Isolevuglandins Promote Mitochondrial Dysfunction and Electrophysiologic Abnormalities in Atrial Cardiomyocytes.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet the cellular and molecular mechanisms underlying the AF substrate remain unclear. Isolevuglandins (IsoLGs) are highly reactive lipid dicarbonyl products that mediate oxidative stress-related injury. In murine hypertension, the lipid dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA) reduced IsoLGs and AF susceptibility. We hypothesized that IsoLGs mediate detrimental pathophysiologic effects in atrial cardiomyocytes that promote the AF substrate. Using Seahorse XFp extracellular flux analysis and a luminescence assay, IsoLG exposure suppressed intracellular ATP production in atrial HL-1 cardiomyocytes. IsoLGs caused mitochondrial dysfunction, with reduced mitochondrial membrane potential, increased mitochondrial reactive oxygen species (ROS) with protein carbonylation, and mitochondrial DNA damage. Moreover, they generated cytosolic preamyloid oligomers previously shown to cause similar detrimental effects in atrial cells. In mouse atrial and HL-1 cells, patch clamp experiments demonstrated that IsoLGs rapidly altered action potentials (AP), implying a direct effect independent of oligomer formation by reducing the maximum Phase 0 upstroke slope and shortening AP duration due to ionic current modifications. IsoLG-mediated mitochondrial and electrophysiologic abnormalities were blunted or totally prevented by 2-HOBA. These findings identify IsoLGs as novel mediators of oxidative stress-dependent atrial pathophysiology and support the investigation of dicarbonyl scavengers as a novel therapeutic approach to prevent AF.

LNK/SH2B3 loss of function increases susceptibility to murine and human atrial fibrillation.

AIMS: The lymphocyte adaptor protein (LNK) is a negative regulator of cytokine and growth factor signalling. The rs3184504 variant in SH2B3 reduces LNK function and is linked to cardiovascular, inflammatory, and haematologic disorders, including stroke. In mice, deletion of Lnk causes inflammation and oxidative stress. We hypothesized that Lnk-/- mice are susceptible to atrial fibrillation (AF) and that rs3184504 is associated with AF and AF-related stroke in humans. During inflammation, reactive lipid dicarbonyls are the major components of oxidative injury, and we further hypothesized that these mediators are critical drivers of the AF substrate in Lnk-/- mice. METHODS AND RESULTS: Lnk-/- or wild-type (WT) mice were treated with vehicle or 2-hydroxybenzylamine (2-HOBA), a dicarbonyl scavenger, for 3 months. Compared with WT, Lnk-/- mice displayed increased AF duration that was prevented by 2-HOBA. In the Lnk-/- atria, action potentials were prolonged with reduced transient outward K+ current, increased late Na+ current, and reduced peak Na+ current, pro-arrhythmic effects that were inhibited by 2-HOBA. Mitochondrial dysfunction, especially for Complex I, was evident in Lnk-/- atria, while scavenging lipid dicarbonyls prevented this abnormality. Tumour necrosis factor-α (TNF-α) and interleukin-1 beta (IL-1ß) were elevated in Lnk-/- plasma and atrial tissue, respectively, both of which caused electrical and bioenergetic remodelling in vitro. Inhibition of soluble TNF-α prevented electrical remodelling and AF susceptibility, while IL-1ß inhibition improved mitochondrial respiration but had no effect on AF susceptibility. In a large database of genotyped patients, rs3184504 was associated with AF, as well as AF-related stroke. CONCLUSION: These findings identify a novel role for LNK in the pathophysiology of AF in both experimental mice and humans. Moreover, reactive lipid dicarbonyls are critical to the inflammatory AF substrate in Lnk-/- mice and mediate the pro-arrhythmic effects of pro-inflammatory cytokines, primarily through electrical remodelling.

Innovations in Undergraduate Research Training Through Multisite Collaborative Programming: American Heart Association Summer Undergraduate Research Experience Syndicate.

Background To support diversity in biomedical science, the American Heart Association launched the Supporting Undergraduate Research Experiences for undergraduate students from underrepresented backgrounds to provide mentorship and high-level exposure at 5 leading medical institutions. Here we describe the initial formation of the partnership and the alteration made in response to the program to accommodate COVID-19 safety precautions. Methods and Results We outline how programming shifted from local, in-person programming in the summer of 2019 to a collaborative, mainly virtual curriculum in 2020 using students' self-reported before and after surveys from both 2019 (n=33) and 2020 (n=42). Students from both in-person (2019) and virtual programs (2020) self-reported significant gains in scientific proficiency. A qualitative-directed content analysis of student open-response questions was performed. Students reported extensive benefits from the 2020 virtual training, including Personal Gains, Research Skills, Thinking and Working Like a Scientist, and Attitudes and Behaviors. Notedly, we observed increases in the Attitudes and Behaviors category. We outline the pros and cons of in-person and virtual programming and make recommendations moving forward in a postpandemic world with hybrid work and learning systems. Conclusions Our effort informs the development of future undergraduate research training programs, significantly maximizing a hybrid training modality. The American Heart Association Supporting Undergraduate Research Experiences serves as a model for building multi-institutional partnerships and providing research experiences that overcome institutional barriers and support students' interests, commitment, and ability to persist in science, technology, engineering, and math fields.

Natriuretic Peptide Oligomers Cause Proarrhythmic Metabolic and Electrophysiological Effects in Atrial Myocytes.

BACKGROUND: With aging, the human atrium invariably develops amyloid composed of ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide). Preamyloid oligomers are the primary cytotoxic species in amyloidosis, and they accumulate in the atrium during human hypertension and a murine hypertensive model of atrial fibrillation susceptibility. We tested the hypothesis that preamyloid oligomers derived from natriuretic peptides cause cytotoxic and electrophysiological effects in atrial cells that promote arrhythmia susceptibility and that oligomer formation is enhanced for a mutant form of ANP linked to familial atrial fibrillation. METHODS: Oligomerization was assessed by Western blot analysis. Bioenergic profiling was performed using the Seahorse platform. Mitochondrial dynamics were investigated with immunostaining and gene expression quantitated using quantitative reverse transcription polymerase chain reaction. Action potentials and ionic currents were recorded using patch-clamp methods and intracellular calcium measured using Fura-2. RESULTS: Oligomer formation was markedly accelerated for mutant ANP (mutANP) compared with WT (wild type) ANP. Oligomers derived from ANP, BNP, and mutANP suppressed mitochondrial function in atrial HL-1 cardiomyocytes, associated with increased superoxide generation and reduced biogenesis, while monomers had no effects. In hypertensive mice, atrial cardiomyocytes displayed reduced action potential duration and maximal dV/dT of phase 0, with an elevated resting membrane potential, compared with normotensive mice. Similar changes were observed when atrial cells were exposed to oligomers. mutANP monomers produced similar electrophysiological effects as mutANP oligomers, likely due to accelerated oligomer formation, while ANP and BNP monomers did not. Oligomers decreased Na+ current, inward rectifier K+ current, and L-type Ca++ current, while increasing sustained and transient outward K+ currents, to account for these effects. CONCLUSIONS: These findings provide compelling evidence that natriuretic peptide oligomers are novel mediators of atrial arrhythmia susceptibility. Moreover, the accelerated oligomerization by mutANP supports a role for these mediators in the pathophysiology of this mutation in atrial fibrillation.

Optimizing transesophageal atrial pacing in mice to detect atrial fibrillation.

Mice are routinely used to investigate molecular mechanisms underlying the atrial fibrillation (AF) substrate. We sought to optimize transesophageal rapid atrial pacing (RAP) protocols for the detection of AF susceptibility in mouse models. Hypertensive and control C57Bl/6J mice were subjected to burst RAP at a fixed stimulus amplitude. The role of parasympathetic involvement in pacing-related atrioventricular (AV) block and AF was examined using an intraperitoneal injection of atropine. In a crossover study, burst and decremental RAP at twice diastolic threshold were compared for induction of AV block during pacing. The efficacy of burst and decremental RAP to elicit an AF phenotype was subsequently investigated in mice deficient in the lymphocyte adaptor protein (Lnk-/-) resulting in systemic inflammation, or the paired-like homeodomain-2 transcription factor (Pitx2+/-) as a positive control. When pacing at a fixed stimulus intensity, pacing-induced AV block with AF induction occurred frequently, so that there was no difference in AF burden between hypertensive and control mice. These effects were prevented by atropine administration, implicating parasympathetic activation due to ganglionic stimulation as the etiology. When mice with AV block during pacing were eliminated from the analysis, male Lnk-/- mice displayed an AF phenotype only during burst RAP compared with controls, whereas male Pitx2+/- mice showed AF susceptibility during burst and decremental RAP. Notably, Lnk-/- and Pitx2+/- females exhibited no AF phenotype. Our data support the conclusion that multiple parameters should be used to ascertain AF inducibility and facilitate reproducibility across models and studies.NEW & NOTEWORTHY Methods were developed to optimize transesophageal rapid atrial pacing (RAP) to detect AF susceptibility in new and established mouse models. High stimulus intensity and pacing rates caused parasympathetic stimulation, with pacing-induced AV block and excessive AF induction in normal mice. For a given model, pacing at twice TH enabled improved phenotype discrimination in a pacing mode and sex-specific manner. Transesophageal RAP should be individually optimized when developing a mouse model of AF.

Highly Reactive Isolevuglandins Promote Atrial Fibrillation Caused by Hypertension.

Oxidative damage is implicated in atrial fibrillation (AF), but antioxidants are ineffective therapeutically. The authors tested the hypothesis that highly reactive lipid dicarbonyl metabolites, or isolevuglandins (IsoLGs), are principal drivers of AF during hypertension. In a hypertensive murine model and stretched atriomyocytes, the dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA) prevented IsoLG adducts and preamyloid oligomers (PAOs), and AF susceptibility, whereas the ineffective analog 4-hydroxybenzylamine (4-HOBA) had minimal effect. Natriuretic peptides generated cytotoxic oligomers, a process accelerated by IsoLGs, contributing to atrial PAO formation. These findings support the concept of pre-emptively scavenging reactive downstream oxidative stress mediators as a potential therapeutic approach to prevent AF.

Cardiomyocyte Homeodomain-Interacting Protein Kinase 2 Maintains Basal Cardiac Function via Extracellular Signal-Regulated Kinase Signaling.

BACKGROUND: Cardiac kinases play a critical role in the development of heart failure, and represent potential tractable therapeutic targets. However, only a very small fraction of the cardiac kinome has been investigated. To identify novel cardiac kinases involved in heart failure, we used an integrated transcriptomics and bioinformatics analysis and identified Homeodomain-Interacting Protein Kinase 2 (HIPK2) as a novel candidate kinase. The role of HIPK2 in cardiac biology is unknown. METHODS: We used the Expression2Kinase algorithm for the screening of kinase targets. To determine the role of HIPK2 in the heart, we generated cardiomyocyte (CM)-specific HIPK2 knockout and heterozygous mice. Heart function was examined by echocardiography, and related cellular and molecular mechanisms were examined. Adeno-associated virus serotype 9 carrying cardiac-specific constitutively active MEK1 (TnT-MEK1-CA) was administrated to rescue cardiac dysfunction in CM-HIPK2 knockout mice. RESULTS: To our knowledge, this is the first study to define the role of HIPK2 in cardiac biology. Using multiple HIPK2 loss-of-function mouse models, we demonstrated that reduction of HIPK2 in CMs leads to cardiac dysfunction, suggesting a causal role in heart failure. It is important to note that cardiac dysfunction in HIPK2 knockout mice developed with advancing age, but not during development. In addition, CM-HIPK2 knockout mice and CM-HIPK2 heterozygous mice exhibited a gene dose-response relationship of CM-HIPK2 on heart function. HIPK2 expression in the heart was significantly reduced in human end-stage ischemic cardiomyopathy in comparison to nonfailing myocardium, suggesting a clinical relevance of HIPK2 in cardiac biology. In vitro studies with neonatal rat ventricular CMscorroborated the in vivo findings. Specifically, adenovirus-mediated overexpression of HIPK2 suppressed the expression of heart failure markers, NPPA and NPPB, at basal condition and abolished phenylephrine-induced pathological gene expression. An array of mechanistic studies revealed impaired extracellular signal-regulated kinase 1/2 signaling in HIPK2-deficient hearts. An in vivo rescue experiment with adeno-associated virus serotype 9 TnT-MEK1-CA nearly abolished the detrimental phenotype of knockout mice, suggesting that impaired extracellular signal-regulated kinase signaling mediated apoptosis as the key factor driving the detrimental phenotype in CM-HIPK2 knockout mice hearts. CONCLUSIONS: Taken together, these findings suggest that CM-HIPK2 is required to maintain normal cardiac function via extracellular signal-regulated kinase signaling.

Microvascular Disease, Peripheral Artery Disease, and Amputation.

BACKGROUND: The mechanism of adverse limb events associated with peripheral artery disease remains incompletely understood. We investigated whether microvascular disease is associated with amputation in a large cohort of veterans to determine whether microvascular disease diagnosed in any location increases the risk of amputation alone and in concert with peripheral artery disease. METHODS: Participants in the Veterans Aging Cohort Study were recruited from April 1, 2003 through December 31, 2014. We excluded participants with known prior lower limb amputation. Using time-updated Cox proportional hazards regression, we analyzed the effect of prevalent microvascular disease (retinopathy, neuropathy, and nephropathy) and peripheral artery disease status on the risk of incident amputation events after adjusting for demographics and cardiovascular risk factors. RESULTS: Among 125 674 veterans without evidence of prior amputation at baseline, the rate of incident amputation over a median of 9.3 years of follow-up was 1.16 per 1000 person-years, yielding a total of 1185 amputations. In time-updated multivariable-adjusted analyses, compared with those without peripheral artery disease or microvascular disease, microvascular disease alone was associated with a 3.7-fold (95% CI, 3.0-4.6) increased risk of amputation; peripheral artery disease alone conferred a 13.9-fold (95% CI, 11.3-17.1) elevated risk of amputation; and the combination of peripheral artery disease and microvascular disease was associated with a 22.7-fold (95% CI, 18.3-28.1) increased risk of amputation. CONCLUSIONS: Independent of traditional risk factors, the presence of microvascular disease increases the risk of amputation alone and synergistically increases risk in patients with peripheral artery disease. Further research is needed to understand the mechanisms by which this occurs.

2017 Meeting of the National Directors of Graduate Studies in Pharmacology and Physiology.

The National Directors of Graduate Studies biennial meeting is a forum for directors from pharmacology and physiology graduate programs to discuss challenges and best practices for programs that are preparing trainees to be successful in the biomedical workforce. The 2017 meeting was held on the campus of Stony Brook University in Stony Brook, NY. Over the course of the 3-day event, several themes evolved, including graduate education training and curricula, diversity and career development, and scientific rigor and communication. Overall, presentations and discussions highlighted the challenges and opportunities for training PhD biomedical scientists and featured best practices from across the country.

Hypoxia Supports Epicardial Cell Differentiation in Vascular Smooth Muscle Cells through the Activation of the TGFß Pathway.

Epicardium-derived cells (EPDCs) are an important pool of multipotent cardiovascular progenitor cells. Through epithelial-to-mesenchymal-transition (EMT), EPDCs invade the subepicardium and myocardium and further differentiate into several cell types required for coronary vessel formation. We previously showed that epicardial hypoxia inducible factor (HIF) signaling mediates the invasion of vascular precursor cells critical for patterning the coronary vasculature. Here, we examine the regulatory role of hypoxia (1% oxygen) on EPDC differentiation into vascular smooth muscle cells (VSMCs). RESULTS: Hypoxia stimulates EMT and enhances expression of several VSMC markers in mouse epicardial cell cultures. This stimulation is specifically blocked by inhibiting transforming growth factor-beta (TGFβ) receptor I. Further analyses indicated that hypoxia increases the expression level of TGFβ-1 ligand and phosphorylation of TGFβ receptor II, suggesting an indispensable role of the TGFβ pathway in hypoxia-stimulated VSMC differentiation. We further demonstrate that the non-canonical RhoA/Rho kinase (ROCK) pathway acts as the main downstream effector of TGFβ to modulate hypoxia’s effect on VSMC differentiation. CONCLUSION: Our results reveal a novel role of epicardial HIF in mediating coronary vasculogenesis by promoting their differentiation into VSMCs through noncanonical TGFβ signaling. These data elucidate that patterning of the coronary vasculature is influenced by epicardial hypoxic signals.

A comparison of best practices for doctoral training in Europe and North America.

The PhD degree was established in Berlin 200 years ago and has since spread across the whole world. While there is general agreement that the degree is awarded in recognition of successfully completed research training, there have been significant differences in the way doctoral training programs have developed in particular countries. There is, however, a clear global tendency to follow the programs currently used either in the United States or in Europe. To determine more clearly how US and European PhD programs are both similar and different, we have used a validated questionnaire to analyze biomedical PhD programs in four representative institutions at Vanderbilt University, University of Manitoba, Karolinska Institutet, and Graz Medical University. The analysis is based on 63 detailed questions concerning the research environment, outcomes, admission criteria, content of programs, mentoring (or supervising), the PhD thesis, assessment of the thesis, and PhD school structure. The results reveal that while there is considerable overlap in the aims and content of PhD programs, there are also considerable differences regarding the structure of PhD programs, mentoring and assessment of PhD theses. These differences are analyzed in detail in order to provide a foundation for discussion of their relative advantages and disadvantages, with a view to providing a platform for discussion of best practices. The results will be of importance in the continued development of global discussion about development of doctoral training.

Plasmin Prevents Dystrophic Calcification After Muscle Injury.

Extensive or persistent calcium phosphate deposition within soft tissues after severe traumatic injury or major orthopedic surgery can result in pain and loss of joint function. The pathophysiology of soft tissue calcification, including dystrophic calcification and heterotopic ossification (HO), is poorly understood; consequently, current treatments are suboptimal. Here, we show that plasmin protease activity prevents dystrophic calcification within injured skeletal muscle independent of its canonical fibrinolytic function. After muscle injury, dystrophic calcifications either can be resorbed during the process of tissue healing, persist, or become organized into mature bone (HO). Without sufficient plasmin activity, dystrophic calcifications persist after muscle injury and are sufficient to induce HO. Downregulating the primary inhibitor of plasmin (α2-antiplasmin) or treating with pyrophosphate analogues prevents dystrophic calcification and subsequent HO in vivo. Because plasmin also supports bone homeostasis and fracture repair, increasing plasmin activity represents the first pharmacologic strategy to prevent soft tissue calcification without adversely affecting systemic bone physiology or concurrent muscle and bone regeneration. © 2016 American Society for Bone and Mineral Research.

Transcriptional Profiling of Cultured, Embryonic Epicardial Cells Identifies Novel Genes and Signaling Pathways Regulated by TGFßR3 In Vitro.

The epicardium plays an important role in coronary vessel formation and Tgfbr3-/- mice exhibit failed coronary vessel development associated with decreased epicardial cell invasion. Immortalized Tgfbr3-/- epicardial cells display the same defects. Tgfbr3+/+ and Tgfbr3-/- cells incubated for 72 hours with VEH or ligands known to promote invasion via TGFßR3 (TGFß1, TGFß2, BMP2), for 72 hours were harvested for RNA-seq analysis. We selected for genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells when incubated with VEH (604), TGFß1 (515), TGFß2 (553), or BMP2 (632). Gene Ontology (GO) analysis of these genes identified dysregulated biological processes consistent with the defects observed in Tgfbr3-/- cells, including those associated with extracellular matrix interaction. GO and Gene Regulatory Network (GRN) analysis identified distinct expression profiles between TGFß1-TGFß2 and VEH-BMP2 incubated cells, consistent with the differential response of epicardial cells to these ligands in vitro. Despite the differences observed between Tgfbr3+/+ and Tgfbr3-/- cells after TGFß and BMP ligand addition, GRNs constructed from these gene lists identified NF-ĸB as a key nodal point for all ligands examined. Tgfbr3-/- cells exhibited decreased expression of genes known to be activated by NF-ĸB signaling. NF-ĸB activity was stimulated in Tgfbr3+/+ epicardial cells after TGFß2 or BMP2 incubation, while Tgfbr3-/- cells failed to activate NF-ĸB in response to these ligands. Tgfbr3+/+ epicardial cells incubated with an inhibitor of NF-ĸB signaling no longer invaded into a collagen gel in response to TGFß2 or BMP2. These data suggest that NF-ĸB signaling is dysregulated in Tgfbr3-/- epicardial cells and that NF-ĸB signaling is required for epicardial cell invasion in vitro. Our approach successfully identified a signaling pathway important in epicardial cell behavior downstream of TGFßR3. Overall, the genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFßR3 signaling.

Common pathways regulate Type III TGFß receptor-dependent cell invasion in epicardial and endocardial cells.

Epithelial-Mesenchymal Transformation (EMT) and the subsequent invasion of epicardial and endocardial cells during cardiac development is critical to the development of the coronary vessels and heart valves. The transformed cells give rise to cardiac fibroblasts and vascular smooth muscle cells or valvular interstitial cells, respectively. The Type III Transforming Growth Factor ß (TGFßR3) receptor regulates EMT and cell invasion in both cell types, but the signaling mechanisms downstream of TGFßR3 are not well understood. Here we use epicardial and endocardial cells in in vitro cell invasion assays to identify common mechanisms downstream of TGFßR3 that regulate cell invasion. Inhibition of NF-κB activity blocked cell invasion in epicardial and endocardial cells. NF-κB signaling was found to be dysregulated in Tgfbr3(-/-) epicardial cells which also show impaired cell invasion in response to ligand. TGFßR3-dependent cell invasion is also dependent upon Activin Receptor-Like Kinase (ALK) 2, ALK3, and ALK5 activity. A TGFßR3 mutant that contains a threonine to alanine substitution at residue 841 (TGFßR3-T841A) induces ligand-independent cell invasion in both epicardial and endocardial cells in vitro. These findings reveal a role for NF-κB signaling in the regulation of epicardial and endocardial cell invasion and identify a mutation in TGFßR3 which stimulates ligand-independent signaling.

Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification.

Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.

Reactive γ-ketoaldehydes promote protein misfolding and preamyloid oligomer formation in rapidly-activated atrial cells.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid ß1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

Type III transforming growth factor beta receptor regulates vascular and osteoblast development during palatogenesis.

BACKGROUND: Cleft palate occurs in up to 1:1,000 live births and is associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palatal shelf elongation, elevation, and fusion. Transforming growth factor ß (TGFß) and bone morphogenetic protein 2 (BMP2) canonical signaling is required during each stage of palate development. The type III TGFß receptor (TGFßR3) binds all three TGFß ligands and BMP2, but its contribution to palatogenesis is unknown. RESULTS: The role of TGFßR3 during palate formation was found to be during palatal shelf elongation and elevation. Tgfbr3(-) (/) (-) embryos displayed reduced palatal shelf width and height, changes in proliferation and apoptosis, and reduced vascular and osteoblast differentiation. Abnormal vascular plexus organization as well as aberrant expression of arterial (Notch1, Alk1), venous (EphB4), and lymphatic (Lyve1) markers was also observed. Decreased osteoblast differentiation factors (Runx2, alk phos, osteocalcin, col1A1, and col1A2) demonstrated poor mesenchymal cell commitment to the osteoblast lineage within the maxilla and palatal shelves in Tgfbr3(-) (/) (-) embryos. Additionally, in vitro bone mineralization induced by osteogenic medium (OM+BMP2) was insufficient in Tgfbr3(-) (/) (-) palatal mesenchyme, but mineralization was rescued by overexpression of TGFßR3. CONCLUSIONS: These data reveal a critical, previously unrecognized role for TGFßR3 in vascular and osteoblast development during palatogenesis.

Type III TGFß receptor and Src direct hyaluronan-mediated invasive cell motility.

During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. This process requires epithelial to mesenchymal transition (EMT) and directed cellular invasion. The Type III Transforming Growth Factor-beta Receptor (TGFßR3) is required for epicardial cell invasion and coronary vessel development. Using primary epicardial cells derived from Tgfbr3(+/+) and Tgfbr3(-/-) mouse embryos, high-molecular weight hyaluronan (HMWHA) stimulated cellular invasion and filamentous (f-actin) polymerization are detected in Tgfbr3(+/+) cells, but not in Tgfbr3(-/-) cells. Furthermore, HMWHA-stimulated cellular invasion and f-actin polymerization in Tgfbr3(+/+) epicardial cells are dependent on Src kinase. Src activation in HMWHA-stimulated Tgfbr3(-/-) epicardial cells is not detected in response to HMWHA. RhoA and Rac1 also fail to activate in response to HMWHA in Tgfbr3(-/-) cells. These events coincide with defective f-actin formation and deficient cellular invasion. Finally, a T841A activating substitution in TGFßR3 drives ligand-independent Src activation. Collectively, these data define a TGFßR3-Src-RhoA/Rac1 pathway that is essential for hyaluronan-directed cell invasion in epicardial cells.