Ocean-Warming During Embryogenesis Programs a Lasting Transcriptomic Signature in Fishes

Exposure to elevated temperatures during embryogenesis has profound acute effects on the cardiac performance, metabolism, and growth of fishes. Some temperature-induced effects may be retained into, or manifest in, later-life through a mechanism termed developmental programming. In this study, we incubated Scyliorhinus canicula embryos at either 15°C or 20°C before transferring the newly hatched sharks to a common set of conditions (15°C) for 5 months. Lasting transcriptomic differences associated with the developmental environment were identified, and interactions between cardiac genes were investigated using hypernetwork modelling. Development at an elevated temperature caused changes in transcriptomic connectivity and entropy, parameters thought to relate to plasticity and fitness. We then validated these observations through a novel re-analysis of published Danio rerio and Dicentrarchus labrax muscle tissue datasets. Together, these data demonstrate a persistent, programmed effect of developmental temperature on the co-ordination of gene expression in three species of fishes, which may relate to altered plasticity and fitness in later-life.

Cardiac muscle disease and therapeutic targets

Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ∼50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, SMP), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, SMN). Gene mutations are most often present in genes encoding the sarcomere proteins myosin heavy chain, myosin-binding protein C, and troponin T. Studies in cardiac tissue samples from patients with obstructive HCM that were obtained during myectomy surgery showed increased myofilament calcium sensitivity, increased kinetics and tension cost, and a reduction of the super-relaxed state of myosin, which is associated with an energy-conserving status of the crossbridges. The increase in myofilament calcium sensitivity is observed at a low dose of mutant protein, while the magnitude of the increase in calcium sensitivity depends on the specific mutation location. These mutation-mediated myofilament changes may underlie inefficient in vivo cardiac performance in mutation carriers. Reduced cardiac efficiency has been observed before onset of cardiac hypertrophy and at advanced disease stages. In addition, impaired diastolic function is an early disease characteristic of HCM. Our recent proteomics studies revealed increased detyrosination of microtubules, which may be a cause of diastolic dysfunction. Recent treatments that target inefficient cardiac performance, such as myosin inhibitors and metabolic drug therapies, may have the potential to prevent, delay, or even reverse disease in HCM-mutation carriers. Treatment response may depend on the specific gene mutation in SMP individuals and may explain diverse response of HCM patients to therapy. While mutation-mediated cardiomyocyte defects have become clear in past years, more research is warranted to define the cellular pathomechanisms of cardiac dysfunction in SMN patients.

Modulation of Myosin by Cardiac Myosin Binding Protein-C Peptides Improves Cardiac Contractility in Ex-Vivo Experimental Heart Failure Models

Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of sarcomeric function. Although reduced phosphorylation of cMyBP-C has been linked to compromised contractility in heart failure patients, direct modulation of cMyBP-C to myosin using small molecules or peptides has not been reported to improve cardiac performance. Here we used previously published cMyBP-C peptides 302A and 302S (surrogates to the regulatory phosphorylation site serine 302) as tool molecules to investigate the role of cMyBP-C in modulating cardiac contraction and relaxation in experimental heart failure (HF) models in vitro. cMyBP-C peptides 302A and 302S were able to increase contractility of papillary muscle fibers isolated from a cMyBP-C phospho-ablation (cMyBP-CAAA) mouse model. In addition, 302A was able to improve the force redevelopment rate (ktr) in papillary muscle fibers from cMyBP-CAAA mice. Consistent with above findings, cMyBP-C peptides 302A and 302S were able to increase the ATPase rates in myofibrils isolated from MI rats but not from sham rats. Furthermore, in cMyBP-CAAA mouse and myocardial infarction (MI) HF models, both cMyBP-C peptides 302A and 302S were able to improve ATPase hydrolysis rates. These changes were not observed in non-transgenic (NTG) mice or sham rats, indicating the specific effects of these peptides in regulating the reduced or unphosphorylated state of cMyBP-C only under pathological conditions of heart failure. Taken together, these studies demonstrate that modulation of cMyBP-C in a reduced phosphorylation or unphosphorylated state can be a therapeutic approach to improve myosin function, sarcomere contractility and relaxation. Therefore, targeting cMyBP-C can be a differentiated approach to improve overall cardiac performance on top of standard care drugs in HF patients.

Loss of toll-like receptor 4 ameliorates cardiovascular dysfunction in aged mice

Background Toll-like receptor 4 (TLR4) is a pattern recognition receptor of the innate immune system. TLR4 contributes to many aging-related chronic diseases. However, whether TLR4 is involved in cardiovascular injury during the aging process has not been investigated. Methods The effects of TLR4 on the cardiovascular system of aged mice were investigated in TLR4−/− mice. An intraperitoneal glucose tolerance test (IPGTT) and insulin sensitivity test (IST) were conducted to evaluate global insulin sensitivity. Echocardiography was used to measure cardiac structure and performance. An isolated artery ring assay was used to measure the vasodilator function of the thoracic aorta. The inflammatory response was reflected by the serum concentration of cytokines. Results TLR4 expression increased in the hearts and aortas of mice in an age-dependent manner. Loss of TLR4 increased insulin sensitivity in aged mice. Moreover, loss of TLR4 improved cardiac performance and endothelium-dependent vascular relaxation in aged mice. Importantly, the increases in serum inflammatory cytokines and oxidative stress in the heart and aorta were also inhibited by TLR4 deficiency. Conclusion In summary, loss of TLR4 improved cardiac performance and endothelium-dependent vascular relaxation in aged mice. The reduced inflammatory responses and oxidative stress may be the reason for the protective effects of TLR4 deficiency during aging. Our study indicates that targeting TLR4 is a potential therapeutic strategy for preventing aging-related cardiovascular disease.

Evaluation of Effects of Ractopamine on Cardiovascular, Respiratory, and Locomotory Physiology in Animal Model Zebrafish Larvae

Ractopamine (RAC) is a beta-adrenoceptor agonist that is used to promote lean and increased food conversion efficiency in livestock. This compound has been considered to be causing behavioral and physiological alterations in livestock like pig. Few studies have addressed the potential non-target effect of RAC in aquatic animals. In this study, we aimed to explore the potential physiological response after acute RAC exposure in zebrafish by evaluating multiple endpoints like locomotor activity, oxygen consumption, and cardiovascular performance. Zebrafish larvae were subjected to waterborne RAC exposure at 0.1, 1, 2, 4, or 8 ppm for 24 h, and the corresponding cardiovascular, respiratory, and locomotion activities were monitored and quantified. In addition, we also performed in silico molecular docking for RAC with 10 zebrafish endogenous β-adrenergic receptors to elucidate the potential acting mechanism of RAC. Results show RAC administration can significantly boost locomotor activity, cardiac performance, oxygen consumption, and blood flow rate, but without affecting the cardiac rhythm regularity in zebrafish embryos. Based on structure-based flexible molecular docking, RAC display similar binding affinity to all ten subtypes of endogenous β-adrenergic receptors, from adra1aa to adra2db, which are equivalent to the human one. This result suggests RAC might act as high potency and broad spectrum β-adrenergic receptors agonist on boosting the locomotor activity, cardiac performance, and oxygen consumption in zebrafish. To validate our results, we co-incubated a well-known β-blocker of propranolol (PROP) with RAC. PROP exposure tends to minimize the locomotor hyperactivity, high oxygen consumption, and cardiac rate in zebrafish larvae. In silico structure-based molecular simulation and binding affinity tests show PROP has an overall lower binding affinity than RAC. Taken together, our studies provide solid in vivo evidence to support that RAC plays crucial roles on modulating cardiovascular, respiratory, and locomotory physiology in zebrafish for the first time. In addition, the versatile functions of RAC as β-agonist possibly mediated via receptor competition with PROP as β-antagonist.

Abstract P307: An Exquisite Cardiac Performance And Protection Package Emerges In Mouse With Cardiac-specific Overexpression Of Adenylyl Cyclase Type 8

Young adult (three-month old) mice with cardiac-specific overexpression of AC type 8 (TGAC8) have a markedly elevated heart rate and markedly enhanced ejection fraction around the clock, mimicking cardiac responses of sympathetic autonomic input during acute exercise on a chronic base, but the TGAC8 mice do not exhibit heart failure or increased mortality up to about a year. Using this incessant young TGAC8 mouse as an ideal model to elucidate a high grade cardiac “Performance and Protection Package” (PPP) in response to chronic cardiac stress, we hypothesized that the TGAC8 heart adjusts to the chronical stress by reprogramming itself at multiple omics scales to code the exquisite cardiac PPP. To this end, we compared three-month old TGAC8 mice and their wildtype (WT) littermates using multiple omics analyses, from transcriptome to proteome, to phosphoproteome. Compared to WT, the phosphorylation level of most proteins was increased in the TGAC8 mice, including transcription factors (TF), kinases and phosphatases that are important in regulating transcription and phosphorylation. Among the 191 TFs identified in phosphoproteome, 91 were increased significantly, in line with the general upregulation in transcriptome. Many important stress response signaling pathways were enriched from phosphoproteome. Wherein, three protein quality control pathways, PI3K/AKT signaling, ERK/MAPK signaling and ubiquitination, were consistently enriched across the three omics scales. Most components in PI3K/AKT signaling pathway were upregulated, especially at protein and phosphorylation levels. Consistently, PI3K/AKT substrate phosphopeptides were increased, and downstream effects and functions of PI3K/AKT signaling were activated, including energy metabolism, protein synthesis, cell growth, cardiovascular functions, and NF-kB mediated functions. In summary, profiling the transcriptome, proteome and phosphoproteome of the TGAC8 heart unraveled the mechanism that controls its PPP. The cardiac overexpression of AC8 activates the AC8-cAMP-PKA axis to increase phosphorylation; widespread phosphorylation of the TFs promotes the transcription of numerous additional molecules that also regulate phosphorylation to reprogram the heart. Thereby, the TGAC8 mouse upregulates many stress response signaling pathways to activate the exquisite cardiac PPP.