Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

Impairment of ubiquitin-proteasome system by E334K cMyBPC modifies channel proteins, leading to electrophysiological dysfunction

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K_v1.5, Na_v1.5, Hcn4, Ca_v3.2, Ca_v1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca²⁺ transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca²⁺ transients were recapitulated by a simulation model. Although a Ca²⁺ antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca²⁺ transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.     

The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2

The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current I_Na and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on I_Na. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca²⁺ and Na⁺ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.     

Mog1 deficiency promotes cardiac contractile dysfunction and isoproterenol-induced arrhythmias associated with cardiac fibrosis and Cx43 remodeling

Our earlier studies identified MOG1 as a Nav1.5-binding protein that promotes Nav1.5 intracellular trafficking to plasma membranes. Genetic studies have identified MOG1 variants responsible for cardiac arrhythmias. However, the physiological functions of MOG1 in vivo remain incompletely characterized. In this study, we generated Mog1 knockout (Mog1^?/?) mice. Mog1^?/? mice did not develop spontaneous arrhythmias at the baseline, but exhibited a prolongation of QRS duration. Mog1^?/? mice treated with isoproterenol (ISO), but not with flecainide, exhibited an increased risk of arrhythmias and even sudden death. Mog1^?/? mice had normal cardiac morphology, however, LV systolic dysfunction was identified and associated with an increase in ventricular fibrosis. Whole-cell patch-clamping and Western blotting analysis clearly demonstrated the normal cardiac expression and function of Nav1.5 in Mog1^?/? mice. Further RNA-seq and iTRAQ analysis identified critical pathways and genes, including extracellular matrix (Mmp2), gap junction (Gja1), and mitochondrial components that were dysregulated in Mog1^?/? mice. RT-qPCR, Western blotting, and immunofluorescence assays revealed reduced cardiac expression of Gja1 in Mog1^?/? mice. Dye transfer assays confirmed impairment of gap-junction function; Cx43 gap-junction enhancer ZP123 decreased arrhythmia inducibility in ISO-treated Mog1^?/? mice. Transmission electron microscopy analysis revealed abnormal sarcomere ultrastructure and altered mitochondrial morphology in Mog1^?/? mice. Mitochondrial dynamics was found to be disturbed, and associated with a trend toward increased mitochondrial fusion in Mog1^?/? mice. Meanwhile, the level of ATP supply was increased in the hearts of Mog1^?/? mice. These results indicate that MOG1 plays an important role in cardiac electrophysiology and cardiac contractile function.     

Reduced Sialylation Impacts Ventricular Repolarization by Modulating Specific K+ Channel Isoforms Distinctly

Voltage-gated K⁺ channels (K_v) are responsible for repolarizing excitable cells and can be heavily glycosylated. Cardiac K_v activity is indispensable where even minimal reductions in function can extend action potential duration, prolong QT intervals, and ultimately contribute to life-threatening arrhythmias. Diseases such as congenital disorders of glycosylation often cause significant cardiac phenotypes that can include arrhythmias. Here we investigated the impact of reduced sialylation on ventricular repolarization through gene deletion of the sialyltransferase ST3Gal4. ST3Gal4-deficient mice (ST3Gal4^−/−) had prolonged QT intervals with a concomitant increase in ventricular action potential duration. Ventricular apex myocytes isolated from ST3Gal4^−/− mice demonstrated depolarizing shifts in activation gating of the transient outward (I_to) and delayed rectifier (I_Kslow) components of K⁺ current with no change in maximum current densities. Consistently, similar protein expression levels of the three K_v isoforms responsible for I_to and I_Kslow were measured for ST3Gal4^−/− versus controls. However, novel non-enzymatic sialic acid labeling indicated a reduction in sialylation of ST3Gal4^−/− ventricular K_v4.2 and K_v1.5, which contribute to I_to and I_Kslow, respectively. Thus, we describe here a novel form of regulating cardiac function through the activities of a specific glycogene product. Namely, reduced ST3Gal4 activity leads to a loss of isoform-specific K_v sialylation and function, thereby limiting K_v activity during the action potential and decreasing repolarization rate, which likely contributes to prolonged ventricular repolarization. These studies elucidate a novel role for individual glycogene products in contributing to a complex network of cardiac regulation under normal and pathologic conditions.     

Inhibition of the Cardiac Na+ Channel Nav1.5 by Carbon Monoxide

Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na⁺ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na⁺ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, l-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor N^ω-nitro-l-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na⁺ current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state.     

Period2 Deficiency Blunts Hypoxia-Induced Mobilization and Function of Endothelial Progenitor Cells

Background

In the clinic, variations in circadian rhythm are evident in patients with cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. In this study, we focused on the role of the circadian gene period2 (per2) in mobilization and function of endothelial progenitor cells (EPCs) in vitro and in vivo after myocardial infarction (MI) in mice.

Methods and Results

MI was produced by surgical ligation of the left anterior descending coronary artery in mice with and without per2 deficiency. Trans-thoracic echocardiography was used to evaluate cardiac function in mice. Per2^−/− mice with MI showed decreased cardiac function and increased infarct size. The number of CD34+ cells and capillary density were decreased in the myocardium of per2^−/− mice on immunohistochemistry. Flow cytometry revealed decreased number of circulating EPCs in per2^−/− mice after MI. In vitro, per2^−/− EPCs showed decreased migration and tube formation capacity under hypoxia. Western blot analysis revealed inhibited activation of extracellular signal-regulated kinase and Akt signaling in the bone marrow of per2^−/− mice and inhibited PI3K/Akt expression in per2^−/− EPCs under hypoxia.

Conclusions

Per2 modulates EPC mobilization and function after MI, which is important to recovery after MI in mice.     

MAIR‐II deficiency ameliorates cardiac remodelling post‐myocardial infarction by suppressing TLR9‐mediated macrophage activation

Macrophages are fundamental components of inflammation in post‐myocardial infarction (MI) and contribute to adverse cardiac remodelling and heart failure. However, the regulatory mechanisms in macrophage activation have not been fully elucidated. Previous studies showed that myeloid‐associated immunoglobulin–like receptor II (MAIR‐II) is involved in inflammatory responses in macrophages. However, its role in MI is unknown. Thus, this study aimed to determine a novel role and mechanism of MAIR‐II in MI. We first identified that MAIR‐II–positive myeloid cells were abundant from post‐MI days 3 to 5 in infarcted hearts of C57BL/6J (WT) mice induced by permanent left coronary artery ligation. Compared to WT, MAIR‐II–deficient (Cd300c2 ^−/−) mice had longer survival, ameliorated cardiac remodelling, improved cardiac function and smaller infarct sizes. Moreover, we detected lower pro‐inflammatory cytokine and fibrotic gene expressions in Cd300c2 ^−/−‐infarcted hearts. These mice also had less infiltrating pro‐inflammatory macrophages following MI. To elucidate a novel molecular mechanism of MAIR‐II, we considered macrophage activation by Toll‐like receptor (TLR) 9–mediated inflammation. In vitro, we observed that Cd300c2 ^−/− bone marrow–derived macrophages stimulated by a TLR9 agonist expressed less pro‐inflammatory cytokines compared to WT. In conclusion, MAIR‐II may enhance inflammation via TLR9‐mediated macrophage activation in MI, leading to adverse cardiac remodelling and poor prognosis.     

Mice with Tissue Inhibitor of Metalloproteinases 4 (Timp4) Deletion Succumb to Induced Myocardial Infarction but Not to Cardiac Pressure Overload

Tissue inhibitor of metalloproteinases 4 (TIMP4) is expressed highly in heart and found dysregulated in human cardiovascular diseases. It controls extracellular matrix remodeling by inhibiting matrix metalloproteinases (MMPs) and is implicated in processes including cell proliferation, apoptosis, and angiogenesis. Timp4-deficient mice (Timp4^−/−) were generated to assess TIMP4 function in normal development and in models of heart disease. We deleted exons 1–3 of the Timp4 gene by homologous recombination. Timp4^−/− mice are born healthy, develop normally, and produce litters of normal size and gender distribution. These mice show no compensation by overexpression of Timp1, Timp2, or Timp3 in the heart. Following cardiac pressure overload by aortic banding, Timp4^−/− mice have comparable survival rate, cardiac histology, and cardiac function to controls. In this case, Timp4 deficiency is compensated by increased cardiac Timp2 expression. Strikingly, the induction of myocardial infarction (MI) leads to significantly increased mortality in Timp4^−/− mice primarily due to left ventricular rupture. The post-MI mortality of Timp4^−/− mice is reduced by administration of a synthetic MMP inhibitor. Furthermore, combining the genetic deletion of Mmp2 also rescues the higher post-MI mortality of Timp4^−/− mice. Finally, Timp4^−/− mice suffer reduced cardiac function at 20 months of age. Timp4 is not essential for murine development, although its loss moderately compromises cardiac function with aging. Timp4^−/− mice are more susceptible to MI but not to pressure overload, and TIMP4 functions in its capacity as a metalloproteinase inhibitor after myocardial infarction.     

KCNE2 and the K+ channel: The tail wagging the dog

KCNE2, originally designated MinK-related peptide 1 (MiRP1), belongs to a five-strong family of potassium channel ancillary (β) subunits that, despite the diminutive size of the family and its members, has loomed large in the field of ion channel physiology. KCNE2 dictates K⁺ channel gating, conductance, α subunit composition, trafficking and pharmacology, and also modifies functional properties of monovalent cation-nonselective HCN channels. The Kcne2^−/− mouse exhibits cardiac arrhythmia and hypertrophy, achlorhydria, gastric neoplasia, hypothyroidism, alopecia, stunted growth and choroid plexus epithelial dysfunction, illustrating the breadth and depth of the influence of KCNE2, mutations which are also associated with human cardiac arrhythmias. Here, the modus operandi and physiological roles of this potent regulator of membrane excitability and ion secretion are reviewed with particular emphasis on the ability of KCNE2 to shape the electrophysiological landscape of both excitable and non-excitable cells.     

Common Genetic Variation Near the Phospholamban Gene Is Associated with Cardiac Repolarisation: Meta-Analysis of Three Genome-Wide Association Studies

To identify loci affecting the electrocardiographic QT interval, a measure of cardiac repolarisation associated with risk of ventricular arrhythmias and sudden cardiac death, we conducted a meta-analysis of three genome-wide association studies (GWAS) including 3,558 subjects from the TwinsUK and BRIGHT cohorts in the UK and the DCCT/EDIC cohort from North America. Five loci were significantly associated with QT interval at P<1×10⁻⁶. To validate these findings we performed an in silico comparison with data from two QT consortia: QTSCD (n = 15,842) and QTGEN (n = 13,685). Analysis confirmed the association between common variants near NOS1AP (P = 1.4×10⁻⁸³) and the phospholamban (PLN) gene (P = 1.9×10⁻²⁹). The most associated SNP near NOS1AP (rs12143842) explains 0.82% variance; the SNP near PLN (rs11153730) explains 0.74% variance of QT interval duration. We found no evidence for interaction between these two SNPs (P = 0.99). PLN is a key regulator of cardiac diastolic function and is involved in regulating intracellular calcium cycling, it has only recently been identified as a susceptibility locus for QT interval. These data offer further mechanistic insights into genetic influence on the QT interval which may predispose to life threatening arrhythmias and sudden cardiac death.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

Identification of a new co-factor, MOG1, required for the full function of cardiac sodium channel Nav 1.5

The cardiac sodium channel Nav 1.5 is essential for the physiological function of the heart and contributes to lethal cardiac arrhythmias and sudden death when mutated. Here, we report that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and modulates the physiological function of Nav 1.5. The yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domains DII and DIII) of Nav 1.5. The interaction was further demonstrated by both in vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation assays in both HEK293 cells with co-expression of MOG1 and Nav1.5 and native cardiac cells. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities. In neonatal myocytes, overexpression of MOG1 increased current densities nearly 2-fold. Western blot analysis revealed that MOG1 increased cell surface expression of Nav1.5, which may be the underlying mechanism by which MOG1 increased sodium current densities. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues with predominant localization at the intercalated discs. In cardiomyocytes, MOG1 is mostly localized in the cell membrane and co-localized with Nav1.5. These results indicate that MOG1 is a critical regulator of sodium channel function in the heart and reveal a new cellular function for MOG1. This study further demonstrates the functional diversity of Nav1.5-binding proteins, which serve important functions for Nav1.5 under different cellular conditions.     

Deletion of PDK1 Causes Cardiac Sodium Current Reduction in Mice

Background

The AGC protein kinase family regulates multiple cellular functions. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is involved in the pathogenesis of arrhythmia, and its downstream factor, Forkhead box O1 (Foxo1), negatively regulates the expression of the cardiac sodium channel, Nav1.5. Mice are known to die suddenly after PDK1 deletion within 11 weeks, but the underlying electrophysiological bases are unclear. Thus, the aim of this study was to investigate the potential mechanisms between PDK1 signaling pathway and cardiac sodium current.

Methods and Results

Using patch clamp and western blotting techniques, we investigated the role of the PDK1-Foxo1 pathway in PDK1 knockout mice and cultured cardiomyocytes. We found that PDK1 knockout mice undergo slower heart rate, prolonged QRS and QTc intervals and abnormal conduction within the first few weeks of birth. Furthermore, the peak sodium current is decreased by 33% in cells lacking PDK1. The phosphorylation of Akt (308T) and Foxo1 (24T) and the expression of Nav1.5 in the myocardium of PDK1-knockout mice are decreased, while the nuclear localization of Foxo1 is increased. The role of the PDK1-Foxo1 pathway in regulating Nav1.5 levels and sodium current density was verified using selective PDK1, Akt and Foxo1 inhibitors and isolated neonatal rat cardiomyocytes.

Conclusion

These results indicate that PDK1 participates in the dysregulation of electrophysiological basis by regulating the PDK1-Foxo1 pathway, which in turn regulates the expression of Nav1.5 and cardiac sodium channel function.     

Mitophagy‐regulated mitochondrial health strongly protects the heart against cardiac dysfunction after acute myocardial infarction

Autophagy including mitophagy serves as an important regulatory mechanism in the heart to maintain the cellular homeostasis and to protect against heart damages caused by myocardial infarction (MI). The current study aims to dissect roles of general autophagy and specific mitophagy in regulating cardiac function after MI. By using Beclin1^+/−, Fundc1 knockout (KO) and Fundc1 transgenic (TG) mouse models, combined with starvation and MI models, we found that Fundc1 KO caused more severe mitochondrial and cardiac dysfunction damages than Beclin1^+/− after MI. Interestingly, Beclin1^+/− caused notable decrease of total autophagy without detectable change to mitophagy, and Fundc1 KO markedly suppressed mitophagy but did not change the total autophagy activity. In contrast, starvation increased total autophagy without changing mitophagy while Fundc1 TG elevated total autophagy and mitophagy in mouse hearts. As a result, Fundc1 TG provided much stronger protective effects than starvation after MI. Moreover, Beclin1^+/−/Fundc1 TG showed increased total autophagy and mitophagy to a level comparable to Fundc1 TG per se, and completely reversed Beclin1^+/−‐caused aggravation of mitochondrial and cardiac injury after MI. Our results reveal that mitophagy but not general autophagy contributes predominantly to the cardiac protective effect through regulating mitochondrial function.     

CREG ameliorates the phenotypic switching of cardiac fibroblasts after myocardial infarction via modulation of CDC42

Phenotype switching of cardiac fibroblasts into myofibroblasts plays important role in cardiac fibrosis following myocardial infarction (MI). Cellular repressor of E1A-stimulated genes (CREG) protects against vascular and cardiac remodeling induced by angiotensin-II. However, the effects and mechanisms of CREG on phenotype switching of cardiac fibroblasts after MI are unknown. This study aimed to investigate the role of CREG on the phenotype switching of cardiac fibroblasts following MI and its mechanism. Our findings demonstrated that, compared with littermate control mice, cardiac function was deteriorated in CREG^+/− mice on day 14 post-MI. Fibrosis size, αSMA, and collagen-1 expressions were increased in the border regions of CREG^+/− mice on day 14 post-MI. Conversely, exogenous CREG protein significantly improved cardiac function, inhibited fibrosis, and reduced the expressions of αSMA and collagen-1 in the border regions of C57BL/6J mice on day 14. In vitro, CREG recombinant protein inhibited αSMA and collagen-1 expression and blocked the hypoxia-induced proliferation and migration of cardiac fibroblasts, which was mediated through the inhibition of cell division control protein 42 (CDC42) expression. Our findings could help in establishing new strategies based on the clarification of the role of the key molecule CREG in phenotype switching of cardiac fibroblasts following MI.Subject terms: Mechanisms of disease, Heart failure     

Results of Genome-Wide Analyses on Neurodevelopmental Phenotypes at Four-Year Follow-Up following Cardiac Surgery in Infancy

Background

Adverse neurodevelopmental sequelae are reported among children who undergo early cardiac surgery to repair congenital heart defects (CHD). APOE genotype has previously been determined to contribute to the prediction of these outcomes. Understanding further genetic causes for the development of poor neurobehavioral outcomes should enhance patient risk stratification and improve both prevention and treatment strategies.

Methods

We performed a prospective observational study of children who underwent cardiac surgery before six months of age; this included a neurodevelopmental evaluation between their fourth and fifth birthdays. Attention and behavioral skills were assessed through parental report utilizing the Attention Deficit-Hyperactivity Disorder-IV scale preschool edition (ADHD-IV), and Child Behavior Checklist (CBCL/1.5-5), respectively. Of the seven investigated, three neurodevelopmental phenotypes met genomic quality control criteria. Linear regression was performed to determine the effect of genome-wide genetic variation on these three neurodevelopmental measures in 316 subjects.

Results

This genome-wide association study identified single nucleotide polymorphisms (SNPs) associated with three neurobehavioral phenotypes in the postoperative children ADHD-IV Impulsivity/Hyperactivity, CBCL/1.5-5 PDPs, and CBCL/1.5-5 Total Problems. The most predictive SNPs for each phenotype were: a LGALS8 intronic SNP, rs4659682, associated with ADHD-IV Impulsivity (P = 1.03×10⁻⁶); a PCSK5 intronic SNP, rs2261722, associated with CBCL/1.5-5 PDPs (P = 1.11×10⁻⁶); and an intergenic SNP, rs11617488, 50 kb from FGF9, associated with CBCL/1.5-5 Total Problems (P = 3.47×10⁻⁷). 10 SNPs (3 for ADHD-IV Impulsivity, 5 for CBCL/1.5-5 PDPs, and 2 for CBCL/1.5-5 Total Problems) had p<10⁻⁵.

Conclusions

No SNPs met genome-wide significance for our three neurobehavioral phenotypes; however, 10 SNPs reached a threshold for suggestive significance (p<10⁻⁵). Given the unique nature of this cohort, larger studies and/or replication are not possible. Studies to further investigate the mechanisms through which these newly identified genes may influence neurodevelopment dysfunction are warranted.