Download Reduction in Muscle Fiber Force Production, Disruption of Muscle

Reduction in Muscle Fiber Force Production, Disruption of Muscle Cell Architecture and Accumulation of Fatty Macrophages in Patients with Chronic Rotator Cuff Tears Asheesh Bedi, MD; Stuart M Roche, BS; Evan B Lynch, BS; Max E Davis, BA; Liz Sibislky Enselman, MEd, ATC; Chris Mendias, PhD, ATC Department of Orthopaedic Surgery University of Michigan Medical School [email protected] Funding Disclosures This study was funded by a research grant from the American Shoulder and Elbow Surgeons Epidemiology of Rotator Cuff Tears and Fatty Degeneration •  >250,000 rotator cuff repair surgeries performed each year –  50% of patients still have symptoms 6 months after RCR –  40% still have symptoms 1 year after RCR •  “Fatty Degeneration”-­‐ The atrophy and concomitant fatty infiltration of rotator cuff muscle following tear –  Despite successful repair, for patients with chronic tears fatty degeneration does not improve –  The molecular etiology of fatty degeneration is still not fully understood Gladstone 2007, Walsworth 2009, Williams 2009, Edwards 2011, Colvin 2012 Fat Accumulation in Muscles Lipid droplets are present in oxida@ve muscle fibers as small, round puncta that can be observed with Oil red O staining G O Nuclei Lipid The lipid droplets that accumulate in torn rotator cuff muscles are much larger than what normally appears in muscle In other @ssues, large lipid droplets can act as the site of produc@on for proinflammatory molecules Uninjured
Torn Cuff
Bossa 2010, Gumucio 2012 Fatty Macrophages/Foam Cells •  Lipid laden macrophages, also known as fatty macrophages or foam cells, accumulate in atherosclerosis and other disease states •  The fat that accumulates in these macrophages serves as a substrate for the production of proinflammatory eicosanoids and prostaglandins •  These fatty macrophages secrete these proinflammatory molecules that then promote local tissue inflammation and fibrosis •  We previously identified fatty macrophages in a rat model of chronic rotator cuff tears, but it was unknown if these cells accumulate in patients with chronic cuff tears Cascieri 2002, Lumeng 2010, Gumucio 2012 Measuring Force Production at the Single Muscle Fiber Level •  Due to the complicated anatomy of the shoulder girdle, it is difficult to isolate specific rotator cuff muscles when performing traditional strength measurements using dynamometers •  The strength of muscle can be measured at the single muscle fiber/cell level from tissue obtained through biopsies •  This in vitro technique allows for a precise and reproducible measurement of muscle strength Muscle Fiber 5
Single Fiber Contrac@lity Measurements CSA = Fiber Cross Sec@onal Area Fo = Maximum Isometric Force sFo = Specific Force (Fo/CSA) CSA =
ai bi
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Current Study Objectives and Design • 
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Study objectives: –  Determine the impact of rotator cuff tear on muscle fiber contractility and the ultrastructure of sarcomeres –  Determine the presence of fatty macrophages in chronically torn rotator cuff muscles Methods –  Biopsy of supraspinatus (SSP) and deltoid (DEL) performed at time of arthroscopic rotator cuff repair •  Biopsies of vastus lateralis (VL) muscles from healthy, age-­‐matched patients served as controls –  Inclusion criteria: Male and female subjects aged > 18, full thickness supraspinatus tear (MRI or US), chronic (>5 years) –  Exclusion criteria: Previous rotator cuff repair or other shoulder surgery; major medical illness, dystrophy or rheumatologic condition Subject demographics –  N=13 (7 ♂, 6 ♀, age 58±3 years) Measures –  Contractility of individual muscle fibers (fast fibers) –  Amount of tendon retraction from MRI (used to stratify force production) –  Histology –  Transmission electron microscopy –  Differences between groups were tested using a one-­‐way ANOVA Specific Force (kPa)
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SSP Specific Force (kPa)
Reduced Muscle Fiber Specific Force (Force per CSA) in Torn Rotator Cuff Muscles 200
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R2=0.40
P=0.0195
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Compared to healthy fibers from the Small
Medium
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VL muscle, there was a reduc@on in (1-2cm)
(2-3cm)
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force produc@on in SSP muscles Tear Size Group
We ini@ally biopsied the deltoid to There was a nega@ve correla@on between muscle serve as a control, but the deltoid fiber specific force produc@on and the size of the also demonstrated pathological tear at the @me of repair changes in force produc@on * P<0.05 Healthy Sarcomere Structure •  An electron micrograph of sarcomeres from healthy muscle fibers is shown to the right •  There is a highly ordered structure of thick and thin filaments and z-­‐discs Thick filament Thin filament Z-­‐disc Devor and Faulkner, 1999 Disordered Sarcomeres and Lysosomes in Torn SSP Muscles 0.5µm
Disordered z-­‐discs Disrupted thick and thin filament overlap 2µm
Large lysosomes Lipid and Macrophage Accumulation in Torn SSP Muscles Nuclei ECM (WGA-­‐Lec4n) BODIPY (neutral lipid) Numerous large lipid droplets and lipid laden macrophages were present in pa@ents with rotator cuff tears Nuclei ECM (WGA-­‐Lec4n) BODIPY (cholesterol) Macrophage (CD68) Conclusions • 
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Compared with healthy muscle fibers from the VL, there was a 29% reduction in specific force in torn SSP muscles, and a 34% reduction in specific force for deltoid muscle fibers –  As healthy muscle undergoes atrophy or hypertrophy, specific force typically does not change –  A reduction in specific force in fibers from torn rotator cuff muscles suggests that bona fide pathological changes are occuring in the fundamental cellular force transmitting structures in rotator cuff muscles –  While the supraspinatus muscle had reduced force production, a reduction in force was also noted in the deltoid which may parallel symptomatic rotator cuff disease Numerous large intramuscular lipid vacuoles and lysosomes were observed in areas of myofibril degradation and streaming of force transmitting Z-­‐disks was often present –  The lipid vacuoles in torn cuff muscles likely directly disrupts force transmission and also serve as a site for proinflammatory eicosanoid and prostaglandin production We have also identified for the first time that a portion of the extramyocellular fat in torn rotator cuff muscles accumulates in fatty macrophages/foam cells –  These fatty macrophages may promote the local inflammation in torn rotator cuff muscles Future therapies that target lysosomal-­‐mediated myofibril degradation or macrophage accumulation may help to ameliorate the severity of fatty degeneration in patients with rotator cuff tears