Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.

Some characteristics of monocarboxylic acid transfer across the cell membrane of epithelial cells from rat small intestine.

1. The translocation of monovalent organic anions (pyruvate, propionate, acetate and butyrate) across the cell membrane of isolated epithelial cells from rat small intestine was studied by measuring competitive inhibition kinetics, exchange diffusion and temperature dependence of the efflux rate. A possible function of a monocarboxylate carrier in intestine will be discussed. 2. Earlier studies on the inhibition of pyruvate transport of fatty acids were extended to propionate and found to show the same characteristics. The kinetics, however, appeared to be more complex by the contribution of several diffusion pathways for propionate. 3. The mechanism of countertransport was most compatible with an "accelerated exchange diffusion" and could be studied at both sides of the membrane. This exchange diffusion exhibited saturation kinetics. It is proposed that different monocarboxylate anions may have different affinities for a common carrier. 4. Temperature dependence of the efflux of pyruvate and propionate was studied. Arrhenius plots obtained were not found to be linear between 0 and 5 degrees C. Between 5 and 15 degrees C activation energies for pyruvate and propionate efflux rates were found to be 19.6 and 12.6 kcal/mol, respectively.

Phosphorylation of low molecular weight proteins in purified preparations of rat heart sarcolemma and sarcoplasmic reticulum.

A rat heart sarcolemmal preparation could be obtained in which both 5'-nucleotidase and adenylate cyclase were enriched approx. 9-fold by subjecting a homogenate to a discontinuous sucrose gradient, without the use of a high salt extraction. After incubation of this fraction with Mg[gamma-32P]ATP, the majority of 32P incorporated was present in 24 000- and 9000-dalton protein components. Only when a heart cytosol fraction or a purified cyclic AMP-dependent protein kinase was added, was enhancement of 32P-incorporaton found by addition of cyclic AMP. The 9000- and 24 000-dalton proteins appeared to be interconvertible. The degree of conversion could be affected by changing the temperature during solubilizaion of the membranes in SDS prior to electrophoresis. This suggested that the 24 000-dalton protein does not correspond to phospholamban, first identified by others in canine heart sarcoplasmic reticulum. Moreover, it could be excluded that the 24 000-dalton protein was derived from contaminating myofibrillar troponin I. When the sarcolemmal fraction was preincubated with Ca2+, Mg2+, ATP and oxalate, contaminating sarcoplasmic reticulum vesicles, loaded with calcium oxalate, settled to a greater density in the sucrose gradient. Membrane constituents other than those with enzymatic activity were monitored to confirm the separation between sarcolemmal and sarcoplasmic reticulum membranes: Coomassie blue staining material, sialic acid, cholesterol and phospholipid. The 24 000- and 9000-dalton proteins were equally distributed among the sarolemmal and sarcoplasmic reticulum fractions present in the sucrose gradient. However, the rate of 32P-incorporation in the presence of heart cytosol fraction was much slowr in the sarcoplasmic reticulum than in the sarcolemmal fraction.

Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine.

1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma.

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na+ + K+)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca2+ transport. The Ca2+ pump was stimulated 1.7- and 2.1-fold by external Na+ and K+, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca2+ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca2+ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca2+ in a medium containing 160 mM NaCl. Ca2+ movements were also studied in the absence of ATP and Mg2+. Vesicles containing an outwardly directed Na+ gradient showed the highest Ca2+ uptake activity. These findings suggested the operation of a Ca2+/Na+ antiporter in addition to the active Ca2+ pump in these sarcolemmal vesicles. A valinomycin-induced inward K+-diffusion potential stimulated the Na+-Ca2+ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg2+ the transmembrane Nai+/Nao+ gradient exceeded 160/15 mM concentrations, Ca2+ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na+ gradient-induced Ca2+ uptake to be a translocation of Ca2+ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na+-Ca2+ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.

The effect of lipid intermediates on Ca2+ and Na+ permeability and (Na+ + K+)-ATPase of cardiac sarcolemma. A possible role in myocardial ischemia.

The effect of fatty acid and acylcarnitine on Ca2+ and Na+ transporting enzymes and carriers was studied in sealed cardiac sarcolemma vesicles of mixed polarity. Palmitoylcarnitine markedly reduced the Na+ gradient-induced Ca2+ uptake. Half-maximal reduction was obtained at 15 microM of the carnitine derivative. In a same concentration range palmitoylcarnitine caused a rapid release of accumulated Ca2+ when added to Ca2+-filled vesicles, which suggests that palmitoylcarnitine increases the permeability of the sarcolemma vesicles to Ca2+. A rapid release of Ca2+ was also observed if Ca2+ was taken up by action of the Ca2+ pump. The (Ca2+ + Mg2+)-ATPase, which most likely drives this active Ca2+ uptake, was 90% increased by 50 microM palmitoylcarnitine and evidence was presented that the acylcarnitine effect again was linked to an alteration of Ca2+ permeability of the vesicles. At the same concentration acylcarnitine was not able to unmask the latent protein kinase, so that probably the sarcolemma ATP permeability was not affected. Palmitoylcarnitine at 25 microM did not affect the ouabain-sensitive (Na+ + K+) -ATPase in native sarcolemma vesicles, however, it inhibited markedly if the enzyme was measured in SDS-treated vesicles. The effect of increased free fatty acid concentration on some of the sarcolemma transporting properties was tested by adding oleate-albumin complexes with different molar ratios to the sarcolemma vesicles. In contrast to molar ratios 1 and 5, the ratio of 7 was able to induce a rapid Ca2+ release and to inhibit (Na+ + K+)-ATPase in either native or SDS-treated vesicles markedly. 22Na release from 22Na-preloaded sarcolemma vesicles was shown to be stimulated by either palmitoylcarnitine (50 microM) or oleate-albumin complex (with a molar ratio of 7). The possible significance of the observed effects of lipid intermediates on ion permeability and (Na+ + K+)-ATPase activity in isolated sarcolemma vesicles for the derangement of cardiac cell function in ischemia is discussed.

Properties of 5'-nucleotidase in rat heart sarcolemma.

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.

Protection by acyl-carnitines and phenylmethylsulfonyl fluoride of rat heart subjected to ischemia and reperfusion.

Perfusion of rat hearts according to the Langendorff technique with micromolar concentrations of palmitoylcarnitine or millimolar concentrations of phenylmethylsulfonyl fluoride protect the heart from deterioration by reperfusion after total-ischemia. This is based on the retention of the cytosolic enzymes determined (lactate dehydrogenase, glycogen phosphorylase and glycogen synthase) and of myoglobin, as well as on the resumption of contractile activity. Palmitoylcarnitine, like phenylmethylsulfonyl fluoride, could protect through plasma membrane stabilization, since more hydrophilic compounds had no effect.

Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction: comparison of a constitutive viral versus an endothelin-1-responsive cardiac promoter.

BACKGROUND: A decrease in sarcoplasmic reticulum Ca(2+) pump (SERCA2) activity is believed to play a role in the impairment of diastolic function of the failing heart. Because the expression ratio of phospholamban (PL) to SERCA2 may be a target to improve contractile dysfunction, a PL antisense RNA strategy was developed under the control of either a constitutive cytomegalovirus (CMV) or an inducible atrial natriuretic factor (ANF) promoter. The latter is upregulated in hypertrophied and failing heart, allowing "induction-by-disease" gene therapy. METHODS AND RESULTS: Part of the PL cDNA was cloned in antisense and sense directions into adenovectors under the control of either a CMV (Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs, respectively) promoter. Infection of cultured rat neonatal cardiomyocytes with Ad5CMVPLas reduced PL mRNA to 30+/-7% of baseline and PL protein to 24+/-3% within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca(2+) sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca(2+) transient. A decrease of PL protein was also achieved by infection with Ad5ANFPLas, and the presence of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation of PL. The adenovectors expressing PL sense RNA had no effect on any of the tested parameters. CONCLUSIONS: Vector-mediated PL antisense RNA expression may become a feasible approach to modulate myocyte Ca(2+) homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective for induction-by-disease gene therapy, ie, selective expression of therapeutic genes in hypertrophied and failing cardiomyocytes.

On the possible role of long chain fatty acylcarnitine accumulation in producing functional and calcium permeability changes in membranes during myocardial ischaemia.

A radioisotope procedure was used to determine long chain fatty acylcarnitine concentrations in fractions of porcine myocardium that had been subjected to different periods of ischaemia (0, 1, 2, and 3 h). In myocardial tissue from non-ligated hearts acylcarnitine concentrations were 0.32(0.03) and 1.53(0.04) nmol.mg-1 protein for homogenate and sarcolemma enriched membrane respectively, which indicates a preferential membrane localisation of long chain fatty acylcarnitine. Both the total and membrane acylcarnitine contents were increased about twofold after 2 h of ischaemia. The accumulation of long chain fatty acylcarnitine was not correlated temporally with changes in adenosine triphosphate dependent calcium uptake activity of homogenates (mainly a function of sarcoplasmic reticulum membranes), sodium gradient induced calcium uptake, and calcium permeability of sarcolemma preparations. Homogenate adenosine triphosphate dependent calcium uptake was decreased by 36% after 3 h of ischaemia. Sodium gradient induced calcium uptake was enhanced about twofold after 1 h of ischaemia, and calcium permeability of sarcolemmal vesicles was decreased by 20% after 3 h of ischaemia. After in vitro incubation of isolated sarcolemma membranes with (1-14C)-palmitoylcarnitine radiolabelled molecules that remained associated with the membrane even after repeated washing were incorporated. No changes were observed in the sodium gradient induced calcium uptake when less than 6 nmol (1(-14)C)- palmitoylcarnitine per mg sarcolemma protein were bound or incorporated into the lipid phase. This exceeded the maximal endogenous concentrations of 3.2(0.6) nmol long chain fatty acylcarnitine per mg sarcolemma protein observed during myocardial ischaemia. The results suggest that the intracellular increase in long chain acylcarnitine during almost zero myocardial flow is not critical to sarcolemmal sodium and calcium permeability and sarcoplasmic reticulum calcium pumping activity.

Phospholipid source and molecular species composition of 1,2-diacylglycerol in agonist-stimulated rat cardiomyocytes.

OBJECTIVE: The aim was to investigate the consequences of simultaneous stimulation of phospholipase C and D by agonists for the molecular species composition of 1,2-diacylglycerol and phospholipids in cardiomyocytes. METHODS: Serum-free cultured neonatal rat cardiomyocytes were stimulated by endothelin-1, phenylephrine or phorbolester. The molecular species of 1,2-diacylglycerol (in mol%) and those derived from phosphatidylcholine and phosphatidylinositol were analyzed by high-performance liquid chromatography and their absolute total concentration (nmol per dish) by gas-liquid chromatography. Phospholipids were labelled with [14C]glycerol or double-labelled with [14C]16:0 and [3H]20:4n6 for measurements of respectively, the amount of or relative rate of label incorporation into 1,2-diacylglycerol. RESULTS: The major molecular species of 1,2-diacylglycerol in unstimulated cells was found to be 18:0/20:4 (57 mol%). The same species was observed predominantly in phosphatidylinositol (73 mol% compared to 11 mol% in phosphatidylcholine). A significant decrease (about 10 mol%) was found for the 18:0/20:4 species of 1,2-diacylglycerol during stimulation (10-40 min) with endothelin-1 or phorbolester, but not phenylephrine. The results of the double-labelling experiments were consistent with the latter finding: the ratio [3H]20:4 over [14C]16:0 in 1,2-diacylglycerol decreased from 1.70 in the control to 1.40 during 10-min endothelin-1 or phorbolester stimulation, but not during phenylephrine stimulation. The [14C]glycerol incorporation into 1,2-diacylglycerol remained relatively constant under agonist-stimulated conditions as did the total concentration of 1,2-diacylglycerol. CONCLUSIONS: 1,2-Diacylglycerol present in unstimulated cardiomyocytes is likely derived from phosphatidylinositol. During stimulation with endothelin-1 and phorbolester, but not phenylephrine, phosphatidylcholine becomes an increasingly important source for 1,2-diacylglycerol due to sustained activation of phospholipase D. The 1,2-diacylglycerol level remains relatively constant during agonist stimulation which strongly indicates that particular molecular species of 1,2-diacylglycerol more than its total concentration determine the activation of protein kinase C isoenzymes.

Sarcoplasmic reticulum Ca2+ ATPase promoter activity during endothelin-1 induced hypertrophy of cultured rat cardiomyocytes.

OBJECTIVES: Characterization of an in vitro model of endothelin-1 induced hypertrophy of cultured neonatal rat ventricular myocytes and subsequent analysis of transcription regulation of the rat promoter of the sarcoplasmic reticulum Ca2+ ATPase gene. METHODS: Neonatal rat ventricular myocytes were cultured in serum free medium and hypertrophy was induced by addition of endothelin-1 to 10(-8) M up to 48 h. Hypertrophy was characterized biochemically, and gene expression regulation was evaluated by Northern blotting. A sarcoplasmic reticulum Ca2+ ATPase promoter fragment, isolated from a rat library was cloned in a reporter vector. Promoter activity during hypertrophy was assessed after transfection of the reporter plasmid to cultured cardiomyocytes. RESULTS: Stimulation with endothelin-1 resulted in increased cell size, as indicated by protein/DNA ratio as well as by augmented protein synthesis. When compared to angiotensin II or alpha 1-adrenergic agonist, endothelin-1 was the strongest inducer of hypertrophy (protein/DNA ratio) after 48 h of stimulation. Endothelin-1 induced hypertrophy was accompanied by a twofold increase in total RNA content per cell as well as to increased glyceraldehydephosphate dehydrogenase mRNA levels. The level of atrial natriuretic factor mRNA was increased more than twofold, relative to glyceraldehydephosphate dehydrogenase, while the expression of the sarcoplasmic reticulum Ca2+ pump and phospholamban genes was decreased (by 26 and 49%, respectively) after induction of hypertrophy by stimulation with endothelin-1. In the same model, a 1.9 kb sarcoplasmic reticulum Ca2+ pump gene promoter fragment (including 0.4 kb of the 5' UTR of the mRNA) directed down-regulation of the expression of the reporter gene to the same magnitude as endogenous Ca2+ pump mRNA relative to glyceraldehydephosphate dehydrogenase mRNA. However, absolute mRNA level per cell did not change for either the reporter gene or the endogenous Ca2+ pump. CONCLUSIONS: Endothelin-1 can induce phenotypic changes in cultured rat ventricular myocytes that are reminiscent of hypertrophy in vivo. In this model, a 1.9 kb sarcoplasmic reticulum Ca2+ pump promoter fragment directed gene expression of a reporter gene identical to the endogenous regulation of the Ca2+ pump. Furthermore, expression of the Ca2+ pump during hypertrophy was only downregulated when compared to (increased levels of) glyceraldehydephosphate dehydrogenase mRNA, but absolute Ca2+ ATPase mRNA amounts remained unchanged. This suggests that the Ca2+ pump promoter is not responding to the increase in transcriptional activity that accompanies hypertrophy.

Alterations in polyunsaturated fatty acid composition of cardiac membrane phospholipids and alpha 1 adrenoceptor mediated phosphatidylinositol turnover.

STUDY OBJECTIVE - The aim of the study was to investigate the steps at which polyunsaturated fatty acids are involved in alpha 1 adrenoceptor mediated phosphatidylinositol turnover. DESIGN - Phosphatidylinositol turnover rates were investigated after preincubating neonatal rat ventricular myocytes with culture media enriched with linoleic acid (18:2n-6) or eicosapentaenoic acid (20:5n-3) to change the polyunsaturated fatty acid composition of their membrane phospholipids. EXPERIMENTAL MATERIAL - Cardiomyocytes were isolated from ventricles of 2-4 d old Wistar rats by trypsinization and were then cultured. Experiments were started 48 h after seeding, when there was a confluent monolayer of beating cardiomyocytes. MEASUREMENTS and RESULTS - In 18:2n-6 treated cells the 18:2n-6 content in the total phospholipid fraction rose from 45 to 68 nmol.mg-1 protein; in 20:5n-6 treated cells the 20:5n-3 content rose from 1.5 to 12.5 nmol.mg-1 protein, and the docosapentaenoic acid (22:5n-3) content rose from 5.1 to 14.7 nmol.mg-1 protein. The major n-3 fatty acid, 22:6n-3 (11.4 nmol.mg-1 protein), did not change after 20:5n-3 treatment. Although the phosphatidylinositol fraction showed changes paralleling those in the total phospholipids, none were significant. In this fraction the major n-3 fatty acid appeared to be 22:5n-3 (0.4 nmol.mg-1 protein). The fatty acid treated cells were prelabelled with [3H]-inositol to estimate the rate of phosphatidylinositol-4,5-bisphosphate turnover. There were no differences in the rate of [3H]-inositolphosphate formation between control, 18:2n-6 treated cells, and 20:5n-3 treated cells. Prolonged alpha 1 adrenergic stimulation of control and treated cells did not change the polyunsaturated fatty acid composition of the total phospholipid and phosphatidylinositol fractions. CONCLUSIONS - The alpha 1 adrenoceptor mediated phosphatidylinositol turnover rate is not affected by changes in polyunsaturated fatty acid composition of membrane phospholipids, neither does prolonged alpha 1 adrenergic stimulation lead to significant depletion of any specific or total polyunsaturated fatty acids in the phosphatidylinositol lipids.

Increased activity of the sarcoplasmic reticular calcium pump in porcine stunned myocardium.

OBJECTIVE: The aim was to determine whether changes in sarcoplasmic reticular Ca2+ transport activity and the degree of phosphorylation of phospholamban of "stunned" myocardium are involved in the reversible depression of contractile function. METHODS: In anaesthetised open chest swine, stunning was induced by subjecting the myocardium perfused by the left anterior descending coronary artery to two cycles of 10 min of occlusion and 30 min of reperfusion. Before and after stunning, systemic haemodynamic variables and regional myocardial function and perfusion were determined, while biopsies were taken for determination of the content of high energy phosphate compounds. Sarcoplasmic reticular function (ATP dependent Ca2+ transport and phosphorylation of phospholamban) of the stunned and control myocardium was determined at the end of the stunning protocol. RESULTS: In the stunned myocardium the segment length shortening decreased from 17.4(SD 4.0)% to 3.5(4.4)%, while perfusion was 38% less than at baseline. ATP and total adenine nucleotide levels of the stunned myocardium were about 35% lower than in the control myocardium, but the energy charge was normal as creatine phosphate levels had increased by 66% over the content determined at baseline. Ca2+ uptake by the sarcoplasmic reticulum isolated from the stunned region was 17% (p < 0.05) higher than Ca2+ uptake from the control region [1240(303) and 1450(280) nmol.min-1.mg-1 protein, respectively]. In the presence of exogenous cyclic AMP dependent protein kinase the amount of 32P incorporated into phospholamban was similar for both myocardial regions. CONCLUSIONS: In this model of stunned porcine myocardium, the phosphorylation state of phospholamban was unchanged, but Ca2+ uptake by the sarcoplasmic reticulum was slightly increased. The results indicate that a change in active Ca2+ transport by the sarcoplasmic reticulum is most likely not to be the principal cause of contractile dysfunction of stunned myocardium.

In vitro cyclic AMP induced phosphorylation of phospholamban: an early marker of long-term recovery of function following reperfusion of ischaemic myocardium?

Changes in myocardial membrane biochemistry and ultrastructure, determined shortly (2 h) after reperfusion of ischaemic myocardium, were compared with the long term (4 wk) recovery of regional myocardial function. Anaesthetised pigs were subjected to 30 min (n = 14, group I) or 60 min (n = 14, group II) of left circumflex coronary artery occlusion. Seven animals of each group were studied 2 h and the others 4 weeks after flow was reinstated. After 2 h of reperfusion, regional myocardial function was absent in both groups. At 4 weeks regional function had returned to normal in group I, but was still significantly depressed in group II. Biochemical studies after 2 h of reperfusion showed that a functional index of the cardiac membrane, the in vitro cyclic AMP dependent 32P incorporation into phospholamban, was 71 (SEM 9)% compared to non-ischaemic myocardium in group I and 31 (6)% in group II (p less than 0.05). After 4 weeks this index had completely recovered in group I, 114 (13)%, but a significant decrease to 79 (2)% could still be observed in group II (p less than 0.05). After 2 h of reperfusion as well as after 4 weeks of recovery the myocytes in group II were more severely damaged than in group I. This study suggests that determination of in vitro phosphorylation of phospholamban shortly after reperfusion of ischaemic myocardium may be of value in the prediction of long term recovery of regional myocardial function.

Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen: evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II.

OBJECTIVE: The hypertrophic response of cardiomyocytes exposed to mechanical stretch is assumed to depend on the release of angiotensin (Ang) II from these cells. Here we studied the synthesis of renin-angiotensin system (RAS) components by cardiac cells under basal conditions and after stretch. METHODS: Myocytes and fibroblasts were isolated by enzymatic dissociation from hearts of 1-3-day-old Wistar rat strain pups, grown for 1 day in serum-supplemented medium and then cultured in a chemically defined, serum-free medium. Medium and cell lysate were collected 5 days later or after exposure of the cells to cyclic stretch for 24 h. Prorenin, renin and angiotensinogen were measured by enzyme-kinetic assay; Ang I and Ang II were measured by radioimmunoassay after SepPak extraction and HPLC separation. RESULTS: Prorenin, but none of the other RAS components, could be detected in the medium of both cell types. However, its levels were low and the Ang I-generating activity corresponding with these low prorenin levels could not be inhibited by the specific rat renin inhibitor CH-732, suggesting that it was most likely due to bovine and/or horse prorenin sequestered from the serum-containing medium to which the cells had been exposed prior to the serum-free period. When incubated with Ang I, both myocytes and fibroblasts generated Ang II in a captopril-inhibitable manner. Myocyte and fibroblast cell lysates did not contain prorenin, renin, angiotensinogen, Ang I or Ang II in detectable quantities. Stretch increased myocyte protein synthesis by 20%, but was not accompanied by Ang II release into the medium. CONCLUSION: Cardiac myocytes and fibroblasts do not synthesize renin, prorenin or angiotensinogen in concentrations that are detectable or, it not detectable, high enough to result in Ang II concentrations of physiological relevance. These cells do synthesize ACE, thereby allowing the synthesis of Ang II at cardiac tissue sites when renin and angiotensinogen are provided via the circulation. Ang II is not a prerequisite to observe a hypertrophic response of cardiomyocytes following stretch.

Oxygen wastage of stunned myocardium in vivo is due to an increased oxygen cost of contractility and a decreased myofibrillar efficiency.

OBJECTIVE: We investigated whether an increased oxygen cost of contractility and/or a decreased myofibrillar efficiency contribute to oxygen wastage of stunned myocardium. Because Ca(2+)-sensitizers may increase myofibrillar Ca(2+)-sensitivity without increasing cross-bridge cycling, we also investigated whether EMD 60263 restores myofibrillar efficiency and/or the oxygen cost of contractility. METHODS: Regional fiber stress and strain were calculated from mesomyocardially implanted ultrasound crystals and left ventricular pressure in anesthetized pigs (n=18). Regional myocardial oxygen consumption (MVO(2)) was measured before contractility (end-systolic elastance, E(es)) and total myofibrillar work (stress-strain area, SSA) were determined from stress-strain relationships. Atrial pacing at three heart rates and two doses of dobutamine were used to vary SSA and E(es), respectively. After stunning (two times 10-min ischemia followed by 30-min reperfusion), measurements were repeated following infusion of saline (n=8) or EMD 60263 (1.5 mg.kg(-1) i.v., n=10). Linear regression was performed using: MVO(2)=alpha.SSA+beta.E(es)+gamma.HR(-1) (alpha(-1), myofibrillar efficiency; beta, oxygen cost of contractility; and gamma, basal metabolism/min). RESULTS: Stunning decreased SSA by 57% and E(es) by 64%, without affecting MVO(2), while increasing alpha by 71% and beta by 134%, without affecting gamma. From the wasted oxygen, 72% was used for myofibrillar work and 18% for excitation-contraction coupling. EMD 60263 restored both alpha and beta. CONCLUSIONS: Oxygen wastage in stunning is predominantly caused by a decreased myofibrillar efficiency and to a lesser extent by an increased oxygen cost of contractility. Considering that EMD 60263 reversed both causes of oxygen wastage, it is most likely that this drug increases myofibrillar Ca(2+)-sensitivity without increasing myofibrillar cross-bridge cycling.

Inhibition of Ca2+-dependent protein kinase and Ca2+/Mg2+-ATPase in cardiac sarcolemma by the anti-calmodulin drug calmidazolium.

Sarcolemma (SL) vesicles, isolated from pig heart, contain both a Ca2+-calmodulin-dependent protein kinase (CaM-PK) and a Ca2+-dependent Mg2+-ATPase (Ca2+/Mg2+)-ATPase). Some of their properties have been compared: their affinity for Ca2+ ions, dependence on exogenous calmodulin (CaM) and sensitivity to the anti-CaM drug calmidazolium (R24571). The properties of Ca2+-CaM-dependent brain phosphodiesterase (PDE) have also been examined. R24571 appeared to be the most potent inhibitor from brain PDE. For the three enzymes studied, exogenously added CaM was able to antagonize the R24571 inhibition, although the efficiency to counteract was rather low in the case of the SL Ca2+/Mg2+-ATPase. R24571 decreased the affinity of the Ca2+/Mg2+-ATPase for Ca2+ ions (KCa 0.35 versus 0.75 microM) and exerted an inhibition non-competitive with Ca2+ ions on the other CaM-dependent enzymes. Membrane-bound CaM, which is involved in controlling the Ca2+/Mg2+-ATPase, appeared to be present in a stoichiometry varying from 1:1 to 1:4 compared to the 32P-intermediate of the ATPase. R24571 treatment of SL vesicles selectively solubilized a number of proteins in the molecular range of 13-20 kD, which may include CaM. The results suggest that different mechanisms are involved in the CaM control of the two SL enzymes studied.

Alpha-adrenergic agonist and endothelin-1 induced intracellular Ca2+ response in the presence of a Ca2+ entry blocker in cultured rat ventricular myocytes.

Previously we demonstrated that stimulation of cultured neonatal rat ventricular myocytes by either alpha 1-adrenergic agonist or endothelin-1 resulted in a rapid formation of total inositolphosphates, although the levels of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate did not rise significantly. The aim of this study was to examine whether stimulation by alpha 1-adrenergic agonist and endothelin-1 could still elicit phosphatidylinositol cycle mediated intracellular Ca2+ mobilization in these cells. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by single cell imaging dual wavelength fluorescence microscopy in Fura-2-loaded cardiomyocytes. The interference of agonist induced [Ca2+]i responses by the beat to beat variation of [Ca2+]i was prevented by arresting the cells with the Ca2+ entry blocker diltiazem (10 microM). The [Ca2+]i response (expressed as % of baseline ratio of fluorescence intensities of Fura-2 at 340 nm and 380 nm excitation wavelength), induced by phenylephrine (10(-4) M) and endothelin-1 (10(-8) M) was small, up to 20% of baseline after 9-20 min. In contrast, Ca(2+)-influx induced by incubation in Na(+)-free buffer caused a steep increase of [Ca2+]i up to 150% of baseline after 30 s. Analysis of single cells following stimulation with phenylephrine or endothelin-1 showed heterogeneity with respect to a rise in [Ca2+]i. However, if rapid Ca(2+)-influx was induced by incubation in Na(+)-free buffer, [Ca2+]i responses in individual myocytes occurred homogeneously. It is concluded that the alpha 1-adrenergic agonist and endothelin-1 induced [Ca2+]i responses are delayed in time, small and quite heterogeneous among cells. The findings are in agreement with earlier observations which revealed no detectable overall increase of the Ca2+ releasing inositolphosphates under these conditions and suggest that other second messengers, such as 1,2-diacylglycerol, are involved in the agonist mediated Ca2+ signals.

Uptake of thyroid hormones in neonatal rat cardiac myocytes.

The uptake and metabolism of T3 and T4 were investigated in cardiomyocytes isolated from 2-day-old rats. Myocytes (2-5 x 10(5) cells/well) were cultured for 1 day in medium with 5% horse serum-5% FCS and subsequently for 4 days without serum; in some cases myocytes were cultured with serum throughout the culture period. Experiments were performed at 37 C in medium with 0.5% BSA for measurement of [125I]T3 (200,000 cpm; 200 pM) uptake and with 0.1% BSA for measurement of [125I]T4 (200,000 cpm; 350 pM) uptake. Uptake of [125I]T3, expressed as femtomoles per picomolar concentration of free hormone, with any incubation time between 15 min and 24 h was at least 2-fold higher than that of [125I]T4. Neither T3 nor T4 was deiodinated within 24 h. This was observed in cells cultured in the absence or presence of serum. After 15 min of incubation, [125I]T3 uptake was 0.048 +/- 0.002 fmol/pM free T3 (n = 9), and [125I]T4 uptake was 0.018 +/- 0.003 fmol/pM free T4 (n = 9). Although [125I]T3 uptake was reduced by 31-40% (P < 0.05) by coincubation with 100 nM to 10 microM unlabeled T3, that of [125I]T4 was not affected by 1 nM to 10 microM unlabeled T4, nor was [125I]T3 uptake reduced by 10 microM unlabeled T4. Preincubation (30 min) and incubation (15 min) with 10 microM oligomycin reduced cellular ATP by 56% (P < 0.05) and [125I]T3 uptake by 73% (P < 0.05), but had no effect on [125I]T4 uptake. Similarly, [125I]T3 uptake, but not [125I]T4 uptake, was dependent on temperature and partly dependent on the Na+ gradient, as shown by the inhibitory effect of 10 microM monensin (27%; P < 0.05). The effect of aromatic amino acids (2 mM) on [125I]T3 uptake increased in the order phenylalanine < tyrosine < tryptophan. It is concluded that T3 is taken up in neonatal cardiomyocytes by an energy-dependent carrier-mediated mechanism that is also partly dependent on the Na+ gradient. Such a transport mechanism for T4 is not present in the neonatal heart, but it may appear later during development.