Alternative Splicing of the Cardiac Sodium Channel in Pulmonary Arterial Hypertension.

Right ventricular (RV) adaptation in pulmonary arterial hypertension (PAH) is poorly understood. Sudden cardiac death (SCD) has been described in PAH,1Tonelli A.R. Arelli V. Minai O.A. et al.Causes and circumstances of death in pulmonary arterial hypertension.Am J Respir Crit Care Med. 2013; 188: 365-369Crossref PubMed Scopus (134) Google Scholar but there are no known predictors. Alternative splicing of sodium voltage-gated channel α subunit 5 (SCN5a), a sodium channel integral to myocardial conduction, results in nonfunctional splice variants (SVs) that induce endoplasmic reticulum and mitochondrial stress via the unfolded protein response in left ventricular (LV) failure.2Gao G. Xie A. Huang S.C. et al.Role of RBM25/LUC7L3 in abnormal cardiac sodium channel splicing regulation in human heart failure.Circulation. 2011; 124: 1124-1131Crossref PubMed Scopus (62) Google Scholar, 3Gao G. Xie A. Zhang J. et al.Unfolded protein response regulates cardiac sodium current in systolic human heart failure.Circ Arrhythm Electrophysiol. 2013; 6: 1018-1024Crossref PubMed Google Scholar, 4Shang L.L. Pfahnl A.E. Sanyal S. et al.Human heart failure is associated with abnormal C-terminal splicing variants in the cardiac sodium channel.Circ Res. 2007; 101: 1146-1154Crossref PubMed Scopus (101) Google Scholar Expression levels of SVs measured from circulating leukocytes track with fold changes and protein expression in ventricular tissue (including the RV) and arrhythmias in LV failure.2Gao G. Xie A. Huang S.C. et al.Role of RBM25/LUC7L3 in abnormal cardiac sodium channel splicing regulation in human heart failure.Circulation. 2011; 124: 1124-1131Crossref PubMed Scopus (62) Google Scholar,5Gao G. Brahmanandam V. Raicu M. et al.Enhanced risk profiling of implanted defibrillator shocks with circulating SCN5A mRNA splicing variants: a pilot trial.J Am Coll Cardiol. 2014; 63: 2261-2269Crossref PubMed Scopus (15) Google Scholar Hypoxia-inducible factor 1α (HIF-1α) induces alternative splicing of SCN5a and is a key regulator of the glycolytic shift in the pulmonary vasculature and RV in PAH.2Gao G. Xie A. Huang S.C. et al.Role of RBM25/LUC7L3 in abnormal cardiac sodium channel splicing regulation in human heart failure.Circulation. 2011; 124: 1124-1131Crossref PubMed Scopus (62) Google Scholar,6Zhang R. Wu Y. Zhao M. et al.Role of HIF-1α in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells.Am J Physiol Lung Cell Mol Physiol. 2009; 297: L631-L640Crossref PubMed Scopus (149) Google Scholar,7Ahmadi A. Ohira H. Mielniczuk L.M. FDG PET imaging for identifying pulmonary hypertension and right heart failure.Curr Cardiol Rep. 2015; 17: 555Crossref PubMed Scopus (13) Google Scholar To test the hypothesis that alternative splicing of SCN5a contributes to metabolic reprogramming in PAH, we performed a pilot study to determine whether patients with PAH had elevated levels of circulating SCN5a SVs and whether these levels correlated with markers of RV function and mitochondrial respiration measured from peripheral blood mononuclear cells (PBMCs) (IRB No. 780729-15). Patients with Group 1 PAH meeting traditional hemodynamic criteria were eligible.8Simonneau G. Gatzoulis M.A. Adatia I. et al.Updated clinical classification of pulmonary hypertension.J Am Coll Cardiol. 2013; 62: D34-D41Crossref PubMed Scopus (1931) Google Scholar Control subjects were recruited from caregivers of patients and from hospital employees. Control subjects were screened by questionnaire to confirm the absence of cardiopulmonary symptoms and cardiac or pulmonary disease, and detailed medical histories were obtained. Expression levels of SVs (E28D), SCN5a, and HIF-1α mRNA were measured.4Shang L.L. Pfahnl A.E. Sanyal S. et al.Human heart failure is associated with abnormal C-terminal splicing variants in the cardiac sodium channel.Circ Res. 2007; 101: 1146-1154Crossref PubMed Scopus (101) Google Scholar SV levels were normalized to absolute values of β-actin and represented as fold changes of E28D/total SCN5a transcripts. PBMCs were concurrently isolated4Shang L.L. Pfahnl A.E. Sanyal S. et al.Human heart failure is associated with abnormal C-terminal splicing variants in the cardiac sodium channel.Circ Res. 2007; 101: 1146-1154Crossref PubMed Scopus (101) Google Scholar and stored immediately in liquid nitrogen. Seahorse XF (Agilent) analysis was performed after batch shipment in seven participants. The first five participants with PAH enrolled underwent same-day fasting 2-deoxy-2-[18F]fluoro-d-glucose (FDG)-PET to detect whether SV expression correlated with FDG uptake in the RV9Lundgrin E.L. Park M.M. Sharp J. et al.Fasting 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography to detect metabolic changes in pulmonary arterial hypertension hearts over 1 year.Ann Am Thorac Soc. 2013; 10: 1-9Crossref PubMed Scopus (74) Google Scholar as an exploratory end point. All analyses were conducted with SAS software 9.4 (SAS Institute Inc.). The associations between ln(E28D/total SCN5a) expression levels, mitochondrial respiration, and case/control subject status, respectively, were examined using generalized linear mixed models and assuming a log-normal distribution. Correlations were examined using Spearman rho (α = 0.05). Eleven participants with PAH and 11 age-matched (within 7 years) and sex-matched control subjects were included (Table 1). Circulating SCN5a SV mRNA levels were 23% higher in PAH subjects as compared with matched control subjects despite lower absolute levels of HIF-1α expression (Table 1). In five subjects with PAH with concurrent PET scans, there was a strong correlation between SV expression levels and RV uptake normalized to uptake in the thoracic aorta blood pool (r = 0.89, P = .04) but no relationship to uptake in the pulmonary arteries or LV. There was no correlation between SV expression levels and RV function assessed by echocardiography and brain natriuretic peptide levels in the total sample (n = 11), although these tests were not performed concurrent with blood draw. There were significant inverse correlations between SV expression levels and baseline mean pulmonary artery pressure (r = –0.75, P = .01), and pulmonary vascular resistance (r = –0.70, P = .04), respectively. Subjects with PAH tended to have lower PBMC basal and maximal oxygen consumption rates (OCRs) as well as a lower postoligomycin extracellular acidification rate (ECAR) as compared with control subjects (Table 1). SV expression levels were significantly correlated with basal OCR in subjects with PAH (r = 0.87, P = .01) and control subjects (r = 0.64, P = .03) (Fig 1B). Among subjects with PAH, SV levels correlated with maximum OCR (r = 0.75, P = .05) and postoligomycin ECAR (r = 0.78, P = .04) (Figs 1C and 1D). Basal OCR/basal ECAR correlated with SV levels in PAH subjects (r = 0.87, P = .01) and control subjects (r = 0.64, P = .03) (Table 1, Fig 1E).Table 1Participant Characteristics and Association of mRNA Expression Levels and Markers of Mitochondrial Respiration by PAH StatusVariablePAHControlP ValueNo.1111…Age, y58 (52-65)60 (48-65)…Female, No. (%)9 (82)10 (91)…White, No. (%)9 (82)9 (82)…BMI, kg/m231 (26-38)29 (24-33)…WBC count, × 10–9/L6.0 (4.7-8.5)6.8 (6.1-7.7)…Diabetes mellitus, No. (%)0 (0)2 (18)…Arrhythmia, No. (%) Atrial fibrillation3 (27)0 (0)… Premature ventricular contractions1 (9)0 (0)…Idiopathic PAH6 (54)……Markers of disease severityaClinical data collected at time point closest to blood draw. 6-min walk distance, m382 (201-472)…… Functional class III/IV, No. (%)3 (27)…… BNP, pg/mL76.8 (14.4-114.8)……Echocardiographic parameters RVSP, mm Hg39 (33-66)…… TAPSE, cm2.1 (1.8-2.3)……Hemodynamics Right atrial pressure, mm Hg7 (5-13)…… Mean PAP, mm Hg40 (32-49)…… PCWP, mm Hg11 (10-15)…… Cardiac output, L/min6.9 (6.5-7.5)…… Cardiac index, L/min/m23.6 (3.3-3.9)…… PVR, Wood units3.9 (3.0-6.4)……PAH therapies, No. (%) Calcium channel blockers2 (18)…… Phosphodiesterase-5 inhibitors8 (72)…… Endothelin receptor antagonists7 (64)…… Prostacyclin analogs6 (55)…… Combination9 (82)……mRNA expression levels ln(E28D/total SCN5a)1.6 (1.2-2.1)1.2 (1.0-1.5).047 ln(HIF-1α/β-actin)0.6 (0.3-1.1)0.9 (0.8-1.1).10Mitochondrial respiration Basal OCR, pmol/min48.7 (32.6-72.8)55.4 (37.2-82.5).28 Maximal OCR, pmol/min214.1 (118.3-387.3)227.8 (124.5-416.8).82 Basal ECAR, mpH/min30.9 (25.4-37.5)28.8 (23.8-34.9).53 Postoligomycin ECAR, mpH/min5.0 (1.1-22.3)6.3 (1.5-26.4).06 Basal OCR/basal ECAR, pmol/mpH1.6 (1.0-2.6)1.9 (1.4-2.6).39BNP = brain natriuretic peptide; ECAR = extracellular acidification rate; HIF = hypoxia inducible factor; OCR = oxygen consumption rate; PAH = pulmonary arterial hypertension; PAP = pulmonary artery pressure; PCWP = pulmonary capillary wedge pressure; PVR = pulmonary vascular resistance; RVSP = right ventricular systolic pressure; TAPSE = tricuspid annular plane systolic excursion.a Clinical data collected at time point closest to blood draw. Open table in a new tab BNP = brain natriuretic peptide; ECAR = extracellular acidification rate; HIF = hypoxia inducible factor; OCR = oxygen consumption rate; PAH = pulmonary arterial hypertension; PAP = pulmonary artery pressure; PCWP = pulmonary capillary wedge pressure; PVR = pulmonary vascular resistance; RVSP = right ventricular systolic pressure; TAPSE = tricuspid annular plane systolic excursion. In this small study, we have shown that prevalent PAH patients have increased circulating SCN5a SV levels as compared with matched control subjects and that these levels track with increased oxygen consumption and markers of glycolysis in PBMCs, and in a small subset, RV FDG uptake. Sudden death and altered cellular energetics have been described in PAH,9Lundgrin E.L. Park M.M. Sharp J. et al.Fasting 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography to detect metabolic changes in pulmonary arterial hypertension hearts over 1 year.Ann Am Thorac Soc. 2013; 10: 1-9Crossref PubMed Scopus (74) Google Scholar,10Xu W. Koeck T. Lara A.R. et al.Alterations of cellular bioenergetics in pulmonary artery endothelial cells.Proc Natl Acad Sci U S A. 2007; 104: 1342-1347Crossref PubMed Scopus (270) Google Scholar but posttranscriptional modification of SCN5a—a possible link between these phenomena—has not been reported. We were likely underpowered to detect differences in HIF-1α expression and mitochondrial respiration between subjects with PAH and control subjects. This may be explained by the inclusion of prevalent PAH patients receiving therapy, discordance between transcript and phenotype, and/or transcriptional and metabolic differences in peripheral PBMCs vs the pulmonary circulation and RV. It is also possible that SCN5a splicing is long-lasting and delayed from upstream triggers and downstream effects (eg, the unfolded protein response, which triggers an interorganelle stress response).3Gao G. Xie A. Zhang J. et al.Unfolded protein response regulates cardiac sodium current in systolic human heart failure.Circ Arrhythm Electrophysiol. 2013; 6: 1018-1024Crossref PubMed Google Scholar The association between SV levels and basal OCR/basal ECAR and RV metabolic activity supports a relationship between increased SV accumulation and increased aerobic glycolysis, consistent with known impairments in glucose oxidation in PAH.10Xu W. Koeck T. Lara A.R. et al.Alterations of cellular bioenergetics in pulmonary artery endothelial cells.Proc Natl Acad Sci U S A. 2007; 104: 1342-1347Crossref PubMed Scopus (270) Google Scholar This may be due to upregulated but inefficient mitochondrial activity to meet adenosine triphosphate demand coupled with increased glycolysis. An increase in mitochondrial mass, fatty acids, and reactive oxygen species could lead to simultaneous increases in both OCR and ECAR. Surprisingly, higher SV levels were inversely correlated with measurements of pulmonary afterload. Clinical data were collected as close as possible to blood draw, but only PET scans were performed on the same day as phlebotomy. We included only well-controlled patients without clinical evidence of RV failure. The RV in PAH may be unique and characterized by different SVs than those measured here; it is also possible that mitochondrial respiration varies by cellular compartment. Limitations of our study include the small sample size, lack of tissue-level mRNA, and missing data. Our knowledge of metabolic markers of disease in humans living with PAH is limited. The results from this study provide data to support a novel and, to our knowledge, not previously reported relationship between alternative splicing and cellular metabolism in PAH. We believe this may be one of the first studies to assess mitochondrial function among living patients with PAH from a peripheral blood draw. This may be the first step toward point-of-care testing for RV metabolic changes (and possibly SCD) in this highly lethal condition. Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of the data, or the preparation of the manuscript. Other contributions: The authors thank all of the participants who volunteered to take part in this study.