About Our Lab

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Left to right: Diego Fraidenraich (PI), Eric Himelman (PhD student), Omar Rafa (NERA student), Kevin Lahey (PhD student), Erik Zhao (Research Associate)

Interest Areas

 

One main focus of my lab is to design paradigms for embryonic stem (ES) and induced pluripotent stem (iPS) cell based repair of cardiac and skeletal muscle defects using mouse models of human disease (thin myocardial syndrome, cardiomyopathy, muscular dystrophy). Another focus is to study paracrine non-myocardial factors in Id KO embryos/mice, a model of congenital heart disease and adult fibrotic cardiomyopathy. The ultimate goal is to identify cardiotrophic and myotrophic factors with rescue potential.

Pluripotent stem cell corrections of Muscular Dystrophy and its associated cardiomyopathy

Duchenne Muscular Dystrophy (DMD) is characterized by the progressive degeneration of contractile muscle and affects 1 out of every 3500 males. The disease develops due to the absence of the Dystrophin protein, a protein critical for contractile muscle integrity. Additionally, due to the absence of Dystrophin, key proteins necessary for cell sarcolemmal stability and cell signaling are no longer localized properly and cannot function. The project I am working on is the analysis of the function and localization of neuronal Nitric Oxide Synthase (nNOS) in cardiac muscle from dystrophic mice. By injecting wild-type embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) into blastocysts prone to develop muscular dystrophy, our lab is able to rescue the dystrophic phenotype and study the mechanisms by which the rescue occurs. This includes the re-localization of proteins such as Nitric Oxide Synthase to their appropriate, wild-type (WT) compartments, thereby restoring functionality of the enzyme.

Limb-Girdle muscular dystrophy-2F (LGMD-2F) is an autosomal recessive disease caused by a mutation in the sarcoglycan-d (SGd) gene.  LGMD-2F causes progressive wasting of the proximal limb muscles and commonly results in cardiomyopathy.   The lack of SGd, which is expressed in both skeletal and cardiac muscle, results in the complete ablation of the sarcoglycan complex (SGC) within the larger dystrophin-glycoprotein complex (DGC).  To study the potential for ESC-treatment, we produced mosaic mice through the injection of wild type (WT) embryonic stem cells (ESCs) into SGd knockout (KO) blastocysts. We show that WT/ SGd chimeric mice, which were allowed to age for 18 months, improved pathology in certain tissues.  Contrary to our previous reports, using mdx mice, which required only minimal ESC incorporation for the rescue of Duchenne muscular dystrophy (DMD), our findings show that >60% ESC incorporation is necessary for the rescue of cardiac and most skeletal muscles, including the pectoralis and the quadriceps (Fig 1).  However, this high degree of WT chimerism was not sufficient to rescue the diaphragm, which also suffered from diminished dystrophin but no elevation of compensatory utrophin.  Thus, we describe that, unlike DMD, LGMD-2F is not amenable to ESC-treatment, especially the diaphragm.

The role of ID genes in the development of the heart and blood

The Inhibitor of DNA binding (Id) genes (Id1 –Id4) play a crucial role in cardiac development and demonstrate overlapping functionality. The Id genes are expressed in nonmyocardial layers (endocardium, endothelium, epicardium) yet the myocardium is also affected. Ablation of both Id1 and Id3 leads to midgestation lethality characterized by multiple cardiac defects. To study postnatal roles of Id genes, our lab generated a conditional knockout mouse model (Id cKO) using the Tie2Cre system to ablate Id1 in the endocardium/endothelium and Id3 globally, while leaving Id expression in the other layers intact. Half of these mice survived to term and ultimately developed dilated cardiomyopathy (Figs 2, 3).  These mice also develop splenomegaly and marked anemia, findings that we suspect play a role in the Id cKO phenotype. Our lab is currently investigating the mechanism and role of paracrine Id signaling in the developing heart.

Legend to Figures 1,2,3

 

Fig. 1. WT ESC incorporation does not prevent fibrosis in WT/SGδδ chimeras with less than 60% mosaicism. Heart (A-D), diaphragm (E-H), quadriceps (I-L) and pectoralis (M-P) muscles were paraffin sectioned and Masson trichrome (MT) stained.  WT (A, E, I, M,), 60-80% WT/SGδ (B, F, J, N), 40-60% WT/SGδ  (C, G, K, O) and <5% WT/SGδ (D, H, L, P) chimeras were analyzed. Magnification of A-D: 200x; E-P: 100x. Yellow arrows (B-D) denote vascular-associated fibrosis. Light blue arrow (A) denotes no vascular-associated fibrosis. Dashed vertical line separates non-phenotypic (left to line) from phenotypic (right to line) tissues.

Figure 2. Newborn Id cKO pups develop multiple cardiac defects. E13.5 Id cKO embryos do not develop a cardiac phenotype; accordingly, cell proliferation is not compromised (A-D, see also Fig1, B and C). However, P1 Id cKO pups display enlarged hearts, with VSDs (E, F and K-N), dilated VS vasculature (K, L insets), trabeculation defects (G, H) and distended epicardial vasculature (I, J). Black dotted line (A, B, E and F): VS thickness; white line (A and B): myocardial wall thickness; green, black and orange arrows (F): VS hollow cavity, myocardial wall interruption and red blood cells in the VS cavity respectively; yellow arrowheads (H): interrupted endocardial lining; white arrowheads (J) endothelialized epicardial vasculature; light blue arrowheads (L inset): dilated VS vasculature. Magnification: A-F and M, N: 40X; E and F, insets: 10X; G and H: 200X, I and J: 400X; K and L: 100X.

Figure 3. P180 Id cKO mice develop cardiac defects. Hearts from adult Tie2Cre+Id1F/FId3-/- or Tie2Cre+Id1F/-Id3-/- but not from Tie2Cre+Id1F/-Id3+/- or WT mice are dilated (A-D). The endo-myocardium of Id cKO mice displays disorganized areas of low or no cellularity filled with fibrotic tissue (E-H). The Id cKO mice have decreased cardiac function (I, J). The endocardial lining of Id cKO mice is interrupted (K, L and K, L insets). Fibrosis is apparent in the large vessels of the heart of Id cKO but not of WT of Mst1 Tg mice (M-O). N inset, shows enlargement in Id cKO hearts (relative to M inset). Abbreviations: end (E-H): endocardium; LV (E-H): left ventricle; sep and post (I): interventricular septal wall and posterior wall of the left ventricle respectively. Red arrows (F, H): disorganized areas of low cellularity in the endomyocardium; yellow arrows (L): regions of endocardial interruption, white arrows (K inset): typical elongated shape of endocardial nuclei (note CD31 staining), absent in L inset; light blue arrowheads (M-O): large vessels; yellow arrowheads (O): fibrotic myocardial interstitium. Magnification: A-D: 10X; E-H: 100X; K and L: 100X; K and L, insets: 400X; M-O: 200X; M and N, insets: 10X.

Beating cardiac myocytes have been derived from mouse induced pluripotent stem cells