Atrial fibrillation (AF), the most prevalent sustained cardiac arrhythmia, often coexists with the related arrhythmia atrial flutter (AFL). Limitations in effectiveness and safety of current therapies make an understanding of the molecular mechanism underlying AF more urgent. Genome-wide association studies implicated a region of human chromosome 4q25 in familial AF and AFL, ≈150 kb distal to the Pitx2 homeobox gene, a developmental left-right asymmetry (LRA) gene. To investigate the significance of the 4q25 variants, we used mouse models to investigate Pitx2 in atrial arrhythmogenesis directly. When challenged by programmed stimulation, Pitx2 null+/− adult mice had atrial arrhythmias, including AFL and atrial tachycardia, indicating that Pitx2 haploinsufficiency predisposes to atrial arrhythmias. Microarray and in situ studies indicated that Pitx2 suppresses sinoatrial node (SAN)-specific gene expression, including Shox2, in the left atrium of embryos and young adults. In vivo ChIP and transfection experiments indicated that Pitx2 directly bound Shox2 in vivo, supporting the notion that Pitx2 directly inhibits the SAN-specific genetic program in left atrium. Our findings implicate Pitx2 and Pitx2-mediated LRA-signaling pathways in prevention of atrial arrhythmias., the most common adult arrhythmia, increases in prevalence with age, eventually afflicting 5% of the population over age 65 years and 10% of those over age 80 years. Moreover, patients with AF have a significantly increased risk of stroke, heart failure, and dementia (1-3). Electrical impulses critical for a normal heartbeat are initiated in the sinoatrial node (SAN) or pacemaker region. In AF, rapid and irregular atrial activity overrides normal SAN function, often resulting in irregular impulse conduction to the ventricles. In many cases, ectopic electrical activity originates in the pulmonary veins and may serve to trigger and maintain AF (1, 4). The related arrhythmia, atrial flutter (AFL), displays more regular and organized electrical activity than does AF (5). Significantly, current treatments for AF are suboptimal because of incomplete effectiveness and deleterious side effects. It also has been recognized that untreated AF results in pathologic remodeling that makes AF more likely to recur (6). Thus, it is critically important to uncover the genetic mechanisms underlying AF to aid in patient management and to develop more safe and effective therapies.The pituitary homeobox (Pitx) family of homeobox genes containing three genes, Pitx1, Pitx2, and Pitx3, is a subgroup within the larger Paired-related superfamily of homeobox genes (7,8). Pitx2 was identified as the gene mutated in Rieger syndrome I, a haploinsufficient disorder that includes ocular, tooth, and anterior body wall defects as primary characteristics (9). Importantly, the Pitx2 gene encodes three isoforms: Pitx2a, Pitx2b, and Pitx2c. The Pitx2c isoform plays a critical role as a late effector in left-right asymmetry (LRA), a fundamental component of organ morphogenesis in vertebrate...

Atrial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca 2+ handling. Diastolic Ca 2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2 R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice.

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2 R176Q/؉ mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2 R176Q/؉ mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2 R176Q/؉ , but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2 R176Q/؉ mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2 R176Q/؉ mice, but not in controls. Isolated cardiomyocytes from RyR2 R176Q/؉ mice exhibited a higher incidence of spontaneous Ca 2؉ oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.arrhythmogenic right ventricular dysplasia ͉ catecholaminergic polymorphic ventricular tachycardia ͉ calcium-release channel T he cardiac ryanodine receptor 2 (RyR2) regulates calcium release from the sarcoplasmic reticulum in cardiomyocytes (1). Two inherited arrhythmogenic syndromes have been linked to mutations in RyR2, arrhythmogenic right ventricular dysplasia (ARVD) and catecholaminergic polymorphic ventricular tachycardia (CPVT) (2, 3). ARVD and CPVT are both characterized by ventricular arrhythmias and a high rate of juvenile sudden death. Patients with CPVT exhibit catecholamine-induced bidirectional ventricular tachycardia (VT) in the setting of a structurally normal heart, whereas patients with ARVD exhibit progressive fibrofatty replacement of the right ventricular myocardium in addition to polymorphic VT. ARVD arising from RyR2 mutations (ARVD2) is typically associated with exercise-induced ventricular arrhythmias and relatively mild structural abnormalities compared with other forms of ARVD and, in some ways, mimics the CPVT phenotype. In fact, the diagnosis of ARVD2 in patients with RyR2 mutations is controversial because of the differences in degree of cardiac structural abnormalities between ARVD2 and other forms of ARVD (4).Disease-causing mutations in RyR2 and the skeletal muscle isoform RyR1 cluster in three highly conserved regions: a cytosolic N-terminal region, a cytosolic central region, and a C-terminal portion containing the transmembrane and pore regions of the channel (5, 6). Multiple mutations in RyR2 have been reported in patients with ...

BACKGROUND-Although defective Ca 2+ homeostasis may contribute to arrhythmogenesis in atrial fibrillation (AF), the underlying molecular mechanisms remain poorly understood. Studies in patients with AF revealed that impaired diastolic closure of SR Ca 2+ -release channels (ryanodine receptors, RyR2) is associated with reduced levels of the RyR2-inhibitory subunit FKBP12.6.

Despite positive animal studies, clinical angiogenesis trials have been disappointing, possibly due to risk factors present in humans but usually unexplored in animals. We recently demonstrated aging causes impaired collateral remodeling and collateral dropout; here, we investigate potential mechanisms responsible for these findings. Four-, 10-, and 18-month-C57BL/6J mice were subjected to femoral artery ligation; flow was measured using laser Doppler perfusion imaging. Endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS were measured in calf muscle. Apoptosis was assessed in endothelial (EC) and smooth muscle (SMC) cells isolated from young and old mice. Angiogenesis was measured using a Matrigel plug assay. Lethally irradiated young and old mice received bone marrow cells (BMC) from either young or old donors and were subjected to femoral artery ligation (FAL). BMC mobilization and homing were assessed. Flow recovery was impaired and less eNOS and phosphorylated eNOS was present in older vs. young mice (p<0.001 and p=0.015, respectively). ECs and SMCs from older mice were more sensitive to an apoptotic stimulus, but were rescued by NO-enhancing drugs. In older mice, angiogenesis (Matrigel plug assay) was impaired, as was mobilization and homing of BM progenitor cells following FAL. Although both mobilization and homing improved when older mice received BMC transplantation from young donors, flow recovery failed to improve. Aging impairs BMC mobilization and homing, collateral responsiveness to angiogenic stimuli, and increases EC and SMC susceptibility to apoptosis via dysfunctional eNOS signaling. The latter could contribute to impaired remodeling and collateral dropout. These finding identify potential obstacles to therapeutic interventions in elderly patients.