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1.  Transgenic swine: expression of human CD39 protects against myocardial injury 
CD39 (Ectonucleoside Triphosphate Diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings require further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury. Transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by three hours of reperfusion. The ischemic area was delineated by perfusion with 5% Phthalo Blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3 % vs. Control: 44.7 ± 5.2 %, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.
doi:10.1016/j.yjmcc.2012.01.002
PMCID: PMC3327755  PMID: 22269791
transgenic pig; ectonucleoside triphosphate diphosphohydrolase 1; CD39; myocardial ischemia; reperfusion
2.  A Novel Core-Shell Microcapsule for Encapsulation and 3D Culture of Embryonic Stem Cells 
In this study, we report the preparation of a novel microcapsule of ~ 100 μm with a liquid (as compared to solid-like alginate hydrogel) core and an alginate-chitosan-alginate (ACA) shell for encapsulation and culture of embryonic stem (ES) cells in the miniaturized 3D space of the liquid core. Murine R1 ES cells cultured in the microcapsules were found to survive (> 90%) well and proliferate to form either a single aggregate of pluripotent cells or embryoid body (EB) of more differentiated cells in each microcapsule within 7 days, dependent on the culture medium used. This novel microcapsule technology allows massive production of the cell aggregates or EBs of uniform size and controllable pluripotency, which is important for the practical application of stem cell based therapy. Moreover, the semipermeable ACA shell was found to significantly reduce immunoglobulin G (IgG) binding to the encapsulated cells by up to 8.2 times, compared to non-encapsulated cardiac fibroblasts, mesenchymal stem cells, and ES cells. This reduction should minimize inflammatory and immune responses induced damage to the cells implanted in vivo becasue IgG binding is an important first step of the undesired host responses. Therefore, the ACA microcapsule with selective shell permeability should be of importance to advance the emerging cell-based medicine.
doi:10.1039/C2TB00058J
PMCID: PMC3596163  PMID: 23505611
3.  A Nonthoracotomy Myocardial Infarction Model in an Ovine Using Autologous Platelets 
BioMed Research International  2013;2013:938047.
Objective. There is a paucity of a biological large animal model of myocardial infarction (MI). We hypothesized that, using autologous-aggregated platelets, we could create an ovine model that was reproducible and more closely mimicked the pathophysiology of MI. Methods. Mepacrine stained autologous platelets from male sheep (n = 7) were used to create a myocardial infarction via catheter injection into the mid-left anterior descending (LAD) coronary artery. Serial daily serum troponin measurements were taken and tissue harvested on post-embolization day three. Immunofluorescence microscopy was used to detect the mepacrine-stained platelet-induced thrombus, and histology performed to identify three distinct myocardial (infarct, peri-ischemic “border zone,” and remote) zones. Results. Serial serum troponin levels (μg/mL) measured 0.0 ± 0.0 at baseline and peaked at 297.4 ± 58.0 on post-embolization day 1, followed by 153.0 ± 38.8 on day 2 and 76.7 ± 19.8 on day 3. Staining confirmed distinct myocardial regions of inflammation and fibrosis as well as mepacrine-stained platelets as the cause of intravascular thrombosis. Conclusion. We report a reproducible, unique model of a biological myocardial infarction in a large animal model. This technique can be used to study acute, regional myocardial changes following a thrombotic injury.
doi:10.1155/2013/938047
PMCID: PMC3866830  PMID: 24367790
4.  CaMKII inhibition rescues pro-arrhythmic phenotypes in model of human ankyrin-B syndrome 
Background
Cardiovascular disease is a leading cause of death worldwide. Arrhythmias are associated with significant morbidity and mortality related to cardiovascular disease. Recent work illustrates that many cardiac arrhythmias are initiated by a pathologic imbalance between kinase and phosphatase activities in excitable cardiomyocytes.
Objective
We tested the relationship between myocyte kinase/phosphatase imbalance and cellular and whole animal arrhythmia phenotypes associated with ankyrin-B cardiac syndrome.
Methods
Using a combination of biochemical, electrophysiological, and in vivo approaches, we tested the ability of CaMKII inhibition to rescue imbalance in kinase/phosphatase pathways associated with human ankyrin-B-associated cardiac arrhythmia.
Results
The cardiac ryanodine receptor (RyR2), a validated target of kinase/phosphatase regulation in myocytes, displays abnormal CaMKII-dependent phosphorylation (pS2814 hyperphosphorylation) in ankyrin-B+/− heart. Notably, RyR2 dysregulation is rescued in myocytes from ankyrin-B+/− mice overexpressing a potent CaMKII-inhibitory peptide (AC3I) and aberrant RyR2 open probability observed in ankyrin-B+/− hearts is normalized by treatment with the CaMKII inhibitor KN-93. CaMKII-inhibition is sufficient to rescue abnormalities in ankyrin-B+/− myocyte electrical dysfunction including cellular afterdepolarizations, and significantly blunts whole animal cardiac arrhythmias and sudden death in response to elevated sympathetic tone.
Conclusions
These findings illustrate the complexity of the molecular components involved in human arrhythmia and define regulatory elements of the ankyrin-B pathway in pathophysiology. Furthermore, the findings illustrate the potential impact of CaMKII-inhibition in the treatment of a congenital form of human cardiac arrhythmia.
doi:10.1016/j.hrthm.2012.08.026
PMCID: PMC3630478  PMID: 23059182
ankyrin; CaMKII; ryanodine receptor; spectrin; arrhythmia
5.  Ankyrin regulates KATP channel membrane trafficking and gating in excitable cells 
Channels (Austin, Tex.)  2010;4(1):55-57.
K(ATP) channels play critical roles in many cellular functions by coupling cell metabolic status to electrical activity. First discovered in cardiomyocytes,1 KATP channels (comprised of Kir6.x and SUR subunits) have since been found in many other tissues, including pancreatic beta cells, skeletal muscle, smooth muscle, brain, pituitary and kidney. By linking cellular metabolic state with membrane potential, KATP channels are able to regulate a number of cellular functions such as hormone secretion, vascular tone and excitability. Specifically, a reduction in metabolism causes a decrease in the ATP:ADP ratio, opening of KATP channels, K+ efflux, membrane hyperpolarization, and suppression of electrical activity. Conversely, increased cellular metabolism causes an increase in the ATP:ADP ratio that leads to closure of the KATP channel, membrane depolarization, and stimulation of cell electrical activity.
PMCID: PMC3671389  PMID: 19901534
ankyrin; spectrin; trafficking; targeting; cytoskeleton; diabetes
6.  Ankyrin-G Participates in INa Remodeling in Myocytes from the Border Zones of Infarcted Canine Heart 
PLoS ONE  2013;8(10):e78087.
Cardiac Na channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 assembly and function are linked to ankyrin-G, gap, and mechanical junction proteins. In this study our objective is to expound the status of the cardiac Na channel, its interacting protein ankyrinG and the mechanical and gap junction proteins at two different times post infarction when arrhythmias are known to occur; that is, 48 hr and 5 day post coronary occlusion. Previous studies have shown the origins of arrhythmic events come from the subendocardial Purkinje and epicardial border zone. Our Purkinje cell (Pcell) voltage clamp study shows that INa and its kinetic parameters do not differ between Pcells from the subendocardium of the 48hr infarcted heart (IZPCs) and control non-infarcted Pcells (NZPCs). Immunostaining studies revealed that disturbances of Nav1.5 protein location with ankyrin-G are modest in 48 hr IZPCs. Therefore, Na current remodeling does not contribute to the abnormal conduction in the subendocardial border zone 48 hr post myocardial infarction as previously defined. In addition, immunohistochemical data show that Cx40/Cx43 co-localize at the intercalated disc (IDs) of control NZPCs but separate in IZPCs. At the same time, Purkinje cell desmoplakin and desmoglein2 immunostaining become diffuse while plakophilin2 and plakoglobin increase in abundance at IDs. In the epicardial border zone 5 days post myocardial infarction, immunoblot and immunocytochemical analyses showed that ankyrin-G protein expression is increased and re-localized to submembrane cell regions at a time when Nav1.5 function is decreased. Thus, Nav1.5 and ankyrin-G remodeling occur later after myocardial infarction compared to that of gap and mechanical junctional proteins. Gap and mechanical junctional proteins remodel in IZPCs early, perhaps to help maintain Nav1.5 subcellular location position and preserve its function soon after myocardial infarction.
doi:10.1371/journal.pone.0078087
PMCID: PMC3796465  PMID: 24155982
7.  Ankyrin-B reduction enhances Ca spark-mediated SR Ca release promoting cardiac myocyte arrhythmic activity 
Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB+/−) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na]i and [Ca]i. Here we investigate how AnkB reduction affects cardiac [Na]i, [Ca]i and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na]i and diastolic [Ca]i in myocytes from AnkB+/− vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB+/− myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB+/− vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB+/− mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB+/− mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB+/− myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB+/− mice.
doi:10.1016/j.yjmcc.2012.02.010
PMCID: PMC3348355  PMID: 22406428
ankyrin-B; Na/K-ATPase; Na/Ca exchanger; intracellular Na; Ca sparks; SR Ca leak
8.  A distal axonal cytoskeleton forms an intra-axonal boundary that controls axon initial segment assembly 
Cell  2012;149(5):1125-1139.
SUMMARY
AnkyrinG (ankG) is highly enriched in neurons at axon initial segments (AIS) where it clusters Na+ and K+ channels and maintains neuronal polarity. How ankG becomes concentrated at the AIS is unknown. Here, we show that as neurons break symmetry, they assemble a distal axonal submembranous cytoskeleton comprised of ankyrinB (ankB), αII spectrin, and βII spectrin that defines a boundary limiting ankG to the proximal axon. Experimentally moving this boundary altered the length of ankG staining in the proximal axon, whereas disruption of the boundary through silencing of ankB, αII spectrin, or βII spectrin expression blocked AIS assembly and permitted ankG to redistribute throughout the distal axon. In support of an essential role for the distal cytoskeleton in ankG clustering, we also found that αII and βII spectrin -deficient mice had disrupted AIS. Thus, the distal axonal cytoskeleton functions as an intra-axonal boundary restricting ankG to the AIS.
doi:10.1016/j.cell.2012.03.039
PMCID: PMC3361702  PMID: 22632975
9.  Reduced Heterogeneous Expression Of Cx43 Results In Decreased Nav1.5 Expression And Reduced Sodium Current Which Accounts For Arrhythmia Vulnerability In Conditional Cx43 Knockout Mice 
Heart Rhythm  2011;9(4):600-607.
Background
Reduced Connexin43 (Cx43), sodium channel (Nav1.5) expression and increased collagen expression (fibrosis) are important determinants of impulse conduction in the heart.
Objective
To study the importance and interaction of these factors at very low Cx43 expression, inducible Cx43 KO mice with and without inducible ventricular tachycardia (VT) were compared by electrophysiology and immunohistochemistry.
Methods
Cx43CreER(T)/fl mice were induced with Tamoxifen and sacrificed after 2 weeks. Epicardial activation mapping was performed on Langendorff-perfused hearts, and arrhythmia vulnerability was tested. Mice were subdivided in VT+ (n=13) and VT− (n=10) and heart tissue was analyzed for Cx43, Nav1.5 and fibrosis.
Results
VT+ mice had decreased Cx43 expression with increased global, but not local, heterogeneity of Cx43, compared to VT− mice. Nav1.5-immunoreactive protein expression was reduced in VT+ versus VT− mice, specifically at sites devoid of Cx43. Levels of fibrosis were similar between VT− and VT+ mice. QRS-duration was increased and epicardial activation was more dispersed in VT+ mice than in VT− mice. The effective refractory period (ERP) was similar between both groups. Premature stimulation resulted in a more severe conduction slowing in VT+ compared to VT− hearts in the right ventricle. Separate patch clamp experiments in isolated rat ventricular myocytes confirmed that loss of Cx43 expression correlated with decreased sodium current amplitude.
Conclusions
Global heterogeneity in Cx43 expression and concomitant heterogeneous downregulation of sodium channel protein expression and sodium current leads to slowed and dispersed conduction, which sensitizes the heart for ventricular arrhythmias.
doi:10.1016/j.hrthm.2011.11.025
PMCID: PMC3336370  PMID: 22100711
Cx43; Nav1.5; heterogeneity; sodium current; arrhythmia
10.  Remodeling of the cardiac sodium channel, Connexin43 and Plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy 
Background
Arrhythmogenic cardiomyopathy (AC) is tightly associated with desmosomal mutations in the majority of patients. Arrhythmogenesis in AC patients is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. The aim of the present study was to assess the immunoreactive signal levels of the sodium channel protein NaV1.5, as well as Connexin43 and Plakoglobin, in myocardial specimens obtained from AC patients.
Methods
Left and right ventricular free wall (LVFW/RVFW) post-mortem material was obtained from 5 AC patients and 5 age and sex-matched controls. RV septal biopsies (RVSB) were taken from another 15 AC patients. All patients fulfilled the 2010 revised Task Force Criteria for AC diagnosis. Immunohistochemical analyses were performed using antibodies against Connexin43 (Cx43), Plakoglobin, NaV1.5, Plakophilin-2 and N-Cadherin.
Results
N-Cadherin and Desmoplakin immunoreactive signals and distribution were normal in AC patients compared to control. Plakophilin-2 signals were unaffected unless a PKP2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to control. Immunoreactive signal levels of PKG, Cx43 and NaV1.5 were disturbed in 74%, 70% and 65% of the patients, respectively.
Conclusions
Reduced immunoreactive signal of PKG, Cx43 and NaV1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Nav1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies.
doi:10.1016/j.hrthm.2012.11.018
PMCID: PMC3608196  PMID: 23178689
11.  Nitric Oxide-Dependent Activation of CaMKII Increases Diastolic Sarcoplasmic Reticulum Calcium Release in Cardiac Myocytes in Response to Adrenergic Stimulation 
PLoS ONE  2014;9(2):e87495.
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 β-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 Ca2+ 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.
doi:10.1371/journal.pone.0087495
PMCID: PMC3911966  PMID: 24498331
12.  Cardiac spectrins: Alternative splicing encodes functional diversity 
doi:10.1016/j.yjmcc.2010.02.002
PMCID: PMC2866816  PMID: 20144617
13.  Defining the Pathways Underlying the Prolonged PR Interval in Atrioventricular Conduction Disease 
PLoS Genetics  2012;8(12):e1003154.
doi:10.1371/journal.pgen.1003154
PMCID: PMC3516548  PMID: 23236297
14.  Transgenic Analysis of the Role of FKBP12.6 in Cardiac Function and Intracellular Calcium Release 
Abstract
FK506 binding protein12.6 (FKBP12.6) binds to the Ca2+ release channel ryanodine receptor (RyR2) in cardiomyocytes and stabilizes RyR2 to prevent premature sarcoplasmic reticulum Ca2+ release. Previously, two different mouse strains deficient in FKBP12.6 were reported to have different abnormal cardiac phenotypes. The first mutant strain displayed sex-dependent cardiac hypertrophy, while the second displayed exercise-induced cardiac arrhythmia and sudden death. In this study, we tested whether FKBP12.6-deficient mice that display hypertrophic hearts can develop exercise-induced cardiac sudden death and whether the hypertrophic heart is a direct consequence of abnormal calcium handling in mutant cardiomyocytes. Our data show that FKBP12.6-deficient mice with cardiac hypertrophy do not display exercise-induced arrhythmia and/or sudden cardiac death. To investigate the role of FKBP12.6 overexpression for cardiac function and cardiomyocyte calcium release, we generated a transgenic mouse line with cardiac specific overexpression of FKBP12.6 using α-myosin heavy chain (αMHC) promoter. MHC-FKBP12.6 mice displayed normal cardiac development and function. We demonstrated that MHC-FKBP12.6 mice are able to rescue abnormal cardiac hypertrophy and abnormal calcium release in FKBP12.6-deficient mice.
doi:10.1089/adt.2011.0411
PMCID: PMC3232634  PMID: 22087651
15.  From Fifth Business to Protagonist 
Proper regulation of cardiac ion channel activity is critical for cellular ion homeostasis and myocyte electrical activity. Recent work has demonstrated that cardiac ion channels are not isolated pores in plasmid membranes, but rather exist within macromolecular signaling complexes. Moreover, within these macro-complexes resides the machinery to finely tune ion channel expression, activity, and signaling. While it is widely-accepted that mutations in ion channel pore-forming genes underlie a number of cardiac arrhythmias, current research is now focusing on the roles of auxiliary subunits in the development of arrhythmia syndromes.
doi:10.1016/j.ddmod.2009.07.003
PMCID: PMC2913888  PMID: 20689672
16.  Defining the Common Disconnect between in vitro Models and Human Arrhythmogenic Disease: Context Matters 
Circulation  2011;124(9):993-995.
doi:10.1161/CIRCULATIONAHA.111.051466
PMCID: PMC3211044  PMID: 21875919
17.  Cardiac ankyrins in health and disease 
Ankyrins are critical components of ion channel and transporter signaling complexes in the cardiovascular system. Over the past five years, ankyrin dysfunction has been linked with abnormal ion channel and transporter membrane organization and fatal human arrhythmias. Loss-of-function variants in the ankyrin-B gene (ANK2) cause “ankyrin-B syndrome” (previously called type 4 long QT syndrome), manifested by a complex cardiac phenotype including ventricular arrhythmias and sudden cardiac death. More recently, dysfunction in the ankyrin-B-based targeting pathway has been linked with a highly penetrant and severe form of human sinus node disease. Ankyrin-G (a second ankyrin gene product) is required for normal expression, membrane localization, and biophysical function of the primary cardiac voltage-gated sodium channel, Nav1.5. Loss of the ankyrin-G/Nav1.5 interaction is associated with human cardiac arrhythmia (Brugada syndrome). Finally, in the past year ankyrin dysfunction has been associated with more common arrhythmia and cardiovascular disease phenotypes. Specifically, large animal studies reveal striking remodeling of ankyrin-B and associated proteins following myocardial infarction. Additionally, the ANK2 locus has been linked with QTc interval variability in the general human population. Together, these findings identify a host of unanticipated and exciting roles for ankyrin polypeptides in cardiac function. More broadly, these findings illustrate the importance of local membrane organization for normal cardiac physiology.
doi:10.1016/j.yjmcc.2009.04.010
PMCID: PMC2745072  PMID: 19394342
18.  Coordinating Electrical Activity of the Heart: Ankyrin Polypeptides in Human Cardiac Disease 
Introduction
Over the past ten years, ankyrin polypeptides have emerged as critical players in cardiac excitation-contraction coupling. Once thought to solely play only a structural role, loss-of-function variants in genes encoding ankyrin polypeptides have highlighted how this protein mediates the proper subcellular localization of the various electrical components of the excitation-contraction coupling machinery. A large body of evidence has revealed how the 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.
Areas Covered
This review details the varied roles that ankyrin polypeptides play in excitation-contraction coupling in the heart and the development of ankyrin-specific cardiomyopathies. It will further discuss how ankyrin polypeptides may be involved in structural and electrical remodeling of the heart, post-myocardial infarct. Attention is given to how ankyrin interactions with membrane bound ion channels may regulate these channels’ response to stimuli. Special attention is given to exciting new data, which may offer the potential for unique therapies, for not only combating heart disease, but which also holds promise for wider applications to various disease states.
Expert Opinion
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.
doi:10.1517/14728222.2011.575363
PMCID: PMC3166622  PMID: 21457127
19.  The Cardiac Cytoskeleton and Arrhythmia: An Unexpected Role for Protein 4.1R in Cardiac Excitability 
Circulation research  2008;103(8):779-781.
doi:10.1161/CIRCRESAHA.108.186460
PMCID: PMC2742414  PMID: 18845816
protein 4.1R; arrhythmia; cytoskeleton; spectrin; Na/Ca exchanger
20.  Defects in Cytoskeletal Signaling Pathways, Arrhythmia, and Sudden Cardiac Death 
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 (AF). 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, AF, and ventricular tachycardia.
doi:10.3389/fphys.2012.00122
PMCID: PMC3343379  PMID: 22586405
ankyrin; spectrin; arrhythmia; cytoskeleton; mouse model
21.  Ankyrin-based Cellular Pathways for Cardiac Ion Channel and Transporter Targeting and Regulation 
The coordinate activities of ion channels and transporters regulate myocyte membrane excitability and normal cardiac function. Dysfunction in cardiac ion channel and transporter function may result in cardiac arrhythmias and sudden cardiac death. While the past fifteen years have linked defects in ion channel biophysical properties with human disease, more recent findings illustrate that ion channel and transporter localization within cardiomyocytes is equally critical for normal membrane excitability and tissue function. Ankyrins are a family of multifunctional adapter proteins required for the expression, membrane localization, and regulation of select cardiac ion channels and transporters. Notably, loss of ankyrin expression in mice, and ankyrin loss-of-function in humans is now associated with defects in myocyte excitability and cardiac physiology. Here, we provide an overview of the roles of ankyrin polypeptides in cardiac physiology, as well as review other recently identified pathways required for the membrane expression and regulation of key cardiac ion channels and transporters.
doi:10.1016/j.semcdb.2010.09.013
PMCID: PMC3035725  PMID: 20934528
ankyrin; NaV1.5; intercalated disc; transverse-tubule; arrhythmia; sodium channel; targeting
22.  Voltage-gated Nav channel targeting in the heart requires an ankyrin-G–dependent cellular pathway 
The Journal of Cell Biology  2008;180(1):173-186.
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.
doi:10.1083/jcb.200710107
PMCID: PMC2213608  PMID: 18180363
23.  Role for CaMKII in cardiovascular health, disease, and arrhythmia 
doi:10.1016/j.hrthm.2010.07.029
PMCID: PMC2988944  PMID: 20673813
calmodulin kinase II; calcium signaling; electrophysiology arrhythmogenesis; heart failure
24.  Proarrhythmic defects in Timothy Syndrome require calmodulin kinase II 
Circulation  2008;118(22):2225-2234.
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.
doi:10.1161/CIRCULATIONAHA.108.788067
PMCID: PMC3226825  PMID: 19001023
action potentials; calcium; ion channels; myocytes
25.  CaMKII-Based Regulation of Voltage-Gated Na+ Channel in Cardiac Disease 
Circulation  2012;126(17):10.1161/CIRCULATIONAHA.112.105320.
Background
Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked with potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite over a decade of investigation. Post-translational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel post-translational modifications and disease. We recently identified a novel pathway for post-translational regulation of the primary cardiac voltage-gated Na+ channel (Nav1.5) by 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 Nav1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Nav1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 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 two human arrhythmia variants that affect Nav1.5 channel activity through direct effects on channel post-translational modification. We propose that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal point for pro-arrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.
doi:10.1161/CIRCULATIONAHA.112.105320
PMCID: PMC3811023  PMID: 23008441
arrhythmia (mechanisms); calmodulin dependent protein kinase II; heart failure; ion channels; long-QT syndrome; myocardial infarction

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