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Hemodynamic stress on the mammalian heart results in compensatory hypertrophy and activation of the unfolded protein response through activating transcription factor 6α (ATF6α) in cardiac myocytes, but the roles of ATF6α or the related transcription factor ATF6β in regulating this hypertrophic response are not well-understood. Here we examined the effects of loss of ATF6α or ATF6β on the cardiac response to pressure overload. Mice gene-deleted for Atf6 or Atf6b were subjected to 2 weeks of transverse aortic constriction, and each showed a significant reduction in hypertrophy with reduced expression of endoplasmic reticulum (ER) stress-associated proteins compared with controls. However, with long-term pressure overload both Atf6 and Atf6b null mice showed enhanced decompensation typified by increased heart weight, pulmonary edema and reduced function compared to control mice. Our subsequent studies using cardiac-specific transgenic mice expressing the transcriptionally active N-terminus of ATF6α or ATF6β revealed that these factors control overlapping gene expression networks that include numerous ER protein chaperones and ER associated degradation components. This work reveals previously unappreciated roles for ATF6α and ATF6β in regulating the pressure overload induced cardiac hypertrophic response and in controlling the expression of genes that condition the ER during hemodynamic stress.

Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC–CRYAB complexes.

Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15 mg/L) while the second method involved a purification approach from 293 cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293 cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293 cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.

Myocilin (MYOC) is the gene with mutations most common in glaucoma. In the eye, MYOC is in trabecular meshwork, ciliary body, and retina. Other tissues with high MYOC transcript levels are skeletal muscle and heart. To date, the function of wild-type MYOC remains unknown and how mutant MYOC causes high intraocular pressure and glaucoma is ambiguous. By investigating mutant MYOC in a non-ocular tissue we hoped to obtain novel insight into mutant MYOC pathology. For this study, we utilized a transgenic mouse expressing human mutant MYOC Y437H protein and we examined its skeletal (gastrocnemius) muscle phenotype. Electron micrographs showed that sarcomeres in the skeletal muscle of mutant CMV-MYOC-Y437H mice had multiple M-bands. Western blots of soluble muscle lysates from transgenics indicated a decrease in two M-band proteins, myomesin 1 (MYOM1) and muscle creatine kinase (CKM). Immunoprecipitation identified CKM as a MYOC binding partner. Our results suggest that binding of mutant MYOC to CKM is changing sarcomere ultrastructure and this may adversely impact muscle function. We speculate that a person carrying the mutant MYOC mutation will likely have a glaucoma phenotype and may also have undiagnosed muscle ailments or vice versa, both of which will have to be monitored and treated.

The endoplasmic reticulum (ER) stress response is one of many signaling events activated in the heart in response to injury and/or cardiac hypertrophy, although the role that this response pathway plays in such events remains under investigation. Recently, our laboratory demonstrated that overexpression of thrombospondin-4 (Thbs4) resulted in a protective ER stress response via activation of activating transcription factor 6 (ATF6), and that mice gene-deleted for Thbs4 demonstrated compromised ER stress signaling and decreased survival after transverse aortic constriction (TAC) or myocardial infarction (MI) surgery. Here we confirm that Thbs4-mediated expansion of the ER compartment requires ATF6 and examine whether the protective ER stress response mediated by transgenic overexpression of Thbs4 is eliminated when crossed with gene-deleted mice lacking ATF6. We also examined cardiac-specific transgenic mice overexpressing the transcriptionally-active ATF6 N-terminus, as well as mice gene-deleted for ATF6, in combination with disease stimuli including TAC and MI surgery. We find that ATF6 proteins are required for compensatory hypertrophy in the mouse heart and that loss of these proteins results in accelerated decompensation and failure, likely due to reductions in ER folding capacity that is required for the increased protein production necessary for cardiac hypertrophy. These results firmly position ATF6 as an essential regulator of compensatory cardiac hypertrophy during disease.

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a novel function for Thbs’ as ER resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury while Thbs4−/− mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. The type-3 repeat domain in Thbs’ bind the ER luminal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4−/−mice failed to show activation of Atf6α and other ER stress response factors with injury, and Thbs4-mediated protection was lost when Atf6α was deleted. Hence, Thbs’ can function inside the cell during disease/remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

To improve contractile function, the myocardium undergoes hypertrophic growth without myocyte proliferation in response to both pathologic and physiologic stimulation. Various membrane-bound receptors and intermediate signal transduction pathways regulate the induction of cardiac hypertrophy, but the cardioprotective regulatory pathways or effectors that antagonize cardiac hypertrophy remain poorly understood. Here we identify the small GTPase Cdc42 as a signaling intermediate that restrained the cardiac growth response to physiologic and pathologic stimuli. Cdc42 was specifically activated in the heart after pressure overload and in cultured cardiomyocytes by multiple agonists. Mice with a heart-specific deletion of Cdc42 developed greater cardiac hypertrophy at 2 and 8 weeks of stimulation and transitioned more quickly into heart failure than did wild-type controls. These mice also displayed greater cardiac hypertrophy in response to neuroendocrine agonist infusion for 2 weeks and, more remarkably, enhanced exercise-induced hypertrophy and sudden death. These pathologies were associated with an inability to activate JNK following stimulation through a MEKK1/MKK4/MKK7 pathway, resulting in greater cardiac nuclear factor of activated T cells (NFAT) activity. Restoration of cardiac JNK signaling with an Mkk7 heart-specific transgene reversed the enhanced growth effect. These results identify what we believe to be a novel antihypertrophic and protective cardiac signaling pathway, whereby Cdc42-dependent JNK activation antagonizes calcineurin-NFAT activity to reduce hypertrophy and prevent transition to heart failure.

Calreticulin is a Ca (2+)-buffering chaperone of the endoplasmic reticulum. The protein is highly expressed in embryonic heart but downregulated in postnatal heart, indicating that expression of calreticulin in the heart is highly regulated. In this study we identify GATA6 and Evi-1 transcription factors as new regulators of the calreticulin gene. In neonatal rat ventricular cardiomyocytes and mouse fibroblasts the calreticulin gene is activated by GATA6 but repressed by Evi-1. Furthermore, transactivation of the calreticulin gene by GATA6 is suppressed by Evi-1, suggesting an antagonistic role between both GATA6 and Evi-1. Using EMSA, ChIP analysis, and site-specific mutagenesis, we showed that GATA6 and Evi-1 bind to site 1 on the calreticulin promoter. GATA6 and Evi-1 are highly expressed early during cardiogenesis of ES cells, suggesting that they may regulate expression of the calreticulin gene during cardiac development.

Calreticulin, a Ca(2+) binding chaperone of the endoplasmic reticulum, is also highly expressed in the embryonic heart, and knockout of the calreticulin gene is lethal during embryogenesis because of impaired cardiac development. The protein is down-regulated after birth, and elevated expression of calreticulin in newborn hearts is associated with severe cardiac pathology and death. Here we show that the transcription factor Nkx2.5 activates expression of the calreticulin gene in the heart. Binding of chicken ovalbumin upstream promoter-transcription factor 1 to the Nkx2.5 binding site suppresses transcription from the calreticulin promoter. Nkx2.5 and chicken ovalbumin upstream promoter-transcription factor 1 play antagonistic roles in regulating the expression of calreticulin during cardiac development. These studies indicate that cardiac-specific transcription factor Nkx2.5 plays a central role in activating calreticulin expression and that there is a cooperation between chicken ovalbumin upstream promoter-transcription factor 1 and Nkx2.5 at the calreticulin promoter.

Changes in the localization of phenolic compounds were investigated in columella cells of embryonic roots in 1-year-old Brassica napus seeds during 48 h of imbibition and germination. In dry, dormant seeds, phenolic compounds were located in the apoplastic compartment between the cell wall and plasmalemma in the outermost layer of the columella. These apoplastic phenolic deposits disappeared during the activation processes associated with imbibition and germination, but new deposits appeared successively in the nucleus, ER cisternae, protein bodies and on the outer surface of the root cap. A large number of phenolic deposits were observed in the outermost part of the columella, becoming less frequent towards the initial centre. Their appearance coincided with restoration of the ER and immediately preceded cytological activation. After the primary root had emerged from the seed coat, deposits of phenolic compounds disappeared from the cytoplasm, but simultaneously appeared in the vacuoles.

Studies on the oral route of nicotine administration in rodents make important contributions to our understanding of human nicotine use, and alternative approaches to smoking cessation. While environmental availability of oral nicotine contributes to voluntary intake and appears to drive consumption initially, solution concentration may exert more control over intake with continued exposure. Further, it is believed that female rodents consume more nicotine and show greater motivation to obtain it than males.The purpose of our study was to determine voluntary oral nicotine intake patterns following continuous exposure to relatively high concentrations in male and female rats, employing a multiple bottle approach, and to describe the relationship between oral nicotine consumption and sera cotinine.Using five bottles, adult Sprague-Dawley rats were given continuous access to water and 15 μg/ml nicotine solutions or water and 15 and 30 μg/ml nicotine solutions for 2 weeks; blood serum was analyzed for cotinine.Rats consistently consumed oral nicotine and female rats ingested more nicotine than males, even at relatively high concentrations. Yet, when both concentrations were presented simultaneously, oral nicotine intake did not exceed that of water, thus overriding an environmental, or multiple-bottle, effect. Cotinine was systemically circulated following first-pass hepatic metabolism of nicotine at early, but not at later stages of nicotine exposure.Our findings suggest rats will readily and voluntarily ingest considerably higher doses of nicotine than previously reported resulting in initial systemic cotinine, and trends toward sex differences are mitigated by solution concentration.

Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo- and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent.

Calreticulin is a Ca 2+ -binding chaperone of the sarcoplasmic/endoplasmic reticulum. It is an important Ca 2+ buffer, a regulator of Ca 2+ homeostasis, and a component of protein quality control processes in the secretory pathway. Calreticulin is essential for cardiac development; its gene is tightly regulated during cardiogenesis, and in the absence of calreticulin, cardiac development is impaired. The protein is highly expressed in the developing heart and down-regulated after birth in the healthy mature heart. Overexpression of calreticulin in postnatal heart leads to bradyarrhythima and complete heart block, followed by sudden death. The calreticulin gene is a target of transcription factors involved in fetal cardiac program (Nkx2.5, myocardin, myocyte enhancer factor 2C, and GATA6). Calreticulin works upstream of calcineurin and myocyte enhancer factor 2C in a Ca 2+ -dependent signal transduction cascade linking the endoplasmic reticulum and the nucleus during cardiac development.

Ca(2+) is a signalling molecule involved in virtually every aspect of cell function. The endoplasmic reticulum (ER) is an important and dynamic organelle responsible for storage of the majority of intracellular Ca(2+). Within the ER lumen are proteins that function as Ca(2+) buffers and/or molecular chaperones including calreticulin, a multifunctional Ca(2+)-binding protein. Calreticulin-deficiency is lethal in utero due to impaired cardiac development. In the absence of calreticulin Ca(2+) storage capacity in the ER and InsP(3) receptor mediated Ca(2+) release from ER are compromised. Remarkably, over-expression of constitutively active calcineurin in the hearts of calreticulin deficient mice rescues them from embryonic lethality and produces live calreticulin deficient animals. These observations provide first evidence that calreticulin is a key upstream regulator of calcineurin in the Ca(2+)-signalling cascade and they highlight the importance of ER during early stages of cellular commitment and tissue development during organogenesis.

Ca2+ is an important secondary messenger and any alteration to intracellular Ca2+ signaling pathways or components of these pathways can have a profound physiological effect on any cell, particularly cardiomyocytes. Early approaches to investigate heart disease focused on many muscle proteins, however recent findings indicate that molecules considered as "non-muscle" proteins may be equally important players in etiology of many cardiac pathologies. Many of these "non-muscle" proteins play a role in Ca2+ cycling or Ca2+ -dependent signaling pathways in the heart. In this review we focus on Ca2+ -dependent pathways in normal, growing, and diseased hearts. Understanding of these unique signaling pathways may hold answers to many cardiac pathologies in children and adults.

Calreticulin is a Ca(2+) binding/storage chaperone resident in the lumen of endoplasmic reticulum (ER). The protein is an important component of the calreticulin/calnexin cycle and the quality control pathways in the ER. In mice, calreticulin deficiency is lethal due to impaired cardiac development. This is not surprising because the protein is expressed at high level at early stages of cardiac development. Overexpression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Recent studies on calreticulin-deficient and transgenic mice revealed that the protein is a key upstream regulator of calcineurin-dependent pathways during cardiac development. Calreticulin and ER may play important role in cardiac development and postnatal pathologies.