With the recent advent of gene therapy, anti-apoptotic therapy has been receiving spotlight as a potential modality to inhibit the deterioration of pump function in the failing heart. We hypothesized that anti-apoptotic therapy using survivin gene delivery will 1) salvage H9c2 cells exposed to doxorubicin toxicity, and 2) ameliorate the progressive decline in left ventricular function in a rat model of doxorubicin-induced cardiomyopathy. The in vitro data suggested that survivin successfully prevented cell death under doxorubicin stress by both direct and indirect/paracrine mechanisms. Doxorubicin-treated animals developed progressive left ventricular dysfunction as evident by echocardiography and invasive pressure-volume loop analysis, which was prevented by ultrasound-mediated survivin plasmid delivery, but not empty plasmid delivery. Post-mortem analysis of myocardial tissue indicated a lowered apoptotic index in survivin-treated hearts, with evidence of decrease in interstitial fibrosis. In conclusion, survivin gene therapy was shown to ameliorate doxorubicin-induced cardiomyopathy, by decreasing apoptosis and preventing adverse remodeling.

Myocardial Infarction (MI) is one of the most prevalent and deadliest diseases in the United States. Since the host myocardium becomes irreversibly damaged following MI, current research is focused on identification of novel, less invasive, and more effective treatment options for patients. Cellular cardiomyopathy, in which viable cells are transplanted into the necrotic tissue, has the potential to regenerate and integrate with the host myocardium. Stem cells, specifically pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC), are ideal candidates for this procedure because they are pluripotent; however, ESCs must be predifferentiated to avoid teratoma formation in vivo. In this dissertation, our goal was improve upon current protocols to direct differentiation of ESCs into cardiomyocytes using an embryoid body (EB) model. We immobilized pro-cardiomyogenic proteins, specifically Sonic Hedgehog (SHH) and Bone Morphogenetic Protein 4 (BMP4) to paramagnetic beads and delivered them in the interior of the EB. While lineage commitment was indiscriminate, the presence of the beads alone appeared to guide differentiation into cardiomyocytes: there were significantly more contracting areas in EBs containing beads than in the presence of SHH or BMP4. To take advantage of this result, we immobilized Arginine-Glycine-Aspartic Acid (RGD) peptides to the beads and magnetized them following incorporation into the EB. Magnetically mediated strain increased the expression of mechanochemical markers, and in combination with BMP4 increased the percentage of cardiomyocytes. Finally, PEGylated fibrin gels were used to investigate the effect of seeding method and fibrinogen concentration on cardiomyocyte behavior and maturation. Cells seeded on top of compliant hydrogels had the most contracting regions compared to stiffer PEGylated fibrin gels, whereas cardiomyocytes seeded within the hydrogels could not remodel the matrix or maintain contractility. As an alternative to 3D culture, we seeded cardiomyocytes within gel layers, which maintained viability as well as contractile activity. We observed that PEGylated fibrin gels can maintain ESC-derived cardiomyocytes; however, the ratio of cardiomyocytes and non-cardiomyocytes should be optimized to maintain contractile phenotypes. Therefore, this dissertation presents novel methods to differentiate ESCs into cardiomyocytes, and subsequently promote their maturation in vitro, for the treatment of MI. / text

An important element of metabolic regulation of cardiac and skeletal muscle energetics is the interaction of mitochondria with cytoskeleton. Mitochondria are in charge of supplying the cells with energy, adjusting its functional activity under conditions of stress or other aspects of life. Mitochondria display a tissue-specific distribution. In adult rat cardiomyocytes, mitochondria are arranged regularly in a longitudinal lattice at the level of A band between the myofibrils and located within the limits of the sarcomeres. In interaction with cytoskeleton, sarcomeres and sarcoplasmic reticulum they form the functional complexes, the intracellular energetic units (ICEUs). The ICEUs have specialized pathways of energy transfer and metabolic feedback regulation between mitochondria and ATPases, mediated by CK and AK. The central structure of ICEUs is the mitochondrial interactosome (MI) containing ATP Synthasome, respiratory chain, mitochondrial creatine kinase and VDAC, regulated by tubulins. The main role of MI is the regulation of respiration and the intracellular energy fluxes via phosophotransfer networks. The regulation of ICEUs is associated with structural proteins. The association of mitochondria with several cytoskeletal proteins described by several groups has brought to light the importance of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To purvey a better understanding of these findings, the present work investigated the mechanism of energy fluxes control and the role of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To show these complex associations in adult cardiac cells several proteins were visualized by confocal microscopy: α-actinin and β-tubulin isotypes. For the first time, it was showed the existence of the specific distribution of β-tubulin isotypes in adult cardiac cells. Respiratory measurements were performed to study the role of tubulins in the regulation of oxygen consumption. These results together confirmed the crucial role of cytoskeletal proteins -i.e. tubulins, α-actinin, plectin, desmin, and others- for the normal shape of cardiac cells as well as mitochondrial arrangement and regulation. In addition, in vivo - in situ mitochondrial dynamics were studied by the transfection of GFP-α-actinin, finding that fusion phenomenon does not occur as often as it is believed in healthy adult cardiac cells. / Un élément important de la régulation du métabolisme énergétique des muscles cardiaque et squelettiques est l'interaction des mitochondries avec le cytosquelette. Les mitochondries sont responsables de l'approvisionnement des cellules en énergie, elles sont capables d'ajuster leur activité fonctionnelle en fonction des conditions de stress ou d'autres aspects de la vie. Les mitochondries ont une distribution spécifique selon les tissus. Dans les cardiomyocytes de rats adultes, les mitochondries sont disposées régulièrement dans un entrelacement longitudinal au niveau des bandes A, entre les myofibrilles et dans les limites des sarcomères. En interaction avec le cytosquelette, le sarcomère et le réticulum sarcoplasmique, elles forment des complexes fonctionnels appelés unités énergétiques intracellulaires (ICEUs). Les ICEUs ont des voies spécialisées de transfert d'énergie et de régulation des feedback métaboliques entre les mitochondries et les ATPases, médiée par la CK et l'AK. La structure centrale des ICEUs est l'interactosome mitochondrial (MI) qui confient l'ATP synthasome, la chaîne respiratoire, la créatine kinase mitochondriale et VDAC, qui pourrait être régulé par les tubulines. Le rôle principal du MI est la régulation de la respiration et des flux d'énergie intracellulaires via les réseaux de phosphotransfert. La régulation des ICEUs est liée aux protéines structurales. L'association des mitochondries avec plusieurs protéines du cytosquelette, décrite par plusieurs groupes, a mis en évidence l'importance de la relation structure-fonction dans la régulation métabolique des cardiomyocytes de rats adultes. Pour fournir une meilleure compréhension de ces résultats, le présent travail étudie le mécanisme de contrôle des flux d'énergie et le rôle des relations structure-fonction dans la régulation métabolique de cardiomyocytes de rats adultes. Pour montrer ces associations complexes dans les cellules cardiaques adultes, plusieurs protéines ont été visualisées par microscopie confocale: l'α-actinine et les isoformes des β-tubulines. Pour la première fois, l'existence d'une distribution spécifique des isoformes de β-tubuline dans les cellules cardiaques adultes a été montré. Des mesures respiratoires ont été réalisées pour étudier le rôle des tubulines dans la régulation de la consommation d'oxygène. Ces résultats ont confirmé le rôle déterminant des protéines du cytosquelette -tubulines, α-actinine, plectine, desmine, et autres- pour le maintien de la forme normale des cellules cardiaques, ainsi que de l'arrangement et de la régulation mitochondrial. En outre, la dynamique mitochondriale a été étudiée in vivo et in situ par la transfection de la GFP-α-actinine, ceci permettant la mise en évidence du fait que le phénomène de fusion ne se produit pas aussi souvent qu'on ne le croit pour des cellules cardiaques adultes en bonne santé.

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13443号 / 論医科博第7号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 長船 健二, 教授 木村 剛, 教授 湊谷 謙司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Background: Cardiovascular diseases are currently the leading cause of death in humans. The high mortality of cardiac diseases is associated with myocardial ischemia and reperfusion (I/R). Recent studies have reported that microRNAs (miRNAs) play important roles in cell apoptosis. However, it is not known yet whether miR-141-3p contributes to the regulation of cardiomyocyte apoptosis. It has been well established that in vitro hypoxia/reoxygenation (H/R) model can follow in vivo myocardial I/R injury. This study aimed to investigate the effects of miR-141-3p and CHD8 on cardiomyocyte apoptosis following H/R. Results: We found that H/R remarkably reduces the expression of miR-141-3p but enhances CHD8 expression both in mRNA and protein in H9c2 cardiomyocytes. We also found either overexpression of miR-141-3p by transfection of miR-141-3p mimics or inhibition of CHD8 by transfection of small interfering RNA (siRNA) significantly decrease cardiomyocyte apoptosis induced by H/R. Moreover, miR-141-3p interacts with CHD8. Furthermore, miR-141-3p and CHD8 reduce the expression of p21. Conclusion: MiR-141-3p and CHD8 play critical roles in cardiomyocyte apoptosis induced by H/R. These studies suggest that miR-141-3p and CHD8 mediated cardiomyocyte apoptosis may offer a novel therapeutic strategy against myocardial I/R injury-induced cardiovascular diseases.

Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.

Doxorubicin (Dox) is a major anticancer chemotherapeutic agent. However, it causes cardiomyopathy due to the side effect of cardiomyocyte apoptosis. We have previously reported that angiopoietin-1 significantly reduced myocardial infarction after ischemic injury and protected cardiomyocytes from oxidative stress-induced apoptosis. It is hypothesized that angiopoietin-1 may protect cardiomyocytes from Dox-induced apoptosis. Cardiomyocytes H9C2 were transfected with adenovirus expressing angiopoietin-1 (Ad5-Ang-1) 24 h before the cells were challenged with Dox at a concentration of 2 μmol/L. Ad5-GFP served as the vector control. Cardiomyocyte apoptosis was evaluated using Annexin V-FITC staining and caspase-3 and caspase-8 activity was determined by Western blotting. The results showed that Dox treatment significantly induced cardiomyocyte apoptosis as evidenced by the greater number of Annexin V-FITC stained cells and increases in caspase-3 and caspase-8 activity. In contrast, overexpression of angiopoietin-1 significantly prevented Dox-induced cardiomyocyte apoptosis. To elucidate the mechanisms by which angiopoietin-1 protected cells from Dox-induced apoptosis, we analyzed both extrinsic and intrinsic apoptotic signaling pathways. We observed that angiopoietin-1 prevented Dox-induced activation of both extrinsic and intrinsic apoptotic signaling pathways. Specifically, angiopoietin-1 prevented DOX-induced in-creases in FasL and Bax levels and cleaved caspase-3 and caspase-8 levels in H9C2 cells. In addition, overexpression of angiopoietin-1 also activated the pro-survival phosphoinositide-3 kinase (PI3K)/Akt signaling pathway and decreased Dox-induced nuclear factor-kappaB (NF-κB) activation. Our data suggest that promoting the expression of angiopoietin-1 could be a potential approach for reducing Dox-induced cardiomyocyte cytoxicity.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13232号 / 論医博第2172号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 江藤 浩之, 教授 木村 剛 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.