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The current risk stratification for long QT syndrome (LQTS) includes clinical parameters such as age, gender and QT interval, but mutation-specific information is rarely used. We investigated whether changes in ion channel characteristics (channel current, current activation rate, current deactivation rate, voltage dependence of activation and maximal conductance) caused by missense mutations linked to LQTS type 1 (LQT1) correlate with increased risk of cardiac events for carriers of the mutations. 387 LQT1 patients carrying 17 different mutations from 4 international LQTS registries were included in this study. The electrophysiological parameters were obtained from expression of mutant ion channels in Xenopus laevis oocytes. Linear regression was used to test for correlation between ion channel characteristics and clinical phenotype, and Cox proportional hazard regression was used to identify independent risk factors for cardiac events.
We found that channels with decreased rate of current activation are associated with increased risk of cardiac events (HR = 2.02) independently of clinical risk factors. In patients with moderate QT prolongation (QTc < 500 ms), slower activation remained as an independent predictor for cardiac events (HR = 2.10), whereas QTc did not. A cardiomyocyte action potential model showed the prolongation of action potential duration for channels with slower activation rates. A pseudo-transmural ECG model predicted T-wave alternans due to early after depolarizations for mutants with slower activation rates.
Our results suggest that slow channel activation impairs cardiac repolarization contributing to arrhythmogenesis. Our results highlight the importance of genotype analysis in the risk stratification of LQT1 patients and its potential use in risk stratification of genetic diseases.
We analyzed cardiac proteins using proteomic analysis and identified two spots as heat shock protein (HSP) 90β. M.W. and pI of one spot, intact-HSP90β, were similar to those of theoretical data, and the other spot, modified-HSP90β, had a different M.W. and pI value from them. In this study, we explored these two spots further.
Western blotting (WB) using two different antibodies to HSP90β were used and changed protein database from Swiss-prot to IPI.
Intact-HSP90β spot was recognized by two antibodies but modified-HSP90β spot was not. Using the IPI protein database, modified-HSP90β was identified as glucose regulated protein (GRP) 94. Antibody to GRP94 recognized the spot and thus we concluded that modified-HSP90β was GRP94. We analyzed correlations between WB data of GRP94 and heart functions. GRP94 contents determined by WB were well correlated with heart functions similar to silver staining data. GRP94 may play a role in the heart failure.
Recently, we have reported that cardioprotective signaling is impaired by increased calcineurin activity and ER stress in OLETF, obese type 2 diabetic rats. The aim of this study was to test whether such dysfunction is common in type 2 diabetes.
A model of non-obese type 2 diabetes, Goto-Kakizaki rat (GK) and its control (WKY), underwent 20 min ischemia/2 hr reperfusion. EPO (5000 units/kg, i.v.) limited infarct size in WKY (45.7±4.4% vs. 62.0±5.7%) but not in GK (52.1±2.7% vs. 55.9±2.6%). EPO-induced phosphorylation of ERK and GSK3β but not Akt in WKY, only ERK was phosphorylated in GK. Although levels of GRP78, GRP94 and ER chaperones, were increased in GK compared with those in WKY, calcineurin activity was similar between the two groups (0.81±0.05 vs. 0.89±0.10 nmol/mg/min). Treatment with losartan (3 mg/kg/day), an ARB, for 2 weeks reduced myocardial GRP78 level and restored cardioprotection by EPO in GK.
Increased ER stress is a common mechanism impairing EPO-induced cardioprotective signaling in diabetic rats, while defects in the signaling by increased calcineurin may be specific to obese diabetic rats.
Wnt signalling controls the balance between stem cell proliferation and differentiation. It has been demonstrated that non-canonical Wnts (Wnt5a or Wnt11) enhance cardiac gene expression in adult progenitor cells (APCs) such as endothelial progenitor cells (EPCs) and mesenchymal stem cells. However the mechanism by which non-canonical Wnts regulate cardiac commitment of APCs is unknown.
Human peripheral blood derived EPCs were stimulated by recombinant Wnt5a. After stimulation, EPCs were analyzed using micro-array analysis, PCR, immunoblots, or chromatine immunoprecipitation and compared with control.
Using microarray analysis of Wnt5a-induced gene expression in EPCs, we discovered that Wnt5a significantly increased several enzymes, which play key roles in epigenetic remodelling by modifying histone methylation such as JmjC-domain containing family, JMJD2B/2C. JMJD2 family demethylate trimethylated histone H3 at lysine 9 (H3K9me3), thereby removing repressive histone modifications. Therefore, we hypothesized that increased JMJD2 might epigenetically modify gene expression thereby opening chromatin structures at silenced promoters and modulate the differentiation of APCs. Indeed, Wnt5a treatment reduced the repressive chromatin mark H3K9me3 as shown by immunoblot and immunohistochemistry, whereas global active chromatin marks (acetyl-H3 and trimethyl-H3K4) remained unchanged indicating that Wnt5a shifts the balance toward active gene expression. In addition, chromatin immunoprecipitation demonstrated that Wnt5a decreased H3K9me3 at the troponin T promoter. In line with removal of repressive chromatin marks, Wnt5a significantly increased differentiation of APCs to cardiomyocytes.
These data demonstrate that non-canonical Wnt5a induces epigenetic remodelling by removing repressive chromatin marks and thereby sensitize APCs for acquiring a cardiac cell fate.
We investigated the effects of pioglitazone, a synthetic PPAR-γ agonist, on the pressure overload-induced atrial fibrosis and atrial fibrillation (AF).
Male SD rats underwent abdominal aorta constriction (AAC). Pioglitazone (3 mg/kg/day) or vehicle was orally administered for 4 weeks.
1) Pioglitazone suppressed AAC-induced LA fibrosis. 2) AAC enhanced the expression of monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta 1 (TGFβ1), and alpha-smooth muscle actin (αSMA), which were suppressed by pioglitazone. 3) Gelatin zymography demonstrated that the activity of matrix metalloproteinase 9 (MMP-9) was increased by AAC, which was suppressed by pioglitazone. 4) In isolated-perfused heart experiments, programmed extrastimuli from RA induced AF in 8/8 AAC-treated rats, which was suppressed by pioglitazone (2/8, p<0.05).
Our results suggest that pioglitazone is effective to prevent pressure overload-induced atrial fibrosis and AF. The anti-fibrotic mechanisms of pioglitazone may be explained at least partly by the attenuation of MCP-1 and TGFβ1 expression and MMP signaling caused by AAC.
Cardiac hypertrophy is the compensatory response of the heart to an elevated workload. However, the changes in Ca2+ handling and contraction in hypertrophied heart induced by pressure-overload remains unclear.
After pulmonary artery banding (PAB) in rats, we measured the intracellular Ca2+ concentration with tension using the aequorin method in the right ventricular papillary muscles.
Four weeks after the operation, the right ventricle weight of the PAB rats was significantly heavier than that of control [PAB; 0.29±0.03 g (n=9), control 0.18±0.01 g (n=9), p<0.05]. We confirmed that peak Ca2+ transient in PAB rats was significantly higher than that in control [PAB; 2.09±0.12 μM (n=9), control 1.46±0.08 μM (n=9), p<0.05]. Interestingly, peak tension in PAB rats and controls did not significantly differ at 4 weeks after the operation [PAB; 28.88±4.40 mN/mm2 (n=9), control; 35.76±4.36 mN/mm2 (n=9)].
Our results suggest that the higher Ca2+ transient in cardiac hypertrophy induced by PAB might be a compensatory response to preserve contraction of the hypertrophied cardiac muscle.
Heart failure is a typical age-associated disease. Although age-related changes in the heart are likely to predispose aged people to heart failure, little is known about the molecular mechanism of cardiac aging.
We analyzed how constitutive activation of the p110-alpha isoform of phosphoinositide 3-kinase (PI3K) modified cardiac aging using a transgenic mouse line expressing a constitutive active PI3K (caPI3K) in a heart-specific manner. Cardiac function was impaired in old caPI3K mice compared to old NTg mice. Fibrosis and a marker of oxidative stress were increased in old caPI3K mice. The expression of senescence associated beta-galactosidase positive cells, lipofuscin, p16 or proinflammatory cytokine was not different between old NTg and caPI3K mice. Expression of genes related to autophagy or protein synthesis was not different.
Constitutive activation of PI3K deteriorated age associated decline of cardiac function, but did not change expression of senescence markers in a transgenic model.
Volume-regulated outwardly rectifying anion channel (VRAC) is activated by cell swelling, and plays a significant role in cell volume homeostasis in cardiac cells. Several signaling molecules, including phosphatidylinositol lipids, are localized to caveolae in cardiac cells, and caveolin-3 is a muscle-specific protein integrated in the caveolae. We found that the cardiac VRAC is functionally associated with caveolin-3.
The Cl− currents through VRAC were measured in single ventricular cells obtained from wild-type (WT) and caveolin-3 knockout (Cav-3 KO) mice, by using a whole-cell voltage-clamp method. In the cells from Cav-3 KO mice, the cell capacitance, an index of cell size, was increased, and the density of VRAC current induced by extracellular hypotonic solution (HYPO) was markedly reduced, compared to those from wild-type (WT) mice. Video-image analysis showed that the degree of HYPO-induced cell swelling in Cav-3 KO mice was significantly bigger than that in WT mice, and the regulatory volume decrease, which was seen in WT cells after osmotic swelling, was lost in myocytes from Cav-3 KO mice. This result is in parallel with the VRAC inhibition. The attenuated VRAC current was restored by intracellular application of a VRAC modulator, phosphatidylinositol 3,4,5-trisphosphate (PIP3). In contrast, other cardiac Cl− currents, acidic extracellular pH-activated Cl− current and extracellular UTP-activated Cl− current, were affected less by the deficiency of caveolin-3.
We postulate that an impairment of PIP3 production is responsible for the attenuation of cardiac VRAC currents in Cav-3 KO mice.
In contrast to Ca2+-dependent contraction of vascular smooth muscle (VSM) which regulates physiological vascular tone, Rho-kinase (ROK)-mediated Ca2+-sensitization of VSM contraction plays a pivotal role in abnormal VSM contractions, including vasospasm and hypertension. As an upstream mediator of such abnormal pathway for Ca2+ sensitization of VSM, we identified sphingosylphosphorylcholine (SPC). Furthermore, we found that eicosapentaenoic acid (EPA), a component of fish oil, can selectively inhibit the SPC-induced ROK-mediated Ca2+ sensitization without affecting physiological Ca2+-dependent contraction. Indeed, EPA clinically had protective effects on cerebral vasospasm. In this study, we aimed to identify plant extracts that selectively inhibit Ca2+ sensitization.
After extensive screening, we found that extracts from Allium cepa L. can strongly inhibit the SPC-induced ROK-mediated Ca2+ sensitization. The extracts of the Allium-blade strongly (60%) inhibited the Ca2+ sensitization, but not physiological Ca2+-dependent contraction. The extracts of basal stems of A. fistulosum abolished Ca2+ sensitization completely (100%), whereas the inhibitory effects on Ca2+-dependent contraction varied between 10% and 80%, depending on the extraction solvents and procedures. These results suggest that blade leaves and basal stems of Allium may contain possible components of novel therapeutic agents for abnormal VSM contraction.
The intercalated disc (IC) contains different junctional complexes (adhesion junction: AJ and gap junction: GJ). We clarified IC remodeling and its potential role in the pathogenesis of arrhythmias during development of heart failure.
We investigated changes of connexin (Cx)43 and AJ proteins in UM-X7.1 cardiomyopathic hamster hearts. UM-X7.1 developed left ventricular (LV) hypertrophy by ages 10w, and showed a moderate reduction in LV contractility at age 20w. LV protein levels of Cx43 in UM-X7.1 were unaffected at age 10–15w, but significantly reduced at 20w with increase of Ser255-phosphorylated Cx43 expression. Electron microscopy revealed IC of UM-X7.1 were more highly convoluted and appeared more electron-dense. The total N-cadherin and β-catenin expression had no change at 10–15w and 20w compared with control, although immunohistochemistry revealed an increase of both protein expression at IC at over 10w. The nuclear β-catenin, which regulates the expression of Cx43, was remarkably decreased at 10–15w. The olmesartan treatment weakened overall electron density of IC and attenuated the decrease of nuclear β-catenin expression.
Alterations in AJ protein precede the GJ remodeling. Treatment with olmesartan might be effective in the treatment of arrhythmias during development of heart failure in cardiomyopathy.
The ATP-binding cassette transporter BCRP1/ABCG2 has been suggested to regulate several tissue defense mechanisms in the heart. However, its clinical significance in cardiac repair after myocardial infarction (MI) remains unknown.
Immunohistochemistry showed that BCRP1/ABCG2 was mainly expressed in endothelial cells of microvessels in the heart. MI was induced in 8- to 12-week-old wild-type (WT) and Bcrp1/Abcg2 knock-out (KO) mice by ligating the left anterior descending artery. At 28 days after MI, survival rate was significantly lower in KO mice than in WT mice due to cardiac rupture. Echocardiographic, hemodynamic and histological assessments showed that ventricular remodeling was more deteriorated in KO mice than in WT mice. Capillary, macrophages and myofibroblasts densities in peri-infarction area at 5 days after MI were significantly reduced in KO mice than in WT mice, although gene expression of proinflammatory, angiogenesis-related and fibrosis-related cytokines was comparable or higher in KO mice than in WT mice. In vitro experiments demonstrated that pharmacological inhibition of BCRP1/ABCG2 resulted in impaired survival of human microvascular endothelial cells from the heart (HMVEC-Cs) under oxidative stress. Moreover, BCRP1/ABCG2 inhibition impaired migration and tube formation properties of HMVEC-Cs.
We demonstrated that BCRP1/ABCG2 plays a critical role in cardiac repair after MI. Thus, BCRP1/ABCG2 might be of interest as a therapeutic target to improve clinical outcomes for MI.
It is generally accepted that Ca2+-induced Ca2+ release is not the predominant mechanism during embryonic stages. However, most studies have been conducted either on primary cultures or acutely isolated cells, in which an apparent reduction of ryanodine receptor density have been reported. The purpose of this study was to analyze Ca2+ transients in murine whole hearts to evaluate the developmental changes of SR function.
Fluo-3 fluorescence signals from stimulated (0.5, 1, 2, 4 Hz) embryonic and neonatal whole hearts were detected using a photomultiplier and stored as Ca2+ transients. We measured the amplitude, time to peak (TTP), and time to 50% relaxation (T50) of Ca2+ transients before and after thapsigargin and ryanodine application.
After thapsigargin application, the T50 of Ca2+ transients was significantly prolonged. After ryanodine application, the amplitude was significantly reduced even at early embryonic stages. The recuperative effect became larger during developmental stages.
Our results indicated that SERCA and RyR began to contribute significantly to Ca2+ homeostasis at early embryonic stages and SR Ca2+ contents increased during developmental stages.
We discovered that prostaglandin D2 (PGD2) protects cardiomyocytes against cell death via activation of ERK during ischemia-reperfusion. PGD2 exerts its effect through high-affinity interactions with two PGD2 receptors; DP1 and CRTH2, both of which are little in cardiomyocytes. This study is designed to investigate how PGD2 transduces signal in cardiomyocytes.
Cultured cardiomyocytes were stimulated with PGD2 (100nM). PGD2 stimulates ERK from 1 min, peaked at 3 min and decreased thereafter. Neither DP1 antagonist (BWA868c), CRTH2 antagonist (CAY10471), nor a combination of the two could block PGD2-induced ERK activation. Conversely, prostaglandin F2α receptor (FP receptor) antagonist (AL8810) completely blocked PGD2-induced ERK activation. FP receptor is abundantly expressed in cardiomyocytes. Next, isolated Langendorff-perfused hearts from wild-type, DP1-knockout, CRTH2-knockout, and FP-knockout mice were treated with PGD2. PGD2-induced ERK activation was blocked in FP-knockout hearts, but not in DP1-, CRTH2-knockout hearts.
PGD2 activates ERK via the FP receptor in cardiomyocytes.
Inflammation plays a key role in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear. Here, we demonstrate that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R. Inflammasome activation triggered by I/R leads to robust interleukin-1β production and subsequent intensive inflammatory responses such as inflammatory cell infiltration and cytokine expression, thereby resulting in infarct development, myocardial fibrosis and dysfunction. In vitro experiments revealed that hypoxia-reoxygenation (H/R) stimulated inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, and that H/R-induced activation was partially mediated through reactive oxygen species (ROS) and potassium efflux. Our results demonstrate the molecular basis for the initial inflammatory response after I/R and suggest the inflammasome to be a novel therapeutic target for myocardial I/R injury.
We hypothesized that cardiomyocyte stiffness in the transverse direction is increased in hypertrophied hearts. We measured the elastic modulus of a single cardiomyocyte by use of an atomic force microscope (AFM) in isoproterenol (ISO)-induced hypertrophied rat hearts.
Male Wistar rats received vehicle (control), ISO (2.4 mg/kg/day) or ISO+beta1-blocker metoprolol (MET) (24 mg/kg/day) subcutaneously (n=5 in each group). After 7 days, compared with those in control and ISO+MET groups, ISO administration had increased left ventricular (LV) wall thickness (P<0.05). Elastic modulus of 10 living cardiomyocytes from LV free wall was measured by parabolic force curves of cantilever deflection/indentation obtained by AFM. Elastic modulus of cardiomyocytes was significantly higher in the ISO group than in control and ISO+MET groups. Next, we added butanedione monoxime (BDM) (20 mM) and blebbistatin (10 μM), an inhibitor of actin-myosin interaction to culture medium. BDM and blebbistatin significantly reduced the stiffness of a cardiomyocyte obtained from an ISO-treated rat.
Cardiomyocyte stiffness in the transverse direction was increased in hearts with ISO-induced hypertrophy along with diastolic dysfunction. This may be caused by incomplete relaxation.
To clarify the role of cardiac remodeling on the pathogenesis of inherited dilated cardiomyopathy (DCM), we used the knock-in mouse model of troponin T mutation (DCM mouse), which shows typical manifestation of human DCM.
Echocardiography was used to monitor cardiac function in vivo. Cardiac fibrosis was identified using Masson Trichrome staining of ventricular tissues. Electrocardiogram was recorded using telemetry system to check electrical remodeling. We also applied ARB (candesartan) to DCM mouse.
DCM mice died suddenly within 6 months after birth. Echocardiography revealed marked dilatation of cardiac chambers and significant reduction of cardiac contraction. Cardiac fibrosis was increased, and QRS width and QT interval in electrocardiogram recordings were prolonged in DCM mice. ARB treatment significantly improved the survival of DCM mice. ARB decreased cardiac chamber size and improved cardiac function. Cardiac fibrosis and altered electrocardiogram parameters were recovered with the application of ARB.
Improvement of structural and electrical remodeling is a promising target for the treatment of DCM.
Clarithromycin (CAM), a major macrolide antibiotic, has many biological functions, including matrix metalloproteinase (MMPs) regulation. However, little is known about the effect of CAM in heart transplantation via MMP-9. The purpose of this study was to clarify the role of MMPs regulated by CAM in the progression of rejection.
We orally administered CAM into murine cardiac allograft recipients. Total allomismatch combination and class II mismatch combinations were used for the analysis of acute and chronic rejection.
Clarithromycin improved acute rejection judged by graft survival and by myocardial cell infiltrating area in a total allomismatch combination. The CAM-treated allografts showed affected expression of T-cells and macrophages and MMP-9 activities. In chronic rejection, CAM suppressed the development of graft arterial disease and myocardial remodeling compared with that of non-treatment. Clarithromycin inhibited the expression of MMP-9 in macrophages and smooth muscle cells.
Clarithromycin is useful to suppress allograft remodeling because it is critically involved in the prevention of cardiac rejection through the suppression of MMP-9.
Endoplasmic reticulum (ER) stress is induced when ER function is impaired. Recent studies have demonstrated that ER stress plays an important role in diseases such as neuro-degenerative disease and diabetes mellitus, and it can also induce cell-specific genes. In this study, we examined whether X-box binding protein (XBP1), a major UPR-linked transcriptional factor, regulated the expression of brain natriuretic peptide (BNP) in cardiomyocytes.
In samples from failing human hearts, extensive splicing of XBP1 was observed along with increased expression of BNP. A pharmacological ER stressor caused a dose-dependent increase in the expression of spliced XBP1 (sXBP1) and BNP in cultured cardiomyocytes. The promoter assay with overexpression of sXBP1 showed that the proximal AP1/CRE-like element in the promoter of BNP was critical for transcriptional regulation of BNP by sXBP1.
These findings suggest that ER stress observed in failing hearts regulates cardiac BNP expression through a novel promoter region of the AP1/CRE-like element.
Post-MI left ventricular function is significantly worse in diabetic compared with non-diabetic patients, but the exact mechanism remains unclear. ERK5, an atypical mitogen activated protein kinase with transcriptional (trans) activity, inhibits apoptosis, and ERK5 trans activity itself is subjected to down regulation by high glucose and reactive oxygen species-dependent SUMOylation. p90RSK activity was significantly increased in DM, and MI-mediated cardiac dysfunction and apoptosis was decreased in non-DM cardiac-specific overexpression of dominant negative-p90RSK mice (DN-p90RSK-Tg). In the current study, we investigated the role of p90RSK activation on DM-mediated ERK5-SUMOylation and subsequent LV dysfunction after MI.
First, we found that H2O2 (30 μM) significantly inhibited ERK5 trans activity by using a mammalian one-hybrid assay, which was reversed by DN-p90RSK, and co-transfection of wild type 90RSK significantly inhibited ERK5 trans activity, suggesting that the inhibition of ERK5 trans activity by H2O2 is, at least partially, p90RSK-dependent. Since transfection of p90RSK wild type also significantly increased ERK5-SUMOylation (co-immunoprecipitation of ERK5 with SUMO), we determined the involvement of ERK5-SUMOylation on the inhibition of p90RSK-mediated ERK5 trans activity. Both ERK5 K6R/K22R double mutant of SUMOylation sites and PIAS1 (E3 ligase) siRNA prevented p90RSK-mediated inhibition of ERK5 trans activity, supporting the idea that the inhibition of ERK5 trans activity by p90RSK was dependent on ERK5-SUMOylation. Finally, we investigated the role of p90RSK activation in DM-mediated exacerbation of LV remodeling and ERK5-SUMOylation. MI was induced in streptozotocin (STZ)-injected (DM + MI group) or vehicle-injected mice (MI group) by ligating the left coronary artery. Although LV remodeling was significantly exacerbated in DM + MI group, diabetic DN-p90RSK-Tg mice showed improved LV function one week after MI compared with diabetic non-transgenic littermate control mice, and the induction of ERK5-SUMOylation in DM + MI group was significantly decreased in DN-p90RSK-Tg mice.
These data suggest that the activation of p90RSK is critical for DM + MI-mediated ERK5-SUMOylation and subsequent exacerbation of LV remodeling.
In many factors to relate with bone marrow cell mobilization, local inflammation induced by cytokines is possible to derive bone marrow cells, resulting in thickened neointima. However, the relationship between the inflammatory reactions and bone marrow cell invasion has not been clarified.
We inserted a wire into the femoral arteries of Balb/c (WT) and TNF-alpha or IL-6 knock out (KO) mice. At 4 weeks after the injury, intima/media ratio was significantly smaller in KO than in WT. The number of inflammatory cells in neointima was much less in both KO. At 1 week, CD34 positive cells were hardly observed in both KO, but some were observed in WT, and a part of them were positive for alpha-smooth muscle actin. Re-endothelialization appeared earlier in both KO than in WT. Moreover, Stat-3 signal was markedly inhibited in KO.
Inflammatory cytokines involved in neointimal formation, possible through their inflammatory effects to induce bone marrow cells.
Skeletal myoblast (SMB) sheets induce myocardial regeneration in failing hearts via the paracrine system. We investigated the effect of adipose tissue derived mesenchymal stem cells (ADMSC) to enhance the paracrine effect of SMB and improve functional performance in a rat myocardial infarction (MI) model.
SMB were isolated from male Lewis rats and ADMSC from human adult female subcutaneous fat samples, then cultured separately or together in temperature-responsive culture dishes to obtain the co-cultured cell sheet (SMB+ADMSC), SMB-single sheet, and ADMSC-single sheet. RT-PCR was used to analyze the expressions of rat HGF (rHGF) and rat VEGF (rVEGF) secreted only from the rat SMB, which revealed their levels were significantly higher in the SMB+ADMSC sheet than the SMB-single sheet. Further, the secretion of these cytokines were significantly greater in the supernatant of the SMB+ADMSC sheets, as shown by ELISA analysis. Next, MI was induced in female athymic rats by ligating the LAD, and they were divided into the following 4 groups: M: SMB+ADMSC sheet; S: SMB sheet; A: ADMSC sheet; and sham groups (n=10 in each). Two weeks after LAD ligation, cell sheets were implanted onto scarred myocardium areas. Echocardiography and pressure-volume analysis showed significant improvement in cardiac function in the M group. Histological examinations revealed cell diameter and percent fibrosis were decreased, while vascular density was increased in the M group. Expression of mRNA and secretion of rHGF and rVEGF in the implanted infarcted hearts were most significantly increased in the M group. More implanted SMB survived in the M group as compared with S group, as assessed by RT-PCR for the Y-chromosome specific Sry gene.
Our results demonstrated that ADMSC enhance the paracrine effect of SMB, thus enhancing angiogenesis, lowering fibrosis, inhibiting cellular hypertrophy, improving cardiac function, and supporting cell survival in MI model rats. Implantation of SMB+ADMSC sheet may be a promising strategy for the treatment of heart failure.
Rat experimental autoimmune myocarditis (EAM) is transferable into naïve syngeneic animals by vitro-activated myocarditogenic T lymphocyte clones (MTL). However, the way in which MTL recruits tissue inside the body and the target tissue have not been clarified. To clarify these questions, unequivocal identification of MTL such as the use of green fluorescent protein (GFP) is required.
MTLs are a lymph node cell (98% GFP-positive, [GFP+]) that is isolated from GFP transgenic rats that can emit green fluorescence, leading to the establishment of a GFP-transgenic MTL bulk culture line (GFP-MTL) (97% GFP+ and 84% CD4+). Intravenous injection of GFP-MTL (1×107/rat) into naïve Lewis rats could unexceptionally induce EAM without any sign of graft vs. host diseases. Real-time confocal laser microscope imaging demonstrated GFP+ and CD4+ cells coincided with inflammatory infiltrates in the myocardium. First, the GFP+ cells gathered at the site of inflammation in the myocardium. Then, GFP-CD4+ cells (cells of host origin) appeared. In addition, the number of GFP+ cells decreased as the myocarditis progressed.
The injected MTL cells infiltrated directly, and caused myocarditis.
AMP-activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK involves allosteric regulation by AMP/ATP ratio as well as phosphorylation by upstream AMPK kinases (AMPKKs), including LKB1 and CaMKKβ. However, it remains unknown whether CaMKKβ is involved in heart failure.
We subjected wild type (WT) mice to transverse aortic constriction (TAC) for 3 weeks. CaMKKβ mRNA and protein were higher in mice hearts after TAC compared to those after sham operation. To examine the pathophysiological role of CaMKKβ in pressure overload-induced heart failure, we generated cardiac-specific transgenic (TG) mice expressing a kinase-dead form of CaMKKβ. The TG mice had normal cardiac function and morphology. However, after TAC, TG mouse hearts showed a significant inhibition of downstream signaling molecules, such as AMPK, CaMKI, and CREB phosphorylation with decreased systolic function. Furthermore, TG mice had signs of heart failure and a higher mortality rate than WT mice after TAC. To assess energy state in the TAC heart, we measured PCr/β-ATP ratios by MR spectroscopy. TG mice demonstrated significantly decreased energy pooling compared to WT mice after TAC (% reduction in PCr/β-ATP ratios were 28% in WT mice and 45% in TG mice).
These data indicate that CaMKKβ exerts cardiac adaptive energy pooling against pressure overload-induced heart failure, and that elevating CaMKKβ activity may have therapeutic potential in the treatment of heart failure.