Transplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR- 1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti-fibrotic protein, will enhance cardiac myocyte differentiation, inhibit native cardiac apoptosis, and reduce fibrosis in the infarcted myocardium. Furthermore, we assess levels of associated pro- (caspase-3, PTEN) and anti-(Akt) apoptotic proteins as well as a pro-fibrotic protein (MMP-9) in the post-MI and cell transplanted heart. microRNAs (miRs) have emerged as critical regulators of various physiological processes including development, differentiation, metabolism, and death. Indeed, miR- 1 plays an integral role in early cardiac development in Drosophila and mice as well as mediates differentiation of cardiac myocytes in vitro. To that end, we generated ES cells overexpressing miR-1 (miR-1-ES cells), transplanted them into the infarcted myocardium, and evaluated their impact on cardiac myocyte differentiation, myocardial repair, and left ventricular dysfunction post-MI. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared to ES cell and culture media transplanted hearts. Assessment of apoptosis revealed overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-Akt and inhibition of caspase-3, PTEN, and superoxide anion production. A significant reduction iv in interstitial and vascular fibrosis was quantified in miR-1-ES and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1- ES cell and ES cell groups was observed. Finally mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls. Our data suggests miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI ultimately giving rise to enhanced cardiac repair, regeneration, and function. Next, we assessed the role of miR-1-ES cells in a chronic model of MI as research has shown that apoptosis occurs not only hours but months following ischemia. 4 weeks following transplantation into the infarcted myocardium, we provide evidence demonstrating reduced cardiac apoptosis in miR-1-ES cell transplanted hearts compared to respective controls. Moreover, we show significant elevation of p-Akt levels and diminished PTEN levels in hearts transplanted with miR-1-ES cells as determined by enzyme-linked immunoassays. Finally, using echocardiography, we reveal mice receiving miR-1-ES cell transplantation post-MI had significantly improved cardiac function compared with animals transplanted with ES cell and culture media. Our data suggests that miR-1, when overexpressed in transplanted ES cells, has the capacity to inhibit apoptosis long term while attenuating contractility loss. In addition to enhancing cardiac-specific donor cell differentiation, improving the efficacy by which stem cells promote cell survival and repair in the host myocardium is imperative in the pursuit of refining and optimizing stem cell therapy. To that end, we overexpressed TIMP-1, an endogenous inhibitor of apoptosis and fibrosis, in ES cells (TIMP-1-ES cells), transplanted them into infarcted myocardium, and evaluated their v impact on adverse cardiac remodeling. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to assess apoptosis, fibrosis, and heart function. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase in p-Akt activity compared with ES cells or culture media controls. Interstitial and vascular fibrosis was significantly decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key pro-fibrotic protein, was significantly reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP- 1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells. Overall, our data suggest that genetic manipulation of ES cells following transplantation in the infarcted heart enhances cardiac myocyte differentiation, inhibits apoptosis and fibrosis as well as improves cardiac function.

Doxorubicin (DOX) is the antineoplastic drug of preference used to treat a wide variety of malignancies, with high survival rates among treated patients. However, the benefits of this drug have become less appealing due to the side effects that occur such as DOX-induced cardiomyopathy (DIC) and an increased risk of myocardial infarction (MI). Therefore, there is an urgent need to explore the therapeutic options to treat DIC. In this context, adult stem cells have been used as a source to reduce cardiomyocyte apoptosis in DIC; however, the effects of transplanted embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in DIC post MI are unknown. As a result, we wanted to understand how transplanted ES and iPS cells and the factors released by them inhibit apoptosis and improve cardiac function in DIC post MI. C57BL/6 mice were divided into five groups: Sham, DOX-MI, DOX-MI+cell culture (CC) media, DOX-MI+ES cells, and DOX-MI+iPS cells. Mice were treated with DOX (12 mg/kg, cumulative dose) followed by left coronary artery ligation to induce MI. ES or iPS cells (5 x 104 ) were delivered into the peri-infarct region. At day 14 post-MI, echocardiography was performed, mice sacrificed, and hearts harvested for further analyses. To investigate if protective effects are provided by factors released from ES and iPS cells in DIC, we performed in vitro studies using condition media (CM) obtained from ES or iPS cells to treat DOX-induced cardiotoxicity in H9c2 cells. Our data reveal that apoptosis was significantly inhibited in the ES and iPS cell transplanted hearts as well as ESCM and iPSCM treated cells compared with the untreated controls. Furthermore, a significant increase in levels of Notch-1, Hes1, and pAkt survival protein were observed. Decreased levels of PTEN, a negative regulator of Akt pathway, along with improved iv heart function were also observed in the stem cell transplanted groups. In conclusion, our data show that transplantation of ES and iPS cells blunt DOX-induced apoptosis in vivo, which is associated with improved cardiac function. Moreover, decreased apoptosis in both in vitro and in vivo models is mediated by the Notch pathway.

Bakgrund: Hjärtinfarkt är en av de vanligaste förekommande sjukdomarna som framför allt drabbar vuxna och är en av de främsta orsakerna till ökad dödlighet i Sverige och över hela världen. Att uppleva en hjärtinfarkt är en händelse som förändrar livet. Genom att beskriva kvinnors upplevelser av hur deras liv påverkas efter en hjärtinfarkt och kan sjuksköterskor ge personcentrerad vård och hjälpa patienter återställa hälsa och välbefinnande. Syfte: Syftet var att beskriva kvinnors upplevda livssituation efter att ha genomgått en hjärtinfarkt. Metod: En litteraturöversikt där 14 artiklar granskades och analyserades. Resultat: Litteraturöversiktens centrala fynd sammanställdes i följande kategorier. Begränsningar i vardagen, känslor av rädsla och osäkerhet, förlust av identitet, förändringar i relationer och det sexuella livet, och anpassning och acceptans till det nya livet. Sammanfattning: Litteraturöversikten visar att många kvinnor upplever förändringar i sin identitet och självbild till följd av sin hjärtinfarkt eftersom att de måste anpassa sig till de nya livssituationerna och inte längre kan leva som de brukade. Förändringar i relationer och sexliv förekommer också hos många kvinnor som konsekvens av den rädsla och begränsningar de upplever efter en hjärtinfarkten. Många kvinnor har också svårt att anpassa sig till sitt nya liv och hitta en balans på grund av alla dessa faktorer. / Background: Heart attack, or myocardial infarction, is one of the most common diseases that mainly affect adults and is one of the most common causes of mortality in both Sweden and globally. Experiencing a heart attack is a life-changing event. By exploring women’s experiences of how their lives are affected by heart attacks nurses can provide person-centered care and help patients restore their health and well-being. Aim: The aim was to describe women’s lived experiences of their life situations after a heart attack. Method: A literature review where 14 articles were reviewed and analyzed. Results: The central findings of this literature review were compiled in the following categories; limitations in the everyday life, emotions of fear and anxiety, loss of identity, changes in relationships and the sexual life, and adaptation and acceptance of the new life. Summary: This literature review indicates that many women experience changes in their identity and self-image as a result of their heart attack because they have to adapt to new life situation and cannot live as they used to. Changes in relationships and the sexual life are also common among women as consequences of the fear and limitations they experience following their heart attacks. Many women also find it difficult to adapt to a new life and find balance because of all these factors.

Rationale: G protein-coupled receptor (GPCR) kinases (GRKs) are important regulators of cardiac function whose primary role is the phosphorylation of GPCRs and attenuation of downstream signaling, and GRK2 is upregulated after cardiac stress, injury, and during heart failure (HF). Recent studies have identified novel non-GPCR roles for GRKs such as cytoskeletal assembly, insulin signaling, fibrosis and pro-death signaling. One of the most compelling discoveries regarding novel roles of GRK2 in HF pathogenesis is its mitochondrial translocation (mtGRK2) following cardiac injury, which is associated with increased ROS production, decreased FA metabolic oxygen consumption, and pro-death signaling. Moreover, this localization is dependent on phosphorylation of serine 670 (S670) by kinases such as ERK1/2. Of note, expression of a GRK2 C-terminus peptide, βARKct, which contains S670 but no kinase domain or activity, is cardioprotective following injury in part due to inhibition of endogenous mtGRK2. This is significant because growing evidence demonstrates that cardiomyocyte metabolism is regulated by levels and activities of individual mitochondrial proteins Objective: This study sought to identify mitochondrial proteins which GRK2 interacts with either basally or after injury- or stress-induced translocation, and to determine whether functional regulation of mitochondrial function via phosphorylation of these proteins contributes to the phenomenon of bioenergetic defects during the development of HF. Methods and Results: Co-immunoprecipitation of GRK2 in vitro from primary ventricular cardiomyocytes and a human transformed cardiomyocyte-derived cell line was followed by liquid-chromatography mass-spectroscopy identification of all GRK2- interacting proteins. We followed this with proteomics analysis using DAVID and iv Ingenuity software to identify main pathways altered by GRK2 during stress or injury, focusing on mitochondrial dysfunction which revealed that GRK2 interacts with all major components of the electron transport chain (ETC). Using recombinant proteins as well as in vitro and in vivo models of myocardial infarction (MI), we demonstrate that GRK2 is able to phosphorylate the catalytic barrel of mitochondrial ATP synthase (complex V). Moreover, reduction of GRK2 levels in vivo using a GRK2 knockdown mouse model appears to protect against injury-induced bioenergetic deficits, whereas increased GRK2 levels in a transgenic GRK2 overexpression mouse model reveals both baseline deficits in ATP production as well as worsened post-MI outcomes. Conclusions: These collective data highlight the significance of the mitochondrial GRK2 interactome as a driver of cardiac bioenergetic deficits, particularly as a response to MI injury which progresses to HF. Given the current lack of effective HF treatments, this highly novel mechanism of GRK2 regulation of the mitochondrial ETC emphasizes the need for GRK2-targeting therapies for treating HF. / Molecular & Cellular Biosciences

Atherosclerosis is a major cause of cardiovascular disease, which is among the leading causes of death globally. Elevated plasma concentration of low density lipoprotein (LDL) cholesterol is a risk factor for atherosclerosis, while a high plasma level of high density lipoprotein (HDL) cholesterol is considered protective. Uptake of HDL cholesterol by hepatocytes during reverse cholesterol transport, and athero-protective signaling induced by HDL in other cells are mediated by the scavenger receptor class B, type 1 (SR-B1). SR-B1 deficiency in mice that are susceptible to atherosclerosis results in exacerbation of atherosclerosis, and in mice with mutations in apolipoprotein E (apoE), renders mice uniquely susceptible to occlusive coronary artery (CA) atherosclerosis and myocardial infarction. In this thesis, the impact of a lack of SR-B1 on the development of atherosclerosis is characterized in otherwise wild type mice, and in mice that also lack the LDL receptor (LDLR). We demonstrate that after prolonged feeding of a high fat, high cholesterol cholate-containing diet, SR-B1 knockout (KO) mice develop similar levels of diet-induced atherosclerosis to LDLR KO mice and apoE KO mice in traditionally susceptible arteries, and significantly more atherosclerosis in arteries that are typically resistant to plaque development, such as the CAs. SR-B1/LDLR double KO mice develop extensive occlusive CA atherosclerosis accompanied by myocardial infarction, and exhibit reduced iv survival compared to LDLR KO control mice when fed a variety of atherogenic diets. In both SR-B1 single KO and SR-BI/LDLR dKO mice, CA atherosclerosis is accompanied by splenomegaly, elevated numbers of circulating leukocytes and increased expression of VCAM-1 in CA endothelium. Interestingly, removal of the spleen has no effect on circulating leukocyte numbers or atherosclerosis in SR-B1/LDLR dKO mice, suggesting the enlarged spleens in SR-B1 deficient mice do not influence atherosclerosis in these animals. We conclude that SR-B1 is important for the protection against atherosclerosis in mice, particularly in CAs. This is likely through roles in multiple cell types including hepatocytes, endothelial cells and bone marrow-derived cells. Future studies should focus on evaluating the impact of cell-specific SR-B1 activity in different cell types on murine atherosclerotic CA disease. / Thesis / Doctor of Philosophy (PhD)

Bakgrund: Fler män än kvinnor drabbas av hjärtinfarkt, vilket har resulterat i att tidigare forskning kring sjukdomen har baserats på mäns upplevelser. Kvinnor upplever ett större spektrum av symtom och har annorlunda riskfaktorer gentemot män. Tydliga förseningar i kvinnors vårdsökande kan ses. Syfte: Syftet var att belysa kvinnors upplevelser av att drabbas av hjärtinfarkt. Metod: En allmän litteraturstudie genomfördes på 11 artiklar. Innehållet av artiklarna analyserades och teman urskildes. Resultat: Fyra teman identifierades: brist på kunskap, känslan av ansvar, bemötandet av vården och livet efter hjärtinfarkten. I resultatet framkom det att kvinnor upplevde prodromala symtom som var atypiska, vilket försvårade kvinnornas förmåga att identifiera orsaken till symtomen. Kvinnorna prioriterade bort att söka vård, då kvinnorna präglades av känslan av ansvar gentemot sina anhöriga och sitt arbete. Många kvinnor kände sig ignorerade när de till slut sökte vård. Konklusion/implikation: Bristen på kunskap kring kvinnors atypiska symtom kan leda till förseningar i vårdsökande. Genom vidare forskning på kvinnors upplevelser, symtom och riskfaktorer kan kunskap spridas och bidra till att kvinnor får möjlighet till adekvat vård. / Background: More men than women suffer from myocardial infarction, which has resulted in previous research being based on men’s experiences. Women experience a greater spectrum of symptoms and have different risk factors than men. A distinct delay can be seen when women seek care. Aim: The purpose was to shed light on women's experiences when suffering a myocardial infarction. Method: A general literature study approach was conducted on 11 articles. The content of the articles was analyzed, and themes were distinguished. Result: Four themes were identified: lack of knowledge, feeling of responsibility, treatment in healthcare services and life after a myocardial infarction. The result showed that women experienced prodromal symptoms that were atypical, which complicated women’s ability to identify the cause of the symptoms. The women did not prioritize seeking care, as the women were influenced by the feeling of responsibility towards their relatives and work. Many women felt ignored when they finally sought care. Conclusion/implication: The lack of knowledge about women's atypical symptoms can lead to delays in seeking care and diagnosis. Through further research into women's experiences, symptoms and risk factors, knowledge can be spread and contribute to women having the opportunity for adequate care.

The underlying pathological process of coronary artery disease (CAD) is the development of coronary artery atherosclerotic occlusions and associated myocardial infarction. Both increased chronic inflammation and plasma low-density lipoprotein (LDL) cholesterol levels promote atherosclerosis. Inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) is widely known for its role in enhancing LDL receptor (LDLR)-mediated cholesterol lowering when the LDLR-apolipoprotein E (APOE) axis is intact and protecting against atherosclerosis progression by reducing plasma cholesterol levels. In this thesis, we sought to test the effects of PCSK9 inhibition mediated cholesterol lowering on pre-existing CAD as well as the plasma cholesterol independent effects of PCSK9 inhibition on CAD by utilizing different mouse models. One year old scavenger receptor class B type I (Sr-b1) knockout (KO) mice which have an intact LDLR-APOE axis, develop coronary artery atherosclerosis and myocardial fibrosis induced by a high fat, high cholesterol and cholate containing (HFCC) diet. Weekly anti-PCSK9 antibody treatment initiated one week before switching to an HFCC diet increased hepatic LDLR protein levels, and reduced plasma cholesterol levels and the progression of atherosclerosis in both the aortic sinus and coronary arteries in one year old Sr-b1 KO mice (maintained on an HFCC diet for 7 weeks). Weekly anti-PCSK9 antibody treatment initiated 7 weeks after switching to an HFCC diet also increased hepatic LDLR protein levels and reduced plasma cholesterol levels in one year old Sr-b1 KO mice (maintained on an HFCC diet for 12 weeks). More importantly, anti-PCSK9 antibody treatment during the last 5 weeks of the 12-week HFCC diet feeding period also slowed down the growth of pre-existing atherosclerosis in both the aortic sinus and coronary arteries and reduced myocardial fibrosis and damage. Mice deficient in both Sr-b1 and ApoE (Sr-b1/ApoE double KO (dKO) mice) spontaneously and rapidly develop features reminiscent of human CAD. Whole body Pcsk9 genetic KO in both female and male Sr-b1/ApoE dKO mice did not affect plasma cholesterol levels despite increased hepatic LDLR protein levels, presumably due to the lack of APOE. However, genetic Pcsk9 inactivation significantly attenuated atherosclerosis in both the aortic sinus and coronary arteries, myocardial fibrosis and damage, left ventricle (LV) dysfunction and cardiac enlargement in both female and male Sr-b1/ApoE dKO mice. Restoring circulating PCSK9 by a recombinant adeno associated virus 8 (AAV8)-mediated hepatic expression of a Pcsk9 cDNA in Pcsk9/Sr-b1/ApoE triple KO mice reversed the plasma cholesterol independent protective effects of genetic PCSK9 KO on aortic sinus and coronary artery atherosclerosis and myocardial fibrosis and damage in both females and males. Treatment of Sr-b1/ApoE dKO mice with an anti-PCSK9 antibody which disrupts the interaction between the LDLR and PCSK9 protected against aortic sinus and coronary artery atherosclerosis in males but not in females and did not protect either males or females against myocardial fibrosis and damage, LV dysfunction or cardiac enlargement. My thesis demonstrates that anti-PCSK9 antibody mediated plasma cholesterol lowering delays the continued development of pre-existing CAD. My thesis also demonstrates that liver-derived, circulating PCSK9 promotes CAD in a plasma cholesterol independent manner in Sr-b1/ApoE dKO mice and these effects appear to be largely independent of the PCSK9-LDLR interaction, particularly in females. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24834号 / 医博第5002号 / 新制||医||1067(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 竹内, 理, 教授 上杉, 志成, 教授 金子, 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The purpose of this thesis investigation was to evaluate the effectiveness of dynamic strength training as an additional mode of exercise rehabilitation, in patients with coronary artery disease and well documented evidence of a previous myocardial infarction. The effects of 10 weeks (20 sessions) of combined weightlifting and aerobic training (WtAer) (n=10) were compared with aerobic training (Aer) (n=8) alone, on indices of strength and aerobic exercise capacity in 18 male patients with coronary artery disease (CAD). Initial test(s) performance was similar between groups. Post Aer, the maximum weightlifting strength (1RM) in single-arm curl, single-leg press, and single-knee extension exercises increased by 13% (x = 11.8 to 13.3 kg; P < 0.01), 4% (x = 97 to 101 kg; N.S.), and 5% (x = 28.2 to 29.7 kg; N.S.) respectively; corresponding gains with AerWt were 43% (x = 12.2 to 17.4 kg; P < 0.01), 21% (x = 99 to 120 kg; P <0.01) , and 24% (x = 29 to 36 kg; P < 0.01) . Following Aer the initial 1RM could be lifted an average of 4 times, compared to 14 times after AerWt. Neither Aer nor AerWt showed significant improvements in peak torque in either isokinetic single-knee extension at 90°/s and 180°/s or single-leg press exercise at 30°/s and 75°/s. Maximum progressive incremental cycle ergometer performance (Wmax)increased by 2% with Aer (x = 1088 to 1113 kpm/min; N.S.) and by 15% (x = 1030 to 1180 kpm/min; P < 0.05) with AerWt.Cycling time at 80% of initial Wmax before attaining a Borg RPE of 7 for the legs, increased by 11% (x = 604 to 672s; N.S.) and by 109% (x = 541 to 1128s; P < 0.05) with Aer and AerWt respectively. In these patients with CAD, AerWt was a more effective method of increasing aerobic performance and strength than Aer alone. In order for cardiac exercise rehabilitation therapy to optimize the strength and functional capacities of CAD patients it may be useful to incorporate appropriately monitored weightlifting training into the traditional aerobic exercise regimen. / Thesis / Master of Science (MS)

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