Focal adhesion kinase is essential for cardiac looping and multichamber heart formation

Abstract

Focal adhesion kinase (FAK) is a critical mediator of matrix- and growth factor-induced signaling during development. Myocyte-restricted FAK deletion in mid-gestation mice results in impaired ventricular septation and cardiac compaction. However, whether FAK regulates early cardiogenic steps remains unknown. To explore a role for FAK in multi-chambered heart formation, we utilized anti-sense morpholinos to deplete FAK in Xenopus laevis. Xenopus FAK morphants exhibited impaired cardiogenesis, pronounced pericardial edema, and lethality by tadpole stages. Spatial-temporal assessment of cardiac marker gene expression revealed that FAK was not necessary for midline migration, differentiation, fusion of cardiac precursors, or linear heart tube formation. However, myocyte proliferation was significantly reduced in FAK morphant heart tubes and these tubes failed to undergo proper looping morphogenesis. Collectively our data imply that FAK plays an essential role in chamber outgrowth and looping morphogenesis likely stimulated by fibroblast growth factors (and possibly other) cardiotrophic factors.

FIG. 1

Depletion of FAK in Xenopus laevis leads to pericardial edema. A: FAK or Control morpholinos (20 and 40 ng) were injected into fertilized oocytes and embryonic FAK protein levels were assessed at the indicated stages by Western blotting. Levels of ERK are shown as a control for loading. B: Western blot analysis for FAK in Con Mo- and FAK Mo-injected embryos (40 ng/embryo) at the indicated stages of development. Levels of ERK are shown as a control for loading. C: Densitometric analysis of Western blots comparing FAK band intensity relative to ERK. Data are presented as FAK levels in FAK Mo-embryos relative to Con Mo-embryos (set to 1) at each developmental stage analyzed. D: Western blot analysis for PYK2 (and ERK) in stage 30 Con Mo- and FAK Mo-injected embryos. E: Gross morphology of Control and FAK morphant tadpoles at Stage 37. FAK morphants exhibit a slightly shortened anteroposterior axis and pericardial edema (arrow).

FIG. 2

FAK morphant embryos exhibit marked cardiac dysmorphogenesis. A: Lateral view of whole-mount immunohistochemistry for tropomyosin reveals a fully looped three-chambered heart in Con Mo-injected embryos while those injected with FAK Mo appear distended and partially looped (middle panel) or unlooped (right panel). Anterior is to the left and dorsal toward the top in all panels. Regions of interest are labeled as follows: i, inflow tract; v, ventricle; o, outflow tract. B: Western blot analysis for FAK in uninjected, Con Mo-injected, FAK Mo-injected, and rescue embryos at Stage 37. Levels of ERK are shown as a control for loading. C: Lateral view of whole-mount immunohisto-chemistry for MHC reveals rescue of the FAK morphant phenotype is achieved by coexpression of 2 ng chicken FAK. D: Heart morphology analysis of Con Mo-injected, FAK Mo-injected, rescue (FAK Mo and 2 ng chicken FAK co-injection), and 2 ng chicken FAK alone demonstrates that while FAK morphant embryos exhibit full looping in only 30% of embryos examined, rescue embryos exhibit full looping morphology in 67%. Total number of embryos analyzed were n = 27 (Con Mo), n = 34 (FAK Mo), n = 42 (Rescue), n = 20 (2 ng c-FAK), collected from two separate experiments.

FIG. 3

FAK depletion does not impact cardiac specification or differentiation. A: In situ hybridization of Stage 30 embryos for TBX5 and NKX2.5. Ventral views (left panels) are oriented with anterior toward the top, lateral views (right panels) are oriented with anterior to the left and dorsal to the top. B, C: RT-PCR analysis at Stages 30 (left panels) and 32 (right panels) for TBX5, TBX20, NKX2.5 (B), and tropomyosin (tm), and Troponin T (TnT) (C). Histone H4 (H4) serves as a control. Data represent results from 10 embryos per condition and experiments were repeated at least twice. D: Ventral and front views of whole-mount immunohistochemistry for MHC reveals a continuous and fused sheet of differentiated myocytes at the ventral midline (indicated by white arrow) in FAK Mo-injected embryos. Images represent 3D reconstructions of confocal z-stack sections.

FIG. 4

FAK depletion impairs looping morphogenesis. Whole mount immunohisto-chemical staining for tropomyosin was performed on Stage 32 (A), and 34 (B) embryos that were injected with either Con Mo (left) or FAK Mo (right) at the one-cell stage. Images represent 3D reconstructions of confocal z-stack sections (top) and Imaris isosurfacing (bottom). Stage 32 view is from the posterior looking through the heart tube toward the anterior end; dorsal is to the top. Stage 34 view is lateral with anterior to the left, dorsal to the top. Note that looping is perturbed in Stage 34 FAK morphants. Arrows point to region of interest where the heart takes on a spiral shape indicative of looping morphogenesis.

FIG. 5

Myocyte mitosis is attenuated in FAK morphant heart tubes. A: Whole mount immunohistochemical staining for tropomyosin (green) and phospho-Histone H3 (red) was performed on prelooped (Stage 32) embryos that were injected with either Con Mo (left) or FAK Mo (right) at the one-cell stage. Images represent 3D reconstructions of confocal z-stack sections (top panels) or a single optical section (bottom panels). B: Total number of pH3 positive myocytes and noncardiac cells were counted in each optical section of Con Mo- and FAK Mo-injected embryos (19 embryos were analyzed per condition, collected from at least three separate experiments).

FIG. 6

Treatment with the FGFR1-inhibitor, SU5402, impairs cardiac looping. Widefield microscopic analysis (A) of gross morphology of untreated control, DMSO-treated (2.5 μM), or SU5402-treated (50 μM) embryos at Stage 37 reveals that SU5402 treatment induces edema in the middle-ventral region of the embryo (top right panel) or pericardial edema (bottom right panel) in some embryos. Arrows point to regions of edema. B: Lateral view of whole-mount immunohistochemistry for MHC reveals a fully looped three-chambered heart in untreated and DMSO-treated embryos while those treated with SU5402 appear unlooped. Anterior is to the left and dorsal is to the top in all panels. Total number of embryos analyzed for heart morphology were: n, 22 (untreated), n = 34 (DMSO), and n = 35 (SU5402) and were collected from three separate experiments.

FIG. 7

FAK is activated by FGF and is necessary for FGF-dependent myocyte proliferation. A: Western blot analysis of cell lysates isolated from primary embryonic rat cardiomyocytes. Cells were maintained in serum free (SF) media and treated with bFGF (100 ng/mL) or vehicle (veh.) for 30 min with or without 10 min pretreatment with the specific FGFR-inhibitor, SU5402 (10 μM). Lysates were immunoblotted with antibodies directed towards phospho-specific Y397-FAK or total FAK. B: BrdU incorporation in isolated embryonic cardiomyocytes infected with GFP- or GFP-FRNK adenovirus (10 m.o.i). Cells were maintained in serum-free medium and treated with vehicle (not shown) or bFGF (100 ng/mL) for 24 h. Costaining with anticardiac troponin T (cTnT) was performed to identify cardiomyocytes. C: Quantification of BrdU- positive cardiomyocytes (means ± SEM; N = 3; minimum of 300 cells/condition). Scale bar is 20 μm.