited &bgr;-adrenergic cardiac hypertrophy. DNA microarray analysis revealed that activation of genes associated with the transcription factor binding site for the nuclear factor-&kgr;B family were inhibited in MAFbx KO mice compared with WT mice after TAC. Although the levels of I&kgr;B-&agr; were significantly decreased after TAC in WT mice, they were increased in MAFbx KO mice. MAFbx regulates ubiquitination and proteasomal degradation of I&kgr;B-&agr; in cardiomyocytes. In primary cultured rat cardiomyocytes, phenylephrine-induced activation of nuclear factor-&kgr;B and hypertrophy were significantly suppressed by MAFbx knockdown but were partially rescued by overexpression of nuclear factor-&kgr;B p65. Conclusions: MAFbx plays an essential role in mediating cardiac hypertrophy in response to pressure overload. Downregulation of MAFbx inhibits cardiac hypertrophy in part through stabilization of I&kgr;B-&agr; and inactivation of nuclear factor-&kgr;B. Taken together, inhibition of MAFbx attenuates pathological hypertrophy, thereby protecting the heart from progression into heart failure.

Autophagy plays an essential role in mediating regression of hypertrophy during unloading of the heart.

Autophagy is a bulk degradation mechanism for cytosolic proteins and organelles. The heart undergoes hypertrophy in response to mechanical load but hypertrophy can regress upon unloading. We hypothesize that autophagy plays an important role in mediating regression of cardiac hypertrophy during unloading. Mice were subjected to transverse aortic constriction (TAC) for 1 week, after which the constriction was removed (DeTAC). Regression of cardiac hypertrophy was observed after DeTAC, as indicated by reduction of LVW/BW and cardiomyocyte cross-sectional area. Indicators of autophagy, including LC3-II expression, p62 degradation and GFP-LC3 dots/cell, were significantly increased after DeTAC, suggesting that autophagy is induced. Stimulation of autophagy during DeTAC was accompanied by upregulation of FoxO1. Upregulation of FoxO1 and autophagy was also observed in vitro when cultured cardiomyocytes were subjected to mechanical stretch followed by incubation without stretch (de-stretch). Transgenic mice with cardiac-specific overexpression of FoxO1 exhibited smaller hearts and upregulation of autophagy. Overexpression of FoxO1 in cultured cardiomyocytes significantly reduced cell size, an effect which was attenuated when autophagy was inhibited. To further examine the role of autophagy and FoxO1 in mediating the regression of cardiac hypertrophy, beclin1+/− mice and cultured cardiomyocytes transduced with adenoviruses harboring shRNA-beclin1 or shRNA-FoxO1 were subjected to TAC/stretch followed by DeTAC/de-stretch. Regression of cardiac hypertrophy achieved after DeTAC/de-stretch was significantly attenuated when autophagy was suppressed through downregulation of beclin1 or FoxO1. These results suggest that autophagy and FoxO1 play an essential role in mediating regression of cardiac hypertrophy during mechanical unloading.

Adipose tissue-derived stem cells as a regenerative therapy for a mouse steatohepatitis-induced cirrhosis model.

Cirrhosis is a chronic liver disease that impairs hepatic function and causes advanced fibrosis. Mesenchymal stem cells have gained recent popularity as a regenerative therapy since they possess immunomodulatory functions. We found that injected adipose tissue‐derived stem cells (ADSCs) reside in the liver. Injection of ADSCs also restores albumin expression in hepatic parenchymal cells and ameliorates fibrosis in a nonalcoholic steatohepatitis model of cirrhosis in mice. Gene expression analysis of the liver identifies up‐ and down‐regulation of genes, indicating regeneration/repair and anti‐inflammatory processes following ADSC injection. ADSC treatment also decreases the number of intrahepatic infiltrating CD11b+ and Gr‐1+ cells and reduces the ratio of CD8+/CD4+ cells in hepatic inflammatory cells. This is consistent with down‐regulation of genes in hepatic inflammatory cells related to antigen presentation and helper T‐cell activation. Conclusion: These results suggest that ADSC therapy is beneficial in cirrhosis, as it can repair and restore the function of the impaired liver. (Hepatology 2013;53:1133–1142)

Adipose tissue derived stromal stem cell therapy in murine ConA-derived hepatitis is dependent on myeloid-lineage and CD4+ T-cell suppression.

Mesenchymal stromal stem cells (MSCs) are an attractive therapeutic model for regenerative medicine due to their pluripotency. MSCs are used as a treatment for several inflammatory diseases, including hepatitis. However, the detailed immunopathological impact of MSC treatment on liver disease, particularly for adipose tissue derived stromal stem cells (ADSCs), has not been described. Here, we investigated the immuno‐modulatory effect of ADSCs on hepatitis using an acute ConA C57BL/6 murine hepatitis model. i.v. administration of ADSCs simultaneously or 3 h post injection prevented and treated ConA‐induced hepatitis. Immunohistochemical analysis revealed higher numbers of CD11b+, Gr‐1+, and F4/80+ cells in the liver of ConA‐induced hepatitis mice was ameliorated after the administration of ADSCs. Hepatic expression of genes affected by ADSC administration indicated tissue regeneration‐related biological processes, affecting myeloid‐lineage immune‐mediating Gr‐1+ and CD11b+ cells. Pathway analysis of the genes expressed in ADSC‐treated hepatic inflammatory cells revealed the possible involvement of T cells and macrophages. TNF‐α and IFN‐γ expression was downregulated in hepatic CD4+ T cells isolated from hepatitis livers co‐cultured with ADSCs. Thus, the immunosuppressive effect of ADSCs in a C57BL/6 murine ConA hepatitis model was dependent primarily on the suppression of myeloid‐lineage cells and, in part, of CD4+ T cells.

Rho-Kinase Activation in Leukocytes Plays a Pivotal Role in Myocardial Ischemia/Reperfusion Injury

The Rho/Rho-kinase pathway plays an important role in many cardiovascular diseases such as hypertension, atherosclerosis, heart failure, and myocardial infarction. Although previous studies have shown that Rho-kinase inhibitors reduce ischemia/reperfusion (I/R) injury and cytokine production, the role of Rho-kinase in leukocytes during I/R injury is not well understood. Mice were subjected to 30-min ischemia and reperfusion. Rho-kinase activity was significantly greater in leukocytes subjected to myocardial I/R compared to the sham-operated mice. Administration of fasudil, a Rho-kinase inhibitor, significantly reduced the I/R-induced expression of the proinflammatory cytokines interleukin (IL)-6, C-C motif chemoattractant ligand 2 (CCL2), and tumor necrosis factor (TNF)-α, in leukocytes, compared with saline as the vehicle. Furthermore, fasudil decreased I/R-induced myocardial infarction/area at risk (IA) and I/R-induced leukocyte infiltration in the myocardium. Interestingly, IA in fasudil-administered mice with leukocyte depletion was similar to that in fasudil-administered mice. I/R also resulted in remarkable increases in the mRNA expression levels of the proinflammatory cytokines TNF-α, IL-6, and CCL2 in the heart. Inhibition of Rho-kinase activation in leukocytes has an important role in fasudil-induced cardioprotective effects. Hence, inhibition of Rho-kinase may be an additional therapeutic intervention for the treatment of acute coronary syndrome.

Optimal Force-Time Integral for Pulmonary Vein Isolation According to Anatomical Wall Thickness Under the Ablation Line.

Background Low contact force and force–time integral (FTI) during catheter ablation are associated with ineffective lesion formation, whereas excessively high contact force and FTI may increase the risk of complications. We sought to evaluate the optimal FTI for pulmonary vein (PV) isolation based on atrial wall thickness under the ablation line. Methods and Results Contact force parameters and FTI during anatomical ipsilateral PV isolation for atrial fibrillation and atrial wall thickness were assessed retrospectively in 59 consecutive patients for their first PV isolation procedure. The PV antrum was divided into 8 segments, and the wall thickness of each segment under the ablation line was determined using multidetector computed tomography. The FTI for each ablation point was divided by the wall thickness of the PV antrum segment where each point was located to obtain FTI/wall thickness. In total, 5335 radiofrequency applications were delivered, and 85 gaps in PV isolation ablation lines and 15 dormant conductions induced by adenosine were detected. The gaps or dormant conductions were significantly associated with low contact force, radiofrequency duration, FTI, and FTI/wall thickness. Among them, FTI/wall thickness had the best prediction value for gaps or dormant conductions by receiver operating characteristic curve analysis. FTI/wall thickness of <76.4 gram‐seconds per millimeter (gs/mm) predicted gaps or dormant conductions with sensitivity (88.0%) and specificity (83.6%), and FTI/wall thickness of <101.1 gs/mm was highly predictive (sensitivity 97.0%; specificity 69.6%). Conclusions FTI/wall thickness is a strong predictor of gap and dormant conduction formation in PV isolation. An FTI/wall thickness ≈100 gs/mm could be a suitable target for effective ablation.

Augmented single‐unit muscle sympathetic nerve activity in heart failure with chronic atrial fibrillation

Non‐technical summary  Augmentation of sympathetic nerve activity plays an important role in the deterioration of heart failure (HF). Atrial fibrillation (AF) is a well known and common complication in chronic HF. However, it remains unclear the relationship between sympathetic nerve activity and irregular ventricular rhythm induced by AF in HF. We examined the different sympathetic firing characteristics between HF patients with and without AF using a single‐unit muscle sympathetic nerve activity (MSNA) method, which is a newly developed assessment of direct recording of sympathetic nerve activity. The firing frequency of single‐unit MSNA was increased in HF patients with AF compared with HF patients without AF; particularly, those with a prolonged long RR interval showed multiple firings of single‐unit MSNA. More intense single‐unit MSNA within one cardiac interval occurred in HF patients with AF than patients without AF, even in AF patients receiving heart rate control therapy. Accordingly, our results suggest that AF per se augments central sympathetic activity and the restoration of sinus rhythm may be more effective in the treatment of HF patients with AF.