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.

TPM3 deletions cause a hypercontractile congenital muscle stiffness phenotype.

OBJECTIVE: Mutations in TPM3, encoding Tpm3.12, cause a clinically and histopathologically diverse group of myopathies characterized by muscle weakness. We report two patients with novel de novo Tpm3.12 single glutamic acid deletions at positions ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with congenital muscle stiffness, rather than weakness, and respiratory failure in one patient. METHODS: The effect of the Tpm3.12 deletions on the contractile properties in dissected patient myofibers was measured. We used quantitative in vitro motility assay to measure Ca(2+) sensitivity of thin filaments reconstituted with recombinant Tpm3.12 ΔE218 and ΔE224. RESULTS: Contractility studies on permeabilized myofibers demonstrated reduced maximal active tension from both patients with increased Ca(2+) sensitivity and altered cross-bridge cycling kinetics in ΔE224 fibers. In vitro motility studies showed a two-fold increase in Ca(2+) sensitivity of the fraction of filaments motile and the filament sliding velocity concentrations for both mutations. INTERPRETATION: These data indicate that Tpm3.12 deletions ΔE218 and ΔE224 result in increased Ca(2+) sensitivity of the troponin-tropomyosin complex, resulting in abnormally active interaction of the actin and myosin complex. Both mutations are located in the charged motifs of the actin-binding residues of tropomyosin 3, thus disrupting the electrostatic interactions that facilitate accurate tropomyosin binding with actin necessary to prevent the on-state. The mutations destabilize the off-state and result in excessively sensitized excitation-contraction coupling of the contractile apparatus. This work expands the phenotypic spectrum of TPM3-related disease and provides insights into the pathophysiological mechanisms of the actin-tropomyosin complex.

Short communication: addition of sodium bicarbonate to maternal colostrum: effects on immunoglobulin G absorption and hematocrit in neonatal calves.

Twenty-six Holstein bull calves born from primiparous and multiparous cows without dystocia were assigned in a randomized complete block design to 1 of 2 treatments: pooled maternal colostrum (PMC) or PMC supplemented with 30 g of sodium bicarbonate (NaHCO(3)). Calves were fed PMC from 9 different batches containing (mean ± SD) 82.05±8.45 g/L of IgG. Calves were fed 2.68 L of PMC at birth (referred to as 0 h) and 1.32 L of PMC 6h later. The total amount of IgG fed was 329.89±34.56 g. Calves were fed 2L of milk replacer at 24, 36, and 48 h postpartum. The addition of NaHCO(3) had no effect on IgG absorption. Serum IgG concentrations at 0, 6, 12, 24, and 48 h postpartum were not different between calves supplemented with or without 30 g of NaHCO(3) to colostrum. Area under the curve, apparent efficiency of absorption, and hematocrit were not affected by the NaHCO(3) treatment.

Short communication: Kelp taste preferences by dairy calves.

Kelp is a common feed additive used on many dairy farms in the United States. However, few data are available supporting the efficacy of its addition to cattle feed. The purpose of this study was to evaluate the taste preferences of calves provided with 0, 30, or 60 g of kelp daily in a sequential elimination experiment. Calves in this study always ranked the control treatment first when given a choice and consumed 34.5% more dry matter from the control treatment in the first 3-d segment of the experiment. During the second feeding segment (d 4 and 5), when the control treatment was removed, daily dry matter consumption was reduced in 4 out of 6 calves compared with control calves when this treatment was available (first feeding segment). However, calves did not differentiate between the 2 amounts of kelp. Results indicated that calves preferred calf starter grains without kelp.

Maximizing profit on New England organic dairy farms: an economic comparison of 4 total mixed rations for organic Holsteins and Jerseys.

The objective of these experiments was to compare 4 total mixed rations fed to USDA-certified organic dairy cows in New England. Forty-eight Jersey cows from the University of New Hampshire (UNH) and 64 Holstein cows from the University of Maine (UMaine) were assigned to a 2 × 2 factorial arrangement of treatments testing the main effects of corn silage versus grass silage as the forage base and commodity concentrates versus a complete pelleted concentrate mixture. Treatment diets were fed as a total mixed ration for 8 wk during the winter and spring months of 2007, 2008, and 2009. Milk yield, component, and quality data were recorded and used to calculate the value of the milk produced for each cow. The dry matter intake (DMI) was recorded and used to calculate the average cost per cow per day of each diet. Income over feed costs were calculated for each diet using milk value and feed cost data. Feed cost and income over feed cost data were resampled using bootstrap methodology to examine potential patterns. Milk yield, milk fat and true protein concentrations, and SCC were similar among treatments. Cows at UNH fed corn silage tended to have higher DMI and lower milk urea nitrogen than did cows fed grass silage, whereas cows fed pellets had higher DMI than cows fed commodities. Cows at UNH fed commodities tended to have higher body condition scores than those fed pellets. Cows at UMaine fed commodities tended to have higher DMI than did cows fed pellets, and cows fed corn silage had lower milk urea nitrogen than did cows fed grass silage. Body weights and body condition scores were not different for cows at UMaine. Feed costs were significantly higher for corn silage diets and diets at UNH containing pellets, but not at UMaine. The calculated value of the milk and income over feed costs did not differ among treatments at either university. Bootstrap replications indicated that the corn silage with commodities diet generally had the highest feed cost at both UNH and UMaine, whereas grass silage diets containing commodities generally had the lowest cost. In contrast, the grass silage with commodities diets had the highest income over feed cost in the majority of the replications at both UNH and UMaine replications, whereas the corn silage with commodities diets had the lowest rank. Similar results were observed when forage prices were increased or decreased by 5, 10, and 25% above or below the actual feed price. Feeding a grass silage-based diet supplemented with commodity concentrates may have an economic advantage for dairy producers in New England operating under an organic system of production.

Anionic salts in the prepartum diet and addition of sodium bicarbonate to colostrum replacer, and their effects on immunoglobulin G absorption in the neonate.

The objectives of this experiment were to determine whether feeding anionic salts to prepartum Holstein cows affected their calf's colostral IgG passive transfer and whether adding sodium bicarbonate to a colostrum replacer (CR) would increase the efficiency of IgG absorption. Forty Holstein cows and their resulting calves were assigned to a 2 x 2 factorial arrangement of treatments in a randomized complete block design based on expected date of calving. Three weeks before the projected due date, cows were placed on 1 of 2 treatments: a diet without anionic salts (dietary cation-anion difference of +77 mEq/kg) or a diet with anionic salts (dietary cation-anion difference of -100 mEq/kg). Within 45 min after birth, all calves received 1 dose of a commercially available CR (132g of IgG) without or with supplemental sodium bicarbonate (19.5 g/dose). A half-dose of CR (66g of IgG) and sodium bicarbonate (9.75g) was fed at 6h of age. Calves received milk replacer at 12, 24, 36, and 48h. Blood samples were obtained from calves at 0, 6, 12, 24, and 48h and were analyzed for IgG concentration. Cows fed the diet supplemented with anionic salts had lower DMI on d 8, 5, 4, and 1 and lower urine pH 2 and 1 wk before parturition compared with cows fed the diet without supplemental anionic salts. Calves born from dams receiving anionic salts had similar IgG concentrations (15.1 vs. 14.4g/L) and apparent efficiency of absorption values (29.2 vs. 28.2%) compared with calves born from dams not fed anionic salts. Calves receiving supplemental sodium bicarbonate in the CR had higher serum IgG concentrations at 12 (14.4 vs. 12.0g/L), 24 (16.3 vs. 13.2g/L), and 48h (14.6 vs. 11.2g/L) and higher apparent efficiency of absorption values (31.2 vs. 26.1%) than calves that did not receive sodium bicarbonate in the CR. Calves receiving sodium bicarbonate also had greater area under the curve values for IgG absorption compared with calves not receiving sodium bicarbonate. There was a trend for an interaction with calves born from dams fed anionic salts having a greater area under the curve when fed supplemental sodium bicarbonate. Of the 40 calves in the study, 90% obtained adequate passive transfer (serum IgG > or = 10g/L). This study indicates that feeding anionic salts to the dam has no effect on passive transfer, whereas adding sodium bicarbonate to the CR increased IgG uptake in calves.

[C-terminal sites of caldesmon drive ATP hydrolysis cycle by shifting actomyosin itermediates from strong to weak binding of myosin and actin].

Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506-793), CaDH2 (residues 683-767), CaDH12 (residues 506-708) and 658C (residues 658-793) on the orientation and mobility of fluorescent label 1.5-IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide, and in the presence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. It was shown that at modelling different intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in orientation and mobility of the oscillator (4 degrees and 30%, respectively) was observed between actomyosin in the presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and B' inhibit formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting the equilibrium between strong and weak forms of actin-myosin binding.

Structure, properties, and probable physiological role of small heat shock protein with molecular mass 20 kD (Hsp20, HspB6).

This review is devoted to critical analysis of data concerning the structure and functions of small heat shock proteins with apparent molecular mass 20 kD (Hsp20). We describe the structure of Hsp20, its phosphorylation by different protein kinases, interaction of Hsp20 with other small heat shock proteins, and chaperone activity of Hsp20. The distribution of Hsp20 in different animal tissues and the factors affecting expression of Hsp20 are also described. Data on the possible involvement of Hsp20 in regulation of platelet aggregation and glucose transport are presented and analyzed. Special attention is paid to literature data describing probable regulatory effect of Hsp20 on contraction of smooth muscle. Two hypotheses postulating direct effect of Hsp20 on actomyosin interaction or its effect on cytoskeleton are compared and analyzed. The most recent data on the effect of Hsp20 on apoptosis and contractile activity of cardiomyocytes are also presented.

Force-velocity relationship of single actin filament interacting with immobilised myosin measured by electromagnetic technique.

The effect of applying an external load to actin filaments moving in the in vitro motility assay is studied. Bead-tailed actin filaments were made by polymerising actin onto 2.8 microm diameter Dynabeads conjugated with gelsolin-G actin. These were introduced into a motility cell coated with 100 microg/ml rabbit fast skeletal myosin in the presence of ATP and 0.5% methylcellulose. The motility cell was inserted between the pole-pieces of an electromagnet and the fluorescent beads and filaments were observed. The force-current relationship of the electromagnet was determined from the velocity of free beads in viscous solution and Stokes' equation. The magnet produced up to 6 pN force on the Dynabeads at 1 A. Many bead-tailed actin filaments stuck to the surface, but the beads that did move moved at the same speed as unloaded f-actin in the same cell. Bead-tailed filaments slowed down under an increasing magnetic load, eventually stalled and then slid backward under increasing load before detaching from the surface. Single-filament force-velocity curves were constructed and a stalling force of about 0.6 pN/mm of actin filament estimated.

Structure and properties of small heat shock proteins (sHsp) and their interaction with cytoskeleton proteins.

The modern classification of small heat shock proteins (sHsp) is presented and peculiarities of their primary structure and the mechanism of formation of oligomeric complexes are described. Data on phosphorylation of sHsp by different protein kinases are presented and the effect of phosphorylation on oligomeric state and chaperone activity of sHsp is discussed. Intracellular location of sHsp under normal and stress conditions is described and it is emphasized that under certain condition sHsp interact with different elements of cytoskeleton. The literature concerning the effect of sHsp on polymerization of actin in vitro is analyzed. An attempt is made to compare effects of sHsp on polymerization of actin in vitro with the results obtained on living cells under normal conditions and after heat shock or hormone action. The literature concerning possible effects of sHsp on cell motility is also analyzed.

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states.

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluorescein-5'-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Phi(A), Phi(E), Theta(1/2) and Nu were calculated. Phi(A) and Phi(E) are angles between the fiber axis and the absorption and emission dipoles, respectively; Theta(1/2) is the angle between the F-actin filament axis and the fiber axis; Nu is the relative number of randomly oriented fluorophores. Actin-tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca(2+)) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca(2+)) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) states in a manner analogous to troponin.

Cardiac and skeletal myopathies: can genotype explain phenotype?

The inherited muscle diseases, skeletal muscle nemaline myopathy and cardiac muscle hypertrophic myopathy (HCM) have been recognised for decades. Recently it has become apparent that mutations in almost any protein component of the sarcomere could cause myopathy. Thus changes in many sarcomeric protein genes can produce a common phenotype. Several recent publications indicate the opposite property: mutations in one sarcomeric protein can produce different muscle disease phenotypes. The most dramatic example of this property is actin, mutations in which are associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, nemaline myopathy and actin myopathy.

Actomyosin cross-linking by caldesmon in non-muscle cells.

The role of myosin-binding in cytoskeletal arrangement of non-muscle low molecular weight caldesmon (l-caldesmon) was studied. The N-terminal myosin-binding domain of caldesmon N152 colocalized with myosin in transiently transfected chicken fibroblasts. When added exogenously to the Triton-insoluble cytoskeleton, N152 enhanced l-caldesmon displacement by exogenous C-terminal actin-binding fragment (H1). Thus, a significant fraction of l-caldesmon cross-links actin and myosin. In contrast, in epithelioid HeLa cells most of l-caldesmon was only actin-bound as H1 alone was enough for its displacement. Phosphorylation by mitogen-activated protein kinase reduced the capability of H1 to displace endogenous l-caldesmon, suggesting it may represent a regulatory mechanism for actin-caldesmon interaction in vivo.

Replacement of Lys-75 of calmodulin affects its interaction with smooth muscle caldesmon.

The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCam) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-induced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants analyzed. SynCam and its double mutant (K75P+KGK) induced nearly complete reversion of caldesmon inhibition of actomyosin ATPase activity with half-maximal reversion achieved at about 1 microM. Two mutants, K75A and K75V, with partially stabilized less positive central domain have higher affinity to caldesmon. These mutants induce 80-85% reversion of caldesmon inhibition of actomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0.4 microM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to caldesmon was the highest among calmodulin mutants tested. These mutants induced complete reversion of caldesmon inhibition with half-maximal effect observed at 0.3-0.4 microM. We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity.

Arthroscopic stabilization of type II SLAP lesions using an absorbable tack.

PURPOSE: To document the outcomes of arthroscopic stabilization of Snyder type II SLAP (superior labrum, anterior and posterior) lesions, using a bioabsorbable tack. TYPE OF STUDY: A case series. METHODS: Twenty-five SLAP lesions were repaired arthroscopically using a bioabsorbable tack. There were 22 recreational, 2 high school, and 1 professional athlete in this group. Shoulder function was surveyed at a mean follow-up of 35 months (range, 24 to 51 months) using the UCLA and ASES shoulder scoring algorithms. RESULTS: Shoulder function improved in 24 of the 25 cases. Follow-up UCLA scores averaged 32 points with 9 patients scoring as excellent, 13 good, 2 fair, and 1 poor, for an overall success rate of 88%. ASES shoulder scores similarly improved from a preoperative average of 45 points to a postoperative average of 92. All but 2 of the athletes had returned to their preinjury level of sports participation. CONCLUSIONS: Detachment of the superior labrum from the glenoid is recognized as a problematic injury in throwing athletes and others who engage in repetitive overhead activities. We conclude from our experience that using an absorbable tack to repair type II SLAP lesions is an effective treatment, even in athletes with high demands and expectations for shoulder function.

A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments.

We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the in vitro motility assay. Immobilized alpha-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of alpha-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing alpha-actinin concentration. The concentration of alpha-actinin needed to stop all filaments from moving (0.8 microg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the 'index of retardation' as the concentration of alpha-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized alpha-actinin on motility in the presence of 10 mM P(i) we found that the index of retardation was 0.62+/-0.07 (n=3) times that in the absence of P(i). This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to P(i). In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9-3.4-fold greater than that of actin with all tropomyosin isoforms.