Saudi Cultural Missions Theses & Dissertations
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Item Restricted The effect of senolytics on cardiac remodelling and repair after injury induced by isoproterenol(King's College London, 2024-04) Altuwaijri, Ahmed; Ellison-Hughes, GeorginaSenescent cells accumulate during ageing and contribute to tissue deterioration, including in the heart. Senescent cells negatively affect an organ's microenvironment by refusing to die and producing a pro-inflammatory senescence-associated secretory phenotype (SASP). Genetic or pharmacologic clearance of senescent cells by senolytics has been shown to improve cardiac recovery and remodelling in aged mice. Moreover, senolytics have improved cardiac recovery after cardiac injury and in heart failure models in aged and young adult mice. Isoproterenol (ISO) has been widely used to induce cardiac injury in rodents, but it is unknown if ISO induces senescence and whether increased senescent cells in the heart contribute to cardiac deterioration and pathophysiology. This PhD aimed to first establish a cardiac injury model which led to increased senescence in the hearts of young adult mice by ISO. Second, to investigate the effects of the senolytics, dasatanib+quercetin (D+Q) on cardiac recovery and remodelling after ISO-injury in young adult and aged mice. To establish the dose of ISO that induced senescence with cardiac injury, ISO 150 mg/kg or 100 mg/kg was subcutaneously administered to ~12-week-old male mice (n=3 per group) for six consecutive days. Echocardiography was conducted from baseline to day 7. On day 7 after ISO, hearts were excised in order to analyse cardiac injury and cellular senescence. Results indicated that 100 mg/kg and 150 mg/kg ISO induced injury mainly in the apex of the heart. However, SA-β-gal staining was evident only in the apex of the ISO 150 mg/kg group. Therefore, ISO 150 mg/kg was chosen over the ISO 100 mg/kg dose. To establish the timeline of increased senescence after ISO-injury, 150 mg/kg ISO was subcutaneously administered to ~12-week-old male mice (n= 3-9 per group) for six consecutive days. Echocardiography was conducted at baseline, day 7 and 14 following ISO administration. Mice were sacrificed and hearts were excised for analysis of cardiac injury and senescence on days 7, 10 and 14. ISO 150 mg/kg successfully induced cardiac injury and senescence that lasted for 14 days. The peak of senescence was observed on day 10 following ISO. Next, ISO 150 mg/kg was subcutaneously administered to ~12-week-old male mice for six consecutive days. On day 10 after ISO, D+Q (5 mg/kg, 50 mg/kg) were administered by oral gavage for five consecutive days. Echocardiography was performed at baseline, day 7 post-ISO, and day 28 after the last D+Q dose. The hearts were excised on day 28 following the last D+Q dose to investigate cardiac injury and cellular senescence. Young adult mice treated with D+Q showed better cardiac recovery and remodelling after ISO injury. D+Q enhanced cardiac function, reduced hypertrophy, and reduced senescence markers. However, levels of collagen and DNA damage were unaffected by D+Q treatment. Aged male mice had four cycles of D+Q (5 mg/kg, 50 mg/kg) via oral gavage. Each cycle was composed of 3 days on and 12 days off. After that, aged mice hearts were injured by subcutaneous injection of 50 mg/kg ISO for six consecutive days. Echocardiography was performed at baseline, 24 hours after D+Q, and 28 days after the last ISO dose. The hearts were excised on day 28 following the last ISO dose to investigate cardiac injury, cellular senescence and remodelling. D+Q did not enhance cardiac function in the aged mice before ISO injury. However, D+Q improved cardiac recovery and survival after ISO injury. In the ISO-injured aged mice, D+Q enhanced cardiac function, reduced hypertrophy, reduced fibrosis, reduced DNA damage and senescence markers, and increased cardiomyocyte DNA synthesis. Clearance of the senescent cells and their SASP factors by D+Q senolytics improved cardiac function and recovery after ISO injury in aged and young adult male mice. These findings encourage the use of senolytics as a potential adjunct therapy for cardiac injury and deterioration with ageing. Senolytics could be used to improve the microenvironment of the heart so that it is more resilient to damage and can recover more effectively.24 0Item Restricted Investigating the effect of cathepsin K inhibition on cardiac function following cardiac ischaemiareperfusion injury(Saudi Digital Library, 2023) Zaeri, Ali Abdullah; Loughrey, Christopher; Nicklin, StuartOne of the main causes of mortality in the world is acute myocardial infarction (MI) due to coronary artery blockage. Obstruction of the coronary arteries prevents cardiac muscle from receiving oxygen and nutrients (ischaemia) and eventually results in cardiac muscle damage or death (MI). Patients with acute MI are mainly treated by primary percutaneous coronary intervention (PPCI) procedure in order to remove the coronary occlusion and restore blood flow to the ischaemic myocardium. Restoring the blood flow to ischaemic tissue is essential to salvage reversibly damaged myocardium and limit the extent of irreversible cell death. This is important because the infarct size is a key determinant of patient outcome and cardiac remodelling progression that leads to heart failure. However, PPCI results in paradoxical cardiomyocyte death caused by myocardial ischaemiareperfusion injury (IRI). The myocardial IRI therefore limits the full effectiveness of PPCI. Currently, it is imperative to discover novel therapeutic targets which can be targeted at the beginning of PPCI to limit myocardial IRI, adverse cardiac remodelling and reduce progression to heart failure. Cysteine cathepsins including cathepsin-K (CatK) are found mostly in lysosomes. Cysteine cathepsins are found to regulate cardiac function and are involved in range of normal processes including hypertrophy, apoptosis, autophagy and extracellular matrix remodelling. Whilst cathepsin are normally located within lysosomes, certain conditions such as IRI can lead to the release of cathepsins from lysosomes and promote adverse cardiac remodelling. The effect of CatK on cardiac function in the context of IRI remains unknown. In this study, we hypothesized that CatK release during IRI is associated with dysfunction of cardiomyocyte calcium handling (a key determinate of LV contraction) and cardiomyocyte survival. To investigate this hypothesis, Langendorff perfusion method was used in the lab to create IRI in rat heart. Isolated ex vivo adult Wister rats hearts were subjected to global ischaemia followed by reperfusion period in the presence and absence of the specific CatK inhibitor; L006235. In this procedure, cardiac function (in particular LV developed pressure, LVDP) was determined and infarct size measured using triphenyltetrazolium chloride (TTC) stain. Additionally, a number of key proteins involved in regulating heart contraction and apoptosis were measured by immunoblotting assay to understand the role of CatK on cardiomyocyte function at the end of the protocol. Hearts treated with 3 μM L006235 showed a 42% reduction in infarct size compared to DMSO (11.4±2.7 vs. 27.2±3.4%; *P<0.05), a reduction in pro-apoptotic Bax expression, and an increase in reciprocal anti-apoptotic Bcl-2. Furthermore, the area under the curve (AUC) of LVDP was higher in the L006235 group compared to DMSO (AUC; 3866±877 SEM vs. 793.5±81 SEM, respectively; *P<0.05). The expression of key calcium handling proteins [such as Sodium-calcium exchanger 1 (NCX1), phosphorylated phospholamban at serine 16 site (p-PLB-s16), phosphorylated phospholamban at threonine 17 site (p-PLB-th17), protein kinase A (PKA) and calcium/calmodulin dependent protein kinase II (CaMKII)] that regulate heart contraction were reduced post-IRI vs. control (without IRI), however, L006235 prevented these changes. This new data warranted investigation of the mechanism by which CatK affects cardiomyocytes. One important signalling pathway that involves an improvement of cardiac contractility and enhancement of cells survival is the Akt downstream pathway. Activation of Akt by phosphorylation Threonine 308 and serine 473 promotes cardiac protection in injured myocardium. Protein expression of Akt phosphorylation at serine 473 (p-Akt-s473) was significantly elevated when L006235 was used in ischaemic hearts relative to DMSO (324±51 vs. 182±36%, *P<0.05) which indicated that L006235 plays a role in Akt cardioprotection mechanism. Furthermore, this result was supported when L006235 combined with an Akt phosphorylation inhibitor (MK2206) in ex vivo IR injury model. This combination resulted in a reduction of cardiac contractility function and increase of infarct size which indicated that inhibiting Akt attenuate the cardiac protection of L006235. Additionally, in isolated neonatal rat cardiomyocytes (NRCMs) using siRNA transfection method to knock down CatK protein resulted in a significant increase of protein expression of p-Akt-s473 along with antiapoptotic Bcl-2 comparing to non-targeted siRNA group. This result suggested that silencing CatK play a role in promoting cell survival through the Akt/Bcl-2 signalling pathway. In conclusion, the data in this thesis demonstrated that inhibition of CatK in ex vivo IRI model suppressed cells death and preserved calcium handling proteins expression and thereby contributed to the improved cardiac function recovery and reduced infarct size observed with L006235. Furthermore, an in vitro study indicated that knocking down of CatK promoted cells survival via Akt/Bcl-2 pathway. Altogether indicating that CatK is a novel therapeutic target with a potential to reduce the deleterious effects of acute IRI after PPCI.10 0