Mohammed A. Matlib

Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc.115, 11799–11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nm) than ruthenium red (IC50 = 6.85 nm) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (K d = 0.34 nm, B max = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nm, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+uptake. Ru360, as high as 10 μm, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 μm) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.

Selectivity of inhibition of Na(+)-Ca2+ exchange of heart mitochondria by benzothiazepine CGP-37157.

The objective was to determine if the benzothiazepine compound CGP-37157 selectively inhibits the Na(+)-Ca2+ exchanger of cardiac mitochondria without affecting the L-type voltage-dependent calcium channel, the Na(+)-Ca2+ exchanger, or the Na(+)-K(+)-ATPase of the cardiac sarcolemma, or the Ca(2+)-ATPase of the cardiac sarcoplasmic reticulum. Mitochondrial Na(+)-Ca2+ exchange activity was determined by monitoring intramitochondrial free [Ca2+] in isolated heart mitochondria loaded with the Ca(2+)-sensitive fluorophore fura-2. CGP-37157 inhibited the activity of mitochondrial Na(+)-Ca2+ exchange in a dose-dependent manner (IC50 0.36 microM). Calcium currents were recorded by whole-cell voltage clamp in isolated neonatal ventricular myocytes. Diltiazem was able to block the recorded current completely, thus confirming the current to be exclusively L-type. CGP-37157 had no effect on the calcium current recorded under identical conditions. CGP-37157, at concentrations < or = 10 microM, had no effect on the activities of the Na(+)-Ca2+ exchanger and Na(+)-K(+)-ATPase in isolated cardiac sarcolemmal vesicles or on activity of the Ca(2+)-ATPase in isolated cardiac sarcoplasmic reticulum vesicles. The data suggest that CGP-37157 is a potent, selective, and specific inhibitor of mitochondrial Na(+)-Ca2+ exchange at concentrations < or = 10 microM.

Specific binding of [3H]nitrendipine to membranes from coronary arteries and heart in relation to pharmacological effects. Paradoxical stimulation by diltiazem

Abstract High affinity binding sites for the calcium channel inhibitor [ 3 H]nitrendipine have been identified in microsomes from pig coronary arteries (K D =1.6 nM; B max =35 fmol/mg) and in purified sarcolemma from dog heart (K D =0.11 nM; B max =230 fmol/mg). [ 3 H]nitrendipine binding to coronary artery microsomes was completely inhibited by nifedipine, partially by verapamil and D600 and, surprisingly, was stimulated by d-cis-diltiazem but not by 1-cis-diltiazem, a less active isomer. Half-maximal relaxation of KCl-depolarized coronary rings occurred in a slow process at 1 nM nitrendipine or 100 nM d-cis-diltiazem. In dog trabecular strips, nitrendipine caused a negative inotropic response (ED 50 =1μM). These results suggest that there may be multiple binding sites for different “subclasses” of calcium channel inhibitors, and that drug binding sites may be different molecular entities from the putative calcium channels.

Compensatory Mechanisms Associated With the Hyperdynamic Function of Phospholamban-Deficient Mouse Hearts

Phospholamban ablation is associated with significant increases in the sarcoplasmic reticulum Ca(2+)-ATPase activity and the basal cardiac contractile parameters. To determine whether the observed phenotype is due to loss of phospholamban alone or to accompanying compensatory mechanisms, hearts from phospholamban-deficient and age-matched wild-type mice were characterized in parallel. There were no morphological alterations detected at the light microscope level. Assessment of the protein levels of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, calsequestrin, myosin, actin, troponin I, and troponin T revealed no significant differences between phospholamban-deficient and wild-type hearts. However, the ryanodine receptor protein levels were significantly decreased (25%) upon ablation of phospholamban, probably in an attempt to regulate the release of Ca2+ from the sarcoplasmic reticulum, which had a significantly higher diastolic Ca2+ content in phospholamban-deficient compared with wild-type hearts (16.0 +/- 2.2 versus 8.6 +/- 1.0 mmol Ca2+/kg dry wt, respectively). The increases in Ca2+ content were specific to junctional sarcoplasmic reticulum stores, as there were no alterations in the Ca2+ content of the mitochondria or A band. Assessment of ATP levels revealed no alterations, although oxygen consumption increased (1.6-fold) to meet the increased ATP utilization in the hyperdynamic phospholamban-deficient hearts. The increases in oxygen consumption were associated with increases (2.2-fold) in the active fraction of the mitochondrial pyruvate dehydrogenase, suggesting increased tricarboxylic acid cycle turnover and ATP synthesis. 31P nuclear magnetic resonance studies demonstrated decreases in phosphocreatine levels and increases in ADP and AMP levels in phospholamban-deficient compared with wild-type hearts. However, the creatine kinase activity and the creatine kinase reaction velocity were not different between phospholamban-deficient and wild-type hearts. These findings indicate that ablation of phospholamban is associated with downregulation of the ryanodine receptor to compensate for the increased Ca2+ content in the sarcoplasmic reticulum store and metabolic adaptations to establish a new energetic steady state to meet the increased ATP demand in the hyperdynamic phospholamban-deficient hearts.

Effects of diltiazem, a calcium antagonist, on regional myocardial function and mitochondria after brief coronary occlusion

Abstract The purpose of the study was to assess the effect of the calcium antagonist diltiazem on mechanical and mitochondrial function of ischemic myocardium of the dog. Persistent depression of developed tension following brief coronary occlusion was measured in anesthetized and thoracotomized dogs. The extent of persistent depression of developed tension during reperfusion depended on the duration of occlusion and also on the pressure-rate index during occlusion. Diltiazem prevented the marked drop in developed tension of the ischemic segment of the myocardium following 5 min or 10 min of coronary occlusion. The inactive optical isomer of diltiazem had no effect on developed tension before or after coronary occlusion. Depression of the state 3 rate of respiration of mitochondria observed following 10 min of occlusion of coronary artery was almost completely reversed by pretreatment with diltiazem. Diltiazem may reduce the damage of ischemic myocardium during occlusion by hemodynamic action of the drug, and possibly by preventing damage to mitochondria.

Modulation of intramitochondrial free Ca2+ concentration by antagonists of Na+-Ca2+ exchange

Evidence has accumulated in the past decade suggesting that Ca2+ acts as a second messenger not only in the cytosol of the heart to regulate contractility, but also within the mitochondria to regulate the rate of oxidative ATP synthesis. Just as elucidation of the second messenger pathways for Ca2+ in the cytosol has led to the development of pharmacological interventions that alter mechanical functioning of the heart, understanding the role of Ca2+ as a second messenger within the mitochondria and the mechanisms by which this organelle transports and regulates Ca2+ has exciting potential for developing pharmacological interventions that alter myocardial energy metabolism. In this article, David Cox and Mohammed Matlib discuss the potential consequences of pharmacologically increasing the intramitochondrial Ca2+ concentration on myocardial energy metabolism, and suggest some pathological conditions in which such an effect may be beneficial.

Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes

1 Ventricular myocytes isolated from ferret or cat were loaded with the acetoxymethyl ester form of indo‐1 (indo‐1 AM) such that ∼75 % of cellular indo‐1 was mitochondrial. The intramitochondrial indo‐1 concentration was 0.5‐2 mm. 2 Myocytes were also voltage clamped (membrane capacitance, Cm= 100 pF) and a typical wash‐out time constant of cytosolic indo‐1 by a patch pipette was found to be ∼300 s. Depolarizations to +110 mV produced graded and progressive cellular Ca2+ load via Na+‐Ca2+ exchange. 3 During these relatively slow Ca2+ transients, cell contraction (ΔL) paralleled fluorescence ratio signals (R) such that ΔL could be used as a bioassay of cytosolic [Ca2+] ([Ca2+]c), where [Ca2+]CL is the inferred signal which is delayed by ∼200 ms from true [Ca2+]c. 4 In myocytes without Mn2+ quench, the kinetics of the total cellular indo‐1 signal, ΔR (including cytosolic and mitochondrial components), match ΔL during stimulations at low basal [Ca2+]i. However, after progressive Ca2+ loading, ΔR kinetics deviate from ΔL dramatically. The deviation can be completely blocked by a potent mitochondrial Ca2+ uniport blocker, Ru360. 5 When cytosolic indo‐1 is quenched by Mn2+, initial moderate stimulation triggers contractions (ΔL), but no change in indo‐1 signal, indicating both the absence of cytosolic Ca2+‐sensitive indo‐1 and unchanged mitochondrial [Ca2+] (Δ[Ca2+]m). Subsequent stronger stimulation evoked larger ΔL and also ΔR. The threshold [Ca2+]c for mitochondrial Ca2+ uptake was 300‐500 nM, similar to that without Mn2+ quench. 6 At high Ca2+ loads where Δ[Ca2+]m is detected, the time course of [Ca2+]m was different from that of [Ca2+]c. Peak [Ca2+]m after stimulation has an ∼1 s latency with respect to [Ca2+]c, and [Ca2+]m decline is extremely slow. 7 Upon a Ca2+ influx which increased [Ca2+]c by 0.4 μm and [Ca2+]m by 0.2 μm, total mitochondrial Ca2+ uptake was ∼13 μmol (l mitochondria)−1. 8 With Mn2+ quench of cytosolic indo‐1, there was no mitochondrial uptake of Mn2+ until the point at which mitochondrial Ca2+ uptake became apparent. However, after mitochondrial Ca2+ uptake starts, mitochondria continually take up Mn2+ even during relaxation, when [Ca2+]c is low. 9 It is concluded that mitochondria in intact myocytes do not take up detectable amounts of Ca2+ during individual contractions, unless resting [Ca2+]c exceeds 300‐500 nM. At high cell Ca2+ loads and [Ca2+]c, mitochondrial Ca2+ transients occur during the twitch, but with much slower kinetics than those of [Ca2+]c.

Noninvasive evaluation of cardiac dysfunction by echocardiography in streptozotocin-induced diabetic rats.

BACKGROUND There has not been a noninvasive in vivo longitudinal evaluation of cardiac function in diabetic rats. The objective of this study is to examine the time course of development of cardiac dysfunction in streptozotocin (STZ)-induced diabetic rats. METHODS AND RESULTS Cardiac function was evaluated by M-mode and Doppler echocardiography in anesthetized Wistar rats at 2, 4, 5, 6, and 8 weeks after injection with 65 mg of STZ/kg and in age-matched control rats before and after the administration of isoproterenol. Body weight (BW) was significantly less and blood glucose level significantly greater in diabetic rats compared with controls at 2 weeks and remained at these levels at all time points. The calculated left ventricular (LV) mass appeared slightly decreased in diabetic rats. However, LV mass-BW ratios were similar in controls and diabetic rats at 2, 4, and 5 weeks, but were significantly greater in diabetic rats at 6 and 8 weeks. Basal heart rate (HR) was significantly lower in diabetic rats at all time points studied. Basal LV systolic and diastolic dimensions, fractional shortening (FS), velocity of circumferential shortening (Vcf), peak emptying rate (PER), peak filling rate (PFR), and aortic peak velocity (APV) were not significantly different between controls and diabetic rats at 2 and 4 weeks. PER and PFR were significantly less in 5-week diabetic rats. However, Vcf, PER, and PFR were significantly less and FS and APV were similar at 6 and 8 weeks. Administration of isoproterenol increased HR, Vcf, FS, PFR, and PER in controls at all time points, but the increases in diabetic rats at 5, 6, and 8 weeks were less compared with those in controls. The increase in APV was significantly less in diabetic rats at all time points studied. CONCLUSION STZ-induced diabetic rats showed bradycardia before contractile dysfunction. Overt and covert contractile dysfunction unmasked by isoproterenol begins at 5 weeks of diabetes. The overt LV systolic and diastolic dysfunction are fully manifested after 6 weeks of diabetes.

Benzodiazepine Ro 5-4864 increases coronary flow

Ro 5-4864 (chlorodiazepam) increased coronary flow in isolated retrograde perfused Langendorff rat heart preparations without affecting heart rate and left ventricular contractility (dP/dt). On the other hand Ro 5-4023 (clonazepam) produced very little effect. PK 11195 which has been shown to inhibit the binding of Ro 5-4864 to cardiac muscle did not antagonize this vasodilatory effect of Ro 5-4864 but increased coronary flow by itself. The data indicate a specific vasodilatory effect of certain benzodiazepines. The mechanism of action remains unknown.

Mitochondria regulate inactivation of L‐type Ca2+ channels in rat heart

1 L‐type Ca2+ channels play an important role in vital cell functions such as muscle contraction and hormone secretion. Both a voltage‐dependent and a Ca2+‐dependent process inactivate these channels. Here we present evidence that inhibition of the mitochondrial Ca2+ import mechanism in rat (Sprague‐Dawley) ventricular myocytes by ruthenium red (RR), by Ru360 or by carbonyl cyanide m‐chlorophenylhydrazone (CCCP) decreases the magnitude of electrically evoked transient elevations of cytosolic Ca2+ concentration ([Ca2+]c). These agents were most effective at stimulus rates greater than 1 Hz. 2 RR and CCCP also caused a significant delay in the recovery from inactivation of L‐type Ca2+ currents (ICa). This suggests that sequestration of cytosolic Ca2+, probably near the mouth of L‐type Ca2+ channels, into mitochondria during cardiac contractile cycles, helps to remove the Ca2+‐dependent inactivation of L‐type Ca2+ channels. 3 We conclude that impairment of mitochondrial Ca2+ transport has no impact on either L‐type Ca2+ currents or SR Ca2+ release at low stimulation frequencies (e.g. 0.1 Hz); however, it causes a depression of cytosolic Ca2+ transients attributable to an impaired recovery of L‐type Ca2+ currents from inactivation at high stimulation frequencies (e.g. 3 Hz). The impairment of mitochondrial Ca2+ uptake and subsequent effects on Ca2+ transients at high frequencies at room temperature could be physiologically relevant since the normal heart rate of rat is around 5 Hz at body temperature. The role of mitochondria in clearing Ca2+ in the micro‐domain near L‐type Ca2+ channels could be impaired during high frequencies of heart beats such as in ventricular tachycardia, explaining, at least in part, the reduction of muscle contractility.