Cigarette smoking is an important etiology for cardiac diseases and electronic cigarettes are increasing in popularity. In the current study, we assessed physiological and transcriptional changes in human induced pluripotent stem cell derived cardiomyocytes (iPS-CMs) after exposure to electronic cigarette extract (ECE) and conventional cigarette extract (CSE). iPS-CMS were plated on multi-electrode array plates (MEA), electrical and contractile properties were determined by Maestro Edge and computer assisted video analysis (MuscleMotion). Transcriptional profiles were accessed by direct digital mRNA detection of heart failure targeted genes. Both ECE and CSE extracts significantly prolong beat period from baseline compared to control after 2 days of exposure (0.74 ± 0.14 s for ECE and 0.78 ± 0.02 s for CSE). Prolonged time to peak in ECE and CSE were also significantly prolonged compared to control with a P < 0.05 (487 ± 44, 373 ± 21 and 312 ± 8 ms respectively). In addition, both ECE and CSE decrease cardiomyocyte contractility in a similar fashion. ECE exposed cells have 25 genes in discovery and 33 genes in validation experiments that were significantly different compared to control with P < 0.05. CSE exposed cells, however, do produce more profound effects on transcriptional profiles of heart failure with 53 genes in discovery and 40 genes in validation compared to control group with P < 0.05. In summary, both ECE and CSE are sufficiently toxic to impair cardiac physiology and alter myocyte gene expression. Transcriptional profiles of heart failure related genes are consistent with cigarettes altering expression of sarcomeric proteins and calcium handling. Human cardiomyocytes can be utilized to study the molecular biology and toxicology of electronic and conventional cigarettes.

Purpose: Dilated cardiomyopathy (DCM) is characterized by transcriptional changes. Clinical recovery after LVAD implant is highly variable and transcriptional changes often persist. Our goal was to identify micro-RNAs (miRNAs) associated with heart failure (HF) and to evaluate responses to LVAD therapy to better understand epigenetic mechanisms regulating differential gene expression (DGE).

Introduction: Micro RNAs (miRNA) are crucial regulators of various biological processes including the pathogenesis of heart failure (HF). In this study, miRNA profiling was compared with pre and post LVAD and non-failing (NF) control heart patients. Our goal was to identify miRNAs involved in HF and responses to LVAD therapy. Methods: Myocardial RNAs were isolated from non-ischemic dilated cardiomyopathy patients (n=22) who went through LVAD placement (pre LVAD) and later transplant (post LVAD) along with 6 normal hearts as controls. NanoString technology was used to quantify miRNA expressions. NanoString miRNAs raw count data were preprocessed and normalized using solver 4.0 Analysis Software. Ingenuity Pathway Analysis (IPA) was done by differentially expressed miRNA to understand the biological pathways involved. Results: Our sample population exhibited 80% male and 95% Caucasian with an average age of 57 and ejection fraction was less than 25% at the time of LVAD placement. The average LVAD duration of patients was 525 days. One ninety-five miRNAs in pre and 120 miRNAs in post LVAD patients were differentially expressed compared to control using cutoff of for the FDR adjusted p-value 0.05 and at least 2-fold changes. There was no significant change in miRNA expression when post LVAD was compared with pre LVAD patients. IPA analysis revealed that there are 15 up-regulated miRNA that promote apoptosis with a net Z score of 2.155 in post LVAD versus control. There are 27 miRNAs with a net of apoptotic with a Z score 0f 0.766 in pre LVAD versus control. It was also noticed from IPA analysis that expression of 8 identical miRNAs in both pre and post LVAD patients were involved in heart fibrosis. LVAD therapy did not change the miRNA expression Conclusions: Our results identify many miRNAs that are associated with apoptosis and fibrosis in end stage heart failure. Molecular mechanisms of heart disease will be identified by miRNA profiling and function as potential biomarkers or therapeutic targets in future. LVAD therapy does not substantially reverse these epigenetic changes in this population.

2012 to 2020.Patients were managed in accordance with their multidisciplinary team who evaluated fever (defined as temperature > 100.4 F) as clinically indicated.Pre-and post-implantation fever curve and infectious work-up, when performed, were reviewed.Summary statistics were reported.Results: A total of 49 patients underwent Impella implantation.The median age was 66 (56-71), and 44/49 (90%) were male.Baseline patient information, including indication for Impella support and clinical outcomes, is summarized in Table 1.Following Impella implantation, 21/49 (43%) patients had a documented fever.Of these, 3/21 (14%) had preimplantation fever with a known source of infection, none of whom had new infections post-implantation.Of the remaining patients with a postimplantation fever, only 1/18 (5.5%) had an identifiable infectious etiology for their fever.An additional 2/18 (11%) patients had positive cultures (1 blood, 1 BAL), but these were clinically managed as contaminants.Conclusion: Although fever after Impella implantation was common, an infectious etiology for a post-implantation fever was rarely identified.Therefore, Impella-related fever should not be considered a barrier to advanced heart failure therapies including transplant or LVAD.Further investigation of post Impella fever is warranted to determine the source of this phenomena.

Introduction: Cigarette smoking is an important etiology for cardiac diseases and electronic cigarettes are increasing in popularity. In the current study, we sought to assess differential gene expression in iPS-cardiomyocytes after exposure to conventional cigarette smoke extract (CSE) and electronic cigarette extracts (ECE) utilizing direct digital mRNA detection of heart failure targeted genes. Hypothesis: Methods: Human cardiomyocytes derived from iPSCs were acutely exposed for two consecutive days with 2.5% of CSE and ECE. RNAs were isolated and NanoString analysis was used to compare gene expression between groups for 126 heart failure specific genes. NanoString mRNA raw count data were preprocessed and normalized using nSolver 4.0 Analysis SoftwareNano-string of customized target panel was run for mRNA expression of cardiomyocytes with direct exposure to CSE (N=3) and ECE (N=3) and was compared to control (N=3). At least a 1.5-fold change in gene expression with FDR adjusted p<0.05 was considered as significant. Results: Five genes were significantly differentially expressed cardiomyocytes exposed to CSE vs control (CETP, ATRLN1, ENOS, C10ORF88 and ERF). In ECE exposed cells, five genes were also significantly differentially expressed compared to control (PDK4, CAV3, PER1, MYH6 and ERF). ERF gene is a transcription factor and protooncogene involved in development, apoptosis, and the regulation of telomerase was significantly overexpressed in cells exposed to both CSE and ECE. Despite different nicotine concentrations in ECE and CSE, no significant gene differences between CSE and ECE were observed. Conclusions: ECE and CSE exposure alters transcriptional profiles in iPS-cardiomyocytes compared to controls in heart failure targeted genes. These genes are involved in transcription and circadian regulation, metabolism and contractile proteins. There were no significant differences in transcriptional profiles between ECE and CSE exposed cells.

Introduction: Mice carrying a homozygous frameshift mutation of the class I major histocompatibility antigen H2-K1 (H2-Kbm1) exhibit impaired development of hypothalamic orexinergic neurons. Similar to mice lacking the hypocretin (orexin) 2 receptor, H2-Kbm1 mice develop dilated cardiomyopathy and systolic heart failure (HF) from an early age. Previous studies suggest response to orexinergic signaling is predictive of patient response to current HF pharmacotherapeutic regimes. Our goal was to characterize the myocardial gene expression profiles of H2-Kbm1 mice. Methods: RNA was isolated from mice hearts (wild type n=5, mutant n=7) via the Qiagen RNeasy Mini Kit. DNase treatment was done to limit contamination of genomic DNA. Gene expression was measured using Illumina nexteq500 and 550 sequencing systems from a DNA library generated via the NuGene library kit (10 ng RNA). RNA-seq data analyses including comparisons, volcano and heatmap plots were completed. Results: H2-Kbm1 mice differentially express 398 myocardial genes compared to control mice. Ingenuity pathway analysis indicates that these genes are involved in necrosis of cardiac muscle (z-score -1.8), cell death (z-score 1.6), and cardiac contractility (z-score -4). Upstream signaling analysis suggest regulation by Mir-122 (z-score 2.4), DNMT3 A and B (z-score 3.3 and 2.5), VEGF (z-score 4.6) and rosiglitazone (z-score -6.8). Canonical pathway analysis is shown in Figure 1. Conclusion: H2-Kbm1 mice develop heart failure with marked myocardial transcriptional abnormalities that lead to impaired cardiac contraction and cell death. Differentially expressed genes are predominantly involved in mitochondrial function, oxidative phosphorylation, sirtuin signaling and the TCA cycle. Regulation of these genes is associated with miR-122, DNA methylations by DNMT3 A & B, VEGF and rosiglitazone signaling. The orexin pathway is a potential target for heart failure therapeutics.

Introduction: Recovery from end-stage heart failure (HF) with left ventricular assist device (LVAD) therapy remains uncommon. In this study we compared targeted gene expression profiles of pre LVAD, post LVAD and non-failing (NF) hearts utilizing direct mRNA detection of HF specific genes. Our goal was to identify molecular mechanisms involved in HF and responses to LVAD therapy. Method: Myocardial RNAs were isolated from non-ischemic dilated cardiomyopathy patients at LVAD placement and during transplant (n=10) along with NF controls (n=6). NanoString analysis was used to compare gene expression between groups for 126 heart failure specific genes. NanoString mRNA raw count data were preprocessed and normalized using nSolver 4.0 Analysis Software. At least a 2 fold change in gene expression with a FDR adjusted p<0.05 was considered as significant. Ingenuity Pathway Analysis (IPA) was completed to understand the biological pathways involved. Results: Our sample population was 90% male, average age of 56 and 100% Caucasian. In the HF populations average ejection fraction was low <25% and average NYHA was 3.5. In HF, expressions of contractile muscle (MYH6), ion channels protein (KCNIP2) and Ca ATPase (SERCA2) were significantly down regulated when compared to NF controls. Natriuretic peptides (NPPA, NPPB), troponin 1 (TNNI3), nuclear kinases (WEE) and metabolic genes (ATRNL1) were upregulated compared to NF hearts. These differential changes in gene expression persisted post LVAD therapy. There were no significant changes in gene expression between pre and post LVAD HF patients. Ingenuity pathway analysis revealed that these changes in gene expression predispose to arrhythmias. Conclusion: Transcriptional changes in HF specific genes persist in many patients post LVAD therapy contributing to incomplete recovery of end stage heart failure and ongoing risk of arrhythmias. These genes are involved in contractile function, calcium handling and ion channels. Direct digital mRNA detection allows for serial analysis of targeted HF specific genes without reverse transcription and amplification of resultant cDNA. New therapies are needed to reverse adverse molecular changes and improve outcomes in heart failure patients who receive LVADs.

3.8 §0.6 mm, p=0.02).Additionally, compared to sham, both LV enddiastolic and end-systolic volumes were increased in the chronic TAC group (Sham: 57 §11 uL v 8-week: 91 §419 uL, p=0.02;Sham: 17 §5 uL v 8-week: 64 §23 uL, p=0.007).Compared to sham, minimum LV pressure was elevated in the chronic TAC cohort (Sham: -4.5 §1.3 mmHg v 8-week: 8.8 §10.3 mmHg, p= 0.04).BMP-10 mRNA expression was elevated in the right atrium, but reduced in the ventricles, at 8 weeks of TAC, while BMP-10 protein expression was increased at 8 weeks of TAC (Figure 1A-B).BMP-9 expression was reduced in the chronic TAC cohort in the left ventricle while endoglin levels were unchanged (Figure 1C).Conclusion: BMP-10 mRNA levels are downregulated, while protein levels are upregulated, in the LV at 8 weeks of TAC.Further studies are needed to fully explore the functional role of BMP-10 in the heart.

Introduction: Diabetes and heart failure are very closely associated. Diabetes patients have an increased risk of developing heart failure and heart failure patients are at higher risk of developing diabetes. Changes occur in myocardial glucose metabolism in end stage heart failure patients and newer therapies for diabetes such as SGLTi improve heart failure outcomes. Our objective was to identify transcriptional changes associated with diabetes in heart failure patients. Methods: Transcriptional profiles were compared in heart tissues obtained at the time of LVAD implant among diabetic heart failure (HF) (n=16), non-diabetic (n=10) HF patients and control (n=3) non failing hearts. RNASeq analysis utilized a custom-targeted panel for 140 genes on the Ion Torrent Personal Genome Machine. Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser SuiteTM. The Bejamini-Hochberg method was used to control the false discovery rate to be no more than 0.05. Ingenuity Pathway Analysis (IPA) was also performed to understand the biological pathways involved. Results: Our sample population was 82% male with an average age of 51 and 84% Caucasian, 14% African American and 2% Asian. Average ejection fraction was quite low <25% and average NYHA was 3.5 in the HF populations. Expressions of contractile muscle and ion channels proteins decreased significantly between HF patients and control. Increased expression of chemokine ligand 4 (CCL4), chemokine ligand 2 (CCL2) and collagen type 1 alpha1 (COL1A1) were observed in diabetic HF compared to non-diabetic HF patients. IPA analysis with log fold change between HF and control revealed activation of both methylation genes DNMT3A and B, necrosis of cardiac muscle and death of cardiomyocytes with 2.0 Z score. Conclusions: Diabetic heart failure patients have increased expression of chemokines and extracellular matrix genes, which may contribute to disease progression. Up regulation of CCL4 and CCL2 in diabetic HF patients may cause additional risk of heart diseases through inflammatory pathways and increased expression of collagen may contribute to myocardial fibrosis. Down regulation of contractile proteins, and ion channels were similar in diabetic and non-diabetic HF patients.

Background Myocardial recovery with Left ventricular assistant device (LVAD) therapy is dichotomous with some patients obtaining remission from end-stage heart failure whereas most require transplantation or remain on pump support long term. Our goal was to determine transcriptional and free radical responses to LVAD treatment. Methods Tissues were collected from patients before and after LVAD placement in non-ischemic dilated cardiomyopathy patients ( n = 14) along with controls ( n = 3). RNA sequencing (RNASeq) analysis quantified transcriptional profiles by using a custom targeted panel of heart failure related genes on the PGM sequencer. The differential expression analysis between groups was conducted using edgeR (Empirical analysis of digital gene expression data in R) package in Bioconductor. Ingenuity Pathway Analysis (IPA) was carried out on differentially expressed genes to understand the biological pathways involved. Electron Paramagnetic Resonance (EPR) Spectroscopy was utilized to measure levels of free radicals in whole blood collected pre- and post-LVAD implantation ( n = 16). Results Thirty-five genes were differentially expressed in pre-LVAD failing hearts compared to controls. In response to LVAD therapy, only Pyruvate dehydrogenase kinase 4 (PDK4) and period circadian protein homolog 1 ( PER1) were altered with 34 heart failure related genes still differentially expressed post-LVAD compared to controls. IPA showed that DNA methylation-related genes were upregulated in both pre- and post-LVAD and was persistent with a Z-score of 2.00 and 2.36 for DNA Methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B), respectively. Inhibition of micro RNA21 (mir21) was also significant on pathway analysis in the post-LVAD population with a Z-score of − 2.00. Levels of free radicals in blood of pre- and post-LVAD patients did not change significantly. Conclusion LVAD therapy does not reverse many of the transcriptional changes associated with heart failure. Persistent changes in gene expression may be related to ongoing oxidative stress, continued DNA methylation, or changes in metabolism. PDK4 is a key regulator of glucose metabolism and its increased expression by LVAD therapy inhibited pyruvate metabolism.

Abstract Background Cigarette smoking is an important risk factor for cardiac diseases. In the current study, we sought to assess the effect of electronic cigarette extract (ECE) and conventional cigarette smoke extract (CSE) on cardiomyocytes. Methods iPSCs-derived cardiomyocytes were used in the study to evaluate cellular toxicities. Cells were exposed to either ECE or CSE for two consecutive days as an acute exposure or every other day for 14 days. Concentration of nicotine in both ECE and CSE were measured by Mass-Spectrometry and Q-Exactive-HF was used to identify other ingredients in both extracts. Fluorescent microscopy was used to measure the oxidative stress after ECE and CSE exposure. Motility and beat frequency of cardiomyocytes were determined using the Sisson-Ammons Video Analysis system. Heart failure target panel genes of exposed cardiomyocytes were compared to control unexposed cells. Results Despite nicotine concentration in CSE being six-fold higher than ECE (50 μg in CSE and 8 μg in ECE), ECE had similar toxic effect on cardiomyocytes. Both CSE and ECE generate significant cellular reactive oxygen species. The Sisson-Ammons Video Analysis (SAVA) analysis showed significant changes in myocyte function with both CSE and ECE slowing beating and increasing cell death. Chronic exposure of both ECE and CSE significantly decreased cardiomyocytes viability long term at all doses. Target panel gene expression profiles of both ECE and CSE exposed cardiomyocytes were different from controls with distinct pattern of genes that involved cell proliferation, inflammation, and apoptosis. Conclusion ECE and CSE produce similar cardiomyocyte toxicities which include generating oxidative stress, negative chronotropic effects, adverse changes in myocardial gene expression and ultimately cell death.

Cigarette smoking is an important etiology for cardiac diseases. In the current study, we sought to assess the effect of electronic cigarette extract (ECE) and cigarette smoke extract (CSE) on cardiomyocyte gene expression and function. Human cardiomyocytes derived from iPSCs were used. Acute and

Purpose Myocardial recovery with LVAD therapy is challenging and unpredictable. Next generation sequencing technologies allow for quick, quantitative, and large scale measurements of gene expression in small amounts of tissue. Our primary goal was identify transcriptional changes in response to LVAD therapy. Methods Tissues were collected from patients before and after LVAD placement (n=14) along with controls (n=3). RNASeq analysis was done by using a custom targeted panel on the PGM sequencer. The RNA-sequencing data was filtered to keep the genes with nonzero reads for at least 13 samples per condition. The expression of 140 genes including cardiac, inflammatory and clock genes were analyzed in with non-ischemic dilated cardiomyopathies. The differential expression analysis was conducted using edgeR (Empirical analysis of digital gene expression data in R) package in Bioconductor. The Bejamini-Hochberg method was used to control the false discovery rate (FDR) to be no more than 0.05. Results After filtering there were 122 genes available for analysis. Differential gene expressions were determined between pre- and post- receiving LVAD after adjusting for the patient effect. PDK4 and PER1 were significantly higher post LVAD compared to pre LVAD tissues. Compared to non-failing control tissues, 25 genes were differentially expressed pre-LVAD. These genes included, MYH6, ABCC9, PLEKHA3, ATRLN1, GLS, UBE2B, CCL4 and WEE1. Conclusion PDK4 and PER1 expressions were significantly higher post LVAD. Pyruvate dehydrogenase kinase 4 (PDK4) is a key regulator of glucose metabolism. PER1 is a clock gene exhibiting rhythmic expression in human hearts. It has been shown that PER1 is involved in BP control and regulates renal sodium transport genes. Recent literature suggests that LVAD therapy improves hemoglobin A1C (HbA1c), fasting plasma glucose, and daily insulin requirements. Glucose metabolism and clock proteins are altered with LVAD therapy but numerous other transcriptional changes persist. Myocardial recovery with LVAD therapy is challenging and unpredictable. Next generation sequencing technologies allow for quick, quantitative, and large scale measurements of gene expression in small amounts of tissue. Our primary goal was identify transcriptional changes in response to LVAD therapy. Tissues were collected from patients before and after LVAD placement (n=14) along with controls (n=3). RNASeq analysis was done by using a custom targeted panel on the PGM sequencer. The RNA-sequencing data was filtered to keep the genes with nonzero reads for at least 13 samples per condition. The expression of 140 genes including cardiac, inflammatory and clock genes were analyzed in with non-ischemic dilated cardiomyopathies. The differential expression analysis was conducted using edgeR (Empirical analysis of digital gene expression data in R) package in Bioconductor. The Bejamini-Hochberg method was used to control the false discovery rate (FDR) to be no more than 0.05. After filtering there were 122 genes available for analysis. Differential gene expressions were determined between pre- and post- receiving LVAD after adjusting for the patient effect. PDK4 and PER1 were significantly higher post LVAD compared to pre LVAD tissues. Compared to non-failing control tissues, 25 genes were differentially expressed pre-LVAD. These genes included, MYH6, ABCC9, PLEKHA3, ATRLN1, GLS, UBE2B, CCL4 and WEE1. PDK4 and PER1 expressions were significantly higher post LVAD. Pyruvate dehydrogenase kinase 4 (PDK4) is a key regulator of glucose metabolism. PER1 is a clock gene exhibiting rhythmic expression in human hearts. It has been shown that PER1 is involved in BP control and regulates renal sodium transport genes. Recent literature suggests that LVAD therapy improves hemoglobin A1C (HbA1c), fasting plasma glucose, and daily insulin requirements. Glucose metabolism and clock proteins are altered with LVAD therapy but numerous other transcriptional changes persist.

PURPOSE:The mechanisms for persistent and progressive loss of myocardial function in advanced heart failure (HF) remain incompletely characterized. In the current study, we sought to determine the impact of TGF-β on fibroblasts transcriptional profiles and assess if exosomes from TGF-β treated fibroblasts could induce a heart failure phenotype in co-cultured cardiomyocytes.METHOD:Normal heart fibroblasts were treated with TGF-β with a final conc. of 2.5 ng/ml in serum free media. HF fibroblasts were also obtained from patients undergoing implantation of left ventricular assist devices. Exosomes were collected using three-step ultracentrifugation. Cardiomyocytes were co-cultured with exosomes from TGF-β-treated, HF and control fibroblasts. RNA was extracted from the fibroblasts, exosomes, and the cardiomyocytes for a targeted panel of genes using Ion AmpliSeq. Fibroblast function was evaluated by collagen gel contraction.RESULTS:Fibroblasts treated with TGF-β differentially express 21 of the 140 genes in our targeted panel. These fibroblasts exhibit enhanced collagen gel contraction similar to HF fibroblasts. Fifty of these targeted genes were also differentially expressed in fibroblast exosomes. Pathway analysis of these transcriptional changes suggest hypertrophic signaling to cardiac muscle. Cardiomyocytes, co-cultured with exosomes from TGF- β treated fibroblasts or heart failure patients, differentially expressed 40 genes compared to controls. Cardiomyocytes co-cultured with exosomes of TGF-β treated fibroblasts induced a molecular phenotype similar to cardiomyocytes co-cultured with exosomes from HF fibroblasts. These changes involve contractile proteins, adrenergic receptors, calcium signaling, metabolism and cell renewal.CONCLUSION:TGF-β induces broad transcriptional changes in fibroblasts as well as their exosomes. These exosomes induce a heart failure phenotype in cardiomyocytes. Exosome signaling from fibroblasts likely contributes to disease progression in heart failure.

The mechanisms for persistent and progressive loss of myocardial function in advanced heart failure remain unknown. In the current study, we sought to assess the role played by TGF-β driven exosome-mediated signaling to cardiac myocytes in HF patients.