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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.  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
3.  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
4.  Ca2+/Calmodulin-Dependent Kinase IIδ Causes Heart Failure by Accumulation of p53 in Dilated Cardiomyopathy 
Circulation  2010;122(9):891-899.
Background
Dilated cardiomyopathy (DCM), characterized by dilatation and dysfunction of the left ventricle, is an important cause of heart failure. Many mutations in various genes, including cytoskeletal protein genes and contractile protein genes, have been identified in DCM patients, but the mechanisms of how such mutations lead to DCM remain unknown.
Methods and Results
We established the mouse model of DCM by expressing a mutated cardiac α-actin gene, which has been reported in patients with DCM, in the heart (mActin-Tg). mActin-Tg mice showed gradual dilatation and dysfunction of the left ventricle, resulting in death by heart failure. The number of apoptotic cardiomyocytes and protein levels of p53 were increased in the hearts of mActin-Tg mice. Overexpression of Bcl-2 or downregulation of p53 decreased the number of apoptotic cardiomyocytes and improved cardiac function. This mouse model showed a decrease in myofilament calcium sensitivity and activation of calcium/calmodulin-dependent kinase IIδ (CaMKIIδ). The inhibition of CaMKIIδ prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function.
Conclusion
CaMKIIδ plays a critical role in the development of heart failure in part by accumulation of p53 and induction of cardiomyocyte apoptosis in the DCM mouse model.
doi:10.1161/CIRCULATIONAHA.109.935296
PMCID: PMC3226824  PMID: 20713897
apoptosis; CaMKII; cardiomyopathy; heart failure; genes; p53
5.  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 1-5 (5)