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.

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.

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.

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.

The chick embryo as an expanding experimental model for cancer and cardiovascular research.

A long and productive history in biomedical research defines the chick as a model for human biology. Fundamental discoveries, including the description of directional circulation propelled by the heart and the link between oncogenes and the formation of cancer, indicate its utility in cardiac biology and cancer. Despite the more recent arrival of several vertebrate and invertebrate animal models during the last century, the chick embryo remains a commonly used model for vertebrate biology and provides a tractable biological template. With new molecular and genetic tools applied to the avian genome, the chick embryo is accelerating the discovery of normal development and elusive disease processes. Moreover, progress in imaging and chick culture technologies is advancing real-time visualization of dynamic biological events, such as tissue morphogenesis, angiogenesis, and cancer metastasis. A rich background of information, coupled with new technologies and relative ease of maintenance, suggest an expanding utility for the chick embryo in cardiac biology and cancer research.

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.

Spatial transcriptional profile of the chick and mouse endocardial cushions identify novel regulators of endocardial EMT in vitro.

Valvular Interstitial Cells (VICs) are a common substrate for congenital and adult heart disease yet the signaling mechanisms governing their formation during early valvulogenesis are incompletely understood. We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 198 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGFß as a nodal point and identified NF-κB as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-κB, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-κB pathway as regulators of endocardial cell EMT in vitro.

Disruption of canonical TGFß-signaling in murine coronary progenitor cells by low level arsenic.

Exposure to arsenic results in several types of cancers as well as heart disease. A major contributor to ischemic heart pathologies is coronary artery disease, however the influences by environmental arsenic in this disease process are not known. Similarly, the impact of toxicants on blood vessel formation and function during development has not been studied. During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types including smooth muscle cells which contribute to the coronary vessels. The TGFß family of ligands and receptors is essential for developmental cardiac epithelial to mesenchymal transition (EMT) and differentiation into coronary smooth muscle cells. In this in vitro study, 18hour exposure to 1.34µM arsenite disrupted developmental EMT programming in murine epicardial cells causing a deficit in cardiac mesenchyme. The expression of EMT genes including TGFß2, TGFß receptor-3, Snail, and Has-2 are decreased in a dose-dependent manner following exposure to arsenite. TGFß2 cell signaling is abrogated as detected by decreases in phosphorylated Smad2/3 when cells are exposed to 1.34µM arsenite. There is also loss of nuclear accumulation pSmad due to arsenite exposure. These observations coincide with a decrease in vimentin positive mesenchymal cells invading three-dimensional collagen gels. However, arsenite does not block TGFß2 mediated smooth muscle cell differentiation by epicardial cells. Overall these results show that arsenic exposure blocks developmental EMT gene programming in murine coronary progenitor cells by disrupting TGFß2 signals and Smad activation, and that smooth muscle cell differentiation is refractory to this arsenic toxicity.

The cytoplasmic domain of TGFßR3 through its interaction with the scaffolding protein, GIPC, directs epicardial cell behavior.

The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor ß Receptor (TGFßR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFßR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFßR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3(-/-) hearts. Tgfbr3(-/-) epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFß1 and TGFß2. Unexpectedly, loss of TGFßR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFßR3 in Tgfbr3(-/-) cells rescued deficits in invasion in vitro in response TGFß1 and TGFß2 as well as FGF2 and HMW-HA. Expression of TGFßR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3(-/-) cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3(+/+) cells decreased invasion in response to TGFß2, FGF2, and HMW-HA. We conclude that TGFßR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3(-/-) mice.

Transforming growth factor beta signaling in adult cardiovascular diseases and repair.

The majority of children with congenital heart disease now live into adulthood due to the remarkable surgical and medical advances that have taken place over the past half century. Because of this, adults now represent the largest age group with adult cardiovascular diseases. It includes patients with heart diseases that were not detected or not treated during childhood, those whose defects were surgically corrected but now need revision due to maladaptive responses to the procedure, those with exercise problems and those with age-related degenerative diseases. Because adult cardiovascular diseases in this population are relatively new, they are not well understood. It is therefore necessary to understand the molecular and physiological pathways involved if we are to improve treatments. Since there is a developmental basis to adult cardiovascular disease, transforming growth factor beta (TGFß) signaling pathways that are essential for proper cardiovascular development may also play critical roles in the homeostatic, repair and stress response processes involved in adult cardiovascular diseases. Consequently, we have chosen to summarize the current information on a subset of TGFß ligand and receptor genes and related effector genes that, when dysregulated, are known to lead to cardiovascular diseases and adult cardiovascular deficiencies and/or pathologies. A better understanding of the TGFß signaling network in cardiovascular disease and repair will impact genetic and physiologic investigations of cardiovascular diseases in elderly patients and lead to an improvement in clinical interventions.

Regulation of the activity and expression of aryl hydrocarbon receptor by ethanol in mouse hepatic stellate cells.

BACKGROUND: During the course of alcohol-induced liver damage, hepatic stellate cells are transformed into proliferative, fibrogenic, and contractile myofibroblasts. Aryl hydrocarbon receptor (AhR) is a transcription factor that controls the expression of genes involved in the metabolism of xenobiotics, inflammation, cell proliferation, and death. METHODS: Immortal mouse hepatic stellate cells (MHSCs) were isolated from transgenic mice that expressed a thermolabile SV40 tumor antigen. Quantitative real-time reverse transcription polymerase chain reaction assays, Western blot analysis, promoter activity assays, and chromatin immunoprecipitation analyses were performed for studying the effect of ethanol (EtOH) on AhR expression and transcriptional activity. RESULTS: Treatment of MHSCs with 50 to 200 mM EtOH for 6 hours induced AhR nuclear translocation, enhanced the promoter activity of cytochrome P450 (CYP) 1A1, increased the amount of AhR bound to the promoter of CYP1A1 and 1B1, and up-regulated the mRNA expression of these AhR target genes in a dose-dependent manner. In contrast, EtOH exposure down-regulated AhR mRNA and protein expression. Similarly, benzo(a)pyrene (BaP) at 10 nM reduced AhR and increased CYP1A1 and 1B1 mRNAs. Pretreatment of MHSCs with 50 mM EtOH for 7 days diminished the capacity of MHSCs to express CYP1A1 and 1B1 induced by a 200 mM EtOH challenge, or by 10 nM BaP. However, the up-regulatory effect of EtOH on solute carrier family 16, member 6 (SLC16a6) was unaffected by EtOH pretreatment. Similar to EtOH, dimethyl sulfoxide (DMSO) at concentrations of 50 to 100 mM down-regulated AhR and up-regulated CYP1A1 mRNA expression in a dose-dependent manner. CONCLUSIONS: These data, for the first time, demonstrate that EtOH activates MHSC AhR and down-regulates its expression. Chronic EtOH pretreatment lowers the availability of AhR, and specifically diminishes the inducibility of CYP genes. The effect on AhR appears to not be an EtOH-specific response, as DMSO alone (and possibly other organic solvents) was also able to activate AhR.

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.

Collagen gel analysis of epithelial-mesenchymal transition in the embryo heart: an in vitro model system for the analysis of tissue interaction, signal transduction, and environmental effects.

The cellular process of epithelial-mesenchymal cell transition (EMT) is a critical event in development that is reiterated in adult pathologies of metastasis and organ fibrosis. An initial understanding of the cellular and molecular events of this process emerged from an in vitro examination of heart valve development. Explants of the chick atrioventricular valve-forming region were placed on collagen gels and removed to show that EMT was regulated by a tissue interaction. Subsequent studies showed that specific TGFß isoforms and receptors were required and steps of activation and invasion could be distinguished. The assay was modified for mouse hearts and has been used to explore signal transduction and gene expression in both species. The principle advantages of the system are a defined temporal window, when EMT takes place and the ability to isolate cells at various stages of the EMT process. These advantages are largely unavailable in other developmental or adult models. As the mesenchymal cells produced by EMT in the heart are involved in defects found in congenital heart disease, there is also a direct relevance of cardiac EMT to human birth defects. This relationship has been explored in relation to environmental exposures and in a number of genetic models. This review provides both an overview of the findings developed from the assay and protocols to enable the use of the assay by other laboratories. The assay provides a versatile platform to explore roles of specific gene products, drugs, and environmental agents on a critical cellular process.

Networked-based characterization of extracellular matrix proteins from adult mouse pulmonary and aortic valves.

A precise mixture of extracellular matrix (ECM) secreted by valvular cells forms a scaffold that lends the heart valve the exact mechanical and tensile strength needed for accurate hemodynamic performance. ECM proteins are a key component of valvular endothelial cell (VEC)-valvular interstitial cell (VIC) communication essential for maintenance of the valve structure. This study reports the healthy adult pulmonary and aortic valve proteomes characterized by LC-MS/MS, resulting in 2710 proteins expressed by 1513 genes, including over 300 abundant ECM proteins. Surprisingly, this study defines a distinct proteome for each semilunar valve. Protein-protein networking (PPN) was used as a tool to direct selection of proteomic candidates for biological investigation. Local PPN for nidogen 1 (Nid1), biglycan (Bgn), elastin microfibril interface-located protein 1 (Emilin-1), and milk fat globule-EGF factor 8 protein (Mfge8) were enriched with proteins essential to valve function and produced biological functions highly relevant to valve biology. Immunofluorescent investigations demonstrated that these proteins are functionally distributed within the pulmonary and aortic valve structure, indicative of important contribution to valve function. This study yields new insight into protein expression contributing to valvular maintenance and health and provides a platform for unbiased assessment of protein alterations during disease processes.

BMP-2 and TGFß2 shared pathways regulate endocardial cell transformation.

Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-ß receptor (TGFßR3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGFßR3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGFß signaling pathways in TGFßR3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGFßR3-dependent EMT stimulated by TGFß2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGFßR3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGFß2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGFßR3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGFßR3-dependent endocardial cell EMT stimulated by either TGFß2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.