Role of cardiac myocyte CXCR4 expression in development and left ventricular remodeling after acute myocardial infarction

Abstract

Rationale: Stromal cell-derived factor (SDF)-1/CXCR4 axis has an instrumental role during cardiac development and has been shown to be a potential therapeutic target for optimizing ventricular remodeling after acute myocardial infarction (AMI) and in ischemic cardiomyopathy. Although a therapeutic target, the specific role of cardiac myocyte CXCR4 (CM-CXCR4) expression following cardiogenesis and survival of cardiac myocyte and left ventricular remodeling after AMI is unknown.

Objective: We hypothesized that cardiac myocyte derived CXCR4 is critical for cardiac development, but it may have no role in adulthood secondary to the short transient expression of SDF-1 and the delayed expression of CM-CXCR4 following AMI. To address this issue, we developed congenital and conditional CM-CXCR4(-/-) mouse models.

Methods and results: Two strains of CM-CXCR4(flox/flox) mice were generated by crossing CXCR4(flox/flox) mice with MCM-Cre(+/-) mouse and MLC2v-Cre(+/-) mouse on the C57BL/6J background, yielding CXCR4(flox/flox) MCM-Cre(+/-) and CXCR4(flox/flox)MLC2v-Cre(+/-) mice. Studies demonstrated recombination in both models congenitally in the MLC2v-Cre(+/-) mice and following tamoxifen administration in the MCM-Cre(+/-) mice. Surprisingly the CXCR4(flox/flox)MLC2v-Cre(+/-) are viable, had normal cardiac function, and had no evidence of ventricular septal defect. CXCR4(flox/flox)MCM(+/-) treated with tamoxifen 2 weeks before AMI demonstrated 90% decrease in cardiac CXCR4 expression 48 hours after AMI. Twenty-one days post AMI, echocardiography revealed no statistically significant difference in the wall thickness, left ventricular dimensions or ejection fraction (40.9+/-7.5 versus 34.4+/-2.6%) in CXCR4(flox/flox) mice versus CM-CXCR4(-/-) mice regardless of strategy of Cre expression. No differences in vascular density (2369+/-131 versus 2471+/-126 vessels/mm(2); CXCR4(flox/flox) versus CM-CXCR4(-/-) mouse), infarct size, collagen content, or noninfarct zone cardiac myocyte size were observed 21 days after AMI.

Conclusions: We conclude that cardiac myocyte-derived CXCR4 is not essential for cardiac development and, potentially because of the mismatch in timings of peaks of SDF-1 and CXCR4, has no major role in ventricular remodeling after AMI.

Figure 1. Characterization of MLC2vCRE^+/−CXCR4^flox/flox mouse

A, Exon 2 of CXCR4 gene is flanked by loxP sites. The primers bind before and after the loxP sites to detect cleaved CXCR4. B, A PCR analysis on genomic DNA from tail and heart was performed on 8-week-old CXCR4^flox/flox, MLC2v-Cre^+/−CXCR4^flox/flox, and MLC2v-Cre^+/−CXCR4^flox/+ mice. i and ii show the presence of CXCR4 floxed allele and Cre into the genome, and iii shows deletion of CXCR4 from ventricular homogenates and not from the tail of the same animal. C, CXCR4 staining (red) and phase contrast pictures of neonatal cardiac myocytes from CXCR4^flox/flox and MLC2v-Cre^+/−CXCR4^flox/flox mice shows deletion of CXCR4 from neonatal cardiac myocytes of MLC2v-Cre^+/−CXCR4^flox/flox mice. White arrows point toward the cardiac myocytes, and the green arrow points toward noncardiac myocyte cells in culture. D, Representative Doppler echocardiography image showing normal outflow and inflow during systole and diastole in MLC2v-Cre^+/−CXCR4^flox/flox mouse without any evidence of a ventricular septal defect. E, Representative image of view through right ventricle to reveal normal IVS in MLC2v-Cre^+/−CXCR4^flox/flox mouse. F, Circumferential and radial strain in MLC2v-Cre^+/−CXCR4^flox/flox and CXCR4^flox/flox mice showing no significant difference (n=4 in each group).

Figure 2. Characterization of MCM^+/−CXCR4^flox/flox mouse

A, PCR analysis on genomic DNA from tail and heart was performed on 8-week-old CXCR4^flox/flox, MCM^+/−CXCR4^flox/flox, and MCM^+/−CXCR4^flox/+ mice. i depicts a wild-type and floxed CXCR4 band; ii depicts CRE band in the genome; and iii depicts deleted band for CXCR4, which was only detected after tamoxifen administration in the ventricular homogenates and not from the tail. B, A dose–response curve for tamoxifen-induced recombination of CXCR4^flox/flox based on quantitative PCR shows maximum deletion was achieved after 4 days of tamoxifen administration. C, Representative images of immunofluorescence for CXCR4 48 hours after AMI shows knockdown of CXCR4 in cardiac myocytes of tamoxifen-treated MCM^+/−CXCR4^flox/flox and endothelial cells positive for CXCR4 (green). Delineated area in CM-CXCR4^−/− image is shown below at higher magnificent to aid in identification of nucleated cardiac myocytes devoid of CXCR4 expression. D, Western blot analysis of CXCR4 48 hours after AMI on infarct region shows ≈90% deletion of CXCR4. E, Representative M-mode recording of CXCR4^flox/flox and MCM^+/−CXCR4^flox/flox after tamoxifen administration.

Figure 3. Ventricular function assessment of cardiac myocyte–specific CXCR4-null hearts after AMI

Echocardiographic analysis of 8- to 10-week-old CXCR4^flox/flox and cardiac-specific CXCR4^−/− mice as a function of time after AMI. A, Anterior wall thickness (AWT) (mm). B through D, Posterior wall thickness (PWT) (mm) (B), LV end-diastolic dimension (LVEDd) (mm) (C), and ejection fraction (%) (D) at 0, 3, and 21 days after AMI (n=7 for CXCR4^flox/flox and n=12 for CM-CXCR4^−/− at each time point). There was no significant difference between the measured parameters at days 3 and 21 after AMI. Data represent means±SEM. E, Representative M-mode recording of CXCR4^flox/flox and cardiac-specific CXCR4^−/− mice 21 days after AMI.

Figure 4

A, Representative photomicrographs of vascular density from CXCR4^flox/flox (left) and CM-CXCR4^−/− (right) mice 21 days after MI (green, isolectin; red, wheat germ agglutinin; blue, DAPI). Vascular density calculated as vessels/mm² (B) and vessels/cardiac myocyte (C) in untreated CM-specific CXCR4^flox/flox (n=4) and treated CM-specific CXCR4^−/− (n=6) mice showed no statistically significant difference among the groups; P>0.05.

Figure 5

A, Masson’s trichrome staining of CXCR4^flox/flox and CM-CXCR4^−/− on mice paraffin-embedded heart sections 21 days after MI. B, Infarct size assessment in mice 21 days after LAD ligation. C, Interstitial fibrosis in viable myocardium 21 days after MI. D, Cardiac myocyte diameter (microns) in posterior wall. Data represent means±SD (n=4 per group).

Figure 6

A, Representative SDF-1α mRNA expression by real-time PCR reaction at 3 days after MI. B, Quantification of quantitative PCR of myocardial SDF-1 expression 3 days after MI. Data represent means±SD (n=3 to 4 animals in each group).