Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins.

OBJECTIVE: The alterations in contractile proteins underlying enhanced Ca(2+)-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca(2+)-responsiveness in human heart failure. METHODS: Isometric force and its Ca(2+)-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca(2+)-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. RESULTS: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca(2+)-sensitivity of the contractile apparatus (pCa(50)) was significantly higher in failing myocardium (deltapCa(50)=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca(2+)-responsiveness. An inverse correlation was found between pCa(50) and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca(2+)-sensitivity to a larger extent in failing (deltapCa(50)=0.20) than in donor (deltapCa(50)=0.03) myocytes, abolishing the difference in Ca(2+)-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca(2+)-responsiveness. CONCLUSIONS: The increased Ca(2+)-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI.

Calcium sensitivity of force in human ventricular cardiomyocytes from donor and failing hearts.

In failing human myocardium changes occur, in particular, in isoform composition and phosphorylation level of the troponin T (TnT) and troponin I (TnI) subunits of the actin filament and the myosin light chains (MLC-1 and -2), but it is unclear to what extent they influence cardiac performance. This overview concentrates on the relation between contractile function, contractile protein composition and phosphorylation levels in small biopsies from control (donor) hearts, from biopsies obtained during open heart surgery (NYHA Class I-IV) and from end-stage failing (explanted, NYHA class IV) hearts. Furthermore, attention is paid to the effect of the catalytic subunit of protein kinase A on isometric force development in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at different resting sarcomere lengths. A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force by approximately 35% both in donor and in failing cardiomyocytes. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50 = 0.22). Our findings indicate that 1) TnI phosphorylation contributes to the differences in calcium sensitivity between donor and end-stage failing hearts, 2) human ventricular myocardium is heterogeneous with respect of the phosphorylation of TnT, MLC-2 and the isoform distribution of MLC-1 and MLC-2, and 3) the Frank-Starling mechanism is preserved in end-stage failing myocardium.

Expression of RGS3, RGS4 and Gi alpha 2 in acutely failing donor hearts and end-stage heart failure.

BACKGROUND: Regulators of G-protein Signalling (RGS) proteins have been shown to limit in vitro signalling of G proteins. In common with end-stage heart failure, we have recently shown that upregulation of the inhibitory G-protein, Gialpha, occurs in acutely failing donor hearts unused for transplantation due to severe myocardial dysfunction. In light of recent data on RGS proteins, we have evaluated mRNA and protein expression of RGS3, RGS4 and Gialpha2 in the myocardium from normal, end-stage failing and acutely failing unused donor hearts. METHODS AND RESULTS: Myocardial samples were obtained from end-stage failing hearts explanted prior to transplantation (n=19), unused donor hearts with ejection fractions < 30% (n=14) and used donor hearts with good function (ejection fraction > 60%) (n=4-7). mRNA levels were quantified using quantitative reverse transcriptase polymerase chain reaction. Levels of RGS3 and RGS4 mRNA were found to be significantly upregulated in unused donor and end-stage failing myocardium (P < 0.05 and 0.01, and P < 0.05 and 0.02, respectively) compared to non-failing hearts. Protein abundance of RGS3 and RGS4 was found to be higher in myocardium from end-stage failing hearts, and relative RGS4 expression higher in unused donor hearts. CONCLUSIONS: We show here that RGS3 and RGS4 mRNA and protein expression is upregulated in human heart failure. These observations suggest that RGS4 may be induced in the heart to regulate cell signalling pathways in response to hypertrophy, and support the existence of a negative feedback loop for the long-term regulation of hypertrophy.

Vascular endothelial growth factor release following coronary artery bypass surgery: extracorporeal circulation versus 'beating heart' surgery.

AIMS: The aim of this study was to examine the circulating levels of vascular endothelial growth factor, following coronary artery bypass graft surgery performed using both standard cardiopulmonary bypass or the 'octopus technique' on the beating heart. BACKGROUND: Vascular endothelial growth factor has a number of effects that are beneficial in the setting of coronary artery bypass graft surgery including cardioprotection, potent angiogenic activity and amelioration of intimal hyperplasia. Hypoxia is a powerful stimulator of vascular endothelial growth factor expression yet the ability of ischaemia, occurring during coronary artery bypass graft surgery, to induce vascular endothelial growth factor production is unknown. METHODS AND RESULTS: Serum vascular endothelial growth factor levels were determined in patients undergoing coronary artery bypass graft surgery with standard cardiopulmonary bypass (CPB-CABG group; n=20), with off-pump coronary artery bypass; (OP-CABG; n=12) and in patients undergoing non-cardiac major surgery (n=6). The effect of hypoxia on vascular endothelial growth factor release by neonatal rat cardiac myocytes in vitro was studied. In the CPB-CABG group vascular endothelial growth factor levels were significantly increased to 78.5+/-39.3 and 110.5+/-16.3 pg. microl(-1)8 and 24 h post-operatively, declining to 14.9+/-9.9 pg. microl(-1)by 48 h to pre-operative values (14.4+/-8.6 pg. microl(-1)). Significantly higher vascular endothelial growth factor levels were also present in the OP-CABG group 3, 6 and 24 h post-operatively (levels 136. 6+/-29.3, 143+/-26.12 pg. microl(-1)and 93.5+/-20.1 pg. microl(-1), respectively). However, non-cardiac major surgery did not result in elevated vascular endothelial growth factor levels post-operatively (46.36+/-9.76 vs pre-surgery levels of 26.84+/-6.1 pg. microl(-1)). Either 15 min or 3 h of hypoxia stimulated vascular endothelial growth factor release from neonatal rat cardiac myocytes in vitro. Twenty-four and 48 h post hypoxia, levels of vascular endothelial growth factor were significantly elevated by approximately 17.5- and 48.5-fold respectively. CONCLUSIONS: These data demonstrate myocardial ischaemia secondary to CPB-CABG and OP-CABG to be a potent stimulator of vascular endothelial growth factor production, which may have implications for graft endothelialization and cardiovascular haemodynamics post-operatively.

Tumor necrosis factor-alpha is expressed in donor heart and predicts right ventricular failure after human heart transplantation.

BACKGROUND-Myocardial failure is an important problem after heart transplantation. Right ventricular (RV) failure is most common, although its mechanisms remain poorly understood. Inflammatory cytokines play an important role in heart failure. We studied the expression of tumor necrosis factor (TNF)-alpha and other cytokines in donor myocardium and their relationship to the subsequent development of RV failure early after transplantation. METHODS AND RESULTS-Clinical details were obtained, and ventricular function was assessed by transesophageal echocardiography in 26 donors before heart retrieval. A donor RV biopsy was obtained immediately before transplantation, and each recipient was followed for the development of RV failure. Reverse transcriptase-polymerase chain reaction was performed to detect TNF-alpha, interleukin-2, interferon-gamma, and inducible nitric oxide synthase expression. Eight of 26 recipients (30.8%) developed RV failure. Seven of these 8 (87.5%) expressed TNF-alpha, but only 4 of the 18 (22.2%) who did not develop RV failure expressed TNF-alpha (P<0.005). As a predictor of RV failure, TNF-alpha mRNA had a sensitivity of 87.5%, a specificity of 83.3%, a positive predictive value of 70%, and a negative predictive value of 93.7%. Western blotting demonstrated more TNF-alpha protein in the myocardium of donor hearts that developed RV failure (658+/-60 versus 470+/-57 optical density units, P<0.05). Immunocytochemistry localized TNF-alpha expression to cardiac myocytes. Reverse transcriptase-polymerase chain reaction detected interferon-gamma in 2 (7.7%), interleukin-2 in 1 (3.8%), and inducible nitric oxide synthase mRNA in 1 (3.8%) of the 26 donor hearts, none of which developed RV failure. CONCLUSIONS-TNF-alpha expression in donor heart cardiac myocytes seems to predict the development of RV failure in patients early after heart transplantation.

Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes.

OBJECTIVE: We investigated whether the Frank-Starling mechanism is absent or preserved in end-stage failing human myocardium and if phosphorylation of contractile proteins modulates its magnitude through the sarcomere length-dependence of calcium sensitivity of isometric force development. METHODS: The effect of phosphorylation of troponin I and C-protein by the catalytic subunit of protein kinase A (3 microg/ml; 40 min at 20 degrees C) was studied in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at sarcomere lengths measured at rest of 1.8, 2.0 and 2.2 microm. Isometric force development was studied at various free-calcium concentrations before and after protein kinase A incubation at 15 degrees C (pH 7.1). RESULTS: Maximal isometric tension at 2.2 microm amounted to 39.6+/-10.4 and 33.7+/-3.5 kN/m2 in donor and end-stage failing cardiomyocytes, respectively. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50=0.22). A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force to 64% and 65% and in pCa50 by 0.10 and 0.08 pCa units in donor and failing cardiomyocytes, respectively. In donor tissue, the effect of protein kinase A treatment was rather small, while in end-stage failing myocardium it was much larger (delta pCa50=0.24) irrespective of sarcomere length. CONCLUSIONS: The data obtained indicate that the Frank-Starling mechanism is preserved in end-stage failing myocardium and suggest that sarcomere length dependence of calcium sensitivity and the effects of phosphorylation of troponin I and C-protein are independent.

Myocardial dysfunction in donor hearts. A possible etiology.

BACKGROUND: Potential cardiac donors show various degrees of myocardial dysfunction, and the most severely affected hearts are unsuitable for transplantation. The cause of this acute heart failure is poorly understood. We investigated whether alterations in calcium-handling proteins, beta-adrenoceptor density, or the inhibitory G protein Gialpha could account for this phenomenon in unused donor hearts (n=4 to 8). We compared these with end-stage failing hearts (n=14 to 16) and nonfailing hearts (n=3 to 12). METHODS AND RESULTS: Myocardial samples were obtained from unused donor hearts displaying ejection fractions <30%. Both trabeculae and isolated myocytes responded as poorly as those from the group of failing hearts to increasing stimulation frequency with regard to inotropic function in vitro. Immunodetectable abundance of sarcoplasmic reticulum calcium-ATPase and sodium calcium exchanger were greater (177%; P<0.01) and smaller (29%; P<0.01), respectively, in the unused donor hearts relative to the failing group, which suggests that alterations of these proteins are not a common cause of contractile dysfunction in the 2 groups. Myocytes from the unused donor group were desensitized to isoprenaline to a similar degree as those from the failing heart group. However, beta-adrenoceptor density was reduced in the failing (P<0.001) but not in the unused donor heart group (P=0.37) relative to the nonfailing heart group (n=5). Gialpha activity was increased in samples from unused donor and failing hearts relative to nonfailing hearts (P<0.05). CONCLUSIONS: Increased activity of the inhibitory G protein Gialpha is a significant contributory factor for impaired contractility in these acutely failing donor hearts.

Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.

Relationship between depolarization-induced force responses and Ca2+ content in skeletal muscle fibres of rat and toad.

1. The relationship between the total Ca2+ content of a muscle fibre and the magnitude of the force response to depolarization was examined in mechanically skinned fibres from the iliofibularis muscle of the toad and the extensor digitorum longus muscle of the rat. The response to depolarization in each skinned fibre was assessed either at the endogenous level of Ca2+ content or after depleting the fibre of Ca2+ to some degree. Ca2+ content was determined by a fibre lysing technique. 2. In both muscle types, the total Ca2+ content could be reduced from the endogenous level of approximately 1.3 mmol l-1 (expressed relative to intact fibre volume) to approximately 0.25 mmol l-1 by either depolarization or caffeine application in the presence of Ca2+ chelators, showing that the great majority of the Ca2+ was stored in the sarcoplasmic reticulum (SR). Chelation of Ca2+ in the transverse tubular (T-) system, either by exposure of fibres to EGTA before skinning or by permeabilizing the T-system with saponin after skinning, reduced the lower limit of Ca2+ content to < or = 0.12 mmol l-1, indicating that 10-20% of the total fibre Ca2+ resided in the T-system. 3. In toad fibres, both the peak and the area (i.e. time integral) of the force response to depolarization were reduced by any reduction in SR Ca2+ content, with both decreasing to zero in an approximately linear manner as the SR Ca2+ content was reduced to < 15% of the endogenous level. In rat fibres, the peak size of the force response was less affected by small decreases in SR content, but both the peak and area of the response decreased to zero with greater depletion. In partially depleted toad fibres, inhibition of SR Ca2+ uptake potentiated the force response to depolarization almost 2-fold. 4. The results show that in this skinned fibre preparation: (a) T-system depolarization and caffeine application can each virtually fully deplete the SR of Ca2+, irrespective of any putative inhibitory effect of SR depletion on channel activation; (b) all of the endogenous level of SR Ca2+ must be released in order to produce a maximal response to depolarization; and (c) a substantial part (approximately 40%) of the Ca2+ released by a depolarization is normally taken back into the SR before it can contribute to force production.

Reduced Mg2+ inhibition of Ca2+ release in muscle fibers of pigs susceptible to malignant hyperthermia.

The inhibitory effect of myoplasmic Mg2+ on Ca2+ release from the sarcoplasmic reticulum (SR) was examined in mechanically skinned skeletal muscle fibers from pigs of different ryanodine-receptor (RyR) genotypes. In fibers from pigs homozygous for the normal RyR allele, the free Mg2+ concentration ([Mg2+]) had to be lowered from the normal resting level of 1 to approximately 0.1 mM to induce Ca2+ release and a force response. Fibers from pigs heterozygous or homozygous for the RyR allele associated with malignant hyperthermia (MH) needed only a smaller reduction in free [Mg2+] to induce Ca2+ release (reduction to 0.1-0.2 and > or = 0.2 mM, respectively). Dantrolene (20 microM) counteracted the effect of this reduced Mg2+ inhibition in MH muscle. The response of muscle fiber bundles to the caffeine-halothane contracture test in the three genotypes correlated well with the responsiveness of single fibers to reduced [Mg2+]. Thus the abnormal responsiveness of MH muscle to various stimuli may largely result from the reduced ability of myoplasmic Mg2+ to inhibit Ca2+ release from the SR.

Effect of low [ATP] on depolarization-induced Ca2+ release in skeletal muscle fibres of the toad.

1. The effect of low [ATP] on depolarization-induced Ca2+ release from the sarcoplasmic reticulum (SR) was examined using mechanically skinned skeletal muscle fibres of the toad. The amount of Ca2+ released was determined by examining the extent of SR Ca2+ depletion following a single depolarization in the presence of specified total [ATP] and free [Mg2+] and 1 mM EGTA (< 10 nM Ca2+). 2. Reducing the total [ATP] from 8 to 2 mM did not significantly affect the extent of depolarization-induced Ca2+ release. When the total [ATP] was lowered to 0.5 mM, depolarization-induced Ca2+ release was markedly reduced. The additional presence of 1.5 mM AMP did not reverse this effect of low [ATP]. 3. At each total [ATP], elevation of the free [Mg2+] from the normal resting level of 1 mM to 3 mM, caused a similar reduction in depolarization-induced Ca2+ release. This effect was not due to the concomitant decrease in free [ATP] and occurred independently of the reduction in Ca2+ release observed at very low total [ATP]. 4. These results show myoplasmic [ATP] modulates depolarization-induced Ca2+ release. Thus, if the total [ATP] near the Ca2+ release channels becomes locally depleted in severely fatigued muscle fibres, it may contribute, in combination with an accompanying increase in free [Mg2+], to the reported reduction in Ca2+ release.

Effects of creatine phosphate and P(i) on Ca2+ movements and tension development in rat skinned skeletal muscle fibres.

1. Mechanically skinned fast-twitch (FT) and slow-twitch (ST) muscle fibres of the rat were used to investigate the effects of fatigue-like changes in creatine phosphate (CP) and inorganic phosphate (P(i)) concentration on Ca(2+)-activation properties of the myofilaments as well as Ca2+ movements into and out of the sarcoplasmic reticulum (SR). 2. Decreasing CP from 50 mM to zero in FT fibres increased maximum Ca(2+)-activated tension (Tmax) by 16 +/- 2% and shifted the mid-point of the tension-pCa relation (pCa50) to the left by 0.28 +/- 0.03 pCa units. In ST fibres, a decrease of CP from 25 mM to zero increased Tmax by 9 +/- 1% and increased the pCa50 by 0.16 +/- 0.01 pCa units. The effect of CP on Tmax was suppressed in both fibre types by prior treatment with 0.3 mM FDNB (1-fluoro-2,4-dinitrobenzene), suggesting that these effects may occur via changes in creatine kinase activity. 3. Increases of P(i) in the range 0-50 mM reduced the pCa50 and Tmax in both fibre types. These effects were more pronounced in ST fibres than in FT fibres in absolute terms. However, normalization of the results to resting P(i) levels appropriate to both fibre types (1 mM for FT and 5 mM for ST fibres) revealed similar decreases in Tmax (approximately 39% at 25 mM P(i) and approximately 48% at 50 mM P(i)) and pCa50 (0.25 pCa units at 25-50 mM P(i)). The depressant action of P(i) on both parameters was considerably reduced when the rise in P(i) was accompanied by an equivalent reduction in [CP]. 4. Tension development in the presence of complex, fatigue-like milieu changes (40 mM P(i) for FT; 20 mM P(i) for ST) was decreased by 35-40% at a constant myoplasmic [Ca2+] of 6 microM in both fibre types. 5. SR Ca2+ loading at a myoplasmic [Ca2+] of 100 nM was found to increase abruptly when the [P(i)] during loading was increased to near 9 mM. At a myoplasmic [Ca2+] of 300 nM, the threshold P(i) for this effect dropped to approximately 3 mM. 6. Tension responses evoked by caffeine in the absence of P(i) were smaller and slower to peak if fibres were exposed to P(i) in a restricted myoplasmic Ca2+ pool after SR Ca2+ loading. This indicated that myoplasmic P(i) can decrease and prolong the rate of Ca2+ release from the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Amniotic fluid tests for fetal maturity in normal and abnormal pregnancies.

Amniotic fluid creatinine, percentage of lipid-positive cells, and L/S ratio were determined on 285 samples from normal pregnancies and 222 samples from abnormal pregnancy states (Rh isoimmunization, diabetes, hypertensive disorders, intrauterine growth retardation, and hydramnios). In normal pregnancy the coefficient of correlation between true gestational age and estimated period of gestation (EPG) based on the three parameters was 0.94, in Rh isoimmunization 0.77, in diabetes 0.67, and in hypertensive disorders 0.59. In intrauterine growth retardation both the L/S ratio and creatinine were depressed, the coefficient was 0.61, and the EPG was consistently less than the true gestational age. The mean L/S ratio in pre-eclampsia was slightly below the normal mean and in diabetes the mean L/S ratio was also depressed. In 150 samples taken within 48 hours of delivery L/S ratios were accurate in assessing fetal pulmonary maturity although there was a 20 per cent incidence over all of false-immature values. There were no false-mature values except in diabetes (2/9).