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1.  Heart Failure Induced by Perinatal Ablation of Cardiac Myosin Light Chain Kinase 
Background: Germline knockout mice are invaluable in understanding the function of the targeted genes. Sometimes, however, unexpected phenotypes are encountered, due in part to the activation of compensatory mechanisms. Germline ablation of cardiac myosin light chain kinase (cMLCK) causes mild cardiac dysfunction with cardiomyocyte hypertrophy, whereas ablation in adult hearts results in acute heart failure with cardiomyocyte atrophy. We hypothesized that compensation after ablation of cMLCK is dependent on developmental staging and perinatal-onset of cMLCK ablation will result in more evident heart failure than germline ablation, but less profound when compared to adult-onset ablation.
Methods and Results: The floxed-Mylk3 gene was ablated at the beginning of the perinatal stage using a single intra-peritoneal tamoxifen injection of 50 mg/kg into pregnant mice on the 19th day of gestation, this being the final day of gestation. The level of cMLCK protein level could no longer be detected 3 days after the injection, with these mice hereafter denoted as the perinatal Mylk3-KO. At postnatal day 19, shortly before weaning age, these mice showed reduced cardiac contractility with a fractional shortening 22.8 ± 1.0% (n = 7) as opposed to 31.4 ± 1.0% (n = 11) in controls. The ratio of the heart weight relative to body weight was significantly increased at 6.68 ± 0.28 mg/g (n = 12) relative to the two control groups, 5.90 ± 0.16 (flox/flox, n = 11) and 5.81 ± 0.33 (wild/wild/Cre, n = 5), accompanied by reduced body weight. Furthermore, their cardiomyocytes were elongated without thickening, with a long-axis of 101.8 ± 2.4 μm (n = 320) as opposed to 87.1 ± 1.6 μm (n = 360) in the controls.
Conclusion: Perinatal ablation of cMLCK produces an increase of heart weight/body weight ratio, a reduction of contractility, and an increase in the expression of fetal genes. The perinatal Mylk3-KO cardiomyocytes were elongated in the absence of thickening, differing from the compensatory hypertrophy shown in the germline knockout, and the cardomyocyte thinning shown in adult-inducible knockout.
PMCID: PMC5080352  PMID: 27833563
heart failure; perinatal; knockout; kinase; Myosin light chain kinase
2.  A Mouse Model of Human Congenital Heart Disease: Progressive Atrioventricular Block Induced by a Heterozygous Nkx2-5 Homeodomain Missense Mutation 
Heterozygous human NKX2-5 homeodomain (DNA binding domain) missense mutations are highly penetrant for varied congenital heart defects, including progressive atrioventricular block (AVB) requiring pacemaker implantation. We recently replicated this genetic defect in a murine knock-in model, in which we demonstrated highly penetrant, pleiotropic cardiac anomalies. In this study, we examined postnatal AV conduction in the knock-in mice.
Methods and Results
A murine knock-in model (Arg52Gly, Nkx2-5+/R52G) in a 129/Sv background was analyzed by histopathology, surface and telemetry ECG, and in vivo electrophysiology studies (EPS), comparing with control Nkx2-5+/+ mice at diverse postnatal stages, ranging from postnatal day 1 (P1) to 17 months. PR-prolongation (1st degree AVB) was present at 4 weeks, 7 months, and 17 months of age but not at P1 in the mutant mice. Advanced AVB was also occasionally demonstrated in the mutant mice. EPS showed that AV nodal function, and right ventricular effective refractory period, were impaired in the mutant mice, while sinus nodal function was not affected. AV nodal size was significantly smaller in the mutant mice compared to their controls at 4 weeks of age, corresponding to the presence of PR-prolongation, but not P1, suggesting, at least in part, that the conduction abnormalities are the result of a morphologically atrophic AV node.
The highly penetrant and progressive AVB phenotype seen in human heterozygous missense mutations in NKX2-5 homeodomain was replicated in mice by knocking-in a comparable missense mutation.
PMCID: PMC4618020  PMID: 26226998
atrioventricular block; congenital heart disease; genetics; animal models; genetics; human; knock-in
3.  Directed Differentiation of Embryonic Stem Cells Into Cardiomyocytes by Bacterial Injection of Defined Transcription Factors 
Scientific Reports  2015;5:15014.
Forced expression of defined transcriptional factors has been well documented as an effective method for cellular reprogramming or directed differentiation. However, transgene expression is not amenable for therapeutic application due to potential insertional mutagenesis. Here, we have developed a bacterial type III secretion system (T3SS)-based protein delivery tool and shown its application in directing pluripotent stem cell differentiation by a controlled delivery of transcription factors relevant to early heart development. By fusing to an N-terminal secretion sequence for T3SS-dependent injection, three transcriptional factors, namely Gata4, Mef2c, and Tbx5 (abbreviated as GMT), were translocated into murine embryonic stem cells (ESCs), where the proteins are effectively targeted to the nucleus with an average intracellular half-life of 5.5 hours. Exogenous GMT protein injection activated the cardiac program, and multiple rounds of GMT protein delivery significantly improved the efficiency of ESC differentiation into cardiomyocytes. Combination of T3SS-mediated GMT delivery and Activin A treatment showed an additive effect, resulting in on average 60% of the ESCs differentiated into cardiomyocytes. ESC derived cardiomyocytes displayed spontaneous rhythmic contractile movement as well as normal hormonal responses. This work serves as a foundation for the bacterial delivery of multiple transcription factors to direct cell fate without jeopardizing genomic integrity.
PMCID: PMC4598736  PMID: 26449528
4.  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 
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. Since 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 knock-in 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’s malformation of the tricuspid valve, and perimembranous and/or muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial septal anomalies, with significant increase in the size of the inter-atrial communication and fossa ovalis, and decrease in the length of the flap valve compared to control Nkx2-5+/+ or Nkx2-5+/− mice.
The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) are highly penetrant, and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knock-in mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.
PMCID: PMC4140955  PMID: 25028484
genetics; heart defects; congenital; noncompaction; knock-in
5.  Compound loss of muscleblind-like function in myotonic dystrophy 
EMBO Molecular Medicine  2013;5(12):1887-1900.
Myotonic dystrophy (DM) is a multi-systemic disease that impacts cardiac and skeletal muscle as well as the central nervous system (CNS). DM is unusual because it is an RNA-mediated disorder due to the expression of toxic microsatellite expansion RNAs that alter the activities of RNA processing factors, including the muscleblind-like (MBNL) proteins. While these mutant RNAs inhibit MBNL1 splicing activity in heart and skeletal muscles, Mbnl1 knockout mice fail to recapitulate the full-range of DM symptoms in these tissues. Here, we generate mouse Mbnl compound knockouts to test the hypothesis that Mbnl2 functionally compensates for Mbnl1 loss. Although Mbnl1−/−; Mbnl2−/− double knockouts (DKOs) are embryonic lethal, Mbnl1−/−; Mbnl2+/− mice are viable but develop cardinal features of DM muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in Mbnl1−/− knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the hypothesis that compound loss of MBNL function is a critical event in DM pathogenesis and provide novel mouse models to investigate additional pathways disrupted in this RNA-mediated disease.
PMCID: PMC3914532  PMID: 24293317
Mbnl1; Mbnl2; muscleblind-like; myotonic dystrophy; RNA-mediated disease
6.  Myosin Light Chain Phosphorylation is Critical for Adaptation to Cardiac Stress 
Circulation  2012;126(22):2575-2588.
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 adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac-MLCK (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 wildtype mice reduced phosphorylated-MLC2v levels by ~40% and cMLCK levels by ~85%. To examine how a reduction in cMLCK and the corresponding reduction in pMLC2v affect function, we generated Mylk3 gene-targeted mice as well as 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.
Our results suggest the novel idea that accelerated cMLCK-protein turnover by the ubiquitin-proteasome system underlie the transition from compensated hypertrophy to decompensated heart failure due to reduced phosphorylation of MLC2v.
PMCID: PMC3510779  PMID: 23095280
myosin light chain; phosphorylation; heart failure
7.  Progressive Anatomical Closure of Foramen Ovale in Normal Neonatal Mouse Hearts 
In the prenatal heart, right-to-left atrial shunting of blood through the foramen ovale is essential for proper circulation. After birth, as the pulmonary circulation is established, the foramen ovale functionally closes as a result of changes in the relative pressure of the two atrial chambers, ensuring the separation of oxygen depleted venous blood in the right atrium from the oxygenated blood entering the left atrium. Little is known regarding the process of anatomical closure of the foramen ovale in the postnatal heart. Genetically engineered mouse models are powerful tools to study heart development and to reveal mechanisms underlying cardiac anomalies, including defects in atrioventricular septation. Using three-dimensional reconstructions of serial sectioned hearts at early postnatal Days 2–7, we show a progressive reduction in the size of the interatrial communication throughout this period and complete closure by postnatal Day 7. Furthermore we demonstrate that fusion of the septum primum and septum secundum occurs between 4 weeks and 3 months of age. This study provides a standard timeline for morphological closure of the right– left atrial communication and fusion between the atrial septa in normal mouse hearts.
PMCID: PMC3336211  PMID: 22354769
mouse; heart defects; congenital; ASD; PFO
8.  Crystal Structure of the Human NKX2.5 Homeodomain in Complex with DNA Target 
Biochemistry  2012;51(32):6312-6319.
NKX2.5 is a homeodomain containing transcription factor regulating cardiac formation and function, and its mutations are linked to congenital heart disease. Here we provide the first report of the crystal structure of the NKX2.5 homeodomain in complex with double-stranded DNA of its endogenous target, locating within the proximal promoter –242 site of the atrial natriuretic factor gene. The crystal structure, determined at 1.8 Å resolution, demonstrates that NKX2.5 homeodomains occupy both DNA binding sites separated by five nucleotides without physical interaction between themselves. The two homeodomains show identical conformation despite the differences in the DNA sequences they bind, and no significant bending of the DNA was observed. Tyr54, absolutely conserved in NK2 family proteins, mediates sequence-specific interaction with the TAAG motif. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.
PMCID: PMC3448007  PMID: 22849347
9.  Ablation of Nkx2-5 at mid-embryonic stage results in premature lethality and cardiac malformation 
Cardiovascular Research  2011;91(2):289-299.
Human congenital heart disease linked to mutations in the homeobox transcription factor, NKX2-5, is characterized by cardiac anomalies, including atrial and ventricular septal defects as well as conduction and occasional defects in contractility. In the mouse, homozygous germline deletion of Nkx2-5 gene results in death around E10.5. It is, however, not established whether Nkx2-5 is necessary for cardiac development beyond this embryonic stage. Because human NKX2-5 mutations are related to septum secundum type atrial septal defects (ASD), we hypothesized that Nkx2-5 deficiency during the processes of septum secundum formation may cause cardiac anomalies; thus, we analysed mice with tamoxifen-inducible Nkx2-5 ablation beginning at E12.5 when the septum secundum starts to develop.
Methods and results
Using tamoxifen-inducible Nkx2-5 gene-targeted mice, this study demonstrates that Nkx2-5 ablation beginning at E12.5 results in embryonic death by E17.5. Analysis of mutant embryos at E16.5 shows arrhythmias, contraction defects, and cardiac malformations, including ASD. Quantitative measurements using serial section histology and three-dimensional reconstruction demonstrate growth retardation of the septum secundum and enlarged foramen ovale in Nkx2-5-ablated embryos. Functional cardiac defects may be attributed to abnormal expression of transcripts critical for conduction and contraction, including cardiac voltage-gated Na+ channel pore-forming α-subunit (Nav1.5-α), gap junction protein connexin40, cardiac myosin light chain kinase, and sarcolipin within 4 days after tamoxifen injection.
Nkx2-5 is necessary for survival after the mid-embryonic stage for cardiac function and formation by regulating the expression of its downstream target genes.
PMCID: PMC3125071  PMID: 21285290
Genetics; Heart defects; Congenital; Conduction; Contractility
10.  Vascular smooth muscle Jak2 mediates angiotensin II-induced hypertension via increased levels of reactive oxygen species 
Cardiovascular Research  2011;91(1):171-179.
Angiotensin II (Ang II) type AT1 receptors expressed on vascular smooth muscle cells (VSMCs) couple to the Jak2 signalling pathway. However, the importance of this tissue-specific coupling is poorly understood. The purpose of this investigation was to determine the importance of VSMC-derived Jak2 in angiotensin II-mediated hypertension.
Methods and results
The Cre-loxP system was used to conditionally eliminate Jak2 tyrosine kinase expression within the smooth muscle cells of mice. Following chronic Ang II infusion, the resulting increase in mean arterial pressure (MAP) was significantly attenuated in the Jak2 null mice when compared with littermate controls. The VSMC Jak2 null mice were also protected from the Ang II-induced vascular remodelling. Aortic rings from the VSMC Jak2 null mice exhibited reduced Ang II-induced contraction and enhanced endothelial-dependent relaxation via increased nitric oxide (NO) bioavailability. When compared with controls, the VSMC Jak2 nulls also had lower levels of hydrogen peroxide, Rho kinase activity, and intracellular Ca2+ in response to Ang II.
The data indicate that VSMC Jak2 expression is involved in the pathogenesis of Ang II-dependent hypertension due to the increased presence of reactive oxygen species (ROS). As such, VSMC-derived Jak2 tyrosine kinase modulates overall vascular tone via multiple, non-redundant mechanisms.
PMCID: PMC3112021  PMID: 21354995
Jak2 tyrosine kinase; Angiotensin II; Vascular smooth muscle cell; Hypertension
11.  Vascular Smooth Muscle Jak2 Deletion Prevents Angiotensin II-mediated Neointima Formation Following Injury in Mice 
The in vitro treatment of vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) causes Janus kinase 2 (Jak2) to interact with the Ang II type 1 receptor (AT1–R) resulting in enhanced cell growth. However, the role that Jak2 plays in AT1-R-mediated vascular cell growth and remodeling in vivo is less clear. We hypothesized that in vivo, Jak2 plays a rate limiting role in Ang II-mediated neointima formation following vascular injury. Using the Cre-loxP system, we conditionally ablated Jak2 from the VSMC of mice. We found that these mice are protected from Ang II-mediated neointima formation following iron chloride-induced vascular injury. In addition, the VSMC Jak2 null mice were protected from injury-induced vascular fibrosis and the pathological loss of the contractile marker, smooth muscle α-actin. Finally, when compared to controls, the VSMC Jak2 null mice exhibited significantly less Ang II-induced VSMC proliferation and migration in vitro and in vivo and more apoptosis. These results suggest that Jak2 plays a central role in the causation of Ang II-induced neointima formation following vascular injury and may provide a novel target for the prevention of neointima formation.
PMCID: PMC3092026  PMID: 21420414
Jak2; Neointima; Angiotensin II; Vascular Injury
12.  Differential Role of Nkx2-5 in Activation of the Atrial Natriuretic Factor Gene in the Developing versus Failing Heart ▿ †  
Molecular and Cellular Biology  2011;31(22):4633-4645.
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.
PMCID: PMC3209251  PMID: 21930795
13.  Heart Failure-Inducible Gene Therapy Targeting Protein Phosphatase 1 Prevents Progressive Left Ventricular Remodeling 
PLoS ONE  2012;7(4):e35875.
The targeting of Ca2+ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca2+ ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca2+ uptake in the SR among the three PP1 isoforms, thereby contributing to Ca2+ downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice.
We created an adeno-associated virus 9 (AAV9) vector encoding PP1β short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×1011 GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months.
In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months.
Heart failure-inducible molecular targeting of PP1β has potential as a novel therapeutic strategy for heart failure.
PMCID: PMC3338799  PMID: 22558250
14.  Mouse and computational models link Mlc2v dephosphorylation to altered myosin kinetics in early cardiac disease 
The Journal of Clinical Investigation  2012;122(4):1209-1221.
Actin-myosin interactions provide the driving force underlying each heartbeat. The current view is that actin-bound regulatory proteins play a dominant role in the activation of calcium-dependent cardiac muscle contraction. In contrast, the relevance and nature of regulation by myosin regulatory proteins (for example, myosin light chain-2 [MLC2]) in cardiac muscle remain poorly understood. By integrating gene-targeted mouse and computational models, we have identified an indispensable role for ventricular Mlc2 (Mlc2v) phosphorylation in regulating cardiac muscle contraction. Cardiac myosin cycling kinetics, which directly control actin-myosin interactions, were directly affected, but surprisingly, Mlc2v phosphorylation also fed back to cooperatively influence calcium-dependent activation of the thin filament. Loss of these mechanisms produced early defects in the rate of cardiac muscle twitch relaxation and ventricular torsion. Strikingly, these defects preceded the left ventricular dysfunction of heart disease and failure in a mouse model with nonphosphorylatable Mlc2v. Thus, there is a direct and early role for Mlc2 phosphorylation in regulating actin-myosin interactions in striated muscle contraction, and dephosphorylation of Mlc2 or loss of these mechanisms can play a critical role in heart failure.
PMCID: PMC3314469  PMID: 22426213
15.  Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells 
PLoS ONE  2011;6(12):e29690.
Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.
Methodology/Principal Findings
Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.
RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.
PMCID: PMC3248424  PMID: 22220215
16.  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 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 re-organization of chromatin structure demonstrated in other tissues. We reasoned that effects of loss of Nkx2–5 in mice may be different after cell-cycle withdrawal compared to perinatal loss of Nkx2–5, which results in rapid conduction and contraction defects within 4 days after 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. Loss of Nkx2–5 beginning at 2 weeks of age resulted in conduction and contraction defects similar to perinatal loss of Nkx2–5, however with substantially slower disease progression demonstrated 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 slower and smaller degree of reduction of several critical Nkx2–5 downstream targets that were observed in mice with 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 substantially distinct level of necessity at 2 weeks of age compared to the perinatal period.
PMCID: PMC2733927  PMID: 19546853
17.  Crystallization and preliminary X-ray analysis of the NKX2.5 homeodomain in complex with DNA 
The NKX2.5 homeodomain has been crystallized in complex with DNA. Diffraction data were collected to 1.7 Å resolution.
As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.
PMCID: PMC2581709  PMID: 18997347
NKX2.5 homeodomain; congenital heart disease
18.  Crystallization and preliminary X-ray analysis of the NKX2.5 homeodomain in complex with DNA 
As part of an effort to elucidate the molecular basis for the pathogenesis of NKX2.5 mutations in congenital heart disease using X-ray crystallography, the NKX2.5 homeodomain has been crystallized in complex with a specific DNA element, the −242 promoter region of atrial natriuretic factor. Crystals of the homeodomain–DNA complex diffracted X-rays to 1.7 Å resolution and belonged to space group P65, with unit-cell parameters a = b = 71.5, c = 94.3 Å. The asymmetric unit contained two molecules of the NKX2.5 homeodomain and one double-stranded oligonucleotide.
PMCID: PMC2581709  PMID: 18997347
19.  Perinatal Loss of Nkx2-5 Results in Rapid Conduction and Contraction Defects 
Circulation research  2008;103(6):580-590.
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 α-subunit (Nav1.5-α), 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.
PMCID: PMC2590500  PMID: 18689573
conduction; contraction; gene targeting; transcription
20.  Chibby, an Antagonist of the Wnt/β-Catenin Pathway, Facilitates Cardiomyocyte Differentiation of Murine Embryonic Stem Cells 
Circulation  2007;115(5):617-626.
Embryonic stem cell (ESC)–derived cardiomyocytes are anticipated to serve as a useful source for future cell-based cardiovascular disease therapies. Research emphasis is currently focused on determining methods to direct the differentiation of ESCs to a large population of cardiomyocytes with high purity. To this aim, understanding the molecular mechanisms that control ESC-to-cardiomyocyte differentiation should play a critical role in the development of this methodology. The Wnt/β-catenin signaling pathway has been implicated in both embryonic cardiac development and in vitro ESC differentiation into cardiomyocytes. Chibby is a recently identified nuclear protein that directly binds to β-catenin and antagonizes its transcriptional activity.
Methods and Results
Chibby was ubiquitously expressed in early stages of ESC differentiation but upregulated during cardiomyocyte specification. Of interest, the Chibby gene promoter has multiple binding sites for the cardiac-specific homeodomain protein Nkx2.5, and its promoter activity was indeed positively regulated by Nkx2.5. Furthermore, overexpression of Chibby increased cardiac differentiation of ESCs, whereas loss of Chibby by RNAi impaired cardiomyocyte differentiation.
These data illustrate the regulation and function of Chibby in facilitating cardiomyocyte differentiation from ESCs. By revealing molecular mechanisms that control ESC-to-cardiomyocyte differentiation, this study will allow for the future development of technologies to improve cardiomyocyte differentiation from ESCs.
PMCID: PMC2565513  PMID: 17261658
myocytes; signal transduction; stem cells
21.  Identification of Cardiac-Specific Myosin Light Chain Kinase 
Circulation research  2008;102(5):571-580.
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.
PMCID: PMC2504503  PMID: 18202317
kinase; transcription; contraction
22.  Myocardin Expression Is Regulated by Nkx2.5, and Its Function Is Required for Cardiomyogenesis 
Molecular and Cellular Biology  2003;23(24):9222-9232.
Nkx2.5 (also known as Csx) is an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Nkx2.5 is required for early heart development, since Nkx2.5-null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed subtractive hybridization by using RNA isolated from wild-type and Nkx2.5-null hearts at embryonic day 8.5. We isolated a mouse cDNA encoding myocardin A, which is an alternative spliced isoform of myocardin and the most abundant isoform in the heart from embryo to adult. The expression of myocardin A and myocardin was markedly downregulated in Nkx2.5-null mouse hearts. Transient-cotransfection analysis showed that Nkx2.5 transactivates the myocardin promoter. Inhibition of myocardin function in the teratocarcinoma cell line P19CL6 prevented differentiation into cardiac myocytes after dimethyl sulfoxide treatment. Myocardin A transactivated the promoter of the atrial natriuretic factor gene through the serum response element, which was augmented by bone morphogenetic protein 2 and transforming growth factor β-activated kinase 1. These results suggest that myocardin expression is regulated by Nkx2.5 and that its function is required for cardiomyogenesis.
PMCID: PMC309615  PMID: 14645532
23.  A Novel Variant of Cardiac Myosin-binding Protein-C That Is Unable to Assemble into Sarcomeres Is Expressed in the Aged Mouse Atrium 
Molecular Biology of the Cell  2003;14(8):3180-3191.
Cardiac myosin-binding protein-C (MyBP-C), also known as C-protein, is one of the major myosin-binding proteins localizing at A-bands. MyBP-C has three isoforms encoded by three distinct genes: fast-skeletal, slow-skeletal, and cardiac type. Herein, we are reporting a novel alternative spliced form of cardiac MyBP-C, MyBP-C(+), which includes an extra 30 nucleotides, encoding 10 amino acids in the carboxyl-terminal connectin/titin binding region. This alternative spliced form of MyBP-C(+) has a markedly decreased binding affinity to myosin filaments and connectin/titin in vitro and does not localize to A-bands in cardiac myocytes. When MyBP-C(+) was expressed in chicken cardiac myocytes, sarcomere structure was markedly disorganized, suggesting it has possible dominant negative effects on sarcomere organization. Expression of MyBP-C(+) is hardly detected in ventricles through cardiac development, but its expression gradually increases in atria and becomes the dominant form after 6 mo of age. The present study demonstrates an age-induced new isoform of cardiac MyBP-C harboring possible dominant negative effects on sarcomere assembly.
PMCID: PMC181559  PMID: 12925755
24.  Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein 
Journal of Clinical Investigation  2001;108(2):189-201.
A DNA nonbinding mutant of the NK2 class homeoprotein Nkx2.5 dominantly inhibits cardiogenesis in Xenopus embryos, causing a small heart to develop or blocking heart formation entirely. Recently, ten heterozygous CSX/NKX2.5 homeoprotein mutations were identified in patients with congenital atrioventricular (AV) conduction defects. All four missense mutations identified in the human homeodomain led to markedly reduced DNA binding. To examine the effect of a DNA binding–impaired mutant of mouse Csx/Nkx2.5 in the embryonic heart, we generated transgenic mice expressing one such allele, I183P, under the β-myosin heavy chain promoter. Unexpectedly, transgenic mice were born apparently normal, but the accumulation of Csx/Nkx2.5(I183P) mutant protein in the embryo, neonate, and adult myocardium resulted in progressive and profound cardiac conduction defects and heart failure. P-R prolongation observed at 2 weeks of age rapidly progressed into complete AV block as early as 4 weeks of age. Expression of connexins 40 and 43 was dramatically decreased in the transgenic heart, which may contribute to the conduction defects in the transgenic mice. This transgenic mouse model may be useful in the study of the pathogenesis of cardiac dysfunction associated with CSX/NKX2.5 mutations in humans.
PMCID: PMC203028  PMID: 11457872
25.  Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease 
Journal of Clinical Investigation  2000;106(2):299-308.
CSX/NKX2.5 is an evolutionarily conserved homeodomain-containing (HD-containing) transcription factor that is essential for early cardiac development. Recently, ten different heterozygous CSX/NKX2.5 mutations were found in patients with congenital heart defects that are transmitted in an autosomal dominant fashion. To determine the consequence of these mutations, we analyzed nuclear localization, DNA binding, transcriptional activation, and dimerization of mutant CSX/NKX2.5 proteins. All mutant proteins were translated and located to the nucleus, except one splice-donor site mutant whose protein did not accumulate in the cell. All mutants that had truncation or missense mutations in the HD had severely reduced DNA binding activity and little or no transcriptional activation function. In contrast, mutants with intact HDs exhibit normal DNA binding to the monomeric binding site but had three- to ninefold reduction in DNA binding to the dimeric binding sites. HD missense mutations that preserved homodimerization ability inhibited the activation of atrial natriuretic factor by wild-type CSX/NKX2.5. Although our studies do not characterize the genotype-phenotype relationship of the ten human mutations, they identify specific abnormalities of CSX/NKX2.5 function essential for transactivation of target genes.
PMCID: PMC314312  PMID: 10903346

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