The objective of the current study is to present the case of an intercollegiate basketball player diagnosed with an ifliopathic cardiomyopathy. The athlete illustrated in the case study experienced ongoing chest discomfort with exercise. Episodic chest pain and syncope in athletes is often alarming and may signal an underlying cardiac condition. Early recognition and maJagement of these athletes is vital to the prevention of sudden cardiac death (SCD). Fortunately, the athlete was referred to a cardiologist by her team physician during her pre-participation physical examination to rule out heart conditions that may lead to SCD. The athlete was presented a mildly reduced ejection fraction during her screening with the cardiologist. In cardiovascular physiology, ejection fraction is the fraction of end-diastolic volume that is ejected from the ventricle with each heart beat. Damage to the myocardium, as seen in cardiomyopathies, decreases the heart's ability to eject blood and therefore reduces the ejection fraction. The athlete underwent VO2max testing as well where it was discovered that her VO2max was exceptionally low for a physically active division-I athlete. This low VO2max suggested that the athlete had an insufficient oxygen uptake during intense exercise. The athlete experienced a treatment protocol consisting of a progressive conditioning regimen of additional cardiovascular exercise that proved to be effective in raising her VO2max by 10%. The athlete returned to full participation and remained asymptomatic throughout the remainder of the season. The pre-participation physical examination is crucial in early detection of events that may lead to sudden cardiac death. A thorough history and physical examination are the most efficient screening methods for detecting cardiovascular abnormalities. Any athlete with episodes of syncope, hypertension, or changes in heart rhythm should be referred to a physician.

Objective The central Appalachian region of the United States is disproportionately burdened with cardiovascular disease (CVD); however, the level of literacy about CVD among residents has not been well studied. This study aimed to examine the prevalence and factors of CVD health literacy (HL) among a sample of patients/caregivers, providers/professionals, and community stakeholders. Methods In 2018, data were collected from central Appalachian residents in six states: Kentucky, North Carolina, Ohio, Tennessee, Virginia, and West Virginia. CVD HL status was determined by assessing correct responses to five interrelated questions about basic knowledge of CVD. Multiple logistic regression analyses were used to examine the associations between potential factors and CVD HL status. Results Of the total respondents (N = 82), <50% correctly answered all 5 CVD HL questions. Multiple logistic regression analyses showed that compared with respondents with advanced college degrees, those with a college degree or less were more likely to have low HL for "typical symptom of CVD,""physical exercise and CVD,""blood pressure and CVD,"and "stress and CVD."In addition, compared with respondents younger than 50 years, those 50 years and older were 3.79 times more likely to have low HL for "physical exercise and CVD."Conclusions These results suggest the incorporation of CVD HL into CVD care and that educational attainments should be part of CVD policies and programs in the region.

Cardiovascular disease is a leading cause of death in the United States. Toll-like receptor (TLR)-mediated pathways have been demonstrated to play a role in myocardial ischemia/reperfusion (I/R) injury. We and others have shown that PI3K/Akt signaling is involved in regulating cellular survival and protecting the myocardium from I/R induced injury. In this dissertation, we provide compelling evidence that miR-125b serves to “fine tune” TLR mediated NF-kB responses by repressing TNF-a and TRAF6 expression. We constructed lentiviral expressing miR-125b, delivered it into the myocardium. The data showed that delivery of lentivirus expressing miR-125b significantly reduces myocardial infarct size and improves cardiac function in I/R hearts. Mechanistic studies demonstrated that miR-125b negatively regulates TLR mediated NF-kB activation pathway by repressing TNF-a and TRAF6 expression in the myocardium. We also observed that transfection of the myocardium with lentivirus expressing miR-214 markedly attenuates I/R induced myocardial infarct size and cardiac dysfunction. We demonstrated that miR-214 activates PI3K/Akt signaling by targeting PTEN expression in the myocardium. We also investigated the role of TLR3 in neonatal heart repair and regeneration following myocardial infarction (MI). Wild type (WT) neonatal mice showed fully cardiac functional recovery and small infarct size, while TLR3 deficient mice exhibited impaired cardiac functional recovery and large infarct area after MI. Poly (I:C), a TLR3 ligand, administration significantly enhances glycolysis, YAP1 activation and the proliferation of WT neonatal cardiomyocytes. 2-deoxyglucose (2-DG), a glycolysis inhibitor treatment abolished cardiac functional recovery and YAP1 activation in neonatal mice after MI. In vitro either inhibition of glycolysis by 2-DG or inhibition of YAP1 activation prevents Poly (I:C) induced YAP1 activation and neonatal cardiomyocyte proliferation. Importantly, YAP1 activation increases miR-152 expression, leading to cardiomyocyte proliferation through suppression P27kip1 and DNMT1 expression. We conclude that microRNAs play an important role in TLR modulation induced protection against myocardial I/R injury by increasing the activation of PI3K/Akt signaling pathway, decreasing TLR/NF-kB mediated inflammatory response, and suppressing activation of apoptotic signaling following myocardial I/R injury. In addition, TLR3 is an essential for neonatal heart repair and regeneration after myocardial infarction. TLR3 modulation could be a novel strategy for heart regeneration and repair.

Paget's disease of the bone is characterized by a focal increase in the rate of bone turnover, which goes through phases of activity and quiescence. Most patients are asymptomatic. The two cardinal features are pain and deformities, and many complications may arise. Diphosphonates and calcitonin are the main therapeutic modalities.

In the United States, individuals of Hispanic ethnicity receive disproportionately lower-quality healthcare. These healthcare disparities exacerbate unequal access to quality healthcare services, including disparities in cardiovascular disease (CVD) and human immunodeficiency virus (HIV) care. Research on the role of ethnicity on the CVD outcomes of people living with HIV (PLWH) has been limited. We hypothesize that immunological (CD4+ cell count) and virological (HIV viral load) control may play a role in the development of acute myocardial infarction (AMI) among PLWH, and that Hispanic ethnicity may worsen these outcomes. To verify our hypotheses, we conducted a retrospective cross-sectional study to investigate the strength and direction of association between CD4+ cell count (immunological cohort, n=513) and HIV viral load (virological cohort, n=261) on AMI among respondents of the All of Us Research Program. Hispanic and non-Hispanic respondents for both cohorts were comparable in terms of demographic characteristics, except for a significantly different distribution by race. While we identified increased proportion of non-Hispanic individuals with AMI in the immunologic (6.0% vs. 1.0%; P=0.04) and virologic (5.8% vs. 0%; P=0.007) cohorts, we were not able to identify CD4+ cell count or viral load as significant predictors significantly increasing the likelihood of AMI. Potential explanations discussed include self-selection bias resulting in incomplete laboratory data and an underpowered sample size. While the sample in this study did not support an increased likelihood of AMI by ethnicity, the results should be interpreted carefully in light of the limitations and the established pathophysiological and epidemiological associations posited, underscoring the importance of future research efforts that better represent ethnic minorities and the associations between HIV infection and CVD.

Background: People from ethnic minority immigrant groups living in host countries are known to have higher risk factors for cardiovascular diseases. The role of acculturation, or assimilation into a different and dominant culture, is often studied from social and medical views when focusing on individuals diagnosed with cardiovascular diseases and their recovery after cardiac events. However, the effects of the complex roles of acculturation and religious coping on these individuals are rarely considered in the research. There is limited knowledge regarding the complex roles of acculturation and religious coping in adopting healthy lifestyle behaviours and managing stress among individuals with cardiovascular diseases from Arab communities in the Ottawa region. This research project’s general purpose was to explore and understand the complex roles of acculturation and religious coping through the experiences of individuals diagnosed with CVD from Arab communities in the Ottawa region. Objectives: The specific objectives were to 1) identify and understand the challenges among Arab immigrants related to acculturation and religiosity in adopting healthy lifestyle behaviours and managing stress; 2) measure and describe the levels of religious beliefs as well as religious coping strategies, acculturation, perceived stress, and healthy lifestyle behaviours among Arab individuals living in the region of Ottawa, Ontario, Canada who have been diagnosed with cardiac events or who are at high risk for cardiovascular diseases; and 3) explore the role of acculturation and religious coping in shaping male Arab individuals’ lived experiences after a cardiac event and to explore their ways of understanding lifestyle behaviours and cardiac rehabilitation during recovery. Methods: A mixed-method approach was adopted in this research, which included three separate studies: Study 1 was a qualitative study (views of key informants with first-hand knowledge) in which three face-to-face focus groups were conducted with 17 Arab health promoters; Study 2 was a cross-sectional survey study was conducted with 63 individuals from local Arab communities who had been diagnosed with cardiac events or who were at high risk for cardiovascular diseases; and Study 3 consisted of a phenomenographic qualitative study, semi-structured in-depth interviews with male Arab individuals (N=10), selected from Study 2, who identified themselves as having had cardiac events while living in Canada. Results: From the perspective of Arab health promoters, there was an overlapping between various aspects of acculturation and religious beliefs that may have impacted the healthy lifestyle of Arab immigrants. These challenges were coded in four themes: “Culture first!”: dominant influence of home country culture; “Religiosity alone does not make you healthy!”: limited religious influence; “It is not easy!”: difficulties adapting to the Canadian lifestyle; and “We are not young!”: generational differences in adopting a healthy lifestyle. Findings from the survey study indicated that most participants were oriented more toward their Arabic culture than Canadian culture. Participants tended to be religious, and their nutritional behaviours were healthier than physical activity behaviours. However, age, gender, and interestingly, length of time living in Canada did not affect the participants’ results in any of the questionnaires. Based on lived experiences of 10 participants in the interview study, five core themes were identified: “Stressful events or cardiac events!”: acculturative stress effects; “It was a dreamlike event!”: dismissing perceptions of cardiac events; “recognizing risk factors is not enough to avoid them”: perceived threat; religious coping outcomes: satisfaction and fatalism; Cardiac Rehabilitation programs: who refers and who participates. Conclusion: This dissertation showed that acculturation level plays an essential role in Arab immigrants’ beliefs and behaviours regarding their health status and their experiences in preventing cardiovascular diseases risk factors or in recovery after cardiac events. Religious coping seems to be a way for less acculturated Arab immigrants to manage stress and mental burdens and find internal peace and satisfaction. From the perspective of Arab health promoters, some religious or cultural beliefs may be barriers to engaging in physical activity, especially for women and older people, and these barriers may be exacerbated by acculturative stress. Religiosity may also play an essential indirect role in managing stress through socialization, family support, and the adoption of coping strategies. Arab individuals living in the region of Ottawa, Ontario, Canada, who have been diagnosed with cardiac events or who are at high risk for cardiovascular diseases may have been more religious and less acculturated in Canadian society. Their lifestyle health behaviours related to physical activity and nutrition may have been influenced by their health status, religious beliefs, and the practices or traditions of their culture of origin. Stress and mental burdens while living in Canada reflected negatively on Arab male individuals’ experiences with cardiac events. Stress was perceived as a potential cause of cardiac events and a factor leading to low self-efficacy in changing lifestyle behaviours. There is a need to promote healthy lifestyle messages and raise awareness about cardiovascular diseases risk factors among Arab communities. Future research is needed to design culturally adapted cardiac rehabilitation programs for Arab individuals and to evaluate the effectiveness of interventions with both physical and mental health components.

Cardiovascular disease is a major cause of morbidity and mortality around the globe, which is linked to athero-thrombosis. The risk factors for atherothrombosis, thus cardiovascular disease is impaired anti-thrombotic and antiinflammatory functions of the endothelium. Thrombosis is a hallmark of cardiovascular disease/complications characterised by increased platelet activation and increased secretion of platelet micro-particles that induce angiogenesis. This study determined the role of platelet micro-particles derived microRNA in the regulation of angiogenesis and migration, with a focus on the regulation of thrombospondin-1 release by platelet micro-particles delivered Let- 7a. The role of thrombospondin-1 receptors (integrin beta-1 and integrin associated protein) and downstream caspase-3 activation were explored by Let-7a inhibition prior to PMP treatment. MicroRNA dependent modulation of proangiogenic proteins including monocyte chemoattractant protein-1 and placental growth factor, and recruitment of activating transcription factor-4 protein to their promoter regions were explored. Main findings are: 1. Platelet micro-particles induce angiogenesis, migration, and release of novel cytokine subsets specific to platelet micro-particle’s RNA content. 2. The targeting of thrombospondin-1 mRNA by platelet micro-particles’ transferred Let-7a chiefly modulate the angiogenic effect on endothelial cells. 3. The inhibition of thrombospondin-1 translation enable platelet micro-particles to increase angiogenesis and migration in the presence of functional integrin beta-1 and integrin associated protein, and reduced cleaving of caspase-3. 4. Platelet micro-particle modulate the transcription of monocyte chemoattractant protein-1 and placental growth factor in a Let-7a dependent manner. 5. Let-7a induce angiogenesis ii independent of other platelet micro-particle’s microRNAs. Platelet micro-particle derived Let-7a is a master regulator of endothelial cell function in this model, which presents an opportunity for the development of new biomarkers and therapeutic approaches in the management of cardiovascular disease. Future studies should aim to confirm these findings in-vivo.

<p>  </p> <p>Heart disease is the leading cause of death for individuals with Duchenne muscular dystrophy (DMD). DMD is a devastating and progressive neuromuscular disease with no known cure. This X-linked genetic disorder affects nearly 1 in 5000 boys and manifests as debilitating muscle weakness and progressive cardiomyopathy (CM). While CM in some individuals with DMD progresses rapidly and fatally in their teenage years, others can live relatively symptom-free into their thirties or forties. Early identification and treatment can improve quality and length of life, but currently, there are no standard imaging biomarkers that can detect early onset or rapidly progressing DMD CM. Addressing this gap, we describe here a novel cardiac image analysis paradigm using 4D cardiac magnetic resonance imaging (CMR) to map left-ventricular kinematics comprehensively in DMD CM. The primary goal of this dissertation work is to introduce novel imaging biomarkers and computational methods to enable earlier diagnosis and precise prognosis for cardiac function in DMD. Central to this goal, we identified myocardial strain biomarkers that predict the early onset and rapid progression of cardiac disease in vulnerable patients. These findings bridge clinical gaps and pave the way for multi-center studies to characterize DMD CM progression and assessment of individual patient risk profiles for improved treatment and outcomes in DMD.</p>

Fetal Alcohol Spectrum Disorders (FASDs) refer to a set of development abnormalities affecting a fetus that can result from prenatal alcohol exposure (PAE). Studies performed by the National Institute of Health estimate that the pervasiveness of FASDs may number as high as 1 to 5 per 100 school children. Congenital heart defects (CHDs) are a subset of these abnormalities and have been observed to occur in 38% of children with FASDs. While there is an association between PAE and CHDs, the exact molecular mechanism as to how it occurs remains unclear. A 2022 RNA sequencing study points to the Wnt7a gene as one of several others to have its mRNA expression significantly decrease in the heart in response to PAE at a critical developmental time point. This gene codes for the Wnt7a protein that can play a role in activating the Wnt signaling pathways. This critical pathway plays a role in cell differentiation, cell proliferation, morphogenesis, embryonic development, and adult tissue homeostasis. While mRNA expression in response to alcohol has been studied, the change in protein expression of Wnt7a is yet to be determined. This thesis serves to demonstrate assay techniques and antibodies that can be used to reliably detect Wnt7a protein. We hypothesize that Wnt7a protein expression can be reliably detected in Human Embryonic Kidney 293 (HEK 293) cells through Western blot and immunofluorescence assays when the protein is overexpressed. Fluorescent, and more effectively, chemiluminescent western blot procedures produced a positive signal for detection of Wnt7a protein at the expected 40-42 kDA molecular weight range. Through Immunofluorescence, the ii Wnt7a+ HEK cells were confirmed to express the protein through Alexa Fluor probing of an anti-Wnt7a antibody, and the DYK HEK cells failed to produce a signal for expression of the protein as expected. The methods and techniques used in this study can be used to detect Wnt7a protein in embryonic hearts and determine how much it is affected by alcohol exposure. This serves toward the larger goal of identifying Wnt7a as a potential biomarker for helping to diagnose alcohol-induced CHDs. Additionally, these future directions can help direct attention to this gene as a useful therapeutic target for preventing and treating CHD formation.

Kardiale Arrhythmien können zum plötzlichen Herzstillstand (SCD) bei Herzinsuffizienz (HF)-Patienten führen und stellen in diesen Zusammenhang die häufigste Todesursache (> 50 %) dar. Aufgrund der schlechten Prognose werden weitere Therapiemöglichkeiten und Wirkstoffe benötigt. Die Phosphodiesterase 2 (PDE2A), ein cAMP und cGMP hydrolysierendes Enzym, ist im Endstadium bei HF-Patienten hochreguliert. Die PDE2A zeichnet sich durch die Möglichkeit aus, durch cGMP allosterisch aktiviert zu werden, welches die PDE2A Hydrolyseaktivität vor allem gegenüber cAMP stark steigert. In vorangegangenen Studien konnte bereits gezeigt werden, dass die herzspezifische PDE2A3-Überexpression in Mäusen eine starke anti-arrhythmische Wirkung vermittelt. Andere Studien wiederum zeigten, dass eine pharmakologische Hemmung der PDE2A dem hypertrophen Wachstum von Kardiomyozyten entgegenwirkt. Das Ziel dieser Arbeit bestand darin, die Rolle der PDE2A für die Herzfunktion unter physiologischen und pathophysiologischen Bedingungen mit Hilfe eines herzspezifischen PDE2A Knockout Modells zu untersuchen. Weiterhin war das Ziel den PDE2A-vermittelten cGMP/cAMP-Crosstalk-Mechanismus unter Verwendung des natriuretischen Peptids Typ C (CNP) in frisch isolierten murinen sowie humanen von induzierten pluripotenten Stammzellen abgeleiteten Kardiomyozyten (hiPSC-KM) zu charakterisieren, um potentielle anti-arrhythmogene kardioprotektive Wirkungen der cGMP-vermittelten PDE2A Stimulation aufzudecken. Zunächst wurde ein neues Mausmodell mit Kardiomyozyten-spezifischer Deletion der PDE2A (PDE2A-KO) unter der Verwendung der „knockout-first“ Strategie generiert und mittels Echokardiographie charakterisiert. Das Fehlen der PDE2A Expression in Kardiomyozyten führte zu keinem veränderten kardialen Phänotyp. Unter chronischen Stressbedingungen nach Isoprenalininfusion (osmotische Minipumpe, 14 Tage) zeigte der PDE2A-KO eine verminderte Kontraktion und Auswurffraktion im Vergleich zu den behandelten Kontrolltieren. Herzfrequenz und Hypertrophie unterschieden sich dabei nicht voneinander. Um den Einfluss der CNP-vermittelten PDE2A Aktivierung auf pro-arrhythmische intrazelluläre Signale zu untersuchen, wurden spontane lokale Ca2+ Freisetzungen aus dem SR (Ca2+ Sparks, CaSp) sowie arrhythmische Ca2+-Wellen (CaW) sowie die Kontraktion von isolierten ventrikulären und atrialen Kardiomyozyten quantifiziert. Interessanterweise reduzierte CNP signifikant die Anzahl an arrhythmogenen CaSp und CaW nach akuter β-adrenerger Stimulation, ohne dabei die zelluläre Kontraktilität zu beeinflussen. Dieser anti-arrhythmogene Effekt wurde in den PDE2A-KO Tieren nicht beobachtet bzw. wurde durch die spezifische PDE2A-inhibition mittels Bay 60-7550 vollständig aufgehoben. Eine simultane Hemmung der cGMP-aktivierten Proteinkinase G hatte keinen Einfluss auf die Anzahl der CaSp. Zusätzlich zeigten Versuche mit isolierten Kardiomyozyten von Mäusen mit herzspezifischen PDE2A3-Überexpression eine deutlich verminderte Anzahl an CaSp nach akuter β-adrenerger Stimulation. Dieser protektive Effekt wurde bei spezifischer PDE2-Hemmung verhindert. Während die alleinige Inhibition der PDE2 keinen Einfluss auf die CaSp Frequenz hatte, steigerte die Hemmung der PDE3 und PDE4 die Anzahl der CaSp in Kardiomyozyten von Kontrolltieren signifikant. Von besonderer Bedeutung ist, dass der CNP/cGMP/PDE2A-vermittelte anti-arrhythmogene Effekt auch in humanen ventrikulären sowie atrialen iPSC-KMs reproduziert werden konnte. Auf zellulärer Ebene wurden die stressinduzierten Ca2+-Lecks in humanen Zellen durch die CNP-vermittelte PDE2A-Stimulation unterdrückt. CNP vermittelte auf zellulärer Ebene starke anti-arrhythmogene Effekte über die Stimulation der PDE2A. Interessanterweise war die Expression von CNP in humanem ventrikulärem Gewebe von Patienten mit Herzinsuffizienz signifikant vermindert, während ANP und BNP hochreguliert waren und damit eine Desensitisierung ihrer Rezeptoren vermitteln könnten. In isolierten murinen Kardiomyozyten induzierte die CNP-vermittelte PDE2 Aktivierung eine signifikante Verminderung der intrazellulären cAMP Level. Die Erkenntnisse aus dieser Arbeit unterstreichen deutlich die klinische Relevanz der PDE2A als potenzielles therapeutisches Ziel bei ventrikulären Herzrhythmusstörungen sowie bei Vorhofflimmern nach akuter Stressstimulation oder bei HF weiter zu untersuchen. Zusammenfassend wurde in dieser Arbeit gezeigt, dass PDE2A eine entscheidende Rolle in der Unterdrückung akuter stressinduzierter arrhythmogener Ca2+-Lecks spielt. Daher könnte die pharmakologisch gesteigerte PDE2A-Aktivität eine neue kardioprotektive Strategie zur Therapie von Arrhythmien bei HF darstellen.:Abkürzungsverzeichnis VI Abbildungsverzeichnis XII Tabellenverzeichnis XVI 1. Einleitung 1 1.1. Herzinsuffizienz und kardiale Arrhythmien 1 1.1.1. Ätiologie und Epidemiologie 1 1.1.2. Pathophysiologie und Klassifikation kardialer Arrhythmien bei HF 3 1.1.3. Zellulärer und molekularer Aspekt 7 1.1.4. Pharmakologische Therapie 9 1.2. Kardiale Signaltransduktion und sekundäre Botenstoffe 11 1.2.1. cAMP-Signalkaskade 12 1.2.2. cGMP-Signalkaskade 13 1.2.3. Natriuretische Peptide und deren klinische Relevanz 14 1.3. Kardiale Phosphodiesterasen 15 1.3.1. Kardiale PDEs mit dualer Substratspezifität 17 1.3.2. Kardiale cAMP und cGMP-spezifische PDEs 18 1.4. Phosphodiesterase 2A 19 1.4.1. Molekularer Aspekt 19 1.4.2. PDE2A-Expression und Aktivität 21 1.4.3. Perspektiven hinsichtlich der Rolle von PDE2A bei Herzerkrankungen 22 2. Zielsetzung 24 3. Material und Methoden 25 3.1. Material 25 3.2. Tiermodel 25 3.2.1. Tierhaltung 25 3.2.2. C57BL/6JRj Wildtyp-Mausmodel 25 3.2.3. Herzspezifische PDE2A-KO-Mausmodel (PDE2A_fl_αMHC_cre) 26 3.2.4. Genotypisierung 27 3.3. Zellkultur und Differenzierung der humanen induced pluripotent stem cell-derived Kardiomyzyten (iPSC-KM) 30 3.4. Gewinnung Patientengewebe 31 3.5. Echokardiografische Langzeitcharakterisierung 32 3.6. Implantierung osmotischer Minipumpen 34 3.7. Zellisolation adulter Mauskardiomyozyten 35 3.7.1. Isolierung ventrikulärer Kardiomyozyten 35 3.7.2. Isolierung atrialer Kardiomyozyten 36 3.8. Kontraktilitätsmessung 37 3.9. Calcium-Imaging 38 3.10. Calcium-Spark Detektion in Kardiomyozyten 41 3.10.1. Calcium-Spark Detektion in Mauskardiomyozyten 41 3.10.2. Calcium-Spark Detektion in humanen iPSC-KM 42 3.11. Proteinexpressionsanalyse aus Mausatrium 42 3.11.1. Proteinisolation und Konzentrationsbestimmung 42 3.11.2. Immunoblot 43 3.12. ELISA 45 3.13. qPCR 45 3.14. Statistische Auswertung 46 4. Ergebnisse 47 4.1. Einfluss des PDE2A-Knockouts auf die Herzfunktion 47 4.1.1. Echokardiografische Charakterisierung im Alter von 3 und 6 Monaten 47 4.1.2. Auswirkung einer chronischen Aktivierung der β-adrenergen Signalkaskade auf die Herzfunktion 51 4.2. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf den intrazellulären Ca2+-Haushalt in PDE2A-Knockout-Kardiomyozyten nach Stressinduktion 56 4.2.1. Auswirkung auf den intrazellulären Ca2+-Zyklus 56 4.2.2. Auswirkung auf den intrazellulären Ca+-Speicher, die fraktionelle und spontane Ca2+-Freisetzung aus dem sarkoplasmatischen Retikulum 60 4.2.3. Auswirkung auf die Geschwindigkeit der Ca2+-Freisetzung und der Ca2+-Wiederaufnahme 63 4.3. Einfluss der cGMP-vermittelten PDE2A-Stimulation durch CNP auf die Kontraktilität ventrikulärer Kardiomyozyten 65 4.3.1. Einfluss auf die zelluläre Kontraktion 65 4.3.2. Einfluss auf die zelluläre Kontraktionsgeschwindigkeit und Relaxationsgeschwindigkeit 67 4.3.3. Einfluss des PDE2A-Knockouts auf die zelluläre Kontraktion 69 4.4. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch natriuretische Peptide auf die spontane Ca2+-Freisetzung (Ca2+-Sparks) in ventrikulären Kardiomyozyten nach Stressinduktion 72 4.4.1. Veränderung der Ca2+-Spark-Frequenz nach CNP-Stimulation 72 4.4.2. Veränderung der Spark-Morphologie nach CNP-Stimulation 73 4.4.3. Veränderung der Ca2+-Spark-Frequenz nach ANP-Stimulation 75 4.4.4. Veränderung der Spark-Morphologie nach ANP 76 4.4.5. Veränderung der Calcium-Spark-Frequenz nach PKG-Inhibition 78 4.4.6. Veränderung der auf Spark-Morphologie nach PGK-Inhibition 80 4.5. Einfluss der cGMP-vermittelten PDE2A-Stimulation durch CNP auf die intrazellulären cAMP-Konzentration in ventrikulären Kardiomyozyten 82 4.6. Auswirkung des Myokardinfarkts auf die murine Genexpression der PDE2A, des CNP und der natriuretischen Peptidrezeptoren 83 4.7. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf den intrazellulären Ca2+-Haushalt atrialer Kardiomyozyten nach Stressinduktion 84 4.7.1. Auswirkung auf den intrazellulären Ca2+-Zyklus 84 4.7.2. Auswirkung auf den intrazellulären Ca2+-Speicher, die fraktionelle und spontane Ca2+-Freisetzung aus dem Sarkoplasmatischen Retikulum 87 4.7.3. Auswirkung auf die Geschwindigkeit der Ca2+-Freisetzung und der Ca2+-Wiederaufnahme 89 4.7.4. Einfluss des PDE2A-Knockouts auf den Ca2+- Zyklus 90 4.8. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf spontane Ca2+-Freisetzung (Ca2+-Sparks) in atrialen Kardiomyozyten 95 4.8.1. Veränderung der Calcium-Spark-Frequenz 95 4.8.2. Veränderung der Spark-Morphologie 96 4.9. Auswirkung des PDE2A-KO auf die Expression Ca2+-regulierender Proteine 98 4.10. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf die spontane Ca2+-Freisetzung (Ca2+-Sparks) in humanen iPSC-Kardiomyozyten nach Stressinduktion 102 4.10.1. Veränderung der Ca2+-Spark-Frequenz nach CNP-Stimulation 102 4.10.2. Auswirkung der CNP-Stimulation auf die Ca2+-Spark-Morphologie 104 4.10.3. Bedeutung der PDE3 und PDE4 Inhibition auf Ca2+-Sparks in ventrikulären hiPSC-KM 106 4.10.4. Einfluss der PDE3 und PDE4 Inhibition auf die Ca2+-Spark-Morphologie 108 4.10.5. Bedeutung der CNP-Stimulation auf Ca2+-Sparks in atrialen hiPSC-KM nach Stressinduktion 109 4.10.6. Einfluss der CNP-Stimulation auf die Ca2+-Spark-Morphologie nach Stressinduktion 111 4.11. Auswirkung dilatativer Kardiomyopathie auf die Genexpression natriuretischer Peptide und deren Rezeptoren 112 5. Diskussion 114 5.1. Der herzspezifische PDE2A-Knockout 115 5.1.1. Basale Herzfunktion konditionaler PDE2A-KO Mäuse 115 5.1.2. Cre-Expression beeinträchtigt die Überlebensfähigkeit der PDE2A-KO Mäuse 116 5.1.3. Der PDE2A-KO führt zur Reduktion der Herzfunktion bei chronischer Stressstimulation 117 5.2. Die CNP-vermittelte PDE2A-Stimulation reduziert arrhythmogene Ca2+-Freisetzungen ohne den intrazellulären Ca2+-Zyklus oder die zelluläre Kontraktion zu beeinflussen 119 5.2.1. PDE2A-Stimulation reduziert stressinduzierte arrhythmogene Ca2+-Wellen 119 5.2.2. Die zelluläre Kontraktilität wird durch die CNP-vermittelte PDE2A-Stimulation nicht beeinträchtigt 121 5.2.3. Natriuretische Peptid-vermittelte PDE2A-Stimualtion unterdrückt die stressinduzierten Ca2+-Lecks aus dem SR 122 5.3. CNP vermittelt einen anti-arrhythmogenen Effekt in humanen iPSC-Kardiomyozyten 124 5.4. Limitationen 125 5.4.1. Einschränkungen des Cre/loxP-Systems beim PDE2A-KO 125 5.4.2. Crosstalk-Mechanismen zyklischer Nukleotide 126 5.4.3. Patientenbasierte Variabilität 127 5.5. Ausblick 128 5.6. Die klinische Relevanz der PDE2A 129 6. Zusammenfassung 131 7. Summary 133 8. Referenzen 135 9. Anhang 162 9.1. Chemikalienliste 162 9.2. Antikörper- und Primerliste 164 9.3. Kits und Verbrauchsmaterialien 166 10. Danksagung 172 11. Erklärung 173 / Cardiac arrhythmias can lead to sudden cardiac death (SCD) in heart failure (HF) patients and are the most common cause of death (> 50%) in this context. Due to the poor prognosis, additional therapeutic options and agents are needed. Phosphodiesterase 2 (PDE2A), an enzyme that hydrolyzes cAMP and cGMP, is upregulated in end-stage HF patients. PDE2A can be allosterically activated by cGMP, which significantly increases its hydrolytic activity, particularly towards cAMP. Previous studies have shown that heart-specific overexpression of PDE2A3 in mice has a strong anti-arrhythmic effect. Conversely, other studies have shown that pharmacological inhibition of PDE2A counteracts the hypertrophic growth of cardiomyocytes. The aim of this study was to investigate the role of PDE2A in heart function under physiological and pathophysiological conditions using a heart-specific PDE2A knockout model. Furthermore, the goal was to elucidate the PDE2A-mediated cGMP/cAMP crosstalk mechanism using C-type natriuretic peptide (CNP) in freshly isolated murine cardiomyocytes and human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CMs) to uncover potential anti-arrhythmic cardioprotective effects of cGMP-mediated PDE2A stimulation. First, a new mouse model with cardiomyocyte-specific deletion of PDE2A (PDE2A-KO) was generated using the 'knockout-first' strategy and characterized via echocardiography. The absence of PDE2A expression in cardiomyocytes did not lead to an altered cardiac phenotype under physiological conditions. However, under chronic stress conditions following isoprenaline infusion (osmotic minipump, 14 days), PDE2A-KO mice showed reduced contraction and ejection fraction compared to treated control animals. Heart rate and hypertrophy did not differ between the groups. To investigate the influence of CNP-mediated PDE2A activation on pro-arrhythmic intracellular signals, spontaneous local Ca2+ releases from the sarcoplasmic reticulum (Ca2+ sparks, CaSp), arrhythmic Ca2+ waves (CaW), and contraction of isolated ventricular and atrial cardiomyocytes were quantified. Interestingly, CNP significantly reduced the number of arrhythmogenic CaSp and CaW following acute β-adrenergic stimulation without affecting cellular contractility. This anti-arrhythmic effect was not observed in PDE2A-KO animals and was completely abolished by specific PDE2A inhibition with Bay 60-7550. Simultaneous inhibition of cGMP-activated protein kinase G had no effect on the number of CaSp. Additionally, experiments with isolated cardiomyocytes from mice with heart-specific PDE2A3 overexpression showed a significantly reduced number of CaSp after acute β-adrenergic stimulation. This protective effect was prevented by specific PDE2A inhibition. While PDE2A inhibition alone had no effect on CaSp frequency, inhibition of PDE3 and PDE4 significantly increased CaSp frequency in cardiomyocytes from control animals. Notably, the CNP/cGMP/PDE2A-mediated anti-arrhythmic effect was also reproduced in human ventricular and atrial iPSC-CMs. At the cellular level, stress-induced Ca2+ leaks in human cells were suppressed by CNP-mediated PDE2A stimulation. CNP mediated strong anti-arrhythmic effects at the cellular level through PDE2A stimulation. Interestingly, CNP expression in human ventricular tissue from HF patients was significantly reduced, while ANP and BNP were upregulated, potentially causing receptor desensitization. In isolated murine cardiomyocytes, CNP-mediated PDE2A activation significantly reduced intracellular cAMP levels. The findings from this study clearly underscore the clinical relevance of PDE2A as a potential therapeutic target for ventricular cardiac arrhythmias and atrial fibrillation following acute stress stimulation or in HF. In summary, this study demonstrated that PDE2A plays a crucial role in suppressing acute stress-induced arrhythmogenic Ca2+ leaks. Therefore, pharmacologically enhancing PDE2A activity could represent a novel cardioprotective strategy for treating arrhythmias in HF.:Abkürzungsverzeichnis VI Abbildungsverzeichnis XII Tabellenverzeichnis XVI 1. Einleitung 1 1.1. Herzinsuffizienz und kardiale Arrhythmien 1 1.1.1. Ätiologie und Epidemiologie 1 1.1.2. Pathophysiologie und Klassifikation kardialer Arrhythmien bei HF 3 1.1.3. Zellulärer und molekularer Aspekt 7 1.1.4. Pharmakologische Therapie 9 1.2. Kardiale Signaltransduktion und sekundäre Botenstoffe 11 1.2.1. cAMP-Signalkaskade 12 1.2.2. cGMP-Signalkaskade 13 1.2.3. Natriuretische Peptide und deren klinische Relevanz 14 1.3. Kardiale Phosphodiesterasen 15 1.3.1. Kardiale PDEs mit dualer Substratspezifität 17 1.3.2. Kardiale cAMP und cGMP-spezifische PDEs 18 1.4. Phosphodiesterase 2A 19 1.4.1. Molekularer Aspekt 19 1.4.2. PDE2A-Expression und Aktivität 21 1.4.3. Perspektiven hinsichtlich der Rolle von PDE2A bei Herzerkrankungen 22 2. Zielsetzung 24 3. Material und Methoden 25 3.1. Material 25 3.2. Tiermodel 25 3.2.1. Tierhaltung 25 3.2.2. C57BL/6JRj Wildtyp-Mausmodel 25 3.2.3. Herzspezifische PDE2A-KO-Mausmodel (PDE2A_fl_αMHC_cre) 26 3.2.4. Genotypisierung 27 3.3. Zellkultur und Differenzierung der humanen induced pluripotent stem cell-derived Kardiomyzyten (iPSC-KM) 30 3.4. Gewinnung Patientengewebe 31 3.5. Echokardiografische Langzeitcharakterisierung 32 3.6. Implantierung osmotischer Minipumpen 34 3.7. Zellisolation adulter Mauskardiomyozyten 35 3.7.1. Isolierung ventrikulärer Kardiomyozyten 35 3.7.2. Isolierung atrialer Kardiomyozyten 36 3.8. Kontraktilitätsmessung 37 3.9. Calcium-Imaging 38 3.10. Calcium-Spark Detektion in Kardiomyozyten 41 3.10.1. Calcium-Spark Detektion in Mauskardiomyozyten 41 3.10.2. Calcium-Spark Detektion in humanen iPSC-KM 42 3.11. Proteinexpressionsanalyse aus Mausatrium 42 3.11.1. Proteinisolation und Konzentrationsbestimmung 42 3.11.2. Immunoblot 43 3.12. ELISA 45 3.13. qPCR 45 3.14. Statistische Auswertung 46 4. Ergebnisse 47 4.1. Einfluss des PDE2A-Knockouts auf die Herzfunktion 47 4.1.1. Echokardiografische Charakterisierung im Alter von 3 und 6 Monaten 47 4.1.2. Auswirkung einer chronischen Aktivierung der β-adrenergen Signalkaskade auf die Herzfunktion 51 4.2. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf den intrazellulären Ca2+-Haushalt in PDE2A-Knockout-Kardiomyozyten nach Stressinduktion 56 4.2.1. Auswirkung auf den intrazellulären Ca2+-Zyklus 56 4.2.2. Auswirkung auf den intrazellulären Ca+-Speicher, die fraktionelle und spontane Ca2+-Freisetzung aus dem sarkoplasmatischen Retikulum 60 4.2.3. Auswirkung auf die Geschwindigkeit der Ca2+-Freisetzung und der Ca2+-Wiederaufnahme 63 4.3. Einfluss der cGMP-vermittelten PDE2A-Stimulation durch CNP auf die Kontraktilität ventrikulärer Kardiomyozyten 65 4.3.1. Einfluss auf die zelluläre Kontraktion 65 4.3.2. Einfluss auf die zelluläre Kontraktionsgeschwindigkeit und Relaxationsgeschwindigkeit 67 4.3.3. Einfluss des PDE2A-Knockouts auf die zelluläre Kontraktion 69 4.4. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch natriuretische Peptide auf die spontane Ca2+-Freisetzung (Ca2+-Sparks) in ventrikulären Kardiomyozyten nach Stressinduktion 72 4.4.1. Veränderung der Ca2+-Spark-Frequenz nach CNP-Stimulation 72 4.4.2. Veränderung der Spark-Morphologie nach CNP-Stimulation 73 4.4.3. Veränderung der Ca2+-Spark-Frequenz nach ANP-Stimulation 75 4.4.4. Veränderung der Spark-Morphologie nach ANP 76 4.4.5. Veränderung der Calcium-Spark-Frequenz nach PKG-Inhibition 78 4.4.6. Veränderung der auf Spark-Morphologie nach PGK-Inhibition 80 4.5. Einfluss der cGMP-vermittelten PDE2A-Stimulation durch CNP auf die intrazellulären cAMP-Konzentration in ventrikulären Kardiomyozyten 82 4.6. Auswirkung des Myokardinfarkts auf die murine Genexpression der PDE2A, des CNP und der natriuretischen Peptidrezeptoren 83 4.7. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf den intrazellulären Ca2+-Haushalt atrialer Kardiomyozyten nach Stressinduktion 84 4.7.1. Auswirkung auf den intrazellulären Ca2+-Zyklus 84 4.7.2. Auswirkung auf den intrazellulären Ca2+-Speicher, die fraktionelle und spontane Ca2+-Freisetzung aus dem Sarkoplasmatischen Retikulum 87 4.7.3. Auswirkung auf die Geschwindigkeit der Ca2+-Freisetzung und der Ca2+-Wiederaufnahme 89 4.7.4. Einfluss des PDE2A-Knockouts auf den Ca2+- Zyklus 90 4.8. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf spontane Ca2+-Freisetzung (Ca2+-Sparks) in atrialen Kardiomyozyten 95 4.8.1. Veränderung der Calcium-Spark-Frequenz 95 4.8.2. Veränderung der Spark-Morphologie 96 4.9. Auswirkung des PDE2A-KO auf die Expression Ca2+-regulierender Proteine 98 4.10. Bedeutung der cGMP-vermittelten PDE2A-Stimulation durch CNP auf die spontane Ca2+-Freisetzung (Ca2+-Sparks) in humanen iPSC-Kardiomyozyten nach Stressinduktion 102 4.10.1. Veränderung der Ca2+-Spark-Frequenz nach CNP-Stimulation 102 4.10.2. Auswirkung der CNP-Stimulation auf die Ca2+-Spark-Morphologie 104 4.10.3. Bedeutung der PDE3 und PDE4 Inhibition auf Ca2+-Sparks in ventrikulären hiPSC-KM 106 4.10.4. Einfluss der PDE3 und PDE4 Inhibition auf die Ca2+-Spark-Morphologie 108 4.10.5. Bedeutung der CNP-Stimulation auf Ca2+-Sparks in atrialen hiPSC-KM nach Stressinduktion 109 4.10.6. Einfluss der CNP-Stimulation auf die Ca2+-Spark-Morphologie nach Stressinduktion 111 4.11. Auswirkung dilatativer Kardiomyopathie auf die Genexpression natriuretischer Peptide und deren Rezeptoren 112 5. Diskussion 114 5.1. Der herzspezifische PDE2A-Knockout 115 5.1.1. Basale Herzfunktion konditionaler PDE2A-KO Mäuse 115 5.1.2. Cre-Expression beeinträchtigt die Überlebensfähigkeit der PDE2A-KO Mäuse 116 5.1.3. Der PDE2A-KO führt zur Reduktion der Herzfunktion bei chronischer Stressstimulation 117 5.2. Die CNP-vermittelte PDE2A-Stimulation reduziert arrhythmogene Ca2+-Freisetzungen ohne den intrazellulären Ca2+-Zyklus oder die zelluläre Kontraktion zu beeinflussen 119 5.2.1. PDE2A-Stimulation reduziert stressinduzierte arrhythmogene Ca2+-Wellen 119 5.2.2. Die zelluläre Kontraktilität wird durch die CNP-vermittelte PDE2A-Stimulation nicht beeinträchtigt 121 5.2.3. Natriuretische Peptid-vermittelte PDE2A-Stimualtion unterdrückt die stressinduzierten Ca2+-Lecks aus dem SR 122 5.3. CNP vermittelt einen anti-arrhythmogenen Effekt in humanen iPSC-Kardiomyozyten 124 5.4. Limitationen 125 5.4.1. Einschränkungen des Cre/loxP-Systems beim PDE2A-KO 125 5.4.2. Crosstalk-Mechanismen zyklischer Nukleotide 126 5.4.3. Patientenbasierte Variabilität 127 5.5. Ausblick 128 5.6. Die klinische Relevanz der PDE2A 129 6. Zusammenfassung 131 7. Summary 133 8. Referenzen 135 9. Anhang 162 9.1. Chemikalienliste 162 9.2. Antikörper- und Primerliste 164 9.3. Kits und Verbrauchsmaterialien 166 10. Danksagung 172 11. Erklärung 173