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Background We sought to determine national trends and long term outcomes of post myocardial infarction (MI) heart failure. An MI can be complicated by heart failure; there are limited data describing the contemporary patterns and clinical implications of post-MI heart failure. Methods and Results We studied patients with an MI aged 65 years or older from 2000 to 2013 in a Medicare database. New-onset heart failure after an MI was defined as either heart failure during the index MI admission or a hospitalization for heart failure within 1 year of the index MI event. A trend analysis of the incidence of heart failure was performed, and differences were examined by Gray tests. The 5-year mortality rates were evaluated and differences among heart failure cohorts were ascertained by Gray tests. There were a total of 1,531,638 patients with an MI and 565,291 patients had heart failure (36.0%). The rate of heart failure during index admission was 32.3% and the frequency of heart failure hospitalization within 1 year was 10.4%. Patients with heart failure were older (81 years vs 77 years). The temporal trend from 2001 to 2012 suggested a decrease in the incidence of heart failure during index admission (2001: 34.7%, 2012: 31.2%, Ptrend Conclusions Post-MI heart failure in older adults occurs in 1 in 3 patients within 1 year; heart failure portends significantly higher long-term mortality.

Visual Abstract, Highlights • The pathogenesis of human heart failure is complex, and the creation of new therapeutic strategies for human heart failure is critical. • Identifying the molecular pathways underlying heart failure is important to define potential new therapeutic targets. • Ankyrin polypeptides serve to target and stabilize membrane proteins in cardiomyocytes. • Ankyrin-G levels are altered in humans and mice with heart failure, and mice lacking ankyrin-G in cardiomyocytes develop cardiomyopathy and systolic dysfunction. • Mechanistically, ankyrin-G is necessary for the expression and localization of critical myocyte proteins essential for regulating cardiac structural and electrical activity., Summary Ankyrin polypeptides are intracellular proteins responsible for targeting cardiac membrane proteins. Here, the authors demonstrate that ankyrin-G plays an unexpected role in normal compensatory physiological remodeling in response to myocardial stress and aging; the authors implicate disruption of ankyrin-G in human heart failure. Mechanistically, the authors illustrate that ankyrin-G serves as a key nodal protein required for cardiac myofilament integration with the intercalated disc. Their data define novel in vivo mechanistic roles for ankyrin-G, implicate ankyrin-G as necessary for compensatory cardiac physiological remodeling under stress, and implicate disruption of ankyrin-G in the development and progression of human heart failure.

Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca2+ channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca2+ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca2+ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction. Background: Endosome-based protein trafficking in the heart is poorly understood. Results: Functional targeting and expression of the T-type Ca2+ channel (TTCC) in the atria requires the endosomal protein, EHD3. Conclusion: Impaired endosomal transport leads to cardiac rhythm and conduction disorders. Significance: Understanding endosome-based protein trafficking in the heart may provide new therapeutic targets for cardiovascular disease.

Rationale: Na v 1.5 ( SCN5A ) is the primary cardiac voltage-gated Na v channel. Na v 1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Na v 1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Na v 1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Na v 1.5 membrane targeting, assembly, and regulation are still largely undefined. Objective: Define the in vivo mechanisms underlying Na v 1.5 membrane regulation. Methods and Results: Here, we define the molecular basis of an Na v channel regulatory platform in heart. Using new cardiac-selective ankyrin-G -/- mice (conditional knock-out mouse), we report that ankyrin-G targets Na v 1.5 and its regulatory protein calcium/calmodulin–dependent kinase II to the intercalated disc. Mechanistically, β IV -spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin–dependent kinase II-δ; however, β IV -spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Na v 1.5 expression/membrane localization and reduced I Na associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Na v channel antagonists. Moreover, we report that ankyrin-G links Na v channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation. Conclusions: Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Na v 1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Na v 1.5.

Rationale : Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. Objective : To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. Methods and Results : We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger–mediated membrane current and Ca v 1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes. Conclusions : These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.

N-type and P/Q-type calcium channels are documented players in the regulation of synaptic function; however, the mechanisms underlying their expression and cellular targeting are poorly understood. Ankyrin polypeptides are essential for normal integral membrane protein expression in a number of cell types, including neurons, cardiomyocytes, epithelia, secretory cells, and erythrocytes. Ankyrin dysfunction has been linked to defects in integral protein expression, abnormal cellular function, and disease. Here, we demonstrate that ankyrin-B associates with Cav2.1 and Cav2.2 in cortex, cerebellum, and brain stem. Additionally, using in vitro and in vivo techniques, we demonstrate that ankyrin-B, via its membrane-binding domain, associates with a highly conserved motif in the DII/III loop domain of Cav2.1 and Cav2.2. Further, we demonstrate that this domain is necessary for proper targeting of Cav2.1 and Cav2.2 in a heterologous system. Finally, we demonstrate that mutation of a single conserved tyrosine residue in the ankyrin-binding motif of both Cav2.1 (Y797E) and Cav2.2 (Y788E) results in loss of association with ankyrin-B in vitro and in vivo. Collectively, our findings identify an interaction between ankyrin-B and both Cav2.1 and Cav2.2 at the amino acid level that is necessary for proper Cav2.1 and Cav2.2 targeting in vivo.

Background Human ANK2 (ankyrin-B) loss-of-function variants are directly linked with arrhythmia phenotypes. However, in atypical non–ion channel arrhythmia genes such as ANK2 that lack the same degree of robust structure/function and clinical data, it may be more difficult to assign variant disease risk based simply on variant location, minor allele frequency, and/or predictive structural algorithms. The human ankyrin-B p.L1622I variant found in arrhythmia probands displays significant diversity in minor allele frequency across populations. Objective The objective of this study was to directly test the in vivo impact of ankyrin-B p.L1622I on cardiac electrical phenotypes and arrhythmia risk using a new animal model. Methods We tested arrhythmia phenotypes in a new "knock-in" animal model harboring the human ankyrin-B p.L1622I variant. Results Ankyrin-B p.L1622I displays reduced posttranslational expression in vivo, resulting in reduced cardiac ankyrin-B expression and reduced association with binding-partner Na/Ca exchanger. Ankyrin-B L1622I/L1622I mice display changes in heart rate, atrioventricular and intraventricular conduction, and alterations in repolarization. Furthermore, ankyrin-B L1622I/L1622I mice display catecholamine-dependent arrhythmias. At the cellular level, ankyrin-B L1622I/L1622I myocytes display increased action potential duration and severe arrhythmogenic afterdepolarizations that provide a mechanistic rationale for the arrhythmias. Conclusion Our findings support in vivo arrhythmogenic phenotypes of an ANK2 variant with unusual frequency in select populations. On the basis of our findings and current clinical data, we support classification of p.L1622I as a "mild" loss-of-function variant that may confer arrhythmia susceptibility in the context of secondary risk factors including environment, medication, and/or additional genetic variation.

β2-Spectrin is critical for integrating membrane and cytoskeletal domains in excitable and nonexcitable cells. The role of β2-spectrin for vertebrate function is illustrated by dysfunction of β2-spectrin-based pathways in disease. Recently, defects in β2-spectrin association with protein partner ankyrin-B were identified in congenital forms of human arrhythmia. However, the role of β2-spectrin in common forms of acquired heart failure and arrhythmia is unknown. We report that β2-spectrin protein levels are significantly altered in human cardiovascular disease as well as in large and small animal cardiovascular disease models. Specifically, β2-spectrin levels were decreased in atrial samples of patients with atrial fibrillation compared with tissue from patients in sinus rhythm. Furthermore, compared with left ventricular samples from nonfailing hearts, β2-spectrin levels were significantly decreased in left ventricle of ischemic- and nonischemic heart failure patients. Left ventricle samples of canine and murine heart failure models confirm reduced β2-spectrin protein levels. Mechanistically, we identify that β2-spectrin levels are tightly regulated by posttranslational mechanisms, namely Ca2+- and calpain-dependent proteases. Furthermore, consistent with this data, we observed Ca2+- and calpain-dependent loss of β2-spectrin downstream effector proteins, including ankyrin-B in heart. In summary, our findings illustrate that β2-spectrin and downstream molecules are regulated in multiple forms of cardiovascular disease via Ca2+- and calpain-dependent proteolysis.

Background— Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked to potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite more than a decade of investigation. Posttranslational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel posttranslational modifications and disease. We recently identified a novel pathway for posttranslational regulation of the primary cardiac voltage-gated Na + channel (Na v 1.5) by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII). However, a role for this pathway in cardiac disease has not been evaluated. Methods and Results— We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Na v 1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Na v 1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Na v 1.5, resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel, resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large-animal model of acquired heart disease and in failing human myocardium. Conclusions— We identify the mechanism for 2 human arrhythmia variants that affect Na v 1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Na v 1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Na v 1.5 in congenital and acquired cardiac disease.

The pace of coatings development is limited by the time required to assess their corrosion protection properties. This study takes a step forward from Part I of this series and correlates the corrosion performance of organic coatings assessed by a series of short-term electrochemical measurement with 18-month beachside exposure results of duplicate panels. A series of 19 coating systems on A36 steel substrates were tested in a blind study using the damage tolerance test (DTT). In the DTT, a through-film pinhole defect is created, and the electrochemical characteristics of the defect are then monitored over the next 4 to 7 days while immersed in 0.5M NaCl. The results from open circuit potential, anodic potentiostatic polarization, and electrochemical impedance spectroscopy were used to characterize the corrosion behavior of the coating systems. The beachside exposure tests were conducted at the Kennedy Space Center according to ASTM D610-01. It was found that for 79% of the coatings systems examined, the 18 month beachside exposure results could be predicted by two independent laboratory tests obtained within 7 days.

Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α(+/-) myocytes resulted in reduced Ca(2+) waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca(2+) regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.

Background Identified genetic variants are insufficient to explain all cases of inherited arrhythmia. We tested whether the integration of whole exome sequencing with well‐established clinical, translational, and basic science platforms could provide rapid and novel insight into human arrhythmia pathophysiology and disease treatment. Methods and Results We report a proband with recurrent ventricular fibrillation, resistant to standard therapeutic interventions. Using whole‐exome sequencing, we identified a variant in a previously unidentified exon of the dipeptidyl aminopeptidase‐like protein‐6 ( DPP 6 ) gene. This variant is the first identified coding mutation in DPP 6 and augments cardiac repolarizing current ( I to ) causing pathological changes in I to and action potential morphology. We designed a therapeutic regimen incorporating dalfampridine to target I to . Dalfampridine, approved for multiple sclerosis, normalized the ECG and reduced arrhythmia burden in the proband by >90‐fold. This was combined with cilostazol to accelerate the heart rate to minimize the reverse‐rate dependence of augmented I to . Conclusions We describe a novel arrhythmia mechanism and therapeutic approach to ameliorate the disease. Specifically, we identify the first coding variant of DPP 6 in human ventricular fibrillation. These findings illustrate the power of genetic approaches for the elucidation and treatment of disease when carefully integrated with clinical and basic/translational research teams.

The ability to dynamically regulate, traffic, retain, and recycle proteins within the cell membrane is fundamental to life and central to the normal function of the heart and cardiovascular system. In the heart, these systems are essential for the regulation of cardiac calcium, both at the level of the plasma membrane, but also at local domains of the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, nucleus, and nuclear envelope. One intracellular pathway often overlooked in relation to cardiovascular calcium regulation and signaling is the endosome-based trafficking pathway. Highlighting its importance, this system and its molecular components are evolutionarily conserved across all metazoans. However, remarkably little is known of how endosome-based protein trafficking and recycling functions within mammalian cells systems, especially in the heart. The vast majority of what is known has been derived from heterologous cell systems. However, recently, more appropriate cell and animal models been developed that have allowed researchers to begin to understand how this system functions within the intact physiological environment. All excitable cells, including cardiomyocytes, depend on the proper expression and organization of multiple ion channels, pumps, exchangers, and transporters within the plasma membrane. As the endosomal system acts to regulate the expression and localization of membrane proteins, understanding the in vivo function of this system in the heart is important. This review will focus on endosome-based protein trafficking in the heart in both health and disease. Special emphasis will be given to the role played by the family of endocytic regulatory proteins, C-terminal Eps15 homology domain -containing proteins (EHDs), as recent data demonstrates that this family of proteins is essential for the proper trafficking and localization and of key proteins involved in excitation-contraction coupling.

Background— The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. Methods and Results— Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin–deficient mice. Conclusions— Our findings identify βII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin–dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF.

It is estimated that nearly seven million individuals within the United States are currently diagnosed with heart failure (HF).1 One in nine deaths in the United States is directly related to HF. Despite great advances in both HF diagnostics and therapies, the 5-year mortality rate is approximately 50%.1 HF is a multifactorial disease state with neither one singular cause, nor one singular progression path. The symptoms of HF can be subtle and practically unnoticeable, or they can be severe as dementia or complete incapacitation. In both humans and animal models, HF is inextricably linked with structural and electrical remodeling of the heart. This remodeling is an adaptive response to maintain cardiac output following insult or injury to the heart. The most common trigger for remodeling is the myocardial infarct (MI). Large tracts of previously functional myocardium are damaged or destroyed ensuing from ischemia and necrosis and are eventually replaced by scar tissue (primarily non-contractile fibrotic tissue). Ultimately, the overall contractile force of the heart is diminished, and the remaining healthy muscle must strengthen (hypertrophy) and electrically remodel in order to compensate for this loss. Electrophysiological changes in the myocardium (both atrial and ventricular) have been observed in HF and hypertrophy for well over 40 years. Slowed conduction and prolongation of the action potential (AP) are characteristics of HF in both the clinical and research setting.2-5 Conduction slowing is thought to be the result of reductions in the expression, subcellular localization, and post-translational modifications of connexin 43, a major cardiac gap junction protein,6-9 while the lengthening of the AP is the complex manifestation of overall changes in the expression and/or function of the various membrane ion channels, transporters, and exchangers expressed in the heart. These proteins mediate depolarizing and repolarizing currents that regulate AP duration and morphology. Increases in the late Na current (INa) and Na/Ca exchanger-mediated current (INCX) are observed in failing and hypertrophied hearts.10-13 Both mediate inward depolarizing currents, and therefore increases in either would be expected to prolong the AP. However, the changes in these proteins are relatively modest and by themselves would only account for a slight increase in AP duration. In fact, it is the reduction in K+ current (IK) density that largely accounts for AP prolongation in HF. The outward whole cell IK is mediated by a numerous K+ channels, each with varying biophysical properties expertly reviewed elsewhere.14, 15 These K+ channels pull against the depolarizing effect of inward currents and are largely responsible for repolarizing the membrane following the initiation of an AP. Therefore, loss of these channels may prolong the AP as the inward depolarizing current would be less opposed. In HF, numerous K+-mediated currents are down-regulated. For example, Ito, IK1, IKr, and IKs are all diminished in human HF and animal models of HF.2, 16, 17 The combination of the prolonged AP duration and the lack of repolarization reserve, resulting from the loss of IK density, greatly increases propensity for arrhythmogenic early afterdepolarizations (EADs) and subsequent ventricular fibrillation or death. Interestingly, contrasting other K+ channels, it was recently reported that the small-conductance calcium-activated potassium current (IKAS) is up-regulated in the ventricles of end stage failing human hearts, where they are normally expressed at extremely low or non-existent levels.18 This increase may in part maintain repolarization reserve and promote well-ordered ventricular repolarization in HF patients. In this issue of The Journal of Cardiovascular Electrophysiology, Lee et al. report that the AP duration in the peri-infarct zone (PZ) and remote zone (RZ) was significantly shorter in a rabbit model of MI.19 Using a pharmacological approach, the authors demonstrate that this decrease in APD is IKAS-dependent. This increase in IKAS was particularly pronounced in the PZ. Notably, this is the first study to report that MI results in an increase in IKAS. The PZ has long been recognized to undergo acute electrical remodeling after MI and is considered a hot spot for arrhythmogenic activity post-MI. Both early- and delayed afterdepolarizations (EADs and DADs) may originate from the PZ depending on the extent and duration of the remodeling process.5 DADs are triggered by spontaneous Ca release events from the sarcoplasmic reticulum (SR) in the form of diastolic SR Ca leak or spontaneous Ca waves.20-22 These spontaneous Ca waves manifest themselves under conditions of cellular Ca overload. It is interesting to speculate that in the infarcted heart, and particularly within the PZ, any electrical remodeling that decreases the AP duration is actually a cardioprotective mechanism. Decreasing the AP duration would limit the amount of Ca entering the cell, thus attenuating Ca overload and arrhythmogenesis. Feeding this speculation further, IKAS channels are Ca-activated, which ideally suits them to respond to Ca overload and blunt this response. The exciting new work of Lee et al. appears to support this hypothesis directly. In fact, the authors show that in the infarcted heart the duration of the [Ca]i transient is significantly shorter when compared with healthy hearts. When the IKAS blocker, apamin, is applied both the APD and [Ca]i transient duration are shifted back to those observed in healthy hearts. These data directly implicate IKAS in the electrical remodeling of the myocardium post-MI. However, as the authors appropriately point out, this potential cardioprotective mechanism is not without its complications. A shortening of the APD that relies on IKAS, while protecting against Ca overload at rest, may actually increase myocardial susceptibility to a reentry circuit during rapid pacing. Tachypacing causes Ca to accumulate in the cytosol leading to increased Ca-dependent activation of IKAS. The resultant shortening of the AP would place the myocardium at higher risk for ventricular fibrillation (VF) especially in the presence of an MI-induced scar. This mechanism has been well-characterized in animal models of HF, and ventricles from failing human hearts exhibit increased IKAS.23,24 Lee et al. observe a more pronounced effect of IKAS-dependent shortening of the APD during rapid pacing. However, they did not observe an increase of VF under the same conditions. This highlights the complex nature of electrical remodeling after MI. Undoubtedly, other ion channels, transporters, and exchangers are also changing their expression and/or function in this model; these changes could conspire to be antiarrhythmogenic and resist any proarrhythmic effect resulting from increased IKAS. It is important to note that the rabbits used in the study by Lee et al. did not yet show signs of overt HF even though the average MI size was 25% of the left ventricle. This suggests that remodeling in these rabbits—both structural and electrical—was incomplete. This leaves open the interesting hypothesis that IKAS remodeling occurs acutely after MI and is cardioprotective during this phase. There are obvious clinical implications in these findings. Potassium channel blockers have been utilized as therapies for arrhythmia. The compensatory increase of IKAS observed could offset the loss of other K+ channels during remodeling and play a critical role in maintaining repolarization reserve. However, this same mechanism may lead to arrhythmogenesis (as noted above). These new data suggest that blockers can be both proarrhythmic and antiarrhythmic depending on disease progression. Furthermore, as the authors point out, these new data demonstrate that IKAS is increased in diseased ventricles; therefore, IKAS blockers should not use considered as atrial-specific antiarrhythmic agent. This study raises important new questions. It focused on one time point for the measurement of IKAS in MI. While outside the scope of this particular study, the design leaves open the possibility for differential IKAS-dependent APD remodeling at further time points of disease progression. The new data imply that IKAS-dependent remodeling had taken place and was maintained in myocardial tissue remote to the infarct zone as soon as 5 weeks post MI. Recent data demonstrate that the shortening of the AP (as observed here) was observed as early as 30 minutes post-MI but was completely normalized by day 60.25 Taken together, these data suggest that the IKAS effect on APD may be transitory, or that other changes in K+ channel expression and function that develop with time tip the scales in favor of APD prolongation. The temporal regulation of individual K channel function post-MI is not well known and has important clinical implications. Electrical remodeling is a broad term encompassing the myriad of changes in expression and function of numerous ion channels, transporters, and exchangers embedded the membrane of cardiomyocytes during hypertrophy and HF. Understanding the role of each individual protein integrated into this response furthers our ability to effectively treat cardiac disease. Of the greater than 250,000 deaths attributed to HF each year in the U.S., approximately 50% of these are sudden and unanticipated.1 This highlights the gaps in our knowledge of both the disease state and our treatment paradigms. The work of Lee et al. draws underscores how revisiting established models and asking simple, novel questions can lead to critical insights.

Channelopathies are a diverse set of disorders associated with defects in ion channel (and transporter) function. Although the vast majority of channelopathies are linked with inherited mutations that alter ion channel biophysical properties, another group of similar disorders has emerged that alter ion channel synthesis, membrane trafficking, and/or posttranslational modifications. In fact, some electrical and episodic disorders have now been identified that are not defects in the ion channel but instead reflect dysfunction in an ion channel (or transporter) regulatory protein. This review focuses on alternative paradigms for physiological disorders associated with protein biosynthesis, folding, trafficking, and membrane retention. Furthermore, the review highlights the role of aberrant posttranslational modifications in acquired channelopathies.

Aims Cardiac function depends on the highly regulated and co-ordinate activity of a large ensemble of potassium channels that control myocyte repolarization. While voltage-gated K+ channels have been well characterized in the heart, much less is known about regulation and/or targeting of two-pore K+ channel (K2P) family members, despite their potential importance in modulation of heart function. Methods and results Here, we report a novel molecular pathway for membrane targeting of TREK-1, a mechano-sensitive K2P channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e.g. arachidonic acid). We demonstrate that βIV-spectrin, an actin-associated protein, is co-localized with TREK-1 at the myocyte intercalated disc, associates with TREK-1 in the heart, and is required for TREK-1 membrane targeting. Mice expressing βIV-spectrin lacking TREK-1 binding ( qv4J ) display aberrant TREK-1 membrane localization, decreased TREK-1 activity, delayed action potential repolarization, and arrhythmia without apparent defects in localization/function of other cardiac potassium channel subunits. Finally, we report abnormal βIV-spectrin levels in human heart failure. Conclusions These data provide new insight into membrane targeting of TREK-1 in the heart and establish a broader role for βIV-spectrin in organizing functional membrane domains critical for normal heart function.

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca2+leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When b-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous C a2+waves in intact cardiomyocytes. Both SCaWs and SR Ca2+leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca2+leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca2+leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis. © 2014 Curran et al.

Atrial fibrillation (AF) is the most commonly observed arrhythmia, and it is estimated to affect more than 2 million adults annually in the United States alone [1]. The risk of stroke, heart failure, and sudden death are all increased in patients with AF. The frequency of AF increases with age and most often underlies other overt cardiomyopathies or cardiovascular diseases such as ischemic heart disease, congestive heart failure, or hypertension [2], [3]. However, for nearly 70 years AF has also been known to be an inherited, Mendelian disorder [4]. Indeed, work from the Framingham Heart Study reported a significant increase in the risk of AF in patients who had just one parent with AF [5]. The genetic mechanisms that lead to AF vary widely as do the degrees of penetrance [6], [7].

Ankyrin polypeptides are cellular adapter proteins that tether integral membrane proteins to the cytoskeleton in a host of human organs. Initially identified as integral components of the cytoskeleton in erythrocytes, a recent explosion in ankyrin research has demonstrated that these proteins play prominent roles in cytoskeletal signaling pathways and membrane protein trafficking/regulation in a variety of excitable and non-excitable cells including heart and brain. Importantly, ankyrin research has translated from bench to bedside with the discovery of human gene variants associated with ventricular arrhythmias that alter ankyrin–based pathways. Ankyrin polypeptides have also been found to play an instrumental role in various forms of sinus node disease and atrial fibrillation. Mouse models of ankyrin deficiency have played fundamental roles in the translation of ankyrin-based research to new clinical understanding of human sinus node disease, atrial fibrillation, and ventricular tachycardia.

Cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca2+ leak is increased in heart failure and during beta-adrenergic (β-AR) stimulation. This increased SR Ca2+ leak could limit the SR [Ca2+] thereby decreasing contractility in HF and could also lead to arrhythmogenic Ca-dependent inward depolarizing current resulting in HF. It has recently been shown in our lab that this leak is dependent on calcium-calmodulin-dependent protein kinase II (CaMKII) and subsequent NOS activation, and that it is independent of either protein kinase A (PKA) activation or an increase in free Ca2+ concentration in the cytosol ([Ca2+]i). Here we investigate whether Akt is also involved in the pathway leading to increased diastolic leak during β-AR stimulation. Methods: [Ca2+]i was measured using fluo-4 in left ventricular myocytes isolated from rabbits. To vary the SR load, myocytes were field-stimulated to steady state at different frequencies. Tetracaine (1mM) was used to rapidly and reversibly block the RyR. The tetracaine-dependent shift of Ca2+ from the cytosol to the SR (decrease in [Ca2+]i and increase in SR Ca2+ content) is proportional to SR Ca2+ leak. To activate β-AR, isoproterenol (ISO; 250 nM) was added to perfuse the myocytes starting 5 min before leak assessment through the end. Results: ISO-induced increase in the SR Ca2+ leak is abolished by the treatment of myocytes with Akt inhibitor X (AIX; 5μM; 30min pre-incubation and perfusion throughout the experiment). When SR Ca2+ load was matched in each group (ISO alone: 155.22 ± 5.1 μM; ISO + AIX: 153.92 ± 3.8 μM), myocytes treated with ISO alone had significantly higher leak (12.43 ± 3.8 μM) vs. those treated with ISO and AIX (1 ± 2.2 μM) (P = 0.01, t-test). This evidence indicates that Akt activation may also be involved in the β-AR-induced SR Ca2+ leak possibly upstream from CaMKII and NOS activation.

Over the past ten years, ankyrin polypeptides have emerged as players in cardiac excitation-contraction coupling. Once thought to solely play a structural role, loss-of-function variants of genes encoding ankyrin polypeptides have highlighted how this protein mediates subcellular localization of various electrical components of the excitation-contraction coupling machinery. Evidence has revealed how disruption of this localization is the primary cause of various cardiomyopathies, ranging from long-QT syndrome 4, to sinus node disease, to more common forms of arrhythmias.The roles of ankyrin polypeptides in excitation-contraction coupling in the heart and the development of ankyrin-specific cardiomyopathies. How ankyrin polypeptides may be involved in structural and electrical remodeling of the heart, post-myocardial infarct. How ankyrin interactions with membrane-bound ion channels may regulate these channels' response to stimuli. New data, which offers the potential for unique therapies, for not only combating heart disease, but also for wider applications to various disease states.The ankyrin family of adapter proteins is emerging as an intimate player in cardiac excitation-contraction coupling. Until recently, these proteins have gone largely unappreciated for their importance in proper cardiac function. New insights into how these proteins function within the heart are offering potentially new avenues for therapies against cardiomyopathy.

Cardiac contraction relies upon the proper expression, trafficking, and retention of integral membrane proteins such as ion channels, transporters, and hormone receptors. These proteins govern cardiac contraction and short and long term adaptations to physiological and pathophysiological stimuli. The profile of expressed proteins is dynamic, and it is regulated to assure the proper response to stress. Yet, despite its obvious importance, little is known regarding even the identity (or the function) of the intracellular molecular pathways underpinning the trafficking and targeting of integral membrane proteins in the context of the native heart. The goal of this work is identify new molecular players that regulate protein targeting and trafficking in the heart. Eps 15 homology domain-containing (EHD) gene products (EHD1-4) are intracellular proteins that are key regulators of endosomal trafficking, lipid homeostasis, membrane protein recycling and trafficking. Previously uncharacterized in the heart, we recently presented evidence which demonstrated that this protein family likely plays indispensable roles in protein trafficking in cardiac muscle. Notably, an essential role for one of these proteins, EHD3, in the membrane trafficking of the Na/Ca exchanger (NCX) in heart was uncovered. The goal of this research program is for the first time to directly test the role of these proteins in cardiac structural and electrical activity using cutting-edge in vivo models of EHD deficiency. We show that EHD3 deficiency in the heart leads to: 1) abnormal cardiac structure at baseline; 2) irregular action potential morphology, heart rhythm, and conduction; 3) depressed β-adrenergic responsiveness; 4) dysfunctional NCX and LTCC trafficking and function; and 5) dysregulated ankyrin-B expression and trafficking. These data strongly support a role for EHD3 in membrane protein trafficking and regulation within the context of the native heart.

Increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak is present in heart failure and in conditions when adrenergic tone is high. Increasing Ca leak from the SR could result in spontaneous Ca wave (SCaW) formation. SCaWs activate the inward Na/Ca exchanger (NCX) current causing a delayed afterdepolarization (DAD), potentially leading to arrhythmia. Here we examine SCaWs in ventricular myocytes isolated from failing and healthy rabbit hearts. Myocytes from healthy hearts did not exhibit SCaWs under baseline conditions versus 43% of those exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of Ca-calmodulin-dependent protein kinase II (CaMKII) by KN93. Inhibition of cAMP-dependent protein kinase (PKA) by H89 had no observed effect. Of myocytes treated with forskolin 50% showed SCaW activity, attributable to a large increase in SR Ca load ([Ca](SRT)) versus control. At similar [Ca](SRT) (121muM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2+/-0.28 vs. 1.1+/-0.28 and 1.29+/-0.39 SCaWs cell(-)(1), respectively). In myocytes isolated from failing hearts ISO induced an increase in the percentage of cells generating SCaWs vs. baseline (74% vs. 11%) with no increase in [Ca](SRT). Inhibiting CaMKII reversed this effect (14%). At similar [Ca](SRT) (71microM) myocytes treated with ISO or ISO plus H89 had significantly more SCaWs per cell vs. untreated (2.5+/-0.5; 1.6+/-0.7 vs. 0.36+/-0.3, respectively). Treatment with ISO plus KN93 completely abolished this effect. The evidence suggests the ISO-dependent increase in SCaW activity in both healthy and failing myocytes is CaMKII-dependent, implicating CaMKII in arrhythmogenesis.

We have previously shown increased cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca leak to be present in heart failure (HF) and in conditions when beta-adrenergic (β-AR) tone is high. This SR Ca leak could contribute to the cause of the observed decreased contractility in HF by limiting SR Ca load. Simultaneously, it could also lead to arrhythmogenic Ca-dependent inward depolarizing current commonly seen in failing hearts. We recently demonstrated that this leak increases in manner dependent on calcium-calmodulin-dependent protein kinase II (CaMKII) and completely independent of either protein kinase A (PKA) activation or an increase in bulk free Ca concentration ([Ca]i). Here we investigate this PKA- and [Ca]i-independent activation of CaMKII. We have found that while stimulating intact myocytes with the β-AR agonist isoproterenol (ISO, 250 nM) the CaMKII-dependent enhancement of SR Ca leak is abolished by treatment of the myocytes with nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μM). When SR Ca load was matched in each group (156 μM), myocytes treated with ISO alone had significantly higher leak (14.2 ± 2.0 μM) vs. those treated with ISO and L-NAME (3.8 ± 1.4 μM) or those left completely untreated (8.0 ± 0.91 μM). When SR Ca leak was matched (9.0 μM) we found the SR Ca load necessary to induce that leak was significantly lower in ISO-treated myocytes (91.6 ± 1.9 μM) vs. those treated with ISO and L-NAME (129.4 ± 16.3 μM) or those left untreated (127.4 ± 2.8 μM). This evidence indicates that NOS activation, and therefore generation of nitric oxide, is necessary for ISO-dependent activation of RyR by CaMKII.

Increased diastolic SR Ca leak can initiate spontaneous Ca waves (SCaWs). SCaWs activate inward Na/Ca exchanger current causing an arrhythmogenic delayed afterdepolarization. Here we examine SCaWs in ventricular myocytes isolated from rabbit hearts. Myocytes did not exhibit SCaWs at baseline conditions, but 43% did when exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of CaMKII by KN93, but not with PKA inhibition by H89. At similar [Ca]SRT (121 μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 & 1.29±0.39 SCaWs cell−1, respectively). We attribute this increase in activity to the previously characterized CaMKII-dependent increase in RyR-dependent leak. We also find that SR Ca leak is increased by the nitric oxide (NO) donor, SNAP; and this NO-dependent effect is also completely reversed by KN-93. We also show the increase in leak to be dependent on nitric oxide synthase 1 (NOS1) activity. At comparable SR Ca load (132 μM) ISO treated myocytes have significantly higher leak vs. control (8.4 vs. 3.8 μM). The ISO-induced leak (at constant SR Ca load) was attenuated by the NOS1 inhibitor, SMLT, but not the NOS3 inhibitor, L-NIO (3.5 vs. 6.8 μM). Moreover, ISO causes an upward trend in myocyte [NO] (sensed by the NO-dependent dye, DAF-2 A), and the NOS inhibitor, L-NAME significantly attenuated the development of SCaWs. Together this data suggests a novel pathway in which β1-adrenergic receptor activation stimulates NO production via NOS1, which in turn activates CaMKII to increase RyR gating, SR Ca leak, SCaWs and delayed afterdepolarizations.

Two potential methods for lunar surface stabilization are: 1) sintering the regolith into a solid and 2) using heat or UV cured polymers. Sintering, a method in which powders are fused into a solid, has been proposed as a way of building lunar launch pads, roads and other building materials. Polymers are currently used by the military to stabilize sandy soils, and adaptations of this technology may be effective for lunar surface stabilization. This paper describes ongoing work at NASA Kennedy Space Center on these two technologies. A solar concentrator has been built to provide the heat source for sintering. Various solvent free polymers have been investigated. Results of physical testing, including load strength and abrasion resistance, on field and laboratory prepared samples are presented.