Abstract

Introduction

G protein coupled receptor 55 (GPR55) belongs to a group of rhodopsin-like seven transmembrane/g-protein coupled receptors and was originally isolated in human striatum [1]. GPR55 has since been shown to be widely distributed in a variety of cell types and in the central nervous, gastrointestinal, and cardiovascular systems, in both humans [2] and rodents [3]. The downstream signalling mechanisms following activation of GPR55 remain unclear although activation of Gαq/11 or Gα13 culminating in an eventual elevation of intracellular calcium (Ca2+) and phosphorylation of extracellular signal-regulated kinase (ERK) and/or nuclear factor of activated T cells (NFAT) has been implicated [4], [5], [6]. Furthermore, while the exact physiological/pathophysiological function of GPR55 remains to be determined, studies have suggested a role in pain, bone development, carcinogenesis, pregnancy, metabolism (reviewed by [2]), and finally in the control of cardiac haemodynamics.

In terms of the cardiovascular system, a role for GPR55 was proposed on the basis of accumulating evidence from a series of studies investigating the profound haemodynamic (hypotension and bradycardia) effects of cannabinoid ligands, which were initially believed to be mediated primarily through the classic cannabinoid receptors, CB1 and CB2, (reviewed by [7]). However, combined evidence from studies using mice deficient in either CB1 or CB2 and from experiments employing various pharmacological agonist/antagonist combinations, have revealed that many cannabinoid-induced haemodynamic responses are mediated by non-CB1/CB2 receptors [8], [9]. Moreover, cannabinoids that have little or no affinity for the CB1/CB2 receptors have also been shown to exert cardiovascular effects, further suggesting a role for additional receptor(s) in mediating these effects [10], [11]. Based on the findings that some vasoactive cannabinoids (e.g. abnormal cannabidiol and O-1602) are potent agonists of GPR55 [3], [5], [12], [13], the latter has been proposed as a possible third cannabinoid receptor [14], [15]. However, a more recent study investigating an array of cannabinoids as possible ligands for GPR55 demonstrated that only lysophosphatidylinositol (LPI), rimonabant, and AM251 are agonists for this receptor, and that neither abnormal cannabidiol nor O-1602 activate GPR55 [16]. Thus, given the incongruous nature of our understanding of the GPR55 receptor and the relative paucity of literature regarding its role in cardiovascular physiology we conducted a study using the previously described homozygous GPR55-deficient (GPR55−/−) mouse [12], [17], to examine the influence of GPR55 on cardiac physiology/function (assessed via pressure volume loop analysis).

Methods

Results

Effect of gene deletion for GPR55 on baseline cardiac function in young mice

Positive staining for GPR55 was detected in ventricular tissue from WT (Figure 1A) but not GPR55−/− mice (Figure 1B). Furthermore, image analysis revealed diffuse staining for GPR55 throughout the cardiomyocytes, suggestive of a role for this receptor in the control of contractile function. However, pressure volume loop analysis revealed that, with the exception of heart rate (HR), which was significantly elevated in 10 week old GPR55−/− mice, none of the other load-dependent indices of left ventricular (LV) systolic and diastolic function; end systolic pressure (ESP), end systolic volume (ESV), stroke volume (SV), cardiac output (CO), ejection fraction (EF), maximum (dP/dtmax) and minimum (dP/dtmin) derivatives of pressure, arterial elastance (Ea; an index of LV afterload and reflective of peripheral vascular resistance), end diastolic pressure (EDP) and volume (EDV), recorded in this study differed significantly when compared to 10 week old WT mice (Table 1). Furthermore, none of the load-independent measurements of LV function, ESPVR, EDPVR, and Emax, differed significantly between both groups of mice (Figure 2).

Figure 1

Expression of GPR55 in ventricular tissue from WT and GPR55−/− mice.

Figure 2

Load-independent (ESPVR, EDPVR, and Emax) haemodynamic parameters in both WT and GPR55−/− mice.

Table 1

Load-dependent haemodynamic parameters in control (WT) mice and those with a genetic deletion for GPR55 (GPR55−/−).

Age dependent changes of GPR55 gene deletion on baseline cardiac function

In terms of load-dependent cardiovascular variables, both indices of systolic and diastolic function were significantly altered with age in both WT and GPR55−/− mice (Table 1). In particular, ESP, ESV, EDV, SV, SW, and CO were all significantly elevated in mature WT mice compared with young mice (Table 1), which in part may be due to the increased circulating blood volume in these larger animals. In contrast, only SV, EDV, and ESV were significantly increased in mature GPR55−/− mice, and in terms of the latter two indices they were also significantly elevated in comparison to age-matched WT mice. Furthermore, mature GPR55−/− mice also exhibited compromised systolic function as EF was significantly decreased in these mice when compared to both young GPR55−/− mice and age-matched WT controls (P<0.001; Table 1). This emerging systolic dysfunction appeared to be due to the significant increase in EDV (P<0.001) recorded in the mature GPR55−/− mice, which was not accompanied by a sufficient increase in SV (to maintain EF), when compared to age-matched WT controls (Table 1). Furthermore, load-independent measurements obtained during transient occlusion of the inferior vena cava (to alter preload), demonstrated a significant downward shift in both the ESPVR slope (P<0.001; Figure 2A) and the time varying elastance (Emax; P<0.0001; Figure 2B) in the mature GPR55−/− mice indicative of decreased contractility/inotropy. In contrast, the slope of the EDPVR (indicative of an increase in LV chamber stiffness; Figure 2C) did not differ significantly between any of the experimental groups examined and thus load-independent diastolic function did not appear to be altered following deletion of the GPR55 gene. Taken together, the changes in both load-dependent and load-independent indices of cardiac function observed in the mature GPR55−/− mice suggest that the emerging systolic dysfunction appears to be due to the deleterious combination of both GPR55 gene deletion and advancing age in these animals (representative pressure volume loops of this cardiac dysfunction are illustrated in Figure 3).

Figure 3

Representative left ventricular pressure volume loops from all experimental groups are included and illustrate the emerging systolic dysfunction (i.e. downward and rightward shift in the ESPVR curve) associated with mature GPR55−/−mice. …

Morphological measurements of cardiac dimensions revealed several significant age-related differences in WT mice. In particular, heart weightbody weight ratio (HWBW; mg/g), cardiomyocyte cross-sectional area (CSA), and left ventricular (LV) wall thickness were all significantly increased in 8 month old WT mice compared to 10 week old WT mice (all P<0.05; Table 2). In contrast, HWBW, LV wall thickness, and nuclei number were all significantly decreased in the mature GPR55−/− mice compared to age-matched WT mice (both P<0.05) and CSA was not significantly altered in comparison to young GPR55−/− mice (Table 2). Furthermore, mature GPR55−/− mice were characterised by significant increases in both interstitial and perivascular cardiac collagen deposition when compared to both the younger knockout mice and age-matched WT controls (P<0.05; Figure 4). While the latter may suggest significant ventricular remodelling (at least at the level of cardiac extracellular matrix composition), as this cardiac ‘fibrosis’ was not coupled with a significant upward shift in the EDPVR slope (Figure 2C) it seems unlikely that this ventricular remodelling influenced LV compliance and/or diastolic function in these mice. Finally, right ventricular wall thickness, interventricular septal wall thickness, and LV chamber area were all unchanged between all four groups of WT and GPR55−/−mice (Table 2).

Figure 4

Influence of GPR55 gene deletion on cardiac collagen deposition.

Table 2

Measurement of ventricular dimensions in WT and GPR55−/−mice.

Effect of GPR55 gene deletion on contractile reserve in young and aged mice

Contractile reserve, assessed by the change from baseline cardiac function in response to the α1/β1-adrenoceptor agonist dobutamine, was significantly attenuated in both young and mature GPR55−/− mice (Table 3). In young GPR55−/− mice, the dobutamine induced increase in dP/dtmax was significantly attenuated compared to WT mice with a resultant reduction in the change in SV and consequently CO and EF (all P<0.01; Table 3). In addition, adrenoceptor mediated changes in dP/dtmin were also significantly decreased in the young GPR55−/−mice (P<0.01; Table 3), suggesting an impaired lusitropic effect of dobutamine. The mature GPR55−/− mice were also characterised by a decreased response to dobutamine in terms of increases in SV, CO, EF and dP/dtmax when compared to age-matched controls (P<0.001; Table 3). These changes occurred concomitant with a somewhat preserved lusitropic effect in terms of the rate of relaxation (i.e. dP/dtmin), however EDV did not increase to a similar extent as that seen in the control group (P<0.001; Table 3). While mature GPR55−/−were characterised by reduced contractile reserve, this was no worse than that observed in young GPR55−/− mice.

Table 3

Effect of GPR55 gene deletion on contractile reserve in young (10 week old) and mature (8 month old) mice.

Discussion

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 & Emax) 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, HWBW 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/dtmax, ESPVR or Emax) 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 HWBW, 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 HWBW 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.

Conclusions

The present study has demonstrated that mature GPR55−/− mice are characterised by a progressive ventricular dysfunction. This intrinsic inability of the heart to maintain systolic function appears to be due to maladaptive adrenergic signalling, which may suggest some interplay/crosstalk between GPR55 and adrenoceptors and a possible role for GPR55 in the pathogenesis and/or progression of heart failure.

Acknowledgments

Funding Statement

This study was funded by a pump-priming grant from the Institute for Health & Wellbeing Research, the Robert Gordon University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References