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We sought to establish a large animal model of inherited hypertrophic cardiomyopathy (HCM) with sufficient disease severity and early penetrance for identification of novel therapeutic strategies. HCM is the most common inherited cardiac disorder affecting 1 in 250-500 people, yet few therapies for its treatment or prevention are available. A research colony of purpose-bred cats carrying the A31P mutation in MYBPC3 was founded using sperm from a single heterozygous male cat. Cardiac function in four generations was assessed by periodic echocardiography and measurement of blood biomarkers. Results showed that HCM penetrance was age-dependent, and that penetrance occurred earlier and was more severe in successive generations, especially in homozygotes. Homozygosity was also associated with progression from preclinical to clinical disease. A31P homozygous cats represent a heritable model of HCM with early disease penetrance and a severe phenotype necessary for interventional studies aimed at altering disease progression. The occurrence of a more severe phenotype in later generations of cats, and the occasional occurrence of HCM in wildtype cats suggests the presence of at least one gene modifier or a second causal variant in this research colony that exacerbates the HCM phenotype when inherited in combination with the A31P mutation., (© 2023. The Author(s).)

Biomedical scientists aim to contribute to further understanding of disease pathogenesis and to develop new diagnostic and therapeutic tools that relieve disease burden. Yet the majority of biomedical scientists do not develop their academic career or professional identity as "translational scientists," and are not actively involved in the continuum from scientific concept to development of new strategies that change medical practice. The collaborative nature of translational medicine and the lengthy process of bringing innovative findings from bench to bedside conflict with established pathways of building a career in academia. This collaborative approach also poses a problem for evaluating individual contributions and progress. The traditional evaluation of scientific success measured by the impact and number of publications and grants scientists achieve is inadequate when the product is a team effort that may take decades to complete. Further, where scientists are trained to be independent thinkers and to establish unique scientific niches, translational medicine depends on combining individual insights and strengths for the greater good. Training programs that are specifically geared to prepare scientists for a career in translational medicine are not widespread. In addition, the legal, regulatory, scientific and clinical infrastructure and support required for translational research is often underdeveloped in academic institutions and funding organizations, further discouraging the development and success of translational scientists in the academic setting. In this perspective we discuss challenges and potential solutions that could allow for physicians, physician scientists and basic scientists to develop a professional identity and a fruitful career in translational medicine.

Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional differences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic differences and their contribution to cell type and depot-specific function. We found that DNA methylomes were notably distinct between different adipocyte depots and were associated with differential gene expression within pathways fundamental to adipocyte function. Most striking differential methylation was found at transcription factor and developmental genes. Our findings highlight the importance of developmental origins in the function of different fat depots.

Cardiac myosin binding protein-C (cMyBP-C) is an essential regulatory protein required for proper systolic contraction and diastolic relaxation. We previously showed that N'-terminal domains of cMyBP-C stimulate contraction by binding to actin and activating the thin filament in vitro. In principle, thin filament activating effects of cMyBP-C could influence contraction and relaxation rates, or augment force amplitude in vivo. cMyBP-C binding to actin could also contribute to an internal load that slows muscle shortening velocity as previously hypothesized. However, the functional significance of cMyBP-C binding to actin has not yet been established in vivo. We previously identified an actin binding site in the regulatory M-domain of cMyBP-C and described two missense mutations that either increased (L348P) or decreased (E330K) binding affinity of recombinant cMyBP-C N'-terminal domains for actin in vitro. Here we created transgenic mice with either the L348P or E330K mutations to determine the functional significance of cMyBP-C binding to actin in vivo. Results showed that enhanced binding of cMyBP-C to actin in L348P-Tg mice prolonged the time to end-systole and slowed relaxation rates. Reduced interactions between cMyBP-C and actin in E330K-Tg mice had the opposite effect and significantly shortened the duration of ejection. Neither mouse model displayed overt systolic dysfunction, but L348P-Tg mice showed diastolic dysfunction presumably resulting from delayed relaxation. We conclude that cMyBP-C binding to actin contributes to sustained thin filament activation at the end of systole and during isovolumetric relaxation. These results provide the first functional evidence that cMyBP-C interactions with actin influence cardiac function in vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background/objectives: There is increasing evidence that metabolic diseases originate in early life, and epigenetic changes have been implicated as key drivers of this early life programming. This led to the hypothesis that epigenetic marks present at birth may predict an individual's future risk of obesity and type 2 diabetes. In this study, we assessed whether epigenetic marks in blood of newborn children were associated with body mass index (BMI) and insulin sensitivity later in childhood., Subjects/methods: DNA methylation was measured in neonatal blood spot samples of 438 children using the Illumina Infinium 450 k BeadChip. Associations were assessed between DNA methylation at birth and BMI z-scores, body fat mass, fasting plasma glucose, insulin and homeostatic model assessment of insulin resistance (HOMA-IR) at age 5 years, as well as birth weight, maternal BMI and smoking status., Results: No individual methylation sites at birth were associated with obesity or insulin sensitivity measures at 5 years. DNA methylation in 69 genomic regions at birth was associated with BMI z-scores at age 5 years, and in 63 regions with HOMA-IR. The methylation changes were generally small (<5%), except for a region near the non-coding RNA nc886 (VTRNA2-1) where a clear link between methylation status at birth and BMI in childhood was observed (P=0.001). Associations were also found between DNA methylation, maternal smoking and birth weight., Conclusions: We identified a number of DNA methylation regions at birth that were associated with obesity or insulin sensitivity measurements in childhood. These findings support the mounting evidence on the role of epigenetics in programming of metabolic health. Whether many of these small changes in DNA methylation are causally related to the health outcomes, and of clinical relevance, remains to be determined, but the nc886 region represents a promising obesity risk marker that warrants further investigation.

Developmental origins of health and disease (DOHaD) is the study of how the early life environment can impact the risk of chronic diseases from childhood to adulthood and the mechanisms involved. Epigenetic modifications such as DNA methylation, histone modifications and non-coding RNAs are involved in mediating how early life environment impacts later health. This review is a summary of the Epigenetics and DOHaD workshop held at the 2016 DOHaD Society of Australia and New Zealand Conference. Our extensive knowledge of how the early life environment impacts later risk for chronic disease would not have been possible without animal models. In this review we highlight some animal model examples that demonstrate how an adverse early life exposure results in epigenetic and gene expression changes that may contribute to increased risk of chronic disease later in life. Type 2 diabetes and cardiovascular disease are chronic diseases with an increasing incidence due to the increased number of children and adults that are obese. Epigenetic changes such as DNA methylation have been shown to be associated with metabolic health measures and potentially predict future metabolic health status. Although more difficult to elucidate in humans, recent studies suggest that DNA methylation may be one of the epigenetic mechanisms that mediates the effects of early life exposures on later life risk of obesity and obesity related diseases. Finally, we discuss the role of the microbiome and how it is a new player in developmental programming and mediating early life exposures on later risk of chronic disease.

Background: Evidence is accumulating that nutritional exposures in utero can influence health outcomes in later life. Animal studies and human epidemiological studies have implicated epigenetic modifications as playing a key role in this process, but there are limited data from large well-controlled human intervention trials. This study utilized a large double-blind randomized placebo-controlled trial to test whether a defined nutritional exposure in utero, in this case docosahexaenoic acid (DHA), could alter the infant epigenome. Pregnant mothers consumed DHA-rich fish oil (800 mg DHA/day) or placebo supplements from 20 weeks' gestation to delivery. Blood spots were collected from the children at birth ( n  = 991) and blood leukocytes at 5 years ( n  = 667). Global DNA methylation was measured in all samples, and Illumina HumanMethylation450K BeadChip arrays were used for genome-wide methylation profiling in a subset of 369 children at birth and 65 children at 5 years., Results: There were no differences in global DNA methylation levels between the DHA and control group either at birth or at 5 years, but we identified 21 differentially methylated regions (DMRs) at birth, showing small DNA methylation differences (<5%) between the treatment groups, some of which seemed to persist until 5 years. The number of DMRs at birth was greater in males (127 DMRs) and in females (72 DMRs) separately, indicating a gender-specific effect., Conclusion: Maternal DHA supplementation during the second half of pregnancy had small effects on DNA methylation of infants. While the potential functional significance of these changes remains to be determined, these findings further support the role of epigenetic modifications in developmental programming in humans and point the way for future studies., Trial Registration: Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12605000569606 and ACTRN12611001127998.

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Scope: Early perturbations in vascular health can be detected by imposing subjects to a high fat (HF) challenge and measure response capacity. Subtle responses can be determined by assessment of whole-genome transcriptional changes. We aimed to magnify differences in health by comparing gene-expression changes in peripheral blood mononuclear cells toward a high MUFA or saturated fatty acids (SFA) challenge between subjects with different cardiovascular disease risk profiles and to identify fatty acid specific gene-expression pathways., Methods and Results: In a cross-over study, 17 lean and 15 obese men (50-70 years) received two 95 g fat shakes, high in SFAs or MUFAs. Peripheral blood mononuclear cell gene-expression profiles were assessed fasted and 4-h postprandially. Comparisons were made between groups and shakes. During fasting, 294 genes were significantly differently expressed between lean and obese. The challenge increased differences to 607 genes after SFA and 2516 genes after MUFA. In both groups, SFA decreased expression of cholesterol biosynthesis and uptake genes and increased cholesterol efflux genes. MUFA increased inflammatory genes and PPAR-α targets involved in β-oxidation., Conclusion: Based upon gene-expression changes, we conclude that an HF challenge magnifies differences in health, especially after MUFA. Our findings also demonstrate how SFAs and MUFAs exert distinct effects on lipid handling pathways in immune cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The increased prevalence of obesity and related comorbidities is a major public health problem. While genetic factors undoubtedly play a role in determining individual susceptibility to weight gain and obesity, the identified genetic variants only explain part of the variation. This has led to growing interest in understanding the potential role of epigenetics as a mediator of gene-environment interactions underlying the development of obesity and its associated comorbidities. Initial evidence in support of a role of epigenetics in obesity and type 2 diabetes mellitus (T2DM) was mainly provided by animal studies, which reported epigenetic changes in key metabolically important tissues following high-fat feeding and epigenetic differences between lean and obese animals and by human studies which showed epigenetic changes in obesity and T2DM candidate genes in obese/diabetic individuals. More recently, advances in epigenetic methodologies and the reduced cost of epigenome-wide association studies (EWAS) have led to a rapid expansion of studies in human populations. These studies have also reported epigenetic differences between obese/T2DM adults and healthy controls and epigenetic changes in association with nutritional, weight loss, and exercise interventions. There is also increasing evidence from both human and animal studies that the relationship between perinatal nutritional exposures and later risk of obesity and T2DM may be mediated by epigenetic changes in the offspring. The aim of this review is to summarize the most recent developments in this rapidly moving field, with a particular focus on human EWAS and studies investigating the impact of nutritional and lifestyle factors (both pre- and postnatal) on the epigenome and their relationship to metabolic health outcomes. The difficulties in distinguishing consequence from causality in these studies and the critical role of animal models for testing causal relationships and providing insight into underlying mechanisms are also addressed. In summary, the area of epigenetics and metabolic health has seen rapid developments in a short space of time. While the outcomes to date are promising, studies are ongoing, and the next decade promises to be a time of productive research into the complex interactions between the genome, epigenome, and environment as they relate to metabolic disease.

Background: Recent technological advances in epigenome profiling have led to an increasing number of studies investigating the role of the epigenome in obesity. There is also evidence that environmental exposures during early life can induce persistent alterations in the epigenome, which may lead to an increased risk of obesity later in life., Method: This paper provides a systematic review of studies investigating the association between obesity and either global, site-specific or genome-wide methylation of DNA. Studies on the impact of pre- and postnatal interventions on methylation and obesity are also reviewed. We discuss outstanding questions, and introduce EpiSCOPE, a multidisciplinary research program aimed at increasing the understanding of epigenetic changes in emergence of obesity., Results: An electronic search for relevant articles, published between September 2008 and September 2013 was performed. From the 319 articles identified, 46 studies were included and reviewed. The studies provided no consistent evidence for a relationship between global methylation and obesity. The studies did identify multiple obesity-associated differentially methylated sites, mainly in blood cells. Extensive, but small, alterations in methylation at specific sites were observed in weight loss intervention studies, and several associations between methylation marks at birth and later life obesity were found., Conclusions: Overall, significant progress has been made in the field of epigenetics and obesity and the first potential epigenetic markers for obesity that could be detected at birth have been identified. Eventually this may help in predicting an individual's obesity risk at a young age and opens possibilities for introducing targeted prevention strategies. It has also become clear that several epigenetic marks are modifiable, by changing the exposure in utero, but also by lifestyle changes in adult life, which implies that there is the potential for interventions to be introduced in postnatal life to modify unfavourable epigenomic profiles.

Mutations in the MYBPC3 gene, encoding cardiac myosin binding protein C (cMyBP-C) are frequent causes of hypertrophic cardiomyopathy (HCM). Previously, we have presented evidence for reduced cMyBP-C expression (haploinsufficiency), in patients with a truncation mutation in MYBPC3. In mice, lacking cMyBP-C cross-bridge kinetics was accelerated. In this study, we investigated whether cross-bridge kinetics was altered in myectomy samples from HCM patients harboring heterozygous MYBPC3 mutations (MYBPC3mut). Isometric force and the rate of force redevelopment (k tr) at different activating Ca(2+) concentrations were measured in mechanically isolated Triton-permeabilized cardiomyocytes from MYBPC3mut (n = 18) and donor (n = 7) tissue. Furthermore, the stretch activation response of cardiomyocytes was measured in tissue from eight MYBPC3mut patients and five donors to assess the rate of initial force relaxation (k 1) and the rate and magnitude of the transient increase in force (k 2 and P 3, respectively) after a rapid stretch. Maximal force development of the cardiomyocytes was reduced in MYBPC3mut (24.5 ± 2.3 kN/m(2)) compared to donor (34.9 ± 1.6 kN/m(2)). The rates of force redevelopment in MYBPC3mut and donor over a range of Ca(2+) concentrations were similar (k tr at maximal activation: 0.63 ± 0.03 and 0.75 ± 0.09 s(-1), respectively). Moreover, the stretch activation parameters did not differ significantly between MYBPC3mut and donor (k 1: 8.5±0.5 and 8.8 ± 0.4 s(-1); k 2: 0.77 ± 0.06 and 0.74 ± 0.09 s(-1); P 3: 0.08 ± 0.01 and 0.09 ± 0.01, respectively). Incubation with protein kinase A accelerated k 1 in MYBPC3mut and donor to a similar extent. Our experiments indicate that, at the cMyBP-C expression levels in this patient group (63 ± 6 % relative to donors), cross-bridge kinetics are preserved and that the depressed maximal force development is not explained by perturbation of cross-bridge kinetics.

Scope: Circulating oxylipins may affect peripheral tissues and are assumed to play an important role in endothelial function. They are esterified in triglyceride-rich lipoproteins that are increased after a high-fat (HF) meal, depending on BMI and fatty acid (FA) type. Yet, it is unclear which oxylipins appear in circulation after HF meals differing in FA composition., Methods and Results: In a double-blind randomized crossover challenge study, we characterized the postprandial oxylipin response after different HF challenges in lean and obese men receiving HF milkshakes, either high in saturated FAs (SFA), monounsaturated FAs (MUFA), or omega 3 (n-3) polyunsaturated FAs (PUFA). Plasma oxylipin profiles were significantly altered at 2 and 4 h after shake consumption when compared to baseline. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) derived oxylipins increased after n-3 PUFA shake consumption. MUFA shake consumption increased levels of cytochrome P450 mediated oxylipins. SFA shake consumption led to strong increases in linoleic acid (LA) derived HODEs. No differences were observed between lean and obese individuals at baseline and after any shake consumption., Conclusion: We are the first demonstrating acute effects on circulating oxylipins after HF meal challenges. These changes were strongly influenced by different dietary FAs and may affect endothelial function., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Myosin binding protein-C (MyBP-C) was first discovered as an impurity during the purification of myosin from skeletal muscle. However, soon after its discovery, MyBP-C was also shown to bind actin. While the unique functional implications for a protein that could cross-link thick and thin filaments together were immediately recognized, most early research nonetheless focused on interactions of MyBP-C with the thick filament. This was in part because interactions of MyBP-C with the thick filament could adequately explain most (but not all) effects of MyBP-C on actomyosin interactions and in part because the specificity of actin binding was uncertain. However, numerous recent studies have now established that MyBP-C can indeed bind to actin through multiple binding sites, some of which are highly specific. Many of these interactions involve critical regulatory domains of MyBP-C that are also reported to interact with myosin. Here we review current evidence supporting MyBP-C interactions with actin and discuss these findings in terms of their ability to account for the functional effects of MyBP-C. We conclude that the influence of MyBP-C on muscle contraction can be explained equally well by interactions with actin as by interactions with myosin. However, because data showing that MyBP-C binds to either myosin or actin has come almost exclusively from in vitro biochemical studies, the challenge for future studies is to define which binding partner(s) MyBP-C interacts with in vivo.

Aims: Familial hypertrophic cardiomyopathy (HCM), frequently caused by sarcomeric gene mutations, is characterized by cellular dysfunction and asymmetric left-ventricular (LV) hypertrophy. We studied whether cellular dysfunction is due to an intrinsic sarcomere defect or cardiomyocyte remodelling., Methods and Results: Cardiac samples from 43 sarcomere mutation-positive patients (HCMmut: mutations in thick (MYBPC3, MYH7) and thin (TPM1, TNNI3, TNNT2) myofilament genes) were compared with 14 sarcomere mutation-negative patients (HCMsmn), eight patients with secondary LV hypertrophy due to aortic stenosis (LVHao) and 13 donors. Force measurements in single membrane-permeabilized cardiomyocytes revealed significantly lower maximal force generating capacity (Fmax) in HCMmut (21 ± 1 kN/m²) and HCMsmn (26 ± 3 kN/m²) compared with donor (36 ± 2 kN/m²). Cardiomyocyte remodelling was more severe in HCMmut compared with HCMsmn based on significantly lower myofibril density (49 ± 2 vs. 63 ± 5%) and significantly higher cardiomyocyte area (915 ± 15 vs. 612 ± 11 μm²). Low Fmax in MYBPC3mut, TNNI3mut, HCMsmn, and LVHao was normalized to donor values after correction for myofibril density. However, Fmax was significantly lower in MYH7mut, TPM1mut, and TNNT2mut even after correction for myofibril density. In accordance, measurements in single myofibrils showed very low Fmax in MYH7mut, TPM1mut, and TNNT2mut compared with donor (respectively, 73 ± 3, 70 ± 7, 83 ± 6, and 113 ± 5 kN/m²). In addition, force was lower in MYH7mut cardiomyocytes compared with MYBPC3mut, HCMsmn, and donor at submaximal [Ca²⁺]., Conclusion: Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca²⁺]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations.

BMI and fatty acid type affect postprandial metabolic TG responses, but whether these factors also affect vascular, inflammatory, and leukocyte adherence responses remains unclear. We therefore compared those postprandial responses between lean and obese men after 3 high-fat challenges differing in fatty acid composition. In a crossover double-blind study, 18 lean (BMI: 18-25 kg/m(2)) and 18 obese (BMI >29 kg/m(2)) middle-aged men received 3 isocaloric high-fat milkshakes containing 95 g fat (88% of energy), either high in SFAs (54% of energy/total fat), MUFAs (83% of energy/total fat), or n3 (omega-3) PUFAs (40% of energy/total fat). Hemodynamics, augmentation index (AIX), leukocyte cell surface adhesion markers, and plasma cytokines involved in vascular adherence, coagulation, and inflammation were measured before and after consumption of the milkshakes. In both groups and after all shakes were consumed, AIX decreased; plasma soluble intercellular adhesion molecule (sICAM) 1, sICAM3, soluble vascular cell adhesion molecule (sVCAM) 1, and interleukin-8 increased; monocyte CD11a, CD11b, and CD621 expression increased; neutrophil CD11a, CD11b, and CD621 expression increased; and lymphocyte CD62l expression increased (P < 0.05). Lymphocyte CD11a and CD11b expression decreased in lean participants after consumption of all shakes but did not change in obese participants (P < 0.05). Obese participants had a less pronounced decrease in heart rate after the consumption of all shakes (P < 0.05). MUFA consumption induced a more pronounced decrease in blood pressure and AIX compared with the other milkshakes in both lean and obese participants (P < 0.05). High-fat consumption initiates an activated state of cellular adherence and an atherogenic milieu. This response was independent of fatty acid type consumed or of being lean or obese, despite the clear differences in postprandial TG responses between the groups and different milkshakes. These findings suggest that in addition to increased TGs, other mechanisms are involved in the high-fat consumption-induced activated state of cellular adherence.

Background: The ability of subjects to respond to nutritional challenges can reflect the flexibility of their biological system. Nutritional challenge tests could be used as an indicator of health status but more knowledge on metabolic and immune responses of different subjects to nutritional challenges is needed. The aim of this study was to compare the responses to high-fat challenges varying in fat type in subjects with different metabolic risk phenotypes., Methodology/principal Findings: In a cross-over design 42 men (age 50-70 y) consumed three high-fat shakes containing saturated fat (SFA), monounsaturated fat (MUFA) or n-3 polyunsaturated (PUFA). Men were selected on BMI and health status (lean, obese or obese diabetic) and phenotyped with MRI for adipose tissue distribution. Before and 2 and 4 h after shake consumption blood was drawn for measurement of expression of metabolic and inflammation-related genes in peripheral blood mononuclear cells (PBMCs), plasma triglycerides (TAG), glucose, insulin, cytokines and ex vivo PBMC immune response capacity. The MUFA and n-3 PUFA challenge, compared to the SFA challenge, induced higher changes in expression of inflammation genes MCP1 and IL1β in PBMCs. Obese and obese diabetic subjects had different PBMC gene expression and metabolic responses to high-fat challenges compared to lean subjects. The MUFA challenge induced the most pronounced TAG response, mainly in obese and obese diabetic subjects., Conclusion/significance: The PBMC gene expression response and metabolic response to high-fat challenges were affected by fat type and metabolic risk phenotype. Based on our results we suggest using a MUFA challenge to reveal differences in response capacity of subjects., Trial Registration: ClinicalTrials.gov NCT00977262.

Background: Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation., Methods and Results: Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3(mut); n=17), mutation negative HCM patients without an identified sarcomere mutation (HCM(mn); n=11), and nonfailing donors (n=12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3(mut) by 33±5%, and similar in HCM(mn) compared with donor. cMyBP-C phosphorylation in MYBPC3(mut) was similar to donor, whereas it was significantly lower in HCM(mn). Troponin I phosphorylation was lower in both patient groups compared with donor. Force measurements in single permeabilized cardiomyocytes demonstrated comparable sarcomeric dysfunction in both patient groups characterized by lower maximal force generating capacity in MYBPC3(mut) and HCM(mn,) compared with donor (26.4±2.9, 28.0±3.7, and 37.2±2.3 kN/m(2), respectively), and higher myofilament Ca(2+)-sensitivity (EC(50)=2.5±0.2, 2.4±0.2, and 3.0±0.2 μmol/L, respectively). The sarcomere length-dependent increase in Ca(2+)-sensitivity was significantly smaller in both patient groups compared with donor (ΔEC(50): 0.46±0.04, 0.37±0.05, and 0.75±0.07 μmol/L, respectively). Protein kinase A treatment restored myofilament Ca(2+)-sensitivity and length-dependent activation in both patient groups to donor values., Conclusions: Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression.

Hypertrophic cardiomyopathy (HCM) is a familial disorder characterized by left ventricular hypertrophy in the absence of other cardiac or systemic disease likely to cause this hypertrophy. HCM is considered a disease of the sarcomere as most causal mutations are identified in genes encoding sarcomeric proteins, although several other disorders have also been linked to the HCM phenotype. The clinical course of HCM is characterized by a large inter- and intrafamilial variability, ranging from severe symptomatic HCM to asymptomatic individuals. The general picture emerges that the underlying pathophysiology of HCM is complex and still scarcely clarified. Recent findings indicated that both functional and morphological (macroscopic and microscopic) changes of the HCM muscle are present before the occurrence of HCM phenotype. This review aims to provide an overview of the myocardial alterations that occur during the gradual process of wall thickening in HCM on a myofilament level, as well as the structural and functional abnormalities that can be observed in genetically affected individuals prior to the development of HCM with state of the art imaging techniques, such as tissue Doppler echocardiography and cardiovascular magnetic resonance imaging. Additionally, present and future therapeutic options will be briefly discussed., (© 2010 The Authors. European Journal of Clinical Investigation © 2010 Stichting European Society for Clinical Investigation Journal Foundation.)

Background: Biomarkers that allow detection of the onset of disease are of high interest since early detection would allow intervening with lifestyle and nutritional changes before the disease is manifested and pharmacological therapy is required. Our study aimed to improve the phenotypic characterization of overweight but apparently healthy subjects and to identify new candidate profiles for early biomarkers of obesity-related diseases such as cardiovascular disease and type 2 diabetes., Methodology/principal Findings: In a population of 56 healthy, middle-aged overweight subjects Body Mass Index (BMI), fasting concentration of 124 plasma proteins and insulin were determined. The plasma proteins are implicated in chronic diseases, inflammation, endothelial function and metabolic signaling. Random Forest was applied to select proteins associated with BMI and plasma insulin. Subsequently, the selected proteins were analyzed by clustering methods to identify protein clusters associated with BMI and plasma insulin. Similar analyses were performed for a second population of 20 healthy, overweight older subjects to verify associations found in population I. In both populations similar clusters of proteins associated with BMI or insulin were identified. Leptin and a number of pro-inflammatory proteins, previously identified as possible biomarkers for obesity-related disease, e.g. Complement 3, C Reactive Protein, Serum Amyloid P, Vascular Endothelial Growth Factor clustered together and were positively associated with BMI and insulin. IL-3 and IL-13 clustered together with Apolipoprotein A1 and were inversely associated with BMI and might be potential new biomarkers. CONCLUSION/ SIGNIFICANCE: We identified clusters of plasma proteins associated with BMI and insulin in healthy populations. These clusters included previously reported biomarkers for obesity-related disease and potential new biomarkers such as IL-3 and IL-13. These plasma protein clusters could have potential applications for improved phenotypic characterization of volunteers in nutritional intervention studies or as biomarkers in the early detection of obesity-linked disease development and progression.

Background and Aims: Diets high in monounsaturated fatty acids (MUFA) such as a Mediterranean diet may reduce the risk of cardiovascular diseases by improving insulin sensitivity and serum lipids. Besides being high in MUFA, a Mediterranean diet also contains abundant plant foods, moderate wine and low amounts of meat and dairy products, which may also play a role. We compared the effects of a high MUFA-diet with a diet high in saturated fatty acids (SFA) and the additional effect of a Mediterranean diet on insulin sensitivity and serum lipids., Methods and Results: A randomized parallel controlled-feeding trial was performed, in 60 non-diabetics (40-65 y) with mild abdominal obesity. After a two week run-in diet high in SFA (19 energy-%), subjects were allocated to a high MUFA-diet (20 energy-%), a Mediterranean diet (MUFA 21 energy-%), or the high SFA-diet, for eight weeks. The high MUFA and the Mediterranean diet did not affect fasting insulin concentrations. The high MUFA-diet reduced total cholesterol (-0.41 mmol/L, 95% CI -0.74, -0.09) and LDL-cholesterol (-0.38 mmol/L, 95% CI -0.65, -0.11) compared with the high SFA-diet, but not triglyceride concentrations. The Mediterranean diet increased HDL-cholesterol concentrations (+0.09 mmol/L, 95% CI 0.0, 0.18) and reduced the ratio of total cholesterol/HDL-cholesterol (-0.39, 95% CI -0.62, -0.16) compared with the high MUFA-diet., Conclusion: Replacing a high SFA-diet with a high MUFA or a Mediterranean diet did not affect insulin sensitivity, but improved serum lipids. The Mediterranean diet was most effective, it reduced total and LDL-cholesterol, and also increased HDL-cholesterol and reduced total cholesterol/HDL-cholesterol ratio., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Cardiomyocyte contraction is regulated by phosphorylation of sarcomeric proteins. Throughout the heart regional and transmural differences may exist in protein phosphorylation. In addition, phosphorylation of sarcomeric proteins is altered in cardiac disease. Heterogeneity in protein phosphorylation may be larger in hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) as it may be caused by multiple mutations in genes encoding different sarcomeric proteins. Moreover, HCM is characterized by asymmetric remodelling of the heart. In the present study we assessed if local differences in sarcomeric protein phosphorylation are more evident in primary HCM or DCM than in non-failing donors. Thereto, phosphorylation of the two main target proteins of the beta-adrenergic receptor pathway, troponin I (cTnI) and myosin binding protein C (cMyBP-C) was analysed in different parts in the free left ventricular wall of end-stage failing HCM and DCM patients and donors obtained during transplant surgery. Intra-patient variability in protein phosphorylation within tissue samples of approximately 2 g wet weight was comparable between donor, HCM and DCM samples and could partly be attributed to the precision of the technique. Thus, our data indicate that within the precision of the measurements small, biopsy-sized cardiac tissue samples are representative for the region of the free left ventricular wall from which they were obtained.

Background: Changes in dietary fat composition could lower the risk of developing metabolic syndrome. Adipose tissue is an interesting tissue in this respect because of its role in lipid metabolism and inflammation., Objective: Our objective was to investigate the effect of a saturated fatty acid (SFA)- and a monounsaturated fatty acid (MUFA)-rich diet on insulin sensitivity, serum lipids, and gene expression profiles of adipose tissue in subjects at risk of metabolic syndrome., Design: A parallel controlled-feeding trial was conducted in 20 abdominally overweight subjects. Subjects received an SFA diet or a MUFA diet for 8 wk. Plasma and subcutaneous adipose tissue samples were obtained, and insulin sensitivity was measured by using a hyperinsulinemic-euglycemic clamp. Adipose tissue samples underwent whole-genome microarray and histologic analysis. Plasma and adipose tissue fatty acid composition and concentrations of serum cholesterol and plasma cytokine were determined., Results: Consumption of the SFA diet resulted in increased expression of genes involved in inflammation processes in adipose tissue, without changes in morphology or insulin sensitivity. The MUFA diet led to a more antiinflammatory gene expression profile, which was accompanied by a decrease in serum LDL-cholesterol concentrations and an increase in plasma and adipose tissue oleic acid content., Conclusions: Consumption of an SFA diet resulted in a proinflammatory "obesity-linked" gene expression profile, whereas consumption of a MUFA diet caused a more antiinflammatory profile. This suggests that replacement of dietary SFA with MUFA could prevent adipose tissue inflammation and may reduce the risk of inflammation-related diseases such as metabolic syndrome. This trial was registered at clinicaltrials.gov as NCT00405197.

Background: Patients on long-term highly active antiretroviral therapy (HAART) were studied to determine persistence, drug resistance development, and evolution of HIV-1 proviral DNA., Methods: Peripheral blood mononuclear cells were obtained by large volume blood drawn (500 mL) from 8 clinically successfully treated patients who had received uninterrupted HAART for up to 8.9 years. HIV-1 load was determined by Taqman real-time polymerase chain reaction. Drug resistance mutations were determined by sequencing and ultrasensitive, allele-specific, reverse transcriptase (RT)-polymerase chain reaction., Results: HIV-1 DNA load was significantly higher in aged memory (CD45RO CD57) when compared with memory (CD45RO CD57) and naive (CD27 CD45RO) CD4 T cells after HAART. Sequencing revealed no major drug resistance mutations in protease in all patients and appearance of resistance mutations in RT in just 1 patient. In 1 of 5 patients with undetectable viremia during treatment, RT M184 substitutions were detected. Phylogenetic analysis showed short genetic distances between patient sequences., Conclusions: During long-term HAART, HIV-1 is able to persist in terminally differentiated CD4 T cells as proviral DNA. Viral evolution was restricted, and in 80% of the patients with undetectable viremia, no sign of viral replication could be detected.

Background: Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3(mut))., Methods and Results: Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864&#95;2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3(mut). Protein expression of cMyBP-C was significantly reduced in MYBPC3(mut) by 33+/-5%. Cardiac MyBP-C phosphorylation in MYBPC3(mut) samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84+/-5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the beta-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3(mut) (20.2+/-2.7 kN/m(2)) compared with donor (34.5+/-1.1 kN/m(2)). Moreover, Ca(2+) sensitivity was higher in MYBPC3(mut) (pCa(50)=5.62+/-0.04) than in donor (pCa(50)=5.54+/-0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic beta-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca(2+) sensitivity between MYBPC3(mut) (pCa(50)=5.46+/-0.03) and donor (pCa(50)=5.48+/-0.02)., Conclusions: Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca(2+) sensitivity in MYBPC3(mut) is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Many changes in morphology, biochemical properties and myocyte function occur during development to heart failure. Most changes may be compensatory, yet unable to prevent cardiac dysfunction in the long run. This illustrates that it is important to carefully dissect the disease causing modifications from cardiac adaptation, in order to obtain a better understanding of the pathophysiological processes leading to heart failure.

Background: Dysregulation of plasma lipoprotein levels may increase the risk for atherosclerosis. Recently, angiopoietin-like protein 4, also known as fasting-induced adipose factor Fiaf, was uncovered as a novel modulator of plasma lipoprotein metabolism. Here we take advantage of the fasting-dependent phenotype of Angptl4-transgenic (Angptl4-Tg) mice to better characterize the metabolic function of Angptl4., Methods and Results: In 24-hour fasted mice, Angptl4 overexpression increased plasma triglycerides (TG) by 24-fold, which was attributable to elevated VLDL-, IDL/LDL- and HDL-TG content. Angptl4 overexpression decreased post-heparin LPL activity by stimulating conversion of endothelial-bound LPL dimers to circulating LPL monomers. In fasted but not fed state, Angptl4 overexpression severely impaired LPL-dependent plasma TG and cholesteryl ester clearance and subsequent uptake of fatty acids and cholesterol into tissues. Consequently, hepatic cholesterol content was significantly decreased, leading to universal upregulation of cholesterol and fatty acid synthesis pathways and increased rate of cholesterol synthesis., Conclusions: The hypertriglyceridemic effect of Angptl4 is attributable to inhibition of LPL-dependent VLDL lipolysis by converting LPL dimers to monomers, and Angptl4 upregulates cholesterol synthesis in liver secondary to inhibition of LPL- and HL-dependent hepatic cholesterol uptake.

The FHH (fawn-hooded hypertensive) rat is a model of hypertension-associated chronic kidney damage. Five interacting quantitative trait loci (QTLs), named Rf-1-Rf-5, determine the high renal susceptibility. The aim of the present study was to investigate a possible interaction between Rf-1 and Rf-3. Differences in renal susceptibility between ACI (August x Copenhagen Irish) controls, Rf-1A and Rf-3 single congenics, and Rf-1A+3 double congenic rats were assessed using four different treatments: two-kidney control (2K), 2K plus N(omega)-nitro-L-arginine methyl ester (L-NAME)-induced hypertension (2K+L-NAME), unilateral nephrectomy (UNX), and UNX plus L-NAME-induced hypertension (UNX+L-NAME). Proteinuria (UPV) and systolic blood pressure (SBP) were assessed after 6, 12, and 18 weeks, while the incidence of glomerulosclerosis (%FGS) was determined at the end of the experiment. In a separate experiment, renal autoregulation was assessed in 13-15-week old 2K rats of all four strains. Compared to ACI rats, small increases in renal susceptibility were found in Rf-1A and Rf-3 single congenics following 2K+L-NAME, UNX, and UNX+L-NAME treatments. However, in the Rf-1A+3 double congenics, a major increase in renal susceptibility was found with all four treatments. Both Rf-1A and Rf-1A+3 congenic rats had an impaired renal autoregulation. In contrast, the Rf-3 had a normal autoregulation, similar to that of the ACI rat. These findings indicate that Rf-1 and Rf-3 alone slightly increase the susceptibility to the development of renal damage. However, a synergistic interaction between these two QTLs markedly enhances renal susceptibility. In contrast to the Rf-1 region, the Rf-3 region does not carry genes influencing renal autoregulation.

Background: Previous studies showed that combining the Rf-1 and Rf-3 or Rf-4 QTLs of FHH induced synergistic interactions markedly enhancing renal susceptibility. The present study aimed to determine the presence of such interaction between the Rf-1 and Rf-5 QTLs., Methods: Renal damage susceptibility was assessed in Rf-1B, Rf-1B+5, Rf-1B+4 congenics and ACI control rats in four situations: two-kidney control (2K), unilateral nephrectomy (UNX), L-NAME-induced hypertension (2K+L-NAME) and UNX+L-NAME. Albuminuria (UAV) and systolic blood pressure (SBP) were measured during 18 weeks of follow-up. In separate experiments, renal autoregulation was assessed in 2K rats., Results: In all four situations, Rf-1B+4 rats developed more severe UAV than ACI, Rf-1B and Rf-1B+5. There were no significant differences in UAV between Rf-1B and Rf-1B+5 rats. In the 2K and UNX situation no differences in SBP were noted between all four strains. With 2K+L-NAME and UNX+L-NAME treatment, SBP in double congenics was higher than that of ACI and Rf-1B rats. Renal autoregulation was similarly impaired in all three congenic strains., Conclusion: We conclude that the Rf-5 region, alone or in the presence of Rf-1B, does not affect the development of renal damage. We cannot substantiate that the Rf-5 region contains genes influencing renal damage susceptibility., (Copyright 2006 S. Karger AG, Basel.)

Background: Five quantitative trait loci (QTLs), Rf-1 to Rf-5, were found in Fawn-Hooded hypertensive (FHH) rats influencing susceptibility to renal damage. Previously, we found that single transfer of the Rf-1 QTL from FHH rats onto the renal-resistant August x Copenhagen Irish (ACI) strain caused a small increase in renal susceptibility. To investigate the separate role of the Rf-4 QTL and its interaction with Rf-1, we generated a single congenic strain carrying Rf-4 and a double congenic carrying both Rf-1 and Rf-4., Methods: Differences in renal susceptibility between ACI, Rf-1A, and Rf-4 single congenics and Rf-1A+4 double congenics were assessed using four different treatments: control (two-kidney), two-kidney with l-arginine analogue N-nitro-l-arginine methyl ester (L-NAME)-induced hypertension, unilateral nephrectomy, and unilateral nephrectomy + L-NAME. In separate experiments, renal blood flow (RBF) autoregulation was compared between two-kidney ACI and congenic rats., Results: Compared to ACI, Rf-1A rats developed more renal damage, while Rf-4 rats did not. The most severe renal damage was found in the Rf-1A+4 double congenic rats. Analysis of variance (ANOVA) demonstrated a significant interaction between the Rf-1A and Rf-4 QTLs. The magnitude of the interaction varied with the type and duration of the treatment. The RBF autoregulation was impaired in Rf-1A single and Rf-1A+4 double congenics, while in Rf-4 single congenics it was similar to that of ACI controls., Conclusion: These findings indicate that the Rf-1 QTL directly influences renal susceptibility and autoregulation. In contrast, the Rf-4 QTL shows no direct effects, but significantly increases susceptibility to renal damage via an interaction with Rf-1.

Background: Linkage analyses of crosses of rats susceptible to renal damage, fawn-hooded hypertensive (FHH), and those resistant to kidney damage, August x Copenhagen Irish (ACI), indicated that five quantitative trait loci (QTLs), Rf-1 to Rf-5, influence proteinuria (UPV), albuminuria (UAV) and focal glomerulosclerosis (FGS). Here we present data obtained in congenic rats to directly assess the role of the Rf-1 and Rf-5 QTLs., Methods: Renal damage (UPV, UAV, and FGS) was assessed in ACI, ACI.FHH-(D1Rat324-D1Rat156)(Rf-1B), and ACI.FHH-(D17Rat117-D17Arb5)(D17Rat180-D17Rat51) (Rf-5) congenic rats in the two-kidney (2K) control situation, and following L-NAME-induced hypertension, unilateral nephrectomy (UNX), and UNX combined with L-NAME. In addition we investigated renal blood flow (RBF) autoregulation in 2K congenic and parental ACI and FHH rats., Results: Compared to ACI, Rf-1B congenic rats showed a significant increase in susceptibility to renal damage after all three treatments. The increase was most pronounced after UNX with L-NAME. In contrast, the degree of renal damage in Rf-5 congenic rats was not different from the ACI. Like FHH, Rf-1B rats had impaired renal autoregulation. In contrast, RBF autoregulation of Rf-5 rats does not differ from ACI., Conclusion: The Rf-5 QTL does not show any direct effect. The Rf-1 QTL carries one or more genes impairing renal autoregulation and influencing renal damage susceptibility. Whether these are the same genes remains to be established., (Copyright 2005 S. Karger AG, Basel.)