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.

Ryanodine Receptors (RyRs) are intracellular Ca channels that mediate Ca flux from the sarco(endo)plasmic reticulum in many cell types. The interaction of RyRs with FK506-binding proteins (FKBPs) has been proposed as an important regulatory mechanism, where the loss of this interaction leads to channel dysfunction. In the heart, phosphorylation of RyR has been suggested to disrupt the RyR-FKBP interaction promoting altered Ca signaling, heart failure and arrhythmias. However, the functional result of FKBP interaction with RyR and how this interaction is regulated remains highly controversial. Recently, high resolution structures of RyR have provided novel aspects to the ongoing debate. This review will discuss the most recent functional data in light of these new structures.

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Our findings suggest a novel mechanism for NO release in cardiomyocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hypotonic swelling.

In different pathological situations, cardiac cells undergo hyperosmotic stress (HS) and cell shrinkage. This change in cellular volume has been associated with contractile dysfunction and cell death. Given that nitric oxide (NO) is a well-recognized modulator of cardiac contractility and cell survival, we evaluated whether HS increases NO production and its impact on the negative inotropic effect observed during this type of stress. Superfusing cardiac myocytes with a hypertonic solution (HS: 440 mOsm) decreased cell volume and increased NO-sensitive DAF-FM fluorescence compared with myocytes superfused with an isotonic solution (IS: 309 mOsm). When cells were exposed to HS in addition to different inhibitors: L-NAME (NO synthase inhibitor), nitroguanidine (nNOS inhibitor), and Wortmannin (eNOS inhibitor) cell shrinkage occurred in the absence of NO release, suggesting that HS activates nNOS and eNOS. Consistently, western blot analysis demonstrated that maintaining cardiac myocytes in HS promotes phosphorylation and thus, activation of nNOS and eNOS compared to myocytes maintained in IS. HS-induced nNOS and eNOS activation and NO production were also prevented by AMPK inhibition with Dorsomorphin (DORSO). In addition, the HS-induced negative inotropic effect was exacerbated in the presence of either L-NAME, DORSO, ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), suggesting that NO provides contractile support via a cGMP/PKG-dependent mechanism. Our findings suggest a novel mechanism of AMPK-dependent NO release in cardiac myocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hyperosmotic stress.