PAK3 overexpression during stress-induced cardiac hypertrophy dysregulates autophagy augmenting acute cell death and accelerating the development of systolic dysfunction
Authors: Ruiz Velasco Hernandez A , Liu W 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation

Introduction
The understanding of stress-induced cardiac hypertrophy remains vital for preventing and better treatment of heart failure. Studies of the p-21activated kinase (PAK) family describe the cardioprotective roles of PAK1 and PAK2 proteins; however, the function of PAK3 is not known. Unlike the first two, the expression of PAK3 increases in failing hearts, suggesting it has distinct regulatory pathways and functions.

Purpose
Elucidate the role of PAK3 during the cardiac stress response and its contribution to the development of cardiac hypertrophy and dysfunction.

Methods
A cardiac-specific PAK3 overexpression mouse model was established by administering an AAV9 vector carrying a PAK3 coding sequence regulated by the Troponin promoter. Acute cardiac stress consisted of two consecutive isoprenaline injections, while the long-term experiment employed subcutaneous minipumps for constant release over two weeks. Cardiac function was assessed by echocardiography, cardiac morphology through histological staining, and protein expression by western blot. For the in vitro experiments, an adenoviral vector was used for overexpression on neonatal rat ventricular myocytes, and starvation was used to induce stress. The tandem mCherry-Gfp-LC3 reporter was used to detect autophagic flux by immunofluorescence and flow cytometry. Cell death was evaluated by measuring lactate dehydrogenase release (LDH) and using the Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay.

Results
After only two days of isoprenaline-induced stress, cardiac PAK3 overexpression in mice resulted in a reduced fractional shortening and enlarged ventricular chamber diameter. Histological analysis showed a rise in interstitial fibrosis and apoptotic cardiomyocytes. Increasing the exposure time to cardiac stress to two weeks aggravated the alterations detected in the acute model. Western blot revealed an mTOR overactivation, possibly through the disassembly of the TSC1 complex, accompanied by the alteration of autophagic markers. In vitro experiments confirmed that PAK3 overexpression led to basal mTOR and autophagosome formation activation but with an impaired flux. One hour of starvation induced LDH release and cardiomyocyte apoptosis, worsened by PAK3 overexpression. These effects were recovered by the MTOR-independent autophagy inducer ABT-737 in vitro and by the recently produced MSL-7.

Conclusion
Contrary to the proteins in the family, PAK3 overexpression is damaging for cardiac function. It dysregulates the autophagic process and renders the cardiomyocytes unable to sustain acute stress. The administration of the autophagy inducer MSL-7 improved muscle tolerance to stress and preserved function showing its therapeutic properties in stress-induced cardiac hypertrophy. The rapidity with which PAK3 overexpression induced systolic dysfunction portrays the importance of further studying its function in cardiac health and disease.