Cody A Rutledge

Mitochondria Oxidative Stress, Connexin43 Remodeling, and Sudden Arrhythmic Death

Background—Previously, we showed that a mouse model (ACE8/8) of cardiac renin–angiotensin system activation has a high rate of spontaneous ventricular tachycardia and sudden cardiac death secondary to a reduction in connexin43 level. Angiotensin-II activation increases reactive oxygen species (ROS) production, and ACE8/8 mice show increased cardiac ROS. We sought to determine the source of ROS and whether ROS played a role in the arrhythmogenesis. Methods and Results—Wild-type and ACE8/8 mice with and without 2 weeks of treatment with L-NIO (NO synthase inhibitor), sepiapterin (precursor of tetrahydrobiopterin), MitoTEMPO (mitochondria-targeted antioxidant), TEMPOL (a general antioxidant), apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), allopurinol (xanthine oxidase inhibitor), and ACE8/8 crossed with P67 dominant negative mice to inhibit the nicotinamide adenine dinucleotide phosphate oxidase were studied. Western blotting, detection of mitochondrial ROS by MitoSOX Red, electron microscopy, immunohistochemistry, fluorescent dye diffusion technique for functional assessment of connexin43, telemetry monitoring, and in vivo electrophysiology studies were performed. Treatment with MitoTEMPO reduced sudden cardiac death in ACE8/8 mice (from 74% to 18%; P<0.005), decreased spontaneous ventricular premature beats, decreased ventricular tachycardia inducibility (from 90% to 17%; P<0.05), diminished elevated mitochondrial ROS to the control level, prevented structural damage to mitochondria, resulted in 2.6-fold increase in connexin43 level at the gap junctions, and corrected gap junction conduction. None of the other antioxidant therapies prevented ventricular tachycardia and sudden cardiac death in ACE8/8 mice. Conclusions—Mitochondrial oxidative stress plays a central role in angiotensin II–induced gap junction remodeling and arrhythmia. Mitochondria-targeted antioxidants may be effective antiarrhythmic drugs in cases of renin–angiotensin system activation.

Caveolin-1 modulates cardiac gap junction homeostasis and arrhythmogenecity by regulating cSrc tyrosine kinase.

Background—Genome-wide association studies have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Methods and Results—Using adult Cav1-/- mice, we revealed a marked reduction in the left ventricular conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the activation of cSrc tyrosine kinase, resulting in the downregulation of connexin 43 and subsequent electric abnormalities. Pharmacological inhibition of cSrc mitigates connexin 43 downregulation, slowed conduction, and arrhythmia inducibility in Cav1-/- animals. Using a transgenic mouse model with cardiac-specific overexpression of angiotensin-converting enzyme (ACE8/8), we demonstrated that, on enhanced cardiac renin–angiotensin system activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation at Cys156, leading to cSrc activation, connexin 43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. Renin–angiotensin system–induced Cav1 S-nitrosation was associated with increased Cav1–endothelial nitric oxide synthase binding in response to increased mitochondrial reactive oxidative species generation. Conclusions—The present studies reveal the critical role of Cav1 in modulating cSrc activation, gap junction remodeling, and ventricular arrhythmias. These data provide a mechanistic explanation for the observed genetic link between Cav1 and cardiac arrhythmias in humans and suggest that targeted regulation of Cav1 may reduce arrhythmic risk in cardiac diseases associated with renin–angiotensin system activation.

Mitochondria and Arrhythmias

There is a clear relationship between cardiac mechanical dysfunction and arrhythmogenesis, and yet the mechanistic link is unknown. Mechanical dysfunction is accompanied by mitochondrial dysfunction, and in this review, we will discuss some of the ways mitochondrial dysfunction can lead to arrhythmogenesis, thereby providing a link between mechanical dysfunction and arrhythmias. Mitochondria occupy around 30% of the mammalian myocardium by volume and are responsible for over 90% of cardiac ATP production1. In addition to energy production, mitochondria have been implicated as critical organelles involved in ion channel regulation, heat maintenance, apoptotic function, and regulation of reactive oxygen species (ROS)2. A growing field of research, coined mitochondrial medicine, is aimed at modifying mitochondrial function, in particular the generation of ROS, to alleviate disease burden attributed to mitochondrial stress2;3. The aim of this review is to discuss the role of mitochondrial dysfunction in arrhythmogenesis and to posit new antiarrhythmic therapies based on ameliorating mitochondrial dysfunction in cardiac disease. New information on mitochondrial regulation of sodium channels, potassium channels, and connexons will be discussed. Calcium handling and the mitochondrial permeability transition pore, both of which contribute to arrhythmogenesis and tissue injury following mitochondrial distress, have been reviewed elsewhere. The mitochondria are organelles with two membranes that create two compartments: the intermembrane space and the mitochondrial matrix. Mitochondria function as key regulators of metabolism, utilizing oxygen and dietary substrates to generate ATP via oxidative phosphorylation (OXPHOS). During OXPHOS, electrons are collected from the oxidation of carbohydrates and fats to allow the production of reducing equivalents NADH and FADH2. These reducing equivalents transfer their electrons to the electron transport chain (ETC) complexes along the inner mitochondrial membrane. As the electrons flow through the complexes of the ETC, H+ is driven out of the mitochondrial matrix and sequestered into the intermembrane space. This creates a strongly negative mitochondrial membrane potential designated as Δψm, that can be utilized to help target drugs to the mitochondria. Movement of H+ down the proton-gradient across the inner membrane drives the final complex of the ETC, ATP synthase, which converts ADP to ATP. As a by-product of OXPHOS, reactive oxygen species (ROS) are often produced. Incomplete reduction or a surplus of electrons in the ETC can result in partially reduced oxygen molecules, creating the reactive intermediate superoxide (O2−). The mitochondrial antioxidant protein, manganese superoxide dismutase (MnSOD), is responsible for converting O2− to H2O2, which can be further broken down by catalase. Mitochondrial ROS production is elevated beyond MnSOD’s antioxidant capacity in a wide range of diseases, including diabetes, metabolic syndrome, cancer, and cardiomyopathy, and aging3. This mitochondrial stress results in the build-up of deleterious metabolites, such as NADH and ADP, and depletion of antioxidant defenses, such as glutathione4;5. Recent works in cardiology have implicated mitochondrial stress in arrhythmogenesis, allowing a potential new avenue for therapeutic approach.