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Dynamics and mediators of acute graft attrition after myoblast transplantation to the heart

Harefield Heart Science Centre, National Heart and Lung Institute
^* Muscle Cell Biology Group, MRC Clinical Science Centre, Hammersmith Hospital, Faculty of Medicine, Imperial College London, UK

¹Correspondence: Cell and Gene Therapy Group, Harefield Heart Science Centre, Harefield, Middlesex, UB9 6JH, UK. E-mail: k.suzuki{at}ic.ac.uk

SPECIFIC AIMS

Survival of skeletal myoblasts within the cardiac environment is crucial for the therapeutic efficacy of myoblast transplantation in treating heart failure. Using an original method, we analyzed the early dynamics of myoblasts implanted into the myocardium and investigated the mechanisms underlying early graft attrition—in particular, the roles of oxidative stress and acute inflammation following myoblast implantation to the heart.

PRINCIPAL FINDINGS

1. Cell counting system to evaluate the number of myoblasts present in the myocardium
To assess early survival and proliferation of myoblasts, we have used an original cell identification system, where male skeletal myoblasts expressing nuclear ß-galactosidase (ß-gal) were radiolabeled with [¹⁴C]-thymidine and grafted into female mouse myocardium. The total (surviving plus proliferated) number of myoblasts derived from the initial graft was evaluated by measuring the amount of Y chromosome using PCR for the male-specific Smcy gene; the surviving proportion of the originally implanted myoblasts was estimated by ¹⁴C radiolabel measurement. The number of proliferated myoblasts could thus be calculated as a subtraction from the total myoblast number by the surviving number. The nuclear expression of ß-gal was useful in histological identification of grafted myoblasts from native cardiac cells.

To draw standard curves to estimate the myoblast number from the amount of Smcy gene and radiolabel present in the myocardium, intact female mice hearts were removed and ex vivo injected with known numbers (0–5.0 x 10⁵) of ¹⁴C-labeled male myoblasts derived from the H-2K^b-tsA58/nlacZ transgenic mice. Highly significant regressions were obtained between the number of grafted myoblast and the intensity of the corresponding bands for Smcy (R²=0.986, P<0.01), as well as between the myoblast number and the ¹⁴C radioactivity present in the myocardium (R²=0.990, P<0.01).

2. Myoblast survival and proliferation after implantation into the heart
A suspension of ¹⁴C-labeled male myoblast (5.0x10⁵) was injected into the left ventricular anterior wall of recipient female mouse hearts in vivo. The number of myoblasts present in the myocardium was calculated using the standard curves shown above. Approximately 60% of the myoblasts originally grafted was lost by 10 min, increasing to 80% by 24 h after implantation (Fig. 1 ). Subsequently, myoblasts showed a gradual but steady loss from the myocardium, giving a surviving myoblast number of only 7.4% at 72 h. Total myoblast numbers were similar at 10 min and 24 h, corresponding to the surviving myoblast number, and 3-fold greater than the surviving myoblast number at 72 h. This indicates that significant proliferation had occurred in surviving myoblasts between 24 and 72 h.

View larger version (22K): [in this window] [in a new window]   Figure 1. Myoblast survival and proliferation after implantation to the heart. After transplantation of 14C-labeled male myoblasts into female hearts, the surviving myoblast number was calculated by 14C measurement and the total myoblast number was evaluated by measuring Smcy. Myoblast number is shown as % of the total number originally grafted. Within the first 10 min of implantation, 60% of myoblasts disappeared and 80% were lost by 24 h. This was followed by gradual death until 72 h. In the latter phase, surviving myoblasts showed proliferation, indicated by the difference between the total and surviving myoblast number. Data shown as mean ± SE, n = 5 at each point.

3. Inflammatory response after myoblast transplantation
Myocardial inflammatory response after myoblast transplantation was evaluated by measuring myeloperoxidase (MPO) activity and by histological study. MPO activity 10 min after implantation was similar (6.1±0.6 IU/g) to that in intact control hearts (4.9±0.1 U/g), but significantly (P<0.05, n=4 at each point) elevated at 24 h (14.9±0.8 U/g) and 72 h (9.2±0.6 U/g). Histological study showed that numerous inflammatory cells, including polymorphonuclear leukocytes, had infiltrated the surrounding areas of grafted myoblasts at 24 h. This was not obvious at 10 min and comparatively reduced at 72 h, corresponding to the change in MPO activity. Multiplex RT-PCR showed strong expression of IL-1ß and TGF-ß, mild up-regulation of TNF- and IL-6, and slight up-regulation of GM-CSF in the myocardium 24 h after myoblast transplantation to the heart, while these were not observed in intact hearts or 10 min samples. The expressions at 24 h—specifically, those of IL-1ß and TGF-ß—apparently diminished by 72 h.

4. Improved survival and attenuated inflammation by SOD treatment
We hypothesized that free radicals derived from the cells (grafted myoblasts and/or native cardiac cells) that were damaged by direct mechanical or ischemia-reperfusion injury in the recipient myocardium could be a mediator of such rapid cell death, as well as a stimulus of subsequent inflammation, which would lead to further graft death. Superoxide dismutase (SOD) is known to provide strong cellular protection by scavenging hazardous superoxide. To investigate the role of free radicals on acute myoblast attrition, recombinant CuZn-SOD (600 U) was added to the myoblast suspension. Both surviving and total myoblast numbers at 10 min were significantly increased (Fig. 2 ) compared with those of untreated myoblasts (refer to Fig. 1 ). Improvement of survival was more marked at 24 and 72 h, when the surviving number was 2-fold greater than that of untreated myoblasts. The total myoblast number showed similar improvement: 2-fold greater than that of untreated myoblasts. MPO activity was significantly reduced (10.5±1.0 U/g at 24 h) in the SOD treatment group compared with untreated myoblast transplants.

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of SOD to improve myoblast survival. To study the role of oxidative stress in myoblast death, CuZn-SOD was administered in myoblast suspension. Surviving and total myoblast numbers were significantly increased by 10 min, with additional improvement at 24 and 72 h compared with untreated myoblasts (see Fig. 1 ). Myoblast number is shown as % of total myoblast number originally grafted. Data shown as mean ± SE, n = 5 at each point, *P< 0.05 vs. relevant time point samples of untreated myoblasts (see Fig. 1 ).

CONCLUSIONS AND SIGNIFICANCE

We have investigated the pattern and mediators of myoblast loss in the acute phase of direct intramyocardial implantation using a novel cell counting system. A large occurrence of death, which appears to be related to oxidative stress, occurred extremely rapidly (even by 10 min), whereas inflammation and associated cytokines were implicated in the subsequent phase of gradual death. This second phase, which occurs between 24 and 72 h, was accompanied by the onset of proliferation of the surviving cells. These data suggest that oxidative stress and inflammatory response with related cytokines may be important mechanisms contributing to graft attrition in the acute phase of implantation, both of which could act as therapeutic targets to improve the efficiency of cell transplantation to the heart.

Our results show that as many as 80% of grafted cells were rapidly lost by 24 h of implantation; notably, 60% disappeared within 10 min. After this initial rapid death, we observed a gradual but continuous loss of myoblasts (a further 8.6%) between 24 and 72 h, so that only 7.4% of the initially grafted myoblasts survived at 72 h. It may be possible that this rapid cell disappearance is contaminated by artifacts to some extent, including myoblast leakage into the extramyocardial space. We did not detect significant amounts of radioactivity in kidney, brain, spleen, or liver. Thus, one could judge myoblast leakage into the LV cavity to be negligible, as leaked myoblasts into this space should have been flushed out into the systemic circulation and entrapped in these organs. Great care and precautions were taken to prevent leakage of cell suspension, and all animals that had a suspicion of leakage to the outside by direct surveillance under a surgical microscope were excluded. However, we cannot exclude the possibility of leakage to the outside myocardium: there is a chance of invisible leakage, or myocardial contraction could cause a delayed release of injected myoblast suspension. Nevertheless, we could confirm that a considerable degree of myoblast death had in fact been induced even within such a short time, because SOD treatment has been shown to significantly attenuate the superacute myoblast disappearance.

It is important to clarify the mechanisms by which such a preponderance of early death of grafted myoblasts occurs. We have shown that CuZn-SOD treatment improves graft survival 10 min after transplantation, from 39.2% to 51.2%. This beneficial effect was even more striking at 24 and 72 h, when survival nearly doubled compared with untreated myoblasts. In addition these results were associated with attenuation of MPO activity at 24 h. We thus speculate that free radicals presumably derived from cells (either myoblasts or native cardiac cells) damaged by mechanical and/or ischemic injury in the recipient myocardium induced by intramuscular direct injection may cause damage to both types of cells. Such damaged cells are then likely to be a source of further free radicals, resulting in more myocardial damage and graft death. In turn, this damage to the myocardium and grafted myoblasts will induce inflammatory response within the myocardium, resulting in further graft death (Fig. 3 ). Factors such as direct mechanical damage during implantation or types of free radicals other than superoxide (in addition to the myoblast leakage discussed above) are likely to be involved in the rapid cell loss mechanism, as SOD treatment was only able to increase survival to 51.2%: 48.8% myoblasts were still lost. On the other hand, it has been suggested that a cytokine-related inflammatory response plays a role in the gradual loss of myoblasts in the latter phase. MPO activity was not significant at 10 min, remarkably elevated at 24 h, and decreased by 72 h. The extent of polymorphonuclear leukocyte infiltration in the grafted area showed the same trend. These changes in inflammatory response corresponded to proinflammatory cytokines such as TGF-ß, IL-1ß, IL-6, and TNF-. IL-1ß was strongly expressed in the myocardium 24 h after myoblast transplantation and the up-regulation was reduced by 60% by 72 h, a change concurrent with the degree of inflammatory response, suggesting that this proinflammatory cytokine plays an important role in myoblast death and associated inflammation in the acute period following myoblast transplantation to the heart.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram for speculated mechanisms of early graft loss. Intramyocardial implantation of myoblasts will cause mechanical damage and/or ischemic injury to cardiomyocytes and grafted myoblasts. These damaged cells will in turn produce free radicals, which result in further damage to cardiomyocytes and grafted myoblasts. These damaged cells can then be a source of more free radicals, resulting in further myocardial damage and graft death. This cellular damage will induce an inflammatory response and related cytokines within the myocardium, which will cause further graft death.

In conclusion, we have demonstrated that the majority of myoblasts grafted to the myocardium were lost in the early phase after implantation. A large portion of the death, which relates to free radicals, occurred rapidly, while inflammation and cytokines are implicated in the subsequent gradual death. Both oxidative stress and inflammatory response may be important mechanisms contributing to the acute graft attrition and thus could provide therapeutic targets to improve the efficiency of cell transplantation to the heart.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1308fje; doi: 10.1096/fj.03-1308fje

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