David J. Crossman

Regeneration of the Heart in Diabetes by Selective Copper Chelation

Heart disease is the major cause of death in diabetes, a disorder characterized by chronic hyperglycemia and cardiovascular complications. Although altered systemic regulation of transition metals in diabetes has been the subject of previous investigation, it is not known whether changed transition metal metabolism results in heart disease in common forms of diabetes and whether metal chelation can reverse the condition. We found that administration of the Cu-selective transition metal chelator trientine to rats with streptozotocin-induced diabetes caused increased urinary Cu excretion compared with matched controls. A Cu(II)-trientine complex was demonstrated in the urine of treated rats. In diabetic animals with established heart failure, we show here for the first time that 7 weeks of oral trientine therapy significantly alleviated heart failure without lowering blood glucose, substantially improved cardiomyocyte structure, and reversed elevations in left ventricular collagen and beta(1) integrin. Oral trientine treatment also caused elevated Cu excretion in humans with type 2 diabetes, in whom 6 months of treatment caused elevated left ventricular mass to decline significantly toward normal. These data implicate accumulation of elevated loosely bound Cu in the mechanism of cardiac damage in diabetes and support the use of selective Cu chelation in the treatment of this condition.

Changes in the organization of excitation-contraction coupling structures in failing human heart

BACKGROUND The cardiac myocyte t-tubular system ensures rapid, uniform cell activation and several experimental lines of evidence suggest changes in the t-tubular system and associated excitation-contraction coupling proteins may occur in heart failure. METHODS AND RESULTS The organization of t-tubules, L-type calcium channels (DHPRs), ryanodine receptors (RyRs) and contractile machinery were examined in fixed ventricular tissue samples from both normal and failing hearts (idiopathic (non-ischemic) dilated cardiomyopathy) using high resolution fluorescent imaging. Wheat germ agglutinin (WGA), Na-Ca exchanger, DHPR and caveolin-3 labels revealed a shift from a predominantly transverse orientation to oblique and axial directions in failing myocytes. In failure, dilation of peripheral t-tubules occurred and a change in the extent of protein glycosylation was evident. There was no change in the fractional area occupied by myofilaments (labeled with phalloidin) but there was a small reduction in the number of RyR clusters per unit area. The general relationship between DHPRs and RyR was not changed and RyR labeling overlapped with 51±3% of DHPR labeling in normal hearts. In longitudinal (but not transverse) sections there was an ∼30% reduction in the degree of colocalization between DHPRs and RyRs as measured by Pearsons correlation coefficient in failing hearts. CONCLUSIONS The results show that extensive remodelling of the t-tubular network and associated excitation-contraction coupling proteins occurs in failing human heart. These changes may contribute to abnormal calcium handling in heart failure. The general organization of the t-system and changes observed in failure samples have subtle differences to some animal models although the general direction of changes are generally similar.

Analysis of ryanodine receptor clusters in rat and human cardiac myocytes

Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and α-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs or “couplons” around the edges of the myofilaments with a nearest-neighbor spacing of 0.66 ± 0.06 μm in rat and 0.78 ± 0.07 μm in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of ≈0.6 μm2 in rat and ≈0.8 μm2 in human. Although the couplons were in reasonable registration with z-lines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was ≈53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca2+ waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca2+-induced Ca2+ release. By quantifying labeling intensity in rat ventricular myocytes, a lower limit of 78 RyRs per cluster (on average) was obtained. By modeling the couplon as a disk wrapping around a t-tubule and fitting cluster images, 95% of couplons contained between 120 and 260 RyRs (assuming that RyRs are tight packed with a spacing of 29 nm). Assuming similar labeling efficiency in human, from the fluorescence intensity alone we estimate that human ventricular myocytes contain ≈30% fewer RyRs per couplon than rat.

4D Super-Resolution Microscopy with Conventional Fluorophores and Single Wavelength Excitation in Optically Thick Cells and Tissues

Background Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. Methodology/Principal Findings We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk. Conclusions/Significance Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

Effect of changes in action potential spike configuration, junctional sarcoplasmic reticulum micro-architecture and altered t-tubule structure in human heart failure

Using a Monte–Carlo model of L-type Ca2+ channel (DHPR) gating, we have examined the effect of changes in the early time course of the action potential as seen in human heart failure on excitation contraction coupling. The time course of DHPR Ca2+ influx was coupled into a simple model of sarcoplasmic reticulum Ca2+ release. Our model shows that the loss of the initial spike in human heart failure should reduce the synchrony of Ca2+ spark production and lead to the appearance of late Ca2+ sparks and greater non-uniformity of intracellular Ca2+. Within the junctional space of the cardiac dyad, a small increase in the mean distance of a DHPR from a RyR results in a marked decrease in the ability of the DHPR-mediated increase in local [Ca2+] concentration to activate RyRs. This suggests that the efficiency of EC coupling may be reduced if changes in micro-architecture develop and such effects have been noted in experimental models of heart failure. High resolution imaging of t-tubules in tachycardia-induced heart failure show deranged t-tubule structure. While in normal human hearts t-tubules run mainly in a radial direction, t-tubules in the heart failure samples were oriented more toward the long axis of the cell. In addition, t-tubules may become dilated and bifurcated. Our data suggest that changes in the micro-architecture of the cell and membrane structures associated with excitation–contraction coupling, combined with changes in early action potential configuration can reduce the efficiency by which Ca2+ influx via DHPRs can activate SR calcium release and cardiac contraction. While the underlying cause of these effects is unclear, our data suggest that geometric factors can play an important role in the pathophysilogy of the human heart in failure.

Comparison of the organization of t‐tubules, sarcoplasmic reticulum and ryanodine receptors in rat and human ventricular myocardium

1. It is apparent from the literature that there are significant differences in excitation–contraction coupling between species, particularly in the density of calcium transporting proteins in the t‐system and sarcoplasmic reticulum (SR) Ca2+ release channels. Unfortunately, there is a lack of information as to how the principal structures that link electrical excitation to the activation of calcium‐induced calcium release (CICR) are different between human and animal models (particularly rat).

Characterization of proteomic changes in cardiac mitochondria in streptozotocin-diabetic rats using iTRAQ™ isobaric tags.

Diabetes now affects more than 5% of the worlds population and heart failure is the most common cause of death amongst diabetic patients. Accumulating evidence supports a view that myocardial mitochondrial structural and functional changes are central to the onset of diabetic heart failure, but the exact nature of these changes at the proteomic level remains unclear.Here we report on proteomic changes in diabetic rat heart mitochondria following 120 days of streptozotocin‐diabetes using the recently developed iTRAQ™ labeling method, which permits quantification of proteins directly from complex mixtures, bypassing the limitations associated with gel‐based methods such as 2‐DE. Of 252 unique proteins identified, 144 were represented in at least three of six individual paired experiments. Relative amounts of 65 proteins differed significantly between the groups, confirming that the cardiac mitochondrial proteome is indeed impacted by diabetes. The most significant changes were increased protein levels of enzymes involved in mitochondrial oxidation of long‐chain fatty acids, which was also confirmed by enzyme assays, and decreased levels of multiple enzymes involved in oxidative phosphorylation and catabolism of short‐chain fatty acids and branched‐chain amino acids. We also found significant changes in levels of several enzymes linked to oxidative stress.

Determination of protein for studies of marine herbivory: a comparison of methods

In this study we compared techniques for the estimation of protein for studies of marine herbivory. As an example we use gut fluid from the temperate marine herbivorous fish Kyphosus sydneyanus. There were two components to this work: (1) to compare the ability of spectrophotometric methods to estimate protein in gut fluid; and (2) to determine the effect of freezing digesta samples on the extraction of gut fluid for the measurement of total hydrolysable amino acids (THAA). The Bradford, Lowry, deoxycholate/trichloroacetic acid (DOC/TCA) Lowry and the bicinchoninic acid (BCA) spectrophotometric protein assays were compared for their ability to estimate protein in pooled gut fluid and compared with protein determined by quantitative amino acid analysis using an automated procedure. The spectrophotometric assays all gave differing estimates of protein content and none correlated well with quantitative amino acid analysis. The Lowry and BCA methods gave similar estimates of protein, which were higher than those measured in the same samples by amino acid analysis. Both the DOC/TCA Lowry and the Bradford assay substantially underestimated protein in pooled gut fluid. Internal standard recovery experiments for the protein assays showed interference of colour formation in the Bradford method but not the other spectrophotometric protein assays. This suggested the low values in the Bradford assay were due to gut fluid interfering with colour formation. Therefore, five algal species known to occur in the diet of Kyphosus sydneyanus were selected to test for their interference in the Bradford assay. The algae included three species of phaeophytes, one rhodophyte and one chlorophyte. Extracts from all five algal species interfered with this protein assay to significant but varying degrees. Two methods of extracting gut fluid were compared: (i) method A, which involved removing the gut fluid from the digesta immediately post capture; and (ii) method B, in which samples of digesta were frozen and gut fluid removed later. The fluid from method B gave significantly higher levels of THAA in some gut segments. Freeze–thawing of digesta in method B was thought to be the basis of the increased THAA. This finding suggests that method A more accurately reflects in vivo gut fluid THAA concentrations. The above methodology provides a framework to compare the ability of different herbivorous species at extracting THAA from their diet. These results highlight that the estimation of protein can be affected by methodology and by sample composition, and shows that methods must be optimized for a particular application.

Reversal of diabetes-evoked changes in mitochondrial protein expression of cardiac left ventricle by treatment with a copper (II)-selective chelator

Cardiac disease is the commonest cause of death amongst diabetic patients. Diabetic cardiomyopathy, which has a poor prognosis, is characterized by left ventricular hypertrophy and impaired cardiac function and mitochondrial damage is said to contribute to its development. We recently showed that treatment with the CuII‐selective chelator, triethylenetetramine (TETA), improved cardiac structure, and function in diabetic subjects without modifying hyperglycemia. Thus, TETA has potential utility for the treatment of heart disease. To further understand the molecular mechanism by which it causes these effects, we have conducted the first study of the effect of oral TETA on protein abundance in the cardiac left ventricle of rats with severe streptozotocin‐induced diabetes. Proteomic methods showed that of 211 proteins changed in diabetes, 33 recovered after treatment. Through MS, 16 proteins were identified which may constitute major targets of drug action. Remarkably, most of these were mitochondrial proteins with roles in energy metabolism. In addition to components of the mitochondrial respiratory chain and enzymes involved in fatty acid oxidation, TETA treatment normalized both myocardial expression and enzymatic activity of carnitine palmitoyltransferase 2. These findings indicate that mitochondria constitute major targets in the mechanism by which TETA restores cardiac structure and function in diabetes.

Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i ≈1 μM; F/F0≈5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.