Anthony W. Herren

Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites.

Background: CaMKII is up-regulated in heart failure and modulates Na+ current (INa), yet the mechanism is unclear. Result: CaMKII phosphorylates several sites in the first intracellular loop of NaV1.5, thereby altering INa gating properties. Conclusion: This multisite phosphorylation may contribute to acquired arrhythmogenesis. Significance: Identification of these regulatory sites is critical for potential therapeutic targeting of CaMKII and NaV1.5 in failing hearts. The cardiac Na+ channel NaV1.5 current (INa) is critical to cardiac excitability, and altered INa gating has been implicated in genetic and acquired arrhythmias. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause INa gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on NaV1.5 that mediate(s) the CaMKII-induced alterations in INa gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of NaV1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δC-CaMKII and the intracellular loop between domains 1 and 2 of NaV1.5 was observed. This region was also phosphorylated by δC-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNaV1.5 were co-expressed with GFP-δC-CaMKII in HEK293 cells, and INa was recorded. As observed in myocytes, CaMKII shifted WT INa availability to a more negative membrane potential and enhanced accumulation of INa into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted INa availability without the need for CaMKII. CaMKII-dependent phosphorylation of NaV1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.

Post-translational modifications of the cardiac Na channel: contribution of CaMKII-dependent phosphorylation to acquired arrhythmias

The voltage-gated Na channel isoform 1.5 (NaV1.5) is the pore forming α-subunit of the voltage-gated cardiac Na channel, which is responsible for the initiation and propagation of cardiac action potentials. Mutations in the SCN5A gene encoding NaV1.5 have been linked to changes in the Na current leading to a variety of arrhythmogenic phenotypes, and alterations in the NaV1.5 expression level, Na current density, and/or gating have been observed in acquired cardiac disorders, including heart failure. The precise mechanisms underlying these abnormalities have not been fully elucidated. However, several recent studies have made it clear that NaV1.5 forms a macromolecular complex with a number of proteins that modulate its expression levels, localization, and gating and is the target of extensive post-translational modifications, which may also influence all these properties. We review here the molecular aspects of cardiac Na channel regulation and their functional consequences. In particular, we focus on the molecular and functional aspects of Na channel phosphorylation by the Ca/calmodulin-dependent protein kinase II, which is hyperactive in heart failure and has been causally linked to cardiac arrhythmia. Understanding the mechanisms of altered NaV1.5 expression and function is crucial for gaining insight into arrhythmogenesis and developing novel therapeutic strategies.

Atherosclerosis exacerbates arrhythmia following myocardial infarction: Role of myocardial inflammation

BACKGROUND Atherosclerotic animal models show increased recruitment of inflammatory cells to the heart after myocardial infarction (MI), which impacts ventricular function and remodeling. OBJECTIVE The purpose of this study was to determine whether increased myocardial inflammation after MI also contributes to arrhythmias. METHODS MI was created in 3 mouse models: (1) atherosclerotic (apolipoprotein E deficient [ApoE(-/-)] on atherogenic diet, n = 12); (2) acute inflammation (wild-type [WT] given daily lipopolysaccharide [LPS] 10 μg/day, n = 7); and (3) WT (n = 14). Sham-operated (n = 4) mice also were studied. Four days post-MI, an inflammatory protease-activatable fluorescent probe (Prosense680) was injected intravenously to quantify myocardial inflammation on day 5. Optical mapping with voltage-sensitive dye was performed on day 5 to assess electrophysiology and arrhythmia susceptibility. RESULTS Inflammatory activity (Prosense680 fluorescence) was increased approximately 2-fold in ApoE+MI and LPS+MI hearts vs WT+MI (P<.05) and 3-fold vs sham (P<.05). ApoE+MI and LPS+MI hearts also had prolonged action potential duration, slowed conduction velocity, and increased susceptibility to pacing-induced arrhythmias (56% and 71% vs 13% for WT+MI and 0% for sham, respectively, P<.05, for ApoE+MI and LPS+MI groups vs both WT+MI and sham). Increased macrophage accumulation in ApoE+MI and LPS+MI hearts was confirmed by immunofluorescence. Macrophages were associated with areas of connexin43 (Cx43) degradation, and a 2-fold decrease in Cx43 expression was found in ApoE+MI vs WT+MI hearts (P<.05). ApoE+MI hearts also had a 3-fold increase in interleukin-1β expression, an inflammatory cytokine known to degrade Cx43. CONCLUSION Underlying atherosclerosis exacerbates post-MI electrophysiological remodeling and arrhythmias. LPS+MI hearts fully recapitulate the atherosclerotic phenotype, suggesting myocardial inflammation as a key contributor to post-MI arrhythmia.

CaMKII-dependent regulation of cardiac Na + homeostasis

Na+ homeostasis is a key regulator of cardiac excitation and contraction. The cardiac voltage-gated Na+ channel, NaV1.5, critically controls cell excitability, and altered channel gating has been implicated in both inherited and acquired arrhythmias. Ca2+/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine kinase important in cardiac physiology and disease, phosphorylates NaV1.5 at multiple sites within the first intracellular linker loop to regulate channel gating. Although CaMKII sites on the channel have been identified (S516, T594, S571), the relative role of each of these phospho-sites in channel gating properties remains unclear, whereby both loss-of-function (reduced availability) and gain-of-function (late Na+ current, INaL) effects have been reported. Our review highlights investigating the complex multi-site phospho-regulation of NaV1.5 gating is crucial to understanding the genesis of acquired arrhythmias in heart failure (HF) and CaMKII activated conditions. In addition, the increased Na+ influx accompanying INaL may also indirectly contribute to arrhythmia by promoting Ca2+ overload. While the precise mechanisms of Na+ loading during HF remain unclear, and quantitative analyses of the contribution of INaL are lacking, disrupted Na+ homeostasis is a consistent feature of HF. Computational and experimental observations suggest that both increased diastolic Na+ influx and action potential prolongation due to systolic INaL contribute to disruption of Ca2+ handling in failing hearts. Furthermore, simulations reveal a synergistic interaction between perturbed Na+ fluxes and CaMKII, and confirm recent experimental findings of an arrhythmogenic feedback loop, whereby CaMKII activation is at once a cause and a consequence of Na+ loading.

CaMKII Phosphorylation of Na(V)1.5: Novel in Vitro Sites Identified by Mass Spectrometry and Reduced S516 Phosphorylation in Human Heart Failure.

The cardiac voltage-gated sodium channel, Na(V)1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca(2+))/calmodulin(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of Na(V)1.5 function through phosphorylation of multiple residues including S516, T594, and S571, and these phosphorylation events may be important for the genesis of acquired arrhythmias, which occur in heart failure. However, phosphorylation of full-length human Na(V)1.5 has not been systematically analyzed and Na(V)1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human Na(V)1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC-MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of Na(V)1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human Na(V)1.5.

Proteomic Analysis of Human Pluripotent Stem Cell-Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation

Background—Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs), but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived VCMs, human fetal and human adult ventricular and atrial CMs. Methods and Results—Using two-dimensional–differential-in-gel electrophoresis, 121 differentially expressed (>1.5-fold; P<0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator–activated receptor &agr; signaling in cardiac maturation. Consistently, WY-14643, a peroxisome proliferator–activated receptor &agr; agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold, signifying their maturation. Conclusions—We conclude that the peroxisome proliferator–activated receptor &agr; and thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

CaMKII comes of age in cardiac health and disease

Almost four decades since its initial discovery in brain (Schulman and Greengard, 1978), the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) has now emerged as a key signaling molecule in the heart. The isoform that predominates in heart, CaMKIId, directly regulates expression and function of several of the main cardiac ion channels and Ca2+ handling proteins (Bers and Grandi, 2009). CaMKII-dependent effects are thought to orchestrate many of the electrophysiologic and contractile adaptations to common cardiac stressors, such as rapid pacing, adrenergic stimulation, and oxidative challenge. In the context of disease, CaMKII has been shown to contribute to a remarkably wide variety of cardiac pathologies, including myocardial hypertrophy, ischemia, heart failure (HF), and arrhythmia. CaMKII expression is increased in patients with HF (Hoch et al., 1999), and elevated CaMKII expression and activity have been implicated in the transition to HF (Zhang et al., 2003). Indeed, inhibiting CaMKII appears to reduce arrhythmias and pathological signaling, which makes this kinase a promising new therapeutic target (Anderson et al., 2011).

Na+ Channel I–II Loop Mediates Parallel Genetic and Phosphorylation-Dependent Gating Changes

In this issue of Circulation , Koval et al1 show that 2 arrhythmogenic human cardiac Na+ channel (hNaV1.5) variants mimic the altered channel gating effects induced by Ca2+-calmodulin–dependent protein kinase (CaMKII). They also show that phosphorylation of an adjacent CaMKII target site on NaV1.5 is enhanced in human heart failure (HF) samples and in the border zone of postinfarcted canine hearts. Article see p 2084 The cardiac Na+ channel, NaV1.5, is responsible for inward Na+ current ( I Na) that drives the cardiac action potential (AP) upstroke and electrical impulse propagation.2 Genetic variants of the SCN5A gene encoding NaV1.5 are associated with long QT syndrome-3 (LQTs; gain of function), Brugada syndrome (BRs; loss of function), conduction system disease, sudden infant death syndrome, sick sinus syndrome, and dilated cardiomyopathy.3,4 These inherited channelopathies have been tremendously important to our understanding of normal NaV1.5 function and arrhythmia mechanisms. However, acquired forms of altered NaV1.5 function attributable to posttranslational modification (eg, phosphorylation or oxidation) may have pathophysiological consequences during ischemia/reperfusion or HF and thus reach a larger patient population. Indeed, half of all HF deaths are sudden and presumed to be attributable to lethal ventricular arrhythmias.5,6 The pore forming α subunit (NaV1.5; ≈220 Kd predicted MW) has 4 homologous domains (I–IV) with 6 transmembrane segments each (S1–S6; Figure 1), is glycosylated, and has auxiliary regulatory β subunits (β1–β4; ≈30–35 Kd).7 The S5–S6 linker includes the P-loops or pore region, the 4 S4 segments serve as voltage sensors (involved in activation gating), whereas an IFM motif in the DIII–IV linker is important for fast inactivation gating. Importantly, NaV1.5 forms a macromolecular complex with interacting proteins that …

Biochemical and biomechanical properties of the pacemaking sinoatrial node extracellular matrix are distinct from contractile left ventricular matrix

Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.

Manipulations of microRNA in human pluripotent stem cells and their derivatives.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells can self-renew while maintaining their pluripotency to differentiate into virtually all cell types. In addition to their potential for regenerative medicine, hESCs and iPSCs can also serve as excellent in vitro models for the study of human organogenesis and disease models, as well as drug toxicity screening. MicroRNAs (miRNAs) are nonencoding RNAs of ∼22 nucleotides that function as negative transcriptional regulators via degradation or inhibition by RNA interference (RNAi). MiRNAs play essential roles in developmental pathways. This chapter provides a description of how miRNAs can be introduced into hESCs/iPSCs or their derivatives for experiments via lentivirus-mediated gene transfer.