CXCR4 gene transfer prevents pressure overload induced heart failure

Abstract

Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, β2-adrenoceptor, and CXCR4 (Cav1.2/β2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-β2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest that AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure.

Fig. 1

TAC Model. (A) The effect of pressure overload on HW:BW ratio at sham, TAC 1day to TAC 8weeks. (B) Transcriptional profile during hypertrophy and heart failure. RNA was isolated from whole ventricular myocardium. Specific mRNA levels were quantified via real-time RT-PCR performed in triplicate (n=5 mice/group *= p<0.05, **=p<0.01, ***=p<0.001)

Fig. 2

AAV9.CXCR4 dose response in vivo. AAV9.CXCR4^WT was injected via the tail vein with increasing viral genomes/mouse, ranging from 2e9 to 3e11. Hearts were harvested four weeks post-injection. (A) CXCR4 mRNA was quantified by RT-PCR. (B) CXCR4 protein expression was assessed by western blot. (C) Immunostaining of CXCR4 in the heart indicated proper membrane localization.

Fig. 3

TAC+AAV9.CXCR4 functional studies. (A) Echocardiographic M-mode images at the level of the papillary muscle in sham operated and TAC 8 week mice injected with AAV9.LacZ and AAV9.CXCR4. (B) In vivo hemodyanmic data were acquired using a pressure-volume conductance catheter via an apical open-chest approach. Pre-load reduction studies were done by transiently occluding the inferior vena cava. AAV9.lacZ (Black) AAV9.CXCR4 (green) at baseline, TAC 2weeks, and TAC 8weeks. (C) HW:BW ratio of AAV9.CXCR4 and AAV9.LacZ during pressure overload. At 12 weeks post injection, AAV9.CXCR4 and AAV.9 Lacz had no effects on cardiac function in an absence of TAC.

Fig. 4

VEGF expression during pressure overload. (A) Cardiac sections were stained with isolectin-488 to identify myocardial capillary density. (B) Quantitative real time PCR data demonstrates an increase in VEGF transcription in TAC+AAV.CXCR4 group. (n=4 mice/group *= p<0.05) as compared to TAC+no AAV or TAC+Lacz groups. (C) VEGF protein expression was quantified using standard ELISA assay.

Fig. 5

CXCL12/CXCR4 expression during cardiac myocyte hypertrophy in vitro. (A) Isolated adult rat cardiac myocytes were treated with isoproterenol (500nM) for 24 hours to induce cellular hypertrophy. Myocytes were immunostained for CXCR4 pre- and post-isoproterenol treatment. Additionally, RNA was harvested from the cell lysates and CXCL12/CXCR4 mRNA content was quantified. (n=4 independent experiments, * p<0.05, ** p<0.01). (B) Isolated adult rat cardiac myocytes were treated with CXC12 (100ng/mL) and isoproterenol (500nM) in the presence or absence of AMD3100 (10µM), CXCR4 antagonist. for 24 hours. When using the CXCR4 inhibitor AMD3100, the cells were pretreated for 45 minutes prior to CXCL12 treatment. Cell size was determined by outlining the myocyte perimeter and H3-leu uptake was measured to detect protein synthesis. (C) Isolated rat ventricular myocytes were treated with isoproterenol as previously described. Endogenous calcineurin acitivity was determined by a colorimetric free-phosphate assay using a calcineurin specific phospho-substrate. Nuclear and cytoplasmic fractions from cardiac myocytes treated with CXCL12 and isoproterenol were analyzed for NFATc3 content. GAPDH was used as a cytoplasmic loading control. RNA was isolated from cardiac myocytes treated with CXCL12 and isoproterenol. The NFAT target and calcineurin inhibitor, MCIP1.4, expression was determined by qPCR. (D) Overexpression of CXCR4 dramatically increases the tolerant threshold of cardiomyocyte to hypertrophic stimuli (isoproterenol at 500 nM) and reduces isoproterenol induced hypertrophy as compared to cells that are not infected. Isolated cardiac myocytes were infected with adenovirus encoding CXCR4 (MOI 100). 48 hours post-infection, cardiac myocytes were treated with isoproterenol as previously described. H3-leu uptake was measured to detect protein synthesis. (E) Whole ventricular tissue was minced, protein isolated and.endogenous calcineurin activity was determined in the lysates of ; no AAV, AAV9.lacZ and AAV9.CXCR4 at TAC 2weeks, and TAC 8weeks by a colorimetric free-phosphate assay using a calcineurin specific phosphosubstrate.

Fig. 6

CXCR4 interacts with β2-AR and Cav1.2 in the cardiac myocyte in vitro and in vivo. (A) Isolated cardiac myocytes were infected with adenovirus encoding flag.CXCR4 (MOI 100). 48 hours post-infection, cardiac myocytes were harvested for protein. Protein was subjected to flag immunoprecipitation using anti-flag coated agarose beads followed by western blot. (B) CXCR4 associates with β2-AR and Cav1.2 in the heart in vivo. Whole ventricular tissue was minced and protein isolated. Anti-Cav1.2 antibody was used for immunoprecipitation and membrane was blotted for CXCR4 and β2-AR. (C) Densitometric analysis for CXCR4 and β2-AR is shown (n=5). *P , 0.05