Cardiac arrhythmias can cause sudden cardiac death (SCD) and add to the current heart failure (HF) health crisis. Nevertheless, the pathological processes underlying arrhythmias are unclear. Arrhythmic conditions are associated with systemic and cardiac oxidative stress caused by reactive oxygen species (ROS). In excitable cardiac cells, ROS regulate both cellular metabolism and ion homeostasis. Increasing evidence suggests that elevated cellular ROS can cause alterations of the cardiac sodium channel (Nav1.5), abnormal Ca2+ handling, changes of mitochondrial function, and gap junction remodeling, leading to arrhythmogenesis. This review summarizes our knowledge of the mechanisms by which ROS may cause arrhythmias and discusses potential therapeutic strategies to prevent arrhythmias by targeting ROS and its consequences.

Rationale Previously, we demonstrated that a deoxycorticosterone acetate (DOCA)-salt hypertensive mouse model produces cardiac oxidative stress and diastolic dysfunction with preserved systolic function. Oxidative stress has been shown to increase late inward sodium current (INa), reducing the net cytosolic Ca2+ efflux. Objective Oxidative stress in the DOCA-salt model may increase late INa resulting in diastolic dysfunction amenable to treatment with ranolazine. Methods and Results Echocardiography detected evidence of diastolic dysfunction in hypertensive mice that improved after treatment with ranolazine (E/E′, sham 31.9 ± 2.8, sham+ranolazine 30.2 ± 1.9, DOCA-salt 41.8 ± 2.6, and DOCA-salt+ranolazine 31.9 ± 2.6, p = 0.018). The end diastolic pressure volume relationship slope was elevated in DOCA-salt mice, improving to sham levels with treatment (sham 0.16 ± 0.01 vs. sham+ranolazine 0.18 ± 0.01 vs. DOCA-salt 0.23 ± 0.2 vs. DOCA-salt+ranolazine 0.17 ± 0.01 mm Hg/L, p < 0.005). DOCA-salt myocytes demonstrated impaired relaxation, τ, improving with ranolazine (DOCA-salt 0.18 ± 0.02, DOCA-salt + ranolazine 0.13 ± 0.01, Sham 0.11 ± 0.01, Sham + ranolazine 0.09 ± 0.02 s, p = 0.0004). Neither late INa nor the Ca2+ transients were different from sham myocytes. Detergent extracted fiber bundles from DOCA-salt hearts demonstrated increased myofilament response to Ca2+ with glutathionylation of myosin binding protein C. Treatment with ranolazine ameliorated the Ca2+ response and cross-bridge kinetics. Conclusions Therefore, diastolic dysfunction could be reversed by ranolazine, likely resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus.

Background Previously, we showed a mouse model (ACE8/8) of cardiac renin-angiotensin system (RAS) activation has a high rate of spontaneous ventricular tachycardia (VT) and sudden cardiac death (SCD) secondary to a reduction in connexin43 (Cx43) 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 if ROS played a role in the arrhythmogenesis. Methods and Results Wild-type and ACE8/8 mice with and without two weeks of treatment with L-NIO (nitric oxide synthase inhibitor), sepiapterin (precursor of tetrahydrobiopterin), MitoTEMPO (mitochondria-targeted antioxidant), TEMPOL (a general antioxidant), apocynin (NADPH oxidase inhibitor), allopurinol (xanthine oxidase inhibitor), and ACE8/8 crossed with P67 dominant negative mice to inhibit the NADPH oxidase were studied. Western blotting, detection of mitochondrial ROS by MitoSOX Red, electron microscopy, immunohistochemistry, fluorescent dye diffusion technique for functional assessment of Cx43, telemetry monitoring, and in-vivo electrophysiology studies were performed. Treatment with MitoTEMPO reduced SCD in ACE8/8 mice (from 74% to 18%, P<0.005), decreased spontaneous ventricular premature beats, decreased VT 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 Cx43 level at the gap junctions, and corrected gap junction conduction. None of the other antioxidant therapies prevented VT and SCD 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 RAS activation.

Background Human heart failure (HF) is associated with decreased cardiac voltage-gated Na+ channel current (encoded by SCN5A), and the changes have been implicated in the increased risk of sudden death in HF. Nevertheless, the mechanism of SCN5A downregulation is unclear. A number of human diseases are associated with alternative mRNA splicing, which has received comparatively little attention in the study of cardiac disease. Splicing factor expression profiles during human HF and a specific splicing pathway for SCN5A regulation were explored in this paper. Methods and Results Gene array comparisons between normal human and heart failure tissues demonstrated that 17 splicing factors, associated with all major spliceosome components, were upregulated. Two of these splicing factors, RBM25 and LUC7L3, were elevated in human heart failure tissue and mediated truncation of SCN5A mRNA in both Jurkat cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). RBM25/LUC7L3-mediated abnormal SCN5A mRNA splicing reduced Na+ channel current 91.1 ± 9.3% to a range known to cause sudden death. Overexpression of either splicing factor resulted in an increase in truncated mRNA and a concomitant decrease in the full-length SCN5A transcript. Conclusions Of the 17 mRNA splicing factors upregulated in HF, RBM25 and LUC7L3 were sufficient to explain the increase in truncated forms and the reduction in full length Na+ channel transcript. Since the reduction in channels was in the range known to be associated with sudden death, interruption of this abnormal mRNA processing may reduce arrhythmic risk in heart failure.

Cardiomyopathy is associated with cardiac Na+ channel downregulation that may contribute to arrhythmias. Previously, we have shown that elevated intracellular NADH causes a decrease in cardiac Na+ current (INa) signaled by an increase in mitochondrial reactive oxygen species (ROS). In this study, we tested whether the NADH–mitochondria ROS pathway was involved in the reduction of INa in a nonischemic cardiomyopathic model and correlated the findings with myopathic human hearts. Nonischemic cardiomyopathy was induced in C57BL/6 mice by hypertension after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and salt water substitution. Sham operated mice were used as controls. After six weeks, heart tissue and ventricular myocytes isolated from mice were utilized for whole cell patch clamp recording, NADH/NAD+ level measurements, and mitochondrial ROS monitoring with confocal microscopy. Human explanted hearts were studied using optical mapping. Compared to the sham mice, the arterial blood pressure was higher, the left ventricular volume was significantly enlarged (104.7±3.9 vs. 87.9±6.1 μL, P<0.05), and the ejection fraction was reduced (37.1±1.8% vs. 49.4±3.7%, P<0.05) in DOCA mice. Both the whole cell and cytosolic NADH level were increased (279±70% and 123±2% of sham, respectively, P<0.01), INa was decreased (60±10% of sham, P<0.01), and mitochondrial ROS overproduction was observed (2.9±0.3-fold of sham, P<0.01) in heart tissue and myocytes of myopathic mice vs. sham. Treatment of myocytes with NAD+ (500 μM), mitoTEMPO (10 μM), chelerythrine (50 μM), or forskolin (5 μM) restored INa back to the level of sham. Injection of NAD+ (100 mg/kg) or mitoTEMPO (0.7 mg/kg) twice (at 24 h and 1 h before myocyte isolation) to animals also restored INa. All treatments simultaneously reduced mitochondrial ROS levels to that of controls. CD38 was found to transduce the extracellular NAD+ signal. Correlating with the mouse model, failing human hearts showed a reduction in conduction velocity that improved with NAD+. Nonischemic cardiomyopathy was associated with elevated NADH level, PKC activation, mitochondrial ROS overproduction, and a concomitant decrease in INa. Reducing mitochondrial ROS by application of NAD+, mitoTEMPO, PKC inhibitors, or PKA activators, restored INa. NAD+ improved conduction velocity in human myopathic hearts.

Background Human heart failure (HF) increases alternative mRNA splicing of the SCN5A cardiac Na+ channel, generating variants encoding truncated, nonfunctional channels that are trapped in the endoplasmic reticulum. In this work, we tested whether truncated Na+ channels activate the unfolded protein response (UPR), contributing to SCN5A electrical remodeling in HF. Methods and Results UPR and SCN5A were analyzed in human ventricular systolic HF tissue samples and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Cells were exposed to angiotensin II (AngII) and hypoxia, known activators of abnormal SCN5A mRNA splicing, or were induced to overexpress SCN5A variants. UPR effectors, PERK, calreticulin, and CHOP, were increased in human HF tissues. Induction of SCN5A variants with AngII or hypoxia or the expression of exogenous variants induced the UPR with concomitant downregulation of Na+ current. PERK activation destabilized SCN5A and, surprisingly, Kv4.3 channel mRNAs but not TRPM7 channel mRNA. PERK inhibition prevented the loss of full-length SCN5A and Kv4.3 mRNA levels resulting from expressing Na+ channel mRNA splice variants. Conclusions UPR can be initiated by Na+ channel mRNA splice variants and is involved in the reduction of cardiac Na+ current during human HF. Since the effect is not entirely specific to the SCN5A transcript, the UPR may play an important role in downregulation of multiple cardiac genes in HF.

Despite the growing number of patients affected, the understanding of diastolic dysfunction and heart failure with preserved ejection fraction (HFpEF) is still poor. Clinical trials, largely based on successful treatments for systolic heart failure, have been disappointing, suggesting that HFpEF has a different pathology to that of systolic dysfunction. In this review, general concepts, epidemiology, diagnosis, and treatment of diastolic dysfunction are summarized, with an emphasis on new experiments suggesting that oxidative stress plays a crucial role in the pathogenesis of at least some forms of the disease. This observation has lead to potential new diagnostics and therapeutics for diastolic dysfunction and heart failure caused by diastolic dysfunction.

heart failure is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increased stiffness. We tested the hypothesis that senescence-prone (SAMP8) mice would develop diastolic dysfunction compared with senescence-resistant controls (SAMR1). Pulsed-wave Doppler imaging of the ratio of blood flow velocity through the mitral valve during early (E) vs. late (A) diastole was reduced from 1.3 Ϯ 0.03 in SAMR1 mice to 1.2 Ϯ 0.03 in SAMP8 mice (P Ͻ 0.05). Tissue Doppler imaging of the early (E') and late (A') diastolic mitral annulus velocities found E' reduced from 25.7 Ϯ 0.9 mm/s in SAMR1 to 21.1 Ϯ 0.8 mm/s in SAMP8 mice and E'/A' similarly reduced from 1.1 Ϯ 0.02 to 0.8 Ϯ 0.03 in SAMR1 vs. SAMP8 mice, respectively (P Ͻ 0.05). Invasive hemodynamics revealed an increased slope of the end-diastolic pressure-volume relationship (0.5 Ϯ 0.05 vs. 0.8 Ϯ 0.14; P Ͻ 0.05), indicating increased left ventricular chamber stiffness. There were no differences in systolic function or mean arterial pressure; however, diastolic dysfunction was accompanied by increased fibrosis in the hearts of SAMP8 mice. In SAMR1 vs. SAMP8 mice, interstitial collagen area increased from 0.3 Ϯ 0.04 to 0.8 Ϯ 0.09% and perivascular collagen area increased from 1.0 Ϯ 0.11 to 1.6 Ϯ 0.14%. Transforming growth factor-␤ and connective tissue growth factor gene expression were increased in the hearts of SAMP8 mice (P Ͻ 0.05 for all data). In summary, SAMP8 mice show increased fibrosis and diastolic dysfunction similar to those seen in humans with aging and may represent a suitable model for future mechanistic studies. heart failure; transforming growth factor; aging HEART FAILURE IS A MAJOR AND growing public health concern in the United States; there are an estimated 5 million people living with this disease, and another 550,000 patients will be diagnosed yearly (6). Approximately one-half of all heart failure patients in the United States suffer from diastolic heart failure, and it is a major cause of mortality in the elderly population (35). Diastolic heart failure describes a group of patients whose clinical manifestation of congestive heart failure is characterized by normal left ventricular diastolic volume, a normal ejection fraction, delayed active relaxation, and increased passive stiffness of the left ventricle (34). Diastolic dysfunction precedes diastolic heart failure and is often clinically silent; it is characterized by abnormal ventricular distensibility, relaxation, and filling (1). Both diastolic dysfunction and diastolic heart failure are most common in the elderly population. In a study that examined the prevalence of this form of heart failure, 50% of patients over the age of 70 showed evidence of diastolic heart failure. Studies (35) indicate that the most important determinants for development of diastolic dysfunction and diastolic heart failure are age and hypertension. Although the exact molecular mechanisms behind diastolic dysfuncti...