Many signals have risen and fallen in the tide of investigation into mechanisms of myocardial hypertrophy and heart failure (HF). In our opinion, the multifunctional Ca and calmodulin-dependent protein kinase II (CaMKII) has emerged as a molecule to watch, in part because a solid body of accumulated data essentially satisfy Koch's postulates, showing that the CaMKII pathway is a core mechanism for promoting myocardial hypertrophy and heart failure. Multiple groups have now confirmed the following: (1) that CaMKII activity is increased in hypertrophied and failing myocardium from animal models and patients; (2) CaMKII overexpression causes myocardial hypertrophy and HF and (3) CaMKII inhibition (by drugs, inhibitory peptides and gene deletion) improves myocardial hypertrophy and HF. Patients with myocardial disease die in equal proportion from HF and arrhythmias, and a major therapeutic obstacle is that drugs designed to enhance myocardial contraction promote arrhythmias. In contrast, inhibiting the CaMKII pathway appears to reduce arrhythmias and improve myocardial responses to pathological stimuli. This brief paper will introduce the molecular physiology of CaMKII and discuss the impact of CaMKII on ion channels, Ca handling proteins and transcription in myocardium. This article is part of a Special Issue entitled “Key Signaling Molecules Special Issue”.

Right ventricular failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, was recently approved for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate 1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; 2) whether the ultrastructural changes are reversible; 3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline (MCT) in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe T-tubule loss and disorganization as well as blunted and dys-synchronous SR Ca2+ release. Sildenafil prevented and reversed the MCT-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling and Ca2+ handling dysfunction. While late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca2+ handling protein and SR Ca2+ release function in MCT-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca2+ handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.

The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (KATP) channels. KATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative KATP channel subunit were compared with littermate controls. Evaluation of KATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80-85% reduction in cardiac KATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal KATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.

Objective

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR).

Rationale

We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca]i) handling.

Methods

Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ−/−, KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 µmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS]i) and [Ca]i handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 µmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 µmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation.

Results

DOX increased [ROS]i and led to significant diastolic [Ca]i overload in rat myocytes. This was associated with reduced [Ca]i transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca]i overload after DOX superfusion and led to partially restored [Ca]i transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca]i overload and Ca spark frequency.

Conclusions

DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca]i homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca]i. In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.

Background

Timothy Syndrome (TS) is a disease of excessive cellular Ca2+ entry and life-threatening arrhythmias due to a mutation in the primary cardiac L-type Ca2+ channel (CaV1.2). The TS mutation causes loss of normal voltage-dependent inactivation (VDI) of CaV1.2 current (ICa). During cellular Ca2+ overload the calmodulin-dependent protein kinase II (CaMKII) causes arrhythmias. We hypothesized that CaMKII is a part of the proarrhythmic mechanism in TS.

Methods and Results

We developed an adult rat ventricular myocyte model of TS (G406R) by lenti virus-mediated transfer of wild type (WT) and TS CaV1.2. The exogenous CaV1.2 contained a mutation (T1066Y) conferring dihydropyridine resistance, so we could silence endogenous CaV1.2 with nifedipine and maintain peak ICa at control levels in infected cells. TS CaV1.2 infected ventricular myocytes exhibited the signature VDI loss under Ca2+ buffering conditions, not permissive for CaMKII activation. In physiological Ca2+ solutions, TS CaV1.2 expressing ventricular myocytes exhibited increased CaMKII activity and a proarrhythmic phenotype that included action potential prolongation, increased ICa facilitation and afterdepolarizations. Intracellular dialysis of a CaMKII inhibitory peptide, but not a control peptide, reversed increases in ICa facilitation, normalized the action potential and prevented afterdepolarizations. We developed a revised mathematical model that accounts for CaMKII-dependent and CaMKII-independent effects of the TS mutation.

Conclusions

In TS the loss of VDI is an upstream initiating event for arrhythmia phenotypes that are ultimately dependent on CaMKII activation.

Patients with systolic left ventricular dysfunction die progressively from congestive heart failure or die suddenly from cardiac arrhythmias. Myocardial hypertrophy is an early event in most forms of heart failure, but the majority of patients with myocardial hypertrophy do not develop heart failure. Developing improved therapies for targeting the cell signaling pathways that enable this deadly transition from early myocardial insult to heart failure and sudden death is a key goal for improving public health. In this issue of the JCI, Ling and colleagues provide new evidence that activation of the multifunctional Ca2+/calmodulin–dependent kinase IIδ is a decisive step on the path to heart failure in mice (see the related article beginning on page 1230).

Physical activity is one of the most important determinants of cardiac function. The ability of the heart to increase delivery of oxygen and metabolic fuels relies on an array of adaptive responses necessary to match bodily demand while avoiding exhaustion of cardiac resources. The ATP-sensitive potassium (KATP) channel has the unique ability to adjust cardiac membrane excitability in accordance with ATP and ADP levels, and up-regulation of its expression that occurs in response to exercise could represent a critical element of this adaption. However, the mechanism by which KATP channel expression changes result in a beneficial effect on cardiac excitability and function remains to be established. Here, we demonstrate that an exercise-induced rise in KATP channel expression enhanced the rate and magnitude of action potential shortening in response to heart rate acceleration. This adaptation in membrane excitability promoted significant reduction in cardiac energy consumption under escalating workloads. Genetic disruption of normal KATP channel pore function abolished the exercise-related changes in action potential duration adjustment and caused increased cardiac energy consumption. Thus, an expression-driven enhancement in the KATP channel-dependent membrane response to alterations in cardiac workload represents a previously unrecognized mechanism for adaptation to physical activity and a potential target for cardioprotection.

Background

Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is both necessary and sufficient to promote lethal ventricular arrhythmias.

Methods and Results

Mice in which the S2814 CaMKII site on RyR2 is constitutively activated (S2814D) develop pathological SR Ca2+ release events resulting in reduced SR Ca2+ load on confocal microscopy. These Ca2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death upon catecholaminergic provocation by caffeine/epinephrine or programmed electrical stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction (TAC) surgery. Finally, genetic ablation of the CaMKII site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild type mice after TAC surgery.

Conclusions

Our results suggest that CaMKII phosphorylation of RyR2 Ca2+ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Background

Dilated cardiomyopathy (DCM), characterized by dilatation and dysfunction of the left ventricle, is an important cause of heart failure. Many mutations in various genes, including cytoskeletal protein genes and contractile protein genes, have been identified in DCM patients, but the mechanisms of how such mutations lead to DCM remain unknown.

Methods and Results

We established the mouse model of DCM by expressing a mutated cardiac α-actin gene, which has been reported in patients with DCM, in the heart (mActin-Tg). mActin-Tg mice showed gradual dilatation and dysfunction of the left ventricle, resulting in death by heart failure. The number of apoptotic cardiomyocytes and protein levels of p53 were increased in the hearts of mActin-Tg mice. Overexpression of Bcl-2 or downregulation of p53 decreased the number of apoptotic cardiomyocytes and improved cardiac function. This mouse model showed a decrease in myofilament calcium sensitivity and activation of calcium/calmodulin-dependent kinase IIδ (CaMKIIδ). The inhibition of CaMKIIδ prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function.

Conclusion

CaMKIIδ plays a critical role in the development of heart failure in part by accumulation of p53 and induction of cardiomyocyte apoptosis in the DCM mouse model.

Rationale

The transverse tubule (t-tubule) system is the ultrastructural substrate for excitation-contraction coupling in ventricular myocytes; t-tubule disorganization and loss are linked to decreased contractility in end stage heart failure (HF).

Objective

To examine 1) if pathological t-tubule remodeling occurs early in compensated hypertrophy and, if so, how it evolves during the transition from hypertrophy to HF; 2) the role of junctophilin-2 in t-tubule remodeling.

Methods and Results

We investigated t-tubule remodeling in relation to ventricular function during HF progression using state-of-the-art confocal imaging of t-tubules in intact hearts, using a thoracic aortic banding (TAB) rat HF model. We developed a quantitative t-tubule power (TTpower) index to represent the integrity of t-tubule structure. We found that discrete local loss and global reorganization of the t-tubule system (leftward shift of TTpower histogram) started early in compensated hypertrophy in left ventricular (LV) myocytes, prior to LV dysfunction, as detected by echocardiography. With progression from compensated hypertrophy to early and late HF, t-tubule remodeling spread from the LV to the right ventricle (RV), and TTpower histograms of both ventricles gradually shifted leftward. The mean LV TTpower showed a strong correlation with ejection fraction and heart weight to body weight ratio. Over the progression to HF we observed a gradual reduction in the expression of a junctophilin protein (JP-2) implicated in the formation of t-tubule / sarcoplasmic reticulum junctions. Furthermore, we found that JP-2 knockdown by gene silencing reduced t-tubule structure integrity in cultured adult ventricular myocytes.

Conclusions

T-tubule remodeling in response to TAB stress begins prior to echocardiographically detectable LV dysfunction and progresses over the development of overt structural heart disease. LV t-tubule remodeling is closely associated with the severity of cardiac hypertrophy and predicts LV function. Thus, t-tubule remodeling may constitute a key mechanism underlying the transition from compensated hypertrophy to HF.

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a ‘voltage clock’ and a Ca2+ dependent process, or ‘Ca2+ clock.’ The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (If) is thought to be particularly important. A Ca2+ dependent process triggers APs when sarcoplasmic reticulum (SR) Ca2+ release activates inward current carried by the forward mode of the electrogenic Na+/Ca2+ exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca2+ clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective If antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest (∼14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca2+ release, but did not affect basal or isoproterenol-enhanced If. Taken together, these results indicate that voltage and Ca2+ dependent automaticity mechanisms coexist in canine SAN cells, and suggest If and SR Ca2+ release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.

Emerging evidence from large animal models implicates Ca2+ regulation, particularly intracellular sarcoplasmic reticulum (SR) Ca2+ release, as essential for sinoatrial node (SAN) automaticity. However, despite the apparent importance of SR Ca2+ release to SAN cell function it is uncertain how SR Ca2+ release is controlled in SAN cells from mouse. Understanding mouse SAN SR Ca2+ release mechanism will allow improved understanding of results in studies on SAN from genetic mouse models of Ca2+ homeostatic proteins. Here we investigated the functional relationship between sarcolemmal Ca2+ influx and SR Ca2+ release at the level of single SAN cell, using simultaneous patch-clamp current recording and high resolution confocal Ca2+ imaging techniques. In mouse SAN cells, both Ca2+ channel currents and triggered SR Ca2+ transients displayed bell-shaped, graded function with the membrane potential. Moreover, the gain function for Ca2+-induced Ca2+ release (CICR) displayed a monotonically decreasing function with strong voltage-dependence, consistent with a ‘local control’ mechanism for CICR. In addition, we observed numerous discrete Ca2+ sparks at the voltage range of diastolic depolarization, in sharp contrast to the much lower frequency of sparks observed at resting potentials. We concluded that the ‘local control’ mechanism of CICR is responsible for both local Ca2+ release during diastolic depolarization and the synchronized Ca2+ transients observed during action potential in SAN cells.

Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltage-gated Na+ channels underlie cellular excitability and control neuronal communication, cardiac excitation-contraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na+ channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified βIV-spectrin as a multifunctional regulatory platform for Na+ channels in mice. We found that βIV-spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant βIV-spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na+ channels, via direct phosphorylation by βIV-spectrin–targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the β2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with β2a in cardiomyocytes and also binds to a domain in β2a that contains Thr498 and exhibits amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter et al., 2006. Mol. Cell 23:641). Here we explore the selectivity of the actions of CaMKII among Ca2+ channel β subunit isoforms. CaMKII phosphorylates the β1b, β2a, β3 and β4 isoforms with similar initial rates and final stoichiometries of 6–12 mole phosphate per mole protein. However, activated/autophosphorylated CaMKII binds to β1b and β2a with similar apparent affinity, but does not bind to β3 or β4. Pre-phosphorylation of β1b and β2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in β2a are highly conserved in β1b, but are different in β3 and β4. Site-directed mutagenesis of this domain in β2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-β2a complexes in vitro and reduces interactions of CaMKII with β2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with β2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the VGCC β subunits with CaMKII.

Calmodulin kinase II (CaMKII) mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ). These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart.

Author Summary

Calmodulin kinase II (CaMKII) is a multifunctional serine/threonine kinase that regulates diverse functions in heart. Recently, a novel pathway for CaMKII activation was discovered where oxidation of the kinase at specific methionine residues produces persistent activity. This alternative oxidation-dependent pathway has important implications for heart disease where oxidative stress is increased (e.g., heart failure and following myocardial infarction). We hypothesized that myocardial infarction caused by occlusion of a coronary artery would increase levels of oxidized CaMKII. Moreover, we hypothesized that oxidative CaMKII activation represents an important mechanistic link between increased oxidative stress and life-threatening heart rhythm disturbances (arrhythmias) in heart disease. We report a dramatic increase in levels of oxidized CaMKII following myocardial infarction in the canine. Based on these experimental data, we developed a novel mathematical model of CaMKII activity to study the role of oxidation-dependent CaMKII activation in regulating cardiac cell excitability. Our findings identify a novel role for oxidation-dependent CaMKII activation following myocardial infarction and provide a mechanistic link between oxidative stress and lethal cardiac arrhythmias in heart disease.

ER stress–induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress–mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress–induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and JNK. Remarkably, CaMKIIγ was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress–induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIγ-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress–induced apoptosis.

Atrial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ 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 Ryr2R176Q/+ 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. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

Transgenic expression of enhanced green fluorescent protein (eGFP) in myocardium can result in cardiac dysfunction and cardiomyopathy, presumably through toxic effects that disrupt normal cellular signaling. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is widely expressed in myocardium and CaMKII activity is increased in human and animal models of cardiomyopathy, so we hypothesized that increased CaMKII activity is important for cardiomyopathy due to transgenic expression of eGFP. Here we report that cardiomyocyte-delimited eGFP over-expression causes increased CaMKII activity that predicts left ventricular dilation and dysfunction. On the other hand, transgenic co-expression of a CaMKII inhibitory peptide with eGFP prevents eGFP-mediated left ventricular dilation and dysfunction. These findings suggest that increased CaMKII activity is a critical pathological signal in transgenic cardiomyopathy due to eGFP over-expression.

Myocardial Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-κB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-κB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb–/– mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.

Voltage-gated Nav channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Nav1.5 is the predominant Nav channel, and Nav1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Nav1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Nav channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Nav1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Nav1.5 expression, abnormal Nav1.5 membrane targeting, and reduced Na+ channel current density. We define the structural requirements on ankyrin-G for Nav1.5 interactions and demonstrate that loss of Nav1.5 targeting is caused by the loss of direct Nav1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Nav channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Nav channel organization in multiple excitable tissues.