Differential sensitivity of atrial and ventricular KATP channels to metabolic inhibition

Objective: The aim is to compare the activation of ATP-sensitive potassium channels (KATP channels) in intact and metabolically impaired atrial and ventricular myocytes. Methods: The KATP channel current is measured by whole cell and gramicidin-perforated patch clamp recordings in 164 cultured neonate rat cardiomyocytes. Results: In whole cell recordings with 84 μmol/l ADP in pipette, spontaneous activity is significantly higher in atrium than ventricle, and EC50 for the KATP channel opener diazoxide is 0.13 μmol/l (atrium) versus 3.1 μmol/l (ventricle). With an ATP-regenerating system in pipette, EC50 for diazoxide is 19.7 μmol/l (atrium) versus 54.9 μmol/l (ventricle). In gramicidin-perforated patch recordings, atrial myocytes respond significantly to 100 nmol/l of the mitochondrial protonophore CCCP, while ventricular myocytes do not. EC50 for diazoxide is 129 μmol/l (atrium) versus <2500 μmol/l (ventricle) for myocytes exposed to CCCP, and 676 versus <2500 μmol/l, respectively, without CCCP. Conclusions: (1) KATP channels are significantly more sensitive to metabolic inhibition in atrial than ventricular myocytes. (2) Sensitivity of atrium versus ventricle to the channel opener diazoxide increases from 3:1 to ≥24:1 with ADP or metabolic inhibition. If extended to intact hearts, the results would predict a higher atrial sensitivity to ischemia, and a high sensitivity of the ischemic atrium to KATP channel opener

Unexpected role for the human Cx37 C1019T polymorphism in tumour cell proliferation

Connexins are a large family of proteins that form gap junction channels allowing exchange of ions and small metabolites between neighboring cells. They have been implicated in pathological processes such as tumourigenesis in which they may act as tumour suppressors. A polymorphism in the human connexin37 (Cx37) gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus (CT) of the Cx37 protein (P319S) has been suggested to be implicated in predisposition to angiosarcomas. In this study, we have used communication-deficient HeLa and SK-HEP-1 cells transfected with Cx37-319S, Cx37-319P or empty vector. We showed that the expression of Cx37-319P limited proliferation of HeLa and SK-HEP-1 cells, whereas Cx37-319S expression was without effect. Using an in vitro kinase assay, we demonstrated phosphorylation of Cx37 CT by glycogen synthase kinase-3 (GSK-3), a kinase known to be implicated in cell proliferation and cancer. GSK-3-induced phosphorylation was associated with reduced gap junctional intercellular communication (GJIC) as measured by microinjection of the tracer neurobiotin. Inhibition of GSK-3 by LiCl or SB415286 reduced phosphorylation of Cx37-319P and increased GJIC. This latter effect on GJIC involved the beta and not the alpha isoform of GSK-3. In contrast, GSK-3 inhibitors were without effect on HeLa cells expressing Cx37-319S. In conclusion, our data indicate functional effects of the Cx37 C1019T polymorphism on GJIC that might contribute to tumour cell growt

Pituitary adenylate cyclase-activating polypeptide activates K ATP current in rat atrial myocytes

International audienc

Intracellular targeting of truncated secretory peptides in the mammalian heart and brain

Secretory polypeptides are vital for nervous system function, sleep, reproduction, growth, and metabolism. Ribosomes scanning the 5'-end of mRNA usually detect the first AUG site for initiating translation. The nascent propeptide chain is then directed via a signal-peptide into the endoplasmic reticulum, processed through the Golgi stacks, and packaged into secretory vesicles. By expressing prepropeptide-EGFP fusion proteins, we observed unusual destinations, mitochondria, nucleus, and cytoplasm, of neuropeptide Y (NPY), atrial natriuretic peptide, and growth hormone in living murine cardiac cells and hypothalamic slices. Subcellular expression was modulated by Zn++ or mutations of N-terminal prohormone sequences but was not due to overexpression in the trans-Golgi network. Mitochondrial targeting of NPY also occurred without the EGFP tag, was enhanced by site-directed mutagenesis of the first AUG initiation site, and abolished by mutation of the second AUG. Immunological methods indicated the presence of N-terminal truncated NPY in mitochondria. Imaging studies showed depolarization of NPY-containing mitochondria. P-SORT software correctly predicted the secondary intracellular destinations and suggested such destinations for many neuropeptides and peptide hormones known. Thus, mammalian cells may retarget secretory peptides from extracellular to intracellular sites by skipping the first translation-initiation codon and thereby alter mitochondrial function, gene expression, and secretion

Sulfonylurea receptor ligands modulate stretch-induced ANF secretion in rat atrial myocyte culture

International audienceStretch-induced atrial natriuretic factor (ANF) secretion was studied in cultures of neonate atrial appendage myocytes. Stretch, applied for 40 min by hypotonic swelling, increased the mean area of 44 individually imaged myocytes by 4.8–8.8% (P < 0.0001) at 6 min and by 2.3–6.2% (P < 0.05) at 35 min. Stretch increased immunoreactive ANF release by 42% (P < 0.05) from a baseline of 315 pg/ml. The ATP-sensitive K+(KATP)-channel blocker tolbutamide (100 μmol/l) increased the stretch-stimulated release to 84% (P < 0.01) over baseline, whereas lower concentrations (1, 10, and 30 μmol/l) had no stimulatory effect. The KATP-channel opener diazoxide (0.1, 1, 10, 30, and 100 μmol/l) inhibited stretch- plus tolbutamide-stimulated ANF release in a concentration-dependent manner, with IC50 = 2.2 μmol/l, although 100 μmol/l diazoxide did not reduce the increase in mean cell area. The stretch-stimulated KATP current, monitored in 82 whole cell recordings with sulfonylurea receptor (SUR) ligands, was inversely correlated with the stretch-induced ANF release (r2 = 0.79,P < 0.0001). In the absence of stretch, the KATP current had no relationship with baseline ANF release, and baseline ANF release was not affected by the KATP-channel modulators. The results show that SUR ligands that open KATP channels inhibit stretch-induced ANF release in atrial myocytes, in correlation with the stretch-activated KATP current. The subcellular site of action of the SUR ligands—plasmalemma or intracellular organelles—remains to be determined

Forkhead transcription factors coordinate expression of myocardial KATP channel subunits and energy metabolism

Coordinate adaptation of myocyte metabolism and function is fundamental to survival of the stressed heart, but the mechanisms for this coordination remain unclear. Bioinformatics led us to discover that Foxs are key transcription factors involved. We performed experiments on the mouse atrial cell line HL-1, neonate rat heart myocytes, and an adult rat model of myocardial infarction. In electrophoretic mobility-shift assays, FoxO1 binds to the FoxO concensus site of the KATP channel subunit KIR6.1 promoter. In primary atrial culture, targeting FoxO1 and FoxO3 with siRNA specifically reduces mRNA expression of FoxO1 and -O3 and KIR6.1. Western blots, confocal immunofluorescence, and quantitative RT-PCR was applied for measuring expression of 10 Fox, 6 KATP channel subunits, and 12 metabolic genes. FoxF2, -O1, and -O3 strongly associate with expression of KATP channel subunits (in particular, KIR6.1, SUR1A and SUR2B) in different heart tissues and in the periinfarct zone of the left ventricle. Patch-clamp recordings demonstrate that molecular plasticity of these channels is matched by pharmacological plasticity and increased sensitivity to a metabolic challenge mimicked by the protonophore CCCP. A balance of FoxF2 and FoxO also regulates expression of at least 9 metabolic genes involved in setting the balance of glycolysis and beta-oxidation. Bioinformatics shows that the transcriptional mechanisms are highly conserved among chicken, mouse, rat, and human, and Fox are intimately linked to other metabolic sensors. Thus, FoxF2 and -O are key transcription factors coordinating expression of KATP channels and energy metabolism