Diet-induced weight loss decreases adipose tissue oxygen tension with parallel changes in adipose tissue phenotype and insulin sensitivity in overweight humans.

BACKGROUND/OBJECTIVES: Although adipose tissue (AT) hypoxia is present in rodent models of obesity, evidence for this in humans is limited. Here, we investigated the effects of diet-induced weight loss (WL) on abdominal subcutaneous AT oxygen tension (pO2), AT blood flow (ATBF), AT capillary density, AT morphology and transcriptome, systemic inflammatory markers and insulin sensitivity in humans. SUBJECTS/METHODS: Fifteen overweight and obese individuals underwent a dietary intervention (DI), consisting of a 5-week very-low-calorie diet (VLCD, 500 kcal day-1; WL), and a subsequent 4-week weight stable diet (WS). Body composition, AT pO2 (optochemical monitoring), ATBF (133Xe washout), and whole-body insulin sensitivity were determined, and AT biopsies were collected at baseline, end of WL (week 5) and end of WS (week 9). RESULTS: Body weight, body fat percentage and adipocyte size decreased significantly during the DI period. The DI markedly decreased AT pO2 and improved insulin sensitivity, but did not alter ATBF. Finally, the DI increased AT gene expression of pathways related to mitochondrial biogenesis and non-mitochondrial oxygen consumption. CONCLUSIONS: VLCD-induced WL markedly decreases abdominal subcutaneous AT pO2, which is paralleled by a reduction in adipocyte size, increased AT gene expression of mitochondrial biogenesis markers and non-mitochondrial oxygen consumption pathways, and improved whole-body insulin sensitivity in humans.

The relationship of peritubular capillary density with glomerular volume and kidney function in living kidney donors.

BACKGROUND: Peritubular capillary rarefaction plays an important role in the progression of chronic kidney disease. Little is known about the relation between peritubular capillary density, glomerular volume and filtration rate in the healthy kidney. METHODS: In this single-center study, we included 69 living kidney donors who donated between 2005 and 2008 and had representative renal biopsies available. In all donors, glomerular filtration rate was measured using 125I-Iothalamate before donation and at five years after donation. Before donation, the increase in glomerular filtration rate after dopamine stimulation was measured. Glomerular volume and peritubular capillary density were determined in biopsies taken at the time of transplantation. Pearson's correlation coefficient and linear regression were used to assess relations between parameters. RESULTS: Mean donor age was 52 ± 11 years and mean measured glomerular filtration rate was 119 ± 22 mL/min before donation and 82 ± 15 mL/min at five years after donation. While peritubular capillary density (measured by either number of peritubular capillaries/50,000 µm2 or number of peritubular capillaries/tubule) was not associated with measured glomerular filtration rate before or after donation, number of peritubular capillaries/tubule was associated with the increase in measured glomerular filtration rate after dopamine stimulation (St.ß = 0.33, p = 0.004), and correlated positively with glomerular volume (R = 0.24, p = 0.047). Glomerular volume was associated with unstimulated measured glomerular filtration rate before donation (St.ß = 0.31, p = 0.01) and at five years (St.ß = 0.30, p = 0.01) after donation, independent of age. CONCLUSIONS: In summary, peritubular capillary density was not related to unstimulated kidney function before or after kidney donation, in contrast to glomerular volume. However, number of peritubular capillaries/tubule correlated with the increase in glomerular filtration rate after dopamine stimulation in healthy kidneys, and with glomerular volume. These findings suggest that peritubular capillary density and glomerular volume differentially affect kidney function in healthy living kidney donors.

Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.

Role of arginine in superficial wound healing in man.

Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.

Cell kinetics of the marine sponge Halisarca caerulea reveal rapid cell turnover and shedding.

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure?

Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the pathogenesis of cardiovascular disease, including atherosclerosis, restenosis, dilated cardiomyopathy, and myocardial infarction. Administration of synthetic MMP inhibitors in experimental animal models of these cardiovascular diseases significantly inhibits the progression of, respectively, atherosclerotic lesion formation, neointima formation, left ventricular remodeling, pump dysfunction, and infarct healing. This review focuses on the role of MMPs in cardiovascular disease, in particular myocardial infarction and the subsequent progression to heart failure. MMPs, which are present in the myocardium and capable of degrading all the matrix components of the heart, are the driving force behind myocardial matrix remodeling. The recent finding that acute pharmacological inhibition of MMPs or deficiency in MMP-9 attenuates left ventricular dilatation in the infarcted mouse heart led to the proposal that MMP inhibitors could be used as a potential therapy for patients at risk for the development of heart failure after myocardial infarction. Although these promising results encourage the design of clinical trials with MMP inhibitors, there are still several unresolved issues. This review describes the biology of MMPs and discusses new insights into the role of MMPs in several cardiovascular diseases. Attention will be paid to the central role of the plasminogen system as an important activator of MMPs in the remodeling process after myocardial infarction. Finally, we speculate on the use of MMP inhibitors as potential therapy for heart failure.

Accelerated atherosclerosis and calcification in vein grafts: a study in APOE*3 Leiden transgenic mice.

Vein grafts fail due to development of intimal hyperplasia and accelerated atherosclerosis. Many murine genetic models in which genes are overexpressed, deleted, or mutated have been introduced recently. Therefore, mouse models are very well suited to dissect the relative contribution of different genes in the development of accelerated atherosclerosis. In the present study, we evaluated whether accelerated atherosclerosis in human vein grafts could be mimicked in hypercholesterolemic APOE*3 Leiden transgenic mice. Venous bypass grafting was performed in the carotid artery in APOE*3 Leiden mice fed either a standard chow diet or a high cholesterol-rich diet for 4 weeks. At several time points (0 hour to 28 days), mice were euthanized and the morphology of the vein grafts was analyzed. In normocholesterolemic mice, vein graft thickening up to 10-fold original thickness, predominantly consisting of alpha-smooth muscle cell actin-positive cells, was observed after 28 days. In hypercholesterolemic mice, accelerated atherosclerosis with accumulation of lipid-loaded foam cells was observed within 7 days after surgery. This accelerated atherosclerosis progressed in time and resulted in significant increase in vein graft thickening up to 50 times original thickness with foam cell-rich lesions and calcification within 28 days after surgery. The atherosclerotic lesions observed in these murine grafts show high morphological resemblance with the atherosclerotic lesions observed in human vein grafts. This accelerated, diet-dependent induction of atherosclerotic-like lesions in murine vein grafts provides a valuable tool in evaluating the mechanisms of accelerated atherosclerosis and therapeutic interventions of vein graft disease.

17beta-estradiol attenuates the development of pressure-overload hypertrophy.

BACKGROUND: Cardiac hypertrophy is an independent risk factor for cardiovascular morbidity and mortality in men and in women. Epidemiological studies indicate that estrogen replacement therapy is cardioprotective; the mechanisms involved in this process, however, are poorly understood. We therefore studied the effect of 17beta-estradiol (E(2)) on the development of pressure-overload hypertrophy. METHODS AND RESULTS: Ovariectomized mice receiving E(2) or placebo underwent transverse aortic constriction (TAC) or sham operation. TAC led to a significant increase in ventricular mass compared with sham operation. E(2) treatment reduced cardiac hypertrophy by 31% and 26% compared with placebo 4 and 8 weeks after TAC, whereas it had no effect on the degree of pressure overload, as determined by hemodynamic measurements. Furthermore, E(2) blocked the increased phosphorylation of p38-mitogen-activated protein kinase (MAPK) observed in the placebo-treated animals with TAC. No differences were observed in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 between the groups. E(2) had no effect on the expression of angiotensin-converting enzyme (ACE) or the angiotensin II type 1 receptor. Ventricular atrial natriuretic peptide (ANP) expression was detected only in the animals with TAC. Compared with placebo, E(2) treatment led to an increased expression of ANP in animals with pressure overload. CONCLUSIONS: Here, we show that E(2) attenuates the hypertrophic response to pressure overload in mice. This observation demonstrates that hormone replacement therapy with E(2) has direct effects on the heart and may be beneficial in the treatment of postmenopausal women to reduce cardiac hypertrophy.

Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.

In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification.

Cardiac angiotensin converting enzyme and myocardial fibrosis in the rat.

OBJECTIVE: The aim was to test the hypothesis that cardiac angiotensin converting enzyme (ACE) is related to the accumulation of fibrous tissue in the heart. METHODS: A model of tissue repair (pericardiotomy with left coronary artery ligation) was used, together with the following: quantitative in vitro autoradiography (125I-351A) to determine ACE binding density; immunohistochemistry (monoclonal ACE antibody, 9B9) to identify cells expressing ACE; and in situ hybridisation to localise cells expressing type I collagen mRNA. Age and sex matched rats receiving this operative procedure without subsequent infarction (sham operated) served as controls to those with left ventricular myocardial infarction. Serial heart sections obtained from each group at 3 days and at 1, 2, 4, and 8 weeks following operation were examined for morphological evidence of injury and inflammatory cells (haematoxylin/eosin) and fibrillar collagen (picrosirius red). RESULTS: Following myocardial infarction: (a) on day 3, neutrophils and macrophages were present at the site of infarction, while fibrillar collagen and ACE binding were not increased compared with control; (b) at week 1, fibrillar collagen and ACE binding were present at the site of infarction and became progressively more advanced at 2, 4, and 8 weeks; (c) at week 2, there was increased ACE binding in the right ventricle and interventricular septum, when perivascular fibrosis of intramural coronary arteries and microscopic scars appeared, together with endomyocardial fibrosis of the septum; (d) there was marked ACE binding in the fibrosed visceral pericardium two weeks after operation in both myocardial infarction and sham operated groups; (e) there was type I collagen mRNA expression on postoperative week 1, localised within fibroblasts or fibroblast-like cells found at infarct and fibrous tissue sites in the right ventricle, septum, and pericardium; and (f) ACE-labelled cells included fibroblast-like cells, as well as macrophages and endothelial cells. CONCLUSIONS: Thus in this model of tissue repair, marked ACE binding density is associated with fibrillar collagen formation that appears within and remote to the site of myocardial infarction, including the pericardium. Cardiac ACE, originating from type I collagen producing cells, therefore represents an integral component of fibrous tissue formation in this rat model of tissue injury.

Angiotensin converting enzyme and kininase-II-like activities in cultured valvular interstitial cells of the rat heart.

OBJECTIVE: The function of angiotensin converting enzyme (ACE) at cell sites of high collagen turnover, such as heart valves, is uncertain. The aim of this study was to assess ACE and kininase-II-like activities and collagen turnover in cultured valvular interstitial cells of the adult rat heart. METHODS: The valvular interstitial cell phenotype was determined by immunolabelling (rhodamine phalloidin, desmin, and Griffonia simplicifolia lectin), and the presence of ACE mRNA and protein was confirmed by reverse transcriptase-polymerase chain reaction analysis, ACE monoclonal antibody and in vitro autoradiography, respectively. ACE and kininase-II-like activities in valvular interstitial cells were analysed by high performance liquid chromatography. Angiotensin II (AT1) and bradykinin receptors in valvular interstitial cell membranes were examined by western immunoblotting and binding assay. Type I collagen and collagenase in valvular interstitial cell culture media were determined by ELISA and zymography, respectively. Type I collagen mRNA expression in cultured valvular interstitial cells was determined by northern blot analysis and in situ hybridisation. RESULTS: In intact valvular interstitial cells or their cell membrane we found: (1) actin microfilaments, but not desmin or lectin labelling; (2) ACE mRNA expression and binding activity; (3) conversion of angiotensin I to angiotensin II, which was completely inhibited by 50 microM lisinopril, while kinase-II-like activity exceeded ACE activity and was not inhibited by lisinopril; (4) AT1 and bradykinin receptors in valvular interstitial cell membrane preparations; (5) type I collagen mRNA expression and collagenase activity; and (6) angiotensin II induced increase in type I collagen synthesis and mRNA expression. CONCLUSIONS: Cultured valvular interstitial cells represent a nonendothelial, non-smooth-muscle cell type that expresses mRNA for ACE and type I collagen. ACE and kininase-II-like activities in valvular interstitial cells may be involved in the regulation of peptides that influence collagen turnover. Angiotensin II stimulates type I collagen synthesis and mRNA expression in these cells.

Connective tissue and repair in the heart. Potential regulatory mechanisms.

The heart is composed of highly differentiated cardiac myocytes, which constitute parenchyma, and stroma or connective tissue. Fibrillar collagen turnover in the heart and its valve leaflets, in particular, is dynamic and essential to tissue repair. Emerging evidence further suggests connective tissue is a metabolically active entity, where peptide hormones are generated and degraded and, in turn, these peptides regulate collagen turnover. This concept arose from quantitative in vitro autoradiography using an iodinated derivative of lisinopril (125I-351A) as ligand to localize angiotensin converting enzyme (ACE) binding density within the heart. A heterogeneous distribution was found: low-density ACE binding within atria and ventricles; high ACE binding density at sites of high collagen turnover, such as valve leaflets, adventitia, and fibrous tissue of diverse etiologic origins. ACE-producing cells at these latter sites were identified by monoclonal ACE antibody. They included valvular interstitial cells (VIC) and fibroblast-like cells each of which also contained alpha-smooth muscle actin and the transcript for type I collagen (in situ hybridization). Substrate utilization in cultured VIC was found to include angiotensin I and bradykinin. Angiotensin II and bradykinin receptor-ligand binding was observed in VIC and at fibrous tissue sites. Connective tissue ACE is independent of circulating angiotensin II. In vivo, fibrous tissue formation is attenuated by ACE inhibition or antagonism of AT1 receptor. Angiotensin II and bradykinin are stimulatory and inhibitory, respectively, to cultured adult cardiac fibroblast collagen synthesis suggesting a paradigm of reciprocal regulation to fibroblast collagen turnover. Stroma and its cellular constituents represent a dynamic metabolic entity that regulates its own peptide hormone composition and turnover of fibrillar collagen. These findings may provide insights that could be used to advantage to either promote or forestall fibrous tissue formation depending on the nature of cardiovascular disease.

Expression of tenascin in perifollicular connective tissue: comparison of normal scalp and alopecia areata.

Tenascin, a recently discovered extracellular matrix protein, was demonstrated in perifollicular connective tissue of normal human scalp using immunohistochemistry. Its localization was different from other well-known extracellular matrix components, like fibronectin, laminin and heparan sulphate proteoglycan. A comparison between alopecia areata and normal scalps did not reveal major qualitative differences, except for an increased expression near heavily infiltrated follicles.

Myocardial collagenase: purification and structural characterization.

Interstitial collagenase (matrix metalloproteinase-1 [MMP-1]) plays an important role in extracellular matrix turnover. Myocardial MMP-1 may contribute to tissue remodelling in the heart. Little is known about collagenase and its regulation in the myocardium. To understand better the nature of this neutral proteinase in the rat myocardium, myocardial collagenase was purified to homogeneity. The purification procedure included a gel-filtration step on Sephacryl S-200 columns and substrate affinity chromatography on type I collagen-Sepharose. Under reducing conditions, collagenase was shown by SDS-PAGE to consist of a single polypeptide chain with a molecular mass of 54 kDa. Purified interstitial collagenase demonstrated a single lytic band on zymography. This band was inhibited by 1,10-phenanthroline (a metal chelator), which indicates that the 54 kDa protein is an MMP. Using a polyclonal antibody to proMMP-1, purified collagenase was characterized by immunoblot analysis. A single band of purified interstitial collagenase was observed on Western blot analysis. This indicated that the purified proenzyme was collagenase. Sequence analysis on cyanogen bromide-digested fragments of latent MMP-1 suggested that the active site sequence of rat myocardial MMP-1 is similar to that of the rat osteoblast collagenase, human skin fibroblast collagenase and Serratia proteinase. The substrate specificity of the purified collagenase was measured against fluorescent-labelled type I collagen. It was observed that after activation, purified collagenase was capable of degrading type I collagen in a time-dependent manner. The half-time for collagen degradation was estimated to be less than 30 s. These results suggest that collagenase is present in the normal adult rat myocardium and that collagen turnover may be regulated by this neutral metalloproteinase. A simple two-step purification protocol is demonstrated for interstitial collagenase. This procedure can be used for routine MMP-1 preparation from tissue sources.

Severity of venous insufficiency is related to the density of microvascular deposition of PAI-1, uPA and von Willebrand factor.

BACKGROUND: Impaired microcirculation in chronic venous insufficiency leads to chronic inflammation and dystrophic changes of the skin, and finally to leg ulceration. The purpose of this study was to investigate in more detail coagulation and fibrinolytic protein response of the capillaries in skin biopsies of the lower extremity. PATIENTS AND METHODS: From eighteen ambulant patients with venous leg ulcer(s) (n = 8) and controls (n = 10) with various degrees of venous insufficiency according to the CEAP classification, we obtained 4 mm punch biopsies. Immunohistochemical staining with tissue derived plasminogen activator (tPA), urokinase derived plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1) and von Willebrand Factor (vWF) was performed and analyzed with bright field microscopy. RESULTS: The amount of staining with vWF (p = 0.04) and uPA (p = 0.02) showed statistically significant differences, PAI-1 (p = 0.09) and tPA (p = 0.50) showed no difference between leg ulcer and control groups. A strong proliferation of capillaries with tortuous capillary loops in the papillary dermis was seen, but no statistically significant difference (M-W test, p = 0.10) was found between the groups. Comparison between CEAP classes 0-6 showed a statistically significant increased staining pattern of vWF (p = 0.06), uPA (p = 0.02) and PAI-1 (p = 0.02), but not from tPA (p = 0.30). CONCLUSIONS: In skin biopsies of the lower extremity of patients with severe venous insufficiency increased deposition of vWF, PAI-1 and uPA were found in the capillaries. These findings point to a local imbalance in coagulation and fibrinolytic status, which might contribute to impaired microcirculation and finally to the development of venous leg ulceration.

Density is in the eye of the beholder: visual versus semi-automated assessment of breast density on standard mammograms.

OBJECTIVES: Visual inspection is generally used to assess breast density. Our study aim was to compare visual assessment of breast density of experienced and inexperienced readers with semi-automated analysis of breast density. METHODS: Breast density was assessed by an experienced and an inexperienced reader in 200 mammograms and scored according to the quantitative BI-RADS classification. Breast density was also assessed by dedicated software using a semi-automated thresholding technique. Agreement between breast density classification of both readers as well as agreement between their assessment versus the semi-automated analysis as reference standard was expressed as the weighted kappa value. RESULTS: Using the semi-automated analysis, agreement between breast density measurements of both breasts in both projections was excellent (ICC >0.9, P < 0.0001). Reproducibility of the semi-automated analysis was excellent (ICC >0.8, P < 0.0001). The experienced reader correctly classified the BI-RADS breast density classification in 58.5% of the cases. Classification was overestimated in 35.5% of the cases and underestimated in 6.0% of the cases. Results of the inexperienced reader were less accurate. Agreement between the classification of both readers versus the semi-automated analysis was considered only moderate with weighted kappa values of 0.367 (experienced reader) and 0.232 (inexperienced reader). CONCLUSION: Visual assessment of breast density on mammograms is inaccurate and observer-dependent.

Impaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodelling.

AIM: To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice. METHODS: Cardiac dimensions and function were determined echocardiographically at 8, 12, 16 and 18 weeks of age. Left ventricular pressure characteristics were measured at 18 weeks under baseline conditions and upon dobutamine infusion. RESULTS: The db/db mice were severely diabetic already at 8 weeks after birth, showing elevated fasting blood glucose levels and albuminuria. Nevertheless, echocardiography revealed no significant changes in cardiac function up to 18 weeks of age. At 18 weeks of age, left ventricular pressure characteristics were not significantly different at baseline between diabetic and control mice. However, dobutamine stress test revealed significantly attenuated cardiac inotropic and lusitropic responses in db/db mice. Post-mortem cardiac tissue analyses showed minor structural remodelling and no significant changes in gene expression levels of the sarcoplasmic reticulum calcium ATPase (SERCA2a) or beta1-adrenoceptor (beta1-AR). Moreover, the phosphorylation state of known contractile protein targets of protein kinase A (PKA) was not altered, indicating unaffected cardiac beta-adrenergic signalling activity in diabetic animals. By contrast, the substantially increased expression of uncoupling protein-3 (UCP3) and angiopoietin-like-4 (Angptl4), along with decreased phosphorylation of AMP-activated protein kinase (AMPK) in the diabetic heart, is indicative of marked changes in cardiac metabolism. CONCLUSION: db/db mice show impaired cardiac functional reserve capacity during maximal beta-adrenergic stimulation which is associated with unfavourable changes in cardiac energy metabolism.