Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts--role of late sodium current and intracellular ion accumulation

The goal of this study was to test the hypothesis that the novel anti-ischemic drug ranolazine, which is known to inhibit late I_Na, could reduce intracellular [Na⁺]_i and diastolic [Ca²⁺]_i overload and improve diastolic function. Contractile dysfunction in human heart failure (HF) is associated with increased [Na⁺]_i and elevated diastolic [Ca²⁺]_i. Increased Na⁺ influx through voltage-gated Na⁺ channels (late I_Na) has been suggested to contribute to elevated [Na⁺]_i in HF. In isometrically contracting ventricular muscle strips from end-stage failing human hearts, ranolazine (10 µmol/L) did not exert negative inotropic effects on twitch force amplitude. However, ranolazine significantly reduced frequency-dependent increase in diastolic tension (i.e., diastolic dysfunction) by ~ 30% without significantly affecting sarcoplasmic reticulum (SR) Ca²⁺ loading. To investigate the mechanism of action of this beneficial effect of ranolazine on diastolic tension, Anemonia sulcata toxin II (ATX-II, 40 nmol/L) was used to increase intracellular Na⁺ loading in ventricular rabbit myocytes. ATX-II caused a significant rise in [Na⁺]_i typically seen in heart failure via increased late I_Na. In parallel, ATX-II significantly increased diastolic [Ca²⁺]_i. In the presence of ranolazine the increases in late I_Na, as well as [Na⁺]_i and diastolic [Ca²⁺]_i were significantly blunted at all stimulation rates without significantly decreasing Ca²⁺ transient amplitudes or SR Ca²⁺ content. In summary, ranolazine reduced the frequency-dependent increase in diastolic tension without having negative inotropic effects on contractility of muscles from end-stage failing human hearts. Moreover, in rabbit myocytes the increases in late I_Na, [Na⁺]_i and [Ca²⁺]_i caused by ATX-II, were significantly blunted by ranolazine. These results suggest that ranolazine may be of therapeutic benefit in conditions of diastolic dysfunction due to elevated [Na⁺]_i and diastolic [Ca²⁺]_i.     

Distinct modulation of myocardial performance,energy metabolism,and [Ca2+]i transients by positive inotropic drugs in normal and severely failing hamster hearts

Summary The present study compared the effects of amrinone, dobutamine, dibutyryl cAMP, digoxin, and isoproterenol on mechanical performance, the high energy phosphate metabolites, and the [Ca²⁺]_i transients in normal and cardiomyopathic hamster hearts with severe heart failure. In normal hearts dobutamine, dibutyryl cAMP, and isoproterenol increased left ventricular developed pressure, while amrinone and digoxin did not. However, the amplitude of [Ca²⁺]_i transients was augmented with all drugs. Diastolic [Ca²⁺]_i level was increased with dobutamine and lowered with dibutyryl cAMP and isoproterenol. In cardiomyopathic hearts with severe heart failure, left ventricular developed pressure, the amplitude of [Ca²⁺]_i transients, the phosphorylation potential, and [cAMP]_i were significantly depressed and left ventricular end-diastolic pressure and diastolic [Ca²⁺]_i were significantly elevated when compared with normal hearts. Amrinone, dibutyryl cAMP, and isoproterenol improved mechanical performance while increasing [cAMP]_i and the amplitude of [Ca²⁺]_i transients, and decreasing diastolic [Ca²⁺]_i. On the other hand, with dobutamine and digoxin diastolic [Ca²⁺]_i was further increased and mechanical performance deteriorated with digoxin. Thus, distinct differences exist in modulation of mechanical performance, high-energy phosphate metabolism, and [Ca²⁺]_i transients by positive inotropic drugs between normal and cardiomyopathic hearts with severe heart failure.Supported in part by the George D. Smith Foundation and NIH grant AA 07413-01. Peter Buser is a recipient of a Career Development Grant (SCORE # 32-29340,90) from the Swiss National Science Foundation.     

Effects of new intravascular contrast agents on [Ca2+]i transients and contraction in cultured ventricular myocytes

Summary We examined the effects of four kinds of intravascular contrast agents (amidtrizoic acid, iohexol, iopamidol, and ioxaglic acid) on [Ca²⁺]_i transients (indo-1 fluorescence) and cell contraction (video motion analyzer), using cultured chick embryo ventricular myocytes. Exposure of ventricular myocytes to amidtrizoic acid (a conventional contrast agent) reduced the [Ca²⁺]_i transients and the sensitivity of the contractile elements to [Ca²⁺]_i. Ioxaglic acid (a low osmotic contrast agent) also reduced the [Ca²⁺]_i transients, but did not significantly change the sensitivity of the contractile elements to [Ca²⁺]_i. Neither iohexol nor iopamidol (nonionic contrast agents) reduced the [Ca²⁺]_i transients, but both significantly decreased the sensitivity of the contractile elements to [Ca²⁺]_i. A marked negative inotropic effect of amidtrizoic acid was caused by both calcium binding and hypertonicity. The less marked depression of contractility produced by ioxaglic acid is possibly the result of calcium binding, but is not caused by hypertonicity. The negative inotropism produced by nonionic contrast agents (iohexol and iopamidol) was due to hypertonicity, but not due to alterations in the [Ca²⁺]_i transients.Exposure of ventricular myocytes to nonionic contrast agents (iohexol and iopamidol) slowed decay in the [Ca²⁺]_i transients with increased end-diastolic [Ca²⁺]_i. After washing out the nonionic contrast agents, these parameters returned to control levels. On the other hand, exposure to amidtrizoic acid decreased end-diastolic [Ca²⁺]_i without changing decay time in the [Ca²⁺]_i transients. After washing out amidtrizoic acid, there was a prolongation of half decay time in [Ca²⁺]_i transients with a significant increase in end-diastolic [Ca²⁺]_i and cell position. Diastolic dysfunction just after washout of amidtrizoic acid was possibly caused by an increase in [Na⁺]_i due to sodium influx during exposure to the contrast agent.     

Glycolysis in heart failure: a31P-NMR and surface fluorometry study

Summary Glycolysis is slow in the heart, especially in the cardiomyopathic heart. Glycolysis is partially rate-limited by phosphofructokinase (PFK), an enzymc which is inhibited by calcium (Ca²⁺)_i and hydrogen ions (H⁺)_i and activated by cAMP. (H⁺)_i and (Ca²⁺)_i are augmented in cardiomyopathy. With glucose as the only substrate (NADH)/(NAD) the phosphorylation potential and developed pressure were significantly lower, and concentrations of phosphomonoester sugars and hydrogen ions (H⁺)_i were significantly higher in isolated cardiomyopathic hearts as compared to healthy hamster hearts. Pyruvate lowered diastolic (Ca²⁺)_i in cardiomyopathic hamster hearts. With pyruvate as the substrate (NADH)/(NAD), the phosphorylation potential and developed pressure incrcased significantly and concentrations of phosphomonoester sugars (PME), (H⁺)_i and diastolic (Ca²⁺)_i decreased significantly in myopathic hamster hearts. The results suggest that late heart failure in the myopathic hamster is associated with calcium and/or hydrogen ion-induced inhibition of glycolysis.Supported in part by the George D. Smith Foundation and NIH Grant AA 07413-01. W. Auffermann supported by # AU 70/2-2 from Deutsche Forschungsgemeinschaft, Bonn, FRG     

Calcium-activated force in a turkey model of spontaneous dilated cardiomyopathy: Adaptive changes in thin myofilament Ca regulation with resultant implications on contractile performance

Using saponin skinned fibers, we investigated whether decreased myofilament calcium responsiveness and contractile activation may in part contribute to heart failure in an animal model of idiopathic spontaneous cardiomyopathy (SCM). We addressed the question as to whether there are adaptive changes at the level of the thin myofilaments in turkey poults with SCM. The calcium concentration ([Ca²⁺]) required for 50% activation ([Ca²⁺]_50%) was 0.80±0.12 μ (n=12) vs. 0.76±0.08 μ (n=12) and the Hill coefficient was 1.98±0.20 (n=12) vs. 2.14±0.38 (n=12) for control and SCM muscles respectively. Maximal Ca²⁺-activated force was not different between control fibers and fibers from failing hearts (3.83±0.88 g/mm² vs. 3.65±0.39 g/mm²). These data indicate there are no differences in calcium-activation between fibers from control and failing myocardium. The effects of caffeine, an agent that increases myofilament Ca²⁺ sensitivity, were also studied. Addition of 10 m caffeine resulted in a 0.06 pCa unit leftward shift of the force-pCa relationship in control hearts and 0.14 pCa units in SCM hearts. Caffeine (30 m ) increased force by 26±2.1% (n=7) in control fibers and 44.5±8.7% (n=8) in myopathic fibers at a pCa of 6.0. The increased responsiveness of muscles from failing hearts to caffeine indicates adaptive changes at the level of the thin myofilaments. Addition of dibutyryl-3′,5′-cyclic-Adenosine Monophosphate (D-cAMP) resulted in a 0.21 pCa rightward shift on the calcium axis to higher intracelluar calcium concentrations in control myocardium and 0.38 pCa units in SCM failing myocardium. The muscles were also sinusoidally oscillated at frequencies ranging between 0.01 and 100 Hz. In this analysis the frequency at which dynamic stiffness is minimum is taken as a measure of cross-bridge cycling rate. In control muscles, the frequency of minimum stiffness (f_min) was 1.20±0.11 (n=4) whereas it was 0.71±0.08 Hz (n=4) in myopathic muscles. The addition of 10 μ D-cAMP shifted f_min from 1.20±0.11 Hz to 1.68±0.09 Hz (Δ=0.48±0.06) in control fibers whereas in SCM fibers it caused greater shift of f_min from 0.71±0.08 Hz to 1.73±0.08 Hz (Δ=1.02±0.07). This differential effect of D-cAMP indicates adaptive changes at the level of the myofilaments. Our studies indicate that adaptive changes at the level of the thin myofilaments can potentially alter the “apparent” calcium-force relationship in failing myocardium and contractile performance, and that failing myocardium has the potential to generate similar levels of force as normal non-failing myocardium.     

Imaging of Ca2+ Transients in Endothelial Cells of Single Perfused Capillaries: Correlation of Peak [Ca2+]i with Sites of Macromolecule Leakage

Objective: To investigate the mechanisms responsible for variation in the macromolecular leakage (formation of localized leaky sites) in venular microvessels with increased permeability, we examined the hypothesis that cytoplasmic calcium concentration [Ca²⁺]_i, does not increase uniformly within microvessel endothelial cells. Methods: We loaded the endothelial cells forming the walls of venular microvessels in frog mesentery with fura-2, and imaged [Ca²⁺]_i using a cooled CCD camera. Results: Control [Ca²⁺]_i was close to 60 nM in all regions. Control permeability was uniformly low in all microvessels. Exposure to ionomycin (5 mM) increased [Ca²⁺]_i in a biphasic manner, but not uniformly. There was variation in both time to peak (bimodal distribution) and peak [Ca²⁺]_i (274 ± 13 nM; mean variation above or below the peak value was 110 nM). Raising extracellular calcium from 1.1 to 5 mM increased the mean variation of [Ca²⁺]_i about peak values. Extravascular leakage of fluorescently labeled albumin or low-density lipoproteins was most prominent at sites where increases in [Ca²⁺]_i were largest. Conclusions: These data indicate that variation in [Ca²⁺]_i within individual endothelial cells or groups of cells could account, at least in part, for the distribution of localized leakage sites for macromolecules in venular microvessels in the high-permeability state.     

H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

H2O2-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia–reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Influence of perfusate calcium concentration on the inotropic insulin effect in isolated guinea pig and rat hearts

Conflicting data on the inotropic effect of insulin are present in the literature suggesting a positive inotropic property or no inotropic effect or even a negative influence. To clarify the reason for these diverging findings, dose-response curves of insulin have been performed in isolated working rat and guinea pig hearts perfused with Krebs-Henseleit buffer containing 0.9, 1.25, 2.5, and 5 mM Ca²⁺ at 37 °C. At 1.25 mM [Ca²⁺], insulin (8 to 16 IU) regularly improved the inotropic state. LVdP/dt_max increased significantly from 1,900 to 2,300 mm Hg/s (+21 %) in guinea pig hearts and from 3,197 to 4,345 mm Hg/s (+36 %) in rat hearts. LVEDP did not change significantly. Myocardial oxygen consumption increased parallel with contractility. Heart rate was not influenced in either species. Coronary flow increased by 16.5 % in guinea pig hearts, but decreased in rat hearts by 13.6 % (p < 0.05 each). With 0.9 mM [Ca²⁺] the positive inotropic effect of insulin did not further augment. At 2.5 mM [Ca²⁺] insulin exhibited in both species no significant change of LVdP/dt_max but very high insulin doses depressed the heart. At 5 mM [Ca²⁺] insulin depressed the heart significantly already at lower concentrations. At 31 °C and 1.25 mM [Ca²⁺] the positive inotropic insulin effect was preserved. We conclude that the positive inotropic insulin effect in rat and guinea pig hearts depends on the extracellular [Ca²⁺], i.e., is maximal around 1.25 mM [Ca²⁺] and is reduced or absent at higher [Ca²⁺] or may even become negative. Received: 3 September 2001/Returned for 1. revision: 24 September 2001/1. Revision received: 20 December 2001/Returned for 2. revision: 2 January 2002/2. Revision received: 21 January 2002/Accepted: 23 January 2002     

K201 improves aspects of the contractile performance of human failing myocardium via reduction in Ca<Superscript>2+</Superscript> leak from the sarcoplasmic reticulum

In heart failure, intracellular Ca²⁺ leak from cardiac ryanodine receptors (RyR2s) leads to a loss of Ca²⁺ from the sarcoplasmic reticulum (SR) potentially contributing to decreased function. Experimental data suggest that the 1,4-benzothiazepine K201 (JTV-519) may stabilise RyR2s and thereby reduce detrimental intracellular Ca²⁺ leak. Whether K201 exerts beneficial effects in human failing myocardium is unknown. Therefore, we have studied the effects of K201 on muscle preparations from failing human hearts. K201 (0.3 μM; extracellular [Ca²⁺]_e 1.25 mM) showed no effects on contractile function and micromolar concentrations resulted in negative inotropic effects (K201 1 μM; developed tension ?9.8 ± 2.5% compared to control group; P < 0.05). Interestingly, K201 (0.3 μM) increased the post-rest potentiation (PRP) of failing myocardium after 120 s, indicating an increased SR Ca²⁺ load. At high [Ca²⁺]_e concentrations (5 mmol/L), K201 increased PRP already at shorter rest intervals (30 s). Strikingly, treatment with K201 (0.3 μM) prevented diastolic dysfunction (diastolic tension at 5 mmol/L [Ca²⁺]_e normalised to 1 mmol/L [Ca²⁺]_e: control 1.26 ± 0.06, K201 1.01 ± 0.03, P < 0.01). In addition at high [Ca²⁺]_e, K201 (0.3 μM) treatment significantly improved systolic function [developed tension +27 ± 8% (K201 vs. control); P < 0.05]. The beneficial effects on diastolic and systolic functions occurred throughout the physiological frequency range of the human heart rate from 1 to 3 Hz. Upon elevated intracellular Ca²⁺ concentration, systolic and diastolic contractile functions of terminally failing human myocardium are improved by K201.     

Respiratory acid-base changes and myocardial contractility: interaction between calcium and hydrogen ions.

The mechanism whereby respiratory acid-base changes alter myocardial contractility is poorly understood. Accordingly, 46 cat right ventricular papillary muscles contracting isometrically at 30°C were studied while varying both the pH and [Ca²⁺] of the bathing medium. The pH changes were induced by varying the PCO₂ of the gassing mixture. A decrease in pH from 7.45 to 7.10 in the presence of 2.5 mm Ca²⁺ caused a rapid fall in active force and rate of force development ($\text{d}\text{F}\text{d}\text{t}$) to levels 76% of control followed by a gradual recovery to approximately 89% of control during a 60 min observation period despite continued acidosis. Increasing the [Ca²⁺] to 5.0 mm markedly attenuated the negative inotropic effects of hypercarbic acidosis. Increasing the pH from either 7.10 to 7.45 or 7.45 to 7.75 caused a rapid 15% increase in active force and $\text{d}\text{F}\text{d}\text{t}$ followed by a return to near control levels after 60 min. The biphasic contractile response to either respiratory acidosis or alkalosis was not influenced by pretreating the muscles with 1 × 10^?6m propranolol. In 10 additional muscles series elastic load-extension relations at varying pH levels were determined using standard quick-release techniques. Respiratory acid-base changes did not alter the measured series elastic properties. These findings indicate that the contractile effects produced by carbon dioxide-induced pH alterations are exerted at the level of the contractile element and that the muscle has the intrinsic capacity to attenuate these inotropic effects with time. Furthermore, our results suggest that the contractile effects produced by changing [H⁺] may involve an interaction with Ca²⁺ at a sarcolemmal or intracellular binding site.     

Endothelin-induced changes in intracellular calcium concentration in rat vascular smooth muscle cells: Effects of extracellular calcium and atrial natriuretic factor

Summary The purpose of this study was to examine whether different mechanisms might underlie the changes in intracellular calcium concentration ([Ca²⁺]_i) stimulated by high and low concentrations of endothelin, and whether atrial natriuretic factor (ANF) has an inhibitory effect on endothelin-induced [Ca²⁺]_i changes in cultured rat vascular smooth muscle cells (VSMCs). In calcium-replete buffer, cultured monolayers of rat VSMCs superfused with endothelin at a high concentration (10 nM) exhibited a marked transient rise in [Ca²⁺]_i, followed by a sustained elevation, whereas a low concentration of endothelin (0.1 nM) induced a sustained monophasic elevation. When calcium-free buffer was used, 10 nM endothelin induced a transient rise in [Ca²⁺]_i of lesser amplitude, whereas 0.1 nM endothelin did not produce a significant rise. Pretreatment of VSMCs with ANF and cosuperfusion with endothelin failed to inhibit either transient or sustained endothelin-induced changes in [Ca²⁺]_i in calcium-replete buffer.     

Reduced tolerance to reperfusion-associated injury in hearts from myopathic hamsters

Summary This study was done to evaluate the response of myopathic hearts from dystrophic hamsters to 30 minutes of ischemia followed by 30 minutes of reperfusion. Hearts from male and female normal animals recovered 77±6% and 64±5% of their contractile force respectively following reperfusion whereas only 34±8% (male) and 34±7% (female) recovery was seen in myopathic hearts (P<0.01). Substantial sustained contractures were observed ruring reperfusion in hearts from dystrophic animals irrespective of gender whereas none were seen with control hearts. Reperfusion produced a rapid release of CPK that peaked at 5 minutes (approximate coronary effluent concentration of 40 mU/ml) and remained elevated for the reperfusion duration. Peak CPK values for normal hearts were reached at 10 minutes following reperfusion, were significantly lower from the myopathic hearts and returned to near control levels at the end of the 30 minute reperfusion period. Reducing Ca²⁺ in the perfusion medium by up to 80% or perfusing the hearts with the Ca²⁺-channel blocker verapamil produced no beneficial effects. Changes in the above parameters produced by ischemia or heart rate alterations throughout the perfusion sequence were not different between normal and myopathic hearts. This study shows a sensitivity of myopathic hearts that is manifested during reperfusion. Possible mechanisms for this reduced tolerance are discussed.     

MCI-154: A Second Generation Ca2+ Sensitizer That Does Not Impair Relaxation — A Novel Approach to the Treatment of Heart Failure

With the negative results of the cyclic AMP-dependent positive inotropic agents in clinical trials, great interest has been focused on the development of agents that directly activate cardiac myofilaments. These drugs are called “Ca²⁺ sensitizers”; they are expected to represent a possible new pharmacological approach to the therapy of chronic heart failure. MCI-154 is one of the most powerful and promising Ca²⁺ sensitizers currently in clinical trials. In preclinical studies, the positive inotropic action of MCI-154 was observed at the concentrations at which the drug did not increase intracellular Ca²⁺. In skinned cardiac muscle fiber bundles from various animal species MCI-154 shifted p_Ca(-log [Ca²⁺] M)-force relation upward and to the left, suggesting that this drug not only increases sensitivity of myofibrils to Ca²⁺, but also enhances cross-bridge interaction. In regard to the molecular mechanism of action of MCI-154, the earliest experiments showed that MCI-154 at the high concentration (10⁻⁴ M) stimulated binding of Ca²⁺ to troponin (Tn) C. However, MCI-154-(at concentrations lower than 10⁻⁵ M)-induced increase in the affinity of TnC to Ca²⁺ led to the complex formation of TnC with TnI and TnT. This effect of the drug on Ca²⁺ regulation and interaction between troponin subunits was discovered using fluorescence spectroscopy. At 10⁻⁴ M MCI-154 decreased binding of Ca²⁺ to TnC. The Ca²⁺ sensitizing effect of MCI-154 disappeared when cardiac TnI was exchanged for skeletal TnI. Taken together, it can be concluded that TnI may represent a target protein for the positive inotropic action of MCI-154. The long-term treatment with MCI-154 prolonged the life span of cardiomyopathic hamsters, Biol 14.6, a model that resembles human heart failure. Unlike PDE inhibitors, MCI-154 did not aggravate arrhythmias generated in the two-stage coronary ligation-, digitalis- and catecholamine-induced canine arrhythmia models. In clinical studies MCI-154 improved contractile function in patients with left ventricular dysfunction after myocardial infarction. Its effect was not associated with an increase in myocardial oxygen consumption or impaired relaxation. In conclusion, MCI-154 could be promising in the treatment of chronic heart failure. Clinical studies with MCI-154 are currently in progress.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Calcium sensitivity, force frequency relationship and cardiac troponin I: Critical role of PKA and PKC phosphorylation sites

Transgenic models with pseudo phosphorylation mutants of troponin I, PKA sites at Ser 22 and 23 (cTnIDD_22,23 mice) or PKC sites at Ser 42 and 44 (cTnIAD_22,23DD_42,44) displayed differential force-frequency relationships and afterload relaxation delay in vivo. We hypothesized that cTnI PKA and PKC phosphomimics impact cardiac muscle rate-related developed twitch force and relaxation kinetics in opposite directions. cTnIDD_22,23 transgenic mice produce a force frequency relationship (FFR) equivalent to control NTG albeit at lower peak [Ca²⁺]_i, while cTnIAD_22,23DD_42,44 TG mice had a flat FFR with normal peak systolic [Ca²⁺]_i, thus suggestive of diminished responsiveness to [Ca²⁺]_i at higher frequencies. Force-[Ca²⁺]_i hysteresis analysis revealed that cTnIDD_22,23 mice have a combined enhanced myofilament calcium peak response with an enhanced slope of force development and decline per unit of [Ca²⁺]_i, whereas cTnIAD_22,23DD_42,44 transgenic mice showed the opposite. The computational ECME model predicts that the TG lines may be distinct from each other due to different rate constants for association/dissociation of Ca²⁺ at the regulatory site of cTnC. Our data indicate that cTnI phosphorylation at PKA sites plays a critical role in the FFR by increasing relative myofilament responsiveness, and results in a distinctive transition between activation and relaxation, as displayed by force-[Ca²⁺]_i hysteresis loops. These findings may have important implications for understanding the specific contribution of cTnI to β-adrenergic inotropy and lusitropy and to adverse contractile effects of PKC activation, which is relevant during heart failure development.     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

Role of mitochondrial dysfunction in cardiac glycoside toxicity

Cardiac glycosides, which inhibit the plasma membrane Na⁺ pump, are one of the four categories of drug recommended for routine use to treat heart failure, yet their therapeutic window is limited by toxic effects. Elevated cytoplasmic Na⁺ ([Na⁺]_i) compromises mitochondrial energetics and redox balance by blunting mitochondrial Ca²⁺ ([Ca²⁺]_m) accumulation, and this impairment can be prevented by enhancing [Ca²⁺]_m. Here, we investigate whether this effect underlies the toxicity and arrhythmogenic effects of cardiac glycosides and if these effects can be prevented by suppressing mitochondrial Ca²⁺ efflux, via inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na⁺]_i in a dose-dependent way, blunted [Ca²⁺]_m accumulation, decreased the NADH/NAD + redox potential, and increased reactive oxygen species (ROS). Concomitant treatment with the mNCE inhibitor CGP-37157 ameliorated these effects. CGP-37157 also attenuated ouabain-induced cellular Ca²⁺ overload and prevented delayed afterdepolarizations (DADs). In isolated perfused hearts, ouabain's positive effects on contractility and respiration were markedly potentiated by CGP-37157, as were those mediated by β-adrenergic stimulation. Furthermore, CGP-37157 inhibited the arrhythmogenic effects of ouabain in both isolated perfused hearts and in vivo. The findings reveal the mechanism behind cardiac glycoside toxicity and show that improving mitochondrial Ca²⁺ retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca²⁺-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.     

Uniformity of calcium channel number and isometric contraction in human right and left ventricular myocardium

Summary We compared contractile performance in trabeculae carneae (n=25) from non-failing right and left ventricles (n=25) of brain dead organ donors without known cardiovascular disease and measured connective tissue content in trabeculae carneae from both non-failing and failing human hearts. Peak twitch force and time-course of contraction were not different between muscles taken from right or left ventricles. Peak twitch force was 13.9±3 vs. 13.7±2.7 mN/mm² for right and left ventricular trabeculae carneae, respectively in 2.5 mM [Ca²⁺]₀ at a 0.33 Hz stimulation frequency. Time to peak tension (405±21 vs. 405±12 ms), time to 50% relaxation from peak contractile response (277±21 vs. 278±14.6 ms) and time to 80% relaxation (428±29 vs. 433±22) were not different between right and left ventricular trabeculae carneae. Calcium channel number determined by [³H]PN200-100 dihydropyridine-radioligand binding assay was also not different (56.2±6.5 fmol/mg protein vs. 58.6±8.4 fmol/mg protein for right and left heart preparations, respectively). However, in myocardium obtained from ischemic hearts the left ventricle showed a reduced number of calcium channels compared to the right ventricle (55.3±3.8 vs. 36.6±3.9 fmol/mg protein for right and left ventricle, respectively p=0.027). No differences were noted in the number of DHP receptor binding sites between right and left ventricular myocardium from patients with idiopathic dilated cardiomyopathy (51.4±7.6 fmol/mg protein vs. 61.8±6.5 fmol/mg protein respectively). Our data indicate that calcium channel number is similar for non-failing left and right human ventricle. Contractile response to changes in [Ca²⁺]₀ and frequency were similar for trabeculae carneae from the left and right ventricles of non-failing human hearts. Studies involving calcium channel activation or inhibition in ischemic human myocardium, where there may be differences in calcium channel number and/or function are warranted. Whether changes in calcium channel number have biological consequences on contractile function remains to be determined. Importantly, careful studies of calcium channel function underin vivo conditions are warranted.Work supported in part by a grant from Glaxo Inc to JKG and the Institute for the Study of the Treatments of Cardiovascular Diseases, Cardiovascular Drug Development and Marketing Consultants, and HL-39091 and HL-36797 to JKG. HL 31117 to JPM. EJG is a fellow of the Stanley J. Sarnoff Society of Fellows for Research in Cardiovascular Science. JKG is an Established Investigator of the American Heart Association.     

Studies on the mechanism of action of the bipyridine milrinone on the heart

Summary Milrinone is a positive inotropic and vasodilator agent when tested in experimental animals and in human heart-failure patients. It is generally believed that milrinone acts by inhibiting phosphodiesterase IV, thus increasing cyclic AMP, [Ca^{+ +}]_i and cardiac contractile force and relaxation. Maximal force produced by milrinone is greater when single-dose response curves are compared to cumulative dose-response curves. In vitro, milrinone produces a tachyphylaxis, the extent of which is both dose- and time-dependent. Recovery of tachyphylaxis is both dose- and time-dependent and is not influenced by inhibitors of protein or RNA synthesis. There is a specific cross-tachyphylaxis between milrinone and amrinone, theophylline, papaverine, and Bay K8644. This tachyphylaxis may explain the low maximal contractile response of the cumulative dose-response observed in isolated tissues. Milrinone increased cyclic AMP in dog and guinea pig cardiac muscle. As previously shown by Endoh et al. [17], milrinone in low doses produced a biphasic effect on cyclic AMP. The early increase (first 60–70 s) in cyclic AMP shows a good correlation with contractile force changes. If cyclic AMP is determined at maximal contractile force this correlation was poor. Here we also present instances where the increase in cyclic AMP after milrinone (determined at maximal effect) does not correlate with the contractile response. The cross-tachyphylaxis of milrinone with Bay K8644 suggests that milrinone has an action on the sarcolemmal Ca⁺⁺ channels. Bay K8644 suppresses the positive inotropic effect of catecholamines by 50%, but not the cyclic AMP response. The inotropic effect of milrinone, in contrast to norepinephrine is highly sensitive to [Ca⁺⁺]₀, stimulation rate, and [K⁺]₀. In this respect milrinone behaves more like Bay K8644. We postulate that the main inotropic action of milrinone is due to a sarcolemmal effect. The early cyclic AMP production described could be in the sarcolemmal compartment and this may explain some of the similarities of milrinone’s actions with those of Bay K8644. The tachyphylaxis observed with the inotropic effect of milrinone does not extend to the decreases in relaxation time. This and other findings to be discussed suggest that the positive inotropic and reduction in relaxation time by milrinone depend on different mechanisms, possibly through differential compartmentalization of cyclic AMP.