Laura E. Briggs

Identification of Cardiac-Specific Myosin Light Chain Kinase

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca2+/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.

Perinatal Loss of Nkx2-5 Results in Rapid Conduction and Contraction Defects

Homeobox transcription factor Nkx2-5, highly expressed in heart, is a critical factor during early embryonic cardiac development. In this study, using tamoxifen-inducible Nkx2-5 knockout mice, we demonstrate the role of Nkx2-5 in conduction and contraction in neonates within 4 days after perinatal tamoxifen injection. Conduction defect was accompanied by reduction in ventricular expression of the cardiac voltage-gated Na+ channel pore-forming &agr;-subunit (Nav1.5-&agr;), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca2+ for contraction (conduction–contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca2+ is released from sarcoplasmic reticulum, was substantially reduced in Nkx2-5 knockout mice. These results indicate that Nkx2-5 function is critical not only during cardiac development but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction.

Myosin Light Chain Phosphorylation is Critical for Adaptation to Cardiac Stress

Background— Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. Methods and Results— We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Conclusions— Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

Slow progressive conduction and contraction defects in loss of Nkx2-5 mice after cardiomyocyte terminal differentiation

Mutations in homeoprotein NKX2-5 are linked to human congenital heart disease, resulting in various cardiac anomalies, as well as in postnatal progressive conduction defects and occasional left ventricular dysfunction; yet the function of Nkx2-5 in the postnatal period is largely unexplored. In the heart, the majority of cardiomyocytes are believed to complete cell-cycle withdrawal shortly after birth, which is generally accompanied by a re-organization of chromatin structure shown in other tissues. We reasoned that the effects of the loss of Nkx2-5 in mice may be different after cell-cycle withdrawal compared with those of the perinatal loss of Nkx2-5, which results in rapid conduction and contraction defects within 4 days after the deletion of Nkx2-5 alleles (Circ Res. 2008;103:580). In this study, floxed-Nkx2-5 alleles were deleted using tamoxifen-inducible Cre transgene (Cre-ER) beginning at 2 weeks of age. The loss of Nkx2-5 beginning at 2 weeks of age resulted in conduction and contraction defects similar to the perinatal loss of Nkx2-5, however, with a substantially slower disease progression shown by 1° atrioventricular block at 6 weeks of age (4 weeks after tamoxifen injections) and heart enlargement after 12 weeks of age (10 weeks after tamoxifen injections). The phenotypes were accompanied by a slower and smaller degree of reduction of several critical Nkx2-5 downstream targets that were observed in mice with a perinatal loss of Nkx2-5. These results suggest that Nkx2-5 is necessary for proper conduction and contraction after 2 weeks of age, but with a substantially distinct level of necessity at 2 weeks of age compared with that in the perinatal period.

Differential Role of Nkx2-5 in activation of the atrial natriuretic factor gene in the developing versus failing heart

ABSTRACT Atrial natriuretic factor (ANF) is abundantly expressed in atrial cardiomyocytes throughout ontogeny and in ventricular cardiomyocytes in the developing heart. However, during cardiac failure and hypertrophy, ANF expression can reappear in adult ventricular cardiomyocytes. The transcription factor Nkx2-5 is one of the major transactivators of the ANF gene in the developing heart. We identified Nkx2-5 binding at three 5′ regulatory elements (kb −34, −31, and −21) and at the proximal ANF promoter by ChIP assay using neonatal mouse cardiomyocytes. 3C analysis revealed close proximity between the distal elements and the promoter region. A 5.8-kb fragment consisting of these elements transactivated a reporter gene in vivo recapitulating endogenous ANF expression, which was markedly reduced in tamoxifen-inducible Nkx2-5 gene knockout mice. However, expression of a reporter gene was increased and expanded toward the outer compact layer in the absence of the transcription repressor Hey2, similar to endogenous ANF expression. Functional Nkx2-5 and Hey2 binding sites separated by 59 bp were identified in the −34 kb element in neonatal cardiomyocytes. In adult hearts, this fragment did not respond to pressure overload, and ANF was induced in the absence of Nkx2-5. These results demonstrate that Nkx2-5 and its responsive cis-regulatory DNA elements are essential for ANF expression selectively in the developing heart.

A Mouse Model of Human Congenital Heart Disease High Incidence of Diverse Cardiac Anomalies and Ventricular Noncompaction Produced by Heterozygous Nkx2-5 Homeodomain Missense Mutation

Background—Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results—We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusions—The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.Background— Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5 , in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results— We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5 +/ R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5 +/ R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5 +/+ or Nkx2-5 +/− mice. Conclusions— The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain ( R52G ) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.

Assessment of Cellular and Functional Biomarkers in Bivalves Exposed to Ecologically Relevant Abiotic Stressors

An understanding of the complex effects of the environment on biomarkers of bivalve health is essential for aquaculturists to successfully select field culture sites and monitor bivalve health in these sites and in hatcheries. We tested several whole-organism (functional) and cellular-level biomarkers as indicators of health of the cultured, stress-tolerant northern quahog (hard clam) Mercenaria mercenaria. We performed single- and dual-stressor experiments that were consistent with available water quality data from a clam culture area on the Gulf coast of Florida. Clams from the culture area were exposed over a 14-d period to low O2 (hypoxia), elevated temperature, hyposalinity, and a combination of elevated temperature and hyposalinity. There was no clear relationship between the functional and cellular-level biomarkers, with most of the treatment effects being detected at the whole-organism level but not the cellular level. Survival and burial ability were significantly affected by elevated temperature and by the combination of elevated temperature and hyposalinity. Glycogen content decreased over the experiment duration and did not differ significantly among treatments. There were no significant changes in expression patterns of eight stress proteins or in the levels of oxidatively damaged RNA. The results highlight the importance of investigating the effects of multiple stressors in short-term, controlled laboratory conditions and suggest that such cellular-level biomarker assays should be paired with functional biomarkers to better understand the responses of highly stress-tolerant species.

A Mouse Model of Human Congenital Heart DiseaseClinical Perspective

Background—Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results—We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusions—The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.Background— Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5 , in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results— We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5 +/ R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5 +/ R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5 +/+ or Nkx2-5 +/− mice. Conclusions— The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain ( R52G ) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.

A Mouse Model of Human Congenital Heart DiseaseClinical Perspective: High Incidence of Diverse Cardiac Anomalies and Ventricular Noncompaction Produced by Heterozygous Nkx2-5 Homeodomain Missense Mutation

Background—Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results—We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusions—The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.Background— Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5 , in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results— We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5 +/ R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5 +/ R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5 +/+ or Nkx2-5 +/− mice. Conclusions— The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain ( R52G ) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.