Gentaro Iribe

Axial Stretch of Rat Single Ventricular Cardiomyocytes Causes an Acute and Transient Increase in Ca2+ Spark Rate

We investigate acute effects of axial stretch, applied by carbon fibers (CFs), on diastolic Ca2+ spark rate in rat isolated cardiomyocytes. CFs were attached either to both cell ends (to maximize the stretched region), or to the center and one end of the cell (to compare responses in stretched and nonstretched half-cells). Sarcomere length was increased by 8.01±0.94% in the stretched cell fraction, and time series of XY confocal images were recorded to monitor diastolic Ca2+ spark frequency and dynamics. Whole-cell stretch causes an acute increase of Ca2+ spark rate (to 130.7±6.4%) within 5 seconds, followed by a return to near background levels (to 104.4±5.1%) within 1 minute of sustained distension. Spark rate increased only in the stretched cell region, without significant differences in spark amplitude, time to peak, and decay time constants of sparks in stretched and nonstretched areas. Block of stretch-activated ion channels (2 &mgr;mol/L GsMTx-4), perfusion with Na+/Ca2+-free solution, and block of nitric oxide synthesis (1 mmol/L L-NAME) all had no effect on the stretch-induced acute increase in Ca2+ spark rate. Conversely, interference with cytoskeletal integrity (2 hours of 10 &mgr;mol/L colchicine) abolished the response. Subsequent electron microscopic tomography confirmed the close approximation of microtubules with the T-tubular–sarcoplasmic reticulum complex (to within ≈10-8m). In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases sarcoplasmic reticulum Ca2+ spark rate via a mechanism that is independent of sarcolemmal stretch-activated ion channels, nitric oxide synthesis, or availability of extracellular calcium but that requires cytoskeletal integrity. The potential of microtubule-mediated modulation of ryanodine receptor function warrants further investigation.

Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): Standardised reporting for model reproducibility, interoperability, and data sharing

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step toward establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Effects of the phosphodiesterase III inhibitors olprinone, milrinone, and amrinone on hepatosplanchnic oxygen metabolism

Objective: To measure the hepatic venous oxygen saturation in patients after cardiac surgery and to compare the effects of olprinone (OLP), a newly synthesized phosphodiesterase III inhibitor, with those of milrinone (MIL) and amrinone (AMR) on hepatosplanchnic oxygen dynamics. Phosphodiesterase III inhibitors are used to improve the hemodynamic state after cardiac surgery. However, the effect of these agents on the hepatosplanchnic circulation has not been investigated thoroughly. Design: Prospective, randomized study. Setting: University hospital intensive care unit (ICU). Patients: Twenty‐nine patients undergoing elective cardiac surgery. Measurements and Main Results: In each patient, a 7.5‐Fr oximeter catheter was placed in the hepatic vein via the right femoral vein. Catheterization was completed before admission to the ICU, and the study was performed 8 to 24 hrs after surgery, after obtaining stable systemic hemodynamics in the ICU. The patients were assigned randomly to three groups, and they received one of three drugs for 2 hrs (OLP group, 0.3 μg/kg/min of OLP; MIL group, 0.5 μg/kg/min of MIL; AMR group, 10 μg/kg/min of AMR). The authors did not change the patients hemodynamic interventions, including catecholamines and vasodilators, throughout the study period. Arterial and hepatic venous blood gas data and hemodynamic data (via a pulmonary artery catheter) were obtained before and after drug infusion. Using these data, the authors calculated systemic oxygen delivery and consumption, the systemic oxygen extraction ratio and the hepatosplanchnic oxygen extraction ratio, and the change in hepatosplanchnic blood flow using Ficks equation. Although the increases in cardiac index were not significantly different among the three groups, hepatic venous oxygen saturation increased significantly only in the OLP group (from 47.1% ± 2.6% to 57.0% ± 1.5% in the OLP group, from 48.4% ± 2.3% to 50.9% ± 2.6% in the MIL group, and from 49.8% ± 3.6% to 50.8% ± 1.7% in the AMR group). The calculated hepatosplanchnic blood flow change was significantly larger in the OLP group than in the other groups (30.1% ± 5.7% in the OLP group, 9.3% ± 5.1% in the MIL group, and 2.6% ± 6.5% in the AMR group). Conclusions: These results suggest that OLP enhances hepatosplanchnic blood flow and thus may be beneficial in protecting the hepatosplanchnic organs after cardiac surgery.

Modulatory effect of calmodulin-dependent kinase II (CaMKII) on sarcoplasmic reticulum Ca2+ handling and interval-force relations: A modelling study

We hypothesize that slow inactivation of Ca2+/calmodulin-dependent kinase II (CaMKII) and its modulatory effect on sarcoplasmic reticulum (SR) Ca2+ handling are important for various interval–force (I–F) relations, in particular for the beat interval dependency in transient alternans during the decay of post-extrasystolic potentiation. We have developed a mathematical model of a single cardiomyocyte to integrate various I–F relations, including alternans, by incorporating a conceptual CaMKII kinetics model into the SR Ca2+ handling model. Our model integrates I–F relations, such as the beat interval-dependent twitch force duration, restitution and potentiation, positive staircase phenomenon and alternans. We found that CaMKII affects more or less all I–F relations, and it is a key factor for integration of the various I–F relations in our model. Alternans arises, in the model, out of a steep relation between SR Ca2+ load and release, owing to SR load-dependent changes in the releasability of Ca2+ via the ryanodine receptor. Beat interval-dependent CaMKII activity, owing to its kinetic properties and amplifying effect on SR Ca2+ load dependency of Ca2+ release, replicated the beat interval dependency of alternans, as observed experimentally. Additionally, our model enabled reproduction of the effects of various interventions on alternans, such as the slowing or accelerating of Ca2+ release and/or uptake. We conclude that a slow time-dependent factor, represented in the model by CaMKII, is important for the integration of I–F relations, including alternans, and that our model offers a useful tool for further analysis of the roles of integrative Ca2+ handling in myocardial I–F relations.

Cardiac myofibroblast engulfment of dead cells facilitates recovery after myocardial infarction

Myocardial infarction (MI) results in the generation of dead cells in the infarcted area. These cells are swiftly removed by phagocytes to minimize inflammation and limit expansion of the damaged area. However, the types of cells and molecules responsible for the engulfment of dead cells in the infarcted area remain largely unknown. In this study, we demonstrated that cardiac myofibroblasts, which execute tissue fibrosis by producing extracellular matrix proteins, efficiently engulf dead cells. Furthermore, we identified a population of cardiac myofibroblasts that appears in the heart after MI in humans and mice. We found that these cardiac myofibroblasts secrete milk fat globule-epidermal growth factor 8 (MFG-E8), which promotes apoptotic engulfment, and determined that serum response factor is important for MFG-E8 production in myofibroblasts. Following MFG-E8–mediated engulfment of apoptotic cells, myofibroblasts acquired antiinflammatory properties. MFG-E8 deficiency in mice led to the accumulation of unengulfed dead cells after MI, resulting in exacerbated inflammatory responses and a substantial decrease in survival. Moreover, MFG-E8 administration into infarcted hearts restored cardiac function and morphology. MFG-E8–producing myofibroblasts mainly originated from resident cardiac fibroblasts and cells that underwent endothelial-mesenchymal transition in the heart. Together, our results reveal previously unrecognized roles of myofibroblasts in regulating apoptotic engulfment and a fundamental importance of these cells in recovery from MI.

Effects of axial stretch on sarcolemmal BKCa channels in post‐hatch chick ventricular myocytes

We have previously reported the electrophysiological properties of sarcolemmal stretch‐activated BKCa (SAKCA) channels cloned from cultured chick embryonic ventricular myocytes. However, the role of BKCa channels in the electrophysiology of the more mature heart is not clear. We have investigated the effects on the BKCa current of axial stretch in post‐hatch ventricular myocytes. Whole‐cell currents of ventricular myocytes isolated from 2‐week‐old chicks were recorded using the patch‐clamp technique, while the cells were either held at resting length or stretched to cause a 10% increase in sarcomere length using a pair of carbon fibres attached to opposite ends of the cell. Stretch did not affect whole‐cell currents immediately after the stretch was applied. However, sustained stretch for 3 min significantly increased outward currents. This stretch‐induced change was reversed by applying 10 nm iberiotoxin, a specific BKCa channel blocker, or a Na+–Ca2+‐free environment. These results were reproduced in a computer simulation study. The present study is the first report about the sarcolemmal BKCa current from post‐hatch ventricular myocytes. The present results suggest that axial stretch activates BKCa channels via a stretch‐induced increase in the cytosolic Na+ concentration followed by an increased Ca2+ influx.

Total Ca handling in canine mild Ca overload failing heart

SummaryWe analyzed total Ca handling of the left ventricle (LV) in the mildly failing heart preparation induced by a temporary intracoronary Ca overloading intervention in eight excised and cross-circulated canine hearts. This Ca intervention consisted of interruption of coronary blood perfusion by Ca-free oxygenated Tyrode perfusion for 10min followed by high-Ca (16 mmol/l) oxygenated Tyrode perfusion for 5 min. This intervention decreased the LV contractility index,Emax (end-systolic maximum elastance), by 40% after restoration of the blood cross-circulation. We expected a Ca overload or paradox failing heart resembling the postischemic stunned heart and being characterized by an increased O2 cost ofEmax. However, LV O2 consumption under mechanically unloading conditions decreased by 30% from control without increasing the O2 cost ofEmax. To obtain a mechanistic view of this failing heart, we investigated cardiac total Ca handling by our integrative analysis method. In this method, we obtained the internal Ca recirculation fraction (RF) from the decay beat constant of the postextrasystolic potentiation following each sporadic spontaneous extrasystole in these failing LVs. We combined the RF with the decreasedEmax and the unchanged O2 cost ofEmax in our recently developed formula of total Ca handling. We found that these failing LVs had a slightly but significantly increased RF accompanied by either a slightly increased futile Ca cycling or a slightly decreased Ca reactivity ofEmax, or both. Any of these three possible changes can account for the unchanged O2 cost ofEmax. This result indicates that the present mildly failing heart has not yet fallen into a typical Ca overload or paradox by the temporary Ca overloading intervention.

Myocardial mechanical restitution and potentiation partly underlie alternans decay of postextrasystolic potentiation : simulation

SummaryWe have reported that the postextrasystolic potentiation (PESP) decays in alternans or monotonically, respectively, depending on whether the first postextrasystolic beat interval has a compensatory pause or not, in the canine left ventricle. To get better mechanistic insight into the alternans PESP decay, we hypothesized that the myocardial mechanical restitution and potentiation could partly account for both types of PESP decay. To test this hypothesis, we simulated PESP decay on a computer using a documented equation combining myocardial mechanical restitution and potentiation. We changed the first postextrasystolic beat interval after a fixed extrasystolic beat interval without changing regular and other postextrasystolic beat intervals. The simulated PESP decayed in alternans or monotonically as a function only of the first postextrasystolic beat interval. Thus, the myocardial mechanical restitution and potentiation could partly account for both alternans and monotonic decay of PESP. We conclude that myocardial mechanical restitution and potentiation may partly underlie the initial two alternating beats, the first beat being the most potentiated and the second beat being the most depressed, of alternans PESP decay in the canine heart.

Effect of azelnidipine and amlodipine on single cell mechanics in mouse cardiomyocytes

Azelnidipine and amlodipine are dihydropyridine-type Ca(2+) channel blockers for the treatment of hypertension. Although these drugs have high vasoselectivity and small negative inotropic effects in vivo, little is known regarding their direct effects on cellular contractility without humoral regulation or the additive effects of these drugs with other antihypertensive drugs on myocardial contractility. To investigate the effects of Ca(2+) channel blockers on single cell mechanics, mouse cardiomyocytes were enzymatically isolated, and a pair of carbon fibers was attached to opposite cell-ends to stretch the cells. Cells were paced at 4 Hz superfused in normal Tyrode solution at 37°C. Cell length and active/passive force calculated from carbon fiber bending were recorded in 6 different preload conditions. Slopes of end-systolic force-length relation curves (maximum elastance) were measured as an index of contractility before and after drugs were administered. Azelnidipine at 10nM and 100 nM did not change maximum elastance, while amlodipine at 100 nM did decrease maximum elastance. The combination of RNH-6270 (active form of angiotensin II receptor blocker, olmesartan, 10nM) and either amlodipine (10nM) or azelnidipine (10nM) did not affect maximum elastance. Although both amlodipine and azelnidipine can be used safely at therapeutically relevant concentrations even in combination with olmesartan, the present results suggest that azelnidipine has a less negative inotropic action compared to amlodipine.

Transmural cellular heterogeneity in myocardial electromechanics

Myocardial heterogeneity is an attribute of the normal heart. We have developed integrative models of cardiomyocytes from the subendocardial (ENDO) and subepicardial (EPI) ventricular regions that take into account experimental data on specific regional features of intracellular electromechanical coupling in the guinea pig heart. The models adequately simulate experimental data on the differences in the action potential and contraction between the ENDO and EPI cells. The modeling results predict that heterogeneity in the parameters of calcium handling and myofilament mechanics in isolated ENDO and EPI cardiomyocytes are essential to produce the differences in Ca2+ transients and contraction profiles via cooperative mechanisms of mechano-calcium-electric feedback and may further slightly modulate transmural differences in the electrical properties between the cells. Simulation results predict that ENDO cells have greater sensitivity to changes in the mechanical load than EPI cells. These data are important for understanding the behavior of cardiomyocytes in the intact heart.