Kai-Chien Yang

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Background— Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). Methods and Results— Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. Conclusions— The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Exenatide improves glucose homeostasis and prolongs survival in a murine model of dilated cardiomyopathy

Background There is growing awareness of secondary insulin resistance and alterations in myocardial glucose utilization in congestive heart failure. Whether therapies that directly target these changes would be beneficial is unclear. We previously demonstrated that acute blockade of the insulin responsive facilitative glucose transporter GLUT4 precipitates acute decompensated heart failure in mice with advanced dilated cardiomyopathy. Our current objective was to determine whether pharmacologic enhancement of insulin sensitivity and myocardial glucose uptake preserves cardiac function and survival in the setting of primary heart failure. Methodology/Principal Findings The GLP-1 agonist exenatide was administered twice daily to a murine model of dilated cardiomyopathy (TG9) starting at 56 days of life. TG9 mice develop congestive heart failure and secondary insulin resistance in a highly predictable manner with death by 12 weeks of age. Glucose homeostasis was assessed by measuring glucose tolerance at 8 and 10 weeks and tissue 2-deoxyglucose uptake at 75 days. Exenatide treatment improved glucose tolerance, myocardial GLUT4 expression and 2-deoxyglucose uptake, cardiac contractility, and survival over control vehicle-treated TG9 mice. Phosphorylation of AMP kinase and AKT was also increased in exenatide-treated animals. Total myocardial GLUT1 levels were not different between groups. Exenatide also abrogated the detrimental effect of the GLUT4 antagonist ritonavir on survival in TG9 mice. Conclusion/Significance In heart failure secondary insulin resistance is maladaptive and myocardial glucose uptake is suboptimal. An incretin-based therapy, which addresses these changes, appears beneficial.

Homeostatic regulation of electrical excitability in physiological cardiac hypertrophy

Pathological biomechanical stresses cause cardiac hypertrophy, which is associated with QT prolongation and arrhythmias. Previous studies have demonstrated that repolarizing K+ current densities are decreased in pressure overload‐induced left ventricular hypertrophy, resulting in action potential and QT prolongation. Cardiac hypertrophy also occurs with exercise training, but this physiological hypertrophy is not associated with electrical abnormalities or increased arrhythmia risk, suggesting that repolarizing K+ currents are upregulated, in parallel with the increase in myocyte size, to maintain normal cardiac function. To explore this hypothesis directly, electrophysiological recordings were obtained from ventricular myocytes isolated from two mouse models of physiological hypertrophy, one produced by swim‐training of wild‐type mice and the other by cardiac‐specific expression of constitutively active phosphoinositide‐3‐kinase‐p110α (caPI3Kα). Whole‐cell voltage‐clamp recordings revealed that repolarizing K+ current amplitudes were higher in ventricular myocytes isolated from swim‐trained and caPI3Kα, compared with wild‐type, animals. The increases in K+ current amplitudes paralleled the observed cellular hypertrophy, resulting in normalized or increased K+ current densities. Electrocardiographic parameters, including QT intervals, as well as ventricular action potential waveforms in swim‐trained animals/myocytes were indistinguishable from controls, demonstrating preserved electrical function. Additional experiments revealed that inward Ca2+ current amplitudes/densities were also increased in caPI3Kα, compared with WT, left ventricular myocytes. The expression of transcripts encoding K+, Ca2+ and other ion channel subunits was increased in swim‐trained and caPI3Kα ventricles, in parallel with the increase in myocyte size and with the global increases in total cellular RNA expression. In contrast to pathological hypertrophy, therefore, the functional expression of repolarizing K+ (and depolarizing Ca2+) channels is increased with physiological hypertrophy, reflecting upregulation of the underlying ion channel subunit transcripts and resulting in increased current amplitudes and the normalization of current densities and action potential waveforms. Taken together, these results suggest that activation of PI3Kα signalling preserves normal myocardial electrical functioning and could be protective against the increased risk of arrhythmias and sudden death that are prevalent in pathological cardiac hypertrophy.

Mechanisms of Sudden Cardiac Death Oxidants and Metabolism

Ventricular arrhythmia is the leading cause of sudden cardiac death (SCD). Deranged cardiac metabolism and abnormal redox state during cardiac diseases foment arrhythmogenic substrates through direct or indirect modulation of cardiac ion channel/transporter function. This review presents current evidence on the mechanisms linking metabolic derangement and excessive oxidative stress to ion channel/transporter dysfunction that predisposes to ventricular arrhythmias and SCD. Because conventional antiarrhythmic agents aiming at ion channels have proven challenging to use, targeting arrhythmogenic metabolic changes and redox imbalance may provide novel therapeutics to treat or prevent life-threatening arrhythmias and SCD.

Combined Deep microRNA and mRNA Sequencing Identifies Protective Transcriptomal Signature of Enhanced PI3Kα Signaling in Cardiac Hypertrophy

The perturbation of myocardial transcriptome homeostasis is the hallmark of pathological hypertrophy, underlying the maladaptive myocardial remodeling secondary to pathological stresses. Classic and novel therapeutics that provide beneficial effects against pathological remodeling likely impact myocardial transcriptome architecture, including miRNA and mRNA expression profiles. Microarray and PCR-based technologies, although employed extensively, cannot provide adequate sequence coverage or quantitative accuracy to test this hypothesis directly. The goal of this study was to develop and exploit next-generation sequencing approaches for comprehensive and quantitative analyses of myocardial miRNAs and mRNAs to test the hypothesis that augmented phosphoinositide-3-kinase-p110α (PI3Kα) signaling in the setting of pathological hypertrophy provides beneficial effects through remodeling of the myocardial transcriptome signature. In these studies, a molecular and bioinformatic pipeline permitting comprehensive analysis and quantification of myocardial miRNA and mRNA expression with next-generation sequencing was developed and the impact of enhanced PI3Kα signaling on the myocardial transcriptome signature of pressure overload-induced pathological hypertrophy was explored. These analyses identified multiple miRNAs and mRNAs that were abnormally expressed in pathological hypertrophy and partially or completely normalized with increased PI3Kα signaling. Additionally, several novel miRNAs potentially linked to remodeling in cardiac hypertrophy were identified. Additional experiments revealed that increased PI3Kα signaling reduces cardiac fibrosis in pathological hypertrophy through modulating TGF-β signaling and miR-21 expression. In conclusion, using the approach of combined miRNA and mRNA sequencing, we identify the protective transcriptome signature of enhanced PI3Kα signaling in the context of pathological hypertrophy, and demonstrate the regulation of TGF-β/miR-21 by which enhanced PI3Kα signaling protects against cardiac fibrosis.

Enhanced cardiac PI3Kα signalling mitigates arrhythmogenic electrical remodelling in pathological hypertrophy and heart failure

AIMS Cardiac hypertrophy and heart failure are associated with QT prolongation and lethal ventricular arrhythmias resulting from decreased K(+) current densities and impaired repolarization. Recent studies in mouse models of physiological cardiac hypertrophy revealed that increased phosphoinositide-3-kinase-α (PI3Kα) signalling results in the up-regulation of K(+) channels and the normalization of ventricular repolarization. The experiments here were undertaken to test the hypothesis that increased PI3Kα signalling will counteract the adverse electrophysiological remodelling associated with pathological hypertrophy and heart failure. METHODS AND RESULTS In contrast to wild-type mice, left ventricular (LV) hypertrophy, induced by transverse aortic constriction (TAC), did not result in prolongation of ventricular action potentials or QT intervals in mice with cardiac-specific expression of constitutively active PI3Kα (caPI3Kα). Indeed, repolarizing K(+) currents and K(+) channel subunit transcripts were increased in caPI3Kα + TAC LV myocytes in proportion to the TAC-induced cellular hypertrophy. Congestive heart failure in a transgenic model of dilated cardiomyopathy model is accompanied by prolonged QT intervals and ventricular action potentials, reduced K(+) currents and K(+) channel transcripts. Increased PI3Kα signalling, but not renin-angiotensin system blockade, in this model also results in increased K(+) currents and improved ventricular repolarization. CONCLUSION In the setting of pathological hypertrophy or heart failure, enhanced PI3Kα signalling results in the up-regulation of K(+) channel subunits, normalization of K(+) current densities and preserved ventricular function. Augmentation of PI3Kα signalling, therefore, may be a useful and unique strategy to protect against the increased risk of ventricular arrhythmias and sudden death associated with cardiomyopathy.

Mitochondria and arrhythmias

Mitochondria are essential to providing ATP, thereby satisfying the energy demand of the incessant electrical activity and contractile action of cardiac muscle. Emerging evidence indicates that mitochondrial dysfunction can adversely affect cardiac electrical functioning by impairing the intracellular ion homeostasis and membrane excitability through reduced ATP production and excessive reactive oxygen species (ROS) generation, resulting in increased propensity to cardiac arrhythmias. In this review, the molecular mechanisms linking mitochondrial dysfunction to cardiac arrhythmias are discussed with an emphasis on the impact of increased mitochondrial ROS on the cardiac ion channels and transporters that are critical to maintaining normal electromechanical functioning of the cardiomyocytes. The potential of using mitochondria-targeted antioxidants as a novel antiarrhythmia therapy is highlighted.

First-Pass Myocardial Perfusion Cardiovascular Magnetic Resonance at 3 Tesla

PURPOSE To test the feasibility of first-pass contrast-enhanced myocardial perfusion imaging at 3 Tesla and to evaluate the change in perfusion index between normal, remote and ischemic myocardium, we obtained perfusion index from healthy subjects and patients with coronary artery stenosis. MATERIALS AND METHODS First-pass contrast-enhanced perfusion imaging was performed on 12 patients and 32 age-matched healthy subjects in both rest and dipyridamole-induced stress states. After bolus injection of contrast agent, Gd-DTPA with dose of 0.025 mmol/kg body weight and injection time of 1.5 s, three short-axis images from apex to base of the left ventricle (LV) were acquired for 80 cardiac cycles using saturation recovery turbo FLASH sequence. The maximal upslope (Upslope) was derived from the signal-time curves of the LV cavity and myocardium to measure myocardial perfusion. Within 72 hours after cardiovascular magnetic resonance examination, patients received coronary angiography, and the results were correlated with cardiovascular magnetic resonance results. RESULTS Using our protocol of contrast agent administration, sufficient perfusion contrast was obtained without susceptibility-induced signal drop-out at the interface between LV cavity and the myocardium. In healthy volunteers, Upslope showed no dependence on myocardial segments or coronary territories. Upslope increased significantly from rest to stress in normal myocardium (0.09 +/- 0.03 vs. 0.16 +/- 0.05, p < 0.001) and remote myocardium (0.09 +/- 0.03 vs. 0.13 +/- 0.03, p < 0.001), whereas in ischemic myocardium the change was insignificant (0.11 +/- 0.03 vs. 0.10 +/- 0.04, p = ns). This resulted in significant difference in the ratio of Upslope at stress to that at rest, representing myocardial perfusion reserve, between ischemic and non-ischemic myocardium (0.96 +/- 0.41 vs. 1.71 +/- 0.42, p < 0.001 for ischemic vs. normal myocardium; 0.96 +/- 0.41 vs. 1.59 +/- 0.40, p < 0.001 for ischemic vs. remote myocardium). CONCLUSIONS First-pass gadolinium-enhanced myocardial perfusion imaging at 3 Tesla is feasible. The Upslope ratio can differentiate ischemic from non-ischemic myocardium.

Unfolded protein response regulates cardiac sodium current in systolic human heart failure

Background— Human heart failure (HF) increases alternative mRNA splicing of the type V, voltage-gated cardiac Na+ channel &agr;-subunit (SCN5A), generating variants encoding truncated, nonfunctional channels that are trapped in the endoplasmic reticulum. In this work, we tested whether truncated Na+ channels activate the unfolded protein response (UPR), contributing to SCN5A electric remodeling in HF. Methods and Results— UPR and SCN5A were analyzed in human ventricular systolic HF tissue samples and human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). Cells were exposed to angiotensin II (AngII) and hypoxia, known activators of abnormal SCN5A mRNA splicing, or were induced to overexpress SCN5A variants. UPR effectors, protein kinase R-like ER kinase (PERK), calreticulin, and CHOP, were increased in human HF tissues. Induction of SCN5A variants with AngII or hypoxia or the expression of exogenous variants induced the UPR with concomitant downregulation of Na+ current. PERK activation destabilized SCN5A and, surprisingly, Kv4.3 channel mRNAs but not transient receptor potential cation channel M7 (TRPM7) channel mRNA. PERK inhibition prevented the loss of full-length SCN5A and Kv4.3 mRNA levels resulting from expressing Na+ channel mRNA splice variants. Conclusions— UPR can be initiated by Na+ channel mRNA splice variants and is involved in the reduction of cardiac Na+ current during human HF. Because the effect is not entirely specific to the SCN5A transcript, the UPR may play an important role in downregulation of multiple cardiac genes in HF.

Invasive Infections of Aggregatibacter (Actinobacillus) Actinomycetemcomitans

BACKGROUND/PURPOSE Aggregatibacter (Actinobacillus) actinomycetemcomitans, part of the normal flora of the mouth, is frequently found in human periodontal cultures and is an important pathogen causing various invasive infections, particularly infective endocarditis. In this study, we describe the clinical course and outcome of patients with A. actinomycetemcomitans infection. METHODS All patients suffering invasive A. actinomycetemcomitans infections at the National Taiwan University Hospital from January 1985 to December 2004 were included in this study. Relevant data regarding clinical presentation, antimicrobial treatment and outcome of these patients were analyzed. RESULTS During the study period, there were 11 patients with invasive A. actinomycetemcomitans infections, including eight patients with infective endocarditis, one with osteonecrosis and two with pneumonia and chest wall lesions. Among the patients with infective endocarditis, four had prosthetic valve replacement, four suffered from rheumatic heart disease and one had undergone surgical repair of ventricular septal defect. Lesions in the oral cavity were the probable portals of entry of the microorganism, and included carious teeth, periodontitis or radiotherapy of the ear-nose-throat field, and were noted in nine patients. Transthoracic echocardiography and/or transesophageal echocardiography were performed on the patients with probable infective endocarditis but growth was demonstrated in only four of these patients. Blood culture yielded A. actinomycetemcomitans after prolonged incubation. Three isolates were resistant to penicillin and two of these were also resistant to ampicillin. CONCLUSION The diagnosis of invasive A. actinomycetemcomitans infection was delayed due to the indolent clinical course, non-specific presentation and slow growth of the organism. Antibiotic therapy using amoxicillin/clavulanic acid, ampicillin, ampicillin/sulbactam, ceftriaxone, clindamycin, cefotaxime, or levofloxacin was successful in all patients. None of the patients demonstrated recurrence of infection 2-36 months following treatment.