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 ␤ 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.

Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and ␣-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 m in rat and 0.78 ؎ 0.07 m in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of Ϸ0.6 m 2 in rat and Ϸ0.8 m 2 in human. Although the couplons were in reasonable registration with zlines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was Ϸ53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca 2؉ waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca 2؉ -induced Ca 2؉ 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 Ϸ30% fewer RyRs per couplon than rat.calcium-induced calcium release ͉ excitation-contraction coupling ͉ sarcoplasmic reticulum I n cardiac ventricular muscle, excitation-contraction (EC) coupling arises from Ca 2ϩ release via clusters of ryanodine receptors (RyRs) in regions of close apposition between the sarcoplasmic reticulum (SR) and surface membranes in functional units called couplons (1, 2). Current work directed at understanding cardiac EC coupling is hindered by uncertainty in the size and 3D distribution of the couplons. Previous detailed analysis from electron micrographs has shown that typically 30-270 RyRs (depending on species) may be present in a couplon (1), but the thin sectioning associated with EM limits analysis of the spatial relationship between nearby and more distant couplons. Such knowledge is important, not only to make sense of the structures that underlie Ca 2ϩ sparks (3, 4) but also for detailed mathematical modeling of cardiac Ca 2ϩ metabolism.In this study, we have used immunocytochemistry combined with 3D imaging and analysis to both reveal the 3D organization of RyR clusters and estimate the numbers of RyRs within the couplon. Our analyses generally support some detailed quantitative measurements from EM (1), but also provide insight into organization in 3D at spatial scales that would be extremely laborious (if not impossible) to achieve by using conventional thin sectioning. In addition, the antibody labe...

Progress in cardiovascular gene therapy has been hampered by concerns over the safety and practicality of viral vectors and the inefficiency of current nonviral transfection techniques. We have previously reported that ultrasound exposure (USE) enhances transgene expression in vascular cells by up to 10-fold after naked DNA transfection, and enhances lipofection by up to three-fold. We report here that performing USE in the presence of microbubble echocontrast agents enhances acoustic cavitation and is associated with approximately 300-fold increments in transgene

BackgroundOptical 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 FindingsWe 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/SignificanceOur 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.

Nominally herbivorous acanthurids (surgeonfishes) and scarids (parrotfishes) have often been considered a 'homogeneous' functional group that consumes and digests algae. Recent work demonstrates that many of these fishes consume detritus. The objective of this study was to investigate the composition of dietary nutrients targeted by these and other fishes in terms of feeding behaviour, diet and short chain fatty acids (SCFA). We undertook a nutritional analysis of a range of species including detritivores, algivores, omnivores and planktivores from north eastern Australia. We calculated assimilation efficiencies for total protein amino acids (TAA), carbohydrate and lipid, and measured TAA in gut fluid along the intestine. Nutrients were assimilated similarly to their dietary proportions, with planktivores assimilating a high proportion of TAA, a moderate proportion of lipid and little carbohydrate. Omnivores assimilated moderate proportions of TAA and carbohydrate, and a low proportion of lipid. Algivores assimilated a low proportion of TAA and lipid, but a high proportion of carbohydrate. Detritivorous scarids and acanthurids differed significantly from algivores, assimilating a high proportion of TAA, a low proportion of carbohydrate and a moderate proportion of lipid. TAA levels in gut fluid of all species were highest in the anterior and lowest in the posterior intestine. Gut segments with highest TAA values were compared between dietary groups and followed a similar trend to TAA assimilation. Planktivores had high concentrations of TAA, while omnivores had intermediate, and algivores the lowest, concentrations. The highest gut fluid TAA concentrations were found in detritivorous scarids and acanthurids, and were significantly higher than in algivores. A significant negative correlation was found between anterior intestinal fluid TAA and posterior intestinal SCFA values. Detritivores had the highest levels of TAA but the lowest levels of SCFA. Planktivorous species had high levels of TAA and low to intermediate levels of SCFA. Omnivores had moderate levels of both TAA and SCFA. Algivores had low levels of TAA but high levels of SCFA. This indicates that there are major differences in the food resources targeted by detritivorous and algivorous fish species on coral reefs.

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BackgroundThe 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 ResultsThe 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 Pearson's correlation coefficient in failing hearts.ConclusionsThe 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.

Algal turf assemblages of the northern Great Barrier Reef, Australia, were sampled to determine the nutritional value of detritus and algae. Samples were collected with a suction apparatus across an exposure gradient from (1) the reef crest at highly exposed outer barrier reefs, (2) the reef crest of moderately exposed midshelf reefs, and (3) the reef slope of sheltered midshelf reefs. The biomass of algae and detritus decreased from sheltered midshelf reefs to moderately exposed midshelf reefs to highly exposed outer barrier reefs. This decrease was significant only for detritus (P Ͻ 0.005). Wave energies were calculated across the exposure gradient with the wave model WAMGBR. Detrital mass was inversely correlated with predicted wave energies and fitted a polynomial relationship (P Ͻ 0.001) and explained 52.8% of the variation. A similar relationship was also found between algal mass and wave energy (P Ͻ 0.001) but only explained 30.0% of the variation. The nutritional value of samples in protein amino acids and starch was assessed. The amino acid composition of detritus and algae was similar and not considered nutritionally different, whereas the concentration of protein amino acids was significantly (P Ͻ 0.001) higher in detritus (21.2 Ϯ 2.0 mg g Ϫ1 ) than in algae (11.8 Ϯ 1.0 mg g Ϫ1 ). Starch content was significantly (P Ͻ 0.05) higher in algae (7.7 Ϯ 0.9 mg g Ϫ1 ) than in detritus (6.0 Ϯ 1.0 mg g Ϫ1). These results demonstrate that detritus is a potentially valuable food source to grazing fishes on coral reefs.Two predominant views of coral reef trophic biology are that shallow-water epilithic algal communities (EAC) are the major sites of primary production and that grazing fish and invertebrates are the predominant consumers of this resource (Hatcher 1997). Grazing fish are classified as herbivores and are assumed to derive the significant component of their nutrition through consumption, digestion, and assimilation of living turf algae