[Sleep apnea automatic detection method based on convolutional neural network].

Sleep apnea (SA) detection method based on traditional machine learning needs a lot of efforts in feature engineering and classifier design. We constructed a one-dimensional convolutional neural network (CNN) model, which consists in four convolution layers, four pooling layers, two full connection layers and one classification layer. The automatic feature extraction and classification were realized by the structure of the proposed CNN model. The model was verified by the whole night single-channel sleep electrocardiogram (ECG) signals of 70 subjects from the Apnea-ECG dataset. Our results showed that the accuracy of per-segment SA detection was ranged from 80.1% to 88.0%, using the input signals of single-channel ECG signal, RR interval (RRI) sequence, R peak sequence and RRI sequence + R peak sequence respectively. These results indicated that the proposed CNN model was effective and can automatically extract and classify features from the original single-channel ECG signal or its derived signal RRI and R peak sequence. When the input signals were RRI sequence + R peak sequence, the CNN model achieved the best performance. The accuracy, sensitivity and specificity of per-segment SA detection were 88.0%, 85.1% and 89.9%, respectively. And the accuracy of per-recording SA diagnosis was 100%. These findings indicated that the proposed method can effectively improve the accuracy and robustness of SA detection and outperform the methods reported in recent years. The proposed CNN model can be applied to portable screening diagnosis equipment for SA with remote server.

Anthocyanin Biosynthesis and Methylation of the MdMYB10 Promoter Are Associated with the Red Blushed-Skin Mutant in the Red Striped-Skin "Changfu 2" Apple.

The color of apple skin, particularly anthocyanin-based coloration, is a key factor determining market acceptance. The mechanisms of anthocyanin accumulation in apples with different skin color patterns (i.e., striped and blushed) were analyzed. In total, 14 anthocyanins and 5 procyanidins were simultaneously assayed in red blushed-skin mutants (CF-B1 and CF-B2) and red striped-skin parents (CF-S1 and CF-S2), and 13 significant differences were revealed. Anthocyanin accumulation was significantly higher in the red blushed-skin apples than it was in the parents. The transcript levels of anthocyanin biosynthesis genes and regulatory factors (MdMYB10, MdbHLH3, and MdWD40) were associated with different skin color patterns during the coloring period at 4, 6, and 8 days after the fruits were debagged. The methylation levels of the MdMYB10 promoter regions -1203 to -779 bp, -1667 to -1180 bp, and -2295 to -1929 bp were associated with different skin color patterns, and there was more methylation in red striped-skin apples. These results improve our understanding of anthocyanin accumulation and its underlying molecular mechanism in apples with different skin color patterns, thereby providing valuable information for apple breeding.

Cardiac Na+ current regulation by pyridine nucleotides.

RATIONALE: Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein reduce cardiac Na+ current (I(Na)) and cause Brugada Syndrome (BrS). GPD1-L has >80% amino acid homology with glycerol-3-phosphate dehydrogenase, which is involved in NAD-dependent energy metabolism. OBJECTIVE: Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Na(v)1.5). METHODS AND RESULTS: HEK293 cells stably expressing Na(v)1.5 and rat neonatal cardiomyocytes were used. The influence of NADH/NAD+ on arrhythmic risk was evaluated in wild-type or SCN5A(+/-) mouse heart. A280V GPD1-L caused a 2.48+/-0.17-fold increase in intracellular NADH level (P<0.001). NADH application or cotransfection with A280V GPD1-L resulted in decreased I(Na) (0.48+/-0.09 or 0.19+/-0.04 of control group, respectively; P<0.01), which was reversed by NAD+, chelerythrine, or superoxide dismutase. NAD+ antagonism of the Na+ channel downregulation by A280V GPD1-L or NADH was prevented by a protein kinase (PK)A inhibitor, PKAI(6-22). The effects of NADH and NAD+ were mimicked by a phorbol ester and forskolin, respectively. Increasing intracellular NADH was associated with an increased risk of ventricular tachycardia in wild-type mouse hearts. Extracellular application of NAD+ to SCN5A(+/-) mouse hearts ameliorated the risk of ventricular tachycardia. CONCLUSIONS: Our results show that Na(v)1.5 is regulated by pyridine nucleotides, suggesting a link between metabolism and I(Na). This effect required protein kinase C activation and was mediated by oxidative stress. NAD+ could prevent this effect by activating PKA. Mutations of GPD1-L may downregulate Na(v)1.5 by altering the oxidized to reduced NAD(H) balance.

NF-kappaB-dependent transcriptional regulation of the cardiac scn5a sodium channel by angiotensin II.

Angiotensin II (ANG II) increases oxidative stress and is associated with increased risk of sudden cardiac death. The cardiac Na(+) channel promoter contains elements that confer redox sensitivity. We tested the hypothesis that ANG II-mediated oxidative stress may modulate Na(+) channel current through altering channel transcription. In H9c2 myocytes treated for 48 h with ANG II (100 nmol/l) or H(2)O(2) (10 micromol/l) showed delayed macroscopic inactivation, increased late current, and 59.6% and 53.8% reductions in Na(+) current, respectively (P < or = 0.01). By quantitative real-time RT-PCR, the cardiac Na(+) channel (scn5a) mRNA abundance declined by 47.3% (P < 0.01) in H9c2 myocytes treated for 48 h with 100 nmol/l ANG II. A similar change occurred with 20 micromol/l H(2)O(2) (46.9%, P < 0.01) after 48 h. Comparable effects were seen in acutely isolated ventricular myocytes. The effects of ANG II could be inhibited by prior treatment of H9c2 cells with scavengers of reactive oxygen species or an inhibitor of the NADPH oxidase. Mutation of the scn5a promoter NF-kappaB binding site prevented decreased activity in response to ANG II and H(2)O(2). Gel shift and chromosomal immunoprecipitation assays confirmed that nuclear factor (NF)-kappaB bound to the scn5a promoter in response to ANG II and H(2)O(2). Overexpression of the p50 subunit of NF-kappaB in H9c2 cells reduced scn5a mRNA (77.3%, P < 0.01). In conclusion, ANG II can decrease scn5a transcription and current. This effect appears to be through production of H(2)O(2) resulting in NF-kappaB binding to the Na(+) channel promoter.

Mutation in glycerol-3-phosphate dehydrogenase 1 like gene (GPD1-L) decreases cardiac Na+ current and causes inherited arrhythmias.

BACKGROUND: Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause approximately 20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. METHODS AND RESULTS: We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by approximately 50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31+/-5% (P=0.01). CONCLUSIONS: GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.

Human heart failure is associated with abnormal C-terminal splicing variants in the cardiac sodium channel.

Heart failure (HF) is associated with reduced cardiac Na+ channel (SCN5A) current. We hypothesized that abnormal transcriptional regulation of this ion channel during HF could help explain the reduced current. Using human hearts explanted at the transplantation, we have identified 3 human C-terminal SCN5A mRNA splicing variants predicted to result in truncated, nonfunctional channels. As compared with normal hearts, the explanted ventricles showed an upregulation of 2 of the variants and a downregulation of the full-length mRNA transcript such that the E28A transcript represented only 48.5% (P<0.01) of the total SCN5A mRNA. This correlated with a 62.8% (P<0.01) reduction in Na+ channel protein. Lymphoblasts and skeletal muscle expressing SCN5A also showed identical C-terminal splicing variants. Variants showed reduced membrane protein and no functional current. Transfection of truncation variants into a cell line stably transfected with the full-length Na+ channel resulted in dose-dependent reductions in channel mRNA and current. Introduction of a premature truncation in the C-terminal region in a single allele of the mouse SCN5A resulted in embryonic lethality. Embryonic stem cell-derived cardiomyocytes expressing the construct showed reductions in Na+ channel-dependent electrophysiological parameters, suggesting that the presence of truncated Na+ channel mRNA at levels seen in HF is likely to be physiologically significant. In summary, chronic HF was associated with an increase in 2 truncated SCN5A variants and a decrease in the native mRNA. These splice variations may help explain a loss of Na+ channel protein and may contribute to the increased arrhythmic risk in clinical HF.

Cardiac-restricted angiotensin-converting enzyme overexpression causes conduction defects and connexin dysregulation.

Renin-angiotensin (RAS) system activation is associated with an increased risk of sudden death. Previously, we used cardiac-restricted angiotensin-converting enzyme (ACE) overexpression to construct a mouse model of RAS activation. These ACE 8/8 mice die prematurely and abruptly. Here, we have investigated cardiac electrophysiological abnormalities that may contribute to early mortality in this model. In ACE 8/8 mice, surface ECG voltages are reduced. Intracardiac electrograms showed atrial and ventricular potential amplitudes of 11% and 24% compared with matched wild-type (WT) controls. The atrioventricular (AV), atrio-Hisian (AH), and Hisian-ventricular (HV) intervals were prolonged 2.8-, 2.6-, and 3.9-fold, respectively, in ACE 8/8 vs. WT mice. Various degrees of AV nodal block were present only in ACE 8/8 mice. Intracardiac electrophysiology studies demonstrated that WT and heterozygote (HZ) mice were noninducible, whereas 83% of ACE 8/8 mice demonstrated ventricular tachycardia with burst pacing. Atrial connexin 40 (Cx40) and connexin 43 (Cx43) protein levels, ventricular Cx43 protein level, atrial and ventricular Cx40 mRNA abundances, ventricular Cx43 mRNA abundance, and atrial and ventricular cardiac Na(+) channel (Scn5a) mRNA abundances were reduced in ACE 8/8 compared with WT mice. ACE 8/8 mice demonstrated ventricular Cx43 dephosphorylation. Atrial and ventricular L-type Ca(2+) channel, Kv4.2 K(+) channel alpha-subunit, and Cx45 mRNA abundances and the peak ventricular Na(+) current did not differ between the groups. In isolated heart preparations, a connexin blocker, 1-heptanol (0.5 mM), produced an electrophysiological phenotype similar to that seen in ACE 8/8 mice. Therefore, cardiac-specific ACE overexpression resulted in changes in connexins consistent with the phenotype of low-voltage electrical activity, conduction defects, and induced ventricular arrhythmia. These results may help explain the increased risk of arrhythmia in states of RAS activation such as heart failure.

A sodium channel pore mutation causing Brugada syndrome.

BACKGROUND: Brugada and long QT type 3 syndromes are linked to sodium channel mutations and clinically cause arrhythmias that lead to sudden death. We have identified a novel threonine-to-isoleucine missense mutation at position 353 (T353I) adjacent to the pore-lining region of domain I of the cardiac sodium channel (SCN5A) in a family with Brugada syndrome. Both male and female carriers are symptomatic at young ages, have typical Brugada-type electrocardiogram changes, and have relatively normal corrected QT intervals. OBJECTIVES: To characterize the properties of the newly identified cardiac sodium channel (SCN5A) mutation at the cellular level. RESULTS: Using whole-cell voltage clamp, we found that heterologous expression of SCN5A containing the T353I mutation resulted in 74% +/- 6% less peak macroscopic sodium current when compared with wild-type channels. A construct of the T353I mutant channel fused with green fluorescent protein failed to traffic properly to the sarcolemma, with a large proportion of channels sequestered intracellularly. Overnight exposure to 0.1 mM mexiletine, a Na(+) channel blocking agent, increased T353I channel trafficking to the membrane to near normal levels, but the mutant channels showed a significant late current that was 1.6% +/- 0.2% of peak sodium current at 200 ms, a finding seen with long QT mutations. CONCLUSIONS: The clinical presentation of patients carrying the T353I mutation is that of Brugada syndrome and could be explained by a cardiac Na(+) channel trafficking defect. However, when the defect was ameliorated, the mutated channels had biophysical properties consistent with long QT syndrome. The lack of phenotypic changes associated with the long QT syndrome could be explained by a T353I-induced trafficking defect reducing the number of mutant channels with persistent currents present at the sarcolemma.

Analysis of arrhythmic potential of embryonic stem cell-derived cardiomyocytes.

By directed differentiation using the hanging drop method, cardiomyocytes (CMs) can be derived from mouse embryonic stem cells. These spontaneously active CMs can then be isolated from the embryoid bodies and studied electrophysiologically for analysis of arrhythmic potential. This method is particularly advantangeous for studying CMs derived from genetically modified stem cells, in which mutations result in embryonic lethality.

Tandem promoters and developmentally regulated 5'- and 3'-mRNA untranslated regions of the mouse Scn5a cardiac sodium channel.

The SCN5A gene encodes a voltage-sensitive sodium channel expressed in cardiac and skeletal muscle. Coding region mutations cause cardiac sudden death syndromes and conduction system failure. Polymorphisms in the 5'-sequence adjacent to the SCN5A gene have been linked to cardiac arrhythmias. We identified three alternative 5'-splice variants (1A, 1B, and 1C) of the untranslated exon 1 and two 3'-variants in the murine Scn5a mRNA. Two of the exon 1 isoforms (1B and 1C) were novel when compared with the published human and rat SCN5A sequences. Quantitative real time PCR results showed that the abundance of the isoforms varied during cardiac development. The 1A, 1B, and 1C mRNA splice variants increased 7.8 +/- 1.7-fold (E1A), 6.0 +/- 1.0-fold (E1B), and 20.6 +/- 3.7-fold (E1C) from fetal to adult heart, respectively. Promoter deletion and luciferase reporter gene analysis using cardiac and skeletal muscle cell lines demonstrated a pattern of distinct cardiac-specific enhancer elements associated with exons 1A and 1C. In the case of exon 1C, the enhancer element appeared to be within the exon. A 5'-repressor preceded each cardiac enhancer element. We concluded that the murine Na(+) channel has both 5'- and 3'-untranslated region mRNA variants that are developmentally regulated and that the promoter region contains two distinct cardiac-specific enhancer regions. The presence of homologous human splicing suggests that that these regions may be fruitful new areas of study in understanding cardiac sodium channel regulation and the genetic susceptibility to sudden death.

Characterization and regulation of T-type Ca2+ channels in embryonic stem cell-derived cardiomyocytes.

T-type Ca2+ channels may play a role in cardiac development. We studied the developmental regulation of the T-type currents (ICa,T) in cardiomyocytes (CMs) derived from mouse embryonic stem cells (ESCs). ICa,T was studied in isolated CMs by whole cell patch clamp. Subsequently, CMs were identified by the myosin light chain 2v-driven green fluorescent protein expression, and laser capture microdissection was used to isolate total RNA from groups of cells at various developmental time points. ICa,T showed characteristics of Cav3.1, such as resistance to Ni2+ block, and a transient increase during development, correlating with measures of spontaneous electrical activity. Real-time RT-PCR showed that Cav3.1 mRNA abundance correlated (r2 = 0.81) with ICa,T. The mRNA copy number was low at 7+4 days (2 copies/cell), increased significantly by 7+10 days (27/cell; P < 0.01), peaked at 7+16 days (174/cell), and declined significantly at 7+27 days (25/cell). These data suggest that ICa,T is developmentally regulated at the level of mRNA abundance and that this regulation parallels measures of pacemaker activity, suggesting that ICa,T might play a role in the spontaneous contractions during CM development.

Hoxc12 expression pattern in developing and cycling murine hair follicles.

We examine the Hoxc12 RNA expression pattern during both hair follicle morphogenesis and cycling in direct comparison to its only upstream neighbor, Hoxc13. Expression of both genes is restricted to the epidermal part of the follicle excluding the outer root sheath and interfollicular epidermis in a distinct stage-dependent and cyclical manner. During the progressive growth phase (anagen) of developing and cycling follicles, the distinct proximo-distal expression domain of Hoxc12 overlaps only proximally, at the upper-most region of the bulb, with the more proximally restricted Hoxc13 domain. This arrangement of the expression domains of the two genes along the proximal-toward-distal axis of increasing follicular differentiation correlates with the sequential expression of first Hoxc13 and then Hoxc12. This indicates a reversal of the typical temporal colinearity of Hox gene activation otherwise observed along the anterior-posterior morphogenetic axis of the embryo (review: Cell 78 (1994) 191).