Enhanced in vivo and in vitro contractile responses to beta(2)-adrenergic receptor stimulation in dogs susceptible to lethal arrhythmias.

The response to beta-adrenergic receptor (beta-AR) stimulation was evaluated in both isolated cardiomyocytes (video edge detection) and the intact animal (echocardiography) in dogs either susceptible (S) or resistant (R) to ventricular fibrillation induced by a 2-min coronary occlusion during the last minute of exercise. In the intact animal, velocity of circumferential fiber shortening (Vcf) was evaluated both before (n = 27, S = 12 and R = 15) and after myocardial infarction. Before infarction, increasing doses of isoproterenol provoked similar contractile and heart rate responses in each group of dogs. Either beta(1)-AR (bisoprolol) or beta(2)-AR (ICI-118551) antagonists reduced the isoproterenol response, with a larger reduction noted after the beta(1)-AR blockade. In contrast, after infarction, isoproterenol induced a significantly larger Vcf and heart rate response in the susceptible animals that was eliminated by beta(2)-AR blockade. The single-cell isotonic shortening response to isoproterenol (100 nM) was also larger in cells obtained from susceptible compared with resistant dogs and was reduced to a greater extent by beta(2)-AR blockade in the susceptible dog myocytes (S, -48%, n = 6; R, -15%, n = 9). When considered together, these data suggest that myocardial infarction provoked an enhanced beta(2)-AR response in susceptible, but not resistant, animals.

Transgenic mice with cardiac-specific expression of activating transcription factor 3, a stress-inducible gene, have conduction abnormalities and contractile dysfunction.

Activating transcription factor 3 (ATF3) is a member of the CREB/ATF family of transcription factors. Previously, we demonstrated that the expression of the ATF3 gene is induced by many stress signals. In this report, we demonstrate that expression of ATF3 is induced by cardiac ischemia coupled with reperfusion (ischemia-reperfusion) in both cultured cells and an animal model. Transgenic mice expressing ATF3 under the control of the alpha-myosin heavy chain promoter have atrial enlargement, and atrial and ventricular hypertrophy. Microscopic examination showed myocyte degeneration and fibrosis. Functionally, the transgenic heart has reduced contractility and aberrant conduction. Interestingly, expression of sorcin, a gene whose product inhibits the release of calcium from sarcoplasmic reticulum, is increased in these transgenic hearts. Taken together, our results indicate that expression of ATF3, a stress-inducible gene, in the heart leads to altered gene expression and impaired cardiac function.

beta(2)-Adrenoceptors and ventricular fibrillation.

beta-Adrenoceptor antagonists significantly reduce the incidence of sudden cardiac death in patients with contractile dysfunction. Contractile dysfunction is associated with a decline in beta(1)-adrenoceptors, no change in the number of beta(2)-adrenoceptors, and an increased responsiveness to beta(2)-adrenoceptor stimulation. Selective beta(2)-adrenoceptor blockade prevents ventricular fibrillation in a canine model of sudden cardiac death. Cardiac beta(2)-adrenoceptor stimulation increases L-type Ca(2+) currents, but unlike beta(1)-adrenoceptor stimulation, it fails to elicit phospholamban phosphorylation. Restoration of resting diastolic [Ca(2+)] following beta(2)-adrenoceptor-mediated increases in Ca(2+) influx is more dependent on Na(+)/Ca(2+) exchange, which generates an arrhythmogenic transient inward current that can trigger ventricular fibrillation.

FK506 alters sarcoplasmic reticulum calcium release in neonatal piglet cardiac myocytes.

Tacrolimus (FK506) is a potent immunosuppressive drug that, when complexed to a family of immunophilin proteins known as FK binding proteins, inhibits calcineurin in T lymphocytes. Although i.v. use of FK506 in pediatric transplant recipients has been linked to development of cardiomyopathies, its mechanism of cardiotoxicity has not been examined in a neonatal animal model. In our study the effects of FK506 were investigated in cardiac myocytes isolated from newborn piglets. The peak amplitude of electrically triggered fura-2 Ca2+ transients was increased in FK506-treated myocytes, but Ca2+ transient duration and baseline fura-2 Ca2+ ratios were not altered. 45Ca2+ uptake by digitonin-lysed piglet cells decreased at pCa < or = 6.0, indicating that sarcoplasmic reticulum efflux channels were leaky. The results suggest that elevated release of sarcoplasmic reticulum Ca2+ during systole contributes to cardiotoxic effects observed in children.

Origin of contractile dysfunction in heart failure: calcium cycling versus myofilaments.

BACKGROUND: Chronic congestive heart failure is a common, often lethal disorder of cardiac contractility. The fundamental pathophysiology of the contractile failure remains unclear, the focus being on abnormal Ca2+ cycling despite emerging evidence for depressed myofilament function. METHODS AND RESULTS: We measured intracellular Ca2+ concentration ([Ca2+]i) and contractile force in intact ventricular muscle from SHHF rats with spontaneous heart failure and from age-matched controls. At physiological concentrations of extracellular Ca2+ ([Ca2+]o), [Ca2+]i transients were equal in amplitude in the 2 groups, but [Ca2+]i peaked later in SHHF muscles. Twitch force peaked slowly and was equivalent or modestly decreased in amplitude relative to controls. Steady-state analysis revealed a much greater (53%) depression of maximal Ca2+-activated force in SHHF muscles, which, had other factors been equal, would have produced an equivalent suppression of twitch force. Phase-plane analysis reveals that the slowing of Ca2+ cycling prolongs the time available for Ca2+ to activate the myofilaments in failing muscle, partially compensating for the marked dysfunction of the contractile machinery. CONCLUSIONS: Our results indicate that myofilament activation is severely blunted in heart failure, but concomitant changes in [Ca2+]i kinetics minimize the contractile depression. These results challenge prevailing concepts regarding the pathophysiology of heart failure: the myofilaments emerge as central players, whereas changes in Ca2+ cycling are reinterpreted as compensatory rather than causative.

31P-NMR analysis of congestive heart failure in the SHHF/Mcc-facp rat heart.

31P-NMR was used to monitor myocardial bioenergetics in compensated and failing SHHF/MCC-fa(cp) (SHF) rat hearts. The SHHF/Mcc-fa(cp) (spontaneous hypertension and heart failure) rat is a relatively new genetic model in which all individuals spontaneously develop congestive heart failure, most during the second year of life. Failing SHF rat hearts displayed a pronounced decrease in resting PCr:ATP ratios (P<0.001), which was explained by a significant (P<0. 0001) drop in total creatine (47.2+/-3.1 nmol/mg protein) v age matched controls (106+/-3 nmol/mg protein). In end stage failure, NMR determined PCr was 2.9+/-0.1 micro mol/g wet weight under basal conditions. In contrast, 6- and 20-month-old controls and compensated SHFs had PCr values of 5.3+/-0.1, and 5.1+/-0.5 and 5. 1+/-0.2 micro mol/g wet weight. Both compensated and failing SHF hearts were metabolically compromised when the rate pressure product (RPP) was increased, as evidenced by an increase in Pi and a drop in PCr. Compensated SHF hearts, however, were able to increase rate pressure products (RRP, mmHg X beats/min) from 44.5+/-1.4 to 66.6+/-3. 4 K with dobutamine infusion, whereas hearts in end-stage failure were able to increase their RPP from baseline values of 27+/-4 K to only 37+/-7 K. The data indicate that a pronounced decline in PCr and total creatine signals the transition from compensatory hypertrophy to decompensation and failure in the SHF rat model of hypertensive cardiomyopathy.

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 (Kd = 0.34 nM, Bmax = 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 microM, 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 microM) 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.

Human myocardial ATP content and in vivo contractile function.

The study was designed to characterize the relationship between the metabolite content of human cardiac muscle and in vivo cardiac function. ATP, total adenine nucleotides, and NAD were quantified in human myocardial biopsies using high performance liquid chromatography. Right ventricular endomyocardial biopsies were obtained from 43 patients with dilated cardiomyopathy, 6 with restrictive cardiomyopathy, 10 with normal systolic and diastolic function, and from 24 cold preserved human donor hearts. Transmural samples of failing right and left ventricular free walls were obtained during cardiac transplantation surgery in 8 patients. ATP, total adenine nucleotides, and NAD were similar in the cold-preserved donor hearts and in right ventricular endomyocardial biopsies from the 10 individuals with normal systolic and diastolic function. In contrast, these values were significantly depressed in tissue samples from patients with dilated or restrictive cardiomyopathy. There was a significant correlation between ATP and pulmonary capillary wedge pressures but not ejection fractions. Declines in the sizes of myocardial ATP, adenine nucleotide, and pyridine nucleotide pools in the human myocardium are associated primarily with diastolic but not systolic dysfunction.

Myocarditis induced by targeted expression of the MCP-1 gene in murine cardiac muscle.

To explore the possible role of monocyte chemotactic protein (MCP-1) in inflammatory diseases of the heart, we expressed the murine MCP-1(JE) gene under the control of the alpha-cardiac myosin heavy chain promoter to attempt to target MCP-1 expression to the adult heart muscle. The five lines of transgenic mice thus produced showed targeted expression of MCP-1 transcripts and protein in the adult heart muscle and pulmonary vein but not in skeletal muscle. MCP-1 level in the transgenic hearts increased up to 30 to 45 days of age, and leukocyte infiltration into interstitium between cardiomyocytes increased up to 60 to 75 days. The infiltrate was mainly macrophages but not T cells. The presence of MCP-1 in the transgenic hearts did not induce cytokine production indicative of leukocyte activation. Echocardiographic analysis of 1-year-old mice that express MCP-1 in the myocardium and of age-matched controls revealed cardiac hypertrophy and dilation, increases in left ventricular (LV) mass, and systolic and diastolic left ventricular internal diameters. A significant decline in M-mode shortening fraction showed depressed contractile function. Transgenic hearts were 65% heavier, and histological analysis showed moderate myocarditis, edema, and some fibrosis. Thus, MCP-1 expression in the heart muscle may provide a model to investigate myocarditis and cardiomyopathy.

Localized cAMP-dependent signaling mediates beta 2-adrenergic modulation of cardiac excitation-contraction coupling.

Recent studies have shown that beta 2-adrenergic receptor (beta 2-AR)-stimulated increases in the intracellular Ca2+ (Cai) transient and contraction in cardiac myocytes are dissociated from the increase in adenosine 3',5'-cyclic monophosphate (cAMP) level and are not accompanied by an increase in phospholamban phosphorylation, an acceleration in relaxation, or a reduction in myofilament Ca2+ response. Thus we hypothesized that the beta 2-AR modulation of cardiac excitation-contraction (EC) coupling may be mediated by either a cAMP-independent mechanism or a compartmentalized cAMP pathway. To directly distinguish between these two possibilities, the responses of the L-type Ca2+ current (ICa), Cai transient, and contraction to beta 2-AR as well as to beta 1-AR stimulation were examined in rat ventricular myocytes in the presence or absence of specific inhibitory cAMP analogs, Rp diastereomers of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) and 8-(4-chlorophenylthio)-cAMP (Rp-CPT-cAMPS). As expected, the positive inotropic effect induced by an adenylyl cyclase activator, forskolin (2 x 10(-7) M), or a beta 1-AR agonist, norepinephrine (5 x 10(-8) M) plus prazosin (10(-6) M), was completely blocked by Rp-CPT-cAMPS. More importantly, the responses of ICa, Cai transient, and contraction to beta 2-AR stimulation by zinterol (10(-5) M) or isoproterenol plus a selective beta 1-AR antagonist, CGP-20712A, were also entirely abolished by Rp-cAMPS (in the patch-pipette solution) or Rp-CPT-cAMPS (in the bath solution). In pertussis toxin-treated cells, although the response of cAMP was not altered, the beta 2-AR-stimulated increase in contraction amplitude was markedly enhanced and accompanied by a hastened relaxation, resulting in a tight association between cAMP and contraction. These results indicate that beta 2-AR modulation of cardiac excitation-contraction coupling requires cAMP. The dissociation of beta 2-AR-stimulated cAMP production and regulation of myofilament and sarcoplasmic reticulum functions is attributable to a functional compartmentation of the cAMP-dependent signaling due to an activation of beta 2-AR-coupled Gi and/or G(o).

Beta2-adrenergic receptor antagonists protect against ventricular fibrillation: in vivo and in vitro evidence for enhanced sensitivity to beta2-adrenergic stimulation in animals susceptible to sudden death.

BACKGROUND: The ventricular myocardium contains functional beta2-adrenergic receptors that when activated increase intracellular Ca2+ transients. Because elevated Ca2+ has been implicated in the induction of ventricular fibrillation (VF), it is possible that the activation of these receptors may also provoke malignant arrhythmias. METHODS AND RESULTS: To test this hypothesis, a 2-minute occlusion of the left circumflex coronary artery was made during the last minute of exercise in 28 dogs with healed anterior myocardial infarctions: 17 had VF (susceptible) and 11 did not (resistant). On a subsequent day, this test was repeated after administration of the beta2-adrenergic receptor antagonist ICI 118,551 (0.2 mg/kg). This drug did not alter the hemodynamic response to the coronary occlusion, yet it prevented VF in 10 of 11 animals tested (P<.001). However, heart rate was reduced in 6 animals. Therefore, the ICI 118,551 exercise-plus-ischemia test was repeated with heart rate held constant by ventricular pacing (n=3). ICI 118,551 still prevented VF when heart rate was maintained. Next, the effects of increasing doses of the beta2-adrenergic receptor agonist zinterol on Ca2+ transient amplitudes were examined in ventricular myocytes. Zinterol elicited significantly greater increases in Ca2+ transient amplitudes at all doses tested (10(-9) to 10(-6) mol/L) in myocytes prepared from susceptible versus resistant animals. The cardiomyocyte response to isoproterenol (10(-7) mol/L) in the presence or absence of the selective beta1- (CGP-20712A, 300 nmol/L) or beta2- (ICI 118,551, 100 nmol/L) adrenergic receptor antagonist was also examined. Isoproterenol elicited larger Ca2+ transient increases in the susceptible myocytes, which were eliminated by ICI but not by CGP. CONCLUSIONS: When considered together, these data demonstrate that canine myocytes contain functional beta2-adrenergic receptors that are activated to a greater extent in the susceptible animals. The resulting cytosolic Ca2+ transient increases may lead to afterpotentials that ultimately trigger VF in these animals.

Calcium handling by sarcoplasmic reticulum of neonatal swine cardiac myocytes.

In recent years, because of similarities to human infants, neonatal piglets have increasingly become the model of choice for studying neonatal heart function. However, the cardiac sarcoplasmic reticulum (SR) has not been thoroughly characterized in this species. Accordingly, Ca2+ pump kinetics, efflux channel characteristics, Ca2+ transients, and contractile movements were examined in isolated newborn piglet cardiac ventricular myocytes. Maximum uptake rate (Vmax) and concentration required to produce a half-maximal effect (K0.5) for oxalate-supported, ATP-dependent 45Ca2+ uptake by the SR of digitonin-lysed myocytes were 285 +/- 17 nmol 45Ca2+.min-1.mg-1 and 0.69 +/- 0.07 microM, respectively. In the absence of phospholamban phosphorylation, Vmax was reduced to 195 +/- 26 nmol 45Ca2+.min-1.mg-1 (P < 0.05 vs. control) and K0.5 increased to 1.28 +/- 0.13 microM (P < 0.05 vs. control). [3H]ryanodine binding studies yielded a maximum binding capacity of 181 +/- 12 fmol/mg and a dissociation constant of 1.7 +/- 0.2 nM. Raising extracellular Ca2+ (0.5-5 mM) increased peak amplitude and decreased the duration of electrically stimulated fura 2 Ca2+ transients and recordings of cell length changes. Both ryanodine and 2,5-di-tert-butylhydroquinone, an inhibitor of SR calcium adenosinetriphosphatase, completely abolished Ca2+ transients in piglet myocytes. These studies indicate that the SR has a significant role in excitation-contraction coupling in neonatal piglet myocytes.

Verapamil accelerates the transition to heart failure in obese, hypertensive, female SHHF/Mcc-fa(cp) rats.

We sought to characterize the effects of the nonselective Ca2+ channel antagonist, verapamil, and the vascular-selective Ca2+ channel antagonist, felodipine, on obese, hypertensive, heart failure-prone, female SHHF/Mcc-fa(cp) rats. Rats were treated for < or = 2 months with verapamil (57 mg/kg/day) or felodipine (24 mg/kg/day). Blood pressures were determined at monthly intervals by the tail-cuff method. Heart weights and myosin isoforms were measured at the end of treatment. Direct cardiac effects of verapamil and felodipine were examined in electrically field stimulated, fura-2/AM-loaded cardiomyocytes. Both Ca2+ channel antagonists reduced systolic blood pressures. Verapamil, but not felodipine, increased heart weights and decreased expression of the myosin V1 isoform. In older animals, 75% of those treated with verapamil developed end-stage congestive heart failure. Age-matched control and felodipine-treated rats remained healthy. In isolated cardiomyocytes, 10(-9) M verapamil significantly reduced Ca2+ transient amplitudes but 10(-9) M felodipine did not. Both Ca2+ channel antagonists reduced blood pressures in obese, hypertensive, female SHHF rats. Verapamil, but not felodipine, produced heart failure in a large number of these animals. Differences between the in vivo effects of the two Ca2+ channel antagonists may be related to the differing effects on sarcolemmal Ca2+ influx.

Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure.

Cardiac hypertrophy and heart failure caused by high blood pressure were studied in single myocytes taken from hypertensive rats (Dahl SS/Jr) and SH-HF rats in heart failure. Confocal microscopy and patch-clamp methods were used to examine excitation-contraction (EC) coupling, and the relation between the plasma membrane calcium current (ICa) and evoked calcium release from the sarcoplasmic reticulum (SR), which was visualized as "calcium sparks." The ability of ICa to trigger calcium release from the SR in both hypertrophied and failing hearts was reduced. Because ICa density and SR calcium-release channels were normal, the defect appears to reside in a change in the relation between SR calcium-release channels and sarcolemmal calcium channels. beta-Adrenergic stimulation largely overcame the defect in hypertrophic but not failing heart cells. Thus, the same defect in EC coupling that develops during hypertrophy may contribute to heart failure when compensatory mechanisms fail.

'Cross talk' between opioid peptide and adrenergic receptor signaling in isolated rat heart.

BACKGROUND: Cardiac myocyte sarcolemma contains both catecholamine and opioid peptide receptors (OPRs). Opioid peptides are coreleased with catecholamines from nerve terminals in the heart. We investigated whether OPR stimulation influences the effects of beta-adrenergic receptor (beta-AR) stimulation in the isolated, isovolumic rat heart and whether the mechanism of such an interaction involves both beta-AR subtypes or an alteration in beta-AR-mediated increase in cAMP. METHODS AND RESULTS: Norepinephrine (NE, 10(-7) mol/L) increased peak left ventricular systolic pressure (LVSP) and cAMP more than twofold compared with controls. The delta-OPR agonist leucine-enkephalin (LE, 10(-8) mol/L) markedly inhibited the beta1-AR-induced positive inotropic effect and increase in cAMP but alone had no effect on basal LVSP or basal cAMP levels. The OPR antagonist naloxone 10(-8) mol/L added to LE+NE perfusate reversed the LE-induced decrease in cAMP and LVSP even though naloxone alone had no effect on LVSP and cAMP levels. LE could not counteract the twofold increase in LVSP produced by the nondegradable cAMP analog CPT-cAMP 2.3x10(-5) mol/L or a high concentration of forskolin (10(-7) mol/L) but did reverse the 173+/-11.8% and 135+/-13.6% increases in LVSP stimulated by 10(-8) and 0.5x10(-8) mol/L forskolin, respectively. LE inhibited cAMP production at all concentrations of forskolin (10(-7), 10(-8), and 0.5x10(-8) mol/L). Pertussis toxin (PTX) pretreatment abolished LE effects on beta1-AR stimulation. Zinterol 10(-5) and 10(-6) mol/L, a specific beta2-AR agonist that elicits a cAMP-independent inotropic effect in rat heart, caused 225+/-14% and 182+/-5% increases in LVSP that could not be reversed by addition of LE. CONCLUSIONS: Potent, inhibitory "cross talk" between delta-OPR and beta1-AR signaling pathways occurs via a PTX-sensitive G(i/o) protein involved in adenylyl cyclase inhibition in rat heart.

Effects of calcium channel antagonists on Ca2+ transients in rat and canine cardiomyocytes.

First-generation Ca2+ channel antagonists depress myocardial contractility, but many of the newer Ca2+ channel blockers have a high degree of "vascular selectivity". This study compares the effects of the Ca2+ antagonists felodipine, amlodipine, mibefradil, verapamil and nifedipine, and the Ca2+ channel agonist. (S)(-)-Bay K-8644 on Ca2+ transient amplitudes in fura-2/AM-loaded rat and canine ventricular cardiomyocytes. At 10(-11) and 10(-10) M, felodipine increased [Ca2+]i transient amplitudes by 10-25% in field-stimulated fura-2-loaded cells from both species while at 10(-6) M it depressed [Ca2+]i transients by 80%. Mibefradil increased [Ca2+]i transient amplitudes by 16% at 10(-11) and 10(-10) M and decreased the transients by 25% at 10(-6) M. The calcium channel agonist, (S)(-)-Bay K-8644 increased [Ca2+]i transient amplitudes at 10(-10)-10(-6) M (maximally 37% at 10(-7) M) but depressed [Ca2+]i transients by 10% at 10(-5) M. Nifedipine was inhibitory at all concentrations tested (10(-11)-10(-6) M) in canine myocytes, but in rat cells. 10(-10) M nifedipine increased [Ca2+]i transient amplitudes by 37%. All concentrations of verapamil and amlodipine (10(-11)-10(-6) M) depressed [Ca2+]i transients in both rat and canine myocytes. We conclude that: (1) felodipine and mibefradil may be positive rather than negative inotropes at low concentrations, which are therapeutically relevant: and (2) low concentrations of nifedipine may have a positive inotropic effect in the rat but not the dog heart.

2,3-Butanedione 2-monoxime (BDM) induces calcium release from canine cardiac sarcoplasmic reticulum.

2,3-Butanedione 2-monoxime (BDM) is a well known inhibitor of skeletal and cardiac muscle contraction. Recently, it has been discovered that BDM has an influence on the sarcoplasmic reticulum (SR). We investigated the effects of BDM on the SR in our digitonin lysed myocyte system, which measures accumulated SR Ca2+. While BDM (30 mM) had no effect on SR Ca2+ uptake (under conditions that included Ca2+ release channel efflux inhibitors), it induced SR Ca2+ release (no efflux inhibitors) with a maximal reduction of 72% of SR Ca2+ at pCa 6.0. A titration showed that even 5 mM BDM resulted in a 45% reduction at that same pCa. Also, a positive correlation was found between the degree of BDM induced Ca2+ release and free Ca2+ concentration. Thus, the use of even low concentrations of BDM as an excitation-contraction uncoupler must be approached with caution.

Metabolic compartmentalization in neonatal swine myocytes.

The transport of metabolites across the mitochondrial membrane is regulated by specific exchange and shuttle systems that are often dependent on the mitochondrial membrane potential. Thus, metabolite concentrations in the cytosol and mitochondrial compartments are largely determined by the energy state of the cardiac muscle cell. The purpose of this study was to investigate metabolic compartmentalization in ventricular myocytes isolated from newborn (< 24 h) swine hearts. Furthermore, the effect of respiratory inhibition on these distribution patterns was examined. Freshly isolated cells contained 33 nmol of ATP and 37 nmol of total adenine nucleotides (AN) per mg of myocyte protein. Rapid digitonin fractionation indicated that 95% of ATP and 86% of AN were cytosolic, whereas > 50% of the pyridine nucleotides were mitochondrial. With 11 mM added glucose, myocytes treated with the respiratory inhibitor, rotenone, maintained ATP at 88% of that of aerobic myocytes, but phosphocreatine declined by 50% over 30 min. Rotenone treatment caused the mitochondrial NAD/NADH ratio to decline from 1.2 to 0.06, whereas the cytosolic pyridine nucleotides remained > 90% oxidized. Total adenine and pyridine nucleotide content and their compartmentalization were unaffected by respiratory inhibition. Comparisons of metabolite content and respiratory activity between isolated piglet mitochondria and the mitochondrial compartment of piglet myocytes indicated that mitochondria account for approximately 30% of total myocyte protein. A similar value (29%) was obtained for the aqueous volume fraction of the in situ mitochondrial matrix using the 4000 Mr 14C-labeled polyethylene glycol-impermeable 3H2O spaces of intact and lysed myocytes. These results are comparable to literature values for myocardium from other species and age groups.