Unraveling the Metabolic Mosaic of Tendon Health and Disease

Abstract

Tendons are cellularly sparse tissues, primarily composed of extracellular matrix (ECM), and play a crucial role in transmitting force from muscle to bone. The mechanical demands placed on tendons require a delicate balance of metabolic processes to maintain homeostasis. However, tendons have limited healing capabilities, making them susceptible to injury and disease. While some progress has been made in understanding tendon homeostasis, much remains unknown about tendon metabolism. This study aimed to explore tendon health and disease by: 1) identifying dysregulation in chronic tendinopathy metabolism; and 2) assessing how tendons respond to metabolic stressors. Tendinopathy affects ~3.5 million people in the U.S. and is characterized by degenerative ECM, reduced cell viability, and a diminished ability to handle mechanical load. Recent research has identified AMP-activated protein kinase (AMPK) as a key regulator of cell viability and ECM remodeling in musculoskeletal tissues. Here, I performed bulk-RNA sequencing on human rotator cuff tendon biopsies from patients with tendinopathy and compared them to healthy hamstring tendon controls and found downregulation of AMPK signaling in tendinopathy. Additionally, I present evidence that in the mouse Achilles tendon, AMPK is necessary for ECM maintenance, cell viability and mechanical function. Using Cre-lox technology, I deleted Prkaa1, encoding AMPKα1, in tendon progenitor cells in mice. At one month of age, this deletion led to dysregulation of cell cycle and ECM-related processes. By three months of age, tendons lacked Prkaa1 exhibited impaired mechanical properties and increased senescence markers. Over time, these tendons developed ectopic calcification (EC) and elevated markers like p16 and p21. To determine if exercise could prevent senescence, I exposed mice to voluntary running wheels. Exercise improved ECM organization and suppressed p16 and p21. These findings highlight the role of AMPKα1 in tendon homeostasis and suggest exercise may delay senescence in tendons lacking AMPK. I also found that EC onset and severity were worse in male mice deficient in AMPK. Using bulk-RNA sequencing, I found a baseline predisposition in males to develop EC, indicated by osteogenic marker transcription. Estradiol, a sex hormone, had a protective effect against this phenotype. In vitro, estradiol increased tendon cell viability, suggesting sex hormones are vital for tendon health, offering insights into therapies for individuals with hormonal imbalances or undergoing hormone therapy. Sex differences are a key risk factor for tendon injury and disease. These findings have implications for gender-affirming care, where the effects of puberty suppression and hormone therapy on tendon health are still under investigation. I found that puberty suppression with and without testosterone supplementation did not negatively impact tendon function or structure. Female mice received treatments with gonadotropin-releasing hormone analogs (GnRHa) and testosterone. I observed that GnRHa, with or without testosterone supplementation, increased tendon ultimate load in female mice. GnRHa also increased cell density at the Achilles enthesis, while testosterone alone did not have this effect. These findings offer early insights into how pubertal suppression and hormone therapy might affect tendon function and structure, providing clinical guidance for gender-diverse patients. The relationship between AMPK and hormonal influences and tendon health highlights the complex ways in which metabolic processes effect tendon homeostasis. Understanding how energy regulation, hormonal treatment, and sex differences intersect is crucial for developing targeted therapies. This research lays the foundation for better care strategies in the treatment of tendon disease and injury.