Investigating the effect of cathepsin K inhibition on cardiac function following cardiac ischaemiareperfusion injury

Abstract

One 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.