Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Background-Digitalis-induced Na ϩ accumulation results in an increase in Ca 2ϩ i via the Na ϩ /Ca 2ϩ exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca 2ϩ load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca 2ϩ i could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and Results-In paced rat ventricular myocytes (0.5 Hz), 50 mol/L ouabain increased contraction amplitude by 160Ϯ5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca 2ϩ waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 mol/L KN93 but not by 2.5 mol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 mol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca 2ϩ content and Ca 2ϩ spark frequency, indicative of enhanced SR Ca 2ϩ leak. KN93 suppressed the ouabain-induced increase in Ca 2ϩ spark frequency without affecting SR Ca 2ϩ content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions-These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca 2ϩ leak from the SR, is the underlying mechanism involved. (Circ Arrhythm Electrophysiol. 2011;4:947-957.) Key Words: cardiotonic steroids Ⅲ arrhythmias Ⅲ CaMKII Ⅲ heart failure C ardiotonic glycosides selectively bind to and inhibit the sarcolemmal Na ϩ /K ϩ -ATPase and cause an increase in intracellular Na ϩ , which in the heart reduces Ca 2ϩ extrusion and/or increases Ca 2ϩ influx through the Na ϩ /Ca 2ϩ exchanger (NCX). This increase in Ca 2ϩ i leads to an increase in sarcoplasmic reticulum (SR) Ca 2ϩ load and to a positive inotropic effect, which explains, at least in part, their thera- Clinical Perspective on p 957peutic use for heart failure treatment 1 ; however, these compounds have associated arrhythmic/toxic effects that conspire against their extensive use in the clinical practice. 2 The arrhythmic effects have been proposed to occur when the SR Ca 2ϩ storage capacity is exceeded so that oscillations of release-uptake cycles arise to re-establish the Ca 2ϩ equilibrium between the cytosol and the SR. These transient increases in Ca 2ϩ i (Ca 2ϩ waves) activate a transient inward (depolarizing) current (I ti ), primarily mediated by the forward-mode ...

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Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca content, Ca transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.

Background: Hypothyroidism, the most common endocrine disease, induces cardiac electrical remodeling that creates a substrate for ventricular arrhythmias. Recent studies report that high thyrotropin (TSH) levels are related to cardiac electrical abnormalities and increased mortality rates. The aim of the present work was to investigate the direct effects of TSH on the heart and its possible causative role in the increased incidence of arrhythmia in hypothyroidism. Methods: A new rat model of central hypothyroidism (low TSH levels) was created and characterized together with the classical propylthiouracil-induced primary hypothyroidism model (high TSH levels). Electrocardiograms were recorded in vivo , and ionic currents were recorded from isolated ventricular myocytes in vitro by the patch-clamp technique. Protein and mRNA were measured by Western blot and quantitative reverse transcription polymerase chain reaction in rat and human cardiac myocytes. Adult human action potentials were simulated in silico to incorporate the experimentally observed changes. Results: Both primary and central hypothyroidism models increased the L-type Ca 2+ current (I Ca-L ) and decreased the ultra-rapid delayed rectifier K + current (I Kur ) densities. However, only primary but not central hypothyroidism showed electrocardiographic repolarization abnormalities and increased ventricular arrhythmia incidence during caffeine/dobutamine challenge. These changes were paralleled by a decrease in the density of the transient outward K + current (I to ) in cardiomyocytes from animals with primary but not central hypothyroidism. In vitro treatment with TSH for 24 hours enhanced isoproterenol-induced spontaneous activity in control ventricular cells and diminished I to density in cardiomyocytes from control and central but not primary hypothyroidism animals. In human myocytes, TSH decreased the expression of KCND3 and KCNQ1 , I to , and the delayed rectifier K + current (I Ks ) encoding proteins in a protein kinase A–dependent way. Transposing the changes produced by hypothyroidism and TSH to a computer model of human ventricular action potential resulted in enhanced occurrence of early afterdepolarizations and arrhythmia mostly in primary hypothyroidism, especially under β-adrenergic stimulation. Conclusions: The results suggest that suppression of repolarizing K + currents by TSH underlies most of the electrical remodeling observed in hypothyroidism. This work demonstrates that the activation of the TSH-receptor/protein kinase A pathway in the heart is...

Hemodynamic collapse and myocardial dysfunction are among the major causes of death in severe sepsis. The purpose of this study was to assess the role played by TLR4 and by the NLRP3 inflammasome in the cardiac dysfunction that occurs after high-grade polymicrobial sepsis. We performed the colon ascendens stent peritonitis (CASP) surgery in Tlr4, Nlrp3 and caspase-1 mice. We also assessed for the first time the electrical heart function in the CASP model. The QJ interval was increased in wild-type C57BL/6J mice after CASP when compared to sham controls, a result paralleled by an increase in the cardiac action potential duration (APD). The decreases in ejection fraction (EF), left-ventricle end diastolic volume (LVEDV), stroke volume, and cardiac output found after CASP were similar among all groups of mice. Similar heart response was found when Nlrp3 mice were submitted to high-grade CLP. Despite developing cardiac dysfunction similar to wild-types after CASP, Nlrp3 mice had reduced circulating levels of IL-1β, IL-6 and TNF-α. Our results demonstrate that the genetic ablation of Tlr4, Nlrp3, and caspase-1 does not prevent the cardiac dysfunction, despite preventing the increase in pro-inflammatory cytokines, indicating that these are not feasible targets to therapy in high-grade sepsis.

Background: It has been shown that carvedilol and its non β-blocking analog, VK-II-86, inhibit spontaneous Ca 2+ release from the sarcoplasmic reticulum (SR). The aim of this study is to determine whether carvedilol and VK-II-86 suppress ouabain-induced arrhythmogenic Ca 2+ waves and apoptosis in cardiac myocytes. Methods and Results: Rat cardiac myocytes were exposed to toxic doses of ouabain (50 µmol/L). Cell length (contraction) was monitored in electrically stimulated and non-stimulated conditions. Ouabain treatment increased contractility, frequency of spontaneous contractions and apoptosis compared to control cells. Carvedilol (1 µmol/L) or VK-II-86 (1 µmol/L) did not affect ouabain-induced inotropy, but significantly reduced the frequency of Ca 2+ waves, spontaneous contractions and cell death evoked by ouabain treatment. This antiarrhythmic effect was not associated with a reduction in Ca 2+ calmodulin-dependent protein kinase II (CaMKII) activity, phospholamban and ryanodine receptor phosphorylation or SR Ca 2+ load. Similar results could be replicated in human cardiomyocytes derived from stem cells and in a mathematical model of human myocytes. Conclusions: Carvedilol and VK-II-86 are effective to prevent ouabain-induced apoptosis and spontaneous contractions indicative of arrhythmogenic activity without affecting inotropy and demonstrated to be effective in human models, thus emerging as a therapeutic tool for the prevention of digitalis-induced arrhythmias and cardiac toxicity.

MicroRNA-150 (miR-150) is downregulated in patients with multiple cardiovascular diseases and in diverse mouse models of heart failure (HF). miR-150 is significantly associated with HF severity and outcome in humans. We previously reported that miR-150 is activated by β-blocker carvedilol (Carv) and plays a protective role in the heart using a systemic miR-150 KO mouse model. However, mechanisms that regulate cell-specific miR-150 expression and function in HF are unknown. Here, we demonstrate that potentially novel conditional cardiomyocyte–specific (CM-specific) miR-150 KO (miR-150 cKO) in mice worsens maladaptive cardiac remodeling after myocardial infarction (MI). Genome-wide transcriptomic analysis in miR-150 cKO mouse hearts identifies small proline–rich protein 1a ( Sprr1a ) as a potentially novel target of miR-150. Our studies further reveal that Sprr1a expression is upregulated in CMs isolated from ischemic myocardium and subjected to simulated ischemia/reperfusion, while its expression is downregulated in hearts and CMs by Carv. We also show that left ventricular SPRR1A is upregulated in patients with HF and that Sprr1a knockdown in mice prevents maladaptive post-MI remodeling. Lastly, protective roles of CM miR-150 are, in part, attributed to the direct and functional repression of proapoptotic Sprr1a . Our findings suggest a crucial role for the miR-150/SPRR1A axis in regulating CM function post-MI.