Cardiovascular Injury: The Role of Adenosine and Cytokines

Abstract

Despite considerable research investigating the role of adenosine in protecting the heart against myocardial ischaemia-reperfusion injury, the specific roles of individual subtypes of adenosine receptors (ARs) remains unclear. Furthermore, the role of inflammation in ischaemia-reperfusion injury, and its interaction with adenosine in the myocardium, is also unclear. This thesis outlines a series of studies aimed at clarifying the role of adenosine (specifically A1ARs) in protecting the ischaemic heart. I also aimed to clarify the role of inflammatory processes in the heart, during both ischaemiareperfusion and exacerbated systemic inflammation. The predominant model employed to study myocardial ischaemia-reperfusion throughout this thesis is the Langendorff perfused isolated mouse heart. Surprisingly, and despite widespread use, relatively few papers have characterised fundamental properties of this valuable model, and perhaps as a result considerable variation exists in both methodological approaches and outcomes with this technically challenging preparation. Initially, we therefore compared responses to ischaemia in hearts perfused with 1.35 and 2 mM free Ca2+, and in hearts paced at 420 and 600 beats per minute (bpm). We also assessed ischaemic tolerance in male vs. female mice over an age range of 8, 16, 20 and 24 wks. The results from this study indicate that differences in Ca2+ concentration in buffer, or variations in heart rate, do not exert major effects on final outcomes from ischaemia-reperfusion but do generate some qualitative shifts in ischaemic responses consistent with predicted actions of both Ca2+ and metabolic (heart) rate. Moreover, we present the first evidence of early (within 16 weeks of age in males) age-dependent changes in ischaemic tolerance in the model, and suggest that tight regulation of the age of mice used in studies of ischaemia-reperfusion will significantly reduce variability in experimental outcomes. Having characterised fundamental properties in the Langendorff isolated heart model, work in Chapters 4,5 and 6 assessed the roles of the A1 adenosine receptor (A1AR) in protecting against ischaemia-reperfusion injuries. The roles of the endogenous and exogenous A1AR activation in protecting against ischaemic contracture (and relationship between contracture and post-ischaemic outcomes) were first assessed, with data revealing that adenosinergic inhibition of contracture is solely A1AR-mediated and is ‘supra-physiological’ (ie. evident only with significant periods of pre-ischaemic AR agonism or enhanced A1AR density). As such, ischaemic contracture appears insensitive to locally generated adenosine, potentially due to rapidity of contracture development vs. the finite time necessary for expression of AR-mediated cardioprotection. Moreover, whilst protection against ischaemic contracture was associated with improved postischaemic outcome, modification of contracture is not a pre-requisite for improved outcome post-ischaemia. Having clarified the role of A1ARs in modifying ischaemic contracture, the impact of A1AR and adenosine deaminase (ADA) knockout or deficiency on ischaemic outcome was characterised (Chapters 5 and 6). Neither genetic manipulation modified baseline contractility, though heart rate was reduced in ADA-deficient mice (in keeping with enhanced adenosine levels). Removal of the A1AR abolished A1AR-mediated bradycardia in response to 2-chloroadenosine (confirming the phenotype), without altering sensitivity of A2AR-mediated coronary dilation. Similarly, functional A1AR and A2AR sensitivities were unaltered by ADA deficiency. Tolerance to ischaemia was limited by the removal of the A1AR, with reduced contractile recovery (by sim25%) and enhanced lactate dehydrogenase (LDH) efflux (by sim100%). Contractile effects of A1AR deficiency involved worsened peak systolic pressure development with no change in diastolic dysfunction. In contrast, ADA deficiency modestly improved tolerance (as did A1AR overexpression), with a primary effect on diastolic dysfunction (but not systolic pressure). Protection with ADA deficiency was eliminated by simultaneous removal of the A1AR. Non-selective agonism (10 mumol/L 2-chloroadenosine) protected wild-type and A1AR deficient hearts, supporting protection via sub-types additional to A1ARs. These data are the first demonstrating that the removal of the A1AR limits intrinsic ischemic tolerance, and that ADA deficiency is protective. The normal function of the A1AR appears to be enhancement of peak contractility and limitation of cell death, with little effect on diastolic dysfunction. Reduced diastolic dysfunction is only apparent with enhanced A1AR agonism or expression. In Chapter 7, the extent to which ‘intrinsic’ inflammatory processes might impact on contractile recovery from ischaemia in isolated myocardium was assessed. Isolated myocardium possess a significant number of cells capable of modulating inflammation, including mast cells, macrophages, and myocytes themselves. Both immunomodulating agents and cytokine knockout mice were used to assess potential contributions of intrinsic inflammatory responses to injury in this model. The pro-inflammatory compounds formyl-Met-Leu-Phe (FMLP) and anti-inflammatory amiprilose HCl, ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL), S-(1,2,-diacarboxyethyl) glutathione (DCE-GS) and benzyloxycarbonyl-Ala-Ser-Thr-Asp-fluoromethylketone (Z-Z-ASTD-FMK) were infused into hearts isolated from C57/BL6 mice. Additionally, functional recoveries were assessed in TNFalpha, IL-10, IL-6, nNOS and eNOS knockout, and IGF transgenic mice. Amiprilose was the only inflammatory modulator to alter outcome: both left ventricular (LV) diastolic and systolic dysfunction were exacerbated by this agent. In terms of gene modified models, baseline coronary flow was significantly higher in IL-6 and IL-10 knockout mice, and IL-6 and eNOS KO mice displayed significant improvements in absolute recovery of LV developed pressure (though neither of these effects were evident when recovery was normalised to baseline function). Both TNFalpha and IL-6 knockout reduced LV diastolic dysfunction during reperfusion. Coronary flow at the end of reperfusion was significantly improved in IL-6 and eNOS knockout mice. IGF transgenic mice displayed a sim25% increase in heart weight:body weight ratio, and significant impairment in post-ischaemic recovery (in both LV diastolic pressure and developed pressure). Results from these studies collectively suggest that there is indeed an influence of inflammatory modulation on isolated myocardium subjected to ischaemia-reperfusion, with a predominantly deleterious functional consequence (with some of these injurious mechanisms amenable to modulation to improve outcome). However, evidence of an inflammatory response, combined with a lack of protection with A2AAR agonism observed in isolated hearts (459) tends to exclude a role for A2AARs in mediating antiinflammatory effects in cells resident to isolated hearts. Finally, in Chapter 8, the role of the A2AAR in modifying exacerbated systemic inflammatory responses was assessed using a murine model of endotoxemia. The impact of Adora2a knockout (A2AAR KO) in a model of acute endotoxemia was studied in both young and aged and male and female mice, in an attempt to assess for age and gender dependencies of inflammatory responses and phenotype effects of A2AAR KO. Young (3-4 months) and aged (10-11 months) mice were injected with 40 mg/Kg LPS or an equivalent volume of normal saline and sacrificed 12 or 24 hours later. The phenotype associated with A2AAR KO (in this setting) was strongly dependant on gender and age. No young animals (male or female, KO or wild-type) died following LPS challenge. However, aged, male, A2AAR KO mice displayed an 80% mortality rate vs. only 20% in aged wild-type mice. This contrasted outcomes in aged females, where mortality was 20% in wild-type mice and 0% in A2AAR KO mice. Thus, while A2AAR KO increased mortality in males, it has little to no effect in females. Gender dependence of the A2AAR KO phenotype was also evident in young mice: haemoglobin and hematocrit levels were both elevated in young male (but not female) A2AAR KO mice treated with LPS while leukocyte and platelet levels were elevated in young male (but not female), A2AAR KO injected with saline. Interestingly, there was no reliable pro- or anti-inflammatory effect associated with A2AAR deletion in response to LPS. Importantly, however, Adora2a-/- mice exhibited a sim6 fold increase in cardiac troponin I (cTnI) levels after LPS challenge indicating a significant cardioprotective role for the A2AAR in the setting of endotoxemia. These data are the first to demonstrate that: males appear less able to compensate for loss of the A2AAR than females (in the context of inflammatory challenge), and this effect becomes more prominent with age; A2AARs play a significant cardioprotective role in sepsis. In summary, the data presented in this thesis reports: a primary role for the A1AR in mediating adenosinergic cardoprotection in isolated mouse hearts, although roles for other adenosine receptor sub-types are also implicated; the existance of an inflammatory response in isolated mouse hearts despite being devoid of blood borne inflammatory cells; significant benefit via the A2AAR in animals and hearts subjected to endotoxaemia, with a significant gender dependancy of the impact of A2AArs in this setting.