Enhancing Plasmin Activity Prevents Diet‐Induced Skeletal Muscle Calcification Following Injury
Authors: Moore Stephanie N , Hawley Gregory D , Gibson Breanne HY , Schoenecker Jonathan G 
Published: November 2023

Introduction

Trauma‐induced skeletal muscle calcification is a spectrum of disease, including dystrophic calcification and heterotopic ossification, that may occur following severe injuries such as burn, blast, neurologic, and musculoskeletal injuries, as well as certain orthopaedic procedures. Under normal physiologic circumstances, when the body undergoes injury, innate protection mechanisms inhibit the formation of calcification within soft tissues such as the skeletal muscle. However, if calcification does form, the body also possesses mechanisms to remove mineral deposits formed in damaged tissues, thereby allowing for proper tissue healing and regeneration. Prevention and removal of calcification from damaged tissues is critical given that if calcification persists, it can result in a chronic inflammatory state, pain, joint dysfunction, and in severe cases necessitation of limb amputation.

Through previous work in our lab, we have demonstrated that without sufficient plasmin activity, due to a partial genetic reduction in its precursor plasminogen, calcification persists within damaged tissues following a localized skeletal muscle injury. As a means to rescue this deficiency, we have subsequently demonstrated that downregulating the primary inhibitor of plasmin, a2□antiplasmin, and thereby enhancing plasmin activity can prevent the development of calcification within injured tissues. Therefore, we now aim to apply this therapeutic approach to a model of trauma‐induced skeletal muscle calcification which is independent of direct alteration to plasmin activity. Specifically, we have investigated the hypothesis that enhancing plasmin activity can prevent high phosphate diet‐induced skeletal muscle calcification following injury.

Methods

Trauma‐induced skeletal muscle calcification was induced by injecting 40μL of 10mM cardiotoxin into the posterior compartment of the lower extremity of male or female mice at 6 weeks of age that were fed a high‐phosphate diet (1.7% phosphate) from the time of weening. Seven days following injury and continuing each week, radiographs were obtained under anesthesia to visualize and longitudinally quantify the amount of skeletal muscle calcification following injury. Radiographs were then quantified using the validated soft tissue calcification scoring system (STiCSS) to longitudinally measure the formation and regression of skeletal muscle calcification following injury. All statistical analysis was conducted by a non‐parametric Mann‐Whitney rank test. Further detailed analysis of skeletal muscle calcification was conducted by endpoint microCT and histological analysis. All animal procedures in this protocol were approved by the Vanderbilt Institutional Animal Care and Use Committee (M/13/099).

Results

Through the use of our validated model of trauma‐induced skeletal muscle calcification in combination with a high phosphate diet, we demonstrated that environment factors such as diet can promote the formation of calcification in injured tissues following injury. We observed marked levels of skeletal muscle calcification at 7 days following injury in mice fed a high phosphate diet as compared to mice fed a standard chow diet (5LOD) (Male chow vs Male High Phosphate: P=0.002; Female chow vs Female high phosphate: P<0.001). Additionally, we observed significantly higher levels of skeletal muscle calcification, as measure by the STiCSS, in female mice as compared to age matched male counterparts (P=0.0091). Following the initial development of skeletal muscle calcification at 7 days post injury, we observed a steady regression of calcification in all diet groups; however given the fact that high phosphate diet animals all began with significantly greater levels of skeletal muscle mineralization at 7 days post injury, these animals still possess marked mineralization at 28 days post injury while chow fed animals had almost completely regressed their calcification by this time.

Next, given our findings that a high phosphate diet can predispose skeletal muscles to calcification following injury, we aimed to test our previously successful therapeutic method of enhancing plasmin activity as a means to prevent the initial formation of skeletal muscle calcification. Starting at the time of weening (3 weeks of age), male mice were placed on a high phosphate diet. At 4 weeks of age, mice were then administered an antisense oligonucleotide (ASO) specific for a2□antiplasmin or a non‐targeting control (100mg/kg/week) beginning two weeks prior to injury and continuing weekly. Following a2□antiplasmin ASO treatment, we observed a marked decrease the development of skeletal muscle calcification following injury at 7 days following injury as compared to control treated animals (P=<0.001). Therefore, these results distinguish plasmin activity as a promising therapeutic target for mitigating the development of skeletal muscle calcification following injury in a plasmin deficiency‐independent model. Moving forward, we aim to test this therapeutic strategy in female mice as well as additional plasmin‐independent models of trauma‐induced skeletal muscle calcification.

Discussion

Through this work, we have demonstrated that skeletal muscle calcification following injury is multifactorial. As such, alteration to multiple pathways, either genetic (such as plasminogen) or environmental (diet) as seen here, can predispose skeletal muscle to calcification. Conversely, we have demonstrated that enhancement of just the fibrinolytic pathway, by targeting plasmin activity, is sufficient to provide cross‐protection against high phosphate diet‐induced skeletal muscle calcification following injury.

Significance

Since plasmin also supports bone homeostasis and fracture repair, increasing plasmin activity represents the first pharmacologic strategy to prevent skeletal muscle calcification without adversely affecting systemic bone physiology or concurrent skeletal muscle and bone regeneration. Therefore, these findings support further therapeutic development and potential bench to bedside translation.

Support or Funding Information

Funding for the project was provided by the Vanderbilt Department of Orthopaedics and Rehabilitation, The Caitlin Lovejoy Fund, and the Predoctoral Pharmacology Training Grant (T32 GM007628).