Search

Cell-cell signaling peptides (e.g., peptide hormones, neuropeptides) are among the largest class of cellular transmitters and regulate a variety of physiological processes. To identify and quantify the relative abundances of cell-cell signaling peptides in different physiological states, liquid chromatography-mass spectrometry-based peptidomics workflows are commonly utilized on freshly dissected tissues. In such animal experiments, the administration of general anesthetics is an important step for many research projects. However, acute anesthetic administration may rapidly change the measured abundance of transmitter molecules and metabolites, especially in the brain and endocrine system, which would confound experimental results. The aim of this study was to evaluate the effect of short-term (5 min) anesthetic administration on the measured abundance of cell-cell signaling peptides, as evaluated by a typical peptidomics workflow. To accomplish this goal, we compared endogenous peptide abundances in the rat pituitary following administration of 5% isoflurane, 200 mg/kg sodium pentobarbital, or no anesthetic administration. Label-free peptidomics analysis demonstrated that acute use of isoflurane changed the levels of a small number of peptides, primarily degradation products of the hormone somatotropin, but did not influence the levels of most other peptide hormones. Acute use of sodium pentobarbital had negligible impact on the relative abundance of all measured peptides. Overall, our results suggest that anesthetics used in pituitary peptidomics studies do not dramatically confound observed results.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that can be cytoprotective, especially in conditions of ischemia/reperfusion injury. However, H2S is also toxic, and unregulated accumulation or exposure to environmental H2S can be lethal. In Caenorhabditis elegans, the hypoxia inducible factor (hif-1) coordinates the initial transcriptional response to H2S, and is essential to survive exposure to low concentrations of H2S. We performed a forward genetic screen to identify mutations that suppress the lethality of hif-1 mutant animals in H2S. The mutations we recovered are specific for H2S, as they do not suppress embryonic lethality or reproductive arrest of hif-1 mutant animals in hypoxia, nor can they prevent the death of hif-1 mutant animals exposed to hydrogen cyanide. The majority of hif-1 suppressor mutations we recovered activate the skn-1/Nrf2 transcription factor. Activation of SKN-1 by hif-1 suppressor mutations increased the expression of a subset of H2S-responsive genes, consistent with previous findings that skn-1 plays a role in the transcriptional response to H2S. Using transgenic rescue, we show that overexpression of a single gene, rhy-1, is sufficient to protect hif-1 mutant animals in H2S. The rhy-1 gene encodes a predicated O-acyltransferase enzyme that has previously been shown to negatively regulate HIF-1 activity. Our data indicate that RHY-1 has novel, hif-1 independent, function that promotes survival in H2S.

Hydrogen sulfide (H(2)S) is an endogenously produced signaling molecule that can be cytoprotective, especially in conditions of ischemia/reperfusion injury. However, H(2)S is also toxic, and unregulated accumulation or exposure to environmental H(2)S can be lethal. In Caenorhabditis elegans, the hypoxia inducible factor (hif-1) coordinates the initial transcriptional response to H(2)S, and is essential to survive exposure to low concentrations of H(2)S. We performed a forward genetic screen to identify mutations that suppress the lethality of hif-1 mutant animals in H(2)S. The mutations we recovered are specific for H(2)S, as they do not suppress embryonic lethality or reproductive arrest of hif-1 mutant animals in hypoxia, nor can they prevent the death of hif-1 mutant animals exposed to hydrogen cyanide. The majority of hif-1 suppressor mutations we recovered activate the skn-1/Nrf2 transcription factor. Activation of SKN-1 by hif-1 suppressor mutations increased the expression of a subset of H(2)S-responsive genes, consistent with previous findings that skn-1 plays a role in the transcriptional response to H(2)S. Using transgenic rescue, we show that overexpression of a single gene, rhy-1, is sufficient to protect hif-1 mutant animals in H(2)S. The rhy-1 gene encodes a predicated O-acyltransferase enzyme that has previously been shown to negatively regulate HIF-1 activity. Our data indicate that RHY-1 has novel, hif-1 independent, function that promotes survival in H(2)S.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that can be cytoprotective, especially in conditions of ischemia/reperfusion injury. However, exposure to exogenous H2S can be toxic, perhaps due to unregulated activation of endogenous H2S signaling pathways. We use the nematode C. elegans to define mechanisms that mediate the physiological effects of H2S in animals. We have previously shown that in C. elegans the hypoxia inducible factor (hif-1) coordinates the initial transcriptional response to H2S and is essential to survive exposure to low concentrations of H2S. In this study, we performed a forward genetic screen to identify mutations that suppress the lethality of hif-1 mutant animals in H2S. The mutations we recovered do not suppress embryonic lethality or reproductive arrest of hif-1 mutant animals in hypoxia, nor can they improve viability of hif-1 mutant animals exposed to hydrogen cyanide, indicating that these are specific for H2S. We found that the hif-1 suppressor mutations activate the skn-1/Nrf2 transcription factor. Activation of SKN-1 by hif-1 suppressor mutations increased the expression of a subset of H2S-responsive genes, consistent with our previous finding that skn-1 plays a role in the transcriptional response to H2S. Using transgenic rescue, we show a single gene, rhy-1, alone is sufficient to protect hif-1 mutant animals in H2S. Our data indicate that RHY-1 acts in concert with CYSL-1, an orthologue of human cystathionine β-synthase, to promote survival in H2S. The rhy-1 gene encodes a predicated O-acyltransferase enzyme that has previously been shown to negatively regulate HIF-1 activity. Our studies reveal a novel function of RHY-1, which is independent of hif-1, that protects against toxic effects of H2S.

Physiological stress occurs when conditions perturb homeostasis. There are a multitude of stressors commonly encountered by organisms, including environmental factors such as temperature, pathogens, toxins, and food or oxygen availability, or internal disturbances caused by genetic defects or damage accumulated over the course of ageing. In this chapter, we discuss the fundamental relationships between stress and homeostasis. We then focus on various stress response strategies and highlight established molecular genetic stress response pathways. Many experiments, particularly those in C. elegans, have highlighted the intimate relationship between stress resistance and longevity. As such, a deeper understanding of the fundamental nature of stress and homeostatic stress responses is essential to fully appreciate the causes and consequences of ageing in animals.

Mutations in the him-5 gene in Caenorhabditis elegans strongly reduce the frequency of crossovers on the X chromosome, with lesser effects on the autosomes. him-5 mutants also show a change in crossover distribution on both the X and autosomes. These phenotypes are accompanied by a delayed entry into pachytene and premature desynapsis of the X chromosome. The nondisjunction, progression defects and desynapsis can be rescued by an exogenous source of double strand breaks (DSBs), indicating that the role of HIM-5 is to promote the formation of meiotic DSBs. Molecular cloning of the gene shows that the inferred HIM-5 product is a highly basic protein of 252 amino acids with no clear orthologs in other species, including other Caenorhabditis species. Although him-5 mutants are defective in segregation of the X chromosome, HIM-5 protein localizes preferentially to the autosomes. The mutant phenotypes and localization of him-5 are similar but not identical to the results seen with xnd-1, although unlike xnd-1, him-5 has no apparent effect on the acetylation of histone H2A on lysine 5 (H2AacK5). The localization of HIM-5 to the autosomes depends on the activities of both xnd-1 and him-17 allowing us to begin to establish pathways for the control of crossover distribution and frequency.

Normal cardiac contraction and relaxation requires efficient operation of the SR Ca2+ ATPase, SERCA2a. In failing hearts, decreased SERCA2a expression and slowed SR Ca2+ reuptake are thought to significantly underlie contractile dysfunction. We have studied the relationship between decreased SERCA2a expression and cardiac function using a mouse model of conditional cardiac Serca2 ablation, the Serca2fl/fl mouse. In this model, Cre activation by tamoxifen injection efficiently deletes Serca2 from the heart; four weeks post-knockout, cardiac SERCA2a protein content is below 5% of normal levels. Serca2 KO mice survive 7-10 weeks post knockout with only mild in vivo impairment prior to this time, suggesting that this loss of SERCA2a protein can be temporarily compensated. We found that isolated SERCA2a KO hearts retained the ability to respond to s-adrenergic stimulation ex vivo despite the loss of most SERCA2a protein. Because the protein regulator of SERCA2a, phospholamban (PLN), is thought to be a major component of the cardiac s-adrenergic response, this finding was unexpected and warranted further detailed investigation. To identify the mechanism underlying this preserved s-adrenergic response in SERCA2-depleted hearts, we bred Serca2fl/fl and PLN-/- mice to generate an inducible Serca2 knockout mouse line lacking PLN. Serca2fl/fl;PLN-/- mice were injected with tamoxifen to induce Serca2 gene disruption, and isolated hearts from Serca2KO;PLN-/- (“DKO”) mice were evaluated by Langendorff perfusion 4-5 weeks after Serca2 knockout. Control PLN-/- hearts show little response to s-adrenergic stimulation, and DKO hearts have impaired contractility under baseline conditions. DKO hearts, however, show improved systolic and diastolic function when perfused with 50 nM isoproterenol, indicating that targets of s-adrenergic signaling beyond PLN are able to support a significant inotropic and lusitropic response when cardiac contractility is impaired. We will discuss how these findings support new mechanisms underlying heart performance during stress and disease.

The hearts of mammalian hibernators maintain contractile function in the face of severe environmental stresses during winter heterothermy. To enable survival in torpor, hibernators regulate the expression of numerous genes involved in excitation-contraction coupling, metabolism, and stress response pathways. Understanding the basis of this transition may provide new insights into treatment of human cardiac disease. Few studies have investigated hibernator heart performance during both summer active and winter torpid states, and seasonal comparisons of whole heart function are generally lacking. We investigated the force-frequency relationship and the response to ex vivo ischemia-reperfusion in intact isolated hearts from 13-lined ground squirrels ( Ictidomys tridecemlineatus) in the summer (active, July) and winter (torpid, January). In standard euthermic conditions, we found that winter hearts relaxed more rapidly than summer hearts at low to moderate pacing frequencies, even though systolic function was similar in both seasons. Proteome data support the hypothesis that enhanced Ca2+handling in winter torpid hearts underlies the increased relaxation rate. Additionally, winter hearts developed significantly less rigor contracture during ischemia than summer hearts, while recovery during reperfusion was similar in hearts between seasons. Winter torpid hearts have an increased glycogen content, which likely reduces development of rigor contracture during the ischemic event due to anaerobic ATP production. These cardioprotective mechanisms are important for the hibernation phenotype and highlight the resistance to hypoxic stress in the hibernator.

Effective contraction and relaxation of the heart requires the robust transport of Ca2+ to and from the sarcoplasmic reticulum (SR). The cardiac SR Ca-ATPase, SERCA2a, is responsible for sequestering Ca2+ to the SR during the contractile cycle, and has diminished expression and function in failing hearts. We have used a model of inducible cardiac-specific Serca2 gene disruption, the Serca2fl/fl mouse, to probe the relationship between decreased SERCA2a expression and cardiac dysfunction. 4 weeks after gene disruption, SERCA2a protein content in KO hearts is less than 5% of control. Despite severely impaired ex vivo function of isolated hearts and cardiomyocytes at this time, in vivo heart function is mildly impaired and KO mice survive until 7-10 weeks post-knockout, indicating that loss of SERCA2a is temporarily compensated. This finding of preserved cardiac function in vivo in the context of severe SERCA2a depletion is surprising and warrants a detailed dissection of potential compensatory mechanisms. Published studies have found that sarcolemmal Ca2+ transport mechanisms such as L-type Ca2+ current, Na-Ca exchange, and plasma membrane Ca-ATPase activity are increased following this loss of SERCA2a protein. These observations prompt the hypothesis that sarcolemmal Ca2+ transport remodeling may partially compensate for diminished SR Ca2+ handling in KO hearts. We have begun to investigate this hypothesis by perfusing isolated Serca2 KO hearts with caffeine to evaluate SR-independent contractile function. During caffeine perfusion, left ventricular (LV) end-diastolic pressures were lower in KO hearts than controls consistent with the reduced SR Ca2+ stores in KO myocytes. Interestingly, during prolonged caffeine perfusion, systolic performance was similar in control and KO hearts. This is functional evidence that trans-sarcolemmal transient adaptations in KO hearts are not of sufficient magnitude to differentiate KO from WT when SR function is disabled by caffeine.

EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca(2+) buffer in many biological systems. Given the centrality of Ca(2+) signaling in health and disease, EF-hand motifs designed to have new biological activities may have widespread utility. Here, an EF-hand motif substitution that had been presumed to destroy EF-hand function, that of glutamine for glutamate at position 12 of the second cation binding loop domain of Parv (ParvE101Q), markedly inverted relative cation affinities: Mg(2+) affinity increased, whereas Ca(2+) affinity decreased, forming a new ultra-delayed Ca(2+) buffer with favorable properties for promoting cardiac relaxation. In therapeutic testing, expression of ParvE101Q fully reversed the severe myocyte intrinsic contractile defect inherent to expression of native Parv and corrected abnormal myocardial relaxation in diastolic dysfunction disease models in vitro and in vivo. Strategic design of new EF-hand motif domains to modulate intracellular Ca(2+) signaling could benefit many biological systems with abnormal Ca(2+) handling, including the diseased heart.

The cardiac SERCA2 Ca(2+) pump is critical for maintaining normal Ca(2+) handling in the heart. Reduced SERCA2a content and blunted Ca(2+) reuptake are frequently observed in failing hearts and evidence implicates poor cardiac Ca(2+) handling in the progression of heart failure. To gain insight into mechanism we investigated a novel genetic mouse model of inducible severe and progressive SERCA2 deficiency (inducible Serca2 knockout, SERCA2 KO). These mice eventually die from overt heart failure 7-10 weeks after knockout but as yet there have been no reports on intrinsic mechanical performance at the isolated whole heart organ level. Thus we studied whole-organ ex vivo function of hearts isolated from SERCA2 KO mice at one and four weeks post-knockout in adult animals. We found that isolated KO heart function was only modestly impaired one week post-knockout, when SERCA2a protein was 32% of normal. At four weeks post-knockout, function was severely impaired with near non-detectable levels of SERCA2. During perfusion with 10 mM caffeine, LV developed pressures were similar between 4-week KO and control hearts, and end-diastolic pressures were lower in KO. When hearts were subjected to ischemia-reperfusion injury, recovery was not different between control and KO hearts at either one or four weeks post-knockout. Our findings indicate that ex vivo function of isolated SERCA2 KO hearts is severely impaired long before symptoms appear in vivo, suggesting that physiologically relevant heart function in vivo can be sustained for weeks in the absence of robust SR Ca(2+) flux.

Diastolic dysfunction is characterized by slow or incomplete relaxation of the ventricles during diastole, and is an important contributor to heart failure pathophysiology. Clinical symptoms include fatigue, shortness of breath, and pulmonary and peripheral edema, all contributing to decreased quality of life and poor prognosis. There are currently no therapies available that directly target the heart pump defects in diastolic function. Calcium mishandling is a hallmark of heart disease and has been the subject of a large body of research. Efforts are ongoing in a number of gene therapy approaches to normalize the function of calcium handling proteins such as sarcoplasmic reticulum calcium ATPase. An alternative approach to address calcium mishandling in diastolic dysfunction is to introduce calcium buffers to facilitate relaxation of the heart. Parvalbumin is a calcium binding protein found in fast-twitch skeletal muscle and not normally expressed in the heart. Gene transfer of parvalbumin into normal and diseased cardiac myocytes increases relaxation rate but also markedly decreases contraction amplitude. Although parvalbumin binds calcium in a delayed manner, it is not delayed enough to preserve full contractility. Factors contributing to the temporal nature of calcium buffering by parvalbumin are discussed in relation to remediation of diastolic dysfunction. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Proper contraction and relaxation of the heart depends on highly orchestrated and rapid release and reuptake of Ca2+ from the sarcoplasmic reticulum (SR). Normal SR Ca2+ reuptake in the rodent heart is accomplished predominately by the cardiac Ca-ATPase, SERCA2a, which in the failing heart exhibits diminished activity and expression. We have utilized a mouse model of inducible cardiac Ca2+ dysregulation, the Serca2fl/fl mouse, to investigate the relationship between diminished SR Ca2+ flux and heart dysfunction. Conditional deletion of Serca2 initiates the progressive loss of SERCA2 protein, allowing us to examine in fine detail the relationship between titrated loss of SR Ca2+ flux and heart dysfunction. By four weeks post-knockout, SERCA2 protein levels are below 5% of baseline and isolated 4-week KO cardiomyocytes have greatly diminished contractility and Ca2+ handling. The in vivo phenotype at this time, however, is unexpectedly mild, and although all KO mice succumb to congestive heart failure, they do so from 7-10 weeks after knockout. This disconnect between the mild in vivo phenotype and severe ex vivo phenotype is intriguing and prompts the hypothesis that a systemic signaling pathway, such as adrenergic signaling, is responsible for sustaining function in KO animals. We found that in isolated Serca2 KO hearts, s-adrenergic stimulation elicits a robust inotropic and lusitropic response despite the near-complete lack of SERCA2 protein. This finding will be discussed as it is at odds with prevailing views of Ca2+ regulation of heart function.