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This study is based on a proposal concerning the feasibility of using aliphatic tertiary amine N-oxides as antiarrhythmic agent prodrugs. Lignocaine was selected as a candidate for prodrug development, because the N-oxide is a non-active, polar derivative of lignocaine and the drug of choice for ventricular arrhythmia, a symptom associated with ischaemic episodes leading to regions of transiently hypoxic heart tissue. An HPLC analytical method was developed to study the metabolism of lignocaine N-oxide. The rapid and sensitive analysis of lignocaine and its metabolites was demonstrated with good reproducibility, stability and high recovery. In this study, it was identified that lignocaine N-oxide can be reduced to its active parent compound, lignocaine with no other metabolites detected in the absence of oxygen. Under anaerobic conditions, no further metabolism of lignocaine was demonstrated in rat liver microsomes and heart S9 fractions suggesting no secondary metabolites were formed. The reduction of lignocaine N-oxide has been shown to be both enzymic and non-enzymic, NADPH dependent, oxygen sensitive and can be suppressed by CO, CN- and protein denaturation. Under anaerobic conditions, in vitro lignocaine N-oxide reduction was found to occur in NADPH supplemented rat liver homogenates, microsomal suspensions; rat heart homogenates, cytosolic solutions; human phenotyped cytochrome P450 isoforms; purified enzymes- cytochrome P450 reductase, xanthine oxidase, deoxymyoglobin and NADPHI ascorbate reduced protohaem (haemin). This reaction can be suppressed through the chemically mediated decrease ofP450 and bs levels in rat liver microsomes. Previous studies demonstrated that lignocaine N-oxide was non-active in aerobic rat heart in vivo and was potent under ischaemic conditions. In this study, high recovery of lignocaine N-oxide was found in the urine of normal rats suggesting low metabolism of the prodrug in oxic tissues. However, in hypoxic isolated rat hearts, lignocaine N-oxide was found to be reduced to lignocaine. The data presented suggested that the bioactivation of lignocaine N-oxide could be regulated by the prevailing oxygen tension in the ischaemic arrhythmic heart. Essentially the pro drug activation of lignocaine N-oxide may be triggered by the ischaemic state of the heart and terminated as the oxygen content in the heart returns to a more normal level. A controlled release and site-specific active drug delivery of lignocaine N-oxide, a hypoxia-mediated antiarrhythmic agent, may thus be achieved.

Abstract Atrial fibrillation (AF) is a common arrhythmia in the clinical setting with a population prevalence of 1–2%. AF significantly increases the risk of stroke and death, worsens coexistent heart diseases and may leave the patient with disabling symptoms. The treatment of AF consists of the control of the underlying conditions, prevention of complications and symptom relief by controlling heart rate (rate control) or by targeting normal rhythm (rhythm control), with the latter achieved either by antiarrhythmic drug (AAD) therapy or catheter ablation (CA). Ablation therapy has generally been applied and studied after failure of AAD therapy. The aim of this study was to evaluate the safety and efficacy of first-line CA in AF. The other objectives were to assess the safety of continuous warfarin therapy during CA and to identify prognostic markers for treatment outcome. A meta-analysis of all randomised studies and a secondary analysis of one randomised study comparing CA and AAD as first-line therapy were performed. In the first study, ablation therapy reduced AF recurrences more than AAD therapy (HR 0.63) when provided as first-line therapy; the rate of complications was similar with both therapies. Some of the complications of ablation therapy were more serious than those encountered with AADs. The second study revealed that the antiarrhythmic efficacy of ablation therapy was more durable. In the third study, the efficacies of continuous and interrupted warfarin therapy were compared in 228 procedures; both strategies were found to be equally safe during a three month follow-up. Furthermore, an analysis of 2317 AF episodes revealed a new electrocardiographic feature at AF initiation, which was associated with AF relapse after the initiation of therapy. Finally, a thorough echocardiographic examination was performed in 49 patients prior to ablation therapy. Mild diastolic dysfunction was associated with AF recurrence. In conclusion, CA was more effective as a first-line therapy than AADs but may cause more severe complications. Continuous warfarin therapy was found to be safe during CA. New electrocardiographic and echocardiographic markers for treatment outcome were recognised. / Tiivistelmä Eteisvärinä on yleinen rytmihäiriö, jonka esiintyvyys väestössä on 1–2 % luokkaa. Eteisvärinä lisää merkittävästi kuolleisuutta ja aivoinfarktiriskiä, vaikeuttaa muiden sydänsairauksien oireita ja saattaa aiheuttaa invalidisoivia oireita. Eteisvärinän hoito keskittyy liitännäissairauksien hoitoon ja komplikaatioiden estoon sekä oireiden lievitykseen joko syketaajuutta säätämällä (sykkeenhallinta) tai pyrkimällä normaaliin rytmiin (rytminhallinta). Rytminhallinnassa käytetään yleisesti joko rytmihäiriölääkkeitä tai katetriablaatiohoitoa. Eteisvärinän katetriablaatiota on useimmiten käytetty ja tutkittu tilanteessa, jossa rytmihäiriölääkitys on osoittautunut tehottomaksi. Tämän tutkimuksen tavoitteena oli arvioida eteisvärinän katetriablaatiohoidon tehoa ja turvallisuutta ensilinjan hoitona. Muina tavoitteina oli katetriablaation turvallisuuden arviointi jatkuvan varfariinihoidon aikana sekä löytää uusia katetriablaatiohoidon tehoa ennustavia tekijöitä. Teimme meta-analyysin kaikista randomisoiduista tutkimuksista ja sekundaarisen analyysin yhdestä randomisoidusta tutkimuksesta, jotka vertasivat rytmihäiriölääke- ja katetriablaatiohoitoa ensilinjan hoitona. Ensimmäisessä työssä ablaatiohoito esti eteisvärinän uusiutumista tehokkaammin (riskisuhde 0.63), eikä komplikaatioiden yleisyydessä ollut eroa hoitojen välillä. Jotkut ablaatiohoitoon liittyvät komplikaatiot olivat kuitenkin luonteeltaan vakavampia kuin lääkehoidossa. Ablaatiohoidon eteisvärinää estävä vaikutus todettiin pidempikestoiseksi toisessa työssämme. Kolmannessa työssä vertasimme jatkuvaa ja tauotettua varfariinihoitoa 228 ablaatiotoimenpiteen aikana. Molemmat lähestymistavat osoittautuivat yhtä turvallisiksi 3 kuukauden seuranta-aikana. Analysoimme edelleen 2317 eteisvärinäkohtausta ja löysimme osalla potilaista uuden eteisvärinäkohtauksen alkuun liittyvän ominaisuuden, joka oli yhteydessä rytminhallinnan tehottomuuteen. Lisäksi teimme 49 potilaalle laajan sydämen ultraäänitutkimuksen ennen katetriablaatiotoimenpidettä. Diastolisen dysfunktion havaittiin olevan yhteydessä eteisvärinän uusiutumiseen. Yhteenvetona totesimme että katetriablaatiohoito on rytmihäiriölääkehoitoa tehokkaampaa ensilinjan hoitona, mutta siihen mahdollisesti liittyvät komplikaatiot olivat luonteeltaan hankalampia. Jatkuva varfariinihoito todettiin turvalliseksi katetriablaation yhteydessä. Löysimme lisäksi sydänsähkökäyrästä ja sydämen ultraäänitutkimuksesta uusia hoidon tehoa ennustavia tekijöitä.

Bakgrund: Arytmi innebär en avvikande rytm hos hjärtat, alltså att det uppstått en avvikelse i impulsbildning och/eller impulsfortledning. Det kan inverka negativt på livskvalitén, orsaka följdsjukdomar eller leda till livshotande tillstånd. Utöver den konventionella behandlingen finns det behov av ytterligare alternativ. Magnesium är ett essentiellt mineralämne som är viktigt för hjärtats elektrofysiologi. Det har i tidigare studier visat sig att magnesium har haft effekt på postoperativt uppkomna arytmier och att låga nivåer av magnesium hos en frisk population har ökat risken för utvecklandet av olika arytmier. Magnesiums föreslagna antiarytmiska egenskaper är därför av intresse att studera vidare.  Syfte: Syftet med studien är att studera om magnesium har någon antiarytmisk effekt hos individer med arytmi.  Metod: En litteraturstudie genomfördes avseende relevanta artiklar publicerade i databasen PubMed från år 2000 och framåt. En relevansbedömning och kvalitetsgranskning genomfördes med utgångspunkt från Statens beredning för medicinsk och social utvärderings (SBU) metodbok.  Resultat: Tolv artiklar inkluderades i litteraturstudien varav sju uppvisade en antiarytmisk effekt av magnesium. En antiarytmisk effekt vid förmaksflimmer uppvisades i samtliga studier där det administrerades mer än 4 gram intravenöst magnesium. Eftersom magnesium i olika former har olika biotillgänglighet skulle formen eventuellt också kunna vara en bidragande faktor till effekten men studiematerialet var för litet gällande olika magnesiumformer. Vidare går det inte att särskilja om magnesium har olika effekt på olika arytmier då representationen av respektive arytmi var för liten. Slutsats: Magnesium har en antiarytmisk effekt vid administrering över 4 gram intravenöst. Huruvida detta gäller för fler arytmier än förmaksflimmer framkommer inte av denna litteraturstudie. Det finns ett behov av fler studier för att dels undersöka vilken som är den eventuella optimala formen och dosen av magnesium, dels för att utreda vilka typer av arytmier som verkar mottagliga för magnesium som behandlingsalternativ. / Background: Arrhythmia means a deviating rhythm in the heart, either in impulse formation and/or impulse conduction. It can adversely affect the quality of life, cause sequelae or lead to life-threatening conditions. In addition to the conventional treatment, there is a need for alternatives. Magnesium is an essential mineral that is important for the electrophysiology of the heart. Previous studies have shown that magnesium has had an effect on postoperative arrhythmias and that low levels of magnesium in a healthy population have increased the risk of developing various arrhythmias. The proposed antiarrhythmic properties of magnesium are therefore of interest for further study.  Aim: The aim of the study is to investigate whether magnesium has any antiarrhythmic effect in individuals with arrhythmias.  Methods: A literature study was conducted regarding relevant articles published in the PubMed database from the year 2000 and until today. Relevance assessment and quality review were carried out on the basis of  Swedish Agency for Health Technology Assessment and Assessment of Social Services (SBU) method book.  Results: Twelve articles were included in the literature study, seven of which showed an antiarrhythmic effect. An antiarrhythmic effect in atrial fibrillation was shown in all studies where more than 4 grams of intravenous magnesium was administered. Since magnesium in different forms has different bioavailability, the form could possibly also be a contributing factor to the effect, but the study material was too small for different forms of magnesium. Furthermore, it is not possible to distinguish whether magnesium has different effects on different arrhythmias as the representation of each arrhythmia was too small.  Conclusions: Magnesium has antiarrhythmic effect when administrated in excess of 4 gram intravenously. Whether this applies to more arrhythmias than atrial fibrillation does not emerge from this literature study. There is a need for more studies to investigate the possible optimal form and dose of magnesium, and to investigate which types of arrhythmias seem susceptible to magnesium as a treatment alternative.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-27T14:33:23Z No. of bitstreams: 2 Tese - Priscilla Regina Nasciutti - 2018.pdf: 3399119 bytes, checksum: 6cdbfee6b9cc1dffd41d48f821659dc3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-28T09:56:34Z (GMT) No. of bitstreams: 2 Tese - Priscilla Regina Nasciutti - 2018.pdf: 3399119 bytes, checksum: 6cdbfee6b9cc1dffd41d48f821659dc3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-11-28T09:56:35Z (GMT). No. of bitstreams: 2 Tese - Priscilla Regina Nasciutti - 2018.pdf: 3399119 bytes, checksum: 6cdbfee6b9cc1dffd41d48f821659dc3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-23 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Mitral valve disease (MVD) is characterized by thickening in the valvular leaflets and may lead to heart failure. Pharmacological treatment of the disease with vasodilators, positive inotropes and diuretics is used in conjunction with dietary management. Omega 3 (ω-3) supplementation has been associated with modulation of blood pressure (BP) and heart rate, improvement of Doppler echocardiography, antiarrhythmic, anti-inflammatory and antidysiphyde effects. In the absence of prospective clinical studies, the objective was to evaluate the influence of ω-3 supplementation in dogs with MVD. For this purpose, 41 dogs were followed up every three months for 12 months by means of clinical evaluation, BP measurement, electrocardiography, Doppler echocardiography, chest radiography, laboratory tests, dosages of inflammatory mediators and cardiac biomarker. The dogs were classified in stages B1, B2 and C, according to the ACVIM consensus. Dogs were randomly divided into the ω-3 group, which received food for dogs with heart diseases supplemented with ω-3 and control group (even food without supplementation). In stage B1 only dogs from the ω-3 group were evaluated. At the end of 12 months, no changes were observed in the parameters evaluated. In dogs stage B2 and C, there was an increase in the serum levels of inflammatory mediators, in a larger amplitude in the control group. The ω-3 preserved body condition score, muscle condition score and reduced by 2.96 times the chance of developing arrhythmias. The DIVEdN and VHS measurements were higher in the control group and correlated with NTproBNP cardiac biomarker concentrations. It is concluded that the supplementation with ω-3 in patients in classes B2 and C, maintains the body condition, helps reduce volume overload, has an antiarrhythmic effect and keeps dogs with MVD in the firsts stages of the disease. / A doença valvar mitral (DVM) é caracterizada pelo espessamento nos folhetos valvares e pode levar ao desenvolvimento de insuficiência cardíaca. O tratamento farmacológico da doença com vasodilatadores, inotrópicos positivos e diuréticos é utilizado em conjunto com o manejo dietético. A suplementação com ômega 3 (ω-3) tem sido associada à modulação da pressão arterial (PA) e da frequência cardíaca, melhora de índices ecodopplercardiográficos, efeitos antiarrítmico, antinflamatório e antidislipidêmico. Diante da ausência de estudos clínicos prospectivos, o objetivo foi avaliar a influência da suplementação com ω-3 em cães portadores de DVM. Para tanto, 41 cães foram acompanhados trimestralmente durante 12 meses por meio de avaliação clínica, mensuração de PA, eletrocardiografia, ecodopplercardiografia, radiografia torácica, exames laboratoriais, dosagens de mediadores inflamatórios e de biomarcador cardíaco. Os cães foram classificados nos estágios B1, B2 e C, segundo o consenso do ACVIM. De maneira aleatória os cães foram dividis no grupo ω-3, que recebeu alimento para cães cardiopatas com suplementação com ω-3 e grupo controle (mesmo alimento sem a suplementação). No estágio B1 foram avaliados apenas os cães do grupo ω- 3. Ao final de 12 meses, não foram observadas alterações nos parâmetros avaliados. Nos cães estágio B2 e C ocorreu aumento nos níveis séricos de mediadores inflamatórios, em maior amplitude no grupo controle. O ω-3 preservou o ECC, ECM e reduziu em 2,96 vezes a chance de desenvolvimento de arritmias. As medidas DIVEdN e VHS foram superiores no grupo controle e se correlacionaram às concentrações do biomarcador cardíaco NT-proBNP. Concluise que a suplementação com ω-3 em pacientes nas classes B2 e C, atua mantendo a condição corporal, auxilia na redução da sobrecarga volumétrica, apresenta efeito antiarrítmico e mantem os cães portadores de DVM em estágios mais brandos da doença.

Background and aim: Patients with heart failure and myocardial infarction have a significant risk of ventricular arrhythmia and sudden cardiac death, contributing to ∼20% of total deaths worldwide. However, pharmacological therapy of life-threatening arrhythmia remains limited and challenging. Consequently, there is a strong medical need for novel antiarrhythmic pharmacotherapy approaches that can effectively address fatal arrhythmias and reduce mortality rates. There is increasing evidence for cardioprotective effects of C-type natriuretic peptide (CNP) generating the intracellular second messenger cyclic guanosine monophosphate (cGMP) via its guanylyl cyclase receptor. While cGMP is proposed to mediate beneficial effects in the diseased myocardium, the second messenger cyclic adenosine monophosphate (cAMP) downstream of the chronically activated β-adrenoceptors provoke detrimental effects, such as cardiac remodelling and arrhythmia generation. Importantly, phosphodiesterase type 2 (PDE2) plays a crucial role in mediating the negative crosstalk between cGMP and cAMP signalling pathways within cardiomyocytes. As the only phosphodiesterase that is allosterically activated by cGMP to increase its cAMP-hydrolysing activity, PDE2 regulates the balance between these two signalling molecules. The aim of this study was to investigate the potential antiarrhythmic effect of CNP-cGMP-induced PDE2 stimulation at both organ and animal level in physiological as well as pathophysiological conditions. Material & methods: Hearts from mouse models with cardiomyocyte-specific PDE2 overexpression (PDE2 OE) or deletion (PDE2 KO) were isolated and subjected to ischemia/reperfusion (I/R) injuries using a Langendorff perfusion system. Thereby, the left descending coronary artery was ligated for 30 minutes to induce ischemia, followed by 30 minutes of reperfusion after removing the occlusion. The incidence of arrhythmic events during the reperfusion phase was examined following PDE2 activation with CNP or PDE2 inhibition with BAY 60-7550 using ex vivo ECG electrodes. To assess the potential antiarrhythmic effects of PDE2 modulation under pathophysiological conditions, two different experimental models were induced in mice: (i) Heart failure (HF) was induced through a 5-week regimen including a high-fat diet and the administration of the NO-synthase inhibitor L-NAME in drinking water to mediate metabolic and hypertensive stress, and (ii) diabetes was induced via 5 consecutive streptozotocin (STZ) i.p. injections, leading to the destruction of pancreatic β-cells and subsequent development of diabetes over a four-week period. In addition to ex vivo arrhythmia quantification after I/R, in vivo arrhythmia development was conducted by implanting ECG telemeters and provoking arrhythmias with double injections of the β adrenoceptor agonist isoprenaline (Iso). Additionally, atrial arrhythmia induction protocols were established to evaluate the development of atrial fibrillation (AF) using two different pacing protocols on the right atrium. The S1S2 protocol was used to detect the effective refractory period of the atria and the burst pacing protocol to determine the inducibility of AF, with simultaneous recording of monophasic action potentials from the left atrium. Results: WT hearts perfused with CNP exhibited a significant reduction in the number of arrhythmic events following I/R compared to control hearts. Importantly, this antiarrhythmic effect of CNP was reversed by pharmacological inhibition of PDE2 with BAY 60-7550 (Cachorro et al., 2023). Interestingly, no differences in arrhythmia development were observed between control and perfusion with BAY (Wagner et al., 2021). In contrast, inhibition of PDE3 resulted in markedly increased number of arrhythmic events compared to control. Hearts with cardiomyocyte-specific PDE2 deletion displayed a significantly higher incidence of arrhythmias after I/R, including ventricular extrasystole, bigeminy, and tachycardia. However, in vivo experiments did not demonstrate increased arrhythmia development after acute β-adrenergic stress in PDE2 KO mice with HF potentially due to desensitization of chronically activated β-adrenergic receptors in this pathophysiological model. Furthermore, hearts from diabetic mice exhibited a significantly higher number of arrhythmic events ex vivo, although those with PDE2 OE were protected against diabetes-induced arrhythmias compared to diabetic controls. However, the established pacing protocols revealed a low AF induction rate in ex vivo perfused mouse hearts, limiting the ability to observe a significant antiarrhythmic effect of CNP. Nonetheless, control hearts perfused with CNP plus BAY or with cardiac-specific deletion of PDE2 showed a trend towards increased AF compared to WT hearts and those perfused with CNP alone. Under diabetic conditions, PDE2 KO mice also displayed a trend to have higher AF inducibility compared to control diabetic hearts. As observed under physiological conditions, simultaneous perfusion of diabetic hearts with CNP plus BAY also showed a trend to have enhanced AF development compared to CNP or control perfusion. Conclusions: In conclusion, the findings of this study provide strong evidence that cGMP-induced PDE2 activation plays a crucial role in protecting the heart from ventricular arrhythmias in both physiological and pathophysiological conditions. Thus, CNP-mediated PDE2 stimulation could provide a novel therapeutic strategy to reduce life-threatening arrhythmias. However, further research should be performed to elucidate the effects of PDE2 stimulation on other tissues expressing PDE2, such as fibroblasts, neurons, endothelial cells, or circulating immune cells. Additionally, the impact of CNP-induced cGMP on the protein kinase G (PKG) pathway in cardiomyocytes should be investigated. The results of this work may provide a new approach for the development of novel antiarrhythmic therapies targeting the CNP-PDE2 axis, improving clinical outcomes for patients with HF and myocardial infarction.:Abbreviations I List of figures V List of tables VII 1. Introduction 1 1.1. Diabetes and diabetic cardiomyopathy 1 1.1.1. Aetiology, epidemiology and classification 1 1.1.2. Pathophysiology mechanisms 2 1.1.3. Diagnosis and treatment 3 1.2. Myocardial Infarction 4 1.2.1. Aetiology, epidemiology and classification 4 1.2.2. Pathophysiology mechanisms 5 1.2.3. Diagnosis and treatment 7 1.3. Heart failure 9 1.3.1. Aetiology, epidemiology and classification 9 1.3.2. Pathophysiology mechanisms 10 1.3.3. Diagnosis and treatment 11 1.4. Cardiac arrhythmia 13 1.4.1. Aetiology, epidemiology and classification 13 1.4.2. Cellular mechanism and pathophysiology 15 1.4.3. Arrhythmia diagnosis and treatment 16 1.5. Cyclic nucleotide signalling in cardiomyocytes 17 1.5.1. cAMP signalling 17 1.5.2. cGMP signalling 19 1.5.3. Compartmentalization of secondary messengers and phosphodiesterase superfamily 20 1.5.4. The increasing clinical significance of CNP 23 1.6. Phosphodiesterase 2 23 1.6.1. Molecular aspects 23 1.6.2. PDE2 regulation in cardiomyocytes 25 1.6.3. PDE2-mediated cGMP/cAMP crosstalk in cardiomyocytes 26 1.6.4. Antiarrhythmic potential of PDE2 27 2. Aims of the study 28 3. Materials & methods 29 3.1. Materials 29 3.1.1. Chemicals 29 3.1.2. PCR primers 30 3.1.3. Kits and reagents 31 3.1.4. Laboratory consumables 31 3.1.5. Devices and experimental hardware 32 3.1.6. Computer software 34 3.2. Methods 34 3.2.1. Animal models 34 3.2.2. Murine pathophysiological models 38 3.2.3. Ex vivo ischemia/reperfusion injuries 39 3.2.4. Characterization of heart hypertrophy and lung congestion 41 3.2.5. TTC staining 42 3.2.6. Ex vivo atrial fibrillation induction 42 3.2.7. In vivo ECG telemetry 43 3.2.8. Acute arrhythmia induction in vivo 44 3.2.9. Echocardiography 45 3.2.10. Biostatistical analysis 45 4. Results 47 4.1. Impact of PDE2 modulation on cardiac arrhythmia following ischemia/reperfusion injuries in ex vivo perfused murine hearts 47 4.1.1. PDE2 modulation and arrhythmogenesis in wild type mice 47 4.1.2. Impact of different phosphodiesterases and protein kinase G on arrhythmia development after ex vivo I/R 52 4.2. Role of PDE2 in murine heart failure model 54 4.2.1. Characterization of the murine heart failure model in different genetic backgrounds 55 4.2.2. Role of PDE2 on arrhythmia development upon acute β-AR stimulation in vivo 61 4.3. Role of PDE2 in mice with STZ-induced diabetic cardiomyopathy 62 4.3.1. Effect of cardiac PDE2 overexpression and deletion on cardiac function and development of STZ-induced diabetes 62 4.3.2. Impact of cardiac-specific overexpression of PDE2 in diabetic mice after I/R injuries 70 4.4. Atrial fibrillation 73 4.4.1. Effects of PDE2 modulation on atrial fibrillation under physiological and pathophysiological conditions after STZ-induced diabetes 73 4.4.2. Effects of cardiomyocyte-specific PDE2 deletion on atrial fibrillation induction in hearts from mice with STZ-induced diabetes 79 5. Discussion 82 5.1. CNP-induced PDE2 stimulation protects from arrhythmia after I/R injuries ex vivo 82 5.2. PDE2-/- mice did not show significant increased arrhythmia development in vivo upon HF induction 86 5.3. PDE2 OE mice are protected against I/R injuries-induced arrhythmia development after diabetes induction with STZ 86 5.4. PDE2 pharmacological inhibition and PDE2 genetic deletion might lead to more atrial fibrillation episodes after burst pacing 90 5.5. Limitations 93 5.6. Conclusions 95 6. Summary 97 7. Zusammenfassung 99 8. Bibliography 102 9. Acknowledgements 124 10. Declaration 126 / Hintergrund und Ziel: Patienten mit Herzinsuffizienz und Myokardinfarkt haben ein hohes Risiko für ventrikuläre Arrhythmien und plötzlichen Herztod, welche weltweit für ∼20 % der Todesfälle verantwortlich sind. Die pharmakologische Therapie lebensbedrohlicher Herzrhythmusstörungen ist jedoch nach wie vor begrenzt und schwierig. Daher besteht ein dringender medizinischer Bedarf an neuen Ansätzen für die antiarrhythmische Pharmakotherapie, um letale Herzrhythmusstörungen zu reduzieren und die Sterblichkeitsrate zu senken. Verschiedene Studien belegen, dass das C-Typ natriuretischem Peptid (CNP) über die Stimulation des Guanylatzyklase-Rezeptors B und die Bildung des intrazellulären Botenstoffs zyklisches Guanosinmonophosphat (cGMP) kardioprotektive Wirkungen vermittelt. Während cGMP im erkrankten Herzmuskel positive Wirkungen vermittelt, fördert zyklisches Adenosinmonophosphat (cAMP) als intrazellulärer Botenstoff der chronisch aktivierten β-Adrenozeptoren schädliche Wirkungen, wie kardiales Remodelling und die Entstehung von Herzrhythmusstörungen. Die Phosphodiesterase 2 (PDE2) spielt eine entscheidende Rolle bei der Vermittlung des negativen Crosstalks zwischen dem cGMP- und dem cAMP-vermittelten Signalweg in Kardiomyozyten. Als einzige Phosphodiesterase wird die PDE2 durch cGMP allosterisch aktiviert, um dann verstärkt cAMP abzubauen und zum Gleichgewicht zwischen beiden Signalmolekülen beizutragen. Ziel dieser Arbeit war es, die potenzielle antiarrhythmische Wirkung der CNP-cGMP-induzierten PDE2-Stimulation unter physiologischen und pathophysiologischen Bedingungen sowohl auf Organebene als auch im Mausmodell in vivo zu untersuchen. Material & Methoden: Für diese Arbeit wurden murine Herzen aus Mäusen mit Kardiomyozyten-spezifischer PDE2-Überexpression (PDE2 OE) oder -Deletion (PDE2 KO) verwendet und ex vivo einer Ischämie/Reperfusion (I/R) am Langendorff-Perfusionssystem unterzogen. Zur Ischämie-Induktion wurde zunächst die linke absteigende Koronararterie für 30 Minuten ligiert. Im Anschluss wurde die Okklusion aufgehoben und für 30 Minuten reperfundiert. Mithilfe von EKG-Elektroden wurde ex vivo während der Reperfusionsphase das Auftreten von Herzrhythmusstörungen nach PDE2-Aktivierung mit CNP oder PDE2-Hemmung mit BAY 60-7550 untersucht. Um die potenziellen antiarrhythmischen Wirkungen der PDE2-Modulation unter pathophysiologischen Bedingungen zu evaluieren, wurden zwei verschiedene experimentelle Modelle in Mäusen induziert: (i) eine Herzinsuffizienz (HF) wurde mittels metabolischem und hypertensivem Stress durch eine 5-wöchige Hochfettdiät und die Verabreichung des NO-Synthase-Inhibitors L-NAME im Trinkwasser induziert, und (ii) Diabetes wurde durch 5 aufeinanderfolgende i. p. -Injektionen von Streptozotocin induziert, was zur Zerstörung der β-Zellen in der Bauchspeicheldrüse und dadurch zur Entstehung eines Diabetes über einen Zeitraum von vier Wochen führte. Zusätzlich zur Quantifizierung der Arrhythmien ex vivo nach I/R wurde die Entstehung von Arrhythmien in vivo nach Provokation durch die zweifache Injektionen des β-Adrenozeptor-Agonisten Isoprenalin mittels implantierter EKG-Telemeter untersucht. Darüber hinaus wurden Protokolle zur Induktion von Vorhofarrhythmien etabliert, um die Entwicklung von Vorhofflimmern (AF) mit zwei verschiedenen Pacing-Protokollen am rechten Vorhof zu untersuchen. Das S1S2-Protokoll wurde verwendet, um die effektive Refraktärzeit der Vorhöfe zu ermitteln, und das Burst-Pacing-Protokoll, um die Induzierbarkeit von Vorhofflimmern bei gleichzeitiger Aufzeichnung monophasischer Aktionspotenziale aus dem linken Vorhof zu bestimmen. Ergebnisse: Wildtyp (WT)-Herzen, die mit CNP perfundiert wurden, wiesen eine signifikant verringerte Anzahl von arrhythmischen Ereignissen nach I/R im Vergleich zu Kontroll-Herzen auf. Diese antiarrhythmische Wirkung von CNP wurde durch pharmakologische PDE2-Hemmung mit BAY 60-7550 (BAY) aufgehoben (Cachorro et al., 2023). Interessanterweise konnten keine Unterschiede in der Entwicklung von Arrhythmien zwischen Kontrollbedingungen und Perfusion mit BAY beobachtet werden (Wagner et al., 2021). Im Gegensatz dazu führte die Hemmung der PDE3 zu einer deutlich erhöhten Anzahl an Arrhythmien verglichen mit der Kontrolle. Herzen mit Kardiomyozyten-spezifischer PDE2-Deletion wiesen eine signifikant höhere Inzidenz von Arrhythmien nach I/R auf, einschließlich ventrikulärer Extrasystolen, Bigemini und Tachykardien. In-vivo-Experimente zeigten dagegen kein verstärktes Auftreten von Arrhythmien nach akuter β-adrenerger Belastung in PDE2 KO-Mäusen mit HF, was möglicherweise auf eine Desensibilisierung chronisch aktivierter β adrenerger Rezeptoren in diesem pathophysiologischen Modell zurückzuführen ist. Darüber hinaus wiesen die Herzen diabetischer Mäuse eine signifikant höhere Anzahl von Arrhythmien ex vivo auf, wobei die Herzen von diabetischen PDE2 OE-Mäusen im Vergleich zu diabetischen Kontroll-Mäusen vor Arrhythmien geschützt waren. Durch die etablierten AF-Pacing-Protokolle konnte insgesamt nur eine niedrige AF-Induktionsrate in ex vivo perfundierten Mausherzen erzielt werden, sodass die antiarrhythmische Wirkung von CNP hier nicht gezeigt werden konnte. Dennoch wiesen gesunde Kontrollherzen, die mit CNP plus BAY perfundiert wurden, und Herzen mit einer kardiospezifischen PDE2 Deletion eine Tendenz zu gesteigertem Auftreten von Vorhofflimmern im Vergleich zu CNP-perfundierten und WT-Herzen auf. Unter diabetischen Bedingungen zeigten PDE2 KO-Herzen im Vergleich zu diabetischen Kontroll-Herzen ebenfalls einen Trend zu höherer AF-Induzierbarkeit. Wie unter physiologischen Bedingungen beobachtet, zeigte sich auch bei der gleichzeitigen Perfusion diabetischer Herzen mit CNP plus BAY eine Tendenz zur verstärkten Entwicklung von Vorhofflimmern im Vergleich zu CNP- oder Kontroll-Perfusion. Schlussfolgerungen: Die Ergebnisse dieser Studie liefern deutliche Hinweise darauf, dass die cGMP-induzierte PDE2-Aktivierung sowohl unter physiologischen als auch pathophysiologischen Bedingungen eine entscheidende Rolle für den Schutz des Herzens vor ventrikulären Arrhythmien spielt. Somit könnte die CNP-vermittelte PDE2-Stimulation eine neue therapeutische Strategie zur Verringerung lebensbedrohlicher Arrhythmien darstellen. Weitere Untersuchungen sollten jedoch durchgeführt werden, um die Auswirkungen der PDE2-Stimulation auf andere Gewebe, welche PDE2 exprimieren, wie Fibroblasten, Neuronen, Endothelzellen oder zirkulierende Immunzellen, zu klären. Darüber hinaus sollten die Auswirkungen von CNP-induziertem cGMP auf den Proteinkinase G-Signalweg in Kardiomyozyten untersucht werden. Die Ergebnisse dieser Arbeit könnten einen Ansatz für die Entwicklung neuer antiarrhythmischer Therapien bieten, welche die CNP-PDE2-Achse modulieren und die klinische Behandlung von Patienten mit Herzinsuffizienz und Herzinfarkt verbessern.:Abbreviations I List of figures V List of tables VII 1. Introduction 1 1.1. Diabetes and diabetic cardiomyopathy 1 1.1.1. Aetiology, epidemiology and classification 1 1.1.2. Pathophysiology mechanisms 2 1.1.3. Diagnosis and treatment 3 1.2. Myocardial Infarction 4 1.2.1. Aetiology, epidemiology and classification 4 1.2.2. Pathophysiology mechanisms 5 1.2.3. Diagnosis and treatment 7 1.3. Heart failure 9 1.3.1. Aetiology, epidemiology and classification 9 1.3.2. Pathophysiology mechanisms 10 1.3.3. Diagnosis and treatment 11 1.4. Cardiac arrhythmia 13 1.4.1. Aetiology, epidemiology and classification 13 1.4.2. Cellular mechanism and pathophysiology 15 1.4.3. Arrhythmia diagnosis and treatment 16 1.5. Cyclic nucleotide signalling in cardiomyocytes 17 1.5.1. cAMP signalling 17 1.5.2. cGMP signalling 19 1.5.3. Compartmentalization of secondary messengers and phosphodiesterase superfamily 20 1.5.4. The increasing clinical significance of CNP 23 1.6. Phosphodiesterase 2 23 1.6.1. Molecular aspects 23 1.6.2. PDE2 regulation in cardiomyocytes 25 1.6.3. PDE2-mediated cGMP/cAMP crosstalk in cardiomyocytes 26 1.6.4. Antiarrhythmic potential of PDE2 27 2. Aims of the study 28 3. Materials & methods 29 3.1. Materials 29 3.1.1. Chemicals 29 3.1.2. PCR primers 30 3.1.3. Kits and reagents 31 3.1.4. Laboratory consumables 31 3.1.5. Devices and experimental hardware 32 3.1.6. Computer software 34 3.2. Methods 34 3.2.1. Animal models 34 3.2.2. Murine pathophysiological models 38 3.2.3. Ex vivo ischemia/reperfusion injuries 39 3.2.4. Characterization of heart hypertrophy and lung congestion 41 3.2.5. TTC staining 42 3.2.6. Ex vivo atrial fibrillation induction 42 3.2.7. In vivo ECG telemetry 43 3.2.8. Acute arrhythmia induction in vivo 44 3.2.9. Echocardiography 45 3.2.10. Biostatistical analysis 45 4. Results 47 4.1. Impact of PDE2 modulation on cardiac arrhythmia following ischemia/reperfusion injuries in ex vivo perfused murine hearts 47 4.1.1. PDE2 modulation and arrhythmogenesis in wild type mice 47 4.1.2. Impact of different phosphodiesterases and protein kinase G on arrhythmia development after ex vivo I/R 52 4.2. Role of PDE2 in murine heart failure model 54 4.2.1. Characterization of the murine heart failure model in different genetic backgrounds 55 4.2.2. Role of PDE2 on arrhythmia development upon acute β-AR stimulation in vivo 61 4.3. Role of PDE2 in mice with STZ-induced diabetic cardiomyopathy 62 4.3.1. Effect of cardiac PDE2 overexpression and deletion on cardiac function and development of STZ-induced diabetes 62 4.3.2. Impact of cardiac-specific overexpression of PDE2 in diabetic mice after I/R injuries 70 4.4. Atrial fibrillation 73 4.4.1. Effects of PDE2 modulation on atrial fibrillation under physiological and pathophysiological conditions after STZ-induced diabetes 73 4.4.2. Effects of cardiomyocyte-specific PDE2 deletion on atrial fibrillation induction in hearts from mice with STZ-induced diabetes 79 5. Discussion 82 5.1. CNP-induced PDE2 stimulation protects from arrhythmia after I/R injuries ex vivo 82 5.2. PDE2-/- mice did not show significant increased arrhythmia development in vivo upon HF induction 86 5.3. PDE2 OE mice are protected against I/R injuries-induced arrhythmia development after diabetes induction with STZ 86 5.4. PDE2 pharmacological inhibition and PDE2 genetic deletion might lead to more atrial fibrillation episodes after burst pacing 90 5.5. Limitations 93 5.6. Conclusions 95 6. Summary 97 7. Zusammenfassung 99 8. Bibliography 102 9. Acknowledgements 124 10. Declaration 126

Aim: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult mammalian ventricular slices as an alternative preparation. Method: Vibratome cut ventricular slices (350 μm thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). Results: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD90, 169.9 ± 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD90 with a half maximal effective concentration (EC50) of 4.5 μM. The IKr blocker E4031 and neuroleptic drug risperidone prolonged APD90 with EC50 values of 31 nM and 0.67 μM, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m ⋅ s-1. Conclusion: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Objectif: Déterminer si l’efficacité de l’amiodarone pour le maintien du rythme sinusal varie selon la fonction systolique ventriculaire gauche. Contexte: Malgré un profil de sécurité établi en insuffisance cardiaque, nous ignorons si l’efficacité de l’amiodarone et son impact sur différentes issues cardiovasculaires sont modulés par la fonction ventriculaire gauche. Méthode: Nous avons effectué une analyse combinée de 3307 patients (âgés de 68,0±0,9 ans ; 31,1% de femmes) recrutés dans les études AFFIRM et AF-CHF qui ont été randomisés au contrôle du rythme par l’amiodarone (N=1107) ou au contrôle de la fréquence cardiaque (N=2200). Résultats: Chez les patients sous amiodarone, la survie sans fibrillation auriculaire était de 84% et de 45% à 1 et 5 ans respectivement, sans égard à la fraction d’éjection ventriculaire gauche (P=0,8754, ajusté). De façon similaire, la proportion moyenne ajustée de temps en fibrillation auriculaire (15,0±1,8%) n’a pas été influencée par la fraction d’éjection (P=0,6094). Durant le suivi, 1963 patients (59,4%) ont requis au moins une hospitalisation, incluant 1401 (42,6%) patients hospitalisés pour cause cardiovasculaire. Les taux ajustés d’hospitalisation pour toutes causes et pour cause cardiovasculaire étaient similaires entre les patients sous amiodarone et ceux dans le groupe de contrôle de la fréquence dans l’ensemble de la population ainsi que dans les sous-groupes de patients avec et sans dysfonction ventriculaire gauche sévère. De façon similaire, les taux ajustés de mortalité globale et cardiovasculaire étaient similaires entre chez les patients sous amiodarone et ceux traités par le contrôle de la fréquence dans l’ensemble de la population ainsi que dans les sous-groupes de patients avec et sans dysfonction ventriculaire gauche sévère. Conclusions: L’efficacité de l’amiodarone pour le maintien du rythme sinusal n’est pas influencée par la fonction ventriculaire gauche. Le contrôle du rythme avec l’amiodarone s’associe à des taux de mortalité et d’hospitalisation comparables au contrôle de la fréquence à la fois chez les patients avec et sans dysfonction ventriculaire gauche sévère. / Objectives: To determine whether amiodarone’s efficacy in maintaining sinus rhythm varies according to left ventricular systolic function. Background: Despite amiodarone’s established safety profile in heart failure, it is unknown whether its impact on cardiovascular outcomes is modulated by ventricular function. Methods: We conducted a pooled analysis of 3307 patients (age 68.0±0.9 years; 31.1% female) enrolled in AFFIRM and AF-CHF trials who were randomized to rhythm control with amiodarone (N=1107) or rate control (N=2200). Results: In amiodarone-treated patients, freedom from recurrent atrial fibrillation was 84% and 45% at 1 and 5 years, respectively, with no differences according to left ventricular function (adjusted P=0.8754). Similarly, the adjusted mean proportion of time in atrial fibrillation (15.0±1.8%) did not vary according to ventricular function (P=0.6094). During follow-up, 1963 (59.4%) patients required at least one hospitalization, with 1401 (42.6%) patients hospitalized for a cardiovascular reason. Adjusted all-cause and cardiovascular hospitalization rates were similar with amiodarone versus rate control in the overall population and in subgroups of patients with and without severe left ventricular dysfunction. Similarly adjusted all-cause and cardiovascular mortality rates were similar with amiodarone versus rate control, overall and in subgroups of patients with and without severe left ventricular dysfunction. Conclusions: Amiodarone’s efficacy in maintaining sinus rhythm and reducing the burden of atrial fibrillation is similar in patients with and without left ventricular dysfunction. Rhythm control with amiodarone was associated with similar mortality and hospitalisation rates when compared to rate control in patients with and without severe left ventricular dysfunction.

Objectif: Déterminer si l’efficacité de l’amiodarone pour le maintien du rythme sinusal varie selon la fonction systolique ventriculaire gauche. Contexte: Malgré un profil de sécurité établi en insuffisance cardiaque, nous ignorons si l’efficacité de l’amiodarone et son impact sur différentes issues cardiovasculaires sont modulés par la fonction ventriculaire gauche. Méthode: Nous avons effectué une analyse combinée de 3307 patients (âgés de 68,0±0,9 ans ; 31,1% de femmes) recrutés dans les études AFFIRM et AF-CHF qui ont été randomisés au contrôle du rythme par l’amiodarone (N=1107) ou au contrôle de la fréquence cardiaque (N=2200). Résultats: Chez les patients sous amiodarone, la survie sans fibrillation auriculaire était de 84% et de 45% à 1 et 5 ans respectivement, sans égard à la fraction d’éjection ventriculaire gauche (P=0,8754, ajusté). De façon similaire, la proportion moyenne ajustée de temps en fibrillation auriculaire (15,0±1,8%) n’a pas été influencée par la fraction d’éjection (P=0,6094). Durant le suivi, 1963 patients (59,4%) ont requis au moins une hospitalisation, incluant 1401 (42,6%) patients hospitalisés pour cause cardiovasculaire. Les taux ajustés d’hospitalisation pour toutes causes et pour cause cardiovasculaire étaient similaires entre les patients sous amiodarone et ceux dans le groupe de contrôle de la fréquence dans l’ensemble de la population ainsi que dans les sous-groupes de patients avec et sans dysfonction ventriculaire gauche sévère. De façon similaire, les taux ajustés de mortalité globale et cardiovasculaire étaient similaires entre chez les patients sous amiodarone et ceux traités par le contrôle de la fréquence dans l’ensemble de la population ainsi que dans les sous-groupes de patients avec et sans dysfonction ventriculaire gauche sévère. Conclusions: L’efficacité de l’amiodarone pour le maintien du rythme sinusal n’est pas influencée par la fonction ventriculaire gauche. Le contrôle du rythme avec l’amiodarone s’associe à des taux de mortalité et d’hospitalisation comparables au contrôle de la fréquence à la fois chez les patients avec et sans dysfonction ventriculaire gauche sévère. / Objectives: To determine whether amiodarone’s efficacy in maintaining sinus rhythm varies according to left ventricular systolic function. Background: Despite amiodarone’s established safety profile in heart failure, it is unknown whether its impact on cardiovascular outcomes is modulated by ventricular function. Methods: We conducted a pooled analysis of 3307 patients (age 68.0±0.9 years; 31.1% female) enrolled in AFFIRM and AF-CHF trials who were randomized to rhythm control with amiodarone (N=1107) or rate control (N=2200). Results: In amiodarone-treated patients, freedom from recurrent atrial fibrillation was 84% and 45% at 1 and 5 years, respectively, with no differences according to left ventricular function (adjusted P=0.8754). Similarly, the adjusted mean proportion of time in atrial fibrillation (15.0±1.8%) did not vary according to ventricular function (P=0.6094). During follow-up, 1963 (59.4%) patients required at least one hospitalization, with 1401 (42.6%) patients hospitalized for a cardiovascular reason. Adjusted all-cause and cardiovascular hospitalization rates were similar with amiodarone versus rate control in the overall population and in subgroups of patients with and without severe left ventricular dysfunction. Similarly adjusted all-cause and cardiovascular mortality rates were similar with amiodarone versus rate control, overall and in subgroups of patients with and without severe left ventricular dysfunction. Conclusions: Amiodarone’s efficacy in maintaining sinus rhythm and reducing the burden of atrial fibrillation is similar in patients with and without left ventricular dysfunction. Rhythm control with amiodarone was associated with similar mortality and hospitalisation rates when compared to rate control in patients with and without severe left ventricular dysfunction.

Abstract Osteoclasts are multinuclear bone degrading cells differentiated from monocytes which can be isolated from bone marrow and peripheral blood. Complex signaling between osteoclast precursors and other bone cells, such as mesenchymal stromal cells (MSC) occurs during the differentiation. Gap junctional communication (GJC) is one of the mechanisms in the cell fusion. GJC can be modulated with several substances such as the specific GJC stimulators, antiarrhythmic peptides (AAP). Due to their promising clinical value in the treatment of cardiac disorders, the effects of AAPs in cardiac tissue are studied extensively. This study was conducted in order to investigate the roles of GJC and AAPs in bone cell cultures. Further, the contribution of the MSCs on the effects of AAPs was studied along with comparison of two types of osteoclastogenesis cultures with differing quantities of MSCs. GJC in osteoclastogenesis was studied with both GJC inhibitors and stimulators in mouse monocyte line RAW 264.7 cells and primary cultures with bone marrow hematopoietic cells. The following studies were made with human monocytes from peripheral blood and bone marrow where the effects of AAP10 were investigated in normal and acidic environments. In addition, comparison of osteoclastogenesis from bone marrow and peripheral blood monocytes was carried out in in vitro cell cultures on bovine or human bone slices. The cells were analyzed with regard to multinuclearity, bone resorption and the expression of several osteoclast markers. The results show that GJC is utilized in osteoclastogenesis, but it is not indispensable. GJC in monocytes can be stimulated with the AAPs during osteoclastogenesis, but the effects depend on the culture conditions as well as on the presence of MSCs in the culture. The AAPs can also activate the MSCs leading to indirect regulation of osteoclastogenesis, as the MSCs produce several molecules affecting the differentiation. Further, monocytes from peripheral blood showed increased potential for osteoclastogenic differentiation compared to bone marrow derived monocytes. This can be explained by the presence of the osteoclastogenesis-controlling MSCs in the bone marrow culture, while the peripheral blood cultures contain only few of these cells and thus lack their regulatory effects. / Tiivistelmä Osteoklastit ovat monitumaisia luuta hajottavia soluja, jotka ovat erilaistuneet monosyyteistä. Monosyyttejä voidaan eristää luuytimestä tai perifeerisestä verestä. Erilaistumisen aikana osteoklastien esiastesolujen sekä muiden luusolujen, kuten mesenkymaalisten stroomasolujen (MSC) välillä tapahtuu monimutkaista signalointia. Aukkoliitoskommunikointi (GJC) on eräs solufuusiossa tapahtuvista mekanismeista. GJC:tä voidaan muunnella useilla aineilla, esimerkiksi spesifisillä stimulaattoreilla, antiarytmisillä peptideillä (AAP). AAP-yhdisteiden vaikutuksia on tutkittu laajalti sydänkudoksessa johtuen niiden lupaavista kliinisistä ominaisuuksista sydänperäisten oireiden hoidossa. Tämän tutkimuksen tarkoituksena oli selvittää GJC:n ja AAP-yhdisteiden roolia luusoluviljelmissä. Lisäksi tutkittiin MSC-solujen osallistumista AAP-yhdisteiden vaikutuksiin sekä vertailtiin kahta erilaista osteoklastogeneesiviljelmää, joissa oli eri määrä MSC-soluja. GJC:tä osteoklastogeneesissä tutkittiin sekä sitä estävillä että stimuloivilla yhdisteillä hiiren monosyyttilinjan RAW 264.7 -soluissa sekä luuytimen hematopoieettisten solujen primääriviljelmissä. Seuraavat tutkimukset tehtiin ihmisen luuytimen ja perifeerisen veren monosyyteillä, ja niissä selvitettiin AAP10-yhdisteen vaikutuksia fysiologisissa sekä happamissa olosuhteissa. Lisäksi vertailtiin luuytimen ja perifeerisen veren monosyyttien osteoklastogeneesiä. In vitro -soluviljelmät tehtiin naudan tai ihmisen luulastujen päällä, ja soluista analysoitiin monitumaisuus, luun resorptio sekä useiden osteoklastimarkkereiden ilmentyminen. Tulokset osoittavat, että GJC:tä hyödynnetään osteoklastogeneesissä, mutta se ei ole korvaamaton mekanismi. GJC:tä voidaan stimuloida AAP-yhdisteillä osteoklastogeneesin aikana, mutta vaikutukset riippuvat viljelyolosuhteista sekä MSC-solujen läsnäolosta. AAP-yhdisteet voivat aktivoida myös MSC-soluja johtaen osteoklastogeneesin epäsuoraan säätelyyn, kun MSC-solut tuottavat useita erilaistumiseen vaikuttavia molekyylejä. Lisäksi perifeerisen veren monosyyteillä havaittiin korkeampi osteoklastogeeninen erilaistumispotentiaali verrattuna luuytimen monosyytteihin. Tulokset voidaan selittää osteoklastogeneesiä säätelevien MSC-solujen läsnäololla luuydinviljelmissä, kun taas perifeerisen veren monosyyttiviljelmissä näitä soluja on vain vähän, jolloin myös niiden säätelyominaisuudet puuttuvat.