Can Cytoprotective Cobalt Protoporphyrin Protect Skeletal Muscle and Muscle-derived Stem Cells From Ischemic Injury?

Abstract

Background Extremity trauma is the most common injury seen in combat hospitals as well as in civilian trauma centers. Major skeletal muscle injuries that are complicated by ischemia often result in substantial muscle loss, residual disability, or even amputation, yet few treatment options are available. A therapy that would increase skeletal muscle tolerance to hypoxic damage could reduce acute myocyte loss and enhance preservation of muscle mass in these situations. Questions/purposes In these experiments, we investigated (1) whether cobalt protoporphyrin (CoPP), a pharmacologic inducer of cytoprotective heme oxygenase-1 (HO-1), would upregulate HO-1 expression and activity in skeletal muscle, tested in muscle-derived stem cells (MDSCs); and (2) whether CoPP exposure would protect MDSCs from cell death during in vitro hypoxia/reoxygenation. Then, using an in vivo mouse model of hindlimb ischemia/reperfusion injury, we examined (3) whether CoPP pharmacotherapy would reduce skeletal muscle damage when delivered after injury; and (4) whether it would alter the host inflammatory response to injury. Methods MDSCs were exposed in vitro to a single dose of 25 lL CoPP and harvested over 24 to 96 hours, assessing HO-1 protein expression by Western blot densitometry and HO-1 enzyme activity by cGMP levels. To generate hypoxia/reoxygenation stress, MDSCs were treated in vitro with phosphate-buffered saline (vehicle), CoPP, or CoPP plus an HO-1 inhibitor, tin protoporphyrin (SnPP), and then subjected to 5 hours of hypoxia (\ 0.5% O 2 ) followed by 24 hours of reoxygenation and evaluated for apoptosis. In vivo, hindlimb ischemia/reperfusion injury was produced in mice by unilateral 2-hour tourniquet application followed by 24 hours of reperfusion. In three postinjury treatment groups (n = 7 mice/group), CoPP was administered intraperitoneally during ischemia, at the onset of reperfusion, or 1 hour later. Two control groups of mice with the same injury received phosphatebuffered saline (vehicle) or the HO-1 inhibitor, SnPP. Myocyte damage in the gastrocnemius and tibialis anterior muscles was determined by uptake of intraperitoneally This work was funded by USAMRMC Award W81XWH-07-01-0246 (MDA) and USAMRMC Award W81XWH-10-01-0789 (MDA Results In MDSCs, a single exposure to CoPP increased HO-1 protein expression and enzyme activity, both of which were sustained for 96 hours. CoPP treatment of MDSCs reduced apoptotic cell populations by 55% after in vitro hypoxia/reoxygenation injury (from a mean of 57.3% apoptotic cells in vehicle-treated controls to 25.7% in CoPP-treated cells, mean difference 31.6%; confidence interval [CI], 28.1-35.0; p \ 0.001). In the hindlimb ischemia/reperfusion model, CoPP delivered during ischemia produced a 38% reduction in myocyte damage in the gastrocnemius muscle (from 86.4% ± 7% EBD + myofibers in vehicle-treated, injured controls to 53.2% EBD + in CoPP-treated muscle, mean difference 33.2%; 95% CI, 18.3, 48.4; p \ 0.001). A 30% reduction in injury to the gastrocnemius was seen with drug...

The role of oxidative stress in skeletal muscle injury and regeneration: focus on antioxidant enzymes

“…In vivo effect of antioxidant enzymes on muscle regeneration can be attributed to the increased viability of myogenic precursors under oxidative stress. Such protective potential was shown for GPX (El et al 2012 ; Lee et al 2006 ; Nishida et al 2007 ), SOD1 (Suzuki et al 2004 ), HO-1 (Kozakowska et al 2012 ; Laumonier et al 2008 ; Wilson et al 2015 ), or CAT (Catani et al 2004 ). Accordingly, decreased level of NQO1 in skeletal muscles of Nrf2 -/- aged mice was correlated with enhanced apoptosis, although it must be noted, that Nrf2 deficiency was accompanied also by decreased expression of CAT, glutathione reductase and GCS (Miller et al 2012 ).…”

“…Aging-induced redox imbalance was associated with aggravated inflammatory reaction after acute damage (Ghaly and Marsh 2010 ). Treatment of skeletal muscle with the non-enzymatic antioxidants (Avci et al 2012 ; Myburgh et al 2012 ), GPX-mimicking compound (Gierer et al 2010 ) and finally pharmacological stimulation of SOD2 (Togliatto et al 2013 ) or HO-1 (Wilson et al 2015 ) decreased inflammation in skeletal muscle after trauma. That may in turn result in reduced muscle injury (Gierer et al 2010 ; Jazwa et al 2013 ; Li et al 2005 ; Myburgh et al 2012 ).…”

“…This phenomenon correlates with increased expression of GPX-3 in quiescent mSC (Pallafacchina et al 2010 ), enhanced SOD activity together with upregulated GSH level in muscle progenitors (Urish et al 2009 ) as well as increased expression of SOD1, SOD2, CAT, and GPX in myoblasts (Franco et al 1999 ) in comparison to their more differentiated descendants. Since high oxidative stress was shown to reduce in vivo and in vitro regenerative potential of myogenic progenitors (Ardite et al 2004 ; Drowley et al 2010 ; Hansen et al 2007 ; Lee et al 2006 ), it is not surprising that changes observed in the levels of antioxidant defence correlate with enhanced regeneration capacity of muscle progenitor cells after transplantation (Urish et al 2009 ) or ischemic injury (Wilson et al 2015 ).

Fig.

…”

Activation of Opioid Receptors Attenuates Ischemia/Reperfusion Injury in Skeletal Muscle Induced by Tourniquet Placement

“…Furthermore, several studies suggested that Oxy can relieve the inflammatory response [ 17 , 18 ]. Thus, it is possible that Oxy treatment reduces the severity of skeletal muscle damage through an anti-inflammatory mechanism to preserve skeletal muscle function [ 8 ].…”

“…Acute limb I/R causes skeletal muscle damage due to several mechanisms [ 8 ]. Skeletal muscle is characterized by its high content of mitochondria, which has a lower threshold for ischemic damage than other tissues such as nerve or bone.…”