Tiannan Wang

Targeted Deletion of MicroRNA-22 Promotes Stress-Induced Cardiac Dilation and Contractile Dysfunction

Background— Delineating the role of microRNAs (miRNAs) in the posttranscriptional gene regulation offers new insights into how the heart adapts to pathological stress. We developed a knockout of miR-22 in mice and investigated its function in the heart. Methods and Results— Here, we show that miR-22–deficient mice are impaired in inotropic and lusitropic response to acute stress by dobutamine. Furthermore, the absence of miR-22 sensitized mice to cardiac decompensation and left ventricular dilation after long-term stimulation by pressure overload. Calcium transient analysis revealed reduced sarcoplasmic reticulum Ca2+ load in association with repressed sarcoplasmic reticulum Ca2+ ATPase activity in mutant myocytes. Genetic ablation of miR-22 also led to a decrease in cardiac expression levels for Serca2a and muscle-restricted genes encoding proteins in the vicinity of the cardiac Z disk/titin cytoskeleton. These phenotypes were attributed in part to inappropriate repression of serum response factor activity in stressed hearts. Global analysis revealed increased expression of the transcriptional/translational repressor purine-rich element binding protein B, a highly conserved miR-22 target implicated in the negative control of muscle expression. Conclusion— These data indicate that miR-22 functions as an integrator of Ca2+ homeostasis and myofibrillar protein content during stress in the heart and shed light on the mechanisms that enhance propensity toward heart failure.

Inhibition of CaMKII Phosphorylation of RyR2 Prevents Induction of Atrial Fibrillation in FKBP12.6 Knockout Mice

Rationale: Abnormal calcium release from sarcoplasmic reticulum (SR) is considered an important trigger of atrial fibrillation (AF). Whereas increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity has been proposed to contribute to SR leak and AF induction, downstream targets of CaMKII remain controversial. Objective: To test the hypothesis that inhibition of CaMKII-phosphorylated type-2 ryanodine receptors (RyR2) prevents AF initiation in FKBP12.6-deficient (−/−) mice. Methods and Results: Mice lacking RyR2-stabilizing subunit FKBP12.6 had a higher incidence of spontaneous and pacing-induced AF compared with wild-type mice. Atrial myocytes from FKBP12.6−/− mice exhibited spontaneous Ca2+ waves (SCaWs) leading to Na+/Ca2+-exchanger activation and delayed afterdepolarizations (DADs). Mutation S2814A in RyR2, which inhibits CaMKII phosphorylation, reduced Ca2+ spark frequency, SR Ca2+ leak, and DADs in atrial myocytes from FKBP12.6−/−:S2814A mice compared with FKBP12.6−/− mice. Moreover, FKBP12.6−/−:S2814A mice exhibited a reduced susceptibility to inducible AF, whereas FKBP12.6−/−:S2808A mice were not protected from AF. Conclusions: FKBP12.6 mice exhibit AF caused by SR Ca2+ leak, Na+/Ca2+-exchanger activation, and DADs, which promote triggered activity. Genetic inhibition of RyR2-S2814 phosphorylation prevents AF induction in FKBP12.6−/− mice by suppressing SR Ca2+ leak and DADs. These results suggest suppression of RyR2-S2814 phosphorylation as a potential anti-AF therapeutic target.

Atrial Identity Is Determined by A COUP-TFII Regulatory Network

Atria and ventricles exhibit distinct molecular profiles that produce structural and functional differences between the two cardiac compartments. However, the factors that determine these differences remain largely undefined. Cardiomyocyte-specific COUP-TFII ablation produces ventricularized atria that exhibit ventricle-like action potentials, increased cardiomyocyte size, and development of extensive T tubules. Changes in atrial characteristics are accompanied by alterations of 2,584 genes, of which 81% were differentially expressed between atria and ventricles, suggesting that a major function of myocardial COUP-TFII is to determine atrial identity. Chromatin immunoprecipitation assays using E13.5 atria identified classic atrial-ventricular identity genes Tbx5, Hey2, Irx4, MLC2v, MLC2a, and MLC1a, among many other cardiac genes, as potential COUP-TFII direct targets. Collectively, our results reveal that COUP-TFII confers atrial identity through direct binding and by modulating expression of a broad spectrum of genes that have an impact on atrial development and function.

Pathogenesis of Lethal Cardiac Arrhythmias in Mecp2 Mutant Mice: Implication for Therapy in Rett Syndrome

Lethal ventricular arrhythmias in a mouse model of Rett syndrome can be prevented by phenytoin, which blocks a persistent sodium current. A Heart-Brain Connection Patients with Rett syndrome, usually girls, have many problems, including impaired brain function and cognition. One of the most unfortunate is the tendency of about 25% of these patients to die suddenly and unexpectedly, likely from cardiac problems. To get to the bottom of this, McCauley et al. examined heart physiology in mice carrying a mutation in methyl-CpG–binding protein 2 (MECP2), the gene that is defective in Rett syndrome. In addition to a Rett-like phenotype, these mice exhibited a long QT interval in their heartbeat tracings and had ventricular tachycardia, and some of the mice died of cardiac causes. Treatment with a common anticonvulsant, phenytoin, normalized these atypical heartbeats, suggesting that this treatment may help to prevent sudden death in patients with Rett syndrome. To determine the underlying cause of the dangerous heartbeats, the authors generated another strain of mice in which MECP2 was only mutated in the nervous system. These mice, which had normal heart MECP2, also showed abnormal heart beating and tachycardia, leading to the conclusion that the heart problems were actually secondary to nervous system deficits. A close look at the cardiac cells showed that even when they were removed from mice and grown in culture, an unusual persistent sodium current was apparent. This current decreased in the presence of phenytoin, consistent with its therapeutic action in the MECP2 mutant mice. The authors hypothesize that nervous system abnormalities cause remodeling of the heart in these patients, including elevation of a persistent sodium current, and suggest that sodium channel blockers such as phenytoin be tested as therapeutic agents. Rett syndrome patients often have recurrent seizures, and a similar situation may occur in patients with epilepsy from other causes. The oddly named SUDEP or “sudden unexpected death in epilepsy” is rare, but is thought also to result from frequent heartbeat abnormalities in these patients, which are similar to those in Rett patients. The connection between seizures and long QT intervals may transcend the exact nature of a patient’s disease. Rett syndrome is a neurodevelopmental disorder typically caused by mutations in methyl-CpG–binding protein 2 (MECP2) in which 26% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction, we characterized the electrocardiograms in 379 people with Rett syndrome and found that 18.5% show prolongation of the corrected QT interval (QTc), an indication of a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function, Mecp2Null/Y, also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice, Mecp2Null/+, show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia, implicating neuronally mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome, β-adrenergic receptor blockers, did not prevent ventricular tachycardia in Mecp2Null/Y mice. To determine whether an alternative therapy would be more appropriate, we characterized cardiomyocytes from Mecp2Null/Y mice and found increased persistent sodium current, which was normalized when cells were treated with the sodium channel–blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2Null/Y mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control, which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current to prevent lethal cardiac arrhythmias.

microRNA-22 promotes heart failure through coordinate suppression of PPAR/ERR-nuclear hormone receptor transcription.

Increasing evidence suggests that microRNAs are intimately involved in the pathophysiology of heart failure. MicroRNA-22 (miR-22) is a muscle-enriched miRNA required for optimum cardiac gene transcription and adaptation to hemodynamic stress by pressure overload in mice. Recent evidence also suggests that miR-22 induces hypertrophic growth and it is oftentimes upregulated in end stage heart failure. However the scope of mRNA targets and networks of miR-22 in the heart failure remained unclear. We analyzed transgenic mice with enhanced levels of miR-22 expression in adult cardiomyocytes to identify important pathophysiologic targets of miR-22. Our data shows that forced expression of miR-22 induces a pro-hypertrophic gene expression program, and it elicits contractile dysfunction leading to cardiac dilation and heart failure. Increased expression of miR-22 impairs the Ca2+ transient, Ca2+ loading into the sarcoplasmic reticulum plus it interferes with transcription of estrogen related receptor (ERR) and PPAR downstream genes. Mechanistically, miR-22 postranscriptionally inhibits peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), PPARα and sirtuin 1 (SIRT1) expression via a synergistic circuit, which may account for deleterious actions of unchecked miR-22 expression on the heart.

Overexpression of cAMP-response element modulator causes abnormal growth and development of the atrial myocardium resulting in a substrate for sustained atrial fibrillation in mice

BACKGROUND AND METHODS Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. The substrate of AF is composed of a complex interplay between structural and functional changes of the atrial myocardium often preceding the occurrence of persistent AF. However, there are only few animal models reproducing the slow progression of the AF substrate to the spontaneous occurrence of the arrhythmia. Transgenic mice (TG) with cardiomyocyte-directed expression of CREM-IbΔC-X, an isoform of transcription factor CREM, develop atrial dilatation and spontaneous-onset AF. Here we tested the hypothesis that TG mice develop an arrhythmogenic substrate preceding AF using physiological and biochemical techniques. RESULTS Overexpression of CREM-IbΔC-X in young TG mice (<8weeks) led to atrial dilatation combined with distension of myocardium, elongated myocytes, little fibrosis, down-regulation of connexin 40, loss of excitability with a number of depolarized myocytes, atrial ectopies and inducibility of AF. These abnormalities continuously progressed with age resulting in interatrial conduction block, increased atrial conduction heterogeneity, leaky sarcoplasmic reticulum calcium stores and the spontaneous occurrence of paroxysmal and later persistent AF. This distinct atrial remodelling was associated with a pattern of non-regulated and up-regulated marker genes of myocardial hypertrophy and fibrosis. CONCLUSIONS Expression of CREM-IbΔC-X in TG hearts evokes abnormal growth and development of the atria preceding conduction abnormalities and altered calcium homeostasis and the development of spontaneous and persistent AF. We conclude that transcription factor CREM is an important regulator of atrial growth implicated in the development of an arrhythmogenic substrate in TG mice.

Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling, Insulin Secretion, and Glucose Tolerance

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca2+-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca2+ concentration ([Ca2+]cyt) of β cells. This elevation in [Ca2+]cyt leads to activation of Ca2+/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca2+-release channel implicated in Ca2+-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca2+ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca2+]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca2+ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.

Genetic Deletion of Rnd3/RhoE Results in Mouse Heart Calcium Leakage Through Upregulation of Protein Kinase A Signaling

Rationale: Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation. The biological function of Rnd3 in the heart remains unexplored. Objective: To define the functional role of the Rnd3 gene in the animal heart and investigate the associated molecular mechanism. Methods and Results: By loss-of-function approaches, we discovered that Rnd3 is involved in calcium regulation in cardiomyocytes. Rnd3-null mice died at the embryonic stage with fetal arrhythmias. The deletion of Rnd3 resulted in severe Ca2+ leakage through destabilized ryanodine receptor type 2 Ca2+ release channels. We further found that downregulation of Rnd3 attenuated &bgr;2-adrenergic receptor lysosomal targeting and ubiquitination, which in turn resulted in the elevation of &bgr;2-adrenergic receptor protein levels leading to the hyperactivation of protein kinase A (PKA) signaling. The PKA activation destabilized ryanodine receptor type 2 channels. This irregular spontaneous Ca2+ release can be curtailed by PKA inhibitor treatment. Increases in the PKA activity along with elevated cAMP levels were detected in Rnd3-null embryos, in neonatal rat cardiomyocytes, and noncardiac cell lines with Rnd3 knockdown, suggesting a general mechanism for Rnd3-mediated PKA signaling activation. &bgr;2-Adrenergic receptor blocker treatment reduced arrhythmia and improved cardiac function. Conclusions: Rnd3 is a novel factor involved in intracellular Ca2+ homeostasis regulation in the heart. Deficiency of the protein induces ryanodine receptor type 2 dysfunction by a mechanism that attenuates Rnd3-mediated &bgr;2-adrenergic receptor ubiquitination, which leads to the activation of PKA signaling. Increased PKA signaling in turn promotes ryanodine receptor type 2 hyperphosphorylation, which contributes to arrhythmogenesis and heart failure.

Long-Term Simulated Microgravity Causes Cardiac RyR2 Phosphorylation and Arrhythmias in Mice

BACKGROUND Long-term exposure to microgravity during space flight may lead to cardiac remodeling and rhythm disturbances. In mice, hindlimb unloading (HU) mimics the effects of microgravity and stimulates physiological adaptations, including cardiovascular deconditioning. Recent studies have demonstrated an important role played by changes in intracellular Ca handling in the pathogenesis of heart failure and arrhythmia. In this study, we tested the hypothesis that cardiac remodeling following HU in mice involves abnormal intracellular Ca regulation through the cardiac ryanodine receptor (RyR2). METHODS AND RESULTS Mice were subjected to HU by tail suspension for 28 to 56 days in order to induce cardiac remodeling (n=15). Control mice (n=19) were treated equally, with the exception of tail suspension. Echocardiography revealed cardiac enlargement and depressed contractility starting at 28 days post-HU versus control. Moreover, mice were more susceptible to pacing-induced ventricular arrhythmias after HU. Ventricular myocytes isolated from HU mice exhibited an increased frequency of spontaneous sarcoplasmic reticulum (SR) Ca release events and enhanced SR Ca leak via RyR2. Western blotting revealed increased RyR2 phosphorylation at S2814, and increased CaMKII auto-phosphorylation at T287, suggesting that CaMKII activation of RyR2 might underlie enhanced SR Ca release in HU mice. CONCLUSION These data suggest that abnormal intracellular Ca handling, likely due to increased CaMKII phosphorylation of RyR2, plays a role in cardiac remodeling following simulated microgravity in mice.

Enhanced impact of SCN5A mutation associated with long QT syndrome in fetal splice isoform.

Congenital long QT syndrome (LQTS) is an inherited syndrome characterized by prolongation of the QT interval on the electrocardiogram and an increased susceptibility to life-threatening ventricular arrhythmias. Mutations in the SCN5A gene, which encodes the α-subunit of the cardiac Na+ channel, represent the third most common cause of LQTS, behind mutations in potassium channel genes KCNQ1 and KCNH2. Moreover, mutations in SCN5A have been linked to other types of inherited channelopathies, including the Brugada syndrome (BRS1), progressive familial heart block type 1 (PFHBI), sick sinus syndrome type 1 (SSS1), idiopathic ventricular fibrillation (IVF), familiar atrial standstill, dilated cardiomyopathy type 1E (CMD1E), and sudden infant death syndrome (SIDS)1. In total, more than 400 unique DNA variants have been reported in SCN5A, of which at least more than 80 mutations were linked to LQTS alone (see inherited arrhythmia data base: http://www.fsm.it/cardmoc/). Mutations in the SCN5A gene associated with LQTS typically cause a gain-of-function phenotype resulting in enhanced Na+ entry into the cardiomyocyte during the repolarization period 2. Each Na+ channel α-subunit (Nav1.5) consists of four structurally homologous domains (DI-DIV), each comprising six transmembrane segments (S1-S6). Most mutations in Nav1.5 disrupt fast inactivation and thereby cause a persistent (or sustained) Na+ current. However, some Na+ channel mutations rather enhance window currents when inactivation occurs at more depolarized potentials, resulting in delayed repolarization in the absence of persistent Na+ current 3. Other biophysical mechanisms of Nav1.5 dysfunction causally linked to LQTS include faster recovery from inactivation, slower inactivation, and a larger peak Na+ current (INa) density 1. Regardless of the underlying mechanism, gain-of-function defects in Nav1.5 disrupt the delicate balance between depolarization and repolarization during the action potential plateau phase, thus delaying repolarization and increasing the risk of lethal ventricular arrhythmias. Postmortem studies have revealed that SCN5A mutations may be the most prevalent genetic cause of sudden infant death syndrome (SIDS), which is the unexpected, sudden death of a child under age 1 in which autopsy does not reveal an explainable cause of death 4. Most SCN5A mutations found in SIDS victims cause biophysical phenotypes similar to those associated with mutations found in older children or adults with LQTS. However, a few SIDS-linked mutations in SCN5A exhibit sustained INa only under acidic conditions, suggesting that environmental factors such as hypoxia or acidosis might contribute to the lethal arrhythmias in susceptible infants 5. In addition, several papers have reported even earlier, prenatal diagnosis of LQTS linked to SCN5A mutations. Such variants were identified in several parts of the channel (e.g., R43Q, L619F, F627L, A1186T, P1332L, F1473C, F1486del, R1623Q, V1763M, N1774D) 6–9. The most common prenatal manifestations of LQTS include sinus bradycardia and atrioventricular block, presumably due to excessive refractory periods related to delayed repolarization. In addition, irregular heart rates due to ventricular ectopy and ventricular tachycardia are commonly observed. In more than half of all published cases, in utero demise occurred during the third trimester 6–9. Previous biophysical analysis of the abovementioned SCN5A variants did not reveal biophysical defects distinct from those described for SCN5A mutations found in individuals with a postnatal diagnosis of LQTS. Therefore, it has remained unclear why fetuses with SCN5A mutations exhibit more severe repolarization defects and higher mortality rates compared to older mutation carriers. In the current issue of HeartRhythm, Murphy et al. 10 described an interesting case report of a fetus carried by a 29-year-old primiparous, otherwise healthy woman, who was diagnosed at 20 weeks of gestation with frequent premature ventricular contractions, which represents the earliest described case of fetal LQTS. The fetus developed episodes of ventricular ectopy, which soon thereafter progressed into polymorphic ventricular tachycardia, extreme QTc interval prolongation, and hydrops fetalis. Because of the extent of the clinical deterioration, pregnancy was terminated at the request of the family. Genetic analysis revealed a novel, de novo, heterozygous missense mutation (L409P) in SCN5A, as well as homozygosity for the common nonsynonymous variant R558 11. The biophysical features of the mutant Na+ channels were studied using whole cell patch clamp of tsA201 cells expressing recombinant Nav1.5 channels with mutation L409P and polymorphism R558. These Nav1.5-L409P/R558 mutant channels exhibited reduced peak current density, depolarized shifts in voltage-dependence of activation and inactivation, and faster recovery from inactivation. In addition, a much larger persistent Na+ current was measured, which is a common feature among most LQTS-linked Na+ channel mutants 1. Next, the authors explored the interesting hypothesis that the severe clinical manifestations of LQTS in the affected fetus were due to alternative splicing of a SCN5A transcript expressed during the fetal period. In human fetal hearts, alternative exon 6A is more abundant than in infant or adult heart. Compared to the adult isoform, fetal Nav1.5-L409P/R558 channels exhibited a more pronounced shift in fast inactivation and an even larger persistent Na+ current. Moreover, the fetal isoform exhibited a slower activation rise time and slower inactivation kinetics, similar to previous reports 12. These exacerbated changes in Na+ channel gating may explain the severity of the clinical phenotype in the fetus with the L409P mutation and R588 polymorphism. The replacement of exon 6 by exon 6a as a result of alternative splicing results in the substitution of 7 amino acids in the fetal Nav1.5 channel. Onkal et al. 12 demonstrated that replacement of a single negatively charged aspartate at position 211 in the adult isoform with a positively charged lysine residue in the fetal isoform introduces a positive charge in the S3 domain adjacent to the S4 voltage sensor of domain I. This particular amino acid substitution was shown to be primarily responsible for the functional effects of exon 6 splicing on Nav1.5 channel parameters. The present study by Murphy et al. 10 revealed that the electrophysiological effects of the R558 polymorphism were similar in the adult and fetal Nav1.5 isoforms. However, when the L409P mutation was added to the R558 polymorphism, more pronounced Na+ channel dysfunction was observed in case of the fetal splice variant. This suggests that alternative splicing of the fetal isoform might be the primary reason for the severe fetal manifestation of arrhythmias in carriers of SCN5A mutations. Since most genes causally linked to LQTS are also subject to alternative splicing, it would be interesting to determine whether the effects of mutations in other cardiac ion channels are also more potent in the fetal splice variants. Finally, it was shown that the R558 polymorphism independently contributed to enhancement of Nav1.5 channel dysfunction caused by the L409P mutation. This observation highlights the importance of SCN5A polymorphisms in terms of Na+ channel electrophysiology. For example, polymorphism S1103Y, which is commonly found in African Americans, has been linked to SIDS 13. Another variant, R1193Q, commonly found in Asians 14 may also increase the risk of SIDS and prenatal death 15. Moreover, polymorphism V1951L found in Latinos 16 also modulates the biophysical effects of SCN5A mutations 17, and has been identified in a victim of SIDS 5. In conclusion, the paper by Murphy et al. 10 suggests that the unusual severity and early onset of ventricular arrhythmias in a fetus with an SCN5A mutation could be attributed to synergistic effects of a disease-causing mutation, a polymorphism, and an alternative splice variant. It would be important to consider the contributions of each of these three factors in future studies of SCN5A variants associated with fetal or perinatal arrhythmias and sudden cardiac death.