Aging-associated bladder wall stiffness and impairment of bladder wall biomechanics are restored to a younger state with mitochondrially-targeted antioxidant therapy

Wolf-Johnston A1, Cheng F1, Watkins S1, Robertson A1, Birder L1

Research Type

Pure and Applied Science / Translational

Abstract Category

Female Lower Urinary Tract Symptoms (LUTS) / Voiding Dysfunction

Abstract 505
Bladder Wall, Microbiome and Nocturia
Scientific Podium Short Oral Session 27
Friday 6th September 2019
09:07 - 09:15
Hall G1
Gerontology Voiding Dysfunction Molecular Biology Animal Study
1.University of Pittsburgh

Amanda Wolf-Johnston



Hypothesis / aims of study
Age-related changes in the extracellular matrix (ECM) may impact the function of the urothelium and other structures in the bladder wall.  Despite having different etiologies, most chronic fibrotic disorders present with a persistent production of similar factors including reactive oxygen species (ROS) that stimulate ECM production, which progressively destroys the organ’s architecture and in turn, its function.  Mitochondria are the primary source of ROS and pathologies associated with mitochondrial dysregulation (such as aging) lead to overproduction of ROS and factors that promote fibrosis. As the bladder fills, the coordinated recruitment of collagen fibers across both the smooth muscle (SM) and lamina propria (LP) layers, essential for the elasticity of the bladder wall, is lost during aging. Our findings reveal that aging leads to decreased recruitment of collagen fibers during bladder stretch which correlates with increased bladder stiffness. Further, treatment of aged rats with the mitochondrially targeted antioxidant MitoTEMPO significantly decreases bladder stiffness and reduces biomarkers for oxidative stress. These findings lead us to hypothesize that aging-related deficits in mitochondrial function impairs bladder wall biomechanics which can impact signalling and bladder dysfunction.
The aim of this study was designed to elucidate the effects of aging on mechanical and structural properties of the ECM and the effect of a mitochondrially-targeted antioxidant treatment.
Study design, materials and methods
This aim was investigated in female Fisher 344 rats (young- 3 mo; aged-25-28 mo). 
•	Some animals were treated for 3 weeks with the mitochondrial-targeted antioxidant, mitoTEMPO (1 mg/kg/day delivered via osmotic pump).
•	Bladders were collected from deeply anesthetized rats and utilized for western blot per previously published methods (minimum of n=5-7 rats/group and n=5-7 tissue samples/experimental group). The volume (intensity) of each protein species was determined and normalized to total protein (Bio-Rad).
•	Concurrent imaging and mechanical testing of collagen fibers (both LP and SM) in excised rat bladders were done under bioaxial mechanical testing conditions coupled with multi-photon microscopy.
Aged bladders were consistently larger than bladders from young rats with significant increased wall stiffness much earlier in the stress/strain-loading curve and altered tortuosity (‘waviness’) of fibers relative to young bladders. Changes in collagen recruitment is a key factor with a decreased recruitment with stretch occurring in both the LP and the SM regions. We further find a significant decrease in mature form of lysyl oxidase (LOX; 37%) as well as phosphorylated adenosine monophosphate (AMP) activated protein kinase (pAMPK; 47%), which is a sensor of cellular energy levels.  A biomarker for oxidative stress and protein damage, nitrotyrosine, was also found to be elevated (81%; mainly in the LP region). Treatment of aged rats with the mitochondrial-targeted antioxidant MitoTEMPO mitigated these dysfunctions resulting in decreased bladder wall stiffness while mimicking findings in a younger bladder (shown in Figure 1A/B).
Interpretation of results
Age-related changes in mitochondrial function as well as dysfunction in energy metabolism due to reduced activity of AMPK can promote the development of lower urinary tract (LUT) dysfunction. Loss of sensitivity of AMPK activation can increase oxidative stress and may even trigger inflammation and metabolic disorders. Together this can lead to defective tissue repair and a dysfunctional remodelling of the bladder wall.  Mitochondria targeted therapies (such as MitoTEMPO) may hold future promise to restore bladder structure in the aging bladder and may contribute to improvement in bladder function, thereby reducing LUT symptoms (LUTS) that typically increase with age.
Concluding message
During filling in a healthy bladder, tortuous (or wavy) collagen fibrils straighten, enabling the bladder to undergo large changes in size.  The aging process is associated with a number of functional changes (such as mitochondrial dysfunction and increased ROS) that can produce alterations in bladder matrix structure, compliance, oxygen tension and biomechanics of the bladder wall. Modifications in physical properties of the ECM proteins have major effects within the bladder wall, resulting in increased intramural tension, decreased wall compliance and mechanosensory dysfunction (leading to an unstable bladder). Our novel findings provide new insights into how increased oxidative stress over time alters fundamental properties of the ECM that may affect bladder relaxation, contractility and even sensation. Treatments that target the mitochondria and decrease oxidative stress (MitoTEMPO) could lead to improvement in these dysregulated pathways that may improve bladder outcomes in older adults.
Figure 1
Figure 2
Funding R37 DK54824, R01 AG056944 Clinical Trial No Subjects Animal Species Rat Ethics Committee Intitutional Animal Care and Use Committee
31/07/2021 02:12:22