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The "postmitotic" phenotype in adult cardiac muscle exhibits similarities to replicative senescence more generally and constitutes a barrier to effective restorative growth in heart disease. Telomere dysfunction is implicated in senescence and apoptotic signaling but its potential role in heart disorders is unknown. Here, we report that cardiac apoptosis in human heart failure is associated specifically with defective expression of the telomere repeatbinding factor TRF2, telomere shortening, and activation of the DNA damage checkpoint kinase, Chk2. In cultured cardiomyocytes, interference with either TRF2 function or expression triggered telomere erosion and apoptosis, indicating that cell death can occur via this pathway even in postmitotic, noncycling cells; conversely, exogenous TRF2 conferred protection from oxidative stress. In vivo, mechanical stress was sufficient to down-regulate TRF2, shorten telomeres, and activate Chk2 in mouse myocardium, and transgenic expression of telomerase reverse transcriptase conferred protection from all three responses. Together, these data suggest that apoptosis in chronic heart failure is mediated in part by telomere dysfunction and suggest an essential role for TRF2 even in postmitotic cells. [ABSTRACT FROM AUTHOR]

H. Oh and M. D. Schneider. The Emerging Role of Telomerase in Cardiac Muscle Cell Growth and Survival.Journal of Molecular and Cellular Cardiology (2002) 34, 717–724. Most mammalian cells—excepting germ cells, tumor cells, and stem cells, that is—possess a finite replicative life span, manifested by the eventual cessation of cell proliferation. Clinically, this is germane not just to the overt derangements of cell growth in cancer, but also to organs such as the heart, in which the capacity for cell replacement and repair is insufficient to maintain organ function following cell death. Among the intrinsic mechanisms that control a conserved program of replicative senescence is the enzyme telomerase, which synthesizes the telomeric repeat for end-capping of each chromosome. The implications of telomerase for cardiac growth have recently begun to be defined. Other functions of telomerase, in maintaining genome integrity, also hold importance for cardiac muscle, as a novel means to suppress apoptosis and, thus, salvage myocardium following ischemic injury. [ABSTRACT FROM AUTHOR]

Reports the promotion of cardiac muscle proliferation, hypertrophy and survival. Role of telomerase reverse transcriptase in the promotion; Limitation of cardiac muscle regeneration by irreversible cell cycle exit; Postulation of the basis to telomere shortening and replicative senescence.

The article discusses the paper "Injectable hydrogel helps bone marrow-derived mononuclear cells restore infarcted myocardium," by X. Y. Li and colleagues, in this issue of the journal.

Hypertrophic growth is a risk factor for mortality in heart diseases. Mechanisms are lacking for this global increase in RNA and protein per cell, which underlies hypertrophy. Hypertrophic signals cause phosphorylation of the RNA polymerase II C-terminal domain, required for transcript elongation. RNA polymerase II kinases include cyclin-dependent kinases-7 (Cdk7) and Cdk9, components of two basal transcription factors. We report activation of Cdk7 and -9 in hypertrophy triggered by signaling proteins (Gαq, calcineurin) or chronic mechanical stress. Only Cdk9 was activated by acute load or, in culture, by endothelin. A preferential role for Cdk9 was shown in RNA polymerase II phosphorylation and growth induced by endothelin, using pharmacological and dominant-negative inhibitors. All four hypertrophic signals dissociated 7SK small nuclear RNA, an endogenous inhibitor, from cyclin T-Cdk9. Cdk9 was limiting for cardiac growth, shown by suppressing its inhibitor (7SK) in culture and preventing downregulation of its activator (cyclin T1) in mouse myocardium. [ABSTRACT FROM AUTHOR]

Abstract: Background: Almost all reports on cardiac regeneration therapy have referred to adults, and only a few have focused on transcoronary infusion of cardiac progenitor cells using the stop‐flow technique in children. Methods: Intracoronary autologous cardiosphere‐derived cell (CDC) transfer was conducted at Okayama University as a phase 1 clinical trial for seven patients with hypoplastic left heart syndrome between January 2011 and December 2012, and as a phase 2 clinical trial for 34 patients with single‐ventricle physiology between July 2013 and March 2015. Results: A total of 41 patients with single‐ventricle physiology underwent transcoronary infusion of CDC with the stop‐flow technique. The median age was 33 months (range, 5–70 months) and the median weight was 10.1 kg (range, 4.1–16.0 kg). Transient adverse events occurred during the procedure, including ST‐segment elevation or depression, hypotension, bradycardia, and coronary artery vasospasm. All patients completely recovered. There were no major procedure‐related adverse events. In this study, transcoronary infusion of CDC using the stop‐flow technique was successfully completed in all patients. Conclusion: Transcoronary infusion of CDC using the stop‐flow technique in children is a feasible and safe procedure. [ABSTRACT FROM AUTHOR]

The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS. [ABSTRACT FROM AUTHOR]

The actions of catecholamines on adrenergic receptors (ARs) induce sympathetic responses, and sustained activation of the sympathetic nervous system results in disrupted circulatory homeostasis. In cardiomyocytes, α1-ARs localize to flask-shaped membrane microdomains known as "caveolae." Caveolae require both caveolin and cavin proteins for their biogenesis and function. However, the functional roles and molecular interactions of caveolar components in cardiomyocytes are poorly understood. Here, we showed that muscle-restricted coiled-coil protein (MURC)/Cavin-4 regulated a1-AR-induced cardiomyocyte hypertrophy through enhancement of ERK1/2 activation in caveolae. MURC/Cavin-4 was expressed in the caveolae and T tubules of cardiomyocytes. MURC/Cavin-4 overexpression distended the caveolae, whereas MURC/Cavin-4 was not essential for their formation. MURC/Cavin-4 deficiency attenuated cardiac hypertrophy induced by a1-AR stimulation in the presence of caveolae. Interestingly, MURC/Cavin-4 bound to α1A- and α1B-ARs as well as ERK1/2 in caveolae, and spatiotemporally modulated MEK/ERK signaling in response to a1-AR stimulation. Thus, MURC/Cavin-4 facilitates ERK1/2 recruitment to caveolae and efficient α1-AR signaling mediated by caveolae in cardiomyocytes, which provides a unique insight into the molecular mechanisms underlying caveola-mediated signaling in cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1). While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER). In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease. [ABSTRACT FROM AUTHOR]

Objectives: This study was designed to determine whether controlled release of basic fibroblast growth factor (bFGF) might improve human cardiosphere-derived cell (hCDC) therapy in a pig model of chronic myocardial infarction. Background: Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation. Methods: We conducted 2 randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. Four weeks after coronary reperfusion, 14 pigs were randomly assigned to receive an intramyocardial injection of placebo medium with or without bFGF-incorporating hydrogel implantation. As a second study, 26 pigs were randomized to receive controlled release of bFGF combined with or without hCDCs or bone marrow–derived mesenchymal stem cell transplantation 4 weeks after reperfusion. Results: Controlled release of bFGF in ischemic myocardium significantly augmented the formation of microvascular networks to enhance myocardial perfusion and contractile function. When combined with cell transplantation, the additive effects of bFGF were confined to hCDC-injected animals, but were not observed in animals receiving human bone marrow–derived mesenchymal stem cell transplantation. This was shown by increased donor-cell engraftment and enhanced cardiomyocyte differentiation in the transplanted hearts, resulting in synergistically improved ventricular function and regional wall motion and reduced infarct size. Conclusions: Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance hCDC engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure. [Copyright &y& Elsevier]

Although the modulated expression of Dicer is documented upon neo-plastic transformation, little is known of the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVEC5). Serum withdrawal induced a time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or sphingosine-l-phosphate. When Dicer expression was silenced by short-hairpin RNA against Dicer, the cells were more prone to apoptosis under serum withdrawal, whereas the rate of apoptosis was comparable with control cells in the serum-containing condition. Real-time PCR-based gene expression profiling identified several genes, the expression of which was modulated by Dicer silencing, including adhesion and matrix-related molecules, caspase-3, and nitric oxide synthase 3 (N053). Dicer silencing markedly impaired migratory functions without affecting cell adhesion and repressed phosphor- ylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in adherent 1{UVECs. Dicer knockdown upregulated caspase-3 and downregulated NOS3 expression, and serum withdrawal indeed in- creased caspase-3 and decreased N053 expression. Furthermore, the overexpression of Dicer in HUVECs resulted in a marked reduction in apoptosis upon serum withdrawal and a decreased caspase-3 and increased NOS3 expression. The inhibition of NOS activity by N°'- nitro-L-arginine methyl ester abrogated the effect of Dicer overexpres- sion to rescue the cells from serum withdrawal-induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to an increased susceptibility to apoptosis through the regulation of caspase-3 and NOS3 expression. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that bone morphogenetic proteins (BMPs) are crucial for cardiac induction, specification, and development. Although signaling of BMPs is tightly regulated through soluble BMP-binding proteins, how they regulate BMP signaling during cardiac differentiation remains unknown. To identify molecules responsible for BMP signaling during early cardiomyocyte differentiation of P19 cells, cDNA subtraction was performed. We found a bimodal expression of the BMP-binding protein Crossveinless-2 (Cv2) during cardiomyocyte differentiation; Cv2 is temporally expressed earlier than cardiac transcription factors such as Nkx2.5 and Tbx5 and acts as a suppressor for BMP signaling in P19 cells. We established a P19 clonal cell line harboring a cardiac alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein gene to monitor cardiac differentiation by flow cytometry. Treatment with BMP2 during the first 2 days of differentiation suppressed cardiomyocyte differentiation through activation of downstream targets Smad1/5/8 protein and Id1 gene, whereas treatment with Cv2 conversely inhibited Smad1/5/8 activation and Id1 expression, leading to increased generation of cardiac cells. RNA interference-mediated knockdown (KD) of endogenous Cv2 showed increased Smad1/5/8 activation and impaired cardiomyocyte differentiation. Expression of cardiac mesoderm markers was reduced, whereas expression of Id1 and endoderm markers such as Sox7, Hnf4, and E-cadherin was induced in Cv2- kinase dead cells. These phenotypes were rescued by the addition of Cv2 protein to the culture media during the first 2 days of differentiation or co-culture with parental cells. These data suggest that Cv2 may specify cardiac mesodermal lineage through inhibition of BMP signaling at early stage of cardiogenesis. [ABSTRACT FROM AUTHOR]

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNA1)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation. [ABSTRACT FROM AUTHOR]

Abstract: The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-β family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle. [Copyright &y& Elsevier]

Abstract: We have reported that skeletal myosphere-derived progenitor cells (MDPCs) can differentiate into vascular cells, and that MDPC transplantation into cardiomyopathic hearts improves cardiac function. However, the autocrine/paracrine molecules and underlying mechanisms responsible for MDPC growth have not yet been determined. To explore the molecules enhancing the proliferation of MDPCs, we performed serial analysis of gene expression and signal sequence trap methods using RNA isolated from MDPCs. We identified osteopontin (OPN), a secretory molecule, as one of most abundant molecules expressed in MDPCs. OPN provided a proliferative effect for MDPCs. MDPCs treated with OPN showed Akt activation, and inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway repressed the proliferative effect of OPN. Furthermore, OPN-pretreated MDPCs maintained their differentiation potential into endothelial and vascular smooth muscle cells. These findings indicate an important role of OPN as an autocrine/paracrine molecule in regulating the proliferative growth of muscle-derived angiogenic progenitor cells via the PI3K/Akt pathway. [Copyright &y& Elsevier]

Abstract: Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant>adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardiomyocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3β may be crucial modulators for hCSC maintenance in human heart. [Copyright &y& Elsevier]

Abstract: Bone marrow cells have been shown to contribute to neovascularization in ischemic hearts, whereas their impaired maturation to restore the δ-sarcoglycan (δ-SG) expression responsible for focal myocardial degeneration limits their utility to treat the pathogenesis of cardiomyopathy. Here, we report the isolation of multipotent progenitor cells from adult skeletal muscle, based on their ability to generate floating-myospheres. Myosphere-derived progenitor cells (MDPCs) are distinguishable from myogenic C2C12 cells and differentiate into vascular smooth muscle cells and mesenchymal progeny. The mutation in the δ-SG has been shown to develop vascular spasm to affect sarcolemma structure causing cardiomyopathy. We originally generated δ-SD knockdown (KD) mice and transplanted MDPCs into the hearts. MDPCs enhanced neoangiogenesis and restored δ-SG expression in impaired vasculatures through trans-differentiation, leading to improvement of cardiac function associated with paracrine effectors secretion. We propose that MDPCs may be the promising progenitor cells in skeletal muscle to treat δ-sarcoglycan complex mutant cardiomyopathy. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are small non-coding RNAs emerging as important post-transcriptional gene regulators. In this study, we examined the role of miR-1, an miRNA specifically expressed in cardiac and skeletal muscle tissue, on the myogenic, osteoblastic, and adipogenic differentiation of C2C12 cells. Upon induction of myogenic differentiation, miR-1 was robustly expressed. Retrovirus-mediated overexpression of miR-1 markedly enhanced expression of muscle creatine kinase, sarcomeric myosin, and α-actinin, while the effects on myogenin and MyoD expression were modest. Formation of myotubes was significantly augmented in miR-1-overexpressing cells, indicating miR-1 expression enhanced not only myogenic differentiation but also maturation into myotubes. In contrast, osteoblastic and adipogenic differentiation was not affected by forced expression of miR-1. Thus, the muscle-specific miRNA, miR-1, plays important roles in controlling myogenic differentiation and maturation in lineage-committed cells, rather than functioning in fate determination. [Copyright &y& Elsevier]

Introduction: We have completed phase 1/2 clinical trials for 48 patients of single ventricle physiology and confirmed the safety and efficacy of cardiosphere-derived cells (CDCs) therapy. On the other hand, the efficacy of CDCs in dilated cardiomyopathy (DCM) remains unknown because of the lack of efficient large animal models to reconstitute the pathogenesis of heart failure. Hypothesis: We hypothesized that microembolization into three coronary arteries may be a stably reproducible approach to create a diffuse hypokinetic heart failure model to investigate the therapeutic efficacy of CDCs infusion in pigs. Methods: Pigs (30 kg, n=13) were treated by intracoronary administration of 1.0х104 microspheres (diameter: 100-300 μm) into three vessels. Two weeks later, 9.0х106 CDCs or placebo were selectively infused into three coronary arteries by non-stop flow technique. Evaluation of ejection fraction (EF) was performed before cell infusion and 1 month after protocol treatment. Results: Ten out of 13 pigs (77%) developed diffuse hypokinetic heart failure (baseline EF 38.2±3.3%) and randomly assigned into two groups (CDCs: n=5, placebo: n=5). No serious adverse events were found during the CDCs infusion. Significant improvement of EF was observed 1 month after CDCs treatment (38.0±3.1% to 43.5±2.5%; P =0.017), in contrast there were no change in placebo group (38.4±3.8% to 37.5±2.2%; P =0.65). Myocardial fibrosis was significantly reduced in CDCs group compared with placebo treatment (9.6±4.2% versus 18.2±4.1%; P <0.01). Immunohistochemistry analysis performed to unveil mechanisms demonstrated that CDCs infusion augmented myocyte cycling and enhanced angiogenesis, those were addressed by increase in Ki67 positive cardiomyocytes (46.9±6.0/106 myocytes versus 27.2±3.6/106 myocytes; P <0.01) and capillary density (876.0±251.2/mm2 versus 644.6±131.5/mm2; P <0.01) at 1 month. Conclusions: We successfully established a novel large animal model of DCM by coronary microembolization technique and confirmed the efficacy of cell therapy. Based on these results, a randomized phase 1 trial of CDCs therapy in 31 cases of children with dilated cardiomyopathy (TICAP-DCM: NCT03129568) is ongoing in Okayama university hospital. [ABSTRACT FROM AUTHOR]

Introduction: Although failing Fontan circulation has led to increased mortality and morbidity in long-term, little experimental model has been established to reconstitute Fontan circulation without any mechanical circulatory assistance in perioperative period in large animals. Hypothesis: We aimed to develop a novel Fontan failure model in pig by connecting inferior vena cava (IVC) with pulmonary artery (PA) and sought to investigate whether preconditioning by inferior vena cava (IVC) banding may improve the survival of IVC-PA connection model. Methods: IVC was connected to PA with14 mm ePTFE graft in 10 Yorkshire pigs (20-24 kg) via median sternotomy. One week before operation, IVC banding was performed as a preconditioning treatment (banding group). Five pigs (20-24 kg) were operated without IVC banding and assigned as control group. Results: Preconditioning by IVC banding showed increased IVC pressure (3.4±0.5 vs. 8.4±0.5 mmHg, P <0.01), decreased blood pressure (P <0.01), and increased heart rate (P =0.02). When pigs received IVC-PA connection , IVC pressure significantly increased after the operation in both groups (control group: 3.2±0.4 vs. 11.4±0.9 mmHg, P <0.01; banding group: 8.0±0.7 vs. 11.8±0.4 mmHg, P <0.01). However, IVC pressure changes were smaller in banding group than control group (control group: 8.2±0.8 vs. banding group: 3.8±0.4 mmHg, P <0.01). One hour after the operation, banding group showed higher systolic blood pressure than control group (control group: 50.8±6.6 vs. banding group: 72.0±7.1 mmHg, P <0.01). These small hemodynamic changes by pre-treatment of IVC banding improved survival after IVC-PA connection (Figure, control group: 0.6±0.1 vs. banding group: 6.2±0.8 days, log-rank P <0.01). Conclusions: Pre-treatment by IVC banding for 1 week may exert preconditioning effects to reduce the acute elevation of venous pressure after IVC-PA connection. This novel surgical strategy may be helpful to establish the Fontan circulation model in large animal to survive without mechanical support. [ABSTRACT FROM AUTHOR]

Introduction: Although failing Fontan circulation leads to increased mortality and morbidity in long-term, little experimental model has been developed to restitute the Fontan circulation without any circulatory support in large animals. Hypothesis: We investigated whether inferior vena cava (IVC) banding represents the abdominal complications in failed Fontan circulation with higher venous pressure and lower cardiac output. Methods: The IVC banding was performed in 5 Yorkshire piglets (20-23kg). The banding was adjusted as the mean IVC pressure increased to 5 mmHg. Sham operation was performed in another 5 piglets. Two months after the operation, models were assessed and sacrificed. Results: The mean IVC pressure increased (pre: 3.3±0.8 mmHg vs. post: 6.3±0.3 mmHg, P <0.01), and left ventricular end diastolic volume decreased (pre: 65.4±3.6 ml vs. post: 51.1±4.2 ml, P <0.01) in IVC banding models after the operation. Ascites was increased in IVC banding models, accompanied by decreased total protein, albumin, total cholesterol, and type 4 collagen in blood examination (all were P <0.01) in IVC banding group. Hepatic sinusoidal dilatation with diffuse fibrosis was observed in hematoxylin and eosin staining (Figure), and increased α-SMA and von Willebrand factor expression were detected by immunostaining. In tissue analysis, TNF-α expression increased in heart and liver, and α-SMA increased only in liver. In addition, type 3 collagen increased in liver and spleen that associated with increased expression of collagenase-related mRNA by RT-PCR. Matrix metalloproteinase 9 increased in spleen and intestine with decreasing in kidney, and CD45 remarkably expressed in spleen. Conclusions: IVC banding in piglet is novel surgical technique to reconstitute abdominal congestive complications. This reproducible procedure may be useful to develop the new therapeutic approaches for late congestive damages after Fontan operation. [ABSTRACT FROM AUTHOR]

We identified a novel muscle-restricted putative coiled-coil protein, MURC, which is evolutionarily conserved from frog to human. MURC was localized to the cytoplasm with accumulation in the Z-line of the sarcomere in the murine adult heart. MURC mRNA expression in the heart increased during the developmental process from the embryonic stage to adulthood. In response to pressure overload, MURC mRNA expression increased in the hypertrophied heart. Using the yeast two-hybrid system, we identified the serum deprivation response (SDPR) protein, a phosphatidylserine-binding protein, as a MURC-binding protein. MURC induced activation of the RhoA/ROCK pathway, which modulated serum response factor-mediated atrial natriuretic peptide (ANP) expression and myofibrillar organization. SDPR augmented MURC-induced transactivation of the ANP promoter in cardiomyocytes, and RNA interference of SDPR attenuated the action of MURC on the ANP promoter. Transgenic mice expressing cardiac-specific MURC (Tg-MURC) exhibited cardiac contractile dysfunction and atrioventricular (AV) conduction disturbances with atrial chamber enlargement, reduced thickness of the ventricular wall, and interstitial fibrosis. Spontaneous episodes of atrial fibrillation and AV block were observed in Tg-MURC mice. These findings indicate that MURC modulates RhoA signaling and that MURC plays an important role in the development of cardiac dysfunction and conduction disturbance with increased vulnerability to atrial arrhythmias. [ABSTRACT FROM AUTHOR]

Recent studies have shown that cardiac stem cells (CSCs) from the adult mammalian heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth are so far unknown. Here, we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult murine hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-like and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERTGFP-positive fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component to promote CSC proliferation and survival to directly facilitate early engraftment, and might indirectly exert the effects on late cardiovascular differentiation after CSC transplantation. [ABSTRACT FROM AUTHOR]