Stretch-induced reactive oxygen species contribute to the Frank-Starling mechanism.

Myocardial stretch physiologically activates NADPH oxidase 2 (NOX2) to increase reactive oxygen species (ROS) production. Although physiological low-level ROS are known to be important as signalling molecules, the role of stretch-induced ROS in the intact myocardium remains unclear. To address this, we investigated the effects of stretch-induced ROS on myocardial cellular contractility and calcium transients in C57BL/6J and NOX2-/- mice. Axial stretch was applied to the isolated cardiomyocytes using a pair of carbon fibres attached to both cell ends to evaluate stretch-induced modulation in the time course of the contraction curve and calcium transient, as well as to evaluate maximum cellular elastance, an index of cellular contractility, which is obtained from the end-systolic force-length relationship. In NOX2-/- mice, the peak calcium transient was not altered by stretch, as that in wild-type mice, but the lack of stretch-induced ROS delayed the rise of calcium transients and reduced contractility. Our mathematical modelling studies suggest that the augmented activation of ryanodine receptors by stretch-induced ROS causes a rapid and large increase in the calcium release flux, resulting in a faster rise in the calcium transient. The slight increase in the magnitude of calcium transients is offset by a decrease in sarcoplasmic reticulum calcium content as a result of ROS-induced calcium leakage, but the faster rise in calcium transients still maintains higher contractility. In conclusion, a physiological role of stretch-induced ROS is to increase contractility to counteract a given preload, that is, it contributes to the Frank-Starling law of the heart. KEY POINTS: Myocardial stretch increases the production of reactive oxygen species by NADPH oxidase 2. We used NADPH oxidase 2 knockout mice to elucidate the physiological role of stretch-induced reactive oxygen species in the heart. We showed that stretch-induced reactive oxygen species modulate the rising phase of calcium transients and increase myocardial contractility. A mathematical model simulation study demonstrated that rapid activation of ryanodine receptors by reactive oxygen species is important for increased contractility. This response is advantageous for the myocardium, which must contract against a given preload.

Myocardial TRPC6-mediated Zn2+ influx induces beneficial positive inotropy through ß-adrenoceptors.

Baroreflex control of cardiac contraction (positive inotropy) through sympathetic nerve activation is important for cardiocirculatory homeostasis. Transient receptor potential canonical subfamily (TRPC) channels are responsible for α1-adrenoceptor (α1AR)-stimulated cation entry and their upregulation is associated with pathological cardiac remodeling. Whether TRPC channels participate in physiological pump functions remains unclear. We demonstrate that TRPC6-specific Zn2+ influx potentiates ß-adrenoceptor (ßAR)-stimulated positive inotropy in rodent cardiomyocytes. Deletion of trpc6 impairs sympathetic nerve-activated positive inotropy but not chronotropy in mice. TRPC6-mediated Zn2+ influx boosts α1AR-stimulated ßAR/Gs-dependent signaling in rat cardiomyocytes by inhibiting ß-arrestin-mediated ßAR internalization. Replacing two TRPC6-specific amino acids in the pore region with TRPC3 residues diminishes the α1AR-stimulated Zn2+ influx and positive inotropic response. Pharmacological enhancement of TRPC6-mediated Zn2+ influx prevents chronic heart failure progression in mice. Our data demonstrate that TRPC6-mediated Zn2+ influx with α1AR stimulation enhances baroreflex-induced positive inotropy, which may be a new therapeutic strategy for chronic heart failure.

High hydrostatic pressure induces slow contraction in mouse cardiomyocytes.

Cardiomyocytes are contractile cells that regulate heart contraction. Ca2+ flux via Ca2+ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca2+]i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca2+]i.

Ischemia Enhances the Acute Stretch-Induced Increase in Calcium Spark Rate in Ventricular Myocytes.

Introduction: In ventricular myocytes, spontaneous release of calcium (Ca2+) from the sarcoplasmic reticulum via ryanodine receptors ("Ca2+ sparks") is acutely increased by stretch, due to a stretch-induced increase of reactive oxygen species (ROS). In acute regional ischemia there is stretch of ischemic tissue, along with an increase in Ca2+ spark rate and ROS production, each of which has been implicated in arrhythmogenesis. Yet, whether there is an impact of ischemia on the stretch-induced increase in Ca2+ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca2+ sparks and ROS that occurs with stretch. Methods: Isolated ventricular myocytes from mice (male, C57BL/6J) were loaded with fluorescent dye to detect Ca2+ sparks (4.6 µM Fluo-4, 10 min) or ROS (1 µM DCF, 20 min), exposed to normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia [15 mM potassium], acidosis [6.5 pH], and metabolic inhibition [1 mM sodium cyanide, 20 mM 2-deoxyglucose]), and subjected to sustained stretch by the carbon fiber technique (~10% increase in sarcomere length, 15 s). Ca2+ spark rate and rate of ROS production were measured by confocal microscopy. Results: Baseline Ca2+ spark rate was greater in SI (2.54 ± 0.11 sparks·s-1·100 µm-2; n = 103 cells, N = 10 mice) than NT (0.29 ± 0.05 sparks·s-1·100 µm-2; n = 33 cells, N = 9 mice; p < 0.0001). Stretch resulted in an acute increase in Ca2+ spark rate in both SI (3.03 ± 0.13 sparks·s-1·100 µm-2; p < 0.0001) and NT (0.49 ± 0.07 sparks·s-1·100 µm-2; p < 0.0001), with the increase in SI being greater than NT (+0.49 ± 0.04 vs. +0.20 ± 0.04 sparks·s-1·100 µm-2; p < 0.0001). Baseline rate of ROS production was also greater in SI (1.01 ± 0.01 normalized slope; n = 11, N = 8 mice) than NT (0.98 ± 0.01 normalized slope; n = 12, N = 4 mice; p < 0.05), but there was an acute increase with stretch only in SI (+12.5 ± 2.6%; p < 0.001). Conclusion: Ischemia enhances the stretch-induced increase of Ca2+ sparks in ventricular myocytes, with an associated enhancement of stretch-induced ROS production. This effect may be important for premature excitation and/or in the development of an arrhythmogenic substrate in acute regional ischemia.

The Effects of Mechanical Preload on Transmural Differences in Mechano-Calcium-Electric Feedback in Single Cardiomyocytes: Experiments and Mathematical Models.

Transmural differences in ventricular myocardium are maintained by electromechanical coupling and mechano-calcium/mechano-electric feedback. In the present study, we experimentally investigated the influence of preload on the force characteristics of subendocardial (Endo) and subepicardial (Epi) single ventricular cardiomyocytes stretched by up to 20% from slack sarcomere length (SL) and analyzed the results with the help of mathematical modeling. Mathematical models of Endo and Epi cells, which accounted for regional heterogeneity in ionic currents, Ca2+ handling, and myofilament contractile mechanisms, showed that a greater slope of the active tension-length relationship observed experimentally in Endo cardiomyocytes could be explained by greater length-dependent Ca2+ activation in Endo cells compared with Epi ones. The models also predicted that greater length dependence of Ca2+ activation in Endo cells compared to Epi ones underlies, via mechano-calcium-electric feedback, the reduction in the transmural gradient in action potential duration (APD) at a higher preload. However, the models were unable to reproduce the experimental data on a decrease of the transmural gradient in the time to peak contraction between Endo and Epi cells at longer end-diastolic SL. We hypothesize that preload-dependent changes in viscosity should be involved alongside the Frank-Starling effects to regulate the transmural gradient in length-dependent changes in the time course of contraction of Endo and Epi cardiomyocytes. Our experimental data and their analysis based on mathematical modeling give reason to believe that mechano-calcium-electric feedback plays a critical role in the modulation of electrophysiological and contractile properties of myocytes across the ventricular wall.

L-type calcium channel modulates mechanosensitivity of the cardiomyocyte cell line H9c2.

The application of mechanical stimuli to cells often induce increases in intracellular calcium, affecting the regulation of a variety of cell functions. Although the mechanism of mechanotransduction-induced calcium increases has not been fully resolved, the involvement of mechanosensitive ion channels in the plasma membrane and the endoplasmic reticulum has been reported. Here, we demonstrate that voltage-gated L-type calcium channels play a critical role in the mechanosensitive calcium response in H9c2 rat cardiomyocytes. The intracellular calcium level in H9c2 cells increased in a reproducible dose-dependent manner in response to uniaxial stretching. The stretch-activated calcium response (SICR) completely disappeared in calcium-free medium, whereas thapsigargin and cyclopiazonic acid, inhibitors of sarcoendoplasmic reticulum calcium ATPase, partially reduced the SICR. These findings suggest that both calcium influx across the cell membrane and calcium release from the sarcoendoplasmic reticulum are involved in the SICR. Nifedipine, diltiazem, and verapamil, inhibitors of L-type calcium channels, reduced the SICR in a dose-dependent manner. Furthermore, small interfering RNA against the L-type calcium channel α1c subunit diminished the SICR dramatically. Nifedipine also diminished the mechanosensitivity of Langendorff-perfused rat heart. These results suggest that the SICR in H9c2 cardiomyocytes involves the activation of L-type calcium channels and subsequent calcium release from the sarcoendoplasmic reticulum.

TRPC3 participates in angiotensin II type 1 receptor-dependent stress-induced slow increase in intracellular Ca2+ concentration in mouse cardiomyocytes.

When a cardiac muscle is held in a stretched position, its [Ca2+] transient increases slowly over several minutes in a process known as stress-induced slow increase in intracellular Ca2+ concentration ([Ca2+]i) (SSC). Transient receptor potential canonical (TRPC) 3 forms a non-selective cation channel regulated by the angiotensin II type 1 receptor (AT1R). In this study, we investigated the role of TRPC3 in the SSC. Isolated mouse ventricular myocytes were electrically stimulated and subjected to sustained stretch. An AT1R blocker, a phospholipase C inhibitor, and a TRPC3 inhibitor suppressed the SSC. These inhibitors also abolished the observed SSC-like slow increase in [Ca2+]i induced by angiotensin II, instead of stretch. Furthermore, the SSC was not observed in TRPC3 knockout mice. Simulation and immunohistochemical studies suggest that sarcolemmal TRPC3 is responsible for the SSC. These results indicate that sarcolemmal TRPC3, regulated by AT1R, causes the SSC.

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.

The effects of load on transmural differences in contraction of isolated mouse ventricular cardiomyocytes.

Mechanical properties of cardiomyocytes from different transmural regions are heterogeneous in the left ventricular wall. The cardiomyocyte mechanical environment affects this heterogeneity because of mechano-electric feedback mechanisms. In the present study, we investigated the effects of the mechanical load (preload and afterload) on transmural differences in contraction of subendocardial (ENDO) and subepicardial (EPI) single cells isolated from the murine left ventricle. Various preloads imposed via axial stretch and afterloads (unloaded and heavy loaded conditions) were applied to the cells using carbon fiber techniques for single myocytes. To simulate experimentally obtained results and to predict mechanisms underlying the cellular response to change in load, our mathematical models of the ENDO and EPI cells were used. Our major findings are the following. Our results show that ENDO and EPI cardiomyocytes have different mechanical responses to changes in preload to the cells. Under auxotonic contractions at low preload (unstretched cells), time to peak contraction (Tmax) and the time constant of [Ca2+]i transient decay were significantly longer in ENDO cells than in EPI cells. An increase in preload (stretched cells) prolonged Tmax in both cell types; however, the prolongation was greater in EPI cells, resulting in a decrease in the transmural gradient in Tmax at high preload. Comparing unloaded and heavy loaded (isometric) contractions of the cells we found that transmural gradient in the time course of contraction is independent of the loading conditions. Our mathematical cell models were able to reproduce the experimental results on the distinct cellular responses to changes in the mechanical load when we accounted for an ENDO/EPI difference in the parameters of cooperativity of calcium activation of myofilaments.

Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches.

AIMS: Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4. METHODS AND RESULTS: Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent Kd = ∼500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+- and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (≥ six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced. CONCLUSIONS: These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.

Mechano-sensitivity of mitochondrial function in mouse cardiac myocytes.

Mitochondria are an important source of reactive oxygen species (ROS). Although it has been reported that myocardial stretch increases cellular ROS production by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), referred to as X-ROS signalling, the involvement of mitochondria in X-ROS is not clear. Mitochondria are organelles that generate adenosine triphosphate (ATP) for cellular energy needs, which are mechanical-load-dependent. Therefore, it would not be surprising if these organelles had mechano-sensitive functions associated with stretch-induced ROS production. In the present study, we investigated the relation between X-ROS and mitochondrial stretch-sensitive responses in isolated mouse cardiac myocytes. The cells were subjected to 10% axial stretch using computer-controlled, piezo-manipulated carbon fibres attached to both cell ends. Cellular ROS production and mitochondrial membrane potential (Δψm) were assessed optically by confocal microscopy. The axial stretch increased ROS production and hyperpolarised Δψm. Treatment with a mitochondrial metabolic uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), at 0.5 µM did not suppress stretch-induced ROS production, whereas treatment with a respiratory Complex III inhibitor, antimycin A (5 µM), blunted the response. Although NOX inhibition by apocynin abrogated the stretch-induced ROS production, it did not suppress stretch-induced hyperpolarisation of Δψm. These results suggest that stretch causes activation of the respiratory chain to hyperpolarise Δψm, followed by NOX activation, which increases ROS production.

Role of TRPC3 and TRPC6 channels in the myocardial response to stretch: Linking physiology and pathophysiology.

Transient receptor potential (TRP) channels constitute a large family of versatile multi-signal transducers. In particular, TRP canonical (TRPC) channels are known as receptor-operated, non-selective cation channels. TRPC3 and TRPC6, two members in the TRPC family, are highly expressed in the heart, and participate in the pathogenesis of cardiac hypertrophy and heart failure as a pathological response to chronic mechanical stress. In the pathological response, myocardial stretch increases intracellular Ca2+ levels and activates nuclear factor of activated T cells to induce cardiac hypertrophy. Recent studies have revealed that TRPC3 and TRPC6 also contribute to the physiological stretch-induced slow force response (SFR), a slow increase in the Ca2+ transient and twitch force during stretch. In the physiological response, a stretch-induced increase in intracellular Ca2+ mediated by TRPC3 and TRPC6 causes the SFR. We here overview experimental evidence of the involvement of TRPC3 and TRPC6 in cardiac physiology and pathophysiology in response to stretch.

Effects of simulated ischemia on the transmural differences in the Frank-Starling relationship in isolated mouse ventricular cardiomyocytes.

The electrical and mechanical functions of cardiomyocytes differ in relation to the spatial locations of cells in the ventricular wall. This physiological heterogeneity may change under pathophysiological conditions, providing substrates for arrhythmia and contractile dysfunctions. Previous studies have reported distinctions in the electrophysiological and mechanical responses to ischemia of unloaded subendocardial (ENDO) and subepicardial (EPI) single cardiomyocytes. In this paper, we briefly recapitulated the available experimental data on the ischemia effects on the transmural cellular gradient in the heart ventricles and for the first time evaluated the preload-dependent changes in passive and active forces in ENDO and EPI cardiomyocytes isolated from mouse hearts subjected to simulated ischemia. Combining the results obtained in mechanically loaded contracting cardiomyocytes with data from previous studies, we showed that left ventricular ENDO and EPI cardiomyocytes are different in their mechanical responses to metabolic inhibition. Simulated ischemia showed opposite effects on the stiffness of ENDO and EPI cells and greatly prolonged the time course of contraction in EPI cells than in ENDO cells, thereby changing the normal transmural gradient in the cellular mechanics.

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.

Celsior preserves cardiac mechano-energetics better than University of Wisconsin solution by preventing oxidative stress.

OBJECTIVES: Identity of the optimal heart preservation solution remains unknown. Because oxidative stress contributes to contractile failure in the ischaemic/reperfused myocardium and the main characteristic of Celsior is its antioxidant effect, it is important to elucidate the relationship between the inhibitory effect on oxidative stress and cardiac mechano-energetics. We therefore evaluated the efficacy of Celsior from both aspects by comparison with the University of Wisconsin solution (UWS). METHODS: We used 18 excised cross-circulated canine hearts. Excised hearts were preserved with UWS (n = 6) or Celsior (n = 6) for 3 h at 4 °C; the remaining six served as controls. Hearts were then cross-circulated and rewarmed. The end-systolic pressure-volume ratio (LV Emax) and the ventricular pressure-volume area, which is a measure of total mechanical energy, were assessed after reperfusion. Biopsies were taken from the endocardium after excising the heart, before reperfusion, after reperfusion and 4 h after reperfusion to assess the inhibitory effect of each agent on oxidative stress. Endo-myocardial biopsy samples were studied immunohistochemically for expression of 4-hydroxy-2-nonenal (HNE)-modified protein, which is a major lipid peroxidation product. RESULTS: Emax in the UWS group was significantly smaller than in the control group, whereas the Emax in the Celsior group was preserved. Oxygen cost of Emax in the UWS group was significantly higher than in the Celsior group. Myocardial HNE-modified protein levels increased gradually, both under preservation and after reperfusion in the UWS group. Myocardial HNE-modified protein levels in the Celsior group were lower, mainly before and 4 h after reperfusion compared with the UWS group. CONCLUSIONS: Celsior may maintain cardiac contractility and conserve oxygen cost by inhibiting oxidative stress.

Load dependency in force-length relations in isolated single cardiomyocytes.

The previously reported pressure-volume (PV) relationship in frog hearts shows that end-systolic PV relation (ESPVR) is load dependent, whereas ESPVR in canine hearts is load independent. To study intrinsic cardiac mechanics in detail, it is desirable to study mechanics in a single isolated cardiomyocyte that is free from interstitial connective tissue. Previous single cell mechanics studies used a pair of carbon fibers (CF) attached to the upper surface of opposite cell ends to stretch cells. These studies showed that end-systolic force-length (FL) relation (ESFLR) is load independent. However, the range of applicable mechanical load using the conventional technique is limited because of weak cell-CF attachment. Therefore, the behavior of ESFLR in single cells under physiologically possible conditions of greater load is not yet well known. To cover wider loading range, we contrived a new method to hold cell-ends more firmly using two pairs of CF attached to both upper and bottom surfaces of cells. The new method allowed stretching cells to 2.2 µm or more in end-diastolic sarcomere length. ESFLR virtually behaves in a load independent manner only with end-diastolic sarcomere length less than 1.95 µm. It exhibited clear load dependency with higher preload, especially with low afterload conditions. Instantaneous cellular elastance curves showed that decreasing afterload enhanced relaxation and slowed time to peak elastance, as previously reported. A simulation study of a mathematical model with detailed description of thin filament activation suggested that velocity dependent thin filament inactivation is crucial for the observed load dependent behaviors and previously reported afterload dependent change in Ca(2+) transient shape.

Energy for myocardial Ca2+ handling per beat increases with heart rate in excised cross-circulated canine heart.

OBJECTIVE: Although tachycardia is well known to increase cardiac oxygen consumption (Vo2) per min, the relationship between Vo2 for excitation-contraction (E-C) coupling per beat and heart rate change over its full working range still remains controversial. METHODS: To elucidate this relationship, we varied heart rate over a reasonably wide range (60-180 beat/min) and studied the relationship between left ventricular (LV) Emax (load-independent contractility index), PVA (pressure-volume area)-independent Vo2, and basal metabolic Vo2 in nine excised, cross-circulated canine hearts. RESULTS: PVA-independent Vo2 per min significantly increased linearly with increasing heart rate while Emax remained unchanged. Basal metabolic Vo2 per min was measured under KCl arrest. E-C coupling Vo2 per min obtained by subtracting the constant basal metabolic Vo2 from the PVA-independent Vo2 also significantly increased linearly with increasing heart rate. However, PVA-independent Vo2 per beat significantly decreased with increasing heart rate. In contrast, E-C coupling Vo2 per beat, as well as that normalized to Emax, slightly but significantly increased with increasing heart rate. CONCLUSION: The E-C coupling energy for myocardial Ca2+ handling increases with heart rate despite constant contractility in the left ventricle of the excised cross-circulated 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.