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. 2012 Summer;17(2):50–58.

International Academy of Cardiovascular Sciences, Japanese Section Meeting

July 7 to 8, 2012, Tokyo, Japan

Nobuakira Takeda 1,2
PMCID: PMC3395455
Exp Clin Cardiol. 2012 Summer;17(2):50.

Y-1: Regulatory Role of Dendritic Cells in Postinfarction Healing and Left Ventricular Remodeling

Atsushi Anzai 1, Toshihisa Anzai 2, Keiichi Fukuda 1

Abstract

BACKGROUND:

An excessive immune-mediated inflammation is associated with cardiac dysfunction and poor clinical outcomes after myocardial infarction (MI). However, the precise regulatory mechanism to control the inflammatory response and subsequent tissue repair following MI is unclear.

METHODS AND RESULTS:

Bone-marrow (BM) cells from CD11c-diphtheria toxin receptor/GFP transgenic mice were transplanted into lethally irradiated wild-type (WT) recipient mice. After reconstitution of BM-derived cells, the recipient mice were treated with either diphtheria toxin (DC ablation) or vehicle (control), and MI was created by left coronary ligation. CD11c+ GFP+ DCs expressing CD11b and MHC class II were recruited into the heart, peaking on day 7 after MI in control group. Mice with DC ablation for 7 days showed deteriorated left ventricular (LV) function and remodeling. The DC-ablated group demonstrated enhanced and sustained expression of inflammatory cytokines, prolonged extracellular matrix degradation associated with a high level of matrix metalloproteinase-9 activity, and diminished expression level of interleukin (IL)-10 and endothelial cell proliferation post-MI compared with control group. In vivo and in vitro analyses revealed that DC-ablated infarcts had an enhanced capacity of monocyte/macrophage recruitment. Among these cells, augmented infiltration of proinflammatory Ly6Chigh monocytes and F4/80+ CD206 M1 macrophages and, conversely, impaired recruitment of anti-inflammatory Ly6Clow monocytes and F4/80+ CD206+ M2 macrophages in the infarcted myocardium were identified in DC-ablated group than in control group. Flow cytometric analysis for inflammatory cells extracted from infarcted tissue revealed that CD11c+ DC is one of the major sources of IL-10. Adoptive transfer of BM-derived DCs from WT, but not IL-10-deficient mice, abrogated the adverse effects of DC depletion on inflammatory response and LV remodeling after MI.

CONCLUSION:

DC could be a potent immunoprotective regulator during the postinfarction healing process, via controlling monocyte/macrophage homeostasis through IL-10 secretion.

Exp Clin Cardiol. 2012 Summer;17(2):50.

Y-2: Cardiac Nuclear High Mobility Group Box 1 Prevents Cardiac Dysfunction Induced by Pressure Overload

Akira Funayama 1, Tetsuro Shishido 1, Isao Kubota 1

Abstract

BACKGROUND:

High mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. In the nucleus, HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription and repair. However, little is known about the effects of nuclear HMGB1 on heart failure. The aim of this study was to examine the impact of nuclear HMGB1 on cardiac dysfunction induced by pressure overload.

METHODS AND RESULTS:

The immnohistochemistry using human biopsy samples revealed that nuclear HMGB1 expression was decreased in hypertrophic and failing hearts compared with non-failing hearts. We found that nuclear HMGB1 was decreased by endothelin-1 (ET-1) stimulation in cultured neonatal rat cardiomyocytes, where nuclear HMGB1 was acetylated and translocated to cytoplasm. Overexpression of nuclear HMGB1 attenuates ET-1 induced cardiomyocyte hypertrophy. Thoracic transverse aortic constriction (TAC) was created in transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) and wild type (WT) mice. Cardiac hypertrophy was attenuated in HMGB1-Tg mice and survival rate after TAC was higher in HMGB1-Tg mice than in WT mice. Induction of fetal cardiac genes was decreased in HMGB1-Tg compared with WT mice. Nuclear HMGB1 expression was preserved in HMGB1-Tg mice compared with WT mice after TAC. Preserved nuclear HMGB1 expression significantly attenuated DNA damage after TAC.

CONCLUSION:

These results suggest that preserved nuclear HMGB1 prevents cardiac dysfunction induced by pressure overload.

Exp Clin Cardiol. 2012 Summer;17(2):50–51.

Y-3: Toll-Like Receptor 2 Mediates Cardiac Adaptive Response to Pressure Overload

Yasutomi Higashikuni 1, Ryozo Nagai 1, Masataka Sata 2

Abstract

BACKGROUND:

Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. TLR2 is a member of the TLR family, and has been shown to recognize not only foreign substances, but also endogenous ligands, which induce an inflammatory response called homeostatic inflammation. Recently, homeostatic inflammation has been revealed to contribute to the pathophysiology of various diseases. However, the role of homeostatic inflammation mediated by TLR2 on pressure overload-induced cardiac hypertrophy remains unclear.

METHODS AND RESULTS:

Pressure overload was induced in eight-to- 12-week-old wild-type (WT) and TLR2 knock-out (KO) mice by transverse aortic constriction (TAC). In the absence of pressure overload, cardiac function and morphology did not differ between WT and KO mice. In WT mice, TLR2 mRNA expression was increased after TAC compared with that before surgery. At two weeks after TAC, KO mice showed reduced cardiac hypertrophy and fibrosis with more left ventricular dilatation and impaired systolic function compared with WT mice, which indicated that the cardiac adaptive response to pressure overload was impaired in KO mice. Gene expression levels of pro-inflammatory cytokines including interleukin-1β (IL-1β) in the heart were reduced in KO mice. Bone marrow transplantation experiments revealed that TLR2 expressed in the heart, not in blood cells, plays an important role in cardiac adaptive response to pressure overload. In vitro experiments demonstrated that Pam3CSK4, a specific TLR2 agonist, induces cardiomyocyte hypertrophy and fibroblast proliferation in a nuclear factor κB (NF-κB) and IL-1β-dependent manner. Systemic administration of a NF-κB inhibitor or IL-1β neutralization antibody to WT mice resulted in impaired cardiac adaptive response to pressure overload.

CONCLUSIONS:

TLR2 mediates cardiac adaptive response to pressure overload. Thus, modulation of TLR2 signaling may provide a novel strategy for treating cardiac hypertrophy and heart failure.

Exp Clin Cardiol. 2012 Summer;17(2):51.

Y-4: Tecnetium Sestamibi Washout, a Potential Indicator for Cardiac Mitochondrial Function, Correlates to BNP Levels in Patients with Chronic Heart Failure

Sayako Hirose 1, Takao Kato 1, Ryuji Nohara 1

Abstract

INTRODUCTION:

In a study of cardiac energy metabolism using a rat model of heart failure, metabolic remodeling accompanied with changes in gene expression related to mitochondrial function with the onset of heart failure (Kato et al. Circulation:Heart Failure, 2010). Technetium-99m sestamibi (99mTc-MIBI), which is widely used in the diagnosis of myocar-dial ischemia, has been reported to show slow clearance in proportion to mitochondrial membrane potential in cultured cardiomyocytes.

HYPOTHESIS:

We assessed the hypothesis that increased 99mTc-MIBI washout (decreased MIBI retention) was observed in patients with heart failure and associated with the severity of heart failure.

METHODS:

After administration of 99mTc-MIBI, 33 patients with chronic heart failure after the treatment of acute exacerbation were examined by myocardial perfusion imaging in the early and delayed phases (interval of 3 h), in which washout rates were calculated. We tested the correlation of washout rate to ejection fraction (EF) on echocardiography, brain natriuretic peptides (BNP), C-reactive protein, estimated glomerular filtration rate, and cardiac events followed for 12 months.

RESULTS AND CONCLUSIONS:

The washout rate of 99mTc-MIBI was positively correlated with BNP levels (r=0.61, P=0.005), but was not correlated with EF. During follow-up, rehospitalized patients tended to show higher washout rates of MIBI during the previous hospitalization. The washout rate of 99mTc-MIBI is thought to be one of markers of myocardial damage or dysfunction. It needs further study to clarify whether it has different roles from established markers, such as BNP or EF, in the management of heart failure.

Exp Clin Cardiol. 2012 Summer;17(2):51.

Y-5: Sirt7-Mediated TGF-β Receptor Stabilization is Essential for Proper wound Healing after Myocardial Infarction

Yasuhiro Izumiya 1, Satoshi Araki 1, Hisao Ogawa 1

Abstract

BACKGROUND AND OBJECTIVES:

Sirt7 is one of the seven members of mammalian sirtuin protein. We investigated the role of Sirt7 in wound healing processes after myocardial infarction (MI).

METHODS:

MI was created in male Sirt7 deficient (Sirt7−/−) and wild-type (WT) mice. Rat neonatal cardiac fibroblasts were used for in vitro experiments.

RESULTS:

Sirt7 expression was increased in the border zone (BZ) of myocardium after MI in WT mice. Sirt7−/− mice were susceptible to cardiac rupture after MI compared with WT mice. Fibrosis and myofibroblast in BZ were decreased in Sirt7−/− mice. In vitro, TGFβ-induced fibroblast proliferation and differentiation into myofibroblast were attenuated by Sirt7 siRNA treatment. Knockdown of Sirt7 accelerated TGFβ receptor I (TβRI) degradation upon TGF-β1 stimulation. These effects were abolished by Smad7 (inhibitory Smad) double knockdown, suggesting that Sirt7 stabilizes TβRI through Smad7. Consistent with these findings, adenovirus-mediated overexpression of Sirt7 could blocked TβRI degradation.

CONCLUSIONS:

Sirt7 is essential for proper wound healing following MI.

Exp Clin Cardiol. 2012 Summer;17(2):51–52.

Y-6: Inhibitory Effect of ADMA on eNOS Phosphorylation and Endothelial Function in CKD Mice

Hidemi Kajimoto 1, Hisashi Kai 2, Tsutomu Imaizumi 3,4

Abstract

BACKGROUND AND OBJECTIVES:

Patients with chronic kidney disease (CKD) show elevation of circulating asymmetric dimethylarginine (ADMA). Recently, it was suggested that ADMA impaired endothelial nitric oxide synthase (eNOS) via additional effects other than the competitive inhibition of L-arginine. The aims of this study were to examine whether increased circulating ADMA causes endothelial dysfunction in CKD mice and if so, to investigate the molecular mechanism.

METHODS AND RESULTS:

In wild-type mice with 5/6 nephrectomy, blood urea nitrogen, serum creatinine and ADMA were increased by 2.5-, 2-, and 1.2-fold, respectively, without any change in blood pressure. Nephrectomy deteriorated endothelium-dependent relaxation and reduced eNOS phosphorylation of isolated aortic rings. In transgenic (TG) mice overexpressing dimethylarginine dimethylaminohydrolase-1, the enzyme that metabolizes ADMA, circulating ADMA was decreased to half that of wild-type mice and was not increased by nephrectomy. The nephrectomy-induced deterioration of endothelium-dependent relaxation and eNOS phosphorylation was abolished in TG mice. In cultured human endothelial cells, the agonist-induced phosphorylations of eNOS and the upstream kinases, such as calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-related kinase (ERK) 1/2 phosphorylation, but not Akt, were decreased by ADMA at concentrations smaller than that of L-arginine in the culture media.

CONCLUSION:

Elevated circulating ADMA is the cause, but not an epiphenomenon, of endothelial dysfunction in CKD. This effect may be attributable to inhibition of the CaMKII- and ERK-mediated eNOS phosphorylation, a novel mechanism by which ADMA impairs eNOS function.

Exp Clin Cardiol. 2012 Summer;17(2):51–52.

Y-7: Optimizing Cardiac Progenitor Cell-Sheet Therapy by Adding Endothelial Progenitor Cell Transplantation for Ischemic Cardiomyopathy

Kamata Sokichi 1, MIyagawa Shigeru 1, Sawa Yoshiki 1

Abstract

BACKGROUND:

Transplantation of cardiac stem cell-sheet (CSC-sheet) is a promising therapeutic strategy for ischemic cardiomyopathy (ICM), while potential ischemia in the transplanted area remains to be overcome. Endothelial progenitor cells (EPCs) reportedly induce angiogenesis in the injected territory. We hypothesized concomitant EPC injection might enhance the therapeutic effects of CSC-sheet in ICM.

METHODS:

A scaffold-free CSC-sheet was generated from human c-kit-positive heart-derived cells. An ICM model was generated by placing the ameroid constrictor around the left coronary artery for four weeks in swine. They then underwent a sham operation, epicardial transplantation of CSC-sheet over the infarct area, intramyocardial injection of EPCs into the infarct and peri-infarct area or CSC-sheet transplantation plus EPC injection, followed by the efficacy assessment for two months.

RESULTS:

Speckle-tracking echocardiography dissected layer-specific regional systolic function by the radial strain (RS). The epicardial RS in the infarct area was similarly greater after the CSC-sheet sole (19±5%) or combination (20±5%) therapy than the EPC sole (9±4%) or sham (7±1%) treatment. The endocardial RS in the infarct area was the greatest after the combination (15±1%) followed by the EPC sole (12±1%), compared to the CSC sole (11±1%) or sham (9±1%) treatment. Consistently, CSC-sheet implantation yielded increased capillary number, reduced fibrosis and minimal cardiomyogenic differentiation via a cell fusion-dependent mechanism in the infarct epicardium, while concomitant EPC injection enhanced therapeutic efficacy of CSC-sheet implantation in the infarct endocardium.

CONCLUSION:

Transplantation of CSC-sheet significantly induced functional recovery of the infarct epicardium, whereas concomitant EPC transplantation achieved transmural improvement in chronic ICM.

Exp Clin Cardiol. 2012 Summer;17(2):52.

Y-8: Involvement of ER-Stress-Mediated Hyperubiquitination of SERCA2a in Alterations in Ventricular Mechanics by Type 2 Diabetes

Hidemichi Kouzu 1, Takayuki Miki 1, Tetsuji Miura 1

Abstract

BACKGROUND:

Although diabetes is well known to induce heart failure, the time course of alterations in ventricular mechanics by diabetes and their molecular mechanisms remain unclear.

OBJECTIVE:

We aimed to characterize changes in ventricular mechanics by type 2 diabetes (T2DM) and the involvement of endoplasmic reticulum (ER) stress.

METHODS AND RESULTS:

Pressure-volume relationships (PVRs) were assessed in OLETF (a rat model of T2DM with modest hypertension), LETO (a non-diabetic control) and LETO with renovascular hypertension (LETOHT) at 30–36 weeks of age. Changes in the slope/position of PVRs during pressure or volume load were assessed by systolic and diastolic volume at pre-defined pressures (ESV125 and EDV20, respectively). Under baseline conditions, OLETF showed lower heart rate, larger left ventricular end-diastolic volume, and larger tau compared with those in LETO and LETOHT, though left ventricular end-diastolic pressure (LVEDP) was similar. Despite the comparable stroke work, pressure-volume area (PVA), an index of myocardial oxygen consumption, was significantly larger in OLETF than in LETO (16.5±1.5 vs 10.5±0.9 mmHg/ml). During afterload challenge by phenylephrine infusion, LEVDP was higher in OLETF than in LETO and LETOHT (14.1±1.8 vs 5.9±2.1 and 6.0±2.1 mmHg, respectively; P<0.01). EDV20 was reduced during phenylephrine infusion in OLETF (from 0.24±0.02 to 0.19±0.02 ml, P<0.01), though such a change was not observed in LETO and LETOHT. Systolic reserve assessed by changes in ESV125 and stroke work was similarly preserved in the three groups. In contrast to afterload challenge, acute volume challenge with saline induced right and downward shift of end-diastolic PVRs in all study groups. Histology-determined interstitial collagen and mRNA levels of types 1 and 3 collagen were similar in OLETF and LETO. There was no difference in mRNA levels of titin, myosin-binding protein C, SERCA2a, phospholamban, ryanodine receptor-2 and Na+-Ca2+ exchanger between OLETF and LETO. Protein level and phosphorylation of phospholamban were similar in OLETF and LETO hearts. However, SERCA2a protein was reduced by 35% and the ubiqutination level of SERCA2a was 85% higher in OLETF than in LETO. An ER stress modulator, 4-phenylbutyric acid, attenuated SERCA2a ubiqutination, restored SERCA2a protein level and reduced the slope of end-diastolic PVR in OLETF, though the increased PVA was unchanged.

CONCLUSIONS:

T2DM induces reduction in ventricular diastolic distensibility, which is unmasked by increased afterload, before development of extracellular remodeling in the heart. Increased degradation of SERCA2a protein by ER-stress-medicated hyperubiqutination underlies the diastolic ventricular dysfunction, though systolic ventricular reserve is preserved at the expense of increased cardiac energy cost.

Exp Clin Cardiol. 2012 Summer;17(2):52.

Y-9: Microtubules are Related to the Regulation of Mitochondrial Permeability Transition Pore in Rat Cardiac Myocytes

Azumi Kumazawa 1, Hideki Katoh 1, Hideharu Hayashi 1

Abstract

BACKGROUND:

Microtubules are the major components of cytoskeletons, and microtubule disorganization is associated with cardiac diseases such as cardiac hypertrophy and reperfusion injury. Microtubules are bound to mitochondria and are related to mitochondrial function such as mitochondrial permeability transition pore (mPTP). We investigated the relationship between microtubule organization and mPTP in cardiac myocytes.

METHODS:

Myocytes isolated from rats were permeabilized with saponin. Microtubule stabilization was achieved with paclitaxel, and microtubule disruption was conducted with nocodazole. We investigated the effects of each drug on mitochondrial membrane potential (ΔΨm; measured with TMRE), and the opening of mPTP (assessed by calcein leakage from mitochondria) with a confocal microscopy.

RESULTS:

(1) The immunolabeling of α-tubulin showed that exposure to paclitaxel (10 μM) or nocodazole (10 μM) for 60 min stabilized or disrupted microtubule network, respectively. (2) Both paclitaxel (10 μM) and nocodazole (10 μM) depolarized ΔΨm (by 57.5±5.52% and 63.1±4.68% of the baseline, respectively, P<0.05) (3) Both paclitaxel and nocodazole induced calcein leakage from mitochondria (by 75.0±1.36% and 75.1±2.78% of the baseline, respectively, P<0.05). (4) Cyclosporin A (an inhibitor of mPTP; 0.4 μM) blocked both ΔΨm depolarization and calcein leakage, induced by paclitaxel or nocodazole. (5) BAPTA (5 mM) and thapsigargin (an inhibitor of SERCA: 10 μM) reduced the effects of paclitaxel on mPTP (86.1±1.33% and 84.2±2.11% of the baseline, respectively, P<0.01 vs paclitaxel), but did not inhibit the effects of nocodazole on mPTP. (6) Ru360 (an inhibitor of mitochondrial uniporter: 10 μM) did not affect the effects of both paclitaxel and nocodazole on mPTP.

CONCLUSION:

Our results showed that both microtubule stabilization with paclitaxel and disruption with nocodazole opened mPTP and depolarized ΔΨm, which indicated that microtubule disorganization plays an important role in the regulation of mitochondrial function in cardiac myocytes. Local Ca2+ releases from the SR regulated paclitaxel-induced mPTP opening from outside of mitochondria, and it was indicated that Ca2+-mediated signaling might be activated at the site of outer mitochondrial membrane. These findings could be related to the mechanism for microtubule-medicated cardiac dysfunction.

Exp Clin Cardiol. 2012 Summer;17(2):52.

Y-10: Biphasic Action of Aldosterone on Akt Signaling in Cardiomyocytes

Takuya Yoshino 1, Tomohisa Nagoshi 1, Michihiro Yoshimura 1

Abstract

Aldosterone and Akt signaling both play pivotal roles in the pathogenesis of heart failure. However, little is known about the correlation between them. We herein investigated whether aldosterone interacts with Akt signaling in a coordinated manner in cardiomyocytes. Neonatal rat cardiomyocytes were stimulated with aldosterone for either a short (10-minute) or long (24-hour) time. The phosphorylation of Akt and its downstream effector, GSK3β, were transiently increased after short-term stimulation, which was blocked by either PI3K or Na+/H+ exchanger inhibitors, but not by the mineralocorticoid receptor (MR) antagonist, eplerenone. Long-term stimulation also significantly increased Akt-GSK3β phosphorylation and this effect was reduced by eplerenone. Thus, these results suggest that aldosterone activates Akt signaling via a biphasic reaction that occurs through different cascades. To understand the significance of the rapid action of aldosterone, cardiomyocytes were exposed to hydrogen peroxide for 10 to 30 minutes. Short-term aldosterone stimulation significantly rescued cardiomyocytes oxidative stress-induced cellular damage. Of note, eplerenone did not abrogate this beneficial effect, while a PI3K inhibitor did. Therefore, during the early phase, aldosterone has favorable effects on cardiomyocytes, partly by acute activation of a MR-independent cascade through the Na+/H+ exchanger, PI3K and Akt. In contrast, its persistent activity produces pathological effects partly by chronic Akt activation in a MR-dependent manner. These results provide new insight about the physiological compensatory role of aldosterone, while still emphasize the usefulness of MR inhibitors for suppressing the unfavorable MR-dependent actions, without blocking out the favorable MR-independent actions.

Exp Clin Cardiol. 2012 Summer;17(2):53.

Y-11: Mitochondrial DNA that Escapes from Autophagy Causes Inflammation and Heart Failure

Oka Takafumi 1, Komuro Issei 1, Otsu Kinya 2

Abstract

BACKGROUND AND OBJECTIVE:

Inflammation has been implicated in the pathogenesis of heart failure. However, the mechanisms underlying the inflammatory responses observed in failing hearts remain to be elucidated. Mitochondrial DNA (mtDNA) contains inflammatogenic unmethylated CpG motif similar to bacterial DNA, which is recognized by endolysosomal TLR9. Damaged mitochondria are degraded by the autophagy/lysosome system. Lysosomes contain degradation enzymes such as deoxyribonuclease II (DNase II), an important enzyme for degradation of engulfed apoptotic nuclei in macrophages. In this study, we hypothesized that accumulation of mtDNA causes inflammatory responses mediated through the TLR9 signaling pathway in the heart and tested this hypothesis.

METHODS:

We generated cardiac-specific DNase II knockout mice (CKO) and DNase II and Toll-like receptor 9 (TLR9) double knockout mice. They were subjected to transverse aortic constriction (TAC) and echocardiographical and histological analyses. Inflammatory responses were evaluated by identifying the infiltrating inflammatory cells and by quantification of proinflammatory cytokine mRNAs using immunohistochemical (IHC) analysis and quantitative RT-PCR, respectively. We performed IHC analysis on mouse heart sections with Picogreen, 5-ethynyl 2’-deoxy uridine (EdU), or anti-lamp2a or anti-LC3 antibody for detection of DNA accumulation in autolysosomes as well as immune-electron microscopy using anti-DNA antibody.

RESULTS:

TAC-operated CKO mice showed increased mortality and heart failure 10 days after TAC. Two days after TAC, CKO mice showed infiltration of inflammatory cells and induction of proinflammatory cytokine mRNAs. We detected the accumulation of Picogreen- or EdU-positive deposits which were positive for lamp2a and LC3, indicating mtDNA deposition in autolysosomes. DNA accumulation in autolysosomes was confirmed by immune-electron microscopy using anti-DNA antibody. Ablation of TLR9 attenuated the inflammatory responses and the development of heart failure in CKO hearts. Furthermore, we observed inflammatory cell infiltration and LAMP2a- or LC3-positive DNA accumulation in wild-type failing hearts and TLR9 KO mice showed better heart function 10 weeks after TAC, suggesting mtDNA/TLR9 is involved in the development of inflammation and heart failure even in wild-type mice.

CONCLUSION:

The mtDNA-TLR9 axis plays a pivotal role in initiating and integrating inflammatory responses in failing hearts in response to pressure overload.

Exp Clin Cardiol. 2012 Summer;17(2):53.

Y-12: Distinct Metabolic Hallmarks Enable Purification of Pluripotent Stem Cell-Derived Cardiomyocytes

Shugo Tohyama 1, Fumiyuki Hattori 1, Keiichi Fukuda 1

Abstract

BACKGROUND:

Mass production of highly purified cardiomyocytes is a critical bottleneck in realizing heart regenerative therapy. Our recently established non-genetic purification method of cardiomyocytes using mitochondrial dye is efficient but not suitable to produce clinical-scale cardiomyocytes due to the usage of FACS. This study was designed to establish a large-scale purification method for pluripotent stem cell (PSC)-derived cardiomyocytes.

METHODS AND RESULTS:

We first approached this issue by focusing on the possible metabolic differences between cardiomyocytes and non-cardiomyocytes including embryonic stem cells (ESCs) by transcriptome and metabolome analyses. Transcriptome and fluxome analysis using [13C]-labeled glucose revealed that proliferating non-cardiomyocytes used glucose mainly for amino acid and nucleotide synthesis, while cardiomyocytes used glucose primarily for ATP production via TCA cycle. In addition, larger amounts of lactate were discarded from proliferating non-cardiomyocytes. On the contrary, it was incorporated into cardiomyocytes. Taken together, we developed a novel method for purifying the bulk of ESC-derived cardiomyocytes using original culture media (glucose-free and lactate-rich environment). This culture media enabled us to obtain >98% pure cardiomyocytes which did not form teratoma when transplanted into immunodeficient mice testes. Electrophysiological analysis of the purified human ESC-derived cardiomyocytes using microelectrode recordings and a multi-electrode array system showed normal properties. We investigated the molecular mechanism to determine why cardiomyocytes can survive for longer periods in glucose-free and lactate-rich environments by fluxome analysis using [13C]-labeled lactate. Surprisingly, they can produce some biomass including nucleotides and amino acids from lactate via gluconeogenic pathway.

CONCLUSIONS:

In this study, we report a novel method to purify a bulk of cardiomyocytes from PSC-derivatives based on the findings from transcriptome and metabolome analyses. We believe that our inexpensive and unique technology will resolve the bottleneck and directly facilitate human heart-regenerative therapies.

Exp Clin Cardiol. 2012 Summer;17(2):53.

Y-13: Nicorandil Enhances Na+/Ca2+ Exchange Current Via Guanylate Cyclase in Isolated Guinea Pig Cardiac Ventricular Myocytes

Jia-zhang Wei 1, Kazuhiko Takeuchi 1, Yasuhide Watanabe 2

Abstract

Nicorandil is a KATP channel opener, a hybrid of a nitrate generator, and an antioxidant, and is used as a drug for the treatment of angina pectoris. The drug induces cardioprotection and vasodilation after ischemia/reperfusion. Lathrop et al., (1990) reported that nicorandil may exert antiarrhythmic actions by abolishing both spontaneous and triggered activity [early after-depolarization (EADs), delayed after-depolarization (DADs)]. In this study, we examined the effect of nicorandil on Na+/Ca2+ exchange current (INCX) in isolated guinea pig ventricular myocytes using the whole-cell patch clamp technique. Nicorandil enhanced INCX in a concentration-dependent manner. The EC50 values of nicorandil were 8.3 μM and 6.6 μM for the outward and inward components of INCX, respectively. 8-Br-cGMP, a membrane permeable analog of cGMP also enhanced INCX in a concentration-dependent manner. ODQ, a guanylate cyclase inhibitor (10 μM) completely abolished the effect of nicorandil on INCX. DADs induced by electrical stimulation with ouabain disappeared in the presence of 100 μM nicorandil. We concluded that nicorandil enhances the function of the Na+/Ca2+ exchanger (NCX) via guanylate cyclase, and this may partially contribute to the cardioprotection of nicorandil by accelerating Ca2+ exit via NCX.

Exp Clin Cardiol. 2012 Summer;17(2):53.

Y-14: A Direct Renin Inhibitor Prevents Ventricular Arrhythmogenic Remodeling and Sudden Cardiac Death in Mice with Dilated Cardiomyopathy

Chinatsu Yamada 1, Koichiro Kuwahara 1, Kazuwa Nakao 1

Abstract

PURPOSE:

Progression of left ventricular (LV) remodeling including fibrosis contributes to the occurrence of lethal ventricular arrhythmias and sudden cardiac death. Further detailed understanding of the molecular mechanism underlying a substrate for arrhythmogenicity in chronic heart failure (CHF) has been desired. To evaluate the contribution of the renin-angiotensin system (RAS) to the generation of this substrate, we administered the direct renin inhibitor aliskiren to a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor transgenic mice (dnNRSF-Tg), which exhibit progressive dilated cardiomyopathy with lethal arrhythmias beginning at 12 weeks of age and 60% of which die suddenly by 30 weeks.

METHODS:

Aliskiren 22 mg/kg/day was administered via osmotic mini-pumps to two group of dnNRSF-Tg: early stage of CHF (from 12-to-18-weeks of age) and late stage of CHF (from 16-to-28 weeks of age). We measured heart rate and blood pressure and performed echocardiographic analyses in both stages. In late stage, we analyzed survival rate, fibrosis in histological sections, ventricular expression of remodeling-related genes, hemodynamic LV parameters by using a catheter-tip micromanometer and arrhythmogenicity by performing in vivo intracardiac electrophysiological study.

RESULTS:

Aliskiren reduced blood pressure in wild-type mice (108±3 mmHg to 94±4 mmHg; P<0.001), but not significantly in dnNRSF-Tg (95±4 mmHg to 89±3 mmHg). Nor did it affect heart rate. In both stages aliskiren improved LV ejection fraction in dnNRSF-Tg (41±2% to 52±4% in early stage, P<0.03; 35±4% to 56±4% in late stage, P<0.005). In late stage aliskiren improved survival rate (vs vehicle; P<0.05), decreased histological LV fibrosis-to-total area ratios (vs vehicle; P<0.05) and attenuated the increase in ventricular mRNA expression of atrial natriuretic peptide, β-myosin heavy chain, transforming growth factor (TGF)-β1, TGF-β3, tissue inhibitor of metalloproteinase-1 and angiotensin converting enzyme (vs vehicle; all P<0.05) in dnNRSF-Tg. Aliskiren also significantly increased the maximal rate of LV pressure change (dP/dt) (vs vehicle; P<0.05) and tended to normalize LV end diastolic pressure. Furthermore, aliskiren strongly prevented ventricular tachycardia in dnNRSF-Tg induced during electrophysiological study compared with vehicle (induction rate of ventricular tachycardia: aliskiren [n=5]; 0% vs. vehicle [n=6]; 83%,P<0.03).

CONCLUSIONS:

Inhibition of RAS by aliskiren significantly improved fibrosis, ventricular remodeling and increased susceptibility to ventricular arrhythmias, thereby reduced the mortality rate without changing blood pressure in dnNRSF-Tg. These results demonstrate the potential contribution of RAS to the generation of arrhythmogenic substrate during the progression of CHF.

Exp Clin Cardiol. 2012 Summer;17(2):54.

O-1: Role of Leptin Signaling in Pathogenesis of Atrial Fibrosis and Fibrillation

Akira Fukui 1, Naohiko Takahashi 1, Takayuki Masaki 2, Chisato Nakada 3, Osamu Kume 2, Naoya Fukunaga 2, Tetsuji Shinohara 2, Yasushi Teshima 1, Kunio Yufu 1, Mikiko Nakagawa 1, Masahide Hara 2, Hironobu Yoshimatsu 2, Tetsunori Saikawa 1

Abstract

BACKGROUND AND OBJECTIVES:

Leptin is known to exert cardiovascular activity, which has not been yet fully understood. We tested the hypothesis as to whether leptin signaling would contribute to atrial fibrosis and atrial fibrillation (AF) evoked by angiotensin II (ATII).

METHODS:

1) Eight week old male C57BL/6 mice (CNT) and age matched ob/ob mice (ob) were subcutaneously infused with ATII (2.0 mg/kg/day) or vehicle (VEH) via an osmotic minipump for two weeks. Transesophageal pacing was preformed to induce AF, and left atrial (LA) appendages were isolated. 2) LA fibroblast isolated from adult Sprague-Dawley (SD) rat and Zucker rat were cultured, followed by the addition of ATII (100 nM) or leptin (100 ng/ml) for 24 hours.

RESULTS:

1) In CNT-ATII, ATII infusion up-regulated leptin expression in the LA. Transesophageal pacing induced AF 87% in CNT-ATII, but it did not induce any AF in ob-ATII. Masson trichrome staining revealed inhomogeneous interstitial fibrosis in CNT-ATII, while such fibrosis was not observed in ob/ob-ATII. The mRNA relating to pro-fibrotic signals (collagen1, collagen3, TGFs1, α-SMA, MCP-1, and RANTES) were up-regulated in CNT-ATII, which was significantly lower in ob-ATII. 2) In cultured SD rat fibroblast, ATII increased leptin expression and leptin increased pro-fibrotic signals including, TGFs1, α-SMA, MCP-1, and RANTES. By contrast, in Zucker fibroblast, leptin did not influence their expressions. Our results demonstrated, for the first time, that leptin signaling plays an important role in the pathogenesis of atrial fibrosis and AF evoked by ATII, and would be a novel therapeutic target.

Exp Clin Cardiol. 2012 Summer;17(2):54.

O-2: Retinal Angiomatous Proliferation, a Severe Form of Exudative Age-Related Macular Degeneration Associated with Risk Alleles of ARMS2/HTRA1 Gene Polymorphisms in Japanese Patients

Takaaki Hayashi 1

Abstract

PURPOSE:

To investigate the association between ARMS2/HTRA1, CFH, and C3 gene polymorphisms and retinal angiomatous proliferation (RAP), an infrequent and severe form of exudative age-related macular degeneration, which is characterized by intraretinal neovascularization.

METHODS:

RAP diagnosis was based on fundus photographs, images of fluorescein and indocyanine green angiographies and optical coherence tomography findings. Six single nucleotide polymorphisms (SNPs), A69S (rs10490924) in ARMS2, rs11200638 in HTRA1, I62V (rs800292) in CFH, Y402H (rs1061170) in CFH, R80G (rs2230199) in C3 and rs2241394 in C3, were genotyped in eight Japanese patients with RAP.

RESULTS:

The two SNPs in the ARMS2/HTRA1 were in complete linkage disequilibrium. The frequency of the risk T allele in ARMS2 (the risk A allele in HTRA1) was 93.8% in the RAP patients. The frequency of homozygosity for the risk genotype TT of ARMS2 (the risk genotype AA of HTRA1) was 87.5%. The frequency of the non-risk allele (A) of I62V was 100%. The frequencies of risk alleles of Y402H, R80G and rs2241394 were 12.5%, 0% and 18.8%, respectively.

CONCLUSIONS:

Our results suggest that the risk alleles of the ARMS2/HTRA1 SNPs are strongly associated with RAP in Japanese patients and have an important role in the pathogenesis of intraretinal angiogenesis.

Exp Clin Cardiol. 2012 Summer;17(2):54.

O-3: Analysis of Technetium-99m Sestamibi Retention in a Rat Model of Heart Failure and Rats Administered a Mitochondrial Uncoupler

Akira Kawamoto 1, Takao Kato 1,2, Tetsuo Shioi 1, Tsuneaki Kawashima 1, Yodo Tamaki 1, Shinichiro Niizuma 1, Junji Okuda 1, Yohei Tanada 1, Michiko Narazaki 3, Tetsuya Matsuda 3, Takeshi Kimura 1

Abstract

BACKGROUND AND OBJECTIVES:

Measurements of technetium-99m sestamibi (99mTc-MIBI) with single-photon emission computed tomography (SPECT) are used for the diagnosis of ischemic heart disease. In addition, the washout of 99mTc-MIBI is reported to be correlated with the severity of heart failure (HF). Since it is difficult to obtain an absolute value of 99mTc-MIBI retention using SPECT, we measured 99mTc-MIBI retention in extracted myocardial tissue in rats with HF or rats administered a mitochondrial uncoupler.

METHODS:

To examine 99mTc-MIBI retention in a failing heart, we administered 99mTc-MIBI to Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet, which develop hypertension and HF. Next, we administered 99mTc-MIBI to rats injected carbonyl cyanide m-chlorophenylhydrazone (CCCP).

RESULTS:

99mTc-MIBI washout was increased in HF rats compared with control rats. 99mTc-MIBI washout was positively correlated with heart weight and ANF gene expression in the heart. 99mTc-MIBI retention positively correlated with 125I-15-(p-iodophenyl)-9-R,S-methylpentadecanoic acid (125I-9MPA), a fatty acid analog, retention. CCCP increased 99mTc-MIBI washout associated with decreased cardiac function and cardiac energy reserve.

CONCLUSIONS:

99mTc-MIBI washout was increased in HF rats and rats administered a mitochondrial uncoupler. Cardiac hypertrophy is likely to be associated with abnormal mitochondrial membrane potential in an animal model of HF.

Exp Clin Cardiol. 2012 Summer;17(2):55.

O-4: The Development of a High-Throughput and Objective Screening System to Validate Candidate Molecules which Mediate Abnormal Vascular Smooth Muscle Contraction

Hiroko Kishi 1, Daisuke Tokumori 1, Katsuko Kajiya 1, Yuichi Takada 1, Ying Zhang 1, Hozumi Kawamichi 1, Kenji Miyanari 1, Tomohiko Kimura 1, Sei Kobayashi 1

Abstract

Rho-kinase (ROK)-mediated Ca2+-sensitization of vascular smooth muscle (VSM) plays a critical role for abnormal VSM contractions such as vasos-pasm. Previously we identified sphingosylphosphorylcholine (SPC)/Fyn/ROK pathway as a novel signaling pathway for abnormal VSM contraction. Furthermore, focused proteomic approaches in human VSM enabled us to identify 21 possible downstream targets of Fyn, including six cytoskeletal proteins. When we tried to examine objectively if those candidate proteins are truly involved in abnormal VSM contraction, analysis of the direct effects of those recombinant candidate proteins on the Ca2+-sensitization in β-escin-permeabilized VSM strips will provide very strong and direct evidence. However, it is a very difficult technique with low throughput. On the other hand, classical microscopic observations of the cell contraction and cytoskeletal rearrangement in VSM cells will have better throughput. However, it would be very difficult to avoid experimenter bias and time-consuming laborious observations of large numbers of cells. Thus in the present study, we aimed to develop a high-throughput screening system using an automated cell imaging device ArrayScanV. Human coronary artery smooth muscle cells (CASMCs) were cultured in a 96-well plate, and stimulated with and without SPC. The images of a cell body, F-actin, and intermediate filaments were automatically acquired. We were able to image 200–450 cells/well in one analysis and, using an original algorithm, to evaluate the SPC-induced morphological change of cell body (cell contraction) and cytoskeletal rearrangement objectively and quantitatively. Furthermore, by the combination of this system and small interfering RNA (siRNA), we successfully obtained evidence that one of the novel candidate molecules may be involved in abnormal VSM contraction.

Exp Clin Cardiol. 2012 Summer;17(2):55.

O-5: Prevention of Doxorubicin-Induced Cardiomyopathy in Cardiac-Specific SOCS3 Deficient Mice by Augmenting STAT3 Signaling Pathway

Sachiko Kyogoku 1, Hideo Yasukawa 1, Takanobu Nagata 1, Toyoharu Oba 1, Hideki Ohshima 1, Tomoko Minami 1, Daisuke Fukui 1, Yusuke Sugi 1, Hiroki Aoki 2, Tsutomu Imaizumi 1

Abstract

BACKGROUND:

Although it has been shown that JAK-STAT activating cytokines prevent the development of doxorubicin (DOX)-induced cardiomyopathy, little is known about the negative regulation of the JAK-STAT pathway in this process. We previously showed that a suppressor of cytokine signaling 3 (SOCS3) is an intrinsic negative regulator of JAK-STAT signaling. We investigated the hypothesis that myocardial SOCS3 would play a role in the development of DOX-induced cardiomyopathy.

METHODS AND RESULTS:

DOX-induced cardiomyopathy was induced in wild-type (WT) mice after a single administration of DOX (15 mg/kg, IP). Western blot analysis revealed that STAT3 was transiently phosphorylated, and SOCS3 was induced in WT mouse hearts after DOX administration. Real-time PCR revealed that JAK-STAT-activating cytokines were increased in mouse hearts two days after DOX administration. The cytokine included G-CSF (8.5-fold), interleukin-11 (3.0-fold), and leukemia inhibitory factor (2.1-fold). The activation of the STAT3-SOCS3 pathway was closely correlated with elevations in serum CK-MB levels after DOX administration. This suggested an important link between the activation of the STAT3-SOCS3 pathway and the development of DOX-induced cardiomyopathy. To clarify the role of myocardial SOCS3, we created a cardiac-specific SOCS3 knockout mouse (SOCS3-CKO). We found that, at 14 days after DOX administration, SOCS3-CKO mice showed improved fractional shortening (FS) and less fibrosis compared to controls. (FS: 33.9% vs 29.4%, P<0.01; cardiac fibrosis area: 2.8% vs 8.6%, respectively; P<0.01). Serum levels of CK-MB were reduced in SOCS3-CKO mice compared to controls (P<0.01). Moreover, the duration and intensity of STAT3 phosphorylation was greater in SOCS3-CKO than controls.

CONCLUSION:

Our results suggested that DOX-induced cardiomyopathy is prevented in SOCS3-CKO mice by augmenting STAT3 mediated cytokine signaling pathway.

Exp Clin Cardiol. 2012 Summer;17(2):55.

O-6: FoxO1 is Involved in Telomere Biology of the Heart Induced by Calorie Restriction

Naoki Makino 1, Tatsuo Furuyama 2, Isao Shimokawa 3, Jun-ichi Oyama 1

Abstract

Calorie restriction (CR) retards the aging processes and extends the lifespan in a wide range of species. Effects of CR on the heart have been suggested, including the improvement of cardiac diastolic function, cardio-protection against ischemia/reperfusion injury and the prevention of cardiac hypertrophy. This study examined whether the forkhead transcription factors of the O group (FoxO1) may be involved in the cardiac remodeling of CR. We used FoxO1 knockout heterozygotic (HT) mice, in which the FoxO1 mRNA level was reduced by 50%, or less, of that in wild-type (WT) mouse tissues. The WT and HT mice were fed ad libitum (AL) or 30% CR diets for 15 weeks. Since telomeres play a role in cellular aging, and telomere attrition has attracted attention in terms of its association to the etiology of cardiovascular disease, this study was conducted to examine telomere length and telomerase activity of the hearts from these mice after CR. The body weight was more increased in WT-AL than WT-CR, but was not between HT-AL and HT-CR. Aging- and CR-related changes in food intake, blood glucose and insulin concentrations were similar among all groups of mice. Telomere attrition in heart tissues was not observed in WT-AL and WT-CR, however, this was reduced in hearts of HT-AL and was in HT-CR. Telomerase activity was not altered in HT-AL and HT-CR, but, was more elevated in WT-CR than WT-AL. Furthermore, to examine cardiac autophagy flux in CR, light chain 3II (LC3II) protein expression and immunostaining were analyzed in those groups of mice. Both expressions were enhanced in WT-CR, but not in HT-CR. The present results suggest that activation of FoxO1 is an important mediator of CR through the induction of genes responsible for a cardioprotective mechanism and is also considered to be important for the process of aging and remodeling in the heart.

Exp Clin Cardiol. 2012 Summer;17(2):55–56.

O-7: Vildagliptin, A Dipeptidyl Peptidase-4 Inhibitor, Prevents the Development of Cardiac Hypertrophy Induced by Chronic Beta-Adrenergic Stimulation in Rats

Daiji Miura 1, Kazufumi Nakamura 1, Toru Miyoshi 1, Masashi Yoshida 1, Hiroki Oe 1, Satoshi Akagi 1, Hiroki Sugiyama 1, Kaoru Akazawa 1, Hiroshi Ito 1

Abstract

BACKGROUND:

Congestive heart failure with left ventricular (LV) diastolic dysfunction and preserved ejection fraction is often observed in hypertensive patients; however, the effective treatment of diastolic heart failure has not been established. Recent studies showed that dipeptidyl peptidase-4 (DPP4) inhibitors, which increase circulating GLP-1 level, have cardio-protective effects. This study elucidated whether a DPP4 inhibitor, vildagliptin, prevents the development of LV hypertrophy in isoproterenol (ISO)-induced hypertrophied rat hearts.

METHODS:

Male Wistar rats (nine-weeks old) received a vehicle (control, n=5), ISO subcutaneously (2.4 mg/kg/day, n=20) or ISO subcutaneously + vildagliptin with oral administration (30mg/kg/day, n=20) for seven days. Cardiac catheterization and echocardiographic study were performed one week after ISO-infusion to evaluate LV diastolic functions. The expression of molecules associated with cardiac hypertrophy and glycemic metabolism were examined by real-time quantitative PCR.

RESULTS:

LV hypertrophy in ISO + vildagliptin was significantly decreased compared with those in ISO after seven days (Heart weight/body weight, Vehicle: 3.19±0.42, ISO: 4.43±0.39, ISO + vildagliptin: 4.14±0.29, P<0.05). Cardiac catheterization showed that increased LV end-diastolic pressure in ISO was ameliorated in ISO + vildagliptin (Vehicle: 2.7±1.1, ISO: 3.5±1.1 or ISO + vildagliptin: 2.1±0.5 mmHg, P<0.05), although heart rate and LV systolic pressure did not differ among three groups. The echocardiographic study showed that the shortened deceleration time as a LV diastolic function in ISO was significantly improved compared with those in ISO + vildagliptin (Vehicle: 43.9±4.3, ISO: 40.2±2.9 or ISO + vildagliptin: 43.0±2.7 ms, P<0.05); however, increased LV wall thickness in ISO did not reduced in ISO + vildagliptin. Real-time quantitative PCR revealed that decreases in glucose transporter type 4 and te alpha heavy chain subunit of cardiac myosin in ISO was improved in ISO + vildagliptin.

CONCLUSION:

This study demonstrated that administration of vildagliptin prevented the development of LV hypertrophy induced by ISO in rat.

Exp Clin Cardiol. 2012 Summer;17(2):56.

O-8: Intermittent Hypoxia-Induced Cardiovascular Remodeling in Diabetes Mellitus.

Atsuo Nomura 1, Satoshi Nishioka 2, Saki Yamaguchi 2, Akiko Tagawa 1, Yuichi Furukawa 1, Ryuji Kato 1, Yoshio Ijiri 1, Yasuo Matsumura 2, Yoshikatsu Okada 3, Michio Asahi 4, Kazuhiko Tanaka 1, Tetsuya Hayashi 1

Abstract

BACKGROUND AND OBJECTIVES:

Diabetes mellitus (DM) is a leading cause of morbidity and mortality because of its cardiovascular complications, and sleep apnea is common among DM patients. We have reported that intermittent hypoxia relevant to sleep apnea increases oxidative stress and causes dyslipidemia in lean mice. The aim of this study was to evaluate the effect of repetitive hypoxic stress on cardiovascular complications in DM.

METHODS:

Male BKS.Cg-Dock7m+/+ Leprdb/J (db/db) mice (n=15) and C57BL/6J control mice (n=20) at seven to 10 weeks of age were exposed to intermittent hypoxia (5 min of 5% O2 followed by 5 min of 21% O2 for 8 h/day during daytime) or normoxia for 14 days.

RESULTS:

In db/db mice, intermittent hypoxic stress significantly increased cardiomyocyte diameter and interstitial fibrosis in LV myocardium. Furthermore, hypoxic stress significantly increased superoxide production in thoracic artery of db/db mice. Our preliminary findings suggest that intermittent hypoxia accelerates cardiovascular remodeling at least partly due to the increased oxidative stress in DM.

Exp Clin Cardiol. 2012 Summer;17(2):56.

IL-1: Multi-Physics, Multi-Scale Heart Simulator ‘UT-Heart’ for Heart Research

Seiryo Sugiura 1, Xiaoke Cui 1, Takumi Washio 1, Jun-ichi Okada 1, Hiroshi Watanabe 1, Toshiaki Hisada 1, Hiroshi Yamashita 1, Taro Kariya 1, Yasushi Imai 1, Ryozo Nagai 1, Yoshimasa Kadooka 2, Akira Hosoi 2, Masahiro Watanabe 2, Takao Hirahara 2, Takashi Yamazaki 2, Takashi Iwamura 2, Machiko Nakagawa 2, Kohei Hatanaka 2, Kazunori Yoneda 2

Abstract

In the past 30 years, reductionist approaches with the power of molecular biology have dominated the biological and medical sciences to discover and reveal the elements of life. However, in the post-genome era, people are also interested in how these elements, ie, genome or proteins, interact through complex crosstalk to maintain physiological or pathophysiological activities. For such integrative approaches in biological science, mathematical modeling boosted by high performance computing technologies are now recognized as a powerful and indispensable tool. We have developed a multi-physics, multi-scale heart simulator, UT-Heart, in which propagation of excitation, contraction and relaxation, and resultant pressure development and blood flow in the heart are reproduced based on the activities of functional proteins in cardiomyocytes. This simulator is based on the finite element method (FEM) and composed of approximately 20,000,000 elements for electrophysiology analysis and 600,000 elements for mechanical analysis arranged in a 3D morphology of the human heart. Because molecular models of excitation-contraction coupling process are implemented in each element, we can study how functional abnormality of a specific molecule in a specific region of the heart tissue affects the behavior of the heart at the organ level. Several examples of the diseased heart model will be shown. Finally, we will also discuss the possibility of a patient specific model and its application for the clinical practice.

Exp Clin Cardiol. 2012 Summer;17(2):57.

IL-2: Morphological and Molecular Consequences in the Myocardium after Left Ventricular Assist=Device Implantation

Hideo A Baba 1

Abstract

Left ventricular assist devices (LVAD) are currently used to either ‘bridge’ patients with terminal congestive heart failure (CHF) until cardiac transplantation is possible or optionally for patients with contraindications for transplantation (‘destination therapy’). Mechanical support is associated with a marked decrease of cardiac dilation and hypertrophy as well as numerous cellular and molecular changes (‘reverse cardiac remodeling’), which can be accompanied by improved cardiac function (‘bridge to recovery’) in a relatively small subset of patients, with heart transplantation no longer necessary even after removal of the device (‘weaning’). In the recent past, novel pharmacological strategies have been developed and are combined with mechanical support, which has increased the percentage of patients with improved clinical status and cardiac performance. Gene expression profiles have demonstrated that individuals who recover after LVAD show different gene expression compared to individuals who do not respond to unloading. This methodology holds promise for the future to develop read out frames to identify individuals who can recover after support. This paper describes our recent observations of DNA alteration in cardiomyocyte nuclei. We present our methodology, discuss the results and propose further experiments to elucidate the underlying mechanisms.

Exp Clin Cardiol. 2012 Summer;17(2):56.

IL-3: Up To Date and Perspective of Translational Research for Failing Heart

Yoshiki Sawa 1

Abstract

Recently cell therapy was introduced to the clinical setting and proved safe and feasible, but te results were inadequate for complete regeneration of heart failure. We developed cell sheet technology and introduced this to the treatment of severely damaged myocardium.

We implanted myoblast sheets to the impaired heart in small and large animal models. In a series of pre-clinical trials, we proved that myoblast sheets could regenerate the impaired heart mainly by a paracrine effect. Evidenced by these pre-clinical trials, we applied myoblast sheets to a DCM patient receiving LVAD and showed the recovery from LVAD.

To achieve the complete myocardial regeneration by a second generation of cell sheet, the delivery of many cells to the impaired myocardium and the development of autologous beating cells are most crucial. The development of cardiomyocyte sheets derived from iPS cells was successful and demonstrated functional recovery in rat MI model. To improve the number of delivered cells, we implanted co-cultured cell sheets combined myoblasts with adipose tissue derived mesenchymal stem cells which activate the paracrine effect of myoblast sheets, leading to the enhancement of myocardial regeneration in a rat MI model. We succeeded in making thick cardiac tissue with a rich vascular network by cell sheet polysurgery technique in porcine model.

More recently, we prepared sheet-shaped cardiomyocyte grafts from mouse iPS cells and measured cardiac performance by cardiomyocyte sheet implantation in a mouse myocardial infarction model. Cardiomyocyte sheets from mouse iPS cells on the heart of mouse myocardial infarction model resulted in improving their heart functions. Cardiomyocyte sheets derived from iPS cells are a viable option as an autologous cell source for cardiac repair and a powerful tool for cardiovascular research.

Newly developed cell sheet technology may be a promising armamentarium for complete regeneration of severely damaged myocardium.

Exp Clin Cardiol. 2012 Summer;17(2):57.

IL-4: Cardiomyopathic Hamsters: Delta-Sarcoglycan and Beyond

Aiji Sakamoto 1

Abstract

BACKGROUND AND OBJECTIVES:

Cardiomyopathic hamsters have distinct sublines: BIO14.6 exhibits hypertrophic cardiomyopathy (CM) whereas TO-2 manifests severe dilated CM. An additional mutation aggravating CM should exist in TO-2, since both hamsters share genetic loss of delta-sarcoglycan.

METHODS:

We analyzed myofibrillar proteins from TO-2, BIO14.6, and normal hamsters and isolated cDNAs for desmin, an intermediate filament specific to Z-disc. We analyzed the potential phosphorylated state of TO-2 desmin in vitro and in vivo. Filament formation of desmin was explored by expressing its cDNA in SW13 cells.

RESULTS:

In TO-2, desmin was progressively lost. While all the hamster desmin cDNAs harbored open reading frames of 1,407 nucleotides, guanine at the 571st nucleotide was replaced by adenine in TO-2, resulting in substitution of alanine at the 191st amino acid to threonine (Ala191Thr). Ala191Thr substitution created a potential phosphorylation site for protein kinase C, but such phosphorylation was not detected. In SW13 cells, the transfected TO-2 desmin formed visible filaments with fragile structure and sparse distribution.

CONCLUSIONS:

From those findings, we consider Ala191Thr a deleterious mutation. We would like to examine whether and how desmin interacts with delta-sarcoglycan in normal cardiac muscles and gain further insight into the pathogenetic mechanisms of CM.

Exp Clin Cardiol. 2012 Summer;17(2):57.

IL-5: Genome-Wide Association Study for the Comprehensive Analysis of Heart Failure Carrying Variable Prevalence and Ethnicity

Licht Toyo-oka 1, Toshihiro Tanaka 2, Sawa Kostin 3, Toru Izumi 4, Jutta Schaper 3, Katsushi Tokunaga 1, Toshiaki Nakajima 5, Teruhiko Toyo-oka 4,5

Abstract

BACKGROUND AND OBJECTIVES:

Heart failure (HF) is a medically and socioeconomically serious syndrome with diverse and ethnic background. A part is heritable, though its underlying genetic factors are still elusive. The Genome-Wide Association Study (GWAS) is promising for identifying not only monogenic, responsible genes but also other polygenic modifier genes that aggravate or ameliorate the clinical process to the advanced HF via a final common pathway in the wide spectrum of heart failure (HF, Toyo-oka et al., PNAS, 2004).

At first, we started GWAS to clarify the genetic effect on HF progression, irrespective of the etiology (congenital, valvular, infectious, ischemic, degenerative, hormonal or metabolic causes etc), covering the wide spectrum of HF, common in or specific to different ethnicity. This study also aimed personalized medicine to clarify responses to the pharmaceutical, physical or surgical therapies.

METHODS AND RESULTS:

The current analyses were conducted in post-genome project in Japan. SNP/SNV/CNV in DNA from peripheral blood or explanted cardiac specimens revealed nearly one million haplo-groups with the invader method (Ota et al., Nature 2004). In the case-controlled study, the number of each allele at P values <10−4 by χ2 test decreased to less than 10,000 SNPs. After restricting to ORF-related regions (exon, exon-intron border, 3′- and 5′-UTR), these SNPs reduced to 1/3, consisting of 93.6%, 3.7%, 1.9% and 0.8% for synonymous, non-synonymous, stop codon and insertion/deletion mutations, respectively. Candidate genes involved adhesion molecules, channel proteins, cell signaling, transcription/translation factors, proteolysis and anonymous proteins. These mutations rarely overlapped with Caucasians with DCM. The ethnicity was confirmed by the full sequencing of the mitochondrial (mt) genome. Furthermore, some mt-gene mutations were closely related with HF, secondary to the oxidative phosphorylation failure (Shin et al., Am J Hum Genet, 2000), suggesting both the cumulative gene-dosing action and cross-talk between nuclear and mt-genomes.

CONCLUSION:

Though the total number of DNA donors is too short now to finalize, GWAS is of a great use for the evaluation of genetic and ethnic background, when the results are physiologically verified with the multidisciplinary bioinformatics. Whole genomic information would be required for shifting gene-based medicine using ES or iPS cells to the clinical level near future.

Exp Clin Cardiol. 2012 Summer;17(2):57.

SL: History of ISHR and IACS: Role of Japan in International Cardiovascular Developments

Naranjan S Dhalla 1
Exp Clin Cardiol. 2012 Summer;17(2):57.

SL-1: Ontogenetic Development of Cardiac Sensitivity to Hypoxia

B Ostadal 1, I Ostadalova 1, Z Charvatova 1, M Milerova 1, Z Drahota 1

Abstract

Immature mammalian heart is highly resistant to ischemia but the tolerance rapidly decreases after birth. The mechanisms of the higher resistance of the immature heart to oxygen deprivation have not yet been satisfactorily clarified. For the explanation of this fact, the physiological changes during the perinatal period should be taken into consideration, particularly the changes of oxygenation. Still unclear is the role of mitochondria in spite of the fact that mitochondria are responsible for cellular oxygen handling. Mitochondrial oxidative phosphorylation is not completely developed in rat heart at birth; cardiac maturation during the first postnatal week is characterized by the increasing content and specific activity of cytochrome c oxidase and the enhanced flux of adenine nucleotides across the inner mitochondrial membrane. Moreover, in newborn animals, a single population of mitochondria with relatively high mitochondrial membrane potential (MMP) was observed; starting with the weaning period, a second population with significantly lower MMP occurs. The collapse of MMP due to the opening of a high conductance mitochondrial permeability transition pore (MPTP) has been implicated in the molecular mechanisms associated with ischemia/reperfusion (I/R) injury of the adult heart. We have observed, however, significant ontogenetic differences in the role of MPTP in the I/R injury. Whereas the blockade of MPTP by sanglifehrin in perfused rat heart had a protective effect on I/R-induced damage in the adult myocardium, it had no effect in the neonatal heart. Furthermore, the extent of Ca-induced swelling of mitochondria from neonatal rats is significantly lower than that from adult animals. All these results support the hypothesis that cardiac mitochondria are deeply involved in the regulation of cardiac tolerance to oxygen deprivation during ontogenetic development. The neonatal period seems to be critical also for the development of possibilities of cardiac protection against ischemia. The most potent protective phenomena, such as ischemic preconditioning or adaptation to chronic hypoxia, failed to increase hypoxic tolerance to oxygen deprivation in the newborn rat heart; their protective effect develops only during the early postnatal period. It seems, therefore, that the decreasing tolerance to oxygen deprivation is counteracted by the development of endogenous protection; the mechanism of protection of the immature heart is still unclear. It may be concluded that the cardiac effect of ischemia is markedly influenced by the age of experimental animals.

Exp Clin Cardiol. 2012 Summer;17(2):58.

SL-3: The Structural Correlate of Cardiac Dysfunction in the Human Heart

Jutta Schaper 1

Abstract

Cardiac dysfunction and cellular alterations in the myocardium are closely linked. On the basis of our results obtained in myocardium from patients with either aortic valve defects or dilated cardiomyopathy, we postulate that diastolic as well as systolic dysfunction are due to specific structural disturbances. Myocyte hypertrophy and a significant degree of reactive fibrosis are the major factors causing early diastolic dysfunction. The equivalents of systolic dysfunction are reduction of myofilaments, occurrence of small mitochondria with less christae and densification of elements of the cytoskeleton, the latter most probably a mechanism compensatory for reduced cellular stability because of loss of sarcomeres. A reduction of the sarcomeric skeleton, consisting of titin, alpha-actinin and myomesin, continues to contribute to sarcomeric instability. Loss of the membrane associated proteins dystrophin, of the vinculin-talin-integrin complex and of spectrin results in an ionic imbalance and in combination with the reduction of the gap junctional protein connexin 43 represents the basis of defects of the excitation-contraction coupling. Accumulation of blood borne cells indicates chronic inflammation. These different processes involving the interstitium as well as almost all cellular components of the cardiomyocytes will finally lead to myocyte death, either autophagic or oncotic but less apoptotic. In conclusion, the structural-functional development of heart failure is a multifactorial event involving the extra-cellular matrix and almost all cellular components of the myocytes. It is postulated that each structural abnormality is characterized by specific functional disturbances.

Exp Clin Cardiol. 2012 Summer;17(2):58.

SL-4: Regeneration of Arterial System Via Collateral Vessels. Arteriogenesis

Wolfgang Schaper 1

Abstract

Under specific conditions, such as early in life, arterial occlusions are relatively well tolerated. Occlusions of the thoracic aorta, as a congenital condition, is often tolerated into early adulthood because intercostal arteries and the internal mammary- and epigastrical arteries had grown into much larger channels. Even in old age, occlusions of coronary, cerebral and peripheral arteries do not lead to infarctions or strokes when the time from stenosis to complete occlusion is slow. We have produced arterial occlusions in animals and have studied the response of the collateral circulation by means of hemodynamic, light microscopic, ultrastructural and molecular biological methods. Collateral vessel growth requires preexistingsmall arterioles, the surrounding tissue need not be ischemic and monocytes must be present to invade the vascular wall. Fluid shear stress (FSS) within preexistent collateral vessels is the prime physical stimulus for growth. This was verified by increasing FSS by AV-shunts. FSS is transmitted from the endothelium to the smooth muscles of the media via ion channels (TRPV4), growth factors and oxygen and nitrogen-based radicals. The NO radical is essential because the combined targeting of all NO producing enzymes totally blocks arteriogenesis. Genome-wide screening for transcriptional expression of peripheral rat collaterals during growth of collaterals showed differential regulation of about 300 genes among which many structural genes like actin binding molecules (abra). Key roles are played by miRNAs that may act as potent inhibitors and stimulators of arteriogenesis. Since only 40% of the normal maximal flow reserve is replaced by collaterals, the complete normalization of reserve is a worthy goal of future research.

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