Three weeks after myocardial infarction with permanent LAD occlusion, a defect in myocardial perfusion was evident, indicating a successful model of myocardial infarction. The group treated with SAX had a significantly better myocardial recovery of perfusion compared to the placebo group, suggesting that this novel CGRP analogue improves myocardial recovery after myocardial infarction. Our study indicates that SAX decreases mortality using this model for myocardial infarction.
SAX is a vasodilatory CGRP analogue that acts on various arteries with a tenfold lower potency than native CGRP in all situations analyzed so far.14,16 In our infarct model, most of the myocardium normally supplied by the occluded LAD will obviously not benefit from a vasodilator. However, at the border of the territory, there will be some supply from “neighboring” non-occluded arteries and, although not addressed in this study, the vasodilatory effect in this non-occluded myocardial vasculature may be hypothesized as part of the mode-of-action of the observed perfusion recovery. Further, in addition to vasodilation, CGRP is known to induce endothelial proliferation and angiogenesis and, given that the SAX-induced recovery of myocardial perfusion is measured three weeks after occlusion and SAX treatment, angiogenesis could also be the mode-of-action.7
A concern regarding SAX and other systemic vasodilator treatments of myocardial ischemia is a coronary steal effect of the coronary arteries 20 in the presence of a flow that limits coronary stenosis. When treating with a vasodilating drug, the vessels in the non-ischemic area dilate and this could divert coronary flow from the ischemic area to the non-ischemic area. This potential effect could therefore reduce the perfusion to the ischemic area. However, since our infarct model was a permanent total occlusion, we would not expect to see any coronary steal.
The most common method of inducing myocardial infarction in animal models is with ligation of the LAD coronary artery. This method has been used since it was first described by Johns and Olson in 1954.21 However, the final infarct size has a large variation ranging from 4 to 59%. This is because of inconsistency in the ligation during surgery. In our study, the variation in infarct size was between 10% and 37%. This variation in infarct size could be because of the placement of ligature or number of ligatures. Srikanth et al. placed the ligature, 8 mm, from the origin of the LAD. This placement seems to give a minor variation in infarct size, but does not induce large infarction, with a reported infarct size variation from 17% to 25%.22 We placed the ligature more proximal, around 5 mm from the origin, to ensure larger infarct size. Despite the fact that our data show a large variation in infarct size, there was no difference in size of the perfusion defect size before first SAX/placebo treatment between the two groups.
This myocardial infarction model has a high early mortality rate. John et al. 21 showed that, in a chronic occlusion model, the surgical mortality was 21% and late death was 12%. In our study, the surgical mortality was 23%, which was expected. We had a late mortality of 28%, which is higher than that seen in other studies. A higher incidence of myocardial infarction could explain this higher mortality. In our study, 100% of the animals had an infarction, compared to only 83% of the animals in the study by John et al.21
SAX showed a significant improvement in perfusion recovery compared to placebo. In the group treated with SAX, there was a median reduction in SRS of 6 points (corresponding to a mean relative reduction of 55%), compared to only 2 points (corresponding to a mean relative reduction of only 11%) in the placebo group. Chronic occlusion of the coronary artery is used in our study; therefore, recovery is not as great as that seen in studies in which a reperfusion infarct model was used. Pfeffer et al. described the development in infarct size in rats with chronic occlusion. The infarct size was 41% after 1 day and 39% after day 106.23 The myocardial infarction was verified using histology and therefore not comparable to our study. However, it shows that the infarct size does not reduce significantly for 106 days when there is no intervention. Therefore, our reduction in final infarct size shows a promising cardioprotective aspect of SAX.
In the placebo group, a majority of rats had negative or no recovery, while none in the SAX-treated group had a negative recovery. An explanation for this could be the physiological effects of SAX regarding the dilation of the epicardial coronary arteries.4
A strength of the model used for determining recovery is the radiopharmaceutical properties of [99mTc]Tc-sestamibi. [99mTc]Tc-sestamibi enters the myocytes easily and accumulates in the mitochondria, because it cannot metabolize further, and redistribution is minimal.24 The injection of [99mTc]Tc-sestamibi in our study was done 20 min before administering SAX or placebo; therefore, the effects of the pharmacological treatment do not affect distribution of the radiotracer. The acute distribution of [99mTc]Tc-sestamibi shows the myocardial area at risk of infarction, and the follow-up SPECT/CT three weeks after intervention shows the final infarct.
In Vivo Quantification Using SPECT
The use of [99mTc]Tc-sestamibi in rats is well established. The half-life, the extraction in the myocardium, and no large positron range are advantages when examining small rodents.18 Conversely, [99mTc]Tc-sestamibi is not the optimal choice in humans when examining myocardial blood flow, because of the long half-life and gamma decay. 82Rubidium is a short-lived positron emission tomography (PET) tracer, used to detect perfusion defects and assess myocardial blood flow. However, the positron range is large and therefore it is hard to discriminate between infarcted and healthy myocardium in small animals. A novel PET tracer, [18F]-flurpiridaz, is promising in assessing perfusion defects and myocardial blood flow.25 The positron range is low, which enables its use in small rodents, with great contrast between infarcted and healthy myocardium. However, so far [18F]-flurpiridaz is not commercially available and was thus not usable in our study.
There was no significant difference in overall survival between the two groups, although there was a tendency toward a higher survival rate in the SAX-treated group. This tendency suggests that SAX does not have a damaging effect on the animals or the myocardium.
In the rats that died before SPECT/CT scan, there was no difference between the two groups regarding treatment with SAX or placebo, or the length of survival. Nilsson et al. have shown that the effect of SAX is most pronounced three hours after treatment, which means that the cardioprotective effect of SAX has not yet been established in rats that had died before the SPECT/CT scan.14 This corresponds to our finding that time to death was similar in the acute phase (first hour after infarction).
In the rats that died in the subacute phase (after a potential effect of SAX), we observed that the SAX group survived significantly longer than the placebo group, which supports the data suggesting a cardioprotective effect of SAX.
In the present study, SAX was the only drug tested and was not compared to other CGRP analogues or other types of vasodilating drugs. There was a significant difference in age and weight between the two groups. The mean difference in age was six days, but we found no indication that this difference affected acute SRS or recovery.
In this study, we did not confirm the infarct size measured with SPECT to either histology or triphenyltetrazolium chloride (TTC) staining. However, the correlation between infarct size measured by [99mTc]Tc-sestamibi and TTC-staining, in a model with permanent ligation of the LAD, has been showed in Sprague-Dawley rats.26
In this study, a chronic occlusion of the coronary artery was made to evaluate the potential cardioprotective effects of SAX with no other kind of intervention. A more clinically relevant model would be a reperfusion model, where the coronary artery is occluded for a limited amount of time, mimicking patients undergoing PCI. However, the reperfusion model shows only small perfusion defect size and the potential myocardial improvement by SAX would be harder to determine.