Early adjustments in mitochondrial structure and function in skeletal muscle to high altitude: design and rationale of the first study from the Kilimanjaro Biobank.

The physiological acclimatisation and adaptation processes in skeletal muscle at high altitude are of high medical and social relevance not only to understand limitations in physical performance at high altitude but also to understand the consequences of hypoxemia and tissue hypoxia in critically ill patients. Of particular importance in these processes are the alterations in content and function of mitochondria and myoglobin. The majority of studies on oxygen delivery to the tissues and utilisation by the cellular metabolism at high altitude were performed after prolonged stay at high altitude and in altitude-adapted highlanders. However, these studies do not provide insight in the sequence of events during the physiological acclimatisation and adaptation processes. Therefore, it is important to identify the early alterations in structure and function of the major determinants of the oxygen transport via myoglobin and oxygen utilisation by the mitochondria in skeletal muscle at high altitude. To achieve this goal, it is of interest to collect, analyse and compare quadriceps muscle biopsies and venous blood samples of climbers, guides and porters before and after climbing Mount Kilimanjaro and in participants of the Kilimanjaro Marathon before and after the run. The samples will be carefully documented and stored in the Kilimanjaro Biobank and will be made available to other research groups.

Correction to: The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease.

In the original version of this article, the name of one of the authors is not correct. The correct name should be W. A. Linke, which is shown correctly in the authorgroup section above.

The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease.

The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.

Pathomechanisms in heart failure: the contractile connection.

Heart failure is a multi-factorial progressive disease in which eventually the contractile performance of the heart is insufficient to meet the demands of the body, even at rest. A distinction can be made on the basis of the cause of the disease in genetic and acquired heart failure and at the functional level between systolic and diastolic heart failure. Here the basic determinants of contractile function of myocardial cells will be reviewed and an attempt will be made to elucidate their role in the development of heart failure. The following topics are addressed: the tension generating capacity, passive tension, the rate of tension development, the rate of ATP utilisation, calcium sensitivity of tension development, phosphorylation of contractile proteins, length dependent activation and stretch activation. The reduction in contractile performance during systole can be attributed predominantly to a loss of cardiomyocytes (necrosis), myocyte disarray and a decrease in myofibrillar density all resulting in a reduction in the tension generating capacity and likely also to a mismatch between energy supply and demand of the myocardium. This leads to a decline in the ejection fraction of the heart. Diastolic dysfunction can be attributed to fibrosis and an increase in titin stiffness which result in an increase in stiffness of the ventricular wall and hampers the filling of the heart with blood during diastole. A large number of post translation modifications of regulatory sarcomeric proteins influence myocardial function by altering calcium sensitivity of tension development. It is still unclear whether in concert these influences are adaptive or maladaptive during the disease process.

Troponin I phosphorylation in human myocardium in health and disease.

Cardiac troponin I (cTnI) is well known as a biomarker for the diagnosis of myocardial damage. However, because of its central role in the regulation of contraction and relaxation in heart muscle, cTnI may also be a potential target for the treatment of heart failure. Studies in rodent models of cardiac disease and human heart samples showed altered phosphorylation at various sites on cTnI (i.e. site-specific phosphorylation). This is caused by altered expression and/or activity of kinases and phosphatases during heart failure development. It is not known whether these (transient) alterations in cTnI phosphorylation are beneficial or detrimental. Knowledge of the effects of site-specific cTnI phosphorylation on cardiomyocyte contractility is therefore of utmost importance for the development of new therapeutic strategies in patients with heart failure. In this review we focus on the role of cTnI phosphorylation in the healthy heart upon activation of the beta-adrenergic receptor pathway (as occurs during increased stress and exercise) and as a modulator of the Frank-Starling mechanism. Moreover, we provide an overview of recent studies which aimed to reveal the functional consequences of changes in cTnI phosphorylation in cardiac disease.

ENerGetIcs in hypertrophic cardiomyopathy: traNslation between MRI, PET and cardiac myofilament function (ENGINE study).

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease mostly due to mutations in genes encoding sarcomeric proteins. HCM is characterised by asymmetric hypertrophy of the left ventricle (LV) in the absence of another cardiac or systemic disease. At present it lacks specific treatment to prevent or reverse cardiac dysfunction and hypertrophy in mutation carriers and HCM patients. Previous studies have indicated that sarcomere mutations increase energetic costs of cardiac contraction and cause myocardial dysfunction and hypertrophy. By using a translational approach, we aim to determine to what extent disturbances of myocardial energy metabolism underlie disease progression in HCM. METHODS: Hypertrophic obstructive cardiomyopathy (HOCM) patients and aortic valve stenosis (AVS) patients will undergo a positron emission tomography (PET) with acetate and cardiovascular magnetic resonance imaging (CMR) with tissue tagging before and 4 months after myectomy surgery or aortic valve replacement + septal biopsy. Myectomy tissue or septal biopsy will be used to determine efficiency of sarcomere contraction in-vitro, and results will be compared with in-vivo cardiac performance. Healthy subjects and non-hypertrophic HCM mutation carriers will serve as a control group. ENDPOINTS: Our study will reveal whether perturbations in cardiac energetics deteriorate during disease progression in HCM and whether these changes are attributed to cardiac remodelling or the presence of a sarcomere mutation per se. In-vitro studies in hypertrophied cardiac muscle from HOCM and AVS patients will establish whether sarcomere mutations increase ATP consumption of sarcomeres in human myocardium. Our follow-up imaging study in HOCM and AVS patients will reveal whether impaired cardiac energetics are restored by cardiac surgery.

In vivo and in vitro investigations of heterozygous nebulin knock-out mice disclose a mild skeletal muscle phenotype.

Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele.

Diaphragm muscle fiber function and structure in humans with hemidiaphragm paralysis.

Recent studies proposed that mechanical inactivity of the human diaphragm during mechanical ventilation rapidly causes diaphragm atrophy and weakness. However, conclusive evidence for the notion that diaphragm weakness is a direct consequence of mechanical inactivity is lacking. To study the effect of hemidiaphragm paralysis on diaphragm muscle fiber function and structure in humans, biopsies were obtained from the paralyzed hemidiaphragm in eight patients with hemidiaphragm paralysis. All patients had unilateral paralysis of known duration, caused by en bloc resection of the phrenic nerve with a tumor. Furthermore, diaphragm biopsies were obtained from three control subjects. The contractile performance of demembranated muscle fibers was determined, as well as fiber ultrastructure and morphology. Finally, expression of E3 ligases and proteasome activity was determined to evaluate activation of the ubiquitin-proteasome pathway. The force-generating capacity, as well as myofibrillar ultrastructure, of diaphragm muscle fibers was preserved up to 8 wk of paralysis. The cross-sectional area of slow fibers was reduced after 2 wk of paralysis; that of fast fibers was preserved up to 8 wk. The expression of the E3 ligases MAFbx and MuRF-1 and proteasome activity was not significantly upregulated in diaphragm fibers following paralysis, not even after 72 and 88 wk of paralysis, at which time marked atrophy of slow and fast diaphragm fibers had occurred. Diaphragm muscle fiber atrophy and weakness following hemidiaphragm paralysis develops slowly and takes months to occur.

Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe.

The cytosolic free Ca(2+) transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca(2+)] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca(2+) dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca(2+)] in enzymatically dissociated flexor digitorum brevis muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca(2+)] did not differ between WT, CSQ-KO, and CSQ-DKO fibers. During sustained contractions, changes were rather small in WT, reflecting powerful buffering of CSQ, whereas in CSQ-KO fibers, significant drops in SR [Ca(2+)] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca(2+), both in CSQ-KO and CSQ-DKO fibers. In CSQ-KO fibers, cytosolic free calcium detected with Fura-2 declined during repetitive stimulation, indicating that SR calcium content was insufficient for sustained contractile activity. SR Ca(2+) reuptake during and after stimulation trains appeared to be governed by three temporally distinct processes with rate constants of 50, 1-5, and 0.3 s(-1) (at 26 °C), reflecting activity of the SR Ca(2+) pump and interplay of luminal and cytosolic Ca(2+) buffers and pointing to store-operated calcium entry (SOCE). SOCE might play an essential role during muscle contractures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.

Impact of temperature on cross-bridge cycling kinetics in rat myocardium.

The dependence of contractile properties on intracellular calcium in cardiac tissue is a highly cooperative process. Here, the temperature and calcium dependence of contractile and energetical properties in permeabilized cardiac trabeculae from rat were studied to provide novel insights into the underlying kinetic processes. Myofilament Ca(2+) sensitivity significantly increased with temperature between 15 and 25 degrees C, whereas its steepness was independent of temperature. A direct proportionality between active tension and Ca(2+)-activated rate of ATP hydrolysis was observed; the slope of this relationship (tension cost) was highly temperature dependent. The rate of tension redevelopment following a quick release-restretch manoeuvre (k(tr)) depended in a complex manner on the level of contractile activation and on temperature. At saturating calcium levels, the temperature dependence (Q(10)) of k(tr) and Ca(2+)-activated ATP hydrolysis rate were similar (Q(10) approximately 3.5), and significantly higher than the Q(10) for maximum tension (T(max); Q(10) approximately 1.3) or tension cost (Q(10) approximately 2.5). In contrast, at a low level of contractile activation ( approximately 5% of T(max)), the Q(10) of k(tr) was similar to that of tension cost, and significantly lower than the Q(10) of Ca(2+)-activated ATP hydrolysis at that level of contractile activation. Our results are consistent with the hypothesis that at high levels of contractile activation, the rates of tension redevelopment and Ca(2+)-activated ATP hydrolysis are determined by both apparent cross-bridge attachment and detachment rates, while at low levels, k(tr) is limited by cross-bridge detachment rate. Tension cost, on the other hand, is determined solely by cross-bridge detachment kinetics at all temperatures and levels of contractile activation.

Alterations in contractile protein composition and function in human atrial dilatation and atrial fibrillation.

The cellular mechanisms responsible for contractile dysfunction associated with atrial fibrillation (AF) are still poorly understood. Atrial fibrillation is often preceded by atrial dilatation. This study aimed to explain contractile alterations associated with AF and their relation to atrial dilatation, by studying the relationships between atrial dimensions, contractile protein composition, force production and Ca(2+)-sensitivity. Force development was determined in mechanically isolated single skinned cardiomyocytes from right atrial appendages from patients with sinus rhythm without (SR;n=9), or with atrial dilation (SR+AD;n=11) or atrial fibrillation (AF;n=16). Echocardiography showed that, compared to the SR group, mean right atrial dimensions were increased by 18% and 35% in the SR+AD and AF group, respectively (P<0.05). Protein composition was determined by 1- and 2-dimensional gel electrophoresis. Compared to the SR group, the AF group exhibited: a reduction in the kinetics of force redevelopment (K(tr)) in isolated atrial cardiomyocytes, enhanced protein expression of the slow myosin heavy chain isoform (beta-MHC), an increase in troponin T (TnT) phosphorylation and a marked increase (70%) of the cytoskeletal protein desmin. Significant correlations were observed between the right atrial major axis (RA(major)) and beta-MHC expression as well as the desmin/actin ratio. Our findings indicate that dilatation may influence cardiomyocyte stability through altered desmin expression, but that it does not predispose to the alterations in contractile function observed in AF.

No direct effect of creatine phosphate on the cross-bridge cycle in cardiac myofibrils.

Creatine phosphate (CP) and creatine kinase (CK) are involved in the rapid resynthesis of ATP and thereby serve to stabilize ATP concentration and to maintain free ADP low inside cardiac muscle cells during contraction. Recently, it has been suggested from experiments in permeabilized multicellular preparations that CP/CK also regulate the kinetics of the actomyosin interaction (cross-bridge cycle) and may explain contractile dysfunction, for instance, during ischemia. However, the reported effects of CP/CK may be confounded by diffusion limitations in multicellular preparations in which inorganic phosphate (P(i)) and ADP may significantly accumulate during contraction. To test this hypothesis, we measured force production and the rates of force development (k (ACT) and k (TR)) in isolated cardiac myofibrils, in which rapid concentration changes of Ca(2+), CP/CK, and P(i) were imposed using a rapid perfusion change system. The results showed that CP/CK did not influence maximum force-generating capacity, whereas P(i) markedly reduced force and increased the rates of force development. No effects of CP/CK on the rates of force development were observed, consistent with the notion that CP/CK do not exert a direct effect on the actomyosin interaction.

Myosin heavy chain composition and the economy of contraction in healthy and diseased human myocardium.

Changes in myosin heavy chain (MHC) isoform expression and protein composition occur during cardiac disease and it has been suggested that even a minor shift in MHC composition may exert a considerable effect on myocardial energetics and performance. Here an overview is provided of the cellular basis of the energy utilisation in cardiac tissue and novel data are presented concerning the economy of myocardial contraction in diseased atrial and ventricular human myocardium. ATP utilisation and force development were measured at various Ca(2+) concentrations during isometric contraction in chemically skinned atrial trabeculae from patients in sinus rhythm (SR) or with chronic atrial fibrillation (AF) and in ventricular muscle strips from non-failing donor or end-stage failing hearts. Contractile protein composition was analysed by one-dimensional gel electrophoresis. Atrial fibrillation was accompanied by a significant shift from the fast alpha-MHC isoform to the slow beta-MHC isoform, whereas both donor and failing ventricular tissue contained almost exclusively the beta-MHC isoform. Simultaneous measurements of force and ATP utilisation indicated that economy of contraction is preserved in atrial fibrillation and in end-stage human heart failure.

Depression of force production and ATPase activity in different types of human skeletal muscle fibers from patients with chronic heart failure.

Isometric force production and ATPase activity were determined simultaneously in single human skeletal muscle fibers (n = 97) from five healthy volunteers and nine patients with chronic heart failure (CHF) at 20 degrees C. The fibers were permeabilized by means of Triton X-100 (1% vol/vol). ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. Calcium-activated actomyosin (AM) ATPase activity was obtained by subtracting the activity measured in relaxing (pCa = 9) solutions from that obtained in maximally activating (pCa = 4.4) solutions. Fiber type was determined on the basis of myosin heavy chain isoform composition by polyacrylamide SDS gel electrophoresis. AM ATPase activity per liter cell volume (+/-SE) in the control and patient group, respectively, amounted to 134 +/- 24 and 77 +/- 9 microM/s in type I fibers (n = 11 and 16), 248 +/- 17 and 188 +/- 13 microM/s in type IIA fibers (n = 14 and 32), 291 +/- 29 and 126 +/- 21 microM/s in type IIA/X fibers (n = 3 and 5), and 325 +/- 32 and 205 +/- 21 microM/s in type IIX fibers (n = 7 and 9). The maximal isometric force per cross-sectional area amounted to 64 +/- 7 and 43 +/- 5 kN/m(2) in type I fibers, 86 +/- 11 and 58 +/- 4 kN/m(2) in type IIA fibers, 85 +/- 6 and 42 +/- 9 kN/m(2) in type IIA/X fibers, and 90 +/- 5 and 59 +/- 5 kN/m(2) in type IIX fibers in the control and patient group, respectively. These results indicate that, in CHF patients, significant reductions occur in isometric force and AM ATPase activity but that tension cost for each fiber type remains the same. This suggests that, in skeletal muscle from CHF patients, a decline in density of contractile proteins takes place and/or a reduction in the rate of cross-bridge attachment of approximately 30%, which exacerbates skeletal muscle weakness due to muscle atrophy.

Myocardial contraction is 5-fold more economical in ventricular than in atrial human tissue.

OBJECTIVE: Cardiac energetics and performance depend on the expression level of the fast (alpha-) and slow (beta-) myosin heavy chain (MHC) isoform. In ventricular tissue, the beta-MHC isoform predominates, whereas in atrial tissue a variable mixture of alpha- and beta-MHC is found. In several cardiac diseases, the slow isoform is upregulated; however, the functional implications of this transition in human myocardium are largely unknown. The aim of this study was to determine the relation between contractile properties and MHC isoform composition in healthy human myocardium using the diversity in atrial tissue. METHODS: Isometric force production and ATP consumption were measured in chemically skinned atrial trabeculae and ventricular muscle strips, and rate of force redevelopment was studied using single cardiomyocytes. MHC isoform composition was determined by one-dimensional SDS-gel electrophoresis. RESULTS: Force development in ventricular tissue was about 5-fold more economical, but nine times slower, than in atrial tissue. Significant linear correlations were found between MHC isoform composition, ATP consumption and rate of force redevelopment. CONCLUSION: These results clearly indicate that even a minor shift in MHC isoform expression has considerable impact on cardiac performance in human tissue.

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.

Effects of Ca2+ -sensitizers in permeabilized cardiac myocytes from donor and end-stage failing human hearts.

During heart failure, alterations occur in contractile protein expression and phosphorylation, which may influence the effects of Ca2+ -sensitizers. To quantify the magnitude of these effects, isometric force was studied in mechanically isolated Triton-skinned myocytes from end-stage failing and non-failing donor hearts under control conditions (pH 7.2; no added inorganic phosphate (Pi)) and under mimicked ischemic conditions (pH 6.5; 10 mM Pi). Two different Ca2+ -sensitizers were used: EMD 53998 (10 microM), which exerts its influence through the actin-myosin interaction, and OR-1896 (10 microM) (the active metabolite of levosimendan), which affects the Ca2+ -sensory function of the thin filaments. The maximal force (Po) measured at saturating Ca2+ concentration and the resting force (Prest) determined in the virtual absence of Ca2+ (pCa 9) did not differ between the failing and non-failing myocytes, but the Ca2+ concentration required to induce the half-maximal force under control conditions was significantly lower in the failing than in the non-failing myocytes (DeltapCa50=0.15). This difference in Ca2+ -sensitivity, however, was abolished during mimicked ischemia. EMD 53998 increased Po and Prest by approximately 15% of Po and greatly enhanced the Ca2+ -sensitivity (DeltapCa50 > 0.25) of force production. OR-1896 did not affect Po and Prest, and provoked a small, but significant Ca2+ -sensitization (DeltapCa50 approximately 0.1). All of these effects were comparable in the donor and failing myocytes, but, in contrast with OR-1896, EMD 53998 considerably diminished the difference in the Ca2+ -sensitivities between the failing and non-failing myocytes. The action of Ca2+ -sensitizers under mimicked ischemic conditions was impaired to a similar degree in the donor and the failing myocytes. Our results indicate that the Ca2+ -activation of the myofibrillar system is altered in end-stage human heart failure. This modulates the effects of Ca2+ -sensitizers both under control and under mimicked ischemic conditions.

The effect of myosin light chain 2 dephosphorylation on Ca2+ -sensitivity of force is enhanced in failing human hearts.

OBJECTIVE: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1+/-2.0%) compared to donor (31.9+/-4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. METHODS: Cooperative effects in isometric force and rate of force redevelopment (K(tr)) were studied in single Triton-skinned human cardiomyocytes at various [Ca(2+)] before and after protein phosphatase-1 (PP-1) incubation. RESULTS: Maximum force and K(tr) values did not differ between failing and donor hearts, but Ca(2+)-sensitivity of force (pCa(50)) was significantly higher in failing myocardium (Deltap Ca(50)=0.17). K(tr) decreased with decreasing [Ca(2+)], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa(50) to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute K(tr) values was found after PP-1 in failing and donor myocytes, while the shape of the K(tr)-Ca(2+) relationships remained unaltered. CONCLUSIONS: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure.

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.

Myosin light chain composition in non-failing donor and end-stage failing human ventricular myocardium.

The increased Ca(2+)-responsiveness in end-stage human heart failure cannot be attributed to contractile protein isoform changes, but rather is the complex resultant of changes in degree of phosphorylation of VLC-2 and TnI. Despite the decreased basal level of VLC-2 phosphorylation the response to VLC-2 dephosphorylation is enhanced in failing myocytes, which might result from differences in endogenous phosphorylation of thin and thick filament proteins between donor and failing hearts. Taken together decreased VLC-2 phosphorylation in end-stage human heart failure might represent a compensatory process leading to an improvement of myocardial contractility by opposing the detrimental effects of increased Ca(2+)-responsiveness of force and impaired Ca(2+)-handling on diastolic function.