Abstract
Sex differences exist in the prevalence, presentation and outcomes of ischemic heart disease (IHD). Females have higher risk of heart failure post-myocardial infarction relative to males and ...are two to three times more likely to die after coronary artery bypass grafting surgery. We examined sex differences in human myocardial gene expression in response to ischemia. Left ventricular biopsies from 68 male/46 female patients undergoing aortic valve replacement surgery were obtained at baseline and after a median 74 min of cold cardioplegic arrest/ischemia. Transcriptomes were quantified by RNA-sequencing. Cell-type enrichment analysis was used to estimate the identity and relative proportions of different cell types in each sample. A sex-specific response to ischemia was observed for 271 genes. Notably, the expression FAM5C, PLA2G4E and CYP1A1 showed an increased expression in females compared to males due to ischemia and DIO3, MT1G and CMA1 showed a decreased expression in females compared to males due to ischemia. Functional annotation analysis revealed sex-specific modulation of the oxytocin signaling pathway and common pathway of fibrin clot formation. Expression quantitative trait locus (eQTL) analysis identified variant-by-sex interaction eQTLs, indicative of sex differences in the genotypic effects on gene expression. Cell-type enrichment analysis showed sex-bias in proportion of specific cell types. Common lymphoid progenitor cells and M2 macrophages were found to increase in female samples from pre- to post-ischemia, but no change was observed in male samples. These differences in response to myocardial ischemia provide insight into the sexual dimorphism of IHD and may aid in the development of sex-specific therapies that reduce myocardial injury.
During cardiac surgery with cardiopulmonary bypass, delivery of cardioplegia solution to achieve electromechanical cardiac quiescence is obligatory. The addition of lidocaine to cardioplegia has ...advantages, although its consequences at a molecular level remain unclear. We performed whole-genome RNA sequencing of the human left ventricular (LV) myocardium to elucidate the differences between whole-blood (WB) cardioplegia with and without addition of lidocaine (LC) on gene expression.
We prospectively enrolled 130 patients undergoing aortic valve replacement surgery. Patients received high-potassium blood cardioplegia either with (n = 37) or without (n = 93) lidocaine. The LV apex was biopsied at baseline, and after an average of 74 minutes of cold cardioplegic arrest. We performed differential gene expression analysis for 18,258 genes between these 2 groups. Clinical and demographic variables were adjusted in the model. Gene ontology (GO) and network enrichment analysis of the retained genes were performed using g:Profiler and Cytoscape.
A total of 1,298 genes were differentially expressed between cardioplegic treatments. Compared with the WB group, genes upregulated in the LC group were identified by network enrichment to play a protective role in ischemic injury by inhibiting apoptosis, increasing transferrin endocytosis, and increasing cell viability. Downregulated genes in the LC group were identified to play a role in inflammatory diseases, oxygen transport, and neutrophil aggregation.
The addition of lidocaine to cardioplegia had pronounced effects on a molecular level with genes responsible for decreased inflammation, reduced intracellular calcium binding, enhanced antiapoptotic protection, augmented oxygen accessibility through transferrins, and increased cell viability showing measurable differences.
Background Human mutations in the X-linked lysosome-associated membrane protein-2 (
) gene can cause a multisystem Danon disease or a primary cardiomyopathy characterized by massive hypertrophy, ...conduction system abnormalities, and malignant ventricular arrhythmias. We introduced an
mutation (denoted L2
) causing human cardiomyopathy, into mouse
gene, to elucidate its consequences on cardiomyocyte biology. This mutation results in
deletion of 41 amino acids, compatible with presence of some defective LAMP2 protein. Methods and Results Left ventricular tissues from L2
and wild-type mice had equivalent amounts of
RNA, but a significantly lower level of LAMP2 protein. By 20 weeks of age male mutant mice developed left ventricular hypertrophy which was followed by left ventricular dilatation and reduced systolic function. Cardiac electrophysiology and isolated cardiomyocyte studies demonstrated ventricular arrhythmia, conduction disturbances, abnormal calcium transients and increased sensitivity to catecholamines. Myocardial fibrosis was strikingly increased in 40-week-old L2
mice, recapitulating findings of human LAMP2 cardiomyopathy. Immunofluorescence and transmission electron microscopy identified mislocalization of lysosomes and accumulation of autophagosomes between sarcomeres, causing profound morphological changes disrupting the cellular ultrastructure. Transcription profile and protein expression analyses of L2
hearts showed significantly increased expression of genes encoding activators and protein components of autophagy, hypertrophy, and apoptosis. Conclusions We suggest that impaired autophagy results in cardiac hypertrophy and profound transcriptional reactions that impacted metabolism, calcium homeostasis, and cell survival. These responses define the molecular pathways that underlie the pathology and aberrant electrophysiology in cardiomyopathy of Danon disease.
Omentin-1, also known as Intelectin-1 (ITLN1), is an adipokine with plasma levels associated with diabetes, obesity, and coronary artery disease. Recent studies suggest that ITLN1 can mitigate ...myocardial ischemic injury but the expression of ITLN1 in the heart itself has not been well characterized. The purpose of this study is to discern the relationship between the expression pattern of ITLN1 RNA in the human heart and the level of circulating ITLN1 protein in plasma from the same patients following myocardial ischemia.
A large cohort of patients (n = 140) undergoing elective cardiac surgery for aortic valve replacement were enrolled in this study. Plasma and left ventricular biopsy samples were taken at the beginning of cardiopulmonary bypass and after an average of 82 min of ischemic cross clamp time. The localization of ITLN1 in epicardial adipose tissue (EAT) was also further characterized with immunoassays and cell fate transition studies.
mRNA expression of ITLN1 decreases in left ventricular tissue after acute ischemia in human patients (mean difference 280.48, p = 0.001) whereas plasma protein levels of ITLN1 increase (mean difference 5.24, p < 0.001). Immunohistochemistry localized ITLN1 to the mesothelium or visceral pericardium of EAT. Epithelial to mesenchymal transition in mesothelial cells leads to a downregulation of ITLN1 expression.
Myocardial injury leads to a decrease in ITLN1 expression in the heart and a corresponding increase in plasma levels. These changes may in part be due to an epithelial to mesenchymal transition of the cells that express ITLN1 following ischemia. Trial Registration Clinicaltrials.gov ID: NCT00985049.
Eighty percent of patients with a diagnosis of tetralogy of Fallot (TOF) do not have a known genetic etiology or syndrome. We sought to identify key molecular pathways and biological processes that ...are enriched in non-syndromic TOF, the most common form of cyanotic congenital heart disease, rather than single driver genes to elucidate the pathogenesis of this disease.
We undertook exome sequencing of 362 probands with non-syndromic TOF and their parents within the Pediatric Cardiac Genomics Consortium (PCGC). We identified rare (minor allele frequency <1 × 10
4),
variants to ascertain pathways and processes affected in this population to better understand TOF pathogenesis. Pathways and biological processes enriched in the PCGC TOF cohort were compared to 317 controls without heart defects (and their parents) from the Simons Foundation Autism Research Initiative (SFARI).
A total of 120 variants in 117 genes were identified as most likely to be deleterious, with
,
,
, and
identified in two probands each. Gene ontology analyses of these variants using multiple bioinformatic tools demonstrated significant enrichment in processes including cell cycle progression, chromatin remodeling, myocyte contraction and calcium transport, and development of the ventricular septum and ventricle. There was also a significant enrichment of target genes of
, which is critical in second heart field development and whose loss results in membranous ventricular septal defects related to disruption of the proximal outlet septum. None of these processes was significantly enriched in the SFARI control cohort.
Innate molecular defects in cardiac progenitor cells and genes related to their viability and contractile function appear central to non-syndromic TOF pathogenesis. Future research utilizing our results is likely to have significant implications in stratification of TOF patients and delivery of personalized clinical care.
BACKGROUND:The exact mechanisms that underlie the pathological processes of myocardial ischemia in humans are unclear. Cardiopulmonary bypass with cardioplegic arrest allows the authors to examine ...the whole transcriptional profile of human left ventricular myocardium at baseline and after exposure to cold cardioplegia-induced ischemia as a human ischemia model.
METHODS:The authors obtained biopsies from 45 patients undergoing aortic valve replacement surgery at baseline and after an average of 79 min of cold cardioplegic arrest. Samples were RNA sequenced and analyzed with the Partek Genomics Suite (Partek Inc., St. Louis, MO) for differential expression. Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA) and Biobase ExPlain (Biobase GmbH, Wolfenbuettel, Germany) systems were used for functional and pathway analyses.
RESULTS:Of the 4,098 genes with a mean expression value greater than 5, 90% were down-regulated and 9.1% were up-regulated. Of those, 1,241 were significantly differentially expressed. Gene ontology analysis revealed significant down-regulation in immune inflammatory response and complement activation categories and highly consistent was the down-regulation of intelectin 1, proteoglycan, and secretory leukocyte peptidase inhibitor. Up-regulated genes of interest were FBJ murine osteosarcoma viral oncogene homolog and the hemoglobin genes hemoglobin α1 (HBA1) and hemoglobin β. In addition, analysis of transcription factor–binding sites revealed interesting targets in factors regulating reactive oxygen species production, apoptosis, immunity, cytokine production, and inflammatory response.
CONCLUSIONS:The authors have shown that the human left ventricle exhibits significant changes in gene expression in response to cold cardioplegia-induced ischemia during cardiopulmonary bypass, which provides great insight into the pathophysiology of ventricular ischemia, and thus, may help guide efforts to reduce myocardial damage during surgery.
The discovery of functional classes of long noncoding RNAs (lncRNAs) has expanded our understanding of the variety of RNA species that exist in cells. In the heart, lncRNAs have been implicated in ...the regulation of development, ischemic and dilated cardiomyopathy, and myocardial infarction. Nevertheless, there is a limited description of expression profiles for these transcripts in human subjects.
We obtained left ventricular tissue from human patients undergoing cardiac surgery and used RNA sequencing to describe an lncRNA profile. We then identified a list of lncRNAs that were differentially expressed between pairs of samples before and after the ischemic insult of cardiopulmonary bypass. The expression of some of these lncRNAs correlates with ischemic time. Coding genes in close proximity to differentially expressed lncRNAs and coding genes that have coordinated expression with these lncRNAs are enriched in functional categories related to myocardial infarction, including heart function, metabolism, the stress response, and the immune system.
We describe a list of lncRNAs that are differentially expressed after ischemia in the human heart. These genes are predicted to function in pathways consistent with myocardial injury. As a result, lncRNAs may serve as novel diagnostic and therapeutic targets for ischemic heart disease.
URL: http://www.clinicaltrials.gov. Unique identifier: NCT00985049.
Arrhythmia Prevention in CPVT. Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmic syndrome caused by mutations in genes encoding the calcium‐regulation ...proteins cardiac ryanodine receptor (RyR2) or calsequestrin‐2 (CASQ2). Mechanistic studies indicate that CPVT is mediated by diastolic Ca2+ overload and increased Ca2+ leak through the RyR2 channel, implying that treatment targeting these defects might be efficacious in CPVT.
Method and results: CPVT mouse models that lack CASQ2 were treated with Ca2+‐channel inhibitors, β‐adrenergic inhibitors, or Mg2+. Treatment effects on ventricular arrhythmia, sarcoplasmic reticulum (SR) protein expression and Ca2+ transients of isolated myocytes were assessed. Each study agent reduced the frequency of stress‐induced ventricular arrhythmia in mutant mice. The Ca2+ channel blocker verapamil was most efficacious and completely prevented arrhythmia in 85% of mice. Verapamil significantly increased the SR Ca2+ content in mutant myocytes, diminished diastolic Ca2+ overload, increased systolic Ca2+ amplitude, and prevented Ca2+ oscillations in stressed mutant myocytes.
Conclusions: Ca2+ channel inhibition by verapamil rectified abnormal calcium handling in CPVT myocytes and prevented ventricular arrhythmias. Verapamil‐induced partial normalization of SR Ca2+ content in mutant myocytes implicates CASQ2 as modulator of RyR2 activity, rather than or in addition to, Ca2+ buffer protein. Agents such as verapamil that attenuate cardiomyocyte calcium overload are appropriate for assessing clinical efficacy in human CPVT. (J Cardiovasc Electrophysiol, Vol. 22, pp. 316‐324, March 2011)
Mutations in the human gene encoding the nucleotide-binding region in the γ-subunit of AMP-activated protein kinase (AMPK) cause cardiomyopathy with preexcitation syndrome. Mutant AMPK showed reduced ...binding affinity to nucleotides in vitro raising the possibility that altered regulation of AMPK activity by AMP/ATP could contribute to the disease phenotype. In this study, we determined the sensitivity of AMPK activity to AMP/ATP in the beating hearts using transgenic mice expressing a mutant (N488I, γ2-mutant) or wild-type γ2-subunit (γ2-TG). The ATP and AMP were unaltered in all hearts but the AMPK activity was increased by 2.5-fold in γ2-mutant hearts freeze-clamped at normal AMP/ATP compared with nontransgenic (WT) or γ2-TG. The increased basal AMPK activity was caused by increased Thr-172 phosphorylation of the α-subunit (p-AMPK, by 4-fold) at normal ATP and was not changed by reducing glycogen content by 60% in the γ2-mutant hearts. A reversal of AMP/ATP, caused by ATP degradation, increased p-AMPK by 7-fold in WT but caused no change in γ2-mutant hearts. These results demonstrate that the mutation renders AMPK insensitive to the inhibitory and stimulatory effects of the regulatory nucleotides ATP and AMP, respectively, suggesting that the pathogenesis of the human disease may not be attributable to a simple loss- or gain-of-function.
Empirical evidence supporting a genetic basis for the etiology of congenital heart disease (CHD) is limited and few disease-causing mutations have been identified. To identify novel CHD genes, we ...performed a forward genetic screen to identify mutant mouse lines with heritable CHD. Lines with recessive N-ethyl-N-nitrsourea-induced CHD-causing mutations were identified using a three-generation backcross. A hierarchical screening protocol was used to test the hypothesis that the fetal-to-neonatal circulatory transition unmasks the specific structural heart defects observed in CHD. Mice with heart defects were efficiently ascertained by selecting for pups exhibiting perinatal lethality and characterizing their cardiac pathology. A marked increase of perinatal lethality was observed in the mutagen-treated cohort compared with an untreated backcross population. Cardiac pathology on perinatal lethals revealed cardiovascular defects in 79 pups from 47 of 321 mutagenized lines. All identified structural abnormalities were analogous to previously described forms of human CHD. Furthermore, the phenotypic recurrence and variance patterns across all lines were similar to human CHD prevalence and recurrence patterns. We mapped the locus responsible for heritable atrioventricular septal defects in six lines (avc1-6). Our screen demonstrated that ‘sporadic’ CHD may have major genetic component and established a practical, efficient approach for identifying CHD candidate genes.
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