[Show abstract][Hide abstract] ABSTRACT: -In myocardial infarction (MI), repolarization alternans is a potent arrhythmia substrate that has been linked to Ca(2+) cycling proteins, such as SERCA2a, located in the sarcoplasmic reticulum (SR). MI is also associated with oxidative stress and increased xanthine oxidase (XO) activity, an important source of reactive oxygen species (ROS) in the SR that may reduce SERCA2a function. We hypothesize that in chronic MI, XO mediated oxidation of SERCA2a is a mechanism of cardiac alternans.
-Male Lewis rats underwent ligation of the LAD (n=54) or sham procedure (n=24). At 4 weeks, optical mapping of intracellular Ca(2+) and ROS were performed. ECG T-wave alternans (ECG ALT) and Ca(2+) transient alternans (Ca(2+)ALT) were induced by rapid pacing (300-120ms) before and after the XO inhibitor allopurinol (ALLO, 50μmol/L). In MI, ECG ALT (2.32±0.41%) and Ca(2+) ALT (22.3±4.5%) were significantly greater compared to sham (0.18±0.08%, p<0.001; 0.79±0.32%, p<0.01). Additionally, ROS was increased by 137% (p<0.01) and oxidation of SERCA2a by 30% (p<0.05) in MI compared to sham. Treatment with ALLO significantly decreased ECG ALT (-77±9%, p<0.05) and Ca(2+) ALT (-56±7%, p<0.05) and, importantly, reduced ROS (-65%, p<0.01) and oxidation of SERCA2a (-38%, p<0.05). CaMKII inhibition and general antioxidant treatment had no effect on ECG ALT and Ca(2+) ALT.
-These results demonstrate, for the first time, that in MI increased ROS from XO is a significant cause of repolarization alternans. This suggests that targeting XO ROS production may be effective at preventing arrhythmia substrates in chronic MI.
[Show abstract][Hide abstract] ABSTRACT: -Brugada syndrome (BrS) is an arrhythmogenic disorder that has been linked to mutations in SCN5A, the gene encoding for the pore-forming α-subunit of the cardiac sodium channel. Typically, BrS mutations in SCN5A result in a reduction of sodium current with some mutations even exhibiting a dominant-negative effect on wild-type (WT) channels thus leading to an even more prominent decrease in current amplitudes. However, there is also a category of apparently benign ("atypical") BrS SCN5A mutations that in vitro demonstrates only minor biophysical defects. It is therefore not clear how these mutations produce a BrS phenotype. We hypothesized that similar to dominant-negative mutations atypical mutations could lead to a reduction in sodium currents when co-expressed with WT to mimic the heterozygous patient genotype.
-WT and "atypical" BrS mutations were co-expressed in HEK293 cells, showing a reduction in sodium current densities similar to typical BrS mutations. Importantly, this reduction in sodium current was also seen when the atypical mutations were expressed in rat or human cardiomyocytes. This decrease in current density was the result of reduced surface expression of both mutant and WT channels.
-Taken together, we have shown how apparently benign SCN5A BrS mutations can lead to the ECG abnormalities seen in BrS patients through an induced defect that is only present when the mutations are co-expressed with WT channels. Our work has implications for risk management and stratification for some SCN5A-implicated BrS patients.
[Show abstract][Hide abstract] ABSTRACT: Background: Emerging evidence suggests that ventricular electrical remodeling (VER) is triggered by regional myocardial strain via mechano-electrical feedback mechanisms, however, the ionic mechanisms underlying strain-induced VER are unknown. Methods and Results: To determine its ionic basis, VER induced by altered electrical activation in dogs undergoing left ventricular pacing (n=6), were compared to unpaced controls (n=4). Action potential duration (APD), ionic currents and calcium transients were measured from canine epicardial myocytes isolated from early-activated (low-strain) and late-activated (high-strain) LV regions. VER in early-activated region was characterized by minimal APD prolongation, but marked attenuation of the action potential phase1 notch attributed to reduced Ito current. By contrast, VER in late-activated region was characterized by significant APD prolongation. Despite marked APD prolongation, there was surprisingly minimal change in ion channel densities, but a two-fold increase in diastolic calcium. Computer simulations demonstrated that changes in sarcolemmal ion channel density could only account for attenuation of phase1 notch observed in early-activated region, but failed to account for APD remodeling in late-activated region. Further, these simulations identified that cytosolic calcium accounted for APD prolongation in late-activated region by enhancing forward mode sodium-calcium exchanger (NCX) activity, and corroborated by increased NCX protein expression. Finally, assessment of skinned fibers following VER identified altered myofilament calcium sensitivity in late-activated regions to be associated with increased diastolic levels of calcium. Conclusions: We identified two distinct ionic mechanisms that underlie VER: strain-independent changes in early-activated regions ion channel remodeling, and strain-induced VER in late-activated regions from sarcomeric calcium handling remodeling.
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca(2+) handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca(2+) leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart. Here we show in the intact heart, reduced NOS1 activity increased Ca(2+)-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca(2+)](i). These arrhythmias arose from increased spontaneous SR Ca(2+) release, resulting from a combination of decreased RyR2 S-nitrosylation (RyR2-SNO) and increased RyR2 oxidation (RyR-SOx) (i.e., increased reactive oxygen species (ROS) from xanthine oxidoreductase activity) and could be suppressed with xanthine oxidoreductase (XOR) inhibition (i.e., allopurinol) or nitric oxide donors (i.e., S-nitrosoglutathione, GSNO). Surprisingly, we found evidence of NOS1 down-regulation of RyR2 phosphorylation at the Ca(2+)/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between nitrosylation and phosphorylation of RyR2. Finally, we show that nitroso-redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype by oxidative stress. Our findings suggest that nitroso-redox imbalance is an important mechanism of ventricular arrhythmias in the intact heart under disease conditions (i.e., elevated [Ca(2+)](i) and oxidative stress), and that therapies restoring nitroso-redox balance in the heart could prevent sudden arrhythmic death.
Proceedings of the National Academy of Sciences 10/2012; 109(44). DOI:10.1073/pnas.1210565109 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, we reported that sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a), the pump responsible for reuptake of cytosolic calcium during diastole, plays a central role in the molecular mechanism of cardiac alternans. Heart failure (HF) is associated with impaired myocardial calcium handling, deficient SERCA2a, and increased susceptibility to cardiac alternans. Therefore, we hypothesized that restoring deficient SERCA2a by gene transfer will significantly reduce arrhythmogenic cardiac alternans in the failing heart.
Adult guinea pigs were divided into 3 groups: control, HF, and HF+AAV9.SERCA2a gene transfer. HF resulted in a decrease in left ventricular fractional shortening compared with controls (P<0.001). As expected, isolated HF myocytes demonstrated slower sarcoplasmic reticulum calcium uptake, decreased Ca(2+) release, and increased diastolic Ca(2+) (P<0.05) compared with controls. Moreover, SERCA2a, cardiac ryanodine receptor 2, and sodium-calcium exchanger protein expression was decreased in HF compared with control (P<0.05). As predicted, HF increased susceptibility to cardiac alternans, as evidenced by decreased heart rate thresholds for both V(m) alternans and Ca alternans compared with controls (P<0.01). Interestingly, in vivo gene transfer of AAV9.SERCA2a in the failing heart improved left ventricular contractile function (P<0.01), suppressed cardiac alternans (P<0.01), and reduced ryanodine receptor 2 P(o) secondary to reduction of ryanodine receptor 2-P(S2814) (P<0.01). This ultimately resulted in a decreased incidence of inducible ventricular arrhythmias (P=0.05).
These data show that SERCA2a gene transfer in the failing heart not only improves contractile function but also directly restores electric stability through the amelioration of key arrhythmogenic substrate (ie, cardiac alternans) and triggers (ie, sarcoplasmic reticulum Ca(2+) leak).
[Show abstract][Hide abstract] ABSTRACT: Brugada syndrome (BrS) is an autosomal-inherited cardiac arrhythmia characterized by an ST-segment elevation in the right precordial leads of the electrocardiogram and an increased risk of syncope and sudden death. SCN5A, encoding the cardiac sodium channel Na(v)1.5, is the main gene involved in BrS. Despite the fact that several mutations have been reported in the N-terminus of Na(v)1.5, the functional role of this region remains unknown. We aimed to characterize two BrS N-terminal mutations, R104W and R121W, a construct where this region was deleted, ΔNter, and a construct where only this region was present, Nter.
Patch-clamp recordings in HEK293 cells demonstrated that R104W, R121W, and ΔNter abolished the sodium current I(Na). Moreover, R104W and R121W mutations exerted a strong dominant-negative effect on wild-type (WT) channels. Immunocytochemistry of rat neonatal cardiomyocytes revealed that both mutants were mostly retained in the endoplasmic reticulum and that their co-expression with WT channels led to WT channel retention. Furthermore, co-immunoprecipitation experiments showed that Na(v)1.5-subunits were interacting with each other, even when mutated, deciphering the mutation dominant-negative effect. Both mutants were mostly degraded by the ubiquitin-proteasome system, while ΔNter was addressed to the membrane, and Nter expression induced a two-fold increase in I(Na). In addition, the co-expression of N-terminal mutants with the gating-defective but trafficking-competent R878C-Na(v)1.5 mutant gave rise to a small I(Na).
This study reports for the first time the critical role of the Na(v)1.5 N-terminal region in channel function and the dominant-negative effect of trafficking-defective channels occurring through α-subunit interaction.
Cardiovascular Research 06/2012; 96(1):53-63. DOI:10.1093/cvr/cvs211 · 5.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Defects in the cardiac sodium channel gene, SCN5A, can cause a broad spectrum of inherited arrhythmia syndromes. After genotyping of a proband who presented with syncope, the SCN5A mutant P2006A and the common polymorphism H558R were identified.
The main objective of this study was to determine whether the SCN5A-H558R polymorphism could modify the defective gating kinetics observed in the P2006A mutation and therefore explain why this gain-of-function mutation has been identified in control populations.
Mutations were engineered using site-directed mutagenesis and heterologously expressed transiently in HEK293 cells. Whole-cell sodium currents were measured at room temperature using the whole-cell patch-clamp technique.
In HEK293 cells, P2006A displayed biophysical defects typically associated with long QT syndrome by increasing persistent sodium current, producing a depolarizing shift in voltage dependence of inactivation, and hastening recovery from inactivation. Interestingly, when coexpressed either on the same or different genes, P2006A and H558R displayed currents that behaved like wild type (WT). We also investigated whether H558R can modulate the gating defects of other SCN5A mutations. The H558R polymorphism also restored the gating defects of the SCN5A mutation V1951L to the WT level.
Our results suggest that H558R might play an important role in stabilization of channel fast inactivation and may provide a plausible explanation as to why the P2006A gain-of-function mutation has been identified in control populations. Our results also suggest that the SCN5A polymorphism H558R might be a disease-modifying gene.
Heart rhythm: the official journal of the Heart Rhythm Society 11/2010; 8(3):455-62. DOI:10.1016/j.hrthm.2010.11.034 · 5.08 Impact Factor