Altered Expression of Zonula occludens-1 Affects Cardiac Na+ Channels and Increases Susceptibility to Ventricular Arrhythmias.

Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1), CRA_e (Transcript Variant 4, TV4), and an additionally expressed (Transcript Variant 3, TV3) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1+/-). ZO-1+/- mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1+/- mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1+/- mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1+/- cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density (INa) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in ICa current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias.

Temporal Trends in Post Myocardial Infarction Heart Failure and Outcomes Among Older Adults.

BACKGROUND: We sought to determine national trends and long term outcomes of post myocardial infarction (MI) heart failure. An MI can be complicated by heart failure; there are limited data describing the contemporary patterns and clinical implications of post-MI heart failure. METHODS AND RESULTS: We studied patients with an MI aged 65 years or older from 2000 to 2013 in a Medicare database. New-onset heart failure after an MI was defined as either heart failure during the index MI admission or a hospitalization for heart failure within 1 year of the index MI event. A trend analysis of the incidence of heart failure was performed, and differences were examined by Gray tests. The 5-year mortality rates were evaluated and differences among heart failure cohorts were ascertained by Gray tests. There were a total of 1,531,638 patients with an MI and 565,291 patients had heart failure (36.0%). The rate of heart failure during index admission was 32.3% and the frequency of heart failure hospitalization within 1 year was 10.4%. Patients with heart failure were older (81 years vs 77 years). The temporal trend from 2001 to 2012 suggested a decrease in the incidence of heart failure during index admission (2001: 34.7%, 2012: 31.2%, Ptrend < .01), as well as heart failure hospitalization within 1 year (2001: 11.3%, 2012: 8.7%, Ptrend < .01). The 5-year mortality rate among patients without heart failure was 38.4% and for patients with any heart failure it was 68.7%. CONCLUSIONS: Post-MI heart failure in older adults occurs in 1 in 3 patients within 1 year; heart failure portends significantly higher long-term mortality.

Beyond Reperfusion: Acute Ventricular Unloading and Cardioprotection During Myocardial Infarction.

Heart failure is a major cause of morbidity and mortality around the world, and myocardial infarction is its leading cause. Myocardial infarction destroys viable myocardium, and this dead tissue is replaced by a non-contractile scar that results in impaired cardiac function and a significantly increased likelihood of the patient developing heart failure. Limiting infarct scar size has been the target of pre-clinical and clinical investigations for decades. However, beyond reperfusion, few therapies have translated into the clinic that limit its formation. New approaches are needed. This review will focus on new clinical and pre-clinical data demonstrating that acute ventricular unloading prior to reperfusion by means of percutaneous left ventricular support devices reduces ischemia-reperfusion injury and limits infarct scar size. Emphasis will be given to summarizing our current mechanistic understanding of this new therapeutic approach to treating myocardial infarction.

Novel Mechanistic Roles for Ankyrin-G in Cardiac Remodeling and Heart Failure.

Ankyrin polypeptides are intracellular proteins responsible for targeting cardiac membrane proteins. Here, the authors demonstrate that ankyrin-G plays an unexpected role in normal compensatory physiological remodeling in response to myocardial stress and aging; the authors implicate disruption of ankyrin-G in human heart failure. Mechanistically, the authors illustrate that ankyrin-G serves as a key nodal protein required for cardiac myofilament integration with the intercalated disc. Their data define novel in vivo mechanistic roles for ankyrin-G, implicate ankyrin-G as necessary for compensatory cardiac physiological remodeling under stress, and implicate disruption of ankyrin-G in the development and progression of human heart failure.

Common human ANK2 variant confers in vivo arrhythmia phenotypes.

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

Dysfunction of the ß2-spectrin-based pathway in human heart failure.

ß2-Spectrin is critical for integrating membrane and cytoskeletal domains in excitable and nonexcitable cells. The role of ß2-spectrin for vertebrate function is illustrated by dysfunction of ß2-spectrin-based pathways in disease. Recently, defects in ß2-spectrin association with protein partner ankyrin-B were identified in congenital forms of human arrhythmia. However, the role of ß2-spectrin in common forms of acquired heart failure and arrhythmia is unknown. We report that ß2-spectrin protein levels are significantly altered in human cardiovascular disease as well as in large and small animal cardiovascular disease models. Specifically, ß2-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, ß2-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 ß2-spectrin protein levels. Mechanistically, we identify that ß2-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 ß2-spectrin downstream effector proteins, including ankyrin-B in heart. In summary, our findings illustrate that ß2-spectrin and downstream molecules are regulated in multiple forms of cardiovascular disease via Ca(2+)- and calpain-dependent proteolysis.

Protein phosphatase 2A regulatory subunit B56α limits phosphatase activity in the heart.

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

Use of whole exome sequencing for the identification of Ito-based arrhythmia mechanism and therapy.

BACKGROUND: Identified genetic variants are insufficient to explain all cases of inherited arrhythmia. We tested whether the integration of whole exome sequencing with well-established clinical, translational, and basic science platforms could provide rapid and novel insight into human arrhythmia pathophysiology and disease treatment. METHODS AND RESULTS: We report a proband with recurrent ventricular fibrillation, resistant to standard therapeutic interventions. Using whole-exome sequencing, we identified a variant in a previously unidentified exon of the dipeptidyl aminopeptidase-like protein-6 (DPP6) gene. This variant is the first identified coding mutation in DPP6 and augments cardiac repolarizing current (Ito) causing pathological changes in Ito and action potential morphology. We designed a therapeutic regimen incorporating dalfampridine to target Ito. Dalfampridine, approved for multiple sclerosis, normalized the ECG and reduced arrhythmia burden in the proband by >90-fold. This was combined with cilostazol to accelerate the heart rate to minimize the reverse-rate dependence of augmented Ito. CONCLUSIONS: We describe a novel arrhythmia mechanism and therapeutic approach to ameliorate the disease. Specifically, we identify the first coding variant of DPP6 in human ventricular fibrillation. These findings illustrate the power of genetic approaches for the elucidation and treatment of disease when carefully integrated with clinical and basic/translational research teams.

Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria.

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

Endosome-based protein trafficking and Ca(2+) homeostasis in the heart.

The ability to dynamically regulate, traffic, retain, and recycle proteins within the cell membrane is fundamental to life and central to the normal function of the heart. In the cardiomyocyte, these pathways are essential for the regulation of Ca(2+), both at the level of the plasma membrane, but also in local cellular domains. One intracellular pathway often overlooked in relation to cardiovascular Ca(2+) regulation and signaling is the endosome-based trafficking pathway. Highlighting its importance, this system and its molecular components are evolutionarily conserved across all metazoans. However, remarkably little is known of how endosome-based protein trafficking and recycling functions within mammalian cells systems, especially in the heart. As the endosomal system acts to regulate the expression and localization of membrane proteins central for cardiac Ca(2+) regulation, understanding the in vivo function of this system in the heart is critical. This review will focus on endosome-based protein trafficking in the heart in both health and disease with special emphasis for the role of endocytic regulatory proteins, C-terminal Eps15 homology domain-containing proteins (EHDs).

Dysfunction in the ßII spectrin-dependent cytoskeleton underlies human arrhythmia.

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 ßII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. ßII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/ßII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac ßII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, ßII 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 ßII spectrin-deficient mice. CONCLUSIONS: Our findings identify ßII 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.

Alternative paradigms for ion channelopathies: disorders of ion channel membrane trafficking and posttranslational modification.

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

Ankyrin-G coordinates intercalated disc signaling platform to regulate cardiac excitability in vivo.

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, ßIV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin-dependent kinase II-δ; however, ßIV-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 ß-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.

EHD3-dependent endosome pathway regulates cardiac membrane excitability and physiology.

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.

Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) leak) through ryanodine receptors. Beta-adrenergic (ß-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 ß-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 Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) 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 ß-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+) 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 Ca(2+) leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of ß-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis.

ß(IV)-Spectrin regulates TREK-1 membrane targeting in the heart.

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 ß(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 ß(IV)-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 ß(IV)-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 ß(IV)-spectrin in organizing functional membrane domains critical for normal heart function.

Ankyrin-B regulates Cav2.1 and Cav2.2 channel expression and targeting.

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

Ca2+/calmodulin-dependent protein kinase II-based regulation of voltage-gated Na+ channel in cardiac disease.

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.