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1.  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
2.  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
3.  Cellular phenotype transformation occurs during thoracic aortic aneurysm development 
Thoracic aortic aneurysms (TAAs) result from dysregulated remodeling of the vascular extracellular matrix (ECM) which may occur as a result of altered resident cellular function. The present study tested the hypothesis that aortic fibroblasts undergo a stable change in cellular phenotype during TAA formation.
Primary murine aortic fibroblasts were isolated from normal and TAA-induced aortas (4-wks post-induction with 0.5M CaCl2 15 min) by outgrowth method. Normal and TAA cultures were examined using a focused PCR array to determine fibroblast-specific changes in gene expression in the absence and presence of biological stimulation (endothelin-1, phorbol-12-myristate-13-acetate, angiotensin II). The relative expression of 38 genes, normalized to 4 housekeeping genes, was determined and genes displaying a minimum 2-fold increase/decrease or genes with significantly different normalized Ct values were considered to have altered expression.
At steady state TAA fibroblasts revealed elevated expression of several MMPs (Mmp2, Mmp11, Mmp14), collagen genes/elastin (Col1a1, Col1a2, Col3a1, Eln), and other matrix proteins, as well as decreased expression of Mmp3, Timp3, and Ltbp1. Moreover, gene expression profiles in TAA fibroblasts were different than normal fibroblasts after equivalent biological stimuli.
This study demonstrated for the first time that isolated primary aortic fibroblasts from TAA-induced mice possess a unique and stable gene expression profile, and when challenged with biological stimuli, induce a transcriptional response that is different from normal aortic fibroblasts. Together, these data suggest that aortic fibroblasts undergo a stable phenotypic change during TAA development which may drive the enhancement of ECM proteolysis in TAA progression.
PMCID: PMC2901416  PMID: 20219212
aneurysm; extracellular matrix; remodeling; phenotype; fibroblast
4.  Chronic Localized High Frequency Electrical Stimulation Within the Myocardial Infarct: Effects on Matrix Metalloproteinases and Regional Remodeling 
Circulation  2010;122(1):20-32.
Disruption of the balance between matrix metalloproteinases (MMP) and MMP inhibitors (TIMPs) within a myocardial infarct (MI) contribute to left ventricular (LV) wall thinning and changes in regional stiffness at the MI region. This study tested the hypothesis that a targeted regional approach through localized high frequency stimulation (LHFS) using low amplitude electrical pulses instituted within a formed MI scar would alter MMP/TIMP levels and prevent MI thinning.
At 3 wks following MI, pigs were randomized for LHFS (n=7, 240bpm, 0.8V, 0.05ms pulses) or unstimulated (UNSTIM, n=10). At 4 wks post-MI, LV wall thickness (echo, 0.89±0.07 vs 0.67±0.08 cm, p<0.05) and regional stiffness (piezoelectric crystals, 14.70±2.08 vs 9.11±1.24, p<0.05) were higher with LHFS than UNSTIM. In vivo interstitial MMP activity (fluorescent substrate cleavage, 943±59 vs 1210±72 units, p<0.05) in the MI region was lower with LHFS than in UNSTIM. In the MI region, MMP-2 levels were lower, while TIMP-1 and collagen levels were higher with LHFS than in UNSTIM (all p<0.05). Transforming growth factor-β (TGF-β) receptor 1 and phosphorylated SMAD-2/3 levels within the MI region were higher with LHFS than in UNSTIM. Electrical stimulation (4Hz) of isolated fibroblasts resulted in a reduction of MMP-2 and MT1-MMP levels, but increased TIMP-1 levels compared to unstimulated fibroblasts.
These unique findings demonstrate that LHFS of the MI region altered LV wall thickness and material properties, likely due to reduced regional MMP activity. Thus, LHFS may provide a novel means to favorably modify LV remodeling post-MI.
PMCID: PMC2946370  PMID: 20566951
Myocardial infarction; extracellular matrix; remodeling; matrix metalloproteinases
5.  Differential Matrix Metalloproteinase Levels in Adenocarcinoma and Squamous Cell Carcinoma of the Lung 
The matrix metalloproteinases (MMPs) have been implicated in the aggressive course of non-small cell lung cancer (NSCLC). However, there are a large number of MMP subtypes with diverse proteolytic substrates and different induction pathways. This study tested the hypothesis that a differential MMP profile would exist between NSCLC and normal lung and that MMP patterns would differ between NSCLC histologic type.
NSCLC samples and remote normal samples were obtained from patients with stage I or II NSCLC with either squamous cell (n=22) or adenocarcinoma (n=19) histology. Absolute concentrations for each of the MMP subclasses; collagenases (MMP-1, 8, -13), gelatinases (MMP-2,-9), lysins (MMP-2, -7) and elastase (MMP-12) were determined by a calibrated and validated multiplex suspension array.
Overall, MMP levels were significantly increased in NSCLC compared to normal. For example, MMP-1 and MMP-7 increased by approximately 10 fold in NSCLC (p<0.05). Moreover, a different MMP portfolio was observed between NSCLC histologic types. For example MMP-1,-8,-9 and -12 increased by over 4-fold in squamous cell versus adenocarcinoma (p<0.05). In those patients who recurred within 3 years of resection, 3-fold higher levels of MMP-8 and -9 were observed (p<0.05).
Increased levels of a number of MMP types occur with NSCLC, but the MMP profile was distinctly different between histologic types and in those patients with recurrence. These different MMP profiles may be important in the mechanistic basis for the natural history of different NSCLC types, as well as identifying potential prognostic and therapeutic targets.
PMCID: PMC2844342  PMID: 20304142
matrix metalloproteinases; lung cancer; multiplex; recurrence

Results 1-5 (5)