Cardiac fibrosis is a common pathologic consequence of stress insult to the heart and is characterized by abnormal deposition of fibrotic extracellular matrix that compromises cardiac function. Cardiac fibroblasts are key mediators of fibrotic remodeling and are regulated by secreted stress-response proteins. The matricellular protein connective tissue growth factor (CTGF), or CCN2, is strongly produced by injured cardiomyocytes and although it is considered a pro-fibrotic factor in many organ systems, its role in cardiac fibrosis is controversial. Here we adopted a cell-specific genetic approach to conditionally delete CCN2 in either cardiomyocytes or activated fibroblasts. Fibrosis was induced by angiotensin II-based neurohumoral stimulation, an insult that strongly induces CCN2 expression from cardiomyocytes and to a lesser extent in fibroblasts. Remarkably, only CCN2 deletion from activated fibroblasts inhibited the fibrotic remodeling while deletion from cardiomyocytes (the main source of CCN2 in the heart) had no effects. In vitro experiments revealed that although efficiently secreted by both fibroblasts and cardiomyocytes, only fibroblast-derived CCN2 is proficient in its ability to fully activate fibroblasts. These results overall indicate that although secreted into the extracellular matrix, CCN2 acts in an autocrine fashion. Secretion of CCN2 by cardiomyocytes is not pro-fibrotic, while fibroblast-derived CCN2 can modulate fibrosis in the heart. In conclusion we found that cardiomyocyte-derived CCN2 is dispensable for cardiac fibrosis, while inhibiting CCN2 induction in activated fibroblasts is sufficient to abrogate the cardiac fibrotic response to angiotensin II. Hence, CCN2 is an autocrine factor in the heart.

The contractile property of the myocardium is maintained by cell-cell junctions enabling cardiomyocytes to work as a syncytium. Alterations in cell-cell junctions are observed in heart failure, a disease characterized by the activation of Transforming Growth Factor beta 1 (TGFβ1). While TGFβ1 has been implicated in diverse biologic responses, its molecular function in controlling cell-cell adhesion in the heart has never been investigated. Cardiac-specific transgenic mice expressing active TGFβ1 were generated to model the observed increase in activity in the failing heart. Activation of TGFβ1 in the heart was sufficient to drive ventricular dysfunction. To begin to understand the function of this important molecule we undertook an extensive structural analysis of the myocardium by electron microscopy and immunostaining. This approach revealed that TGFβ1 alters intercalated disc structures and cell-cell adhesion in ventricular myocytes. Mechanistically, we found that TGFβ1 induces the expression of neural adhesion molecule 1 (NCAM1) in cardiomyocytes in a p38-dependent pathway, and that selective targeting of NCAM1 was sufficient to rescue the cell adhesion defect observed when cardiomyocytes were treated with TGFβ1. Importantly, NCAM1 was upregulated in human heart samples from ischemic and non-ischemic cardiomyopathy patients and NCAM1 protein levels correlated with the degree of TGFβ1 activity in the human cardiac ventricle. Overall, we found that TGFβ1 is deleterious to the heart by regulating the adhesion properties of cardiomyocytes in an NCAM1-dependent mechanism. Our results suggest that inhibiting NCAM1 would be cardioprotective, counteract the pathological action of TGFβ1 and reduce heart failure severity.

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-L1622L/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 Ca(2+)- and calpain-dependent proteases. Furthermore, consistent with this data, we observed Ca(2+)- 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 Ca(2+)- and calpain-dependent proteolysis.

Background— Voltage-gated Na + channels (Na v ) are essential for myocyte membrane excitability and cardiac function. Na v current ( I Na ) is a large-amplitude, short-duration spike generated by rapid channel activation followed immediately by inactivation. However, even under normal conditions, a small late component of I Na ( I Na,L ) persists because of incomplete/failed inactivation of a subpopulation of channels. Notably, I Na,L is directly linked with both congenital and acquired disease states. The multifunctional Ca 2+ /calmodulin-dependent kinase II (CaMKII) has been identified as an important activator of I Na,L in disease. Several potential CaMKII phosphorylation sites have been discovered, including Ser571 in the Na v 1.5 DI-DII linker, but the molecular mechanism underlying CaMKII-dependent regulation of I Na,L in vivo remains unknown. Methods and Results— To determine the in vivo role of Ser571, 2 Scn5a knock-in mouse models were generated expressing either: (1) Na v 1.5 with a phosphomimetic mutation at Ser571 (S571E), or (2) Na v 1.5 with the phosphorylation site ablated (S571A). Electrophysiology studies revealed that Ser571 regulates I Na,L but not other channel properties previously linked to CaMKII. Ser571-mediated increases in I Na,L promote abnormal repolarization and intracellular Ca 2+ handling and increase susceptibility to arrhythmia at the cellular and animal level. Importantly, Ser571 is required for maladaptive remodeling and arrhythmias in response to pressure overload. Conclusions— Our data provide the first in vivo evidence for the molecular mechanism underlying CaMKII activation of the pathogenic I Na,L . Relevant for improved rational design of potential therapies, our findings demonstrate that Ser571-dependent regulation of Na v 1.5 specifically tunes I Na,L without altering critical physiological components of the current.

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.

Aims: Ventricular arrhythmias are a common cause of death in patients with heart failure (HF). Structural and electrical abnormalities in the heart provide a substrate for such arrhythmias. Canine tachypacing-induced HF models of 4-6 weeks duration are often used to study pathophysiology and therapies for HF. We hypothesized that a chronic canine model of HF would result in greater electrical and structural remodeling than a short term model, leading to a more arrhythmogenic substrate.Main methods: HF was induced by ventricular tachypacing for one (short-term) or four (chronic) months to study remodeling.Key findings: Left ventricular contractility was progressively reduced, while ventricular hypertrophy and interstitial fibrosis were evident at 4 month but not 1 month of HF. Left ventricular myocyte action potentials were prolonged after 4 (p < 0.05) but not 1 month of HF. Repolarization instability and early afterdepolarizations were evident only after 4 months of HF (p < 0.05), coinciding with a prolonged QTc interval (p < 0.05). The transient outward potassium current was reduced in both HF groups (p < 0.05). The outward component of the inward rectifier potassium current was reduced only in the 4 month HF group (p < 0.05). The delayed rectifier potassium currents were reduced in 4 (p < 0.05) but not 1 month of HF. Reactive oxygen species were increased at both 1 and 4 months of HF (p < 0.05).Significance: Reduced I-to, outward In, I-Ks, and I-Kr in HF contribute to EAD formation. Chronic, but not short term canine HF, results in the altered electrophysiology and repolarization instability characteristic of end-stage human HF. (C) 2015 Elsevier Inc. All rights reserved.

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 Cav1.2-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.

The role of I(KCa) in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular electrophysiologic effects of I(KCa) blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The I(KCa) blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by I(KCa) blockade in all of the groups studied. I(KCa) blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. I(KCa) blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, I(KCa) blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for I(KCa) in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles.

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 (K-2P) 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 K-2P channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e. g. arachidonic acid). We demonstrate that beta(IV)-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 bIV-spectrin lacking TREK-1 binding (qv(4J)) 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 bIV-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 beta(IV)-spectrin in organizing functional membrane domains critical for normal heart function.

Rationale: Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 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 Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined. Objective: Define the in vivo mechanisms underlying Nav1.5 membrane regulation. Methods and Results: Here, we define the molecular basis of an Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G-/- mice (conditional knock-out mouse), we report that ankyrin-G targets Nav1.5 and its regulatory protein calcium/calmodulin–dependent kinase II to the intercalated disc. Mechanistically, &bgr;IV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin–dependent kinase II-&dgr;; however, &bgr;IV-spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Nav1.5 expression/membrane localization and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to &bgr;-adrenergic stimulation. Conclusions: Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.

Because structural remodeling of several proteins, including ion channels, may underlie the abnormal action potentials of Purkinje cells (PCs) that survive in the 48 hr infarcted zone of the canine heart (IZPCs), we sought to determine the subcellular structure and function of the KV1.5 (KCNA5) protein in single IZPCs. Clustering of the Kv1.5 subunit in axons is regulated by a synapse-associated protein, SAP97, and is linked to an actin-binding protein, cortactin, and an intercellular adhesion molecule, N-cadherin. To understand the functional remodeling of the Kv1.5 channel and its regulation in IZPCs, Kv1.5 currents in PCs were measured as the currents blocked by 10 µM RSD1379 using patch-clamp techniques. Immunocytochemistry and confocal imaging were used for both single and aggregated IZPCs vs normal PCs (NZPCs) to determine the relationship of Kv1.5 with SAP-97, cortactin and N-cadherin. In IZPCs, both the sarcolemma (SL) and intercalated disk (ID) Kv1.5 protein are abundant, and the amount of cytosolic Kv1.5 protein is greatly increased. SAP-97 is also increased at IDs and has notable cytosolic localization suggesting that SAP-97 may regulate the functional expression and stabilization of Kv1.5 channels in IZPCs. Cortactin, which is located with N-cadherin at IDs in NZPCs, remains at IDs but begins to dissociate from N-cadherin, often forming ring structures and colocalizing with Kv1.5 within IZPCs. At the same time, cortactin/Kv1.5 colocalization is increased at the ID, suggesting an ongoing active process of membrane trafficking of the channel protein. Finally, the Kv1.5 current, measured as the RSD1379-sensitive current, at +40 mV did not differ between NZPCs (0.81±0.24 pA/pF, n = 14) and IZPCs (0.83±0.21 pA/pF, n = 13, NS). In conclusion, the subcellular structural remodeling of Kv1.5, SAP97 and cortactin maintained and normalized the function of the Kv1.5 channel in Purkinje cells that survived myocardial infarction.

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.

The mechanisms responsible for exercise-induced reductions in baseline heart rate (HR), known as training bradycardia, remain controversial. Therefore, changes in cardiac autonomic regulation and intrinsic sinoatrial nodal (SAN) rate were evaluated using dogs randomly assigned to either a 10- to 12-wk exercise training (Ex, n = 15) or an equivalent sedentary period (Sed, n = 10). Intrinsic HR was revealed by combined autonomic nervous system (ANS) blockade (propranolol + atropine, iv) before and after completion of the study. At the end of the study, SAN function was further evaluated by examining the SAN recovery time (SNRT) following rapid atrial pacing and the response to adenosine in anesthetized animals. As expected, both the response to submaximal exercise and baseline HR significantly (P < 0.01) decreased, and heart rate variability (HRV; e.g., high-frequency R-R interval variability) significantly (P < 0.01) increased in the Ex group but did not change in the Sed group. Atropine also induced significantly (P < 0.01) greater reductions in HRV in the Ex group compared with the Sed group; propranolol elicited similar HR and HRV changes in both groups. In contrast, neither intrinsic HR (Ex before, 141.2 ± 6.7; Ex after, 146.0 ± 8.0 vs. Sed before, 143.3 ± 11.1; Sed after, 141.0 ± 11.3 beats per minute), the response to adenosine, corrected SNRT, nor atrial fibrosis and atrial fibrillation inducibility differed in the Ex group vs. the Sed group. These data suggest that in a large-animal model, training bradycardia results from an enhanced cardiac parasympathetic regulation and not from changes in intrinsic properties of the SAN.

Altered myocyte electrophysiology in heart failure contributes to cardiac arrhythmias. In chronic human heart failure atrial repolarization is shortened while ventricular repolarization is prolonged. The calcium activated potassium (I KCa ) current has been suggested as a novel atrial-selective pharmacologic target to modulate repolarization and arrhythmogenesis. We hypothesized that chamber-specific differences in I KCa current contribute to HF-induced repolarization changes. Methods: End-stage failing hearts obtained at the time of transplant were used to isolate left ventricular (LV) midwall and left atrial appendage (LAA) myocytes. Action potential durations (APDs) were recorded at 0.5, 1 and 2 Hz before and after the application of 100nM apamin, an I KCa blocker. Results: In LAA cells, apamin had no effect on APD50 or APD90 and no cellular arrhythmias (i.e. early afterdepolarizations, EADs) were observed. In the ventricular cells, apamin resulted in significant prolongation of the APD50 (p<0.05) and APD90 (p<0.05) at all rates; there was also a 40% increase in EADs, consistent with proarrhythmic potential. Conclusions: In end-stage human HF, I KCa modulates LV repolarization and I KCa inhibition induces cellular arrhythmias. I KCa does not appear to modulate LAA myocyte electrophysiology in end-stage HF; our results do not support I KCa as a pharmacologic target for AF associated with heart failure. Potential explanations for the chamber-specific effects we observed include differential HF-induced alterations in APD, channel expression or calcium cycling. Thus, we suggest that inhibition of I KCa as a pharmacologic strategy in HF may be detrimental, as it may favor the initiation of triggered ventricular arrhythmias.

Heart failure (HF) is a chronic disease that develops over months to years. In HF, ventricular repolarization is prolonged. We tested the hypothesis that I KCa modulates repolarization only when ventricular repolarization reserve is reduced as occurs in chronic HF, but not short term HF. Methods: A tachypacing - induced HF canine model was used, and LV midwall myocytes were isolated from 1 and 4 month HF groups, and compared to normal controls. Action potential duration at 50 (APD50) and 90% (APD90) repolarization was measured before and after the application of 100nM apamin (I KCa blocker, n=5-9 per group). Adjacent tissue (n=4 per group) was collected to measure the proteins encoding cardiac I KCa (SK2 and SK3). Results: One and 4 month HF had similar severity of HF (LVFS: 19.00 ± 1.36 vs. 15.9 ± 2.45, respectively P=NS). APD50 or APD90 in 1 month HF was no different from controls. 4 Mo HF resulted in a significant (P<0.05) APD90 prolongation compared to both control and 1 Mo HF groups, consistent with reduced repolarization reserve. Apamin significantly increased (P<0.05) APD50 and APD90 only in the 4 Mo HF group. Similarly proarrhythmic repolarization instability (beat-to-beat variability) was evident after apamin in the 4 month, but not the 1 month HF group (p<0.05). SK2 expression was unchanged between groups; SK3 was increased ~ 4 fold in both 1 month and 4 month HF group (P<0.05 vs control). Conclusions: Changes in SK protein expression do not fully explain I KCa -induced repolarization modulation. Rather, I KCa inhibition prolongs repolarization only when repolarization reserve is decreased, as occurs in chronic HF. The safety of targeting I KCa in chronic HF needs to be carefully evaluated as I KCa inhibition may increase susceptibility to ventricular arrhythmias.

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.

Kinase/phosphatase balance governs cardiac excitability in health and disease. Although detailed mechanisms for cardiac kinase regulation are established, far less is known regarding cardiac protein phosphatase 2A (PP2A) regulation. This is largely due to the complexity of the PP2A holoenzyme structure (combinatorial assembly of three subunit enzyme from >17 subunit genes) and the inability to segregate "global" PP2A function from the activities of multiple "local" holoenzyme populations. Here we report that PP2A catalytic, regulatory, and scaffolding subunits are tightly regulated at transcriptional, translational, and post-translational levels to tune myocyte function at base line and in disease. We show that past global read-outs of cellular PP2A activity more appropriately represent the collective activity of numerous individual PP2A holoenzymes, each displaying a specific subcellular localization (dictated by select PP2A regulatory subunits) as well as local specific post-translational catalytic subunit methylation and phosphorylation events that regulate local and rapid holoenzyme assembly/disassembly (via leucine carboxymethyltransferase 1/phosphatase methylesterase 1 (LCMT-1/PME-1). We report that PP2A subunits are selectively regulated between human and animal models, across cardiac chambers, and even within specific cardiac cell types. Moreover, this regulation can be rapidly tuned in response to cellular activation. Finally, we report that global PP2A is altered in human and experimental models of heart disease, yet each pathology displays its own distinct molecular signature though specific PP2A subunit modulatory events. These new data provide an initial view into the signaling pathways that govern PP2A function in heart but also establish the first step in defining specific PP2A regulatory targets in health and disease.

Significance This study defines a functional role for β IV -spectrin in pancreas, expands the pathways for calcium/calmodulin-dependent protein kinase II (CaMKII) local control in excitable cells, and identifies a mechanism for CaMKII-dependent regulation of K ATP channels.

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.