Our findings demonstrate that genetic deletion of GPR55 in mice leads to the development of cardiac dysfunction with age and cardiac decompensation in response to adrenoceptor stimulation. While basal cardiac function was unaffected in young mice with a genetic deletion for GPR55, mature GPR55^−/− mice, which would still be considered young adult mice and unlikely to be affected by senescent heart dysfunction [20], were characterised by significantly comprised systolic function. In particular, both load-independent (ESPVR & E _max) and load-dependent (ejection fraction) indices of systolic function are significantly decreased in the mature GPR55^−/− mice. Furthermore, mature GPR55^−/− mice were also characterised by significant myocardial remodelling; including reductions in left ventricular free wall thickness, HW∶BW and ventricular cell number, and increased collagen deposition. Taken together, these changes are indicative of the presence of some form of cardiomyopathy (an all-encompassing term referring to alterations in both cardiac structure and function, that lead to a deterioration in cardiac function and ultimately heart failure), and possibly one that possesses several of the features of dilated cardiomyopathy (DCM) i.e. LV wall thinning, comprised systolic function, and reduced contractile reserve.

Possible explanations for the emerging systolic dysfunction observed in the mature GPR55^−/− mice may include either impaired Ca²⁺ signalling in cardiomyocytes and/or altered central control of cardiac function. Studies have demonstrated GPR55 induced elevations in intracellular Ca²⁺ in both endothelial cells [21], and more recently cardiomyocytes [22]. In the latter study, Yu et al. [22] demonstrated that LPI applied both extra- and intracellularly induced elevations in [Ca²⁺]_i in a GPR55 dependent manner. Furthermore, these findings led them to suggest that the receptor was expressed both at the sarcolemma and endo-lysosomal compartment, which may explain the extensive expression of GPR55 observed in ventricular tissue in the present study. As the previous study has demonstrated a role for GPR55 in Ca²⁺ signalling in the cardiomyocyte [22], it is possible that GPR55 gene deletion could adversely affect excitation-contraction coupling in the cardiomyocyte, and consequently the contractile ability of both the cell and myocardium as a whole. However, as none of the Ca²⁺ dependent indices of contractility (dP/dt_max, ESPVR or E _max) differed between young GPR55^−/− and WT mice this seems unlikely.

An alternative explanation may be an affect on the central control of cardiac contractility. It is well established that in the early stages of systolic dysfunction, compensatory mechanisms are initiated to maintain systolic function and meet metabolic demands, including sympathoexcitation (reviewed by [23]). In particular, catecholamines acting on β₁-adrenoceptors on pacemaker cells of the sinoatrial node, serve to increase action potential firing rate and induce a positive chronotropic response [24], [25]. In the present study, mature GPR55^−/− mice were not characterised by positive chronotropy (in an attempt to maintain systolic function), which may suggest impaired sympathetic control of the myocardium. In support of this, previous studies have demonstrated that activation of GPR55 leads to both increased excitability of dorsal root ganglion neurons [4], and enhanced pre-synaptic signalling in the hippocampus [26]. Although GPR55 expression in the nucleus tractus solitarius has yet to be demonstrated, it is possible that GPR55 may have a role in the regulation of synaptic transmission between preganglionic and postganglionic sympathetic efferents, and thus deletion of this GPCR may adversely affect sympathetic outflow. However, as basal systolic dysfunction appeared to be due to a chronic effect of GPR55 gene deletion (i.e. only evident at 8 months) and associated with significant ventricular remodelling it seems unlikely that GPR55 has a direct role in the control of cardiac function.

In the absence of a direct role for GPR55 in the control of cardiac contractility it is possible that this GPCR regulates the activity of another cardiac receptor responsible for regulating systolic function. Cardiac adrenoceptors, and β-adrenoceptors in particular, are the predominant GPCR in the heart and the chief modulators of both cardiac chronotropy and inotropy (reviewed by [27]). In the present study, all GPR55^−/− mice exhibited significantly attenuated positive inotropic responses to (±)-dobutamine (an agonist which directly stimulates cardiac adrenoceptors) when compared to WT mice, suggesting a pivotal role for GPR55 in the regulation of adrenoceptor activity in the myocardium. This proposed maladaptive adrenergic signalling may in part explain the progressive cardiac dysfunction associated with these GPR55^−/− mice. Accumulating evidence has shown that chronic stimulation of cardiac β-adrenoceptors activation leads to receptor phosphorylation via GPCR kinases i.e. βARK1 (desensitization), subsequent internalization of desensitized receptors via β-arrestin (downregulation), a loss of β-adrenoceptor mediated signalling, and finally the development of systolic heart failure (reviewed by [28]). Indeed, preservation of β-adrenergic signalling, via gene delivery of a βARK1 inhibitor, can reverse and/or prevent the development of cardiac dysfunction [29], [30]. Thus it's possible that the systolic dysfunction evident in the mature GPR55^−/− mice may be due to the progressive loss of cardiac adrenoceptors. In line with this it might have been expected that the impaired positive inotropy to dobutamine observed in the young GPR55^−/− mice would be further attenuated in the mature knockout mice, however this was not the case.

A possible explanation for this may involve α₁-adrenoceptors, as although β₁-adrenoceptors are thought to be primarily responsible for catecholamine induced increases in cardiomyocyte contractility, α₁-adrenoceptors have been shown to induce cardiac contraction [31], [32], [33]. Furthermore, they have previously been shown to mediate part of the (±)-dobutamine induced positive inotropy in the rodent heart [34]. Although α₁-adrenoceptor expression in healthy murine and human hearts is considerably less than that of the β-adrenoceptor subtypes [35], β-adrenoceptors are downregulated in heart failure whereas α₁-adrenoceptors are not [36], [37]. Thus α₁-adrenoceptor-mediated responses may contribute substantially to the compensatory positive inotropy in failing hearts.

In addition to altered cardiac function, mature GPR55^−/− mice were also characterised by significant ventricular remodelling including decreased HW∶BW, a thinning of the LV wall, a reduction in myocardial cell number, and increased collagen deposition. While there is currently no direct evidence for a functional role for GPR55 in the control of fibroblast activity, a recent study has demonstrated that this receptor is expressed on cells, which likely include fibroblasts, in the adventitial layer of rodent vasculature [38], and that LPI is synthesised by transformed mouse BALB/3T3 fibroblasts [39], thus it is possible GPR55 may play a role in fibrogenesis. Indirectly, GPR55 may regulate fibrogenesis by altering the activity of another GPCR i.e. CB₁, as it has recently been demonstrated that GPR55 can form a heteromer with CB₁ allowing the former to alter the signalling mechanisms/activity of the latter, and vice versa [40]. Therefore it is possible that the profibrogenic effect of CB₁, previously documented in an experimental model of doxorubicin-induced cardiomyopathy [41], may be unimpeded in the mature mice lacking GPR55 thus resulting in the development of mild cardiac fibrosis. Finally, if mature GPR55^−/− mice are in fact characterised by increased cardiac α₁-adrenoceptor activity (as discussed in the previous section) then this may account for the increased myocardial collagen deposition as cardiac fibrosis has previously been demonstrated in mice overexpressing α₁-adrenoceptors [42].

The observation that mature GPR55^−/− mice are characterised by cardiac fibrosis seems somewhat incongruous with the significant reductions in both HW∶BW and LV wall thickness (indicative of a ‘lighter’ heart) observed in these animals. Therefore rather then increased fibrogenesis being the culprit for the increased cardiac collagen deposition, the latter may simply be ‘increased’ in the face of increased cell death/loss from the heart. In the present study, the left ventricles of mature GPR55^−/− were characterised by a signficant reduction in stained nuclei indicating an increase in myocardial cell loss. However, as this data was acquired from H&E stained tissue, which is not specific for cardiomyocytes, we cannot conclusively say that all of the cell loss was due to cardiomyocyte apoptosis and additional studies are required. Previous work has suggested both anti-inflammatory [43] and anti-oxidant [44] roles for GPR55, thus loss of the receptor may lead to a chronic upregulation of both inflammation and oxidative stress in the mature GPR55^−/−, both of which are chief instigators of cardiomyocyte cell death. However as the present study did not examine either the inflammatory or oxidative status of these animals these proposed mechanisms remain to be confirmed.

Exactly how the deletion of the GPR55 gene affects cardiac adrenoceptor signalling/function in the present study is unclear. However, accumulating evidence has demonstrated that co-localised GPCRs, not limited to adrenoceptor subtypes alone [45], [46], [47], but adrenoceptors and other GPCRs, can interact and regulate surface expression of each other via a process termed dimerization (reviewed by [48]). In particular, data from isolated ventricular cardiomyocytes has demonstrated cross-regulation between adrenergic and adenosinergic receptors, where stimulation of one inhibited the activity of the other and vice versa [49]. Furthermore, as previously discussed GPR55 can form heteromers with CB₁ enabling both GPCR's to alter the signalling mechanisms/activity of the other [40]. In heart failure, the crosstalk between α-adrenoceptors and β-adrenoceptors is well established, in that expression of the former is elevated in the response to the downregulation of the latter as a means of sustaining positive inotropism of the contractile apparatus [50], [51]. While studies have yet to demonstrate co-expression of GPR55 and adrenoceptors within the same cardiomyocytes (as was demonstrated in murine vascular cells [38]), it is possible that there is some level of co-localisation in the myocardium that may facilitate crosstalk between these GPCRs influencing their function/expression, although this requires investigation. Finally, as the present study only examined the impact of GPR55 gene deletion on the function of adrenoceptors, we cannot rule out the possibilty that other GPCR's are similarly adversely affected. Furthermore, rather than GPR55 having a direct effect in terms of modulating other GPCR's function it is possible that the absence of this receptor may result in a more generalised adverse effect i.e. defective G protein-coupled signalling, culminating in the dysfunction of numerous GPCRs particularly those involved in stress-sensitive pathways. Thus the effect of GPR55 gene deletion on other GPCRs capable of inducing both inotropic and chronotropic responses should also be investigated in the future.