Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca2+ Leakage through the Ryanodine Receptor by Suppressing Mitochondrial Reactive Oxygen Species Production Induced by Isoproterenol in Failing Cardiomyocytes.

Catecholamines induce intracellular reactive oxygen species (ROS), thus enhancing diastolic Ca2+ leakage through the ryanodine receptor during heart failure (HF). However, little is known regarding the effect of atrial natriuretic peptide (ANP) on ROS generation and Ca2+ handling in failing cardiomyocytes. The aim of the present study was to clarify the mechanism by which an exogenous ANP exerts cardioprotective effects during HF. Cardiomyocytes were isolated from the left ventricles of a canine tachycardia-induced HF model and sham-operated vehicle controls. The degree of mitochondrial oxidized DNA was evaluated by double immunohistochemical (IHC) staining using an anti-VDAC antibody for the mitochondria and an anti-8-hydroxy-2'-deoxyguanosine antibody for oxidized DNA. The effect of ANP on ROS was investigated using 2,7-dichlorofluorescin diacetate, diastolic Ca2+ sparks assessed by confocal microscopy using Fluo 4-AM, and the survival rate of myocytes after 48 h. The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in the mitochondria in HF and that the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP. ROS production and Ca2+ spark frequency (CaSF) were increased in HF compared to normal controls, and were further increased in the presence of ISO. Notably, ANP significantly suppressed both ISO-induced ROS and CaSF without changing sarcoplasmic reticulum Ca2+ content in HF (p<0.01, respectively). The survival rate after 48 h in HF was significantly decreased in the presence of ISO compared with baseline (p<0.01), whereas it was significantly improved by the co-presence of ANP (p<0.01). Together, our results suggest that ANP strongly suppresses ISO-induced mitochondrial ROS generation, which might correct aberrant diastolic Ca2+ sparks, eventually contributing to the improvement of cardiomyocyte survival in HF.

A low-dose ß1-blocker in combination with milrinone improves intracellular Ca2+ handling in failing cardiomyocytes by inhibition of milrinone-induced diastolic Ca2+ leakage from the sarcoplasmic reticulum.

OBJECTIVES: The purpose of this study was to investigate whether adding a low-dose ß1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism. BACKGROUND: The molecular mechanism underlying how the combination of low-dose ß1-blocker and milrinone affects intracellular Ca(2+) handling in heart failure remains unclear. METHODS: We investigated the effect of milrinone plus landiolol on intracellular Ca(2+) transient (CaT), cell shortening (CS), the frequency of diastolic Ca(2+) sparks (CaSF), and sarcoplasmic reticulum Ca(2+) concentration ({Ca(2+)}SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB). RESULTS: In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca(2+)}SR. Co-administration of ß1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca(2+)}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes. CONCLUSION: A low-dose ß1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca(2+) leak.

Assessment of the aortic valve annular geometry by real-time three-dimensional transthoracic echocardiography: comparison with two-dimensional transthoracic echocardiography and multidetector computed tomography.

BACKGROUND: Real-time three-dimensional transthoracic echocardiography (3DTTE) has been developed and provides detailed 3D information, noninvasively. However, the accuracy and usefulness of 3DTTE in the evaluation of aortic root geometry are still not clear. METHODS: 2DTTE and 3DTTE were performed in 161 patients with various cardiac diseases. Multidetector computed tomography (MDCT) was performed in 35 of the 161 patients. The diameters and areas of the aortic annulus were evaluated by these three methods and compared. To evaluate the shape of the aortic annuli, eccentricity index (EI) (1 - minimum diameter/long-axis diameter) were calculated. RESULTS: Maximum dimensions of the aortic annulus measured by MDCT were significantly larger than those by 3DTTE and 2DTTE. The aortic annular areas measured by MDCT and 3DTTE were significantly larger than areas by 2DTTE. A good correlation (r = 0.85) was observed between the areas obtained by 3DTTE and MDCT; however, the correlation between the values by 2DTTE and MDCT was rough (r = 0.44). EI values in 46 % of the patients were greater than 0.1, i.e., the aortic annulus was elliptical. CONCLUSION: The images obtained by 3DTTE provided accurate values of the aortic annular area, which were equal to the values measured by MDCT. 3DTTE is a useful method to evaluate the aortic annular geometry.

Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts.

AIMS: The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure. METHODS AND RESULTS: The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels. CONCLUSION: Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

Low-dose ß-blocker in combination with milrinone safely improves cardiac function and eliminates pulsus alternans in patients with acute decompensated heart failure.

BACKGROUND: The purpose of this study was to determine whether a low-dose ß-blocker, in combination with milrinone, improves cardiac function in acute decompensated heart failure (ADHF) with tachycardia. METHODS AND RESULTS: Twenty ADHF patients (New York Heart Association classification III, n=1, and IV, n=19; heart rate [HR], 107±12 beats/min; left ventricular ejection fraction, 24±7%; cardiac index [CI], 2.2±0.6 L·min(-1)·m(-2); pulmonary capillary wedge pressure [PCWP], 26±8 mmHg) were enrolled in this study. The patients first underwent conventional therapy with milrinone, vasodilators and diuretics; landiolol (1.5-6.0 µg·kg(-1)·min(-1); i.v.), which is an ultra-short-acting ß(1)-selective blocker, was then added to the treatment regimen to study its effect on hemodynamics. Low-dose landiolol (1.5 µg·kg(-1)·min(-1)) significantly reduced HR by 11% without changing blood pressure (BP) and CI, whereas higher doses (≥3.0 µg·kg(-1)·min(-1)) tended to decrease BP and CI while increasing PCWP and systemic vascular resistance. After treatment with landiolol (1.5 µg·kg(-1)·min(-1)), hemodynamic parameters such as PCWP, stroke volume index, SvO(2), rate pressure product, filling time/RR, E/e', and Tei index were significantly improved. CONCLUSIONS: A low-dose ß-blocker in combination with milrinone improved cardiac function in ADHF patients with tachycardia; therefore, it may be considered as an adjunct therapy for use when standard therapy with milrinone is not effective at slowing HR.

Simultaneous Doppler tracing of transmitral inflow and mitral annular velocity as an estimate of elevated left ventricular filling pressure in patients with atrial fibrillation.

BACKGROUND: The time interval between the onset of early transmitral flow velocity (E) and that of early diastolic mitral annular velocity (e') (T(E-e')) is a good predictor of elevated left ventricular (LV) filling pressure in patients with sinus rhythm. Although the evaluation of LV filling pressure using E/e' has been challenging in atrial fibrillation (AF), the usefulness of T(E-e') is unknown. METHODS AND RESULTS: E and e' were simultaneously recorded using dual Doppler echocardiography in 45 AF patients (30 men; mean age, 69 ± 9 years). E/e' and T(E-e') were calculated and compared with the pulmonary capillary wedge pressure (PCWP), which was measured invasively. E/e' and T(E/e') correlated with PCWP (E/e', r=0.57, P<0.001; T(E-e'), r=0.77, P<0.001). Using receiver operating characteristic analysis, the optimal cut-off for T(E-e') was 34 ms (sensitivity, 95%; specificity, 88%) and that for E/e' was 14.6 (sensitivity, 50%; specificity, 84%) in order to predict >12-mmHg PCWP. When the combined cut-offs of T(E-e') >34 ms and E/e' >14.6 were used, the sensitivity and specificity of predicting elevated PCWP were improved to 100% and 88%, respectively. CONCLUSIONS: In AF patients, the simultaneous recording of E and e' using dual Doppler echocardiography and the analysis of T(E-e'), in addition to E/e', improved the accuracy of evaluation of LV filling pressure.

Urinary 8-hydroxy-2'-deoxyguanosine as a novel biomarker for predicting cardiac events and evaluating the effectiveness of carvedilol treatment in patients with chronic systolic heart failure.

BACKGROUND: The authors recently reported that urinary 8-hydroxy-2'-deoxyguanosine (U8-OHdG) derived from cardiac tissue reflects clinical status and cardiac dysfunction severity in patients with chronic heart failure (CHF). The aim of the present study was to investigate whether U8-OHdG levels can accurately predict cardiac events in CHF patients and their response to ß-blocker treatment. METHODS AND RESULTS: Plasma brain natriuretic peptide (BNP) and U8-OHdG levels were measured in 186 consecutive CHF patients before discharge. Patients were then prospectively followed (median follow-up, 649 days) with endpoints of cardiac death or hospitalization due to progressive heart failure. From receiver operating characteristic curve analysis, cut-offs were 12.4ng/mg creatinine (Cr) for U8-OHdG and 207pg/ml for BNP. On multivariate Cox analysis, U8-OHdG and BNP were independent predictors of cardiac events. Patients were classified into 4 groups according to U8-OHdG and BNP cut-offs. The hazard ratio for cardiac events in patients with BNP ≥207pg/ml and U8-OHdG ≥12.4ng/mg Cr was 16.2 compared with approximately 4 for patients with only 1 indicator above its respective cut-off. Furthermore, carvedilol therapy was initiated in 30 CHF patients. In responders (≥10% increase in left ventricular ejection fraction [LVEF] or ≥1 class decrease in New York Heart Association [NYHA] class), U8-OHdG levels decreased significantly along with improved NYHA class, LVEF, and BNP levels after treatment. CONCLUSIONS: U8-OHdG may be a useful biomarker for predicting cardiac events and evaluating ß-blocker therapy effectiveness in CHF patients.

Urinary 8-hydroxy-2'-deoxyguanosine reflects symptomatic status and severity of systolic dysfunction in patients with chronic heart failure.

AIMS: Oxidative stress is known to play a crucial role in the pathogenesis of heart failure (HF). We investigated whether urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), a product of oxidative DNA damage, is a clinically useful biomarker of the severity of chronic heart failure (CHF) and oxidative stress levels in failing hearts. METHODS AND RESULTS: We measured 8-OHdG in the serum obtained from the coronary sinus (CS) and aortic root (Ao) in small groups of control subjects and CHF patients. We then measured urinary 8-OHdG and other biomarkers (brain natriuretic peptide, 8-isoplastane, high-sensitivity C-reactive protein, interleukin-6, and tumour necrosis factor-α) in 31 control subjects and 194 patients with CHF [left-ventricular ejection fraction (LVEF): 28.3 ± 8.1%]. Serum 8-OHdG was significantly higher in the CS than the Ao in CHF patients only. Urinary 8-OHdG was also significantly higher in CHF patients than in control subjects, and urinary 8-OHdG became higher as New York Heart Association class increased. Moreover, there was a significant correlation between urinary 8-OHdG and LVEF (r = -0.27), pulmonary capillary wedge pressure (r = 0.31), or left-ventricular end-diastolic volume index (r = 0.22). In contrast, there was poor correlation between the severity of CHF and the other neurohumoral biomarkers. CONCLUSION: In HF, urinary 8-OHdG seems to reflect the level of oxidative stress and various parameters related to symptomatic status and functional severity of CHF.

A case of mitochondrial disease with severe left ventricular hypertrophy.

A 35-year-old woman was admitted for progressive dyspnea with lower limb edema. Transthoracic echocardiography showed severe left ventricular hypertrophy (LVH) and heart failure with preserved ejection fraction (HFPEF). Electron microscopy of an endomyocardial biopsy sample revealed a high density of mitochondria of abnormal size and shape. We report a case of mitochondrial disease with severe LVH and HFPEF.

Heterogeneity of apex-to-base dispersion in diastolic lengthening is related to impaired global left ventricular relaxation in patients with hypertrophic cardiomyopathy.

BACKGROUND: The presence of apex-to-base disparity in diastolic left ventricle (LV) endocardial lengthening, based on an electromechanical activation sequence, has been recognized as an important determinant of LV diastolic properties. However, the behavior of LV apical and basal diastolic lengthening and its relationship to LV filling in hypertrophic cardiomyopathy (HCM) are unknown. METHODS: We obtained basal and apical LV short-axis views in 27 patients with non-obstructive HCM and 25 healthy volunteers. The patients with HCM were subdivided into two groups; those with apical hypertrophy [APH(+)] or those without apical hypertrophy [APH(-)]. Eight equiangular points on the endo-myocardium at end diastole were placed in each view, and the movements of these points were automatically tracked using a two-dimensional echocardiographic tissue tracking system. Time-LV internal diameter curves were obtained and averaged. The time intervals from the aortic valve closure to the point of the first 40% of peak diastolic lengthening (T 40) were measured in each view. The standard deviation of the time to peak systolic circumferential shortening at the base and apex were calculated to assess the heterogeneity of LV contraction. RESULTS: The time difference in the T 40 between the apex and base (dt-T 40) in the HCM-APH(+) and HCM-APH(-) groups was greater than that in the control group. The heterogeneities in LV apical systolic shortening in the HCM groups were greater than those in the control group. There were good linear correlations between the dt-T 40 and the LV early diastolic echo-parameters and the LV mass index. CONCLUSIONS: Delayed apical relaxation and filling in patients with HCM is related to LV diastolic dysfunction and systolic dyssynchronous contraction.

Dantrolene, a therapeutic agent for malignant hyperthermia, inhibits catecholaminergic polymorphic ventricular tachycardia in a RyR2(R2474S/+) knock-in mouse model.

BACKGROUND: Dantrolene, a specific agent for the treatment of malignant hyperthermia, was found to inhibit Ca(2+) leak through not only the skeletal ryanodine receptor (RyR1), but also the cardiac ryanodine receptor (RyR2) by correcting the defective inter-domain interaction between N-terminal (1-619 amino acid) and central (2,000-2,500 amino acid) domains of RyRs. Here, the in vivo anti-arrhythmic effect of dantrolene in a human catecholaminergic polymorphic ventricular tachycardia (CPVT)-associated RyR2(R2474S/+) knock-in (KI) mouse model was investigated. METHODS AND RESULTS: ECG was monitored in KI mice (n=6) and wild-type (WT) mice (n=6), before and after an injection of epinephrine (1.0mg/kg) or on exercise using a treadmill. In all KI (but not WT) mice, bi-directional ventricular tachycardia (VT) was induced after an injection of epinephrine or on exercise. Pre-treatment with dantrolene (for 7-10 days) significantly inhibited the inducible VT (P<0.01). In KI cardiomyocytes, Ca(2+) spark frequency (SpF; s(-1)·100µm(-1): 5.8±0.3, P<0.01) was much more increased after the addition of isoproterenol than in WT cardiomyocytes (SpF: 3.6±0.2). The increase in SpF seen in KI cardiomyocytes was attenuated by 1.0µmol/L dantrolene (SpF: 3.6±0.5, P<0.01). CONCLUSIONS: Dantrolene prevents CPVT, presumably by inhibiting Ca(2+) leak through the RyR2.

Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca(2+) release in heart failure.

AIMS: Calmodulin (CaM) is well known to modulate the channel function of the cardiac ryanodine receptor (RyR2). However, the possible role of CaM on the aberrant Ca(2+) release in diseased hearts remains unclear. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions in pacing-induced failing hearts. METHODS AND RESULTS: The characteristics of CaM binding to RyR2 and the role of CaM on the aberrant Ca(2+) release were assessed in normal and failing canine hearts. The affinity of CaM binding to RyR2 was lower in failing sarcoplasmic reticulum (SR) than in normal SR. Addition of FK506, which dissociates FKBP12.6 from RyR2, to normal SR reduced the CaM-binding affinity. Dantrolene restored a normal level of the CaM-binding affinity in either FK506-treated (normal) SR or failing SR, suggesting that the defective inter-domain interaction between the N-terminal domain and the central domain of RyR2 (the therapeutic target of dantrolene) is involved in the reduction of the CaM-binding affinity in failing hearts. In saponin-permeabilized cardiomyocytes, the frequency of spontaneous Ca(2+) sparks was much more increased in failing cardiomyocytes than in normal cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. CONCLUSION: The defective inter-domain interaction between N-terminal and central domains within RyR2 reduces the binding affinity of CaM to RyR2, thereby causing the spontaneous Ca(2+) release events in failing hearts. Correction of the defective CaM binding may be a new strategy to protect against the aberrant Ca(2+) release in heart failure.

The impact of intermittent pneumatic compression devices on deep venous flow velocity in patients with congestive heart failure.

BACKGROUND: Intermittent pneumatic compression (IPC) has been used to prevent deep venous thrombosis (DVT), but the effects of IPC on the hemodynamics of popliteal and soleal veins, especially in patients with congestive heart failure (CHF) have not been evaluated. The aim of this study was to evaluate the effects of IPC on the flow velocity of deep veins in the lower extremities and to compare the efficacy of two different types of IPC in deep venous flow enhancement in patients with CHF. METHODS: Flow velocities of popliteal and soleal veins were recorded in 19 patients with CHF and in 19 control subjects using a high-resolution linear probe. Peak and mean flow velocities were measured (1) at rest, (2) with sequential foot and calf IPC (SFC-IPC) which consists of an electrically driven air compressor and four air chambers, and (3) with impulse foot IPC (IF-IPC) which consists of a pneumatic impulse generator operated at an applied pressure of 130 mmHg. RESULTS: In the resting condition, popliteal venous flow velocity in the CHF group was attenuated (12.8+/-4.7 cm/s vs. 21.1+/-13.5 cm/s; p<0.05). Both SFC-IPC and IF-IPC increased venous velocity, but the increase with IF-IPC in CHF patients was lower than that in control subjects. In the soleal veins, after applying SFC-IPC, the peak and mean velocity in CHF increased to the same extent as in the control group. IF-IPC increased soleal venous velocity in control subjects, but there was no increase in CHF patients. CONCLUSION: Two-dimensional Doppler scanning revealed a significant increase in the mean and peak velocities in the soleal and popliteal veins with SFC-IPC but not with IF-IPC in patients with CHF. These results indicate that SFC-IPC could have favorable effects in preventing DVT in patients with CHF.

Impact of intraoperative transesophageal echocardiography in cardiac and thoracic aortic surgery: experience in 1011 cases.

BACKGROUND: Although intraoperative transesophageal echocardiography (IOTEE) has been widely used in cardiovascular surgery, the exact incidence of abnormalities detected by IOTEE in each type of surgical procedure is still unclear. The aim of this study was to review our experiences of IOTEE, in patients who underwent different types of cardiovascular surgery and to evaluate the clinical usefulness of IOTEE. METHODS AND RESULTS: Our database of 1011 consecutive patients, who underwent cardiovascular surgery and IOTEE monitoring was reviewed. The incidence of abnormal findings was 115 of 1011 patients (11.4%), and the highest incidence was the appearance of new wall motion abnormalities after cardiopulmonary bypass. These findings influenced surgical decision-making in 59 of the evaluated 1011 patients (5.8%). CONCLUSIONS: IOTEE provides important intraoperative and postoperative information that may influence surgical decision-making in various cardiovascular surgeries.