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Background. During atherosclerotic plaque formation smooth muscle cells (SMCs) switch from a contractile/differentiated to a synthetic/dedifferentiated phenotype We previously isolated differentiated spindle-shaped (S) and dedifferentiated rhomboid (R) SMCs from porcine coronary artery. R-SMCs express S100A4, a calcium binding protein. Aim. We investigated the role of apelin in this phenotypic conversion, as well as its relationship with S100A4. Results. We found that apelin was highly expressed in R-SMCs compared with S-SMCs. We observed a nuclear expression of apelin in SMCs within experimentally-induced intimal thickening of the porcine coronary artery and rat aorta. Plasmids targeting apelin to the nucleus (N. Ap) and to the secretory vesicles (S. Ap) were transfected into S-SMCs where apelin was barely detectable. Both plasmids induced the SMC transition towards a R-phenotype. Overexpression of N. Ap, and to a lesser degree S. Ap, led to a nuclear localization of S100A4. Stimulation of S-SMCs with platelet-derived growth factor-BB, known to induce the transition toward the R-phenotype, yielded nuclear expression of both apelin and S100A4. Conclusion. Apelin induces a SMC phenotypic transition towards the synthetic phenotype. These results suggest that apelin acts via nuclear re-localization of S100A4, raising the possibility of a new complex pro-atherogenic relationship between apelin and S100A4.

The aim is to compare the activation of ATP-sensitive potassium channels (K(ATP) channels) in intact and metabolically impaired atrial and ventricular myocytes.The K(ATP) channel current is measured by whole cell and gramicidin-perforated patch clamp recordings in 164 cultured neonate rat cardiomyocytes.In whole cell recordings with 84 micromol/l ADP in pipette, spontaneous activity is significantly higher in atrium than ventricle, and EC(50) for the K(ATP) channel opener diazoxide is 0.13 micromol/l (atrium) versus 3.1 micromol/l (ventricle). With an ATP-regenerating system in pipette, EC(50) for diazoxide is 19.7 micromol/l (atrium) versus 54.9 micromol/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. EC(50) for diazoxide is 129 micromol/l (atrium) versus2500 micromol/l (ventricle) for myocytes exposed to CCCP, and 676 versus2500 micromol/l, respectively, without CCCP.(1) K(ATP) 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 toor = 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 K(ATP) channel openers.

Objectives: The aim of this study was to test whether adenosine triphosphate-sensitive K+ (KATP) channel expression relates to mechanical and hypoxic stress within the left human heart.Background: ...

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K+(KATP) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC50= 0.28 nmol/l ( r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective ( P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC1(≥2 isoforms), VPAC1, and VPAC2. PACAP-38 dose dependently activates the whole cell atrial KATPcurrent with EC50= 1–3 nmol/l ( n = 44). Maximal effects occur at 10 nmol/l (91 ± 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% ( P < 0.05; n = 6). H89(500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated KATPcurrent to 17.8 ± 9.6% ( n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced KATPcurrent. A protein kinase C (PKC) inhibitor peptide (50 μmol/l) in the pipette reduces the PACAP-38-induced KATPcurrent to 33 ± 17 pA/pF ( P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC1, VPAC1, and VPAC2are present in atrial myocytes; and 2) PACAP-38 activates the atrial KATPchannels through both PKA and PKC pathways.

The role of endothelin (ET) receptors was tested in volume-stimulated atrial natriuretic factor (ANF) secretion in conscious rats. Mean ANF responses to slow infusions (3 × 3.3 ml/8 min) were dose dependently reduced ( P < 0.05) by bosentan (nonselective ET-receptor antagonist) from 64.1 ± 18.1 (SE) pg/ml (control) to 52.6 ± 16.1 (0.033 mg bosentan/rat), 16.1 ± 7.6 (0.33 mg/rat), and 11.6 ± 6.5 pg/ml (3.3 mg/rat). The ET-A-receptor antagonist BQ-123 (1 mg/rat) had no effect relative to DMSO controls, whereas the putative ET-B antagonist IRL-1038 (0.1 mg/rat) abolished the response. In a second protocol, BQ-123 (≥0.5 mg/rat) nonsignificantly reduced the peak ANF response (106.1 ± 23.0 pg/ml) to 74.0 ± 20.5 pg/ml for slow infusions (3.5 ml/8.5 min) but reduced the peak response (425.3 ± 58.1 pg/ml) for fast infusions (6.6 ml/1 min) by 49.9% ( P < 0.001) and for 340 pmoles ET-1 (328.8 ± 69.5 pg/ml) by 83.5% ( P < 0.0001). BQ-123 abolished the ET-1-induced increase in arterial pressure (21.8 ± 5.2 mmHg at 1 min). Changes in central venous pressure were similar for DMSO and BQ-123 (slow: 0.91 and 1.14 mmHg; fast: 4.50 and 4.13 mmHg). The results suggest 1) ET-B receptors mainly mediate the ANF secretion to slow volume expansions of

Background: The main circulatory form of Apelin is a highly conserved 13 amino acid hypotensive peptide derived from a 77 amino acid prepropeptide. The very low plasma levels suggest autocrine, paracrine and possibly intracrine functions of Apelin. It is synthesized in smooth muscle cells (SMCs), and we found a nuclear expression in rhomboid type (R), poorly differentiated SMCs. The biological features of R-SMCs (i.e. enhanced proliferative and migratory activities) explain their capacity to accumulate in the intima. Objectives: Investigate the role of intracrine Apelin in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation. Methods and Results: As shown in our previous work, a 3’-shift in translation can lead to N-truncated peptides with new address sequences (FASEB J. 20(6): 732-4, 2006). P-SORT software analysis of preproApelin sequence suggests that N-terminal truncated Apelin may target the nucleus, and we confirm this in many cell types by overexpression of mutated preproApelin-EGFP and preproApelin-His-tag. Transfection of mutated preproApelin-His-tag encoding plasmid in differentiated spindle-shaped (S) SMCs induces a transition towards a R-phenotype associated with increased proliferative activity, as well as downregulation of SMC differentiation markers (i.e. α-smooth muscle actin) and increased nuclear expression of S100A4 (a marker typical of R-SMCs). In contrast, transfection of S-SMCs with wild type preproApelin-His-tag encoding plasmid does not induce nuclear targeting of Apelin or S100A4, and does not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB also induces nuclear targeting of both Apelin and S100A4 and transition to the R-phenotype. In vivo, Apelin is expressed in SMC nuclei of stent-induced intimal thickening, while its expression in SMCs of the media is mainly cytoplasmic. Conclusions: Altogether, theses preliminary studies provide evidence for nuclear targeting of Apelin in SMCs and suggest actions on gene expression, proliferation, and differentiation. The pathophysiological consequences of this retargeting could be of importance in the understanding of artherosclerosis.

ATP-sensitive potassium channels couple cell excitability to energy metabolism, thereby providing life-saving protection of stressed cardiomyocytes. The signaling for ATP-sensitive potassium channel expression is still unknown. We tested involvement of biochemical and biophysical parameters and potential transcription factors Forkhead box (FOX) and hypoxia-inducible factor (HIF-1α). Right atrial tissues were obtained during surgery from 28 children with heart disease. Expression of K + -inward-rectifier subunits Kir6.1/Kir6.2; sulfonyl urea receptors (SURs) SUR1A/B and SUR2A/B; and FOX class O (FOXO) 1, FOXO3, FOXF2, and HIF-1α were related to 31 parameters, including personal data, blood chemistry, and echocardiography. Venous hypoxemia (but not other ischemia indicators, such as venous hypercapnia or low glucose) predicts increased Kir6.1 ( P P P P P P P

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 β-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.

ANP (atrial natriuretic peptide) is widely recognized as an important vasorelaxant, diuretic, and cardioprotective hormone. Little is known, however, about how ANP-secretory vesicles form within the atrial myocytes. Secretory vesicles were visualized by fluorescence microscope imaging in live rat atrial myocytes expressing proANP–enhanced green fluorescent protein (EGFP), or N-terminal–mutated fusion proteins thought to suppress the calcium-dependent aggregation of proANP. Results showed the following: (1) aggregates of proANP and coexpressed proANP-EGFP recruited peptidylglycine α-amidating monooxygenase (PAM)-1, an abundant atrial integral vesicle membrane protein; (2) coexpressed N-terminal–mutated (Glu23,24→Gln23,24) and N-terminal–deleted proANP-EGFP inhibited recruitment of PAM-1 by up to 60%; (3) 4-phenyl-3-butenoic acid (PBA) (10 μmol/L), a pharmacological inhibitor of the lumenal peptidylglycine α-hydroxylating monooxygenase domain of PAM proteins, inhibited recruitment of endogenous PAM-1 and of coexpressed pro-EGFP–PAM-1; (4) PBA had no effect on exocytosis of the potassium inward rectifier KIR2.1; (5) PBA induced a deformation of the secretory vesicles but did not inhibit docking. These findings suggest that recruitment of PAM-1 to secretory vesicles depends on intact N-terminal proANP and on the lumenal domain of PAM-1. Conversely, PAM-1 participates in shaping the proANP-secretory vesicles. The full text of this article is available online at http://circres.ahajournals.org.

Cardiomyocytes express mRNA for all major subunits of ATP-sensitive potassium (K(ATP)) channels: KIR6.1, KIR6.2, SUR1A, SUR2A, and SUR2B. It has remained controversial as to whether KIR6.1 may associate with KIR6.2 to form the tetrameric pore of K(ATP) channels in cardiomyocytes. To explore this possibility, cultured rat cardiomyocytes were examined for an inhibition of K(ATP) current by overexpression of pore loop-mutated (inactive) KIR6.x. Bicistronic plasmids were constructed encoding loop-mutated (AFA or SFG for GFG) rat KIR6.x followed by EGFP. In ventricular myocytes, the overexpression of KIR6.1SFG-pIRES(2)-EGFP or KIR6.2AFA-pIRES(2)-EGFP DNA caused, after 72 h, a major decrease of K(ATP) current density of 85.8% and 82.7%, respectively (P0.01), relative to EGFP controls (59 +/- 9 pA/pF). In atrial myocytes, overexpression of these pore-mutated KIR6.x by 6.0-fold and 10.6-fold, as assessed by quantitative immunohistochemistry, caused a decrease of K(ATP) current density of 73.7% and 58.5%, respectively (P0.01). Expression of wild-type rat KIR6.2 increased the ventricular and atrial K(ATP) current density by 58.3% and 42.9%, respectively (P0.01), relative to corresponding EGFP controls, indicating a reserve of SUR to accommodate increased KIR6.x trafficking to the sarcolemma. The results favor the view that KIR6.1 may associate with KIR6.2 to form heterotetrameric pores of native K(ATP) channels in cardiomyocytes.

The relative role of plasmalemmal and mitochondrial ATP-sensitive K+(KATP) channels in calcium homeostasis of the atrium is little understood. Electrically triggered (1 Hz) cytoplasmic calcium transients were measured by 340-to-380-nm wavelength fura 2 emission ratios in cultured rat atrial myocytes. CCCP, a mitochondrial protonophore (100–400 nmol/l), dose dependently reduced the transient amplitude by up to 85%, caused a slow rise in baseline calcium, and reduced the recovery time constant of the transient from 143 to 91 ms ( P < 0.05). However, neither 5-hydroxydecanoate, a mitochondrial KATPchannel blocker, nor diazoxide (500 μmol/l) affected the amplitude, baseline, or time constant in CCCP-treated cells. HMR-1098 (30 μmol/l), a plasmalemmal KATPchannel blocker, and glibenclamide (1 μmol/l) increased the amplitude in CCCP-treated myocytes by 69–82%, sharply elevated the calcium baseline, and prolonged the recovery time constant to 181–193 ms ( P < 0.01). Thus opening of plasmalemmal but not mitochondrial KATPchannels reduces the calcium overload in metabolically compromised but otherwise intact atrial myocytes. Mitochondrial KATPchannels probably operate through a different mechanism to afford ischemic protection.

How vesicles are born in the trans -Golgi network and reach their docking sites at the plasma membrane is still largely unknown and is investigated in the present study on live, primary cultured atrial cardiomyocytes. Secretory vesicles (n=422) are visualized by expressing fusion proteins of proatrial natriuretic peptide (proANP) and green fluorescent protein. Myocytes expressing fusion proteins with intact proANP display two populations of fluorescent vesicles with apparent diameters of 120 and 175 nm, moving at a top velocity of 0.3 μm/s. The number of docked vesicles is significantly correlated with the number of mobile vesicles ( r =0.71, P 23,24 →gln 23,24 ) change size and shape—but not velocity—of the vesicles, and, strikingly, abolish their docking at the plasma membrane. The shapes thus change from spheres to larger, irregular floppy bags or vesicle trains. Deletion of the C-terminal proANP[45-127], where the ANP and its disulfide bond reside, does not change size, shape, docking, or velocity of the mobile vesicles. The N-terminal acid calcium-binding sequence of proANP is known to cause protein aggregation at the high calcium concentration prevailing in the trans -Golgi network. Therefore, these results indicate that amino acid residues favoring cargo aggregation are critically important in shaping the secretory vesicles and determining their fate—docking or not docking—at the plasma membrane. The full text of this article is available at http://www.circresaha.org.

Stretch-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 ( r 2 = 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.