Transgenic rescue of defective Cd36 enhances myocardial adenylyl cyclase signaling in spontaneously hypertensive rats

Abstract

Dysfunction or abnormalities in the regulation of fatty acid translocase Cd36, a multifunctional membrane protein participating in uptake of long-chain fatty acids, has been linked to the development of heart diseases both in animals and humans. We have previously shown that the Cd36 transgenic spontaneously hypertensive rat (SHR-Cd36), with a wild type Cd36, has higher susceptibility to ischemic ventricular arrhythmias when compared to spontaneously hypertensive rat (SHR) carrying a mutant Cd36 gene, which may have been related to increased β-adrenergic responsiveness of these animals (Neckar et al., 2012 Physiol. Genomics 44:173-182). The present study aimed to determine whether the insertion of the wild type Cd36 into SHR would affect the function of myocardial G protein-regulated adenylyl cyclase (AC) signaling. β-Adrenergic receptors (β-ARs) were characterized by radioligand-binding experiments and the expression of selected G protein subunits, AC, and protein kinase A (PKA) was determined by RT-PCR and Western blot analyses. There was no significant difference in the amount of trimeric G proteins, but the number of β-ARs was higher (by about 35 %) in myocardial preparations from SHR-Cd36 as compared to SHR. Besides that, transgenic rats expressed increased amount (by about 20 %) of the dominant myocardial isoforms AC5/6 and contained higher levels of both nonphosphorylated (by 11 %) and phosphorylated (by 45 %) PKA. Differently stimulated AC activity in SHR-Cd36 significantly exceeded (by about 18-30 %) the enzyme activity in SHR. Changes at the molecular level were reflected by higher contractile responses to stimulation by the adrenergic agonist dobutamine. In summary, it can be concluded that the increased susceptibility to ischemic arrhythmias of SHR-Cd36 is attributable to upregulation of some components of the β-AR signaling pathway, which leads to enhanced sensitization of AC and increased cardiac adrenergic responsiveness.

Excess ischemic tachyarrhythmias trigger protection against myocardial infarction in hypertensive rats

“…Adult male rats of SHR and SHR-CRP strains [16][17][18][19][20] weeks old, n=99 per strain, body weight (BW) 300-370 g] were used. Transgenic SHR-CRP were derived by microinjections of SHR fertilized ova with a construct containing cDNA for human CRP under control of the apoE promoter with the objective of driving expression of the CRP transgene in the liver where CRP is normally produced [10,15].…”

“…In the separate sets of SHR and SHR-CRP, evaluation of geometrical and functional parameters of the hearts was performed using echocardiographic system GE Vingmed System Seven with 14 MHz linear matrix probe [19]. Anesthesia was induced with 3% of isoflurane (Aerrane, Baxter SA) and then maintained at 2% during the ultrasound procedure.…”

β-Adrenergic signaling in rat heart is similarly affected by continuous and intermittent normobaric hypoxia

“…Myocardial β-ARs were determined by radioligand binding assay with the β-antagonist [ 3 H]CGP 12177 as described previously (Klevstig et al 2013). Briefly, samples of myocardial membranes (100 µg protein) were incubated in a buffer B (50 mM Tris-Cl, 10 mM MgCl 2 and 1 mM ascorbic acid; pH 7.4) containing 4 nM [ 3 H]CGP 12177 at 37°C for 1 h (total volume of 0.5 ml).…”

The cardioprotective effect persisting during recovery from cold acclimation is mediated by the β2-adrenoceptor pathway and Akt activation

“…The total number of myocardial b-ARs was determined by the radioligand binding assay with the b-AR antagonist [ 3 H] CGP-12177 as previously described (21). Briefly, samples of crude membrane fraction (100 μg protein) were incubated in the medium (total volume of 0.5 mL) containing 50 mM Tris-HCl, 10 mM MgCl 2 , and 1 mM ascorbic acid at pH 7.4 with the b-AR antagonist [ 3 H]CGP 12177 (ARC) in descending concentrations from 5 nM to 0.15 nM at 37 C for 1 h. The reaction was terminated by adding 3 mL of ice-cold washing buffer (50 mM Tris-HCl and 10 mM MgCl 2 ; pH 7.4) and subsequent filtration through GF/C filter pre-soaked for 1 h with 0.3% polyethyleneimine.…”