Effects of myocardial infarction on the distribution and transport of nutrients and oxygen in porcine myocardium

Abstract

One of the primary limitations of cell therapy for myocardial infarction is the low survival of transplanted cells, with a loss of up to 80% of cells within 3 days of delivery. The aims of this study were to investigate the distribution of nutrients and oxygen in infarcted myocardium and to quantify how macromolecular transport properties might affect cell survival. Transmural myocardial infarction was created by controlled cryoablation in pigs. At 30 days post-infarction, oxygen and metabolite levels were measured in the peripheral skeletal muscle, normal myocardium, the infarct border zone, and the infarct interior. The diffusion coefficients of fluorescein or FITC-labeled dextran (0.3-70 kD) were measured in these tissues using fluorescence recovery after photobleaching. The vascular density was measured via endogenous alkaline phosphatase staining. To examine the influence of these infarct conditions on cells therapeutically used in vivo, skeletal myoblast survival and differentiation were studied in vitro under the oxygen and glucose concentrations measured in the infarct tissue. Glucose and oxygen concentrations, along with vascular density were significantly reduced in infarct when compared to the uninjured myocardium and infarct border zone, although the degree of decrease differed. The diffusivity of molecules smaller than 40 kD was significantly higher in infarct center and border zone as compared to uninjured heart. Skeletal myoblast differentiation and survival were decreased stepwise from control to hypoxia, starvation, and ischemia conditions. Although oxygen, glucose, and vascular density were significantly reduced in infarcted myocardium, the rate of macromolecular diffusion was significantly increased, suggesting that diffusive transport may not be inhibited in infarct tissue, and thus the supply of nutrients to transplanted cells may be possible. in vitro studies mimicking infarct conditions suggest that increasing nutrients available to transplanted cells may significantly increase their ability to survive in infarct.

Fig. 1

(a) Contrast-enhanced MRI confirming the presence of infarcted myocardium (arrow) at 3 weeks post cryoinjury. (b) Masson's trichrome stain of the 30 day old myocardial infarct site showing full transmural infarct. (c) Endogenous alkaline phosphatase staining, with eosin counterstain, showing endothelial cells within the uninjured myocardium, infarct border zone, and infarct center.

Fig. 2

(a) Masson's trichrome stain of myocardial infarct showing the location of microdialysis and oxygen probes (marked by reverse perfusion (microdialysis) or the injection (oxygen probe) of 0.1% Evans blue dye). (b) Oxygen concentration is significantly lower in the myocardial infarct scar than in skeletal muscle, uninjured heart, or border zone (*p < 0.05). (c) Glucose concentration in myocardial infarct scar was significantly lower than in the border zone, plasma, or uninjured heart (*p < 0.05). (d) Lactate concentrations measured in the skeletal muscle, uninjured heart, myocardial infarct scar, infarct border zone, and blood plasma varied widely in individual measurements within groups; there were no significant differences between groups. All bars are mean + SEM.

Fig. 3

The mean diffusion coefficients of 0.3 kD and 10 kD molecules were significantly increased in the infarct center and border zone when compared to uninjured myocardium (*p < 0.05, †p < 0.1). The mean calculated diffusion coefficients at 40 kD and 70 kD tended to increase in the infarct center and border zone, relative to uninjured heart, however, the results are not statistically significant. All bars are mean + SEM.

Fig. 4

(a) Micrographs showing varying degrees of differentiation of the C₂C₁₂ myoblasts under control, hypoxia, low glucose, or ischemia conditions. Myotubes are evident in both the control and hypoxia groups by day 6. (b) Survival of myoblasts over time under control, hypoxia, or low glucose was greater than under ischemia conditions. (c) Lactate dehydrogenase (LDH) levels released into the media by cells under experimental conditions (normalized to day 0 levels). The LDH levels of cells under ischemia conditions followed closely with the peak in cell death. The LDH levels in hypoxia and control conditions rose only when the cultures were primarily composed of myotubes. (d) A count of fully differentiated myoblasts under control, hypoxia, starvation, or ischemia conditions over a period of 14 days. Myoblasts were evident only after differentiation under control or ischemia conditions.