Fumitaka Nakamura

Roles of transient receptor potential canonical (TRPC) channels and reverse-mode Na+/Ca2+ exchanger on cell proliferation in human cardiac fibroblasts: Effects of transforming growth factor β1

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca2+ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca(2+)-mediated [Ca2+]i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca2+ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca2+ entry. KB-R7943, a reverse-mode Na+/Ca2+ exchanger (NCX) inhibitor, and/or replacement of Na+ with NMDG+ inhibited thapsigargin-, OAG- and Ang II-induced Ca2+ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca2+ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca2+ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca2+ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca2+ influx pathways requiring Ca2+ signals for its effect on fibroblast proliferation.

Diverse contribution of bone marrow-derived late-outgrowth endothelial progenitor cells to vascular repair under pulmonary arterial hypertension and arterial neointimal formation

AIMS It is still controversial whether bone marrow (BM)-derived endothelial progenitor cells (EPCs) can contribute to vascular repair and prevent the progression of vascular diseases. We aimed to characterize BM-derived EPC subpopulations and to evaluate their therapeutic efficacies to repair injured vascular endothelium of systemic and pulmonary arteries. METHODS AND RESULTS BM mononuclear cells of Fisher-344 rats were cultured under endothelial cell-conditions. Early EPCs appeared on days 3-6. Late-outgrowth and very late-outgrowth EPCs (LOCs and VLOCs) were defined as cells forming cobblestone colonies on days 9-14 and 17-21, respectively. Among EPC subpopulations, LOCs showed the highest angiogenic capability with enhanced proliferation potential and secretion of proangiogenic proteins. To investigate the therapeutic effects of these EPCs, Fisher-344 rats underwent wire-mediated endovascular injury in femoral artery (FA) and were concurrently injected intraperitoneally with 60mg/kg monocrotaline (MCT). Injured rats were then treated with six injections of one of three EPCs (1×10(6) per time). After 4weeks, transplanted LOCs, but not early EPCs or VLOCs, significantly attenuated neointimal lesion formation in injured FAs. Some of CD31(+) LOCs directly replaced the injured FA endothelium (replacement ratio: 11.7±7.0%). In contrast, any EPC treatment could neither replace MCT-injured endothelium of pulmonary arterioles nor prevent the progression of pulmonary arterial hypertension (PAH). LOCs modified protectively the expression profile of angiogenic and inflammatory genes in injured FAs, but not in MCT-injured lungs. CONCLUSION BM-derived LOCs can contribute to vascular repair of injured systemic artery; however, even they cannot rescue injured pulmonary vasculature under MCT-induced PAH.

High-sensitivity cardiac troponin T for earlier diagnosis of acute myocardial infarction in patients with initially negative troponin T test--comparison between cardiac markers.

BACKGROUND AND PURPOSE An early diagnosis is essential for therapeutic decision and risk stratification in patients with suspected acute myocardial infarction (AMI). We analyzed and compared the diagnostic value of high-sensitivity troponin T (hs-TnT) and other cardiac markers in patients with an initially negative troponin T test at presentation. METHODS AND SUBJECTS The present study was a prospective, multicenter study including five participating emergency cardiovascular tertiary centers in Japan. From November 2009 through January 2011, patients with suspected AMI at the emergency room with an initial troponin T (c-TnT) test <100 ng/L were enrolled. RESULTS A total of 85 patients were prospectively assigned from five participating emergency cardiovascular tertiary centers in Japan. The median time from the onset of chest pain to the T0 sampling was 165 min [IQR 120-180]. The final diagnoses according to the recent universal definition and other standard cut-off values were AMI in 47, unstable angina in 12, and non AMI in 38 patients. The overall ROC-AUC value of hs-TnT, c-TnT, creatine kinase MB, and heart-type fatty acid-binding protein were 0.810, 0.716, 0.782, and 0.880, respectively. The diagnostic sensitivity and negative predictive value of hs-TnT were both 100% for the patients admitted more than 120 min from the onset, however the specificity was limited for the whole time-windows (71%). The absolute value change of the hs-TnT from T0 to T3 sampling 3h later improved the ROC-AUC up to 0.972 and the specificity was 92% at 22 ng/L (ROC-optimized cut-off) and 100% at 105 ng/L (rule-in cut-off). CONCLUSIONS The hs-TnT displayed 100% sensitivity and negative predictive value for the patients admitted more than 120 min from the onset, however the specificity was limited. The absolute change from T0 to T3 (22 ng/L) improved the total diagnostic performance.

Effects of methylglyoxal on human cardiac fibroblast: Roles of transient receptor potential ankyrin 1 (TRPA1)channels

Cardiac fibroblasts contribute to the pathogenesis of cardiac remodeling. Methylglyoxal (MG) is an endogenous carbonyl compound produced under hyperglycemic conditions, which may play a role in the development of pathophysiological conditions including diabetic cardiomyopathy. However, the mechanism by which this occurs and the molecular targets of MG are unclear. We investigated the effects of MG on Ca(2+) signals, its underlying mechanism, and cell cycle progression/cell differentiation in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, Western blot, immunocytochemical analysis, and intracellular Ca(2+) concentration [Ca(2+)]i measurement were applied. Cell cycle progression was assessed using the fluorescence activated cell sorting. MG induced Ca(2+) entry concentration dependently. Ruthenium red (RR), a general cation channel blocker, and HC030031, a selective transient receptor potential ankyrin 1 (TRPA1) antagonist, inhibited MG-induced Ca(2+) entry. Treatment with aminoguanidine, a MG scavenger, also inhibited it. Allyl isothiocyanate, a selective TRPA1 agonist, increased Ca(2+) entry. The use of small interfering RNA to knock down TRPA1 reduced the MG-induced Ca(2+) entry as well as TRPA1 mRNA expression. The quantitative real-time RT-PCR analysis showed the prominent existence of TRPA1 mRNA. Expression of TRPA1 protein was confirmed by Western blotting and immunocytochemical analyses. MG promoted cell cycle progression from G0/G1 to S/G2/M, which was suppressed by HC030031 or RR. MG also enhanced α-smooth muscle actin expression. The present results suggest that methylglyoxal activates TRPA1 and promotes cell cycle progression and differentiation in human cardiac fibroblasts. MG might participate the development of pathophysiological conditions including diabetic cardiomyopathy via activation of TRPA1.

Usefulness of contrast computed tomography to detect left ventricular apical thrombus associated with takotsubo cardiomyopathy

Left ventricular (LV) apical thrombus can rarely occur during the early phase of takotsubo cardiomyopathy. We report such a case that was depicted clearly in contrast computed tomography (CT) but not in initial echocardiography. Because LV thrombus may lead to thromboembolic events, we should evaluate all patients with takotsubo cardiomyopathy for the presence of a LV thrombus. LV thrombus is generally recognized with echocardiography in the course of follow-up, but limited depiction of the LV apex with echocardiography can make evaluation of LV thrombus difficult. Contrast CT is useful to detect LV apical thrombus associated with takotsubo cardiomyopathy.

Noncompaction of the Ventricular Myocardium and Polycystic Kidney Disease: A Case Report.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary disorders, characterized by the formation of multiple cysts in the kidneys and other organs, as well as noncystic manifestations such as cerebral aneurysm. The most common cardiovascular disorders associated with ADPKD include valvular abnormalities and aortic aneurysm. An association between ADPKD and impaired left ventricular function has occasionally been reported. We describe a 74-year-old woman with ADPKD and exertional dyspnea. Impaired left ventricular function resulting from noncompaction of the ventricular myocardium (NVM) and secondary left ventricular aneurysm were diagnosed. Cardiac sarcoidosis and ischemic heart disease were ruled out. Myocardial ischemia resulting from NVM was the presumptive cause of the ventricular aneurysm. To our knowledge, this is the first report of concurrent isolated NVM and left ventricular aneurysm in a patient with ADPKD. ADPKD and various cardiomyopathies, including NVM, are all reported to involve mutations of sarcomere genes, suggesting a possible link between the conditions.

Heat induces interleukin-6 in skeletal muscle cells via TRPV1/PKC/CREB pathways

Interleukin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms whereby skeletal muscle cells sense and respond to heat. When mouse myoblast cells were exposed to 37-42°C for 2 h, mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura 2 fluorescence dual-wavelength excitation method showed that heat increased intracellular calcium flux in a temperature-dependent manner. Intracellular calcium flux and IL-6 mRNA expression were increased by the TRPV1 agonists capsaicin and N-arachidonoyldopamine and decreased by the TRPV1 antagonists AMG9810 and SB366791 and siRNA-mediated knockdown of TRPV1. TRPV2, 3, and 4 agonists did not change intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased phosphorylation levels of TRPV1, followed by PKC and cAMP response element-binding protein (CREB). PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and knockdown of CREB suppressed the heat-induced increases in IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.NEW & NOTEWORTHY Heat increases the release of interleukin-6 (IL-6) from skeletal muscle cells. IL-6 has been shown to serve immune responses and metabolic functions in muscle. It can be anti-inflammatory as well as proinflammatory. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Here we show that heat increases IL-6 in skeletal muscle cells through the transient receptor potential vannilloid 1, PKC, and cAMP response element-binding protein signal transduction pathway.

Acute Myocardial Infarction Caused by Thrombotic Microangiopathy Complicated With Myelodysplastic Syndrome.

Thrombotic microangiopathy (TMA) is a rare but lethal multisystem disease characterized by peripheral thrombocytopenia, microangiopathic hemolytic anemia, fever, and various stages of renal and neurological dysfunctions.(1,2)) The causes of TMA are mainly thrombotic thrombocytopenic purpura (TTP) or hemolytic-uremic syndrome (HUS), and cases of TMA related to myelodysplastic syndrome (MDS) are quite rare. Herein, we report a case of acute myocardial infarction (AMI) caused by TMA which is strongly suspected to have a relationship to MDS, and discuss the treatment of our patient who needed antiplatelet or anticoagulant therapy after AMI, while on the other hand, had pancytopenia and a bleeding event due to MDS.

Muscle hypertrophy following blood flow-restricted, low-force isometric electrical stimulation in rat tibialis anterior: role for muscle hypoxia

Low-force exercise training with blood flow restriction (BFR) elicits muscle hypertrophy as seen typically after higher-force exercise. We investigated the effects of microvascular hypoxia [i.e., low microvascular O2 partial pressures (P mvO2)] during contractions on muscle hypertrophic signaling, growth response, and key muscle adaptations for increasing exercise capacity. Wistar rats were fitted with a cuff placed around the upper thigh and inflated to restrict limb blood flow. Low-force isometric contractions (30 Hz) were evoked via electrical stimulation of the tibialis anterior (TA) muscle. The P mvO2 was determined by phosphorescence quenching. Rats underwent acute and chronic stimulation protocols. Whereas P mvO2 decreased transiently with 30 Hz contractions, simultaneous BFR induced severe hypoxia, reducing P mvO2 lower than present for maximal (100 Hz) contractions. Low-force electrical stimulation (EXER) induced muscle hypertrophy (6.2%, P < 0.01), whereas control group conditions or BFR alone did not. EXER+BFR also induced an increase in muscle mass (11.0%, P < 0.01) and, unique among conditions studied, significantly increased fiber cross-sectional area in the superficial TA ( P < 0.05). Phosphorylation of ribosomal protein S6 was enhanced by EXER+BFR, as were peroxisome proliferator-activated receptor gamma coactivator-1α and glucose transporter 4 protein levels. Fibronectin type III domain-containing protein 5, cytochrome c oxidase subunit 4, monocarboxylate transporter 1 (MCT1), and cluster of differentiation 147 increased with EXER alone. EXER+BFR significantly increased MCT1 expression more than EXER alone. These data demonstrate that microvascular hypoxia during contractions is not essential for hypertrophy. However, hypoxia induced via BFR may potentiate the muscle hypertrophic response (as evidenced by the increased superficial fiber cross-sectional area) with increased glucose transporter and mitochondrial biogenesis, which contributes to the pleiotropic effects of exercise training with BFR that culminate in an improved capacity for sustained exercise. NEW & NOTEWORTHY We investigated the effects of low microvascular O2 partial pressures (P mvO2) during contractions on muscle hypertrophic signaling and key elements in the muscle adaptation for increasing exercise capacity. Although demonstrating that muscle hypoxia is not obligatory for the hypertrophic response to low-force, electrically induced muscle contractions, the reduced P mvO2 enhanced ribosomal protein S6 phosphorylation and potentiated the hypertrophic response. Furthermore, contractions with blood flow restriction increased oxidative capacity, glucose transporter, and mitochondrial biogenesis, which are key determinants of the pleiotropic effects of exercise training.

Clinical Outcomes After Treatment with ELCA for In-Stent Restenosis of DES

R ecently, access to the coronary arteries has become possible with excimer laser coronary angioplasty (ELCA) because of a new technique that utilizes a pulsed laser source and multifiber guided catheters. The potential advantages of excimer laser revascularization include concomitant plaque debulking, thrombus removal, and facilitation of adjunct stenting by softening of the plaque. Improved catheter designs, improved techniques, and firmer indications have led to the expansion of applications for ELCA. These include long lesions, ostial disease, saphenous veins, chronic total occlusions, moderately calcified lesions, thrombus rich lesions, failed balloon angioplasty, and in-stent restenosis (ISR). ELCA is thought to be advantageous for ISR treatment by debulking and softening of the neointima, but the detailed mechanism of its advantages and optimal treatment strategies using it remain unknown. Furthermore, there is little information available concerning the clinical outcomes after treatment for ISR of drug-eluting stents (DES). To approach this question, in the current issue of International Heart Journal, Ichimoto, et al evaluated the long-term clinical outcomes after percutaneous coronary intervention (PCI) with ELCA for ISR of DES.