Objectives We studied activating autoantibodies to β1-adrenergic (AAβ1AR) and M2 muscarinic receptors (AAM2R) in the genesis of atrial fibrillation (AF) in Graves’ hyperthyroidism. Background AF frequently complicates hyperthyroidism. AAβ1AR and AAM2R have been described in some patients with dilated cardiomyopathy and AF. We hypothesized their co-presence would facilitate AF in autoimmune Graves’ hyperthyroidism. Methods IgG purified from 38 patients with Graves’ hyperthyroidism with AF (n=17) or sinus rhythm (n=21) and 10 healthy controls was tested for its effects on isolated canine Purkinje fiber contractility with and without atropine and nadolol. IgG electrophysiologic effects were studied using intracellular recordings from isolated canine pulmonary veins. Potential cross-reactivity of AAβ1AR and AAM2R with stimulating thyrotropin receptor (TSHR) antibodies was evaluated before and after adsorption to CHO cells expressing human TSHRs using flow cytometry and enzyme-linked immunosorbent assays. Results The frequency of AAβ1AR and/or AAM2R differed significantly between patients with AF and sinus rhythm (AAβ1AR = 94% vs. 38%, p<0.001; AAM2R = 88% vs. 19%, p<0.001; and AAβ1AR+AAM2R = 82% vs. 10%, p<0.001). The co-presence of AAβ1AR and AAM2R was the strongest predictor of AF (odds ratio 33.61, 95% CI 1.17 - 964.11, p=0.04). IgG from autoantibody-positive patients induced hyperpolarization, decreased action potential duration, enhanced early afterdepolarization formation and facilitated triggered firing in pulmonary veins by local autonomic nerve stimulation. Imunoadsorption studies demonstrated that AAβ1AR and AAM2R were immunologically distinct from TSHR antibodies. Conclusions AAβ1AR and AAM2R when present in patients with Graves’ hyperthyroidism facilitate development of AF.

Arrhythmia-prone subepicardial border zone (EBZ) tissue demonstrates decreased G protein receptor kinase 2 (GRK2) activity and increased sensitivity to isoproterenol 6 -24 h after coronary artery ligation (CAL) in the dog. With the use of a semiquantitative immunofluorescence technique, the relative fluorescence intensity (RF) of GRK2 in EBZ decreased to 24% of that in a remote site (RS) (P Ͻ 0.01, n ϭ 30 cells from 3 dogs), whereas GRK5 RF did not change. Confocal studies of cardiac tissue from transgenic mice overexpressing GRK2 validated the use of a semilogarithmic relationship between RF and GRK2 activity. As shown with the use of quantitative real-time RT-PCR, both GRK2 and GRK5 mRNA were not decreased at 24 h in EBZ (n ϭ 6 dogs) relative to RS control, indicating that the decrease of GRK2 in the EBZ is likely due to posttranscriptional degradation following CAL. Pretreatment of six dogs with the selective proteasome inhibitor bortezomib provided 100% (EBZ) and 50% (infarct) protection against loss of GRK2 at 24 h. There was an absence of rapid (Ͼ300 beats/min) and very rapid (Ͼ360 beats/min) ventricular triplets that are highly predictive of sudden cardiac death during ECG monitoring in the bortezomib-pretreated animals in contrast to nonpretreated infarcted animals. We have demonstrated that the dramatic decrease in GRK2 in cardiac ischemic tissue can be largely blocked by prior proteasome blockade and that this is associated with significant cardioprotection against malignant ventricular tachyarrhythmias.

Background-Activating autoantibodies to β-adrenergic receptors (AAβ1/2AR) and M2 muscarinic receptors (AAM2R) have been reported in several cardiac diseases and may have pathophysiologic relevance. However, the interactions and relative effects of AAβ1AR, AAβ2AR and AAM2R on contractile function have not been characterized. Methods-The inotropic effects of IgG from 18 selected patients with cardiomyopathy and/or atrial tachyarrhythmias positive by ELISA for antibodies to β1/2AR were studied using an isolated canine Purkinje fiber contractility assay. M2R-blockade was tested using atropine while selective β1AR and β2AR blockade used CGP-20712A and ICI-118551 respectively.Results-Fifteen of the 18 anti-β1/2AR ELISA-positive samples demonstrated evidence for negative inotropic muscarinic effects which were blocked using atropine. Atropine failed to uncover a positive inotropic response in 2 of the 18 IgG samples (false positive ELISA for AAβAR). In the remaining 16 AAβAR true-positive subjects, the β1AR-induced increase in contractility (concurrent M2/β2 blockade) was augmented to 140.5 ± 12.2% of baseline compared to 127.4 ± 7.2% of baseline with M2 blockade (atropine) only (p<0.001, n = 16). The β2AR-induced increase in contractility (concurrent M2/β1 blockade) was only 114.5 ± 4.3% of baseline (p<0.001, n = 16). Combined M2 and β1/β2 blockade eliminated any increase in contractility.Conclusions-The inherently positive inotropic effect of AAβ1AR was negatively modulated by AAM2R and AAβ2AR. These opposing effects of receptor-activating autoantibodies may alter cardiac performance and influence clinical outcome depending on their receptor type and relative contractile activity.

Background Orthostatic hypotension (OH) is characterized by an abnormal autonomic response to upright posture. Activating autoantibodies to β1/2-adrenergic (AAβ1/2AR) and M2/3 muscarinic receptors (AAM2/3R) produce vasodilative changes in the vasculature that may contribute to OH. Methods and Results IgG from 6 patients with idiopathic OH harboring autoantibodies and from 10 healthy control subjects were examined for: 1) β1AR and M2R activity with a perfused Purkinje fiber assay and PKA assay in H9c2 cells, and 2) vasodilator β2AR and M3R activity using a pressurized cremaster resistance arteriole assay. Changes of IgG activity with and without propranolol, atropine, and L-NAME were used to estimate AAβAR, AAM2R and AAM3R activation of their respective functions. All 6 patients had elevated ELISA titers to at least one of the receptors compared to controls. βAR-mediated contractility activity and M2R activity were increased in 5 of the 6 patients. IgG from all 6 patients produced a direct vasodilator effect upon cremaster arterioles. βAR and nitric oxide synthase blockade led to near normalization of IgG-induced vasodilation. Conclusion Aβ1/2AR and AAM2/3R are present in some patients with idiopathic OH compatible with an in vivo effect. These autoantibodies and their cardiovascular effects provide new mechanistic insights into the pathophysiology of OH.

Etanercept (2 mg/kg), a TNFalpha sequestrant, was administered 24 hours and 1 hour before LAD coronary artery ligation to examine the role of TNFalpha on lethal ventricular tachyarrhythmias and myocardial necrosis. Dogs treated with etanercept had decreased very rapid (>360 bpm) ventricular triplets (6 +/- 1/h, n = 8) 2 to 24 hours following coronary artery ligation compared with saline (21 +/- 6/h, n = 10, P < 0.05). This was concordant with 8-fold salvage of beta-adrenergic receptor kinase 1 (betaARK) activity compared with control (33.8 +/- 7.2% versus 4.3 +/- 2.2% of unoperated control tissue, P < 0.01, n = 5). Salvage of betaARK occurred without change in the thickness of the epicardial tissue overlying the infarct. In dogs pretreated with etanercept before a 2-hour occlusion/4-hour reperfusion of the LAD coronary artery, infarct mass decreased by 61% (% area at risk) and 55% (% left ventricular mass) in the etanercept group (n = 8) compared with saline (n = 9, P < 0.05). This was concordant with an etanercept-mediated six-fold decrease in leukocyte accumulation within ischemically injured myocardium. TNFalpha antagonism decreases malignant ventricular tachyarrhythmias and may relate to partial protection of normal betaARK-mediated desensitization of beta-adrenergic receptors. TNFalpha sequestration also decreases infarct size in an occlusion/reperfusion model of myocardial ischemia.