Euy Myoung Jeong

Ranolazine Improves Cardiac Diastolic Dysfunction Through Modulation of Myofilament Calcium Sensitivity

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 versus sham+ranolazine, 0.18±0.01 versus DOCA-salt, 0.23±0.2 versus DOCA-salt+ranolazine, 0.17±0.0 1 mm Hg/L; P<0.005). DOCA-salt myocytes demonstrated impaired relaxation, &tgr;, 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 seconds; 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: Diastolic dysfunction could be reversed by ranolazine, probably resulting from a direct effect on myofilaments, indicating that cardiac oxidative stress may mediate diastolic dysfunction through altering the contractile apparatus.

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.

Tetrahydrobiopterin improves diastolic dysfunction by reversing changes in myofilament properties

Despite the increasing prevalence of heart failure with preserved left ventricular function, there are no specific treatments, partially because the mechanism of impaired relaxation is incompletely understood. Evidence indicates that cardiac relaxation may depend on nitric oxide (NO), generated by NO synthase (NOS) requiring the co-factor tetrahydrobiopterin (BH(4)). Recently, we reported that hypertension-induced diastolic dysfunction was accompanied by cardiac BH(4) depletion, NOS uncoupling, a depression in myofilament cross-bridge kinetics, and S-glutathionylation of myosin binding protein C (MyBP-C). We hypothesized that the mechanism by which BH(4) ameliorates diastolic dysfunction is by preventing glutathionylation of MyBP-C and thus reversing changes of myofilament properties that occur during diastolic dysfunction. We used the deoxycorticosterone acetate (DOCA)-salt mouse model, which demonstrates mild hypertension, myocardial oxidative stress, and diastolic dysfunction. Mice were divided into two groups that received control diet and two groups that received BH(4) supplement for 7days after developing diastolic dysfunction at post-operative day 11. Mice were assessed by echocardiography. Left ventricular papillary detergent-extracted fiber bundles were isolated for simultaneous determination of force and ATPase activity. Sarcomeric protein glutathionylation was assessed by immunoblotting. DOCA-salt mice exhibited diastolic dysfunction that was reversed after BH(4) treatment. Diastolic sarcomere length (DOCA-salt 1.70±0.01 vs. DOCA-salt+BH(4) 1.77±0.01μm, P<0.001) and relengthening (relaxation constant, τ, DOCA-salt 0.28±0.02 vs. DOCA-salt+BH(4) 0.08±0.01, P<0.001) were also restored to control by BH(4) treatment. pCa(50) for tension increased in DOCA-salt compared to sham but reverted to sham levels after BH(4) treatment. Maximum ATPase rate and tension cost (ΔATPase/ΔTension) decreased in DOCA-salt compared to sham, but increased after BH(4) treatment. Cardiac MyBP-C glutathionylation increased in DOCA-salt compared to sham, but decreased with BH(4) treatment. MyBP-C glutathionylation correlated with the presence of diastolic dysfunction. Our results suggest that by depressing S-glutathionylation of MyBP-C, BH(4) ameliorates diastolic dysfunction by reversing a decrease in cross-bridge turnover kinetics. These data provide evidence for modulation of cardiac relaxation by post-translational modification of myofilament proteins.

Mitochondrial Dysfunction Causing Cardiac Sodium Channel Downregulation in Cardiomyopathy

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 (I(Na)) 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 I(Na) 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), I(Na) 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 I(Na) back to the level of sham. Injection of NAD(+) (100mg/kg) or mitoTEMPO (0.7 mg/kg) twice (at 24h and 1h before myocyte isolation) to animals also restored I(Na). 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 I(Na). Reducing mitochondrial ROS by application of NAD(+), mitoTEMPO, PKC inhibitors, or PKA activators, restored I(Na). NAD(+) improved conduction velocity in human myopathic hearts.

Unfolded protein response regulates cardiac sodium current in systolic human heart failure

Background— Human heart failure (HF) increases alternative mRNA splicing of the type V, voltage-gated cardiac Na+ channel &agr;-subunit (SCN5A), 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 electric 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, protein kinase R-like ER kinase (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 transient receptor potential cation channel M7 (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. Because 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.

Role of Mitochondrial Oxidative Stress in Glucose Tolerance, Insulin Resistance, and Cardiac Diastolic Dysfunction

Background Diabetes mellitus (DM) is associated with mitochondrial oxidative stress. We have shown that myocardial oxidative stress leads to diastolic dysfunction in a hypertensive mouse model. Therefore, we hypothesized that diabetes mellitus could cause diastolic dysfunction through mitochondrial oxidative stress and that a mitochondria‐targeted antioxidant (MitoTEMPO) could prevent diastolic dysfunction in a diabetic mouse model. Methods and Results C57BL/6J mice were fed either 60 kcal % fat diet (high‐fat diet [HFD]) or normal chow (control) for 8 weeks with or without concurrent MitoTEMPO administration, followed by in vivo assessment of diastolic function and ex vivo studies. HFD mice developed impaired glucose tolerance compared with the control (serum glucose=495±45 mg/dL versus 236±30 mg/dL at 60 minutes after intraperitoneal glucose injection, P<0.05). Myocardial tagged cardiac magnetic resonance imaging showed significantly reduced diastolic circumferential strain (Ecc) rate in the HFD mice compared with controls (5.0±0.3 1/s versus 7.4±0.5 1/s, P<0.05), indicating diastolic dysfunction in the HFD mice. Systolic function was comparable in both groups (left ventricular ejection fraction=66.4±1.4% versus 66.7±1.2%, P>0.05). MitoTEMPO‐treated HFD mice showed significant reduction in mitochondria reactive oxygen species, S‐glutathionylation of cardiac myosin binding protein C, and diastolic dysfunction, comparable to the control. The fasting insulin levels of MitoTEMPO‐treated HFD mice were also comparable to the controls (P>0.05). Conclusions MitoTEMPO treatment prevented insulin resistance and diastolic dysfunction, suggesting that mitochondrial oxidative stress may be involved in the pathophysiology of both conditions.

Diastolic Dysfunction:– Potential New Diagnostics and Therapies –

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.

Association of low plasma adiponectin with early diastolic dysfunction.

Diastolic dysfunction (DD) with preserved left ventricular (LV) ejection fraction (EF) has been linked to obesity. Adiponectin is a cytokine related to obesity and obesity-linked cardiovascular complications. The authors aimed to determine the independent association of DD with adiponectin. Fifty patients with impaired relaxation DD and a normal EF and age-matched normal controls were recruited. Plasma levels of total and high molecular weight (HMW) adiponectin were measured. Mid and low molecular weight (MMW+LMW) fractions of adiponectin were calculated by subtracting HMW fraction from total adiponectin. The DD group had significantly lower total (median, 4.4 vs 12.7 μg/mL; P=.001), HMW fraction (median, 1.3 vs 3.4 μg/mL; P=.02), and MMW+LMW fraction of adiponectin (median, 3.8 vs 7.2 μg/mL; P=.01). Body mass index (BMI) negatively correlated with total (r:-0.46, P=.003), HMW (r:-0.32, P=.038), and MMW+LMW (r:-0.40, P=.006) fractions of adiponectin. DD had an independent association with both BMI (P<.05) and total adiponectin (P<.001) in linear regression model using sex, BMI, blood pressure, and total adiponectin as covariates. DD was associated with BMI (P=.02), HMW fraction (P=.03), and MMW+LMW fraction (P=.004) in similar linear regression analyses. Adiponectin deficiency may be one explanation for the adiposity-related cardiac oxidation known to be involved in the pathogenesis of DD.

Effect of α7 nicotinic acetylcholine receptor activation on cardiac fibroblasts: A mechanism underlying RV fibrosis associated with cigarette smoke exposure

Right ventricular (RV) dysfunction is associated with numerous smoking-related illnesses, including chronic obstructive pulmonary disease (COPD), in which it is present even in the absence of pulmonary hypertension. It is unknown whether exposure to cigarette smoke (CS) has direct effects on RV function and cardiac fibroblast (CF) proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to CS and determined mechanisms of smoke-induced changes in CF signaling and fibrosis. AKR mice were exposed to CS for 6 wk followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat CFs exposed to CS extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. Mice exposed to CS had significantly decreased RV function, as determined by tricuspid annular plane systolic excursion. There were no changes in left ventricular parameters. RV collagen content was significantly elevated, but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased CF proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-δ, and reduced p38-MAPK phosphorylation. CS and nicotine have direct effects on CFs to induce proliferation and fibrosis, which may negatively affect right heart function.

Renin-Angiotensin Activation and Oxidative Stress in Early Heart Failure with Preserved Ejection Fraction.

Animal models have suggested a role of renin-angiotensin system (RAS) activation and subsequent cardiac oxidation in heart failure with preserved ejection fraction (HFpEF). Nevertheless, RAS blockade has failed to show efficacy in treatment of HFpEF. We evaluated the role of RAS activation and subsequent systemic oxidation in HFpEF. Oxidative stress markers were compared in 50 subjects with and without early HFpEF. Derivatives of reactive oxidative metabolites (DROMs), F2-isoprostanes (IsoPs), and ratios of oxidized to reduced glutathione (E h GSH) and cysteine (E h CyS) were measured. Angiotensin converting enzyme (ACE) levels and activity were measured. On univariate analysis, HFpEF was associated with male sex (p = 0.04), higher body mass index (BMI) (p = 0.003), less oxidized E h CyS (p = 0.001), lower DROMs (p = 0.02), and lower IsoP (p = 0.03). Higher BMI (OR: 1.3; 95% CI: 1.1–1.6) and less oxidized E h CyS (OR: 1.2; 95% CI: 1.1–1.4) maintained associations with HFpEF on multivariate analysis. Though ACE levels were higher in early HFpEF (OR: 1.09; 95% CI: 1.01–1.05), ACE activity was similar to that in controls. HFpEF is not associated with significant systemic RAS activation or oxidative stress. This may explain the failure of RAS inhibitors to alter outcomes in HFpEF.