Geoffrey G. Hesketh

Electrophysiological Consequences of Dyssynchronous Heart Failure and Its Restoration by Resynchronization Therapy

Background— Cardiac resynchronization therapy (CRT) is widely applied in patients with heart failure and dyssynchronous contraction (DHF), but the electrophysiological consequences of CRT in heart failure remain largely unexplored. Methods and Results— Adult dogs underwent left bundle-branch ablation and either right atrial pacing (190 to 200 bpm) for 6 weeks (DHF) or 3 weeks of right atrial pacing followed by 3 weeks of resynchronization by biventricular pacing at the same pacing rate (CRT). Isolated left ventricular anterior and lateral myocytes from nonfailing (control), DHF, and CRT dogs were studied with the whole-cell patch clamp. Quantitative polymerase chain reaction and Western blots were performed to measure steady state mRNA and protein levels. DHF significantly reduced the inward rectifier K+ current (IK1), delayed rectifier K+ current (IK), and transient outward K+ current (Ito) in both anterior and lateral cells. CRT partially restored the DHF-induced reduction of IK1 and IK but not Ito, consistent with trends in the changes in steady state K+ channel mRNA and protein levels. DHF reduced the peak inward Ca2+ current (ICa) density and slowed ICa decay in lateral compared with anterior cells, whereas CRT restored peak ICa amplitude but did not hasten decay in lateral cells. Calcium transient amplitudes were depressed and the decay was slowed in DHF, especially in lateral myocytes. CRT hastened the decay in both regions and increased the calcium transient amplitude in lateral but not anterior cells. No difference was found in CaV1.2 (α1C) mRNA or protein expression, but reduced CaVβ2 mRNA was found in DHF cells. DHF reduced phospholamban, ryanodine receptor, and sarcoplasmic reticulum Ca2+ ATPase and increased Na+-Ca2+ exchanger mRNA and protein. CRT did not restore the DHF-induced molecular remodeling, except for sarcoplasmic reticulum Ca2+ ATPase. Action potential durations were significantly prolonged in DHF, especially in lateral cells, and CRT abbreviated action potential duration in lateral but not anterior cells. Early afterdepolarizations were more frequent in DHF than in control cells and were reduced with CRT. Conclusions— CRT partially restores DHF-induced ion channel remodeling and abnormal Ca2+ homeostasis and attenuates the regional heterogeneity of action potential duration. The electrophysiological changes induced by CRT may suppress ventricular arrhythmias, contribute to the survival benefit of this therapy, and improve the mechanical performance of the heart.

Cyclic GMP/PKG-Dependent Inhibition of TRPC6 Channel Activity and Expression Negatively Regulates Cardiomyocyte NFAT Activation: Novel Mechanism of Cardiac Stress Modulation by PDE5 Inhibition

Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phospho-mimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKG-dependent channel phosphorylation.

Ultrastructure and Regulation of Lateralized Connexin43 in the Failing Heart

Rationale: Gap junctions mediate cell-to-cell electric coupling of cardiomyocytes. The primary gap junction protein in the working myocardium, connexin43 (Cx43), exhibits increased localization at the lateral membranes of cardiomyocytes in a variety of heart diseases, although the precise location and function of this population is unknown. Objective: To define the subcellular location of lateralized gap junctions at the light and electron microscopic level, and further characterize the biochemical regulation of gap junction turnover. Methods and Results: By electron microscopy, we characterized gap junctions formed between cardiomyocyte lateral membranes in failing canine ventricular myocardium. These gap junctions were varied in structure and appeared to be extensively internalizing. Internalized gap junctions were incorporated into multilamellar membrane structures, with features characteristic of autophagosomes. Intracellular Cx43 extensively colocalized with the autophagosome marker GFP-LC3 when both proteins were exogenously expressed in HeLa cells, and endogenous Cx43 colocalized with GFP-LC3 in neonatal rat ventricular myocytes. Furthermore, a distinct phosphorylated form of Cx43, as well as the autophagosome-targeted form of LC3 (microtubule-associated protein light chain 3) targeted to lipid rafts in cardiac tissue, and both were increased in heart failure. Conclusions: Our data demonstrate a previously unrecognized pathway of gap junction internalization and degradation in the heart and identify a cellular pathway with potential therapeutic implications.

Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes

AIMS Calmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood. METHODS AND RESULTS Purified Na(V)1.5-glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I-II linker. Whole-cell voltage-clamp was used to measure Na+ current (I(Na)) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na+ channel availability by approximately +5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change I(Na) decay. These CaMKII-induced changes of Na+ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca2+/CaM alone reproduced the CaMKII-induced changes of I(Na) availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of I(Na) function. CONCLUSION Ca2+/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na+ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na+ channel gating.

A 14-3-3 Mode-1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.

IK1 Heterogeneity Affects Genesis and Stability of Spiral Waves in Cardiac Myocyte Monolayers

Previous studies have postulated an important role for the inwardly rectifying potassium current (IK1) in controlling the dynamics of electrophysiological spiral waves responsible for ventricular tachycardia and fibrillation. In this study, we developed a novel tissue model of cultured neonatal rat ventricular myocytes (NRVMs) with uniform or heterogeneous Kir2.1expression achieved by lentiviral transfer to elucidate the role of IK1 in cardiac arrhythmogenesis. Kir2.1-overexpressed NRVMs showed increased IK1 density, hyperpolarized resting membrane potential, and increased action potential upstroke velocity compared with green fluorescent protein–transduced NRVMs. Opposite results were observed in Kir2.1-suppressed NRVMs. Optical mapping of uniformly Kir2.1 gene-modified monolayers showed altered conduction velocity and action potential duration compared with nontransduced and empty vector-transduced monolayers, but functional reentrant waves could not be induced. In monolayers with an island of altered Kir2.1 expression, conduction velocity and action potential duration of the locally transduced and nontransduced regions were similar to those of the uniformly transduced and nontransduced monolayers, respectively, and functional reentrant waves could be induced. The waves were anchored to islands of Kir2.1 overexpression and remained stable but dropped in frequency and meandered away from islands of Kir2.1 suppression. In monolayers with an inverse pattern of IK1 heterogeneity, stable high frequency spiral waves were present with IK1 overexpression, whereas lower frequency, meandering spiral waves were observed with IK1 suppression. Our study provides direct evidence for the contribution of IK1 heterogeneity and level to the genesis and stability of spiral waves and highlights the potential importance of IK1 as an antiarrhythmia target.

Mechanisms of Gap Junction Traffic in Health and Disease

Gap junctions (GJs) allow direct communication between cells. In the heart, GJs mediate the electrical coupling of cardiomyocytes and as such dictate the speed and direction of cardiac conduction. A prominent feature of acquired structural heart disease is remodeling of GJ protein expression and localization concomitant with increased susceptibility to lethal arrhythmias, leading many to hypothesize that the two are causally linked. Detailed understanding of the cellular mechanisms that regulate GJ localization and function within cardiomyocytes may therefore uncover potential therapeutic strategies for a significant clinical problem. This review will outline our current understanding of GJ cell biology with the intent of highlighting cellular mechanisms responsible for GJ remodeling associated with cardiac disease.

Phosphodiesterase-5A (PDE5A) is localized to the endothelial caveolae and modulates NOS3 activity

AIMS It has been well demonstrated that phosphodiesterase-5A (PDE5A) is expressed in smooth muscle cells and plays an important role in regulation of vascular tone. The role of endothelial PDE5A, however, has not been yet characterized. The present study was undertaken to determine the presence, localization, and potential physiologic significance of PDE5A within vascular endothelial cells. METHODS AND RESULTS We demonstrate primary location of human, mouse, and bovine endothelial PDE5A at or near caveolae. We found that the spatial localization of PDE5A at the level of caveolin-rich lipid rafts allows for a feedback loop between endothelial PDE5A and nitric oxide synthase (NOS3). Treatment of human endothelium with PDE5A inhibitors resulted in a significant increase in NOS3 activity, whereas overexpression of PDE5A using an adenoviral vector, both in vivo and in cell culture, resulted in decreased NOS3 activity and endothelium-dependent vasodilation. The molecular mechanism responsible for these interactions is primarily regulated by cGMP-dependent second messenger. PDE5A overexpression also resulted in a significant decrease in protein kinase 1 (PKG1) activity. Overexpression of PKG1 rapidly activated NOS3, whereas silencing of the PKG1 gene with siRNA inhibited both NOS3 phosphorylation (S1179) and activity, indicating a novel role for PKG1 in direct regulation of NOS3. CONCLUSION Our data collectively suggest another target for PDE5A inhibition in endothelial dysfunction and provide another physiologic significance for PDE5A in the modulation of endothelial-dependent flow-mediated vasodilation. Using both in vitro and in vivo models, as well as human data, we show that inhibition of endothelial PDE5A improves endothelial function.

A Mutation Causing Brugada Syndrome Identifies a Mechanism for Altered Autonomic and Oxidant Regulation of Cardiac Sodium Currents

Background—The mechanisms of the electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the sodium channel gene, SCN5A, and regulatory proteins that reduce or eliminate sodium current (INa) have been linked to BrS. We studied the properties of a BrS-associated SCN5A mutation in a protein kinase A (PKA) consensus phosphorylation site, R526H. Methods and Results—In vitro PKA phosphorylation was detected in the I-II linker peptide of wild-type (WT) channels but not R526H or S528A (phosphorylation site) mutants. Cell surface expression of R526H and S528A channels was reduced compared with WT. Whole-cell INa through all channel variants revealed no significant differences in the steady-state activation, inactivation, and recovery from inactivation. Peak current densities of the mutants were significantly reduced compared with WT. Infection of 2D cultures of neonatal rat ventricular myocytes with WT and mutant channels increased conduction velocity compared with noninfected cells. PKA stimulation significantly increased peak INa and conduction velocity of WT but not mutant channels. Oxidant stress inhibits cardiac INa; WT and mutant INa decreases with the intracellular application of reduced nicotinamide adenine dinucleotide (NADH), an effect that is reversed by PKA stimulation in WT but not in R526H or S528A channels. Conclusions—We identified a family with BrS and an SCN5A mutation in a PKA consensus phosphorylation site. The BrS mutation R526H is associated with a reduction in the basal level of INa and a failure of PKA stimulation to augment the current that may contribute to the predisposition to arrhythmias in patients with BrS, independent of the precipitants.

Cardiac Resynchronization Therapy Improves Altered Na Channel Gating in Canine Model of Dyssynchronous Heart Failure

Background—Slowed Na+ current (INa) decay and enhanced late INa (INa-L) prolong the action potential duration (APD) and contribute to early afterdepolarizations. Cardiac resynchronization therapy (CRT) shortens APD compared with dyssynchronous heart failure (DHF); however, the role of altered Na+ channel gating in CRT remains unexplored. Methods and Results—Adult dogs underwent left-bundle branch ablation and right atrial pacing (200 beats/min) for 6 weeks (DHF) or 3 weeks followed by 3 weeks of biventricular pacing at the same rate (CRT). INa and INa-L were measured in left ventricular myocytes from nonfailing, DHF, and CRT dogs. DHF shifted voltage-dependence of INa availability by −3 mV compared with nonfailing, enhanced intermediate inactivation, and slowed recovery from inactivation. CRT reversed the DHF-induced voltage shift of availability, partially reversed enhanced intermediate inactivation but did not affect DHF-induced slowed recovery. DHF markedly increased INa-L compared with nonfailing. CRT dramatically reduced DHF-induced enhanced INa-L, abbreviated the APD, and suppressed early afterdepolarizations. CRT was associated with a global reduction in phosphorylated Ca2+/Calmodulin protein kinase II, which has distinct effects on inactivation of cardiac Na+ channels. In a canine AP model, alterations of INa-L are sufficient to reproduce the effects on APD observed in DHF and CRT myocytes. Conclusions—CRT improves DHF-induced alterations of Na+ channel function, especially suppression of INa-L, thus, abbreviating the APD and reducing the frequency of early afterdepolarizations. Changes in the levels of phosphorylated Ca2+/Calmodulin protein kinase II suggest a molecular pathway for regulation of INa by biventricular pacing of the failing heart.