Myocyte specific overexpression of myoglobin impairs angiogenesis after hind-limb ischemia

Abstract

Objective: In preclinical models of peripheral arterial disease the angiogenic response is typically robust, though it can be impaired in conditions such as hypercholesterolemia and diabetes where the endothelium is dysfunctional. Myoglobin (Mb) is expressed exclusively in striated muscle cells. We hypothesized that myocyte specific overexpression of myoglobin attenuates ischemia-induced angiogenesis even in the presence of normal endothelium.

Methods and results: Mb overexpressing transgenic (MbTg, n=59) and wild-type (WT, n=56) C57Bl/6 mice underwent unilateral femoral artery ligation/excision. Perfusion recovery was monitored using Laser Doppler. Ischemia-induced changes in muscle were assessed by protein and immunohistochemistry assays. Nitrite/nitrate and protein-bound NO, and vasoreactivity was measured. Vasoreactivity was similar between MbTg and WT. In ischemic muscle, at d14 postligation, MbTg increased VEGF-A, and activated eNOS the same as WT mice but nitrate/nitrite were reduced whereas protein-bound NO was higher. MbTg had attenuated perfusion recovery at d21 (0.37+/-0.03 versus 0.47+/-0.02, P<0.05), d28 (0.40+/-0.03 versus 0.50+/-0.04, P<0.05), greater limb necrosis (65.2% versus 15%, P<0.001), a lower capillary density, and greater apoptosis versus WT.

Conclusions: Increased Mb expression in myocytes attenuates angiogenesis after hind-limb ischemia by binding NO and reducing its bioavailability. Myoglobin can modulate the angiogenic response to ischemia even in the setting of normal endothelium.

Figure 1

A, Myoglobin(Mb) and Mb-transgene (hemagluttinin-tagged, HA-Mb) expression in Western blots of gastrocnemus muscle from transgenic (MbTg) and wild-type (wt) mice. Total myoglobin is higher in MbTg vs WT mice by ELISA (n=8 per group *P<0.001), as was (B) VEGF-A (n=8 per group; *P<0.05). There was no difference in (C) capillary density or (D) phophorylated endothelial nitric oxide synthase (peNOS) to total eNOS. E, In aortic rings, there was no difference in endothelium-dependent (ACh-mediated) and endothelium-independent (SNP-mediated) relaxation between groups.

Figure 2

A, Time course (left) and image (right) of laser Doppler perfusion imaging (LDPI) shows myoglobin-transgenic (MbTx) had impaired perfusion recovery compared to wild-type (WT) mice, day 21 and 28 after hind-limb ischemia (n=20 per group, *P<0.05). B, Ischemic muscle from MbTg mice (I, 28 days after ischemia) had lower capillary density compared to WT, indicating an attenuated angiogenic response (n=8 per group, *P<0.05) with more apoptosis by (C) caspase-3 activity (n=8 per group, *P<0.01) and (D) Tunel staining (n=8 per group, *P<0.05).

Figure 3

A, At 14 days postoperatively, both myoglobin-transgenic (MbTg) and wild-type (WT) mice had comparable increases in VEGF-A in ischemic (I) vs nonischemic (NI) limbs (n=10 per group). B, The expression of p-eNOS/eNOS (eNOS activation) was similar (n=6 per group) and (C) there was no difference in total NOS activity (representative picture from n=4 per group, see methods for details).

Figure 4

A, The sum of stable NO products nitrate and nitrite was measured as an index of the available NO. At 14 days postoperatively, ischemic skeletal muscle from myoglobin-transgenic mice (MbTg) had significantly less NO compared to wild-type (WT) mice (n=10 per group; *P<0.05). B, cGMP levels did not differ in the nonischemic limb, but were significantly lower in the ischemic muscle from MbTg vs wt mice (n=8 per group, P<0.05). C, MbTg muscle had significantly higher levels of protein-bound NO compared to WT mice; indicating NO scavenging by myoglobin (n=9 per group; P<0.05).