Noncoding RNAs and Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Cardiac Arrhythmic Brugada Syndrome.

Hundreds of thousands of people die each year as a result of sudden cardiac death, and many are due to heart rhythm disorders. One of the major causes of these arrhythmic events is Brugada syndrome, a cardiac channelopathy that results in abnormal cardiac conduction, severe life-threatening arrhythmias, and, on many occasions, death. This disorder has been associated with mutations and dysfunction of about two dozen genes; however, the majority of the patients do not have a definite cause for the diagnosis of Brugada Syndrome. The protein-coding genes represent only a very small fraction of the mammalian genome, and the majority of the noncoding regions of the genome are actively transcribed. Studies have shown that most of the loci associated with electrophysiological traits are located in noncoding regulatory regions and are expected to affect gene expression dosage and cardiac ion channel function. Noncoding RNAs serve an expanding number of regulatory and other functional roles within the cells, including but not limited to transcriptional, post-transcriptional, and epigenetic regulation. The major noncoding RNAs found in Brugada Syndrome include microRNAs; however, others such as long noncoding RNAs are also identified. They contribute to pathogenesis by interacting with ion channels and/or are detectable as clinical biomarkers. Stem cells have received significant attention in the recent past, and can be differentiated into many different cell types including those in the heart. In addition to contractile and relaxational properties, BrS-relevant electrophysiological phenotypes are also demonstrated in cardiomyocytes differentiated from stem cells induced from adult human cells. In this review, we discuss the current understanding of noncoding regions of the genome and their RNA biology in Brugada Syndrome. We also delve into the role of stem cells, especially human induced pluripotent stem cell-derived cardiac differentiated cells, in the investigation of Brugada syndrome in preclinical and clinical studies.

Novel Transcriptomic Interactomes of Noncoding RNAs in the Heart under Altered Thyroid Hormonal States.

Noncoding RNAs are emerging as vital players in cardiovascular diseases. Thyroid hormones (THs) are crucial for cardiovascular survival; however, correction of systemic hypothyroidism (low serum THs) may not improve cardiac tissue-level hypothyroidism or cardiac function. Mechanistically, the understanding of noncoding transcriptomic interactions influencing TH-mediated cardiac effects is unclear. Adult C57BL/6J mixed-sex mice were randomized into Control, Hypothyroid (HypoTH), Hyperthyroid (HyperTH), and HypoTH-Triiodothyronine restoration groups. Physiological, morphological, biochemical, molecular, and whole transcriptomic studies and appropriate statistical analyses were performed. HypoTH showed significant atrophy, depressed cardiac function, and decreased serum THs versus controls, and Triiodothyronine supplementation restored them. HyperTH significantly increased serum THs with hypertrophy. Real-time PCR showed significantly altered inflammatory and immune lncRNAs. The transcriptomic sequencing revealed significant differential expressions of lncRNAs, miRNAs, and mRNAs. Eleven novel circRNAs significantly decreased with increased THs. Multiple pathways were GO-/KEGG-enriched, including cardiac, thyroid, cancer, mitochondrial, inflammatory, adrenergic, metabolic, immune-mediated, vesicular, etc. We also uncovered significant novel co-expression and interactions of lncRNA-miRNA, lncRNA-miRNA-mRNA, lncRNA-mRNA, circRNA-miRNA, and miRNA-mRNA, and splicing events. This includes a novel pathway by which the predominant cardiac TH receptor alpha may interact with specific lncRNAs and miRNAs. This is the first study reporting a comprehensive transcriptome-wide interactome in the cardiac-thyroid axis.

Cardioprotection by triiodothyronine following caloric restriction via long noncoding RNAs.

Severe caloric-restriction compromises thyroid hormone (TH) status, apparently to save energy and proteins for enduring stress stimulus. However, a persistent decrease in TH levels may compromise heart function. We hypothesized that supplementation of low dose active TH or targeting hypoxia-inducible factor-1-alpha, HIF-1α (a strong activator of deiodinase enzyme that degrades peripheral active THs) will prevent deterioration of cardiac performance. Adult mice were subjected to acute fasting based on institutional animal protocols with ad libitum access to water. The following groups were studied: Control mice with free access to food; severe caloric restriction fasting only group; Fasting with Triiodo-l-Thyronine (T3); Fasting with HIF-1α inhibitor (BAY). Cardiac hemodynamic and electrophysiological studies were performed and role of long noncoding RNAs were explored. Following severe caloric-restriction, we found that body weights, and heart weights to a partial extent, were decreased. Low-dose T3 treatment attenuated left ventricular hemodynamic impairment in indices of cardiac contractility and relaxation. In electrophysiology studies, fasting mice developed atrial tachyarrhythmias upon induction. This reverted to control levels following T3 treatment. There was a significant increase in atrioventricular conduction time and significant decrease in heart rate following fasting. Both these changes were attenuated following T3 treatment. Furthermore, BAY partially improved hemodynamics. Compared to the severe caloric-restriction group, both T3 and BAY reduced MALAT1 and GAS5 long noncoding RNA expression. These new findings indicate that T3 and BAY protect from cardiac decompensation secondary to acute severe caloric-restriction partly mediated by long noncoding RNAs.

Comparison of Therapeutic Triiodothyronine Versus Metoprolol in the Treatment of Myocardial Infarction in Rats.

BACKGROUND: Beta blockers are standard therapy for myocardial infarction (MI). Preclinical studies have shown efficacy and safety of thyroid hormone (TH) treatment of cardiovascular disorders. Since THs interact with the sympathoadrenergic system, this study aimed to compare triiodothyronine (T3) and metoprolol (Met) in the treatment of rats with MI on pathophysiology and TH-adrenergic signaling. METHODS: Female Sprague-Dawley rats aged 12 weeks underwent left anterior descending coronary artery ligation (MI) or sham surgeries. T3 (5 µg/kg/day) or Met (100 mg/kg/day) was given in drinking water immediately after surgery for eight weeks. At the terminal of the experiments, the rats were subjected to morphological, functional, and molecular examination. RESULTS: T3 and Met significantly enhanced left ventricular contractility (left ventricular fractional shortening 21.37 ± 2.58% and 21.14 ± 3.71%, respectively) compared to untreated MI (17.88 ± 1.23%), and decreased the incidence of inducible atrial tachyarrhythmia by 87.5% and 62.5%, respectively. Although both treatments showed efficacy, T3 but not Met showed statistically significant improvements compared to MI in arrhythmia duration, left atrial diameter (T3 vs. MI 4.33 ± 0.63 vs. 5.65 ± 1.32 mm; p < 0.05), fibrosis (6.1 ± 0.6%, 6.6 ± 0.6% vs. 8.2 ± 0.7%, T3, Met vs. MI, respectively), and aortic vasorelaxation responsiveness to acetylcholine (pD2 6.97 ± 0.22, 6.83 ± 0.21 vs. 6.66 ± 0.22, T3, Met vs. MI, respectively). Quantitative polymerase chain reaction showed that T3 and Met attenuated expression of genes associated with inflammation and oxidative stress and restored expression of ion channels and contractile proteins. CONCLUSION: These results support comparable efficacy of T3 and Met treatments, suggesting that T3 may provide a therapeutic alternative to standard ß-receptor blockade, especially for patients intolerant to treatment with ß-blockers after MI.

Modified Low-Dose Triiodo-L-thyronine Therapy Safely Improves Function Following Myocardial Ischemia-Reperfusion Injury.

Background: We have shown that thyroid hormones (THs) are cardioprotective and can be potentially used as safe therapeutic agents for diabetic cardiomyopathy and permanent infarction. However, no reliable, clinically translatable protocol exists for TH treatment of myocardial ischemia-reperfusion (IR) injury. We hypothesized that modified low-dose triiodo-L-thyronine (T3) therapy would confer safe therapeutic benefits against IR injury. Methods: Adult female rats underwent left coronary artery ligation for 60 min or sham surgeries. At 2 months following surgery and T3 treatment (described below), the rats were subjected to functional, morphological, and molecular examination. Results: Following surgery, the rats were treated with T3 (8 µg/kg/day) or vehicle in drinking water ad libitum following IR for 2 months. Oral T3 significantly improved left ventricular (LV) contractility, relaxation, and relaxation time constant, and decreased beta-myosin heavy chain gene expression. As it takes rats ~6 h post-surgery to begin drinking water, we then investigated whether modified T3 dosing initiated immediately upon reperfusion confers additional improvement. We injected an intraperitoneal bolus of T3 (12 µg/kg) upon reperfusion, along with low-dose oral T3 (4.5 µg/kg/day) in drinking water for 2 months. Continuous T3 therapy (bolus + low-dose oral) enhanced LV contractility compared with oral T3 alone. Relaxation parameters were also improved compared to vehicle. Importantly, these were accomplished without significant increases in hypertrophy, serum free T3 levels, or blood pressure. Conclusions: This is the first study to provide a safe cardiac therapeutic window and optimized, clinically translatable treatment-monitoring protocol for myocardial IR using commercially available and inexpensive T3. Low-dose oral T3 therapy supplemented with bolus treatment initiated upon reperfusion is safer and more efficacious.

Safe Oral Triiodo-L-Thyronine Therapy Protects from Post-Infarct Cardiac Dysfunction and Arrhythmias without Cardiovascular Adverse Effects.

BACKGROUND: A large body of evidence suggests that thyroid hormones (THs) are beneficial for the treatment of cardiovascular disorders. We have shown that 3 days of triiodo-L-thyronine (T3) treatment in myocardial infarction (MI) rats increased left ventricular (LV) contractility and decreased myocyte apoptosis. However, no clinically translatable protocol is established for T3 treatment of ischemic heart disease. We hypothesized that low-dose oral T3 will offer safe therapeutic benefits in MI. METHODS AND RESULTS: Adult female rats underwent left coronary artery ligation or sham surgeries. T3 (~6 µg/kg/day) was available in drinking water ad libitum immediately following MI and continuing for 2 month(s) (mo). Compared to vehicle-treated MI, the oral T3-treated MI group at 2 mo had markedly improved anesthetized Magnetic Resonance Imaging-based LV ejection fraction and volumes without significant negative changes in heart rate, serum TH levels or heart weight, indicating safe therapy. Remarkably, T3 decreased the incidence of inducible atrial tachyarrhythmias by 88% and improved remodeling. These were accompanied by restoration of gene expression involving several key pathways including thyroid, ion channels, fibrosis, sympathetic, mitochondria and autophagy. CONCLUSIONS: Low-dose oral T3 dramatically improved post-MI cardiac performance, decreased atrial arrhythmias and cardiac remodeling, and reversed many adverse changes in gene expression with no observable negative effects. This study also provides a safe and effective treatment/monitoring protocol that should readily translate to humans.

Role of thyroid hormones in ventricular remodeling.

Cardiac remodeling includes alterations in molecular, cellular, and interstitial systems contributing to changes in size, shape, and function of the heart. This may be the result of injury, alterations in hemodynamic load, neurohormonal effects, electrical abnormalities, metabolic changes, etc. Thyroid hormones (THs) serve as master regulators for diverse remodeling processes of the cardiovascular system-from the prenatal period to death. THs promote a beneficial cardiomyocyte shape and improve contractility, relaxation, and survival via reversal of molecular remodeling. THs reduce fibrosis by decreasing interstitial collagen and reduce the incidence and duration of arrhythmias via remodeling ion channel expression and function. THs restore metabolic function and also improve blood flow both by direct effects on the vessel architecture and decreasing atherosclerosis. Optimal levels of THs both in the circulation and in cardiac tissues are critical for normal homeostasis. This review highlights TH-based remodeling and clinically translatable strategies for diverse cardiovascular disorders.

Low-dose T3 replacement restores depressed cardiac T3 levels, preserves coronary microvasculature and attenuates cardiac dysfunction in experimental diabetes mellitus.

Thyroid dysfunction is common in individuals with diabetes mellitus (DM) and may contribute to the associated cardiac dysfunction. However, little is known about the extent and pathophysiological consequences of low thyroid conditions on the heart in DM. DM was induced in adult female Sprague Dawley (SD) rats by injection of nicotinamide (N; 200 mg/kg) followed by streptozotocin (STZ; 65 mg/kg). One month after STZ/N, rats were randomized to the following groups (N = 10/group): STZ/N or STZ/N + 0.03 µg/mL T3; age-matched vehicle-treated rats served as nondiabetic controls (C). After 2 months of T3 treatment (3 months post-DM induction), left ventricular (LV) function was assessed by echocardiography and LV pressure measurements. Despite normal serum thyroid hormone (TH) levels, STZ/N treatment resulted in reductions in myocardial tissue content of THs (T3 and T4: 39% and 17% reduction versus C, respectively). Tissue hypothyroidism in the DM hearts was associated with increased DIO3 deiodinase (which converts THs to inactive metabolites) altered TH transporter expression, reexpression of the fetal gene phenotype, reduced arteriolar resistance vessel density, and diminished cardiac function. Low-dose T3 replacement largely restored cardiac tissue TH levels (T3 and T4: 43% and 10% increase versus STZ/N, respectively), improved cardiac function, reversed fetal gene expression and preserved the arteriolar resistance vessel network without causing overt symptoms of hyperthyroidism. We conclude that cardiac dysfunction in chronic DM may be associated with tissue hypothyroidism despite normal serum TH levels. Low-dose T3 replacement appears to be a safe and effective adjunct therapy to attenuate and/or reverse cardiac remodeling and dysfunction induced by experimental DM.

Both hypothyroidism and hyperthyroidism increase atrial fibrillation inducibility in rats.

BACKGROUND: Evidence indicates that cardiac hypothyroidism may contribute to heart failure progression. It is also known that heart failure is associated with an increased risk of atrial fibrillation (AF). Although it is established that hyperthyroidism increases AF incidence, the effect of hypothyroidism on AF is unclear. This study investigated the effects of different thyroid hormone levels, ranging from hypothyroidism to hyperthyroidism on AF inducibility in thyroidectomized rats. METHODS AND RESULTS: Thyroidectomized rats with serum-confirmed hypothyroidism 1 month after surgery were randomized into hypothyroid (N=9), euthyroid (N=9), and hyperthyroid (N=9) groups. Rats received placebo, 3.3-mg l-thyroxine (T4), or 20-mg T4 pellets (60-day release form) for 2 months, respectively. At the end of treatment, hypothyroid, euthyroid, and hyperthyroid status was confirmed. Hypothyroid animals showed cardiac atrophy and reduced cardiac systolic and diastolic functions, whereas hyperthyroid rats exhibited cardiac hypertrophy and increased cardiac function. Hypothyroidism and hyperthyroidism produced opposite electrophysiological changes in heart rates and atrial effective refractory period, but both significantly increased AF susceptibility. AF incidence was 78% in hypothyroid, 67% in hyperthyroid, and the duration of induced AF was also longer, compared with 11% in the euthyroid group (all P<0.05). Hypothyroidism increased atrial interstitial fibrosis, but connexin 43 was not affected. CONCLUSIONS: Both hypothyroidism and hyperthyroidism lead to increased AF vulnerability in a rat thyroidectomy model. Our results stress that normal thyroid hormone levels are required to maintain normal cardiac electrophysiology and to prevent cardiac arrhythmias and AF.

Altered ubiquitin-proteasome signaling in right ventricular hypertrophy and failure.

Alterations in the ubiquitin-proteasome system (UPS) have been described in left ventricular hypertrophy and failure, although results have been inconsistent. The role of the UPS in right ventricular (RV) hypertrophy (RVH) and RV failure (RVF) is unknown. Given the greater percent increase in RV mass associated with RV afterload stress, as present in many congenital heart lesions, we hypothesized that alterations in the UPS could play an important role in RVH/RVF. UPS expression and activity were measured in the RV from mice with RVH/RVF secondary to pulmonary artery constriction (PAC). Epoxomicin and MG132 were used to inhibit the proteasome, and overexpression of the 11S PA28α subunit was used to activate the proteasome. PAC mice developed RVH (109.3% increase in RV weight to body weight), RV dilation with septal shift, RV dysfunction, and clinical RVF. Proteasomal function (26S ß5 chymotrypsin-like activity) was decreased 26% (P < 0.05). Protein expression of 19S subunit Rpt5 (P < 0.05), UCHL1 deubiquitinase (P < 0.0001), and Smurf1 E3 ubiquitin ligase (P < 0.01) were increased, as were polyubiquitinated proteins (P < 0.05) and free-ubiquitins (P = 0.05). Pro-apoptotic Bax was increased (P < 0.0001), whereas anti-apoptotic Bcl-2 decreased (P < 0.05), resulting in a sixfold increase in the Bax/Bcl-2 ratio. Proteasomal inhibition did not accelerate RVF. However, proteasome enhancement by cardiac-specific proteasome overexpression partially improved survival. Proteasome activity is decreased in RVH/RVF, associated with upregulation of key UPS regulators and pro-apoptotic signaling. Enhancement of proteasome function partially attenuates RVF, suggesting that UPS dysfunction contributes to RVF.

Loss of adenomatous poliposis coli-α3 integrin interaction promotes endothelial apoptosis in mice and humans.

RATIONALE: Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/ß-catenin pathway, we proposed that ß-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH. OBJECTIVE: This study aims to establish the role of ß-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury. METHODS AND RESULTS: To assess the impact of ß-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (Apc(Min/+)) mouse, where reduced APC causes constitutive ß-catenin elevation. Surprisingly, hypoxic Apc(Min/+) mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from Apc(Min/+) demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC. CONCLUSIONS: We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.

Dynamic microRNA expression during the transition from right ventricular hypertrophy to failure.

MicroRNAs (miRs) are small, noncoding RNAs that are emerging as crucial regulators of cardiac remodeling in left ventricular hypertrophy (LVH) and failure (LVF). However, there are no data on their role in right ventricular hypertrophy (RVH) and failure (RVF). This is a critical question given that the RV is uniquely at risk in patients with congenital right-sided obstructive lesions and in those with systemic RVs. We have developed a murine model of RVH and RVF using pulmonary artery constriction (PAC). miR microarray analysis of RV from PAC vs. control demonstrates altered miR expression with gene targets associated with cardiomyocyte survival and growth during hypertrophy (miR 199a-3p) and reactivation of the fetal gene program during heart failure (miR-208b). The transition from hypertrophy to heart failure is characterized by apoptosis and fibrosis (miRs-34, 21, 1). Most are similar to LVH/LVF. However, there are several key differences between RV and LV: four miRs (34a, 28, 148a, and 93) were upregulated in RVH/RVF that are downregulated or unchanged in LVH/LVF. Furthermore, there is a corresponding downregulation of their putative target genes involving cell survival, proliferation, metabolism, extracellular matrix turnover, and impaired proteosomal function. The current study demonstrates, for the first time, alterations in miRs during the process of RV remodeling and the gene regulatory pathways leading to RVH and RVF. Many of these alterations are similar to those in the afterload-stressed LV. miRs differentially regulated between the RV and LV may contribute to the RVs increased susceptibility to heart failure.

Cardiac pressure overload hypertrophy is differentially regulated by ß-adrenergic receptor subtypes.

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not ß-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of ß-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of ß-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of ß(1), ß(2), or both ß(1)- and ß(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, ß(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas ß(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both ß-ARs were ablated (ß(1)ß(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in ß(1)ß(2)(-/-) compared with the other genotypes, whereas transforming growth factor-ß(2), a positive mediator of hypertrophy was upregulated in all genotypes except the ß(1)ß(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both ß(1)- and ß(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.

Cardiac ErbB-1/ErbB-2 mutant expression in young adult mice leads to cardiac dysfunction.

Multiple factors lead to the development and maintenance of chronic heart failure. Blockade of ErbB-2 or ErbB-4 tyrosine kinase receptor signaling leads to dilated cardiomyopathy. ErbB-1 may protect the heart against stress-induced injury and its ligand; epidermal growth factor (EGF) increases myocardial contractility, whereas heparin-binding EGF is essential for normal cardiac function. However, the role of ErbB-1 in control of cardiac function is not clear. We hypothesized that ErbB-1 is essential for maintaining adult cardiac function. Using the ecdysone-inducible gene expression system, we expressed humanized cardiomyocyte-specific dominant-negative ErbB-1 mutant receptors (hErbB-1-mut) in young adult mice that block endogenous cardiac ErbB-1 signaling. Molecular, morphological, and physiological tests (under anesthesia) were performed. As a result, hErbB-1-mut was expressed selectively in cardiomyocytes leading to the blockade of endogenous ErbB-1 phosphorylation and ErbB-2 transphosphorylation. An increase in left ventricular mass, atrial natriuretic factor expression, and histological changes were indicative of cardiac hypertrophy. Cardiac dilation, numerous cardiac lesions, and the loss of the clear boundary between cardiac fibrils were noted histologically. Early and long-term hErbB-1-mut induction led to a significant decrease in fractional shortening and to significant increases in left ventricular end-systolic diameter and volume. The treatment of adenylyl cyclase activator (forskolin analog) normalized the depressed cardiac function. Resting cardiac function returned to normal after reversing mutant expression. A 4-day survival rate of transverse-aortic constricted hErbB-1-mut mice was only 20% compared with 100% in controls. In conclusion, these observations indicate that the blockade of cardiac ErbB-1 signaling leads to the blockade of ErbB-2 signaling and that together they result in cardiac dysfunction.