Cristobal G. dos Remedios

Cardiomyocyte proliferation contributes to heart growth in young humans

The human heart is believed to grow by enlargement but not proliferation of cardiomyocytes (heart muscle cells) during postnatal development. However, recent studies have shown that cardiomyocyte proliferation is a mechanism of cardiac growth and regeneration in animals. Combined with evidence for cardiomyocyte turnover in adult humans, this suggests that cardiomyocyte proliferation may play an unrecognized role during the period of developmental heart growth between birth and adolescence. We tested this hypothesis by examining the cellular growth mechanisms of the left ventricle on a set of healthy hearts from humans aged 0–59 y (n = 36). The percentages of cardiomyocytes in mitosis and cytokinesis were highest in infants, decreasing to low levels by 20 y. Although cardiomyocyte mitosis was detectable throughout life, cardiomyocyte cytokinesis was not evident after 20 y. Between the first year and 20 y of life, the number of cardiomyocytes in the left ventricle increased 3.4-fold, which was consistent with our predictions based on measured cardiomyocyte cell cycle activity. Our findings show that cardiomyocyte proliferation contributes to developmental heart growth in young humans. This suggests that children and adolescents may be able to regenerate myocardium, that abnormal cardiomyocyte proliferation may be involved in myocardial diseases that affect this population, and that these diseases might be treatable through stimulation of cardiomyocyte proliferation.

Antibody arrays: an embryonic but rapidly growing technology.

Protein arrays are now an attractive proposition as they can measure a diverse range of protein interactions not possible with traditional DNA arrays. Antibody arrays are a specific subset of this technology. Originally conceived as multi-analyte detectors, antibody arrays are now used in a wide variety of applications. For instance, the potential of this technology to diagnose human diseases, such as leukemia, breast cancer and, potentially, heart failure, has stimulated much interest. Furthermore, identification of new protein targets in particular disease states will prove to be an invaluable tool in drug discovery and development. Patient prognosis and treatment are also potential applications of the technology. Antibody arrays have proved to be dynamic in response to these broad range of possibilities. This review examines variations in antibody array design and discusses current and potential applications of this novel and interesting technology.

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.

Multiple Reaction Monitoring to Identify Site-Specific Troponin I Phosphorylated Residues in the Failing Human Heart

Background— Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples. Methods and Results— On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry–based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy. Conclusions— Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.

Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca2+/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca2+-handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin. Objective: To elucidate whether CaMKII phosphorylates titin and modulates Fpassive in normal and failing myocardium. Methods and Results: Titin phosphorylation was assessed in CaMKII&dgr;/&ggr; double-knockout (DKO) mouse, transgenic CaMKII&dgr;C-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using stable isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. Fpassive of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. Fpassive was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered Fpassive of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts. Table. Titin Phosphosites Downregulated in CaMKII&dgr;/&ggr; DKO vs WT Mouse Hearts, Identified by Quantitative Mass Spectrometry Conclusions: CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering Fpassive. Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.

Current Status of Biomarkers for Prostate Cancer

Prostate cancer (PCa) is a leading cause of cancer-related death of men globally. Since its introduction, there has been intense debate as to the effectiveness of the prostate specific antigen (PSA) test as a screening tool for PCa. It is now evident that the PSA test produces unacceptably high rates of false positive results and is not prognostic. Here we review the current status of molecular biomarkers that promise to be prognostic and that might inform individual patient management. It highlights current efforts to identify biomarkers obtained by minimally invasive methods and discusses current knowledge with regard to gene fusions, mRNA and microRNAs, immunology, and cancer-associated microparticles.

Differential variability analysis of gene expression and its application to human diseases

Motivation: Current microarray analyses focus on identifying sets of genes that are differentially expressed (DE) or differentially coexpressed (DC) in different biological states (e.g. diseased versus non-diseased). We observed that in many human diseases, some genes have a significantincrease or decrease in expression variability (variance). Asthese observed changes in expression variability may be caused by alteration of the underlying expression dynamics, such differential variability (DV) patterns are also biologically interesting. Results: Here we propose a novel analysis for changes in gene expression variability between groups of amples, which we call differential variability analysis. We introduce the concept of differential variability (DV), and present a simple procedure for identifying DV genes from microarray data. Our procedure is evaluated with simulated and real microarray datasets. The effect of data preprocessing methods on identification of DV gene is investigated. The biological significance of DV analysis is demonstrated with four human disease datasets. The relationships among DV, DE and DC genes are investigated. The results suggest that changes in expression variability are associated with changes in coexpression pattern, which imply that DV is not merely stochastic noise, but informative signal. Availability: The R source code for differential variability analysis is available from the contact authors upon request. Contact: joshua@it.usyd.edu.au; mcharleston@it.usyd.edu.au

Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation.

SummaryThe contractile proteins actin and myosin are of considerable biological interest. They are essential for muscle contraction and in eukaryotic cells they play a crucial role in most contractile phenomena. Over the years since the first fluorescence resonance energy transfer (FRET) paper appeared, an extensive body of literature has accumulated on this technique using actin, myosin and the actomyosin complex. These papers are reviewed with several aims in mind: (i) we assess the reliability and consistency of intra- and inter-molecular distances measured between the fluorescent probes attached to specific sites on these proteins; (ii) we determine whether the measurements can be assembled into an internally consistent model which can be fitted to the known dimensions of the actomyosin complex; (iii) several of the FRET distances are consistent with the available structural data from crystallographic and electron microscopic dimensions; (iv) the modelled FRET distances suggest that the assumed value of the orientation factor (K2 = 2/3) is reasonable; (v) we conclude that the model has a predictive value, i.e. it suggests that a small number of the published dimensions may be incorrect and predicts the magnitude of a larger number of measurements which have not yet been reported; and finally (vi) we discuss the contribution of FRET determinations to the current debate on the molecular mechanism of contraction.

Lanthanide ion probes of calcium-binding sites on cellular membranes

Abstract The chemical basis for the similarity between the lanthanide series of ions and calcium is outlined together with the experimental difficulties associated with the use of these ions. A number of properties of the lanthanide ions are highlighted which make them potentially valuable probe elements. In this context the use of lanthanum and the lanthanide ions in probing calcium sites on cellular membranes is reviewed. In most instances, the lanthanide ions displace membranebound Ca and inhibit Ca-mediated membrane function, but, unlike Ca, these ions do not appear to be transported across cellular membranes (mitochondria may be an exception). Generally two relationships can be demonstrated between the inhibition of the Ca-mediated function and the atomic number of the lanthanide ion. Extracellular membranes appear to respond selectively to Tm(III) while intracellular membranes lack the Tm peak and instead exhibit a broad trend in which the smaller ions are the least effective inhibitors.

Neuregulin stimulation of cardiomyocyte regeneration in mice and human myocardium reveals a therapeutic window

The growth factor neuregulin stimulates heart muscle repair in newborn mice and heart muscle from human infants if given during a specific therapeutic time period. Young at heart: Restoring cardiac function in children When children are given adult roles in TV commercials, the results range from adorable to brilliant to simply hilarious. But when children with heart disease were given adult medicines in clinical trials, the results were disappointing—and the need for pediatric-specific treatment regimens became clear. In adult mice, the recombinant growth factor neuregulin-1 (rNRG1) stimulates heart regeneration by driving the proliferation of heart muscle cells (cardiomyocytes). Because young mice bear more proliferation-competent cardiomyocytes than do adult animals, Polizzotti et al. asked whether rNRG1 might put cardiomyocyte proliferation into overdrive if given to mice during the neonatal period. To test their hypothesis, the authors treated newborn mice with rNRG1 at various times after heart injury and found that early treatment starting at 1 day of age boosted cardiomyocyte cell division and heart function in a persistent manner relative to treatment regimens that began at 4 days after birth. rNRG1 also drove cardiomyocyte proliferation in heart muscle isolated from human infants with heart disease who were less than 6 months of age, but not in tissue from older pediatric patients. These findings suggest that rNRG1 administration during the neonatal period might be a new therapeutic strategy for pediatric heart disease. Now that would be brilliant. Therapies developed for adult patients with heart failure have been shown to be ineffective in pediatric clinical trials, leading to the recognition that new pediatric-specific therapies for heart failure must be developed. Administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates regeneration of heart muscle cells (cardiomyocytes) in adult mice. Because proliferation-competent cardiomyocytes are more abundant in growing mammals, we hypothesized that administration of rNRG1 during the neonatal period might be more effective than in adulthood. If so, neonatal rNRG1 delivery could be a new therapeutic strategy for treating heart failure in pediatric patients. To evaluate the effectiveness of rNRG1 administration in cardiac regeneration, newborn mice were subjected to cryoinjury, which induced myocardial dysfunction and scar formation and decreased cardiomyocyte cell cycle activity. Early administration of rNRG1 to mice from birth to 34 days of age improved myocardial function and reduced the prevalence of transmural scars. In contrast, administration of rNRG1 from 4 to 34 days of age only transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). We also assessed the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease who were less than 6 months of age. Our results identify an effective time period within which to execute rNRG1 clinical trials in pediatric patients for the stimulation of cardiomyocyte regeneration.