Our experiment shed light on new effects exerted by chronic upregulation of ECS in relation to cardiomiocytes’ intracellular signaling pathways and lipid metabolism. This novel insight in the action of ECS seems to be relevant since previously we have found that enhanced endocannabinoid tone significantly alters cardiac muscle energetic milieu in both normotensive and hypertensive conditions [20,21,22]. It is important to note that present study, which was based on a genetic model of primary hypertension (SHRs), has revealed the link between chronic activation of ECS by URB597 treatment (2-week) and insulin signaling pathway in cardiomyocytes subjected to the increased afterload. Our data suggest that ECS upregulation ameliorates impaired myocardial insulin signal transduction in SHR rats. Nevertheless, in previous studies [21,22,32] we have reported that chronic FAAH inhibition in a model of secondary hypertension considerably elevated intramyocardial lipid and glycogen deposition along with palmitic acid uptake in order to facilitate adjustment in fuel demands of overloaded left ventricle. We provide evidence upon different routes of URB597 action in SHRs, including significant reduction of BP which corresponds with elevated palmitate uptake and its esterification as well as an enhancement in the expression of selected insulin signaling proteins (e.g., IRS-1, AKT, GSK-3, MAPK). Taken together, our experimental model also confirms the existence of a dynamic connection between cardiovascular parameters and metabolic status of the myocardium.

Current study has shown an increased intramyocardial content of DAG and CER fractions in the spontaneously hypertensive rats compared to the normotensive rats. Recent data provide several lines of evidence to indicate that the above lipid fractions, when accumulated, can trigger lipotoxicity and are implicated in intracellular signaling impairments, including insulin pathway (in cardiac and skeletal muscles or liver) [8,9,33,34]. Therefore, DAG and CER are referred as lipid mediators of insulin resistance (IR). In a number of studies it was shown that increased levels of bioactive lipid species such as DAG and CER can interfere with downstream cellular insulin transduction through dysregulation of major proteins involved in this transmission, i.e., IRS-1, AKT/PKB or atypical protein kinases C (PKC) activation [35,36]. This, in turn leads to energetic and nutrient imbalance in the heart due to reduced glucose uptake as well as glycogen storage. Furthermore, it was demonstrated that elevated contents of DAG and CER fractions together with reactive oxygen species (ROS) and other metabolites are engaged in the development of changes in the cardiac muscle’s structure, mitochondrial activity, endoplasmic reticulum stress and apoptosis [37]. Importantly, in this respect our results exhibit a probable protective role of ECS due to observed reduction in the content of DAG and lack of an increase in CER fraction in SHRs (in the presence of chronic URB597 treatment). Moreover, the above observations coincide with enhancement of insulin signaling transmission in the left ventricle compared to the control hypertensive rats. Consequently, we have noticed that prolonged activation of ECS in SHRs was parallel with markedly enhanced phosphorylation of proteins involved in the myocardial insulin action, i.e., IRS-1, AKT/PKB, GSK-3, and MAPK, which may indicate and reflect causal relationship. What is more, probably the above alternations arise from plasma measurements, which revealed that enhanced ECS tone ameliorated insulin sensitivity by reducing significantly insulin concentration in hypertensive rats. Even though, studies on the ground of ECS control over insulin secretion from pancreatic islets are still contradictory [38,39,40]. The above changes are in line with our previous investigation, where it was demonstrated that URB597 treatment had restored myocardial glycogen level in DOCA-salt hypertensive rats [22], which is also controlled by insulin. However, we have to bear in mind that the ability of SHRs to maintain energetic homeostasis (including glucose metabolism) in the cardiac tissue decreases with age [32]. Moreover, we are aware of the fact that the limitation of this study is lack of direct cardiac muscle stimulation by insulin for instance in isolated primary cardiomyocytes after chronic URB597 treatment. Nonetheless, the obtained results undoubtedly indicate causal relationship between ECS upregulation and improvement in overloaded cardiac muscle metabolic pathways, including insulin. In the study conducted by Zhang et al. [33] on mice fed a high fat diet there was a proposed mechanism by which accumulated DAGs, through translocation of protein kinase C-α to the plasma membrane and impaired IRS-1 phosphorylation, led to the development of cardiac insulin resistance. Furthermore, in our previous study [20], we have shown that chronic URB597 administration to SHRs promoted palmitic acid incorporation into neutral lipid fractions in the heart, i.e., triacylglycerol (TAG), free fatty acid (FFA) and cholesterol rather than DAG, as it was demonstrated in the present study. What is more, in the same research we revealed that chronic URB597 treatment enhances fatty acid oxidation only in normotensive conditions, which was linked with an elevation in peroxisome proliferator-activated receptor alpha (PPARα) expression, with parallel no effects in SHRs [20]. Additionally, as it was indicated in current experiments, a significant decline in the myocardial DAG content in SHRs treated with URB597 was followed by alternations in the expression of DGAT2 and ATGL, two major enzymes involved in the lipid metabolism. Therefore, we can presume that enhanced activation of ECS can serve as a cardioprotective tool due to diminishing potentially harmful lipotoxic level of DAG and CER.

As we mentioned earlier one of our goals was to examine intramyocardial level of ceramide, as a potentially detrimental lipid derivative, in a genetic model of primary hypertension. Hence, we have investigated major pathways of sphingolipid metabolism under the conditions of high BP and chronic activation of ECS. To our knowledge, for the first time we have shown that SHRs exhibit a substantially increased CER content in the heart. The increase was a result of greater de novo ceramide synthesis pathway activation, since simultaneous rise in SFA content compared to the normotensive rats was observed (with concomitant no change in SM content). Interestingly, the same group of hypertensive rats displayed a considerably lower value of S1P/CER ratio in comparison with the normotensive conditions, which may have a relationship with decreased activation of cardiomyocytes’ signaling pathways (IRS-1 and AKT/PKB) and changed apoptotic status as well. However, the most profound alternations were observed in the myocardial sphingolipid metabolism in SHRs when ECS tone was elevated. Surprisingly, 2-week URB597 treatment prevented overloaded cardiac muscle against CER accumulation and caused further increase in sphingosine-1-phosphate as well as S1P/CER ratio compared to the hypertensive rats, which may confirm protective role of ECS in the hypertensive heart in respect of lipotoxicity. Spijkers et al. [41] demonstrated that S1P/CER ratio in SHRs was shifted towards ceramide accumulation in carotid artery. Moreover, in the same study it was revealed that plasma level of CER was elevated in both SHR and hypertensive patients [41], which also correlated with progression of hypertension. Nevertheless, the above mentioned change in intramyocardial S1P level in our model of hypertension seems to be relevant due to the fact that compared to CER and SFO this sphingolipid derivative has potential cardioprotective properties [42,43]. Moreover, in the previous research it was found that S1P acts as a vasoactive substance and triggers vasodilation depending on activation of endothelial nitric oxide synthase [44,45], thereby, affecting metabolic status of the tissue.