Patrick Y. Jay

Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system

Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40(-)/connexin45(+) cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system.

Protective effects of exercise and phosphoinositide 3-kinase(p110α) signaling in dilated and hypertrophic cardiomyopathy

Physical activity protects against cardiovascular disease, and physiological cardiac hypertrophy associated with regular exercise is usually beneficial, in marked contrast to pathological hypertrophy associated with disease. The p110α isoform of phosphoinositide 3-kinase (PI3K) plays a critical role in the induction of exercise-induced hypertrophy. Whether it or other genes activated in the athletes heart might have an impact on cardiac function and survival in a setting of heart failure is unknown. To examine whether progressive exercise training and PI3K(p110α) activity affect survival and/or cardiac function in two models of heart disease, we subjected a transgenic mouse model of dilated cardiomyopathy (DCM) to swim training, genetically crossed cardiac-specific transgenic mice with increased or decreased PI3K(p110α) activity to the DCM model, and subjected PI3K(p110α) transgenics to acute pressure overload (ascending aortic constriction). Life-span, cardiac function, and molecular markers of pathological hypertrophy were examined. Exercise training and increased cardiac PI3K(p110α) activity prolonged survival in the DCM model by 15–20%. In contrast, reduced PI3K(p110α) activity drastically shortened lifespan by ≈50%. Increased PI3K(p110α) activity had a favorable effect on cardiac function and fibrosis in the pressure-overload model and attenuated pathological growth. PI3K(p110α) signaling negatively regulated G protein-coupled receptor stimulated extracellular responsive kinase and Akt (via PI3K, p110γ) activation in isolated cardiomyocytes. These findings suggest that exercise and enhanced PI3K(p110α) activity delay or prevent progression of heart disease, and that supraphysiologic activity can be beneficial. Identification of genes important for hypertrophy in the athletes heart could offer new strategies for treating heart failure.

Capping protein levels influence actin assembly and cell motility in dictyostelium

Actin assembly is important for cell motility, but the mechanism of assembly and how it relates to motility in vivo is largely unknown. In vitro, actin assembly can be controlled by proteins, such as capping protein, that bind filament ends. To investigate the function of actin assembly in vivo, we altered the levels of capping protein in Dictyostelium cells and found changes in resting and chemoattractant-induced actin assembly that were consistent with the in vitro properties of capping protein in capping but not nucleation. Significantly, overexpressers moved faster and underexpressers moved slower than control cells. Mutants also exhibited changes in cytoskeleton architecture. These results provide insights into in vivo actin assembly and the role of the actin cytoskeleton in motility.

Mice lacking sister chromatid cohesion protein PDS5B exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome

PDS5B is a sister chromatid cohesion protein that is crucial for faithful segregation of duplicated chromosomes in lower organisms. Mutations in cohesion proteins are associated with the developmental disorder Cornelia de Lange syndrome (CdLS) in humans. To delineate the physiological roles of PDS5B in mammals, we generated mice lacking PDS5B (APRIN). Pds5B-deficient mice died shortly after birth. They exhibited multiple congenital anomalies, including heart defects, cleft palate, fusion of the ribs, short limbs, distal colon aganglionosis, abnormal migration and axonal projections of sympathetic neurons, and germ cell depletion, many of which are similar to abnormalities found in humans with CdLS. Unexpectedly, we found no cohesion defects in Pds5B-/- cells and detected high PDS5B expression in post-mitotic neurons in the brain. These results, along with the developmental anomalies of Pds5B-/- mice, the presence of a DNA-binding domain in PDS5B in vertebrates and its nucleolar localization, suggest that PDS5B and the cohesin complex have important functions beyond their role in chromosomal dynamics.

Dosage Effects of Cohesin Regulatory Factor PDS5 on Mammalian Development: Implications for Cohesinopathies

Cornelia de Lange syndrome (CdLS), a disorder caused by mutations in cohesion proteins, is characterized by multisystem developmental abnormalities. PDS5, a cohesion protein, is important for proper chromosome segregation in lower organisms and has two homologues in vertebrates (PDS5A and PDS5B). Pds5B mutant mice have developmental abnormalities resembling CdLS; however the role of Pds5A in mammals and the association of PDS5 proteins with CdLS are unknown. To delineate genetic interactions between Pds5A and Pds5B and explore mechanisms underlying phenotypic variability, we generated Pds5A-deficient mice. Curiously, these mice exhibit multiple abnormalities that were previously observed in Pds5B-deficient mice, including cleft palate, skeletal patterning defects, growth retardation, congenital heart defects and delayed migration of enteric neuron precursors. They also frequently display renal agenesis, an abnormality not observed in Pds5B−/− mice. While Pds5A−/− and Pds5B−/− mice die at birth, embryos harboring 3 mutant Pds5 alleles die between E11.5 and E12.5 most likely of heart failure, indicating that total Pds5 gene dosage is critical for normal development. In addition, characterization of these compound homozygous-heterozygous mice revealed a severe abnormality in lens formation that does not occur in either Pds5A−/− or Pds5B−/− mice. We further identified a functional missense mutation (R1292Q) in the PDS5B DNA-binding domain in a familial case of CdLS, in which affected individuals also develop megacolon. This study shows that PDS5A and PDS5B functions other than those involving chromosomal dynamics are important for normal development, highlights the sensitivity of key developmental processes on PDS5 signaling, and provides mechanistic insights into how PDS5 mutations may lead to CdLS.

Heterogeneity of genetic modifiers ensures normal cardiac development

Background— Mutations of the transcription factor Nkx2-5 cause pleiotropic heart defects with incomplete penetrance. This variability suggests that additional factors can affect or prevent the mutant phenotype. We assess here the role of genetic modifiers and their interactions. Methods and Results— Heterozygous Nkx2-5 knockout mice in the inbred strain background C57Bl/6 frequently have atrial and ventricular septal defects. The incidences are substantially reduced in the Nkx2-5+/− progeny of first-generation (F1) outcrosses to the strains FVB/N or A/J. Defects recur in the second generation (F2) of the F1×F1 intercross or backcrosses to the parental strains. Analysis of >3000 Nkx2-5+/− hearts from 5 F2 crosses demonstrates the profound influence of genetic modifiers on disease presentation. On the basis of their incidences and coincidences, anatomically distinct malformations have shared and unique modifiers. All 3 strains carry susceptibility alleles at different loci for atrial and ventricular septal defects. Relative to the other 2 strains, A/J carries polymorphisms that confer greater susceptibility to atrial septal defect and atrioventricular septal defects and C57Bl/6 to muscular ventricular septal defects. Segregation analyses reveal that ≥2 loci influence membranous ventricular septal defect susceptibility, whereas ≥2 loci and at least 1 epistatic interaction affect muscular ventricular and atrial septal defects. Conclusions— Alleles of modifier genes can either buffer perturbations on cardiac development or direct the manifestation of a defect. In a genetically heterogeneous population, the predominant effect of modifier genes is health.

Exenatide improves glucose homeostasis and prolongs survival in a murine model of dilated cardiomyopathy

Background There is growing awareness of secondary insulin resistance and alterations in myocardial glucose utilization in congestive heart failure. Whether therapies that directly target these changes would be beneficial is unclear. We previously demonstrated that acute blockade of the insulin responsive facilitative glucose transporter GLUT4 precipitates acute decompensated heart failure in mice with advanced dilated cardiomyopathy. Our current objective was to determine whether pharmacologic enhancement of insulin sensitivity and myocardial glucose uptake preserves cardiac function and survival in the setting of primary heart failure. Methodology/Principal Findings The GLP-1 agonist exenatide was administered twice daily to a murine model of dilated cardiomyopathy (TG9) starting at 56 days of life. TG9 mice develop congestive heart failure and secondary insulin resistance in a highly predictable manner with death by 12 weeks of age. Glucose homeostasis was assessed by measuring glucose tolerance at 8 and 10 weeks and tissue 2-deoxyglucose uptake at 75 days. Exenatide treatment improved glucose tolerance, myocardial GLUT4 expression and 2-deoxyglucose uptake, cardiac contractility, and survival over control vehicle-treated TG9 mice. Phosphorylation of AMP kinase and AKT was also increased in exenatide-treated animals. Total myocardial GLUT1 levels were not different between groups. Exenatide also abrogated the detrimental effect of the GLUT4 antagonist ritonavir on survival in TG9 mice. Conclusion/Significance In heart failure secondary insulin resistance is maladaptive and myocardial glucose uptake is suboptimal. An incretin-based therapy, which addresses these changes, appears beneficial.

Homeostatic regulation of electrical excitability in physiological cardiac hypertrophy

Pathological biomechanical stresses cause cardiac hypertrophy, which is associated with QT prolongation and arrhythmias. Previous studies have demonstrated that repolarizing K+ current densities are decreased in pressure overload‐induced left ventricular hypertrophy, resulting in action potential and QT prolongation. Cardiac hypertrophy also occurs with exercise training, but this physiological hypertrophy is not associated with electrical abnormalities or increased arrhythmia risk, suggesting that repolarizing K+ currents are upregulated, in parallel with the increase in myocyte size, to maintain normal cardiac function. To explore this hypothesis directly, electrophysiological recordings were obtained from ventricular myocytes isolated from two mouse models of physiological hypertrophy, one produced by swim‐training of wild‐type mice and the other by cardiac‐specific expression of constitutively active phosphoinositide‐3‐kinase‐p110α (caPI3Kα). Whole‐cell voltage‐clamp recordings revealed that repolarizing K+ current amplitudes were higher in ventricular myocytes isolated from swim‐trained and caPI3Kα, compared with wild‐type, animals. The increases in K+ current amplitudes paralleled the observed cellular hypertrophy, resulting in normalized or increased K+ current densities. Electrocardiographic parameters, including QT intervals, as well as ventricular action potential waveforms in swim‐trained animals/myocytes were indistinguishable from controls, demonstrating preserved electrical function. Additional experiments revealed that inward Ca2+ current amplitudes/densities were also increased in caPI3Kα, compared with WT, left ventricular myocytes. The expression of transcripts encoding K+, Ca2+ and other ion channel subunits was increased in swim‐trained and caPI3Kα ventricles, in parallel with the increase in myocyte size and with the global increases in total cellular RNA expression. In contrast to pathological hypertrophy, therefore, the functional expression of repolarizing K+ (and depolarizing Ca2+) channels is increased with physiological hypertrophy, reflecting upregulation of the underlying ion channel subunit transcripts and resulting in increased current amplitudes and the normalization of current densities and action potential waveforms. Taken together, these results suggest that activation of PI3Kα signalling preserves normal myocardial electrical functioning and could be protective against the increased risk of arrhythmias and sudden death that are prevalent in pathological cardiac hypertrophy.

Molecular genetics of congenital diaphragmatic defects

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is accompanied by malformations of the lung, heart, testis, and other organs. Patients with CDH may have any combination of these extradiaphragmatic defects, suggesting that CDH is often a manifestation of a global embryopathy. This review highlights recent advances in human and mouse genetics that have led to the identification of genes involved in CDH. These include genes for transcription factors, molecules involved in cell migration, and extracellular matrix components. The expression patterns of these genes in the developing embryo suggest that mesenchymal cell function is compromised in the diaphragm and other affected organs in patients with CDH. We discuss potential mechanisms underlying the seemingly random combination of diaphragmatic, pulmonary, cardiovascular, and gonadal defects in these patients.

Loss of glypican-3 Function Causes Growth Factor-dependent Defects in Cardiac and Coronary Vascular Development

Glypican-3 (Gpc3) is a heparan sulfate proteoglycan (HSPG) expressed widely during vertebrate development. Loss-of-function mutations cause Simpson-Golabi-Behmel syndrome (SGBS), a rare and complex congenital overgrowth syndrome with a number of associated developmental abnormalities including congenital heart disease. We found that Gpc3-deficient mice display a high incidence of congenital cardiac malformations like ventricular septal defects, common atrioventricular canal and double outlet right ventricle. In addition we observed coronary artery fistulas, which have not been previously reported in SGBS. Coronary artery fistulas are noteworthy because little is known about the molecular basis of this abnormality. Formation of the coronary vascular plexus in Gpc3-deficient embryos was delayed compared to wild-type, and consistent with GPC3 functioning as a co-receptor for fibroblast growth factor-9 (FGF9), we found a reduction in Sonic Hedgehog (Shh) mRNA expression and signaling in embryonic mutant hearts. Interestingly, we found an asymmetric reduction in SHH signaling in cardiac myocytes, as compared with perivascular cells, resulting in excessive coronary artery formation in the Gpc3-deficient animals. We hypothesize that the excessive development of coronary arteries over veins enables the formation of coronary artery fistulas. This work has broad significance to understanding the genetic basis of coronary development and potentially to molecular mechanisms relevant to revascularization following ischemic injury to the heart.