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.
transgenic pig; ectonucleoside triphosphate diphosphohydrolase 1; CD39; myocardial ischemia; reperfusion
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.
ankyrin; spectrin; trafficking; targeting; cytoskeleton; diabetes
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.
ankyrin-B; Na/K-ATPase; Na/Ca exchanger; intracellular Na; Ca sparks; SR Ca leak
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.
Reduced Connexin43 (Cx43), sodium channel (Nav1.5) expression and increased collagen expression (fibrosis) are important determinants of impulse conduction in the heart.
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.
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.
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.
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.
Cx43; Nav1.5; heterogeneity; sodium current; arrhythmia
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.
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.
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.
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.
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.
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.
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.
ankyrin; spectrin; arrhythmia; cytoskeleton; mouse model
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.
ankyrin; NaV1.5; intercalated disc; transverse-tubule; arrhythmia; sodium channel; targeting
protein 4.1R; arrhythmia; cytoskeleton; spectrin; Na/Ca exchanger
calmodulin kinase II; calcium signaling; electrophysiology arrhythmogenesis; heart failure
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.
In TS the loss of VDI is an upstream initiating event for arrhythmia phenotypes that are ultimately dependent on CaMKII activation.
action potentials; calcium; ion channels; myocytes
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.
Over the past fifteen years, gene mutations in cardiac ion channels have been linked with a host of potentially fatal human arrhythmias including long QT syndrome, short QT syndrome, Brugada syndrome, and catecholaminergic polymorphic ventricular tachycardia. More recently, a new paradigm for human arrhythmia has emerged based on gene mutations that affect the activity of cardiac ion channel- and transporter- associated proteins. As part of the Circulation Research thematic series on Inherited Arrhythmias, this review will focus on the emerging field of human arrhythmias due to dysfunction in cytosolic gene products (including ankyrins, yotiao, syntrophin, and caveolin-3) that regulate the activities of key membrane ion channels and transporters.
arrhythmia; cytoskeleton; ankyrin; AKAP; yotiao; syntrophin; caveolin-3
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.
sinoatrial node cells; action potentials; funny current; sarcoplasmic reticulum; pacemaker
The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function.
Connexin43; Zonula occludens protein-1; Nav1.5; Intercalated disc
During the classic “fight-or-flight” stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca2+). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A+/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca2+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A+/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A+/+ mice resulted in impaired exercise capacity. RyR2-S2808A+/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation.
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.
sinoatrial node; automaticity; diastolic depolarization; Ca2+ sparks; local control
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.