Increased expression of adenosine triphosphate-sensitive K+ channels in mitral dysfunction: mechanically stimulated transcription and hypoxia-induced protein stability?

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: The KATP channels play a vital role in preserving the metabolic integrity of the stressed heart. However, the mechanisms that govern the expression of their subunits (e.g., potassium inward rectifier [Kir] 6.2) in adult pathologies are mostly unknown. METHODS: We collected biopsies from the 4 cardiac chambers and 50 clinical parameters from 30 surgical patients with severe mitral dysfunction. Proteins and messenger ribonucleic acids (mRNAs) of KATP pore subunits and mRNAs of their known transcriptional regulators (forkhead box [FOX] F2, FOXO1, FOXO3, and hypoxia inducible factor [HIF]-1α) were measured respectively by Western blotting, immunohistochemistry, and quantitative real-time polymerase chain reaction, and submitted to statistical analysis. RESULTS: In all heart chambers, Kir6.2 mRNA correlated with HIF-1α mRNA. Neither Kir6.1 nor Kir6.2 proteins positively correlated with their respective mRNAs. The HIF-1α mRNA related in the left ventricle to aortic pressure, in the left atrium to left atrial pressure, and in all heart chambers to a decreased Kir6.2 protein/mRNA ratio. Interestingly, in the left heart, Kir6.2 protein and its immunohistochemical detection in myocytes were maximal at low venous PO(2). In the left ventricle, the Kir6.2 protein/mRNA ratio was also significantly higher at low venous PO(2), suggesting that tissue hypoxia might stabilize the Kir6.2 protein. CONCLUSIONS: Results suggest that post-transcriptional events determine Kir6.2 protein expression in the left ventricle of patients with severe mitral dysfunction and low venous PO(2). Mechanical stress mainly affects transcription of HIF-1α and Kir6.2. This study implies that new therapies could aim at the proteasome for stabilizing the left ventricular Kir6.2 protein.

Central venous hypoxemia is a determinant of human atrial ATP-sensitive potassium channel expression: evidence for a novel hypoxia-inducible factor 1alpha-Forkhead box class O signaling pathway.

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-1alpha). 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-1alpha 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<0.003) and Kir6.2 (P<0.03) protein. Kir6.1 associates with SUR2A/B mRNA (P<0.05) and correlates with FOXOs (P<0.002). FOXOs correlate with HIF-1alpha (P<0.01) and HIF-1alpha with venous hypoxemia (P<0.003). Electrophoretic mobility-shift assays suggest causal links among hypoxia, HIF-1alpha, FOXO1, and Kir6.1. To mimic mild ischemia encountered in some patients, cultured rat atrial myocytes were tested in hypoxia, hypercapnia, or low glucose, with normal conditions serving as the control. Mild hypoxia (24-hour) increases expression of HIF-1alpha, FOXO1, and SUR2A/B/Kir6.1 in culture (P<0.01), whereas hypercapnia and low glucose have no or opposite effects. Gene knockdown of HIF-1alpha or FOXO1 by small-interfering RNAs abolishes hypoxia-induced expression of FOXO1 and SUR2A/B/Kir6.1. These results suggest that low tissue oxygen determines increased expression of the atrial SUR2A/B/Kir6.1 gene via activation of HIF-1alpha-FOXO1. Because increased SUR2A/B/Kir6.1 has known survival benefits, this pathway offers novel therapeutic targets for children with heart disease.

Angiotensin II and tumour necrosis factor alpha as mediators of ATP-dependent potassium channel remodelling in post-infarction heart failure.

AIMS: Angiotensin II (Ang II) and tumour necrosis factor alpha (TNFalpha) are involved in the progression from compensated hypertrophy to heart failure. Here, we test their role in the remodelling of ATP-dependent potassium channel (K(ATP)) in heart failure, conferring increased metabolic and diazoxide sensitivity. METHODS AND RESULTS: We observed increased expression of both angiotensinogen and TNFalpha in the failing rat myocardium, with a regional gradient matching that of the K(ATP) subunit Kir6.1 expression. Both angiotensinogen and TNFalpha expression correlated positively with Kir6.1 and negatively with Kir6.2 expression across the post-infarction myocardium. To further identify a causal relationship, cardiomyocytes isolated from normal rat hearts were exposed in vitro to Ang II or TNFalpha. We observed increased Kir6.1 and SUR subunit and reduced Kir6.2 subunit mRNA expression in cardiomyocytes cultured with Ang II or TNFalpha, similar to what was observed in failing hearts. In patch-clamp experiments, cardiomyocytes cultured with Ang II or TNFalpha exhibited responsiveness to diazoxide, in terms of both K(ATP) current and action potential shortening. This was not observed in untreated cardiomyocytes and resembles the diazoxide sensitivity of failing cardiomyocytes that also overexpress Kir6.1. Ang II exerted its effect through induction of TNFalpha expression, because TNFalpha-neutralizing antibody abolished the effect of Ang II, and in failing hearts, regional expression of angiotensinogen matched TNFalpha expression. Finally, Ang II and TNFalpha regulated K(ATP) subunit expression, possibly through differential expression of Forkhead box transcription factors. CONCLUSION: This study identifies Ang II and TNFalpha as mediators of the remodelling of K(ATP) channels in heart failure.

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.

Expression and function of ATP-dependent potassium channels in late post-infarction remodeling.

Myocardial remodeling late after infarction is associated with increased incidence of fatal arrhythmias. Heterogeneous prolongation of the action potential in the surviving myocardium is one of the predominant causes. Sarcolemmal ATP-dependent potassium (K(ATP)) channels are important metabolic sensors regulating electrical activity of cardiomyocytes and are capable of considerably shortening the action potential. We determined whether ATP-dependent potassium channels generate or, on the contrary prevent the heterogeneity in action potential prolongation. Cardiomyocytes were obtained from the infarct border zone, the septum and the right ventricle of rat hearts 20 weeks after coronary occlusion when rats developed signs of heart failure. Expression of the conductance subunit Kir6.1, but not Kir6.2, and of all SUR regulatory subunits was increased up to 3-fold in cardiomyocytes from the infarct border zone. Concomitantly, there was a prominent response of the K(ATP) current to diazoxide that was not detectable in control cardiomyocytes. The action potential was prolonged in cardiomyocytes from the infarct border zone (74 ms) relative to sham (41 ms). However, activation of the K(ATP) channels by diazoxide reduced action potential duration to 42 ms. In myocytes of the septum and right ventricle, expression of channel subunits, duration of action potential, and sensitivity to diazoxide were only slightly increased relative to shams. In conclusion, the myocardium remodeled after infarction displays alterations of K(ATP) expression and function with spatial heterogeneity matching that of the action potential prolongation. Drugs selectively activating diazoxide-sensitive sarcolemmal K(ATP) channels should be considered in the prevention of arrhythmias in post-infarction heart failure.

ApoO, a novel apolipoprotein, is an original glycoprotein up-regulated by diabetes in human heart.

Obesity is an independent risk factor for cardiac failure. Obesity promotes excessive deposition of fat in adipose and nonadipose tissues. Intramyocardial lipid overload is a relatively common finding in nonischemic heart failure, especially in obese and diabetic patients, and promotes lipoapoptosis that contributes to the alteration of cardiac function. Lipoprotein production has been proposed as a heart-protective mechanism through the unloading of surplus cellular lipids. We previously analyzed the heart transcriptome in a dog nutritional model of obesity, and we identified a new apolipoprotein, regulated by obesity in heart, which is the subject of this study. We detected this new protein in the following lipoproteins: high density lipoprotein, low density lipoprotein, and very low density lipoprotein. We designated it apolipoprotein O. Apolipoprotein O is a 198-amino acid protein that contains a 23-amino acidlong signal peptide. The apolipoprotein O gene is expressed in a set of human tissues. Confocal immunofluorescence microscopy colocalized apolipoprotein O and perilipins, a cellular marker of the lipid droplet. Chondroitinase ABC deglycosylation analysis or cell incubation with p-nitrophenyl-beta-d-xyloside indicated that apolipoprotein O belongs to the proteoglycan family. Naringenin or CP-346086 treatments indicated that apolipoprotein O secretion requires microsomal triglyceride transfer protein activity. Apolipoprotein O gene expression is up-regulated in the human diabetic heart. Apolipoprotein O promoted cholesterol efflux from macrophage cells. To our knowledge, apolipoprotein O is the first chondroitin sulfate chain containing apolipoprotein. Apolipoprotein O may be involved in myocardium-protective mechanisms against lipid accumulation, or it may have specific properties mediated by its unique glycosylation pattern.

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.

Uncomplicated human obesity is associated with a specific cardiac transcriptome: involvement of the Wnt pathway.

A dramatic increase in obesity prevalence and cardiovascular morbidity is expected for the coming years. However, with relevance to the heart, little is known about the specific contribution of obesity on associated morbidity. Consequently, global analysis of gene regulations in human heart was undertaken to monitor molecular regulations related to obesity or to obesity-related hypertension. Transcriptome analysis using cDNA arrays was performed in right appendage biopsies from obese patients (n=5), from patients with arterial hypertension with (n=5) or without obesity (n=5), and from 5 leans. All biopsies came from patients that had cardiac surgery and coronary bypass. Statistical analysis of the data revealed 2686 differentially expressed genes out of 11,500 when compared with lean tissues. Differential expression was verified by real-time PCR in 84% of 50 randomly chosen genes. Among genes encountered, 397 were specifically regulated in obese, 1,299 in non-obese hypertensive, and 355 in obese hypertensive patients, respectively, whereas an additional set of 153 genes was differentially expressed in all these situations. Ontology analysis, hierarchical clustering, and molecular pathway analysis indicated that the heart molecular picture of obesity differs clearly from that observed for obesity-related hypertension or arterial hypertension. Clearly, the Wnt pathway known to be involved in cardiac hypertrophy mechanisms, showed opposite regulation in obese heart compared with hypertensive heart and potentially prevented the development of cardiac remodeling in obese patients. All over, this work shows that uncomplicated obesity has a strong impact on cardiac gene expression, which could be considered as precursor signs for future cardiac disease and also demonstrates that obesity-related hypertension generates a heart-molecular-distinct phenotype that cannot be predicted by a simple sum of the impact of obesity and arterial hypertension on gene expression.

Kinetic analysis of cardiac transcriptome regulation during chronic high-fat diet in dogs.

In the present study, we investigated, using custom dog cDNA arrays, the time course of transcriptional changes in the left ventricle of dogs fed a normal diet or a high-fat diet (HFD) for 9-24 wk. Array hybridizations were performed with complex probes representing mRNAs expressed in left ventricles from obese hypertensive and lean control dogs. We identified 63 differentially expressed genes, and expression of 17 of 20 randomly chosen genes was confirmed by real-time PCR. Transcripts were categorized into groups involved in metabolism, cell signaling, tissue remodeling, ionic regulation, cell proliferation, and protein synthesis. Hierarchical clustering indicated that the pattern of coregulated genes depends on duration of the HFD, suggesting that HFD-induced obesity hypertension is associated with continuous cardiac transcriptome adaptation despite stability of both body weight and blood pressure. GenMAPP analysis of the data pointed out the crucial importance of the ventricle TGF-beta pathway. Our results suggest that this system may be involved in molecular remodeling during HFD and in changes observed in the transcription profile, reflecting functional and morphological abnormalities that arise during prolonged HFD. These results also suggest some novel regulatory pathways for cardiac adaptation to obesity.

Adrenomedullin upregulates M2-muscarinic receptors in cardiomyocytes from P19 cell line.

1. The effects of AM on expression of muscarinic (M) receptors from P19-derived cardiomyocytes were examined. 2. RT-PCR experiments revealed expression of M(1)-M(4) receptor genes. Immuno-histochemistry indicated that M(2) expression is restricted to contractile cells. Carbachol inhibition of isoprenaline-induced increase in beating rate was prevented by atropine and methoctramine (pA(2): 8.1). Inhibition of [(3)H]-NMS binding by atropine (pK(i): -8.4+/-0.2) and methoctramine (pK(i): -8.3+/-0.2) suggests that M(2) is the functional expressed isoform. 3. [(3)H]-NMS binding and semiquantitative RT-PCR studies showed a dome shaped time course of M(2) expression with a maximum at 7 days of differentiation followed by a progressive decline. 4. AM concentration-dependently upregulated M(2) receptor mRNA during late differentiation stages in P19 cells but also in rat atrial cardiomyocytes. This effect was potentiated by factor H. AM (100 nM) plus factor H (50 nM) treatment of P19 cells for 24 h significantly increased [(3)H]-NMS-specific binding (B(max): 81+/-7 vs 31+/-6 fmol mg(-1) prot). The effect of AM on mRNA levels was prevented by AM receptor antagonist AM(22-52) (1 micro M) but not by CGRP antagonist, CGRP(8-37) (1 micro M). 5. The mRNA levels encoding CRLR receptor declined with culture duration, whereas those encoding L1/G10D receptor remained stable. 6. Our findings demonstrate that AM regulates M(2) receptors expression in cardiomyocytes probably through a mechanism involving L1/G10D receptors. The 'in vivo' significance of this phenomenon remains to be demonstrated.

Cardiac transcriptome analysis in obesity-related hypertension.

Obesity is associated with volumetric arterial hypertension and with early increase in heart rate and decreased heart rate variability. The consequences of obesity-related hypertension on heart gene regulation are poorly known and were investigated in a model of obesity-related hypertension induced by high fat diet in dogs. When compared with control animals (n=6), a 9-week high fat diet (n=6) provoked significant weight gain and increased blood pressure load and heart rate but failed to significantly change left ventricular mass assessed by echocardiography. Subtractive hybridization of dog heart cDNA libraries were used to generate sublibraries containing differentially expressed cDNAs that were in turn spotted onto membranes to create custom microarrays. Hybridizations of these microarrays with complex probes representing mRNAs expressed in right atria and left ventricles from obese hypertensive and control dogs were performed. Thirty-eight differentially expressed genes were identified; altered expression was confirmed by Northern blot analysis in 15. In addition, real-time quantitative polymerase chain reaction confirmed differential expression for 80% of the randomly chosen tested genes. Once identified, transcripts were categorized into groups involved in metabolism, cell signaling, ionic regulation, cell proliferation, protein synthesis, and tissue remodeling. In addition, we found a set of 11 cDNAs encoding proteins with unknown functions. This study clearly shows that obesity-related hypertension, lasting for only 9 weeks, causes marked changes in gene expression in right atrium as well as the left ventricle that may contribute to early functional changes in heart function and to long-term structural changes such as left ventricular hypertrophy and remodeling.