Etiology-dependent impairment of relaxation kinetics in right ventricular end-stage failing human myocardium.

BACKGROUND: In patients with end-stage heart failure, the primary etiology often originates in the left ventricle, and eventually the contractile function of the right ventricle (RV) also becomes compromised. RV tissue-level deficits in contractile force and/or kinetics need quantification to understand involvement in ischemic and non-ischemic failing human myocardium. METHODS AND RESULTS: The human population suffering from heart failure is diverse, requiring many subjects to be studied in order to perform an adequately powered statistical analysis. From 2009-present we assessed live tissue-level contractile force and kinetics in isolated myocardial RV trabeculae from 44 non-failing and 41 failing human hearts. At 1 Hz stimulation rate (in vivo resting state) the developed active force was not different in non-failing compared to failing ischemic nor non-ischemic failing trabeculae. In sharp contrast, the kinetics of relaxation were significantly impacted by disease, with 50% relaxation time being significantly shorter in non-failing vs. non-ischemic failing, while the latter was still significantly shorter than ischemic failing. Gender did not significantly impact kinetics. Length-dependent activation was not impacted. Although baseline force was not impacted, contractile reserve was critically blunted. The force-frequency relation was positive in non-failing myocardium, but negative in both ischemic and non-ischemic myocardium, while the ß-adrenergic response to isoproterenol was depressed in both pathologies. CONCLUSIONS: Force development at resting heart rate is not impacted by cardiac pathology, but kinetics are impaired and the magnitude of the impairment depends on the underlying etiology. Focusing on restoration of myocardial kinetics will likely have greater therapeutic potential than targeting force of contraction.

Deferiprone inhibits iron overload-induced tissue factor bearing endothelial microparticle generation by inhibition oxidative stress induced mitochondrial injury, and apoptosis.

Iron overload-induced cardiovascular toxicity is one of the most common causes of morbidity and mortality in beta-thalassemia major patients. We have previously shown that iron overload-induced systemic arterial changes characterized by endothelial dysfunction are associated with increased endothelial microparticle (EMP) release. In this study, we further demonstrate how EMP release is associated with iron-induced mitochondrial injury and apoptosis of endothelial cells. Iron increased the production of reactive oxygen species (ROS) and calcium influx into mitochondria [Ca2+]m. Iron also disturbed mitochondrial respiration function and eventually led to loss of mitochondrial membrane potential (ΔΨm). A significant increase in apoptotic cells and EMPs were found under iron treatment. EMPs contained tissue factor (TF), which has potential clinical impact on thromboembolic phenomenon. Then, we investigated the salvaging effect of deferiprone (L1) on endothelial cell damage and EMP release. We found that L1 could inhibit iron-induced ROS generation, and decrease mitochondrial damage with the resultant effect of less endothelial cell apoptosis and EMP release. L1 could protect endothelial cells from iron-induced toxic effects and minimize EMP release, which could be potentially helpful in a subgroup of thalassemia patients who have increased thromboembolic complications.

Carvedilol protects against iron-induced microparticle generation and apoptosis of endothelial cells.

BACKGROUND: Increased circulating endothelial microparticles (EMPs) have been shown to associate with endothelial dysfunction. We explored the effect of iron on EMP generation by human umbilical vein endothelial cells (HUVECs) and the potential protective effect of carvedilol. METHODS: FeCl 3 was added to HUVEC culture. Iron entry into cells was monitored using fluorescent microscopic imaging, while the quantity of EMPs that was released was determined by flow cytometry. The apoptosis of HUVECs was assessed by annexin V/propidium iodide assay and caspase-3 expression. Membrane bleb formation was visualized using electron microscopy. Intracellular production of reactive oxygen species (ROS) was also monitored. The effects of beta-blockers, carvedilol and propranolol on these processes were determined by co-incubation in a dose-dependent manner. Iron entry into HUVECs was not blocked by either beta-blocker. Iron induced the generation of EMPs, the formation of membrane blebs, the apoptosis of HUVECs and the production of ROS, each in a dose-dependent manner. Carvedilol, but not propranolol, ameliorated all of these processes. RESULTS: Our result indicates that iron induces EMP generation and apoptosis of endothelial cells in association with increased oxidative stress. CONCLUSION: The protective effects of carvedilol, via its antioxidant effect, may have therapeutic potential in patients with iron overload.

Left ventricular torsional mechanics and myocardial iron load in beta-thalassaemia major: a potential role of titin degradation.

BACKGROUND: Iron may damage sarcomeric proteins through oxidative stress. We explored the left ventricular (LV) torsional mechanics in patients with beta-thalassaemia major and its relationship to myocardial iron load. Using HL-1 cell and B6D2F1 mouse models, we further determined the impact of iron load on proteolysis of the giant sarcomeric protein titin. METHODS AND RESULTS: In 44 thalassaemia patients aged 25 ± 7 years and 38 healthy subjects, LV torsion and twisting velocities were determined at rest using speckle tracking echocardiography. Changes in LV torsional parameters during submaximal exercise testing were further assessed in 32 patients and 17 controls. Compared with controls, patients had significantly reduced LV apical rotation, torsion, systolic twisting velocity, and diastolic untwisting velocity. T2* cardiac magnetic resonance findings correlated with resting diastolic untwisting velocity. The increments from baseline and resultant LV torsion and systolic and diastolic untwisting velocities during exercise were significantly lower in patients than controls. Significant correlations existed between LV systolic torsion and diastolic untwisting velocities in patients and controls, both at rest and during exercise. In HL-1 cells and ventricular myocardium of B6D2F1 mice overloaded with iron, the titin-stained pattern of sarcomeric structure became disrupted. Gel electrophoresis of iron-overloaded mouse myocardial tissue further showed significant decrease in the amount of titin isoforms and increase in titin degradation products. CONCLUSIONS: Resting and dynamic LV torsional mechanics is impaired in patients with beta-thalassaemia major. Cell and animal models suggest a potential role of titin degradation in iron overload-induced alteration of LV torsional mechanics.

Stretching single titin molecules from failing human hearts reveals titin's role in blunting cardiac kinetic reserve.

AIMS: Heart failure (HF) patients commonly experience symptoms primarily during elevated heart rates, as a result of physical activities or stress. A main determinant of diastolic passive tension, the elastic sarcomeric protein titin, has been shown to be associated with HF, with unresolved involvement regarding its role at different heart rates. To determine whether titin is playing a role in the heart rate (frequency-) dependent acceleration of relaxation (FDAR). W, we studied the FDAR responses in live human left ventricular cardiomyocytes and the corresponding titin-based passive tension (TPT) from failing and non-failing human hearts. METHODS AND RESULTS: Using atomic force, we developed a novel single-molecule force spectroscopy approach to detect TPT based on the frequency-modulated cardiac cycle. Mean TPT reduced upon an increased heart rate in non-failing human hearts, while this reduction was significantly blunted in failing human hearts. These mechanical changes in the titin distal Ig domain significantly correlated with the frequency-dependent relaxation kinetics of human cardiomyocytes obtained from the corresponding hearts. Furthermore, the data suggested that the higher the TPT, the faster the cardiomyocytes relaxed, but the lower the potential of myocytes to speed up relaxation at a higher heart rate. Such poorer FDAR response was also associated with a lesser reduction or a bigger increase in TPT upon elevated heart rate. CONCLUSIONS: Our study established a novel approach in detecting dynamic heart rate relevant tension changes physiologically on native titin domains. Using this approach, the data suggested that the regulation of kinetic reserve in cardiac relaxation and its pathological changes were associated with the intensity and dynamic changes of passive tension by titin.

Iron overload and apoptosis of HL-1 cardiomyocytes: effects of calcium channel blockade.

BACKGROUND: Iron overload cardiomyopathy that prevails in some forms of hemosiderosis is caused by excessive deposition of iron into the heart tissue and ensuing damage caused by a raise in labile cell iron. The underlying mechanisms of iron uptake into cardiomyocytes in iron overload condition are still under investigation. Both L-type calcium channels (LTCC) and T-type calcium channels (TTCC) have been proposed to be the main portals of non-transferrinic iron into heart cells, but controversies remain. Here, we investigated the roles of LTCC and TTCC as mediators of cardiac iron overload and cellular damage by using specific Calcium channel blockers as potential suppressors of labile Fe(II) and Fe(III) ingress in cultured cardiomyocytes and ensuing apoptosis. METHODS: Fe(II) and Fe(III) uptake was assessed by exposing HL-1 cardiomyocytes to iron sources and quantitative real-time fluorescence imaging of cytosolic labile iron with the fluorescent iron sensor calcein while iron-induced apoptosis was quantitatively measured by flow cytometry analysis with Annexin V. The role of calcium channels as routes of iron uptake was assessed by cell pretreatment with specific blockers of LTCC and TTCC. RESULTS: Iron entered HL-1 cardiomyocytes in a time- and dose-dependent manner and induced cardiac apoptosis via mitochondria-mediated caspase-3 dependent pathways. Blockade of LTCC but not of TTCC demonstrably inhibited the uptake of ferric but not of ferrous iron. However, neither channel blocker conferred cardiomyocytes with protection from iron-induced apoptosis. CONCLUSION: Our study implicates LTCC as major mediators of Fe(III) uptake into cardiomyocytes exposed to ferric salts but not necessarily as contributors to ensuing apoptosis. Thus, to the extent that apoptosis can be considered a biological indicator of damage, the etiopathology of cardiosiderotic damage that accompanies some forms of hemosiderosis would seem to be unrelated to LTCC or TTCC, but rather to other routes of iron ingress present in heart cells.