Statin Therapy Negatively Impacts Skeletal Muscle Regeneration and Cutaneous Wound Repair in Type 1 Diabetic Mice

Those with diabetes invariably develop complications including cardiovascular disease (CVD). To reduce their CVD risk, diabetics are generally prescribed cholesterol-lowering 3-hydroxy-methylglutaryl coenzyme A reductase inhibitors (i.e., statins). Statins inhibit cholesterol biosynthesis, but also reduce the synthesis of a number of mevalonate pathway intermediates, leading to several cholesterol-independent effects. One of the pleiotropic effects of statins is the reduction of the anti-fibrinolytic hormone plasminogen activator inhibitor-1 (PAI-1). We have previously demonstrated that a PAI-1 specific inhibitor alleviated diabetes-induced delays in skin and muscle repair. Here we tested if statin administration, through its pleiotropic effects on PAI-1, could improve skin and muscle repair in a diabetic rodent model. Six weeks after diabetes onset, adult male streptozotocin-induced diabetic (STZ), and WT mice were assigned to receive control chow or a diet enriched with 600 mg/kg Fluvastatin. Tibialis anterior muscles were injured via Cardiotoxin injection to induce skeletal muscle injury. Punch biopsies were administered on the dorsal scapular region to induce injury of skin. Twenty-four days after the onset of statin therapy (10 days post-injury), tissues were harvested and analyzed. PAI-1 levels were attenuated in statin-treated diabetic tissue when compared to control-treated tissue, however no differences were observed in non-diabetic tissue as a result of treatment. Muscle and skin repair were significantly attenuated in Fluvastatin-treated STZ-diabetic mice as demonstrated by larger wound areas, less mature granulation tissue, and an increased presence of smaller regenerating muscle fibers. Despite attenuating PAI-1 levels in diabetic tissue, Fluvastatin treatment impaired cutaneous healing and skeletal muscle repair in STZ-diabetic mice

Impaired Macrophage and Satellite Cell Infiltration Occurs in a Muscle-Specific Fashion Following Injury in Diabetic Skeletal Muscle

<div><p>Background</p><p>Systemic elevations in PAI-1 suppress the fibrinolytic pathway leading to poor collagen remodelling and delayed regeneration of tibialis anterior (TA) muscles in type-1 diabetic Akita mice. However, how impaired collagen remodelling was specifically attenuating regeneration in Akita mice remained unknown. Furthermore, given intrinsic differences between muscle groups, it was unclear if the reparative responses between muscle groups were different.</p><p>Principal Findings</p><p>Here we reveal that diabetic Akita muscles display differential regenerative responses with the TA and gastrocnemius muscles exhibiting reduced regenerating myofiber area compared to wild-type mice, while soleus muscles displayed no difference between animal groups following injury. Collagen levels in TA and gastrocnemius, but not soleus, were significantly increased post-injury versus controls. At 5 days post-injury, when degenerating/necrotic regions were present in both animal groups, Akita TA and gastrocnemius muscles displayed reduced macrophage and satellite cell infiltration and poor myofiber formation. By 10 days post-injury, necrotic regions were absent in wild-type TA but persisted in Akita TA. In contrast, Akita soleus exhibited no impairment in any of these measures compared to wild-type soleus. In an effort to define how impaired collagen turnover was attenuating regeneration in Akita TA, a PAI-1 inhibitor (PAI-039) was orally administered to Akita mice following cardiotoxin injury. PAI-039 administration promoted macrophage and satellite cell infiltration into necrotic areas of the TA and gastrocnemius. Importantly, soleus muscles exhibit the highest inducible expression of MMP-9 following injury, providing a mechanism for normative collagen degradation and injury recovery in this muscle despite systemically elevated PAI-1.</p><p>Conclusions</p><p>Our findings suggest the mechanism underlying how impaired collagen remodelling in type-1 diabetes results in delayed regeneration is an impairment in macrophage infiltration and satellite cell recruitment to degenerating areas; a phenomena that occurs differentially between muscle groups.</p></div

Characteristics of wild type and Akita diabetic mice.

<p>Data collected at 6 weeks of diabetes (6 weeks group), or at 5, 10, or 35 days following cardiotoxin muscle injury at 8 weeks of diabetes (9–13 weeks group). All measures except for soleus fiber area were found to be significantly altered in the diabetic mice. All muscle mass and fiber area measures presented here are from the uninjured muscle; the cardiotoxin injured contralateral leg muscle data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070971#pone-0070971-g001" target="_blank">Figure 1</a>. NEFA, non-esterified fatty acids; PAI-1, plasminogen activator inhibitor-1; TA, tibialis anterior; GPS, gastrocnemius-plantaris-soleus complex; GAS, gastrocnemius; SOL, soleus. * denotes significant difference in Akita compared to matching wild type (WT) value as assessed by t-test (P<0.05).</p

Necrosis of muscle fibers persists throughout muscle regeneration in Akita tibialis anterior muscle.

<p>(A) Uninjured (left), necrotic (center), and actively regenerating (right) regions of skeletal muscle are easily identified in collagen type I immunostain (green) with DAPI (blue) counterstain. Note the presence of centrally located nuclei in muscle fibers in regenerating muscle, indicative of new myofiber formation. (B) TA of diabetic mice have clearly defined areas of necrosis remaining at 10 days post-injury (significant interaction [P<0.05]; * denotes post-hoc difference). Similarly, gastrocnemius (GAS) (C) follows this pattern although not statistically significant (P = 0.21). In contrast, both WT and Akita soleus (SOL) display no areas of necrosis at 5 or 10 days post-injury (not shown). (D) Representative image of TA muscle undergoing regeneration at 10 days post-injury in WT and Akita TA. Note the distinct area of necrosis in the Akita TA. Scale bar represents 500 um.</p

Soleus muscle is resilient to the regenerative defects found in other muscles from Akita mice.

<p>(A) H&E stained cryosections of tibialis anterior (TA), gastrocnemius (GAS), and soleus (SOL) illustrate decrements in muscle regeneration in diabetic Akita mouse muscles. (B) TA demonstrates the worst impairment in regeneration, as determined by cross sectional area of regenerating (centrally-nucleated) fibers (significant main effect of diabetes and interaction [P<0.05]). GAS (C) also demonstrates impaired regeneration (significant main effect of diabetes and interaction [P<0.05]), while SOL (D) does not. In panels E–F, TA (E), GAS (F), and SOL (G) CTX-injured fiber area data are expressed relative to fiber area from the contralateral uninjured muscles. The TA (significant main effect of diabetes and interaction [P<0.05]) and GAS (significant main effect of diabetes [P<0.05]) both demonstrate poor regeneration even when expressed in these relative terms. The mass of muscles that had been injured with cardiotoxin further illustrate poor regeneration. The muscle mass of the injured TA (H; significant main effect of diabetes and interaction [P<0.05]) and gastrocnemius-plantaris-soleus (GPS) complex (I; significant main effect of diabetes [P<0.05]) are illustrated here. * denotes significant post-hoc analysis differences (P<0.05). Scale bar represents 50 um.</p