Neutrophil PAD4 negatively influences cardiac function and remodeling with increasing age
Authors: Van Bruggen S , Van Wauwe J , Carai P , Frederix L , Vangilbergen L , Witsch T , Martinod K 
Published: October 2021

Abstract

Background
Aging can be viewed as a status of chronic inflammation, in which neutrophils have a lower threshold for activation. The enzyme peptidylarginine deiminase 4 (PAD4), which catalyzes the conversion of arginine to citrulline, will be activated in a certain population of neutrophils. When this conversion takes place on the histones, neutrophils can form neutrophil extracellular traps (NETs), which are both prothrombotic and proinflammatory. Mice lacking this enzyme systemically were previously reported to be protected from age-related fibrosis.

Purpose
We aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, and in consequence NETs, are involved in cardiac fibrosis development, which in turn will result in impaired cardiac function.

Methods
We generated a mouse model of impaired NET release capability via deletion of PAD4, a NET-essential gene, under the neutrophil-specific promoter (PAD4fl/flMRP8Cre+). In order to study heart failure (HF) development, these specific deletion mice and their littermate controls were aged for a period of two years (coinciding with approximately 70 years of age in the human population; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated by echocardiography and histology, respectively. A separate set of young mice (12 weeks) were evaluated in parallel.

Results
We performed a comprehensive echocardiography analysis including both structural and functional parameters. As for systolic function, we could see that in old wild type (WT) mice, ejection fraction (EF) significantly decreased as compared to EF in young and healthy (YH) mice (YH - 67±6%, WT - 53±10%; p&lt;0.0001) (Figure 1B). However, this decrease in systolic function was absent in the old PAD4fl/flMRP8Cre+ mice, with EF being comparable to the YH group (PAD4fl/flMRP8Cre+ - 67±7%; p=0.9169) (figure 1 A,B). As for diastolic function, again we could see a marked decrease in E/A ratio in the WT as compared to the YH population (YH- 1.50±0.23, WT – 1.21±0.17; p&lt;0.0001), while this functional deterioration was absent in aged PAD4fl/flMRP8Cre+ animals (PAD4fl/flMRP8Cre+ - 1.38±0.21; p=0.0837) (Figure 1 C,D). To link this decline in heart function to tissue remodeling, we quantified collagen deposition in the heart. We saw that natural aging resulted in an increase in cardiac collagen deposition in the WT population as compared to YH mice (YH – 0.86±0.63%, WT – 4.02±1.71%). This increased collagen deposition was absent in the neutrophil deletion mice (PAD4fl/flMRP8Cre+ - 1.7±0.76%). Additionally, when comparing WT to PAD4 deletion-mice, we saw that the increase in collagen deposition is significantly higher in the WT mice (p&lt;0.0001).

Conclusion
Our data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac fibrosis, resulting in cardiac dysfunction.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fonds Wetenschappelijk Onderzoek (FWO) - Vlaanderen