Novel and Annotated Long Noncoding RNAs Associated with Ischemia in the Human Heart.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been implicated in the pathogenesis of cardiovascular diseases. We aimed to identify novel lncRNAs associated with the early response to ischemia in the heart. METHODS AND RESULTS: RNA sequencing data gathered from 81 paired left ventricle samples from patients undergoing cardiopulmonary bypass was collected before and after a period of ischemia. Novel lncRNAs were validated with Oxford Nanopore Technologies long-read sequencing. Gene modules associated with an early ischemic response were identified and the subcellular location of selected lncRNAs was determined with RNAscope. A total of 2446 mRNAs, 270 annotated lncRNAs and one novel lncRNA differed in response to ischemia (adjusted p < 0.001, absolute fold change >1.2). The novel lncRNA belonged to a gene module of highly correlated genes that also included 39 annotated lncRNAs. This module associated with ischemia (Pearson correlation coefficient = -0.69, p = 1 × 10-23) and activation of cell death pathways (p < 6 × 10-9). A further nine novel cardiac lncRNAs were identified, of which, one overlapped five cis-eQTL eSNPs for the gene RWD Domain-Containing Sumoylation Enhancer (RWDD3) and was itself correlated with RWDD3 expression (Pearson correlation coefficient -0.2, p = 0.002). CONCLUSION: We have identified 10 novel lncRNAs, one of which was associated with myocardial ischemia and may have potential as a novel therapeutic target or early marker for myocardial dysfunction.

Design and implementation of the infection prevention program into risk management: Managing high level disinfection and sterilization in the outpatient setting.

Reusable, invasive medical devices within the outpatient setting pose a risk for patient harm. Ineffective disinfection of medical devices can potentially lead to transmission of pathogens between patients; and improper handling can lead to patient injury. A risk assessment was conducted, and the results strongly supported the necessity to develop a robust infection prevention program within the risk management department. This exclusive program was a proactive approach to preventing patient exposure within our healthcare system. Designing and integrating an Infection Prevention program into the Risk Management Department presented challenges, especially with the magnitude of devices and lack of standardization throughout our 33 clinics. Key components of the program included: capturing an accurate inventory of devices throughout the system, hiring a sterile processing expert, engaging support from senior leadership, adhering to rigorous auditing processes, and establishing a staff competency training structure. Since the program was launched 2 years ago, outcomes include: identification of high-risk practices with immediate resolution, increase in average clinic compliance to device reprocessing standards from 88% to 99%, elimination of 71% of scope reprocessing and 39% of instrument sterilization by clinic staff with allocation to central sterile processing departments, and development of a staff competency training structure.

Cardiac CRFR1 Expression Is Elevated in Human Heart Failure and Modulated by Genetic Variation and Alternative Splicing.

Corticotropin-releasing factor (CRF) and the CRF-related peptides, urocortin (Ucn)-1, Ucn2, and Ucn3 signal through receptors CRFR1 and CRFR2 to restore homeostasis in response to stress. The Ucns exert potent cardioprotective effects and may have clinical utility in heart failure. To explore the activity of this system in the heart, we measured the levels of myocardial gene expression of the CRF/Ucn family of ligands/receptors and investigated genetic variation and alternative splicing of CRFR1 in 110 heart failure patients and 108 heart donors. Using quantitative real-time PCR, we detected CRFR1, CRFR2, CRF, Ucn1, Ucn2, and Ucn3 in all samples. CRFR2α was the most abundant receptor and Ucn3 the most abundant ligand, both in patients and donors. Compared with donors, cardiac expression of CRFR1, CRF, and Ucn3 was higher (P < .001) and CRFR2α lower (P = .012) in patients. In patients and donors, genetic variation within CRFR1, represented by the chromosome 17q21.31 inversion polymorphism, was associated with markedly higher CRFR1 expression (P < .001), making CRFR1 and CRFR2α expression almost equivalent in some patients. A novel, truncated splice variant of CRFR1, designated CRFR1j, was identified and shown to exert a dominant-negative effect on CRFR1 signaling in vitro. The novel variant was expressed in a greater proportion of patients (60%) than donors (3%, P < .001). In summary, cardiac expression of CRFR1, CRF, and Ucn3 genes is elevated in heart failure and may contribute to the activation of the CRF/Ucn system in these patients. A common variant within the CRFR1 gene and a novel CRFR1 splice variant may modulate CRFR1 expression and signaling.

The rs11515 Polymorphism Is More Frequent and Associated With Aggressive Breast Tumors with Increased ANRIL and Decreased p16 (INK4a) Expression.

Chromosome position 9p21 encodes three-tumor suppressors p16(INK4a), p14(ARF), and p15(INK4b) and the long non-coding RNA ANRIL (antisense non-coding RNA in the INK4 locus). The rs11515 single-nucleotide polymorphism in the p16 (INK4a) /p14 (ARF) 3'-untranslated region is associated with glioblastoma, melanoma, and other cancers. This study investigated the frequency and effect of rs11515 genotypes in breast cancer. Genomic DNA samples from 400 women (200 with and 200 without a diagnosis of breast cancer) were genotyped for the rs11515 major (C) and minor (G) alleles. The rs11515 polymorphism was also investigated in 108 heart tissues to test for tissue-specific effects. Four 9p21 transcripts, p16 (INK4a) , p14 (ARF) , p15 (INK4b) , and ANRIL were measured in breast tumors and myocardium using quantitative PCR. Heterozygotes (CG genotype) were more frequent in women with breast cancer compared to the control population (P = 0.0039). In those with breast cancer, the CG genotype was associated with an older age (P = 0.016) and increased lymph node involvement (P = 0.007) compared to homozygotes for the major allele (CC genotype). In breast tumors, the CG genotype had higher ANRIL (P = 0.031) and lower p16 (INK4a) (P = 0.006) expression compared to the CC genotype. The CG genotype was not associated with altered 9p21 transcripts in heart tissue. In breast cancer, the rs11515 CG genotype is more frequent and associated with a more aggressive tumor that could be due to increased ANRIL and reduced p16 (INK4a) expression. The absence of association between rs11515 genotypes and 9p21 transcripts in heart tissue suggests this polymorphism has tissue- or disease-specific functions.

Association of noninvasively measured left ventricular mechanics with in vitro muscle contractile performance: a prospective study in hypertrophic cardiomyopathy patients.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a primary myopathic process in which regional left ventricular dysfunction may exist without overt global left ventricular dysfunction. In obstructive HCM patients who underwent surgical myectomy (SM), we sought to determine if there is a significant association between echocardiographic longitudinal strain, histopathology, and in vitro myocardial performance (resting tension and developed tension) of the surgical specimen. METHODS AND RESULTS: HCM patients (n=122, 54±14 years, 54% men) undergoing SM were prospectively recruited. Longitudinal systolic strain and diastolic strain rates were measured at that basal septum (partially removed at SM) by using velocity vector imaging on preoperative echocardiography. Semiquantitative histopathologic grading of myocyte disarray and fibrosis and in vitro measurements of resting tension and developed tension were made in septal tissue obtained at SM. Mean basal septal systolic strain and diastolic strain rate were -8.3±5% and 0.62±0.4/s, while mild or greater degree of myocyte disarray and interstitial fibrosis were present in 85% and 87%, respectively. Mean resting tension and developed tension were 2.8±1 and 1.4±0.8 g/mm(2). On regression analysis, basal septal systolic strain, diastolic strain rate, disarray, and fibrosis were associated with developed tension (ß=0.19, 0.20, -0.33, and -0.40, respectively, all P<0.01) and resting tension (ß=0.21, 0.22, -0.25, and -0.28, respectively, all P<0.01). CONCLUSION: In obstructive HCM patients who underwent SM, left ventricular mechanics (echocardiographic longitudinal systolic strain and diastolic strain rates), assessed at the basal septum (myocardium removed during myectomy) and histopathologic findings characteristic for HCM (disarray and fibrosis) were significantly associated with in vitro myocardial resting and developed contractile performance.

S100A1 in human heart failure: lack of recovery following left ventricular assist device support.

BACKGROUND: We hypothesized that S100A1 is regulated during human hypertrophy and heart failure and that it may be implicated in remodeling after left ventricular assist device. S100A1 is decreased in animal and human heart failure, and restoration produces functional recovery in animal models and in failing human myocytes. With the potential for gene therapy, it is important to carefully explore human cardiac S100A1 regulation and its role in remodeling. METHODS AND RESULTS: We measured S100A1, the sarcoplasmic endoplasmic reticulum Ca(2+)ATPase, phospholamban, and ryanodine receptor proteins, as well as ß-adrenergic receptor density in nonfailing, hypertrophied (left ventricular hypertrophy), failing, and failing left ventricular assist device-supported hearts. We determined functional consequences of protein alterations in isolated contracting muscles from the same hearts. S100A1, sarcoplasmic endoplasmic reticulum Ca(2+)ATPase and phospholamban were normal in left ventricular hypertrophy, but decreased in failing hearts, while ryanodine receptor was unchanged in either group. Baseline muscle contraction was not altered in left ventricular hypertrophy or failing hearts. ß-Adrenergic receptor and inotropic response were decreased in failing hearts. In failing left ventricular assist device-supported hearts, S100A1 and sarcoplasmic endoplasmic reticulum Ca(2+)ATPase showed no recovery, while phospholamban, ß-adrenergic receptor, and the inotropic response fully recovered. CONCLUSIONS: S100A1 and sarcoplasmic endoplasmic reticulum Ca(2+)ATPase, both key Ca(2+)-regulatory proteins, are decreased in human heart failure, and these changes are not reversed after left ventricular assist device. The clinical significance of these findings for cardiac recovery remains to be addressed.

An animal model of stress-induced cardiomyopathy utilizing the social defeat paradigm.

Stress-induced cardiomyopathy (SIC) is a form of acute heart disease triggered by extreme psychological stress. In patients who develop SIC, the outward symptoms are almost indistinguishable from acute myocardial infarction (AMI). However, some important criteria differentiate patients with SIC from those with AMI. Patients with SIC: (1) experience some form of extreme psychological stress from minutes to hours before developing heart disease, (2) do not suffer from atherosclerosis or coronary artery obstruction, and 3) exhibit abnormal ballooning of the left ventricle. In the present study, the resident-intruder (RI) social defeat test was investigated as a potential rat model for stressed-induced cardiomyopathy. Adult Long-Evans rats were implanted with a biotelemetry transmitter for ECG recordings and habituated for two weeks. An intruder rat was placed in the cage of a resident rat behind a wire-mesh partition for 5 min. The partition was then removed for 5 min to allow direct contact between the intruder and resident rats. After this interval, the wire-mesh partition was replaced and the intruder rat remained behind the partition for an additional 50 min. Behavioral responses were noted and ECG recordings were collected during the entire 60-min testing period. Upon completion of the test, the intruder rat was removed from the cage of the resident rat and sacrificed. The heart was examined and blood was collected. Heart weight/body weight ratio, left ventricle/body weight ratio, heart length, plasma corticosterone levels, and plasma troponin I levels of intruder rats were significantly higher as compared to control rats. Intruder rats significantly increased their heart rate during the first 5 min of the RI test. It is concluded that the RI test to induce social defeat is a novel rodent paradigm for modeling stress-induced cardiomyopathy in the human.

The chromosome 9p21.3 coronary heart disease risk allele is associated with altered gene expression in normal heart and vascular tissues.

Genome-wide association studies have identified a coronary artery disease (CAD) risk locus in a non-coding region at 9p21.3, the nearest genes being CDKN2A and CDKN2B. To understand the pathways by which this locus might influence CAD susceptibility, we investigated associations between the 9p21.3 risk genotype and global gene expression in heart tissue from donors with no diagnosed heart disease (n = 108, predominant cause of death, cerebral vascular accident) and in carotid plaque (n = 106), aorta (n = 104) and mammary artery (n = 88) tissues from heart valve and carotid endarterectomy patients. Genotyping was performed with Taqman assays and Illumina arrays, and gene expression profiles generated with Affymetrix microarrays. Associations were analyzed with an additive genetic model. In heart tissue, 46 genes were putatively altered in association with the 9p21.3 risk allele (70% down-regulated, fold-change >1.1 per allele, p<0.05 adjusted for age, gender, ethnicity, cause of death). These genes were enriched for biomarkers of myocardial infarction (p = 1.53×10(-9)), response to wounding (p = 2.65×10(-10)) and inflammatory processes (p<1.97×10(-7)). Among the top 10 most down-regulated genes, 7 genes shared a set of transcription factor binding sites within conserved promoter regions (p<1.14×10(-5)), suggesting they may be co-regulated. Canonical pathway modelling of the most differentially expressed transcripts across all tissues (154 genes, 60% down-regulated, fold-change >1.1 per allele, p<0.01) showed that 75% of the genes could be transcriptionally regulated through the cell cycle G1 phase progression pathway (p<1.08×10(-258)), in which CDKN2A and CDKN2B play a regulatory role. These data suggest that the cell cycle G1 phase progression pathway is activated in individuals with the 9p21.3 risk allele. This may contribute to a proliferative phenotype that promotes adverse cardiac hypertrophy and vascular remodeling, leading to an increased CAD risk.

Pulmonary hypertension associated with advanced systolic heart failure: dysregulated arginine metabolism and importance of compensatory dimethylarginine dimethylaminohydrolase-1.

OBJECTIVES: This study sought to examine the hemodynamic determinants of dysregulated arginine metabolism in patients with acute decompensated heart failure and to explore possible mechanisms of arginine dysregulation in human heart failure. BACKGROUND: Accumulating methylated arginine metabolites and impaired arginine bioavailability have been associated with heart failure, but the underlying pathophysiology remains unclear. METHODS: This study prospectively determined plasma levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and global arginine bioavailability ratio [GABR = arginine/(ornithine + citrulline)] by tandem mass spectrometry in subjects with advanced decompensated heart failure in the intensive care unit (n = 68) and with stable chronic heart failure (n = 57). RESULTS: Compared with chronic heart failure subjects, plasma ADMA was significantly higher (median [interquartile range]: 1.29 [1.04 to 1.77] µmol/l vs. 0.87 [0.72 to 1.05] µmol/l, p < 0.0001), and global arginine bioavailability ratio significantly lower (median [interquartile range]: 0.90 [0.69 to 1.22] vs. 1.13 [0.92 to 1.37], p = 0.002) in advanced decompensated heart failure subjects. Elevated ADMA and diminished global arginine bioavailability ratio were associated with higher systolic pulmonary artery pressure (sPAP) and higher central venous pressure, but not with other clinical or hemodynamic indices. We further observed myocardial levels of dimethylarginine dimethylaminohydrolase-1 were increased in chronic heart failure without elevated sPAP (<50 mm Hg), but diminished with elevated sPAP (≥50 mm Hg, difference with sPAP <50 mm Hg, p = 0.02). CONCLUSIONS: Dysregulated arginine metabolism was observed in advanced decompensated heart failure, particularly with pulmonary hypertension and elevated intracardiac filling pressures. Compared with hearts of control subjects, we observed higher amounts of ADMA-degradation enzyme dimethylarginine dimethylaminohydrolase-1 (but similar amounts of ADMA-producing enzyme, protein methyltransferase-1) in the human failing myocardium.

Duration of left ventricular assist device support: Effects on abnormal calcium cycling and functional recovery in the failing human heart.

BACKGROUND: Intracellular Ca(2+) handling is abnormal in human heart failure. Studies have demonstrated that left ventricular assist device (LVAD) support reverses phenotypic alterations, suggesting that, in select patients, LVAD support may be a bridge to recovery. Few studies have related support duration to phenotypic recovery. We hypothesized that reversal of impaired sarcoendoplasmic reticulum (SR) Ca(2+) cycling following LVAD implantation is duration-dependent. METHODS: We used post-rest potentiation to assess SR function, and Western blot analysis to measure Ca(2+)-cycling proteins. Left ventricular tissue from 10 non-failing hearts, 8 failing hearts and 10 LVAD-supported hearts was analyzed. Support ranged from 7 to 334 days. The median duration, 115 days, divided patients into short- and long-term support groups. RESULTS: Post-rest potentiation (PRP) response recovered after short-term LVAD support to a level (116.8 +/- 12.1%; n = 5) close to non-failing (123.4 +/- 12.0%; n = 10) hearts, but recovery after long-term support (23.5 +/- 7.0%; n = 5) remained closer to that of failing hearts (13.5 +/- 5.6%). We found a similar pattern of normalization for SR Ca(2+)-ATPase protein and the phospholamban/SR Ca(2+)-ATPase ratio (non-failing: 0.66 +/- 0.11; failing: 1.21 +/- 0.23; short-duration LVAD: 0.68 +/- 0.14; long-duration LVAD: 1.67 +/- 0.30; correlation p < 0.001; r = 0.93). The ratio also tended to correlate with the PRP response after unloading (p = 0.05; r = -0.65). CONCLUSIONS: SR Ca(2+) handling improved during early LVAD support, but long-term support was associated with abnormal Ca(2+) cycling. These findings cast doubt on strategies designed to wean patients after complete unloading with an LVAD.

Genomic selection of reference genes for real-time PCR in human myocardium.

BACKGROUND: Reliability of real-time PCR (RT-qPCR) data is dependent on the use of appropriate reference gene(s) for normalization. To date, no validated reference genes have been reported for normalizing gene expression in human myocardium. This study aimed to identify validated reference genes for use in gene expression studies of failed and non-failed human myocardium. METHODS: Bioinformatic analysis of published human heart gene expression arrays (195 failed hearts, 16 donor hearts) was used to identify 10 stable and abundant genes for further testing. The expression stability of these genes was investigated in 28 failed and 28 non-failed human myocardium samples by RT-qPCR using geNorm software. RESULTS: Signal recognition particle 14 kDa (SRP14), tumor protein, translationally-controlled 1 (TPT1) and eukaryotic elongation factor 1A1 (EEF1A1) were ranked the most stable genes. The commonly used reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was ranked the least stable of the genes tested. The normalization strategy was tested by comparing RT-qPCR data of both normalized and raw expression levels of brain natriuretic peptide precursor (NPPB), a gene known to be up-regulated in heart failure. Non-normalized levels of NPPB exhibited a marginally significant difference between failed and non-failed samples (p = 0.058). In contrast, normalized NPPB expression levels were significantly higher in heart-failed patients compared with controls (p = 0.023). CONCLUSION: This study used publicly available gene array data to identify a strategy for normalization involving two reference genes in combination that may have broad application for accurate and reliable normalization of RT-qPCR data in failed and non-failed human myocardium.