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1.  A technique for quantifying intracellular free sodium ion using a microplate reader in combination with sodium-binding benzofuran isophthalate and probenecid in cultured neonatal rat cardiomyocytes 
BMC Research Notes  2013;6:556.
Background
Intracellular sodium ([Na+]i) kinetics are involved in cardiac diseases including ischemia, heart failure, and hypertrophy. Because [Na+]i plays a crucial role in modulating the electrical and contractile activity in the heart, quantifying [Na+]i is of great interest. Using fluorescent microscopy with sodium-binding benzofuran isophthalate (SBFI) is the most commonly used method for measuring [Na+]i. However, one limitation associated with this technique is that the test cannot simultaneously evaluate the effects of several types or various concentrations of compounds on [Na+]i. Moreover, there are few reports on the long-term effects of compounds on [Na+]i in cultured cells, although rapid changes in [Na+]i during a period of seconds or several minutes have been widely discussed.
Findings
We established a novel technique for quantifying [Na+]i in cultured neonatal rat cardiomyocytes attached to a 96-well plate using a microplate reader in combination with SBFI and probenecid. We showed that probenecid is indispensable for the accurate measurement because it prevents dye leakage from the cells. We further confirmed the reliability of this system by quantifying the effects of ouabain, which is known to transiently alter [Na+]i. To illustrate the utility of the new method, we also examined the chronic effects of aldosterone on [Na+]i in cultured cardiomyocytes.
Conclusions
Our technique can rapidly measure [Na+]i with accuracy and sensitivity comparable to the traditional microscopy based method. The results demonstrated that this 96-well plate based measurement has merits, especially for screening test of compounds regulating [Na+]i, and is useful to elucidate the mechanisms and consequences of altered [Na+]i handling in cardiomyocytes.
doi:10.1186/1756-0500-6-556
PMCID: PMC3879185  PMID: 24369990
Intracellular sodium; Cardiomyocyte; SBFI; Probenecid; Microplate reader
2.  Rapid Induction of Aldosterone Synthesis in Cultured Neonatal Rat Cardiomyocytes under High Glucose Conditions 
BioMed Research International  2013;2013:161396.
In addition to classical adrenal cortical biosynthetic pathway, there is increasing evidence that aldosterone is produced in extra-adrenal tissues. Although we previously reported aldosterone production in the heart, the concept of cardiac aldosterone synthesis remains controversial. This is partly due to lack of established experimental models representing aldosterone synthase (CYP11B2) expression in robustly reproducible fashion. We herein investigated suitable conditions in neonatal rat cardiomyocytes (NRCMs) culture system producing CYP11B2 with considerable efficacy. NRCMs were cultured with various glucose doses for 2–24 hours. CYP11B2 mRNA expression and aldosterone concentrations secreted from NRCMs were determined using real-time PCR and enzyme immunoassay, respectively. We found that suitable conditions for CYP11B2 induction included four-hour incubation with high glucose conditions. Under these particular conditions, CYP11B2 expression, in accordance with aldosterone secretion, was significantly increased compared to those observed in the cells cultured under standard-glucose condition. Angiotensin II receptor blocker partially inhibited this CYP11B2 induction, suggesting that there is local renin-angiotensin-aldosterone system activation under high glucose conditions. The suitable conditions for CYP11B2 induction in NRCMs culture system are now clarified: high-glucose conditions with relatively brief period of culture promote CYP11B2 expression in cardiomyocytes. The current system will help to accelerate further progress in research on cardiac tissue aldosterone synthesis.
doi:10.1155/2013/161396
PMCID: PMC3830858  PMID: 24288663
3.  An Immunohistochemical Analysis of Tissue Thrombin Expression in the Human Atria 
PLoS ONE  2013;8(6):e65817.
Objective
Thrombin, the final coagulation product of the coagulation cascade, has been demonstrated to have many physiological effects, including pro-fibrotic actions via protease-activated receptor (PAR)-1. Recent investigations have demonstrated that activation of the cardiac local coagulation system was associated with atrial fibrillation. However, the distribution of thrombin in the heart, especially difference between the atria and the ventricle, remains to be clarified. We herein investigated the expression of thrombin and other related proteins, as well as tissue fibrosis, in the human left atria and left ventricle.
Methods
We examined the expression of thrombin and other related molecules in the autopsied hearts of patients with and without atrial fibrillation. An immunohistochemical analysis was performed in the left atria and the left ventricle.
Results
The thrombin was immunohistologically detected in both the left atria and the left ventricles. Other than in the myocardium, the expression of thrombin was observed in the endocardium and the subendocardium of the left atrium. Thrombin was more highly expressed in the left atrium compared to the left ventricle, which was concomitant with more tissue fibrosis and inflammation, as detected by CD68 expression, in the left atrium. We also confirmed the expression of prothrombin in the left atrium. The expression of PAR-1 was observed in the endocardium, subendocardium and myocardium in the left atrium. In patients with atrial fibrillation, strong thrombin expression was observed in the left atrium.
Conclusions
The strong expression levels of thrombin, prothrombin and PAR-1 were demonstrated in the atrial tissues of human autopsied hearts.
doi:10.1371/journal.pone.0065817
PMCID: PMC3681873  PMID: 23785453
4.  Effects of co-administration of candesartan with pioglitazone on inflammatory parameters in hypertensive patients with type 2 diabetes mellitus: a preliminary report 
Background
Angiotensin receptor blockers (ARBs) are reported to provide direct protection to many organs by controlling inflammation and decreasing oxidant stress. Pioglitazone, an anti-diabetic agent that improves insulin resistance, was also reported to decrease inflammation and protect against atherosclerosis. This study aimed to evaluate the utility of combination therapy with both medicines from the viewpoint of anti-inflammatory effects.
Methods
We administered candesartan (12 mg daily) and pioglitazone (15 mg daily) simultaneously for 6 months to hypertensive patients with type 2 diabetes mellitus (T2DM) and evaluated whether there were improvements in the serum inflammatory parameters of high-molecular-weight adiponectin (HMW-ADN), plasminogen activator inhibitor-1 (PAI-1), highly sensitive C-reactive protein (Hs-CRP), vascular cell adhesion molecule-1 (VCAM-1), and urinary-8-hydroxydeoxyguanosine (U-8-OHdG). We then analyzed the relationship between the degree of reductions in blood pressure and HbA1c values and improvements in inflammatory factors. Furthermore, we analyzed the relationship between pulse pressure and the degree of lowering of HbA1c and improvements in inflammatory factors. Finally, we examined predictive factors in patients who received benefits from the co-administration of candesartan with pioglitazone from the viewpoint of inflammatory factors.
Results
After 6 months of treatment, in all patients significant improvements from baseline values were observed in HMW-ADN and PAI-1 but not in VCAM-1, Hs-CRP, and U-8-OHdG. Changes in HbA1c were significantly correlated with changes in HMW-ADN and PAI-1 in all patients, but changes in blood pressure were not correlated with any of the parameters examined. Correlation and multilinear regression analyses were performed to determine which factors could best predict changes in HbA1c. Interestingly, we found a significant positive correlation of pulse pressure values at baseline with changes in HbA1c.
Conclusions
Our data suggest that the pulse pressure value at baseline is a key predictive factor of changes in HbA1c. Co-administration of candesartan with pioglitazone, which have anti-inflammatory (changes in HMW-ADN and PAI-1) effects and protective effects on organs, could be an effective therapeutic strategy for treating hypertensive patients with type 2 diabetes mellitus.
Trial registration
UMIN-CTR: UMIN000010142
doi:10.1186/1475-2840-12-71
PMCID: PMC3663745  PMID: 23635096
Candesartan; Angiotensin receptor blockers; Type 2 diabetes mellitus; Inflammatory parameters; Pulse pressure
5.  Transient decrease in serum potassium level during ischemic attack of acute coronary syndrome: Paradoxical contribution of plasma glucose level and glycohemoglobin 
Background
Although a decrease in serum potassium level has been suggested to be a fairly common observation in acute coronary syndrome (ACS), there have so far been no definitive reports directly demonstrating the transient potassium decrease (the potassium dip) during ischemic attack of ACS compared to stable phase in individual patients. To understand the pathophysiological significance of the potassium dip, we examined the changes in serum potassium level throughout ischemic attack and evaluated the clinical factors affecting it.
Methods
The degree of the potassium dip during ischemic attack (as indicated by ΔK, ΔK = K at discharge − K on admission) was examined in 311 consecutive patients with ACS who required urgent hospitalization in our institution.
Results
Serum potassium level during ischemic attack was significantly decreased compared to that during stable phase (P < 0.001). Multiple regression analysis revealed that plasma glucose level during attack was the sole factor which was positively correlated with ΔK (P < 0.01), while HbA1c level was negatively correlated (P < 0.05). The medication profiles and renal function had no impact on ΔK. A longer hospitalization period, higher incidence of myocardial infarction and higher peak creatine kinase level were observed in patients with a larger ΔK.
Conclusions
We have clearly demonstrated that there is a transient decrease in serum potassium level during ischemic attack of ACS compared to stable phase. The degree of the potassium dip was tightly correlated with glucose level, which overwhelmed the diabetic condition, and it also indicates the disease severity. The present study therefore promotes awareness of the significance of monitoring potassium level in parallel with glucose level in patients with ACS.
doi:10.1186/1475-2840-12-4
PMCID: PMC3561250  PMID: 23289667
Potassium level; Acute coronary syndrome; Glucose level; Diabetes
6.  Effect of rosuvastatin on systemic blood pressure in patients with hypercholesterolemia 
OBJECTIVE:
To investigate whether rosuvastatin reduces blood pressure (BP) in patients with hypercholesterolemia.
METHODS:
The present study investigated the effect of rosuvastatin on lipids and clinical parameters in 25 patients with a mean (± SD) age of 58.4±10.6 years over a three-month period.
RESULTS:
Rosuvastatin (2.5 mg/day to 5.0 mg/day) reduced systolic BP from 136.3±13.1 mmHg to 130.8±10.7 mmHg (P<0.01), along with a significant reduction in serum low-density lipoprotein cholesterol level (P<0.01). The patients were divided into two groups: 13 responders whose BP decreased by >5 mmHg with rosuvastatin treatment and 12 nonresponders who showed a BP reduction of ≤5 mmHg. Baseline systolic BP was significantly higher in responders than nonresponders (143.6±9.6 mmHg versus 128.4±11.9 mmHg, respectively; P<0.01). Responders also had a lower serum concentration of high-sensitivity C-reactive protein compared with nonresponders (0.11±0.07 mg/dL versus 0.40±0.28 mg/dL; P<0.01). The extent of BP reduction was positively correlated with baseline systolic BP (r=0.585; P=0.0021) but not with the reduction of low-density lipoprotein cholesterol level. Among the patients with baseline systolic BP >130 mmHg, all 11 responders (138.3 mmHg) were nonsmokers, while five of six nonresponders (145.7 mmHg) were smokers.
CONCLUSION:
Rosuvastatin had an additive antihypertensive effect in patients with poorly controlled hypertension that was independent of its lipid-lowering effect, which may be related to an inflammatory mechanism.
PMCID: PMC3627279  PMID: 23592940
Blood pressure; Hypercholesterolemia; Hypertension; Hydroxymethylglutaryl-CoA reductase inhibitors
7.  Effects of candesartan in hypertensive patients with type 2 diabetes mellitus on inflammatory parameters and their relationship to pulse pressure 
Background
Angiotensin receptor blockers (ARBs) are reported to provide direct protection to many organs by controlling inflammation and decreasing oxidant stress in patients without arteriosclerosis. This study aimed to evaluate (1) whether an ARB (candesartan) decreases values for inflammatory parameters in hypertensive patients with type 2 diabetes mellitus of long duration accompanied by arteriosclerosis and (2) whether there any predictors of which patients would receive the benefits of organ protection by candesartan.
Methods
We administered candesartan therapy (12 mg daily) for 6 months and evaluated whether there was improvement in serum inflammatory parameters high molecular weight adiponectin (HMW-ADN), plasminogen activator inhibitor-1 (PAI-1), highly sensitive C-reactive protein (Hs-CRP), vascular cell adhesion molecule-1 (VCAM-1) in serum and urinary-8-hydroxydeoxyguanosine (U-8-OHdG). We then analyzed the relationship between the degree of lowering of blood pressure and inflammatory factors and the relationship between pulse pressure and inflammatory factors. Finally, we analyzed predictive factors in patients who received the protective benefit of candesartan.
Results
After 6 months of treatment, significant improvements from baseline values were observed in all patients in HMW-ADN and PAI-1 but not in Hs-CRP, VCAM-1 and U-8-OHdG. Multilinear regression analysis was performed to determine which factors could best predict changes in HMW-ADN and PAI-1. Changes in blood pressure were not significant predictors of changes in metabolic factors in all patients. We found that the group with baseline pulse pressure <60 mmHg had improved HMW-ADN and PAI-1 values compared with the group with baseline pulse pressure ≥ 60 mmHg. These results suggest that pulse pressure at baseline could be predictive of changes in HMW-ADN and PAI-1.
Conclusions
Candesartan improved inflammatory parameters (HMW-ADN and PAI-1) in hypertensive patients with type 2 diabetes mellitus of long duration independent of blood pressure changes. Patients with pulse pressure <60 mmHg might receive protective benefits by candesartan.
Trial registration
UMIN000007921
doi:10.1186/1475-2840-11-118
PMCID: PMC3489584  PMID: 23034088
Candesartan; Angiotensin receptor blockers; Type 2 diabetes mellitus; Inflammatory parameters; Pulse pressure
8.  Serial measurements associated with an amelioration of acute heart failure: an analysis of repeated quantification of plasma BNP levels 
Background:
The magnitude of improvement of acute heart failure achieved during treatment varies greatly among patients. We examined changes in the plasma B-type natriuretic peptide (BNP) levels of patients with acute heart failure and attempted to elucidate the clinical factors associated with amelioration of acute heart failure.
Methods and results:
The study population consisted of 208 consecutive patients admitted to our institution with acute heart failure. We measured plasma BNP levels before and after treatment of acute heart failure and evaluated these levels based on median age, body mass index (BMI), creatinine (Cr) level, and left ventricular ejection fraction (EF). Plasma BNP levels before treatment were equivalent between the younger and older age groups; however, plasma BNP levels after treatment were higher in the older age group (p<0.01). Plasma BNP levels before treatment were significantly high in the lower BMI group (p<0.05) and the higher Cr group (p<0.01). Similarly, plasma BNP levels after treatment were high in both the lower BMI and higher Cr groups (p<0.01 for both). In the low EF group, plasma BNP levels before treatment were significantly high (p<0.01), while plasma BNP levels after treatment were equivalent to those in the high EF group. A multiple linear regression analysis revealed that Cr was positively correlated and BMI and EF were negatively correlated with plasma BNP levels before treatment; however, the contributions of age, BMI, and Cr in reducing plasma BNP levels were more significant after treatment.
Conclusions:
The contributions of clinical factors working against amelioration of heart failure vary before and after treatment. Regarding plasma BNP levels, older age, very low BMI, and the presence of renal dysfunction eventually act to prevent amelioration of acute heart failure. Systolic dysfunction does not act against amelioration of acute heart failure.
doi:10.1177/2048872612458580
PMCID: PMC3760539  PMID: 24062913
Body mass index; B-type natriuretic peptide; creatinine; diastolic dysfunction; ejection fraction
9.  The role of Na+/H+ exchanger in Ca2+ overload and ischemic myocardial damage in hearts from type 2 diabetic db/db mice 
Background
A higher increase in intracellular Na+ via Na+/H+ exchanger (NHE) during ischemia has been reported in type 2 diabetic mouse hearts. We investigated the role of NHE in inducing changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and alterations in ventricular function during ischemia-reperfusion in type 2 diabetic mouse hearts.
Methods
Hearts from male type 2 diabetic db/db (12-15 weeks old) and age-matched control db/+ mice were subjected to Langendorff perfusion and loaded with 4μM of the Ca2+ indicator fura-2. The hearts were exposed to no-flow ischemia for 15 minutes and then reperfused. [Ca2+]i was measured by monitoring fura-2 fluorescence at 500 nm (excitation wavelengths of 340 and 380 nm), while left ventricular (LV) pressure was simultaneously measured.
Results
db/db hearts exhibited a lower recovery of LV developed pressure than db/+ hearts during reperfusion following ischemia. Diastolic [Ca2+]i was increased to a greater level in diabetic hearts than in the control hearts during ischemia and reperfusion. Such an increase in cytoplasmic Ca2+ overload during ischemia-reperfusion in diabetic hearts was markedly reduced in the presence of the NHE inhibitor cariporide. This was accompanied by a significantly improved recovery of ventricular function on reperfusion, as shown by a lower increase in diastolic pressure and increased recovery of developed pressure.
Conclusion
NHE plays a key role in enhancing cytoplasmic Ca2+ overload during ischemia-reperfusion and severely impairing post-ischemic cardiac function in hearts from type 2 diabetic db/db mice.
doi:10.1186/1475-2840-11-33
PMCID: PMC3366908  PMID: 22490613
10.  Optimization of Cardiac Metabolism in Heart Failure 
Current Pharmaceutical Design  2011;17(35):3846-3853.
The derangement of the cardiac energy substrate metabolism plays a key role in the pathogenesis of heart failure. The utilization of non-carbohydrate substrates, such as fatty acids, is the predominant metabolic pathway in the normal heart, because this provides the highest energy yield per molecule of substrate metabolized. In contrast, glucose becomes an important preferential substrate for metabolism and ATP generation under specific pathological conditions, because it can provide greater efficiency in producing high energy products per oxygen consumed compared to fatty acids. Manipulations that shift energy substrate utilization away from fatty acids toward glucose can improve the cardiac function and slow the progression of heart failure. However, insulin resistance, which is highly prevalent in the heart failure population, impedes this adaptive metabolic shift. Therefore, the acceleration of the glucose metabolism, along with the restoration of insulin sensitivity, would be the ideal metabolic therapy for heart failure. This review discusses the therapeutic potential of modifying substrate utilization to optimize cardiac metabolism in heart failure.
doi:10.2174/138161211798357773
PMCID: PMC3271354  PMID: 21933140
myocardial glucose and fatty acid metabolism; insulin resistance; metabolic therapy; heart failure.
11.  Vascular Endothelial-Cadherin Stabilizes at Cell–Cell Junctions by Anchoring to Circumferential Actin Bundles through α- and β-Catenins in Cyclic AMP-Epac-Rap1 Signal-activated Endothelial Cells 
Molecular Biology of the Cell  2010;21(4):584-596.
Vascular endothelial (VE)-cadherin is a cell-cell adhesion molecule involved in endothelial barrier function. Here, we show that initial circumferential actin bundling induced by cyclic AMP-Epac-Rap1 signal and its linkage to VE-cadherin through α- and β-catenins lead to the stabilization of VE-cadherin at cell-cell contacts.
Vascular endothelial (VE)-cadherin is a cell–cell adhesion molecule involved in endothelial barrier functions. Previously, we reported that cAMP-Epac-Rap1 signal enhances VE-cadherin–dependent cell adhesion. Here, we further scrutinized how cAMP-Epac-Rap1 pathway promotes stabilization of VE-cadherin at the cell–cell contacts. Forskolin induced circumferential actin bundling and accumulation of VE-cadherin fused with green fluorescence protein (VEC-GFP) on the bundled actin filaments. Fluorescence recovery after photobleaching (FRAP) analyses using VEC-GFP revealed that forskolin stabilizes VE-cadherin at cell–cell contacts. These effects of forskolin were mimicked by an activator for Epac but not by that for protein kinase A. Forskolin-induced both accumulation and stabilization of junctional VEC-GFP was impeded by latrunculin A. VE-cadherin, α-catenin, and β-catenin were dispensable for forskolin-induced circumferential actin bundling, indicating that homophilic VE-cadherin association is not the trigger of actin bundling. Requirement of α- and β-catenins for forskolin-induced stabilization of VE-cadherin on the actin bundles was confirmed by FRAP analyses using VEC-GFP mutants, supporting the classical model that α-catenin could potentially link the bundled actin to cadherin. Collectively, circumferential actin bundle formation and subsequent linkage between actin bundles and VE-cadherin through α- and β-catenins are important for the stabilization of VE-cadherin at the cell–cell contacts in cAMP-Epac-Rap1 signal-activated cells.
doi:10.1091/mbc.E09-07-0580
PMCID: PMC2820423  PMID: 20032304
12.  Cardiac 12/15 lipoxygenase–induced inflammation is involved in heart failure 
The Journal of Experimental Medicine  2009;206(7):1565-1574.
To identify a novel target for the treatment of heart failure, we examined gene expression in the failing heart. Among the genes analyzed, Alox15 encoding the protein 12/15 lipoxygenase (LOX) was markedly up-regulated in heart failure. To determine whether increased expression of 12/15-LOX causes heart failure, we established transgenic mice that overexpressed 12/15-LOX in cardiomyocytes. Echocardiography showed that Alox15 transgenic mice developed systolic dysfunction. Cardiac fibrosis increased in Alox15 transgenic mice with advancing age and was associated with the infiltration of macrophages. Consistent with these observations, cardiac expression of monocyte chemoattractant protein 1 (MCP-1) was up-regulated in Alox15 transgenic mice compared with wild-type mice. Treatment with 12-hydroxy-eicosatetraenoic acid, a major metabolite of 12/15-LOX, increased MCP-1 expression in cardiac fibroblasts and endothelial cells but not in cardiomyocytes. Inhibition of MCP-1 reduced the infiltration of macrophages into the myocardium and prevented both systolic dysfunction and cardiac fibrosis in Alox15 transgenic mice. Likewise, disruption of 12/15-LOX significantly reduced cardiac MCP-1 expression and macrophage infiltration, thereby improving systolic dysfunction induced by chronic pressure overload. Our results suggest that cardiac 12/15-LOX is involved in the development of heart failure and that inhibition of 12/15-LOX could be a novel treatment for this condition.
doi:10.1084/jem.20082596
PMCID: PMC2715088  PMID: 19546247

Results 1-12 (12)