In the current study, we conducted a study on the mechanism by which ADRB1 gene polymorphism affected myocardial dysfunction caused by metabolic syndrome at both cellular and animal levels, and we found that the occurrence of myocardial insufficiency with MS and ADRB1 gene polymorphism have inseparable relationship. Firstly, we used point mutation to construct β1AR-49M (HA-β1AR-S49G) and β1AR-389M (HA-β1AR-R389G) mutant particles in HEK-293 cells, and analyzed the differences in downstream signaling pathway changes caused by six β1-AR inhibitors (Esmolol, Carvediolol, Landiolol, Labetolol, Isoproterenol and Metoprolol) on β1-AR wild type and mutants. Secondly, we used palmitic acid (PA) to treat H9C2 myocardial cells to simulate myocardial insufficiency caused by metabolic syndrome, detecting β-AR changes and its downstream signaling molecule proteins through transfection of two mutants. Finally, we constructed an animal model of metabolic syndrome by high-fat diet to explore the relationship between myocardial dysfunction with metabolic syndrome (MS) and β-adrenergic receptor (β-AR) signaling pathway.
In HEK-293 cells, all six β1-AR inhibitors can produce good inhibition of activation by either wild-type or both mutants. Of the six inhibitors, the selective inhibitor, Bisoprolol, acted best on wild-type (WT) and mutant R389G, while the other selective inhibitor, Metoprolol, was the least effective inhibitor. For the mutant S49G, the β1-AR non-selective inhibitor Carvedilol showed the best inhibition, and Metoprolol showed the worst inhibition. The study by Rochais et al. [40] also reported that the two mutants at position 389 showed different reactivity to Bisoprolol, Metoprolol and Carvedilol, suggesting that the two genotypes at position 389 were differential [41]. Our results also indicated that carrying different SNP sites may lead to differences in responsiveness to the same drug. Carrying the same SNP site may have different efficacy to different types of β-AR blockers, and lay the foundation for the precise treatment of myocardial dysfunction with MS.
We used PA to investigate the mechanism of lipotoxicity to H9C2 cardiomyocytes in a model of myocardial insufficiency with metabolic syndrome. According to the literature, the use of the cultured cells can simulate a variety of cardiomyocyte injury cell models, and can avoid the interference of multiple factors in the somatic situation with strong stability and repetition. The H9C2 ventricular myoblasts of rats are similar to primary cardiomyocytes: phenotype, energy metabolism and gene expression patterns, and the response of the cell to metabolism and gene expression remodeling under different stimuli [42–43]. Therefore, H9C2 cell line was selected in this study to construct a model of myocardial injury caused by MS. Various ways in the literature to induce myocardial injury, such as doxorubicin, hydrogen peroxide, hypoxia/ reoxygenation, high glucose/high fat and isoproterenol. High glucose is not the most obvious change under the metabolic syndrome condition [44]. Palmitic acid (PA) is a saturated fatty acid that can induce apoptosis or injury in cardiomyocytes, which provides a suitable cell model for cardiovascular disease induced by MS by SFA [45–46]. We evaluated the effect of PA on H9C2 cell viability, suggesting that PA200µM could reduce cell viability, which is consistent with previous studies showing that higher concentrations of PA (200µM-800µM) inhibited cell proliferation and promote cell apoptosis in many cell types [47]. Therefore, it is feasible to select PA100µM to treat H9C2 cell to construct the metabolic syndrome model. At this concentration, (PA100µM) cells can still maintain good vitality and state, while cardiac troponin T (cTnT) is significantly increased, which showed that the myocardial injury occurred. At this concentration, we conducted subsequent experimental studies.
Currently, the prevalence of metabolic syndrome and type 2 diabetes is increasing, and patients with metabolic syndrome also have a higher risk of cardiovascular-related complications [48]. Each component of MS is an independent risk factor for cardiovascular disease. Although some progress has been made in understanding MS combined with myocardial insufficiency, the underlying pathophysiological mechanisms remain incompletely understood [49]. In this study, we used 45% high-fat diet feeding 16w to induce metabolic syndrome model. The DBP, T-Chol, HDL and LDL, the TG, HR, SBP and MAP levels of MS rats were higher than those of Control group, indicating that the rats had obesity, hyperlipidemia and hypertension, similar to that reported in the literature [50]. In MS rats, TG, HR, SBP, MABP, leptin, and C peptide levels were higher than Control rats and the altered C peptide indicates a degree of insulin resistance in the rats. All the above results indicate the success of constructing an animal model of metabolic syndrome.
For decades, a high-fat diet has been used to model obesity, dyslipidemia, and insulin resistance in rodents. Complications caused by a high-fat diet are similar to the human metabolic syndrome, and these complications can extend to cardiac hypertrophy and myocardial fibrosis [51]. We performed pathological staining of heart samples and found that high-fat diet could lead to myocardial structure disorder and cardiac fibrosis in the heart, indicating that high-fat diet will cause a certain degree of structural changes in the myocardium. Rider et al proposed that cardiac remodeling is an adaptive feature of obesity [52]. Our data are consistent with previous studies that a high fat diet leads to the appearance of structural remodeling in the heart [53]. Our result also suggests that carrying different SNP sites may lead to differences in their responsiveness to the same drug. Of course, carrying the same SNP site may produce different efficacy against different types of β-AR blockers, this partly implied the individual variability shown by the pathophysiological condition and the effect of drug treatment, which lay the foundation for the precision treatment of myocardial insufficiency with MS. The β1AR-R389G mutation is located in the cytoplasmic tail of the receptor carboxyl-terminal region, which is thought to be an important position for the coupling of the receptor to Gs proteins. The β1AR-S49G mutation is located in the extracellular amino terminal region of the receptor protein, which plays an important role in receptor membrane localization and receptor transduction, and two common ADRB1 gene polymorphisms have significant effect on cellular signal transduction system [54], which may be the basis for individual differences in pathological characteristics and different responses to β1-AR antagonists in cardiovascular diseases.
Recent findings have identified the majority of obese patients with concentric left ventricular hypertrophy as well as mild (subclinical) diastolic and / or systolic dysfunction with normal or elevated ejection fraction [55]. We evaluated cardiac echocardiography in both groups. The LV systolic and diastolic function were not significantly changed in MS rats compared with the Control group. In addition, we observed the response of rats to β-adrenergic stimulation (ISO administration) by echocardiography. The β-adrenergic stimulation triggered a positive inotropic effect in both groups of rats, but showed no significant difference between the two groups. Therefore, our results suggest that the positive inotropic effect was not impaired in MS rats. However, a recent study showed that animals with MS had reduced ventricular myocyte contraction and relaxation function, impaired myocyte contractile reserve, and impaired response to increased extracellular Ca2+ and β-adrenergic stimulation [56]. Cardiac dysfunction in the MS group can be explained at least in part by changes in the intrinsic properties of cardiomyocyte contractility and β-adrenergic response due to alterations in Ca 2+ processing [57].
To further investigate the muscle force changes in rats, we isolated the left ventricular papillary muscle to further analyze the myocardial contractility at baseline and after pharmacological intervention. In vitro, the papillary muscles could reflect the mechanical and elastic properties of the heart tissue in the body, as well as the functional electrophysiological unity of myocardial cells, choosing the left ventricular papillary muscle to study myocardial contractility and drug intervention is a scientific choice. In general, β -AR receptor activity was assessed by measuring the dose-response relationship between Isoproterenol and papillary muscle mechanical parameters. Echocardiography results showed that no systolic or diastolic dysfunction through echocardiography was detected in vivo, however, we found that the positive inotropic effect in MS rats was significantly weaker than that in the Control group, indicating that the myocardial function in MS rats was still impaired in vitro. After all, the animal body is a whole, however, the conditions and environment of the muscle are relatively single and controllable in the isolated situation, and the characteristics of myocardial injury are more likely to showcase, hence, there was a phenomenon of inconsistency between in vitro and in vivo conditions. Some studies have shown that under baseline conditions, the muscle force of MS rats was not changed, and functional parameters were similar between groups except for diastolic dysfunction [58]. Although there are some differences between our results and the data reported in the literature, we speculated that this may be caused by the influence of animal mold making methods, detection methods and experimental operation, and it needs to be further explored in the future.
In the current literature, few studies evaluate altered β-AR in experimental models of high-fat-diet-induced obesity [59–60]. Some studies have shown that impairment of cardiac function is associated with the β-AR system alteration [60–61], while others have reported there was no relationship with β-AR reactivity [62]. Our study demonstrated that high-fat feeding can cause elevated β1-or β2-AR expression in both rat myocardium. Studies have shown that the expression of both β1-AR and β2-AR in gonadal adipose tissue (gonadal white adipose tissue, GWAT) is upregulated in high-fat diet feeding. Of course, this changing trend varies somewhat from that reported in part of the literature, possibly related to leptin levels, diet type, animal model and catecholamine levels [62–64]. Llano-Diez M, et al. showed that the distribution of β1-or β2-AR was not altered in cardiomyocytes of mice with metabolic syndrome fed by a high-fat diet (45% fat) for 8 weeks, and immunofluorescence showed that β1-AR was mainly distributed to the membrane surface and T tube region, while β2-AR was more restricted to the T tubular region of the membrane [65]. Little is known about the cardiomyopathy associated with the metabolic syndrome, especially about the role and contribution of β-AR subtypes. Thus, the function of each receptor subtype varies significantly depending on cardiac tissue and pathophysiological status, and alterations in β-adrenergic signaling pathways can be regulated at the level of receptor expression or activation of downstream signaling molecules, such as PKA, adenylyl cyclase activation, cAMP, CaMKII and other molecular proteins or genes [66].
In physiological conditions, β1-AR produces positive inotropic effect through the activation of the Gs-AC-cAMP-PKA signaling pathway in the heart. However, when β1-AR is continuously overstimulated, signal transduction pathway switched from the Gs-AC-cAMP-PKA pathway to the Gs-Ca2+-CaMKII pathway. Cardiomyocyte intracellular CaMKII activation leads to desensitization of PKA signaling. Similarly, our results indicated that in metabolic syndrome conditions, β1-AR and β2-AR expression increased, p-PKA/ PKA ratio decreased, and p-CaMKII/CaMKII expression increased, indicating that in the pathological situation of metabolic syndrome, PKA sensitivity decreased in cardiac muscle, while CaMKII is activated in the heart under hyperlipidemia conditions.
Regarding the expression of downstream signaling molecule proteins, our study found that the expression of p-PKA/PKA in the group carrying mutant β1AR- R389G is opposite to that of the wild-type, and its expression in the group carrying mutant S49G is the same as that in the wild-type group. After intervention with Bisoprolol, the expression of p-PKA/PKA increased in both the R389G mutant group and the wild-type group, with enhanced PKA phosphorylation and Bisoprolol played the strongest effect on this mutant. The results showed that Bisoprolol had the strongest effect on the β1AR-R389G mutant and wild type. The WB results of LV myocardium also showed that the phosphorylation of PKA was reduced in the wild-type group, we speculated that Bisoprolol played a similar mechanism to wild-type, mediated by affecting the phosphorylation of PKA.PA intervention caused a rising trend in p-CaMKII / CaMKII expression. After Bisoprolol intervention, the expression of p-CaMKII / CaMKII was decreased in the mutants (β1AR-R389G and β1AR-S49G), and its expression in the β1AR-R389G mutant group was lower than that of the β1AR-S49G mutant group. Based on the above results, we suggest that the phosphorylation of CaMKII was different in both wild-type and mutant groups. Moreover, Bisoprolol had a stronger effect on cells carrying the R389G mutant. Similarly, we found that SERCA2a expression in the H9C2 cell coincides with the expression trend in the rat LV tissue. In conclusion, the differences between mutants and wild-type caused by palmitic acid or Bisoprolol intervention are mainly influenced by the expression of β1-AR and SERCA2a, as well as the phosphorylation of PKA. And the inhibitory effect of Bisoprolol on cell carrying the β1AR-R389G mutant was stronger than that on the β1AR-S49G mutant. Therefore, it also proved that the molecular mechanism of carrying ADRB1 polymorphism can lead to MS with myocardial insufficiency, and the β1AR-R389G mutant favors the effectiveness of Bisoprolol.
Of course, there are some limitations to this study. The overall impact of multiple changes in extracellular environment and cellular signaling related to metabolic syndrome on myocardial cell function is complex and we can’t conduct a comprehensive exploration of these mechanisms. In the study, PKA and CaMKII inhibitors or siRNAs treatment were also not used to analyze the role of PKA and CaMKII. We didn’t explore the different pharmacological effects of using various β1-AR inhibitors on the presence of ADRB1 gene polymorphisms.