Programme Contents

Manipulating the innate immune system as a cardioprotective strategy

Myocardial ischaemia-reperfusion injury (IRI) occurs when blood flow is restored to the myocardium following a period of ischaemia, such as after revascularisation after infarction, or reperfusion after cardioplegia. Despite intense research, strategies to abrogate or minimise IRI are not effective. The mechanisms underlying IRI include damage to mitochondria, generation of ROS, disturbance of ion fluxes (particularly Ca²⁺ overload), and contractile protein dysfunction. Inflammation, via activation of the immune system, also plays a large role. The innate immune system is the more “primitive” part of immune defence, which recognises a range of small molecules rather than specific antigens. Part of this recognition is by Toll-like receptors (TLRs), which are expressed in the heart. It has been proposed that during reperfusion activation of TLR4 receptors contributes to IRI, based on the finding that knockdown of TLR4 receptors reduces IRI (Oyama et al. 2004).

A proven non-pharmacological method to protect the myocardium from IRI is afforded by ischaemic preconditioning, whereby exposure of the heart to brief transient periods of ischaemia can reduce the damage caused by a subsequent longer period of ischaemia (Murry et al., 1986). However, no dominant single mechanism seems to be involved, and attempts to produce “pharmacological preconditioning”, or cardioprotection, have been unsuccessful. We have therefore examined whether TLR4 receptors are involved in ischaemic preconditioning.

It has been shown that LPS (an agonist of TLR4 receptors) is cardioprotective (Rowland et al., 1997). We hypothesized that this is due to desensitisation of TLR4 receptors, and that similar activation/desensitisation of TLR4 receptors by transient ischaemia may be a mechanism of preconditioning. If so, pharmacological block of TLR4 during preconditioning should abrogate the protective effect. To test this idea, we used amytripliyline, an antagonist at TLR4 receptors, in a preconditioning protocol in isolated rat hearts. Surprisingly, amitriptyline (10 μM) did not affect preconditioning, but was itself cardioprotective. We ascribe this to off-target actions (Lee et al., 2015).

We have therefore recently used more specific compounds, naloxone and naltrexone in the same way. The (+) isomers of these compounds do not interact with opiod receptors, but they are antagonists at the TLR4 receptor (Hutchinson et al 2008). In an isolated mouse heart model of preconditioning, we find that either naltrexone or naloxone (20 μM) did not affect IRI itself, but did impede recovery of LVDP after ischaemic preconditioning if they were present during the preconditioning period.

We conclude that TLR4 receptors may play a role in ischaemic preconditioning.

Hutchinson MR, Zhang YN, Brown K, Coates BD, Shridhar M, Sholar PW, Patel SJ, Crysdale NY, Harrison JA, Maier SF, Rice KC & Watkins LR. (2008). Non-stereoselective reversal of neuropathic pain by naloxone and naltrexone: involvement of toll-like receptor 4 (TLR4). Eur J Neurosci 281, 20-29.

Lee S, Hutchinson MR & Saint DA. (2015). Amitriptyline pharmacologically preconditions rat hearts against cardiac ischemic-reperfusion injury. Int J Cardiol 190, 353-359.

Murry CE, Jennings RB & Reimer KA. (1986). Preconditioning with ischemia – a delay of lethal cell injury in ischaemic myocardium. Circulation 74, 1124-1136.

Oyama J, Blais C, Liu XL, Pu My, Kobzik L, Kelly RA & Bourcier T. (2004). Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice. Circulation 109, 784-789.

Rowland RT, Meng X, Cleveland JC Jr, Meldrum DR, Harken AH, Alden H & Brown JM. (1997). LPS-induced delayed myocardial adaptation enhances acute preconditioning to optimize postischemic cardiac function. Am J Physiol 272, H2708-H2715.