An eNAMPT-neutralizing mAb reduces post-infarct myocardial fibrosis and left ventricular dysfunction

Abstract

Myocardial infarction (MI) triggers adverse ventricular remodeling (VR), cardiac fibrosis, and subsequent heart failure. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is postulated to play a significant role in VR processing via activation of the TLR4 inflammatory pathway. We hypothesized that an eNAMPT specific monoclonal antibody (mAb) could target and neutralize overexpressed eNAMPT post-MI and attenuate chronic cardiac inflammation and fibrosis. We investigated humanized ALT-100 and ALT-300 mAb with high eNAMPT-neutralizing capacity in an infarct rat model to test our hypothesis. ALT-300 was ^99mTc-labeled to generate ^99mTc-ALT-300 for imaging myocardial eNAMPT expression at 2 hours, 1 week, and 4 weeks post-IRI. The eNAMPT-neutralizing ALT-100 mAb (0.4 mg/kg) or saline was administered intraperitoneally at 1 hour and 24 hours post-reperfusion and twice a week for 4 weeks. Cardiac function changes were determined by echocardiography at 3 days and 4 weeks post-IRI. ^99mTc-ALT-300 uptake was initially localized to the ischemic area at risk (IAR) of the left ventricle (LV) and subsequently extended to adjacent non-ischemic areas 2 hours to 4 weeks post-IRI. Radioactive uptake (%ID/g) of ^99mTc-ALT-300 in the IAR increased from 1 week to 4 weeks (0.54 ± 0.16 vs. 0.78 ± 0.13, P < 0.01). Rats receiving ALT-100 mAb exhibited significantly improved myocardial histopathology and cardiac function at 4 weeks, with a significant reduction in the collagen volume fraction (%LV) compared to controls (21.5 ± 6.1% vs. 29.5 ± 9.9%, P < 0.05). Neutralization of the eNAMPT/TLR4 inflammatory cascade is a promising therapeutic strategy for MI by reducing chronic inflammation, fibrosis, and preserving cardiac function.

Keywords: Cardiac fibrosis; Cardioprotective effect; DAMP; Myocardial infarction; eNAMPT; mAb.

Fig. 1.

Experimental protocol illustration in IRI-exposed rats receiving ALT-100 mAb or saline.

Fig. 2.

Uptake of ^99mTc-ALT-300 in ischemic-reperfused myocardium. Top panel (A1-A3): Photographs of transverse slices from representative rat hearts with Evans blue-staining at 6 h (A1), 1 week (A2), and 4 weeks (A3) post-IRI. Middle panel (B1-B3): Corresponding iQID images of ^99mTc-ALT-300 uptake in the tissue slices of A1-A3. Focal radioactive lesions were prominently visible in the anterior wall, lateral wall, and apex of the left ventricle. At 6 hours post-IRI, the radioactive distribution notably corresponded to the IAR regions as outlined by dashed lines (A1 and B1). This radioactive distribution was expanded beyond the IAR margins at 1 week (A2 and B2) and was observed in areas of non-ischemic myocardium by 4 weeks (A3 and B3). Bottom panel (C): Ex vivo data on the uptake of ^99mTc-ALT-300 (%ID/g) taken 4 hours post-injection in the intact LV myocardium, the dissected non-ischemic segment (No-IAR), and the IAR of rat hearts at 1 week and 4 weeks post-IRI. * P < 0.05 compared to the No-IAR. ** P < 0.05 comparison between groups at 1 week and 4 weeks post-IRI.

Fig. 3.

Results of cardiac functional measurements. A and B: Representative long-axis echocardiographic images obtained from a sham-operated rat heart (A) and an infarcted rat heart at 4 weeks post-IRI (B). Extensive hypokinesis of the anterior LV wall and ventricular dilation were observed in the rat with MI, as illustrated by the green outlines, in comparison to the control heart. C: FS, LVEF, and CO values as determined by echocardiography in rats from the ALT-100 treatment and control groups. * P < 0.05 indicates a significant difference compared to the baseline. ** P < 0.05 denotes a significant difference between the ALT-100 treatment and saline control groups. *** P < 0.05 observed consistently from 3 to 28 days.

Fig. 4.

Measurements of myocardial ischemic area at risk (IAR) and infarct size. A and C: Photographs of Evans blue-stained transverse LV slices from two respective rat hearts treated with ALT-100 (A) and saline (C) at 4 weeks post-IRI. Nonischemic areas are stained deep blue, while the IAR appears unstained. B and D: Photographs of TTC-stained corresponding LV slices from A and C. Normal myocardial tissues appear brick red, whereas infarct segments exhibit a pale or white color. E: Summarized results from the planimetry analysis of IAR and infarct size in the two experimental groups of rats treated with ALT-100 and saline. * P < 0.05 indicates statistical significance compared to the saline control.

Fig. 5.

Histological analysis of remote and ischemic-reperfused myocardium. A-C: Representative integrated photographs of an H&E-stained LV section (A), ROI over the entire LV wall (B), and ROI located on the infarcted area of the LV section (C) established by the Olympus CellSens Dimension software for quantitative analysis of myocardial infarct size. D and E: Histological morphology (40X) of the healthy myocardium from the remote LV septal wall (D) and the infarcted border area (E). Varied inflammatory cell infiltrates and fibrous scar tissue are visible in the infarction zone and infarcted border zone. Myocardial cells lost their normal ordered structure in the border zone. F: Measurement results of myocardial infarcts over the LV sections of mixed-gender, female, and male rats received ALT-100 treatment and saline as control. * P < 0.05 vs. the saline control. ** P < 0.05 in the difference between the females and males which received ALT-100 treatment. *** P < 0.05 in the difference between the females and males received saline as control.

Fig. 6.

Representative images of cardiac sections with Masson’s Trichrome staining. A-D: Integrated microphotographs of representative cardiac sections with H&E (A and B) and Masson’s Trichrome staining (C and D) from two rat hearts treated by saline control (A and C) and ALT-100 (B and D), respectively, at 4 weeks after MI. E and F: Microphotographs (40X) derived from the infarcted border zones of Trichrome-stained cardiac sections (C and D) to show the histopathologic features of myocardial fibrosis indicated by the blue or gray Trichrome stain. Cardiac sections from rats treated by ALT-100 showed less interstitial fibrosis, perivascular fibrosis, and replacement fibrosis.

Fig. 7.

Results of myocardial fibrosis measurement based on Trichrome (TC) staining. A and B: The area (mm²) with positive TC staining (A) and CVF value (B) estimated by the CellSens Dimension software on the cardiac sections of the infarcted hearts at 4 weeks post-MI. * P < 0.05 ALT-100 treatment vs. saline control. C: Sex-related difference in the results of CVF estimations on the infarcted hearts of rats treated with ALT-100 and saline. ^# P < 0.05 males vs. females.

Fig. 8.

Microphotographs (40X) of LV sections showing inflammatory cell infiltration, identified by IHC staining for anti-CD8 (A-C) and anti-CD68 (D-F). CD8-positive lymphocytes and CD68-positive macrophages, stained brown, are predominantly located in the peri-infarct zones of rat hearts at 6 hours(A and D), 1 week (B and E), and 4 weeks (C and F) post-IRI. Insets in panels C and F show nonischemic areas remote from the ischemic zone, appearing histologically normal with minimal to no staining for CD8 and CD68. Compared to CD8-positive lymphocytes, there is a higher presence of CD68-positive macrophages at 1 week and 4 weeks post-IRI, indicating the evolving post-infarct inflammatory response.

Fig. 9.

Microphotographs (40X) of left ventricular (LV) sections illustrate NAMPT (A-D) and TLR4 (E-H) expression as revealed by IHC staining in both healthy and infarcted rat hearts. The nonischemic myocardium from a rat heart subjected to sham surgery (A) exhibited low, yet detectable, cytoplasmic expression of NAMPT, and an absence of TLR4 expression. IHC staining showed substantial brown precipitates of anti-NAMPT and anti-TLR4 in the peri-infarct zones of infarcted rat hearts at 6 hours (B and F), 1 week (C and G), and 4 weeks (D and H) post-IRI, indicative of the upregulation of NAMPT and TLR4.

Fig. 10.

NAMPT IHC staining images of rat hearts subjected to IRI and treated with ALT-100 or saline. Panels A–B and C–D display cardiac sections (10X) illustrating Masson’s Trichrome staining (A & C) and anti-NAMPT IHC staining (B & D), respectively. Panels A & B are from a control heart treated with saline, while panels C & D are from an ALT-100-treated rat heart at 4 weeks post-IRI. The numbered boxes on the IHC images indicate areas from distinct zones selected for 40X magnification: the remote non-infarct area (“1”), the peri-infarct zone (“2”), and the fibrotic infarct region (“3”). Panels B1–B3 and D1–D3 are magnified microphotographs (40X) from the corresponding boxed areas on panels B and D, respectively. The brown staining represents NAMPT expression in the cardiac tissues. No discernible difference in the distribution patterns of NAMPT expression was qualitatively observed between the heart treated with ALT-100 and the saline control. The intensity of positive anti-NAMPT staining is lower in the cardiac section from the rat treated with ALT-100 compared to that in the saline control.