Objectives: Myocardial ischemic injury triggers a rapid inflammatory response that prompts subsequent cardiac fibroblast activation and transdifferentiation to myofibroblasts. During this transitional process fibroblasts upregulate expression of the fibroblast activation protein (FAP) [1]. Clinical imaging of FAP after acute myocardial infarction has established ⁶⁸Ga-FAPI-46 positron emission tomography as a unique cellular substrate from conventional fibrosis imaging that exceeds the perfusion defect and regional scar [2,3]. The relationship between FAP expression and severity of ventricle remodeling remains unclear. We hypothesized that molecular imaging of FAP could depict the dynamics of cardiac fibroblast activation, predict functional outcome and monitor response to therapy.  

Methods: Binding of the FAP-targeted tracer ⁶⁸Ga-FAPI-46 was assessed in vitro in human cardiac fibroblasts under basal conditions and after exposure to medium from monocyte-derived polarized macrophages. C57Bl/6 mice underwent ischemia/reperfusion (I/R, n=22) or sham surgery (n=7) and serial ⁶⁸Ga-FAPI-46 imaging at 1, 3, 7, 14 and 42 days. Functional outcome was determined by cardiac magnetic resonance imaging and perfusion SPECT at 6 weeks. Additional groups of mice were blindly treated with oral enalapril or vehicle to modulate acute inflammation after I/R. Serial ⁶⁸Ga-FAPI-46 imaging assessed the molecular response to the intervention and was compared to subsequent functional outcome.

Results: Exposure of human cardiac fibroblasts to supernatant from M1-like macrophages elevated uptake of ⁶⁸Ga-FAPI-46 by 60% relative to exposure to supernatant from non-polarized macrophages (p=0.01) or reparative M2-like macrophages. Workup of supernatant identified elevated expression of TGF-β (+14%, p=0.003), and pro-inflammatory cytokines IL-1β (p<0.001) and TNF-α (p<0.001). No difference in these cytokines was observed in medium from M2-like macrophages.  I/R injury in mice resulted in moderate perfusion defect at 6wk (18.1±5.3%) and increased ventricle dimensions compared to sham (end diastolic volume: 69±9 vs 49±4µL; end systolic volume: 38±7 vs 21±4µL; p<0.001). Serial FAPI-46 imaging described biphasic upregulation after I/R with increased signal emanating from the infarct territory during the acute inflammatory phase at 3d post-I/R (% injected dose (ID)/g, 1.3±0.3 vs 0.7.±0.1, p<0.001) and the intermediate phase at 14d post-I/R (%ID/g, 0.9±0.1 vs 0.7±0.1, p=0.05). A similar elevation was observed in remote non-infarcted myocardium. Tracer signal at both timepoints significantly correlated with subsequent left ventricle function (ejection fraction) at 6 weeks (3d: r=-0.43, p<0.05; 14d: r=-0.51, p<0.05). To evaluate the sensitivity of FAPI-46 imaging to anti-inflammatory therapy, we treated mice with enalapril to suppress monocyte mobilization from the spleen. FAPI-46 signal at 3d was reduced by 52% relative to untreated MI (p=0.048). By contrast the FAPI-46 signal was modestly higher in the global myocardium at 14d after enalapril therapy compared to untreated mice (+%, p=0.04). Notably, treatment improved chronic outcome as measured by ejection fraction (values, p<0.001).

Conclusions: FAP expression by cardiac fibroblasts increased in response to inflammatory stimuli derived from macrophages. Biphasic FAPI signal suggests distinct patterns of FAP expression, reflecting both inflammation-associated fibroblast activation in response to the initial insult as well as later activation during ventricle remodeling. Taken together, FAPI imaging may provide a sensitive indicator of downstream response to anti-inflammatory therapy, illuminating the immune-fibrosis axis.

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Image/Figure Caption:

Figure. Serial imaging of fibroblast activation protein (68Ga-FAPI-46, colourscale) shows increased signal in the infarct and border zone territory defined by 18F-FDG (greyscale). Quantitative analysis identifies biphasic elevation of FAPI-46 uptake at 3d and 14d after myocardial ischemia/reperfusion (I/R) injury (lower left).  The intensity of the FAPI-46 PET signal corresponds to worse left ventricle ejection fraction (LVEF) at 6 weeks after injury (lower right).

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