Shinichiro Takashima

G12/13 and Gq mediate S1P2-induced inhibition of Rac and migration in vascular smooth muscle in a manner dependent on Rho but not Rho kinase

AIMS The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs. METHODS AND RESULTS S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of Galpha subunits (Galpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Galpha(12)-CT, Galpha(13)-CT, and Galpha(q)-CT, but not that of Galpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the S1P inhibition. The expression of Galpha(q)-CT as well as Galpha(12)-CT and Galpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT(1), which robustly couples to G(q), did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G(q) alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to Galpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway. CONCLUSION These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.

Clinical Study Using Adipose-Derived Mesenchymal-Like Stem Cells in Acute Myocardial Infarction and Heart Failure

Adipose tissue represents an abundant, accessible source of regenerative cells that can be easily obtained in sufficient amount for therapy. Adipose-derived regenerative cells (ADRC) are comprised of leukocytes, smooth muscles, endothelial cells, and mesenchymal stem cells. In contrast to bone-marrow-derived MSC, the abundance of adipose tissue in patients and the higher frequency per unit mass of regenerative cells allow for the isolation of cells in therapeutic meaningful amounts in less than 2h after donor tissue acquisition.Harvest of adipose tissue can thus follow primary PCI, allowing efficient treatment within 24h. This obviates the need for extensive cell culturing in GMP clean room facilities and makes ADSCs a promising and practical autologous cell source. In the following chapter, we will describe the liposuction procedure for stem cell harvest, two cell delivery techniques, and pressure/volume loop analysis for the follow-up of our patients enrolled in the clinical studies.

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.

Effect of pioglitazone on muscle sympathetic nerve activity in type 2 diabetes mellitus with alpha-glucosidase inhibitor

Activation of the sympathetic nervous system is augmented in patients with type 2 diabetes mellitus (DM). Pioglitazone, an anti-diabetic drug, improves insulin resistance, but its influence on sympathetic nerve activity is not clear. To identify the relationship between insulin resistance and sympathetic activity, we examined muscle sympathetic nerve activity (MSNA) in controlled type 2 DM patients with alpha-glucosidase inhibitor (GI). We measured MSNA and calculated homeostasis model assessment of insulin resistance index (HOMA-IR) in twelve DM patients treated with alpha-GI and thirteen age-matched healthy subjects. In DM patients with alpha-GI, all parameters were reexamined after three months of treatment with pioglitazone. MSNA and HOMA-IR were significantly greater in DM patients with alpha-GI compared to healthy subjects. Hemoglobin A1c did not differ in DM patients before and after pioglitazone. However, pioglitazone significantly decreased MSNA in DM patients compared with alpha-GI (21.7±5.2 vs. 32.0±6.8 burst/min, p<0.01). Furthermore, MSNA level in pioglitazone was similar to that in healthy subjects. HOMA-IR significantly decreased after pioglitazone, and a significant relationship was found between the absolute change in MSNA and HOMA-IR (r=0.65, p<0.05). These results suggest that improved insulin resistance with pioglitazone provides an additional effect on inhibition of sympathetic nerve activity.

Benidipine reduces ischemia reperfusion-induced systemic oxidative stress through suppression of aldosterone production in mice

Aldosterone is implicated in the pathogenesis of several cardiovascular diseases, including ischemia reperfusion (I/R) and myocardial infarction, and also causes oxidative stress and inflammation in cardiovascular systems. Benidipine, a long-acting T- and L-type calcium channel blocker, reduces infarct size following myocardial I/R in rabbits. Benidipine also inhibits the production of aldosterone in vitro. However, the precise mechanism of this phenomenon in vivo remains unknown. We therefore evaluated whether benedipine has a beneficial role through the regulation of oxidative stress in myocardial I/R. C57BL/6J mice were subjected to 30 min of left ascending coronary I/R. Benidipine was administered orally at 3 mg kg−1 daily for 3 weeks without any changes in hemodynamic variables. Benidipine significantly reduced infarction size (13.4±2.5%) compared with controls (25.5±3.6%). Urinary 8-hydroxy-2′ deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, increased significantly after I/R. I/R induced increases in 8-OHdG were significantly lower with benidipine. Local myocardial 8-OHdG was also elevated in I/R, but this augmentation was significantly suppressed with benidipine. The plasma aldosterone concentration (PAC) significantly increased 2 days after I/R and remained elevated at least 7 days after I/R. Treatment with benidipine significantly decreased I/R-induced elevation of the PAC. I/R-induced markers of fibrosis in hearts also reduced in benidipine. These results suggest that the administration of benidipine reduces myocardial infarct size as well as systemic oxidative stress after I/R. These phenomena are partially linked to reduced plasma aldosterone levels.

Augmented sphingosine 1 phosphate receptor-1 signaling in cardiac fibroblasts induces cardiac hypertrophy and fibrosis through angiotensin II and interleukin-6

Background: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1–S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II—AT1 and IL-6 are involved.

Sodium 4-Phenylbutyrate Attenuates Myocardial Reperfusion Injury by Reducing the Unfolded Protein Response.

Background: The unfolded protein response (UPR) plays a pivotal role in ischemia–reperfusion (I/R) injury in various organs such as heart, brain, and liver. Sodium 4-phenylbutyrate (PBA) reportedly acts as a chemical chaperone that reduces UPR. In the present study, we evaluated the effect of PBA on reducing the UPR and protecting against myocardial I/R injury in mice. Methods: Male C57BL/6 mice were subjected to 30-minute myocardial I/R, and were treated with phosphate-buffered saline (as a vehicle) or PBA. Results: At 4 hours after reperfusion, mice treated with PBA had reduced serum cardiac troponin I levels and numbers of apoptotic cells in left ventricles (LVs) in myocardial I/R. Infarct size had also reduced in mice treated with PBA at 48 hours after reperfusion. At 2 hours after reperfusion, UPR markers, including eukaryotic initiation of the factor 2α-subunit, activating transcription factor-6, inositol-requiring enzyme-1, glucose-regulated protein 78, CCAAT/enhancer-binding protein (C/EBP) homologous protein, and caspase-12, were significantly increased in mice treated with vehicle compared to sham-operated mice. Administration of PBA significantly reduced the I/R-induced increases of these markers. Cardiac function and dimensions were assessed at 21 days after I/R. Sodium 4-phenylbutyrate dedicated to the improvement of cardiac parameters deterioration including LV end-diastolic diameter and LV fractional shortening. Consistently, PBA reduced messenger RNA expression levels of cardiac remodeling markers such as collagen type 1α1, brain natriuretic peptide, and α skeletal muscle actin in LV at 21 days after I/R. Conclusion: Unfolded protein response mediates myocardial I/R injury. Administration of PBA reduces the UPR, apoptosis, infarct size, and preserved cardiac function. Hence, PBA may be a therapeutic option to attenuate myocardial I/R injury in clinical practice.

Cable externalization at the proximal portion of the superior vena cava coil in Riata implantable cardioverter defibrillator leads

Many Riata (St. Jude Medical, St. Paul, MN, USA) implantable cardioverter defibrillator (ICD) leads have reportedly developed cable externalization. The most likely cause of cable externalization is insulation abrasion, which often occurs at the can or between the right ventricular coil and superior vena cava (SVC) coil. We report a rare case of an adult male whose ICD lead cable was externalized at the proximal portion of the SVC coil. This lead became fixed to the wall at the subclavian vein and SVC and became bent between these adhesions. Furthermore, the motion of this lead was affected by pulsation of the aortic arch. The ICD lead might develop inside-out abrasion due to mechanical stress evoked by pulsation of the aortic arch at this site. <Learning objective: Cable externalization of the implantable cardioverter defibrillator lead at the proximal portion of the superior vena cava (SVC) coil has rarely been reported. Externalization might be the result of deformation of the left brachiocephalic vein and the anatomical relationship with the aortic arch. The anatomical pathway of the lead should be carefully considered during the procedure, especially when a dual-coil lead is selected. Moreover, possible cable externalization at both the proximal and distal portions of the SVC coil should be kept in mind during follow-up.>.