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Functional and histopathological improvement of the post-infarcted rat heart upon myoblast cell grafting and relaxin therapy

Although the myocardium contains progenitor cells potentially capable of regenerating tissue upon lethal ischaemic injury, their actual role in post-infarction heart healing is negligible. Therefore, transplantation of extra-cardiac stem cells is a promising therapeutic approach for post-infarction heart dysfunction. Paracrine cardiotropic factors released by the grafted cells, such as the cardiotropic hormone relaxin (RLX), may beneficially influence remodelling of recipient hearts. The current study was designed to address whether grafting of mouse C2C12 myoblasts, genetically engineered to express green fluorescent protein (C2C12/GFP) or GFP and RLX (C2C12/RLX), are capable of improving long-term heart remodelling in a rat model of surgically induced chronic myocardial infarction. One month after myocardial infarction, rats were treated with either culture medium (controls), or C2C12/GFP cells, or C2C12/RLX cells plus exogenous RLX, or exogenous RLX alone. The therapeutic effects were monitored for 2 further months. Cell transplantation and exogenous RLX improved the main echocardiographic parameters of cardiac function, increased myocardial viability (assessed by positron emission tomography), decreased cardiac sclerosis and myocardial cell apoptosis and increased microvascular density in the post-infarction scar tissue. These effects were maximal upon treatment with C2C12/RLX plus exogenous RLX. These functional and histopathological findings provide further experimental evidence that myoblast cell grafting can improve myocardial performance and survival during post-infarction heart remodelling and dysfunction. Further, this study provides a proof-of-principle to the novel concept that genetically engineered grafted cells can be effectively employed as cell-based vehicles for the local delivery of therapeutic cardiotropic substances, such as RLX, capable of improving adverse heart remodelling.

myocardium and improving the clinical symptoms of heart failure [5]. Therefore, the attention of many researchers has been long-focused on extra-cardiac stem cells with the ability, at least in principle, to differentiate into the cardiac muscle phenotype [6]. In this context, stem cell grafting for cardiac repair is viewed as the new frontier in the treatment of post-infarction heart failure [7][8][9]. Skeletal myoblasts, for example, have been considered promising candidates [10], mainly because they are resistant to ischaemia and hence may survive within the poorly vascularized myocardial scar. Further, these cells can be isolated and expanded ex vivo from skeletal muscle biopsies of the same patients scheduled for cell grafting therapy, with obvious immunological and ethical advantages [6,10]. Although engrafted skeletal myoblasts do not exhibit the potential to differentiate into myocardial-like cells [11], numerous studies have demonstrated a significant improvement of the heart's contractile performance upon myoblast transplantation [12,13]. A possible explanation for this ostensible paradox has come from the results of recent experimental studies, suggesting that the grafted cells may release paracrine cardiotropic factors capable of inducing collagen remodelling and neoangiogenesis in the recipient hearts [14,15].
Based on the above-reported observations, it appears that further investigations are needed to shed light on the mechanisms underlying the functional advantages of myoblast grafting in the post-ischaemic heart and to better define the possible therapeutic protocols. A novel approach may be the combination of myoblast cell grafting and cellular therapy, whereby cells can act as carriers for genes encoding cardiotropic substances that can be delivered locally [10]. Indeed, a recent study from our group showed that the cardiotropic hormone relaxin (RLX) is capable of improving the contractile performance of postinfarcted swine hearts by positively influencing cardiac remodelling and neo-angiogenesis [15]. RLX is a member of a peptide hormone family, which includes three distinct RLX molecules (denoted H1, H2 and H3, respectively), as well as insulin-like peptides (INSL) 3 to 6 [16,17]. In human beings, three separate RLX genes have been found and designated RLN1, RLN2 and RLN3 [16,17]. , encoded by the RLN2 gene, is the major circulating form and accounts for most of the known biological effects of RLX in human beings and experimental animals. RLX acts on both reproductive and non-reproductive tissues [17][18][19][20][21][22] and exerts major effects on the cardiovascular system [17,21,22]. Some of these effects, namely increased heart perfusion [23], neo-angiogenesis [24] and reduced cardiac fibrosis by increased extracellular matrix (ECM) turnover [25], are potentially relevant to cell grafting therapy. In fact, it may be speculated that H2 RLX could be employed, both as a drug and as a functional transgene, for cellular-or gene-based therapy of postinfarction cardiac disease. Therefore, we designed the current study to determine whether skeletal myoblasts, wild-type or engineered to expressed RLX, and exogenously administered RLX are capable of improving the longterm sequelae in a rat model of chronic myocardial infarction and to investigate the possible mechanisms involved.

Surgery and experimental protocols
The experimental protocol complied with the Declaration of Helsinki and the recommendations of the European Economic Community (86/609/CEE) on animal experimentation. Wistar male rats, weighing 250-300 g, under general anaesthesia and artificially ventilated, underwent ligation of the left anterior descending coronary artery after the first diagonal branch to induce myocardial infarction, as previously described [27]. The animals were then placed in cages under controlled temperature and high pO2 to facilitate post-operative recovery until achievement of a normal respiratory activity and cardiac beating rate (350-450 beats/min.). Rats were housed under standard conditions until the next experimental step.
Thirty days after surgery, the animals were divided in four experimental groups.
To prevent reflow, in all animals, the vena cardiaca magna was tied to the infusion catheter for 10 min. In the previous studies used as reference for the design of the present experimental protocol, a 5-min. occlusion of the catheterized vena cardiaca magna allowed the settlement of about 30% of total grafted myoblasts within the heart, evaluated as soon as 10 min. after delivery [27,28].
Following recovery, rats were housed under standard conditions for further 60 days. The animals of all groups underwent daily oral administration of cyclosporine (15 mg/kg b.wt.) to obtain immune suppression and enhance the success of C2C12 cell xenografting. Rat mortality upon the first intervention for induction of myocardial infarction was 20-30% and mortality upon re-operation ranged between 40% and 50%. The experiment was terminated when each group comprised at least five animals.
Circulating levels of RLX in groups 2 and 4 were measured 1 month (T2) and 2 months (T3) after osmotic pump implantation using a commercial human H2 RLX ELISA kit (Immundiagnostik, Bensheim, Germany). In the former case, animals were anaesthesized by isoflurane inhalation and 0.5 ml blood samples were collected by tail vein puncture and centrifuged at 500 g for 15 min. at 20ЊC to separate plasma. In the latter case, blood samples were taken from the superior vena cava before heart removal for tissue sampling.

Echocardiographic assessment of cardiac function
The animals of the four groups were anaesthesized and subjected to transthoracic bi-dimensional M-mode echocardiography using a Sequoia 512 device (Siemens Acuson, Erlangen, Germany) equipped with a 15-MHz probe to evaluate overall and segmental left ventricular dynamics and hence heart contractile performance [29,30]. The following parameters were measured or calculated: (i) left ventricular end-diastolic diameter, LVEDD; (ii) left ventricular end-systolic diameter, LVESD; (iii) fractional shortening, FS; (iv) left ventricular ejection fraction, LVEF; (v) myocardial performance index, MPI, the sum of isovolumetric contraction and relaxation times/ejection time. This assay was carried out prior (T0) and 1 month after (T1) the induction of myocardial infarction, as well as 1 (T2) and 2 (T3) months after DMEM, RLX or cell delivery.

Micro-PET analysis of myocardial viability and performance
Positron emission tomography (PET) scans on the rats of the different groups were performed after administration of the viability tracer 18 fluorodeoxyglucose ( 18 F-FDG), as previously described [31,32]. 18

Determination of microvessel density
Microvascular density (MVD) in the cardiac scar tissue was measured by point counting on 2Ϫm-thick semi-thin sections of resin-embedded samples, as described [15].

In all the animals, surgical induction of myocardial infarction caused a marked decay of the assayed echocardiographic parameters of cardiac function. Namely, 1 month after surgery (T1), LVEF and FS were significantly reduced, whereas LVEDD, LVESD and MPI were significantly increased in comparison with the preoperative (T0) values (Fig. 1). In the untreated controls (group 1), these parameters remained substantially unchanged in the successive measurements carried out 1 month (T2) and 2 months (T3) after re-operation, at variance with significant improvements in the rats subjected to the therapeutic treatments. In particular, (i) LVEF was significantly increased in the rats which received C2C12/GFP (group 3) and even more in those which received C2C12/RLXϩRLX (group 4); (ii) FS was significantly increased in the rats given both RLX alone (group 2) and C2C12/RLXϩRLX (group 4) at T2, and in all the treated animal groups at T3: at both time-points, the best performance was achieved by the rats of group 4; (iii) LVEDD and LVESD were significantly reduced in all the treated animal groups at T2 and even more at T3; LVEDD was similarly improved in all the three groups, whereas LVESD was particularly reduced in the animals receiving C2C12 engraftment;
(iv) MPI were significantly and similarly reduced in all the treated animal groups at T2 and even more at T3.

Micro-PET analysis
Quantitative tomographic analysis of cardiac 18 F-FDG uptake showed that, 1 month after surgical induction of myocardial infarction (T1), the rats of all the experimental groups had a marked reduction of the overall volume of viable myocardium (Fig. 2)

Localization of the grafted cells in the host hearts
Relatively few immunoreactive eGFP-expressing C2C12 were found in the scar tissue from groups 3 and 4 ( Fig. 3A and B)

Histological features of post-infarcted hearts
By modified Azan staining (Fig. 4, upper panels), the post-infarction cardiac scar from the untreated control animals showed densely packed bundles of collagen fibres. Conversely, samples from rats subjected to the therapeutic treatments (groups 2 to 4) exhibited a reduction in the amount of collagen fibres, confirmed by morphometric analysis of tissue collagen content expressed as sclerosis index (Fig. 4, centre panel). By TEM (Fig. 4, lower   appeared as activated cells: their cytoplasms contained abundant RER and large Golgi complexes and their plasma membranes were characterized by sites of apposition to thin electron-dense filaments, featuring collagen microfibrils undergoing pericellular processing and assembly. Moreover, large collagen bundles were present in the ECM. In rats receiving C2C12/RLXϩRLX, as well as in those treated with H2 RLX alone, fibroblasts featured predominantly quiescent cells, with no evidence of pericellular collagen microfibril assembly, and were immersed in a loose, electronlucent ECM with few, thin collagen fibrils. In contrast, the samples from rats receiving C2C12/GFP showed ultrastructural features not substantially different from those of the untreated controls. To explore the effects of RLX on scar tissue vascularization and angiogenesis, microvascular density (MVD) was measured. Sections of cardiac tissue harvested from rats of groups 2, 3 and 4 exhibited a significant increase in MVD compared to the untreated controls of group 1. No significant differences were observed between the therapeutic treatments (Fig. 5).

Determination of apoptotic and proliferating cardiac cells
To provide further insight into the mechanisms of the observed improvement of the post-infarcted hearts subsequent to therapeutic treatments, we examined and quantified the amount of apoptotic and cycling cells in the myocardium surrounding the scar. By TUNEL assay (Fig. 6), the untreated controls showed a relatively large amount of apoptotic cardiomyocytes. Apoptotic cells were significantly reduced in the hearts from rats subjected to the therapeutic treatments, especially those of groups 2 and 4. By Ki-67 immunocytochemistry, very few positive cycling cells were encountered: they were mostly isolated cells of the scar tissue or sparse endothelial cells of microvessels in the scar or the border-line myocardium (data not shown). No differences were observed between the different experimental groups.

Plasma RLX levels
One month after the therapeutic treatments (T2), in the rats treated with C2C12/RLXϩRLX and H2 RLX alone, plasma RLX levels were 82 Ϯ 37 and 75 Ϯ 29 ng/ml, respectively. Two months after the treatments (T3), RLX plasma concentration fell below the sensitivity of the assay. As expected, RLX levels were undetectable in all samples from untransplanted controls and C2C12/GFP-treated animals.

Discussion
Using a rat model of chronic myocardial infarction suitable for long-term investigation of heart remodelling, we performed a detailed functional analysis by combining echocardiographic assessment of cardiac performance and PET-based metabolic imaging of myocardial viability. This approach allowed us to achieve a careful time-course evaluation of cardiac failure and of the benefits afforded by the different therapeutic treatments. Taken together, the results of the functional and histopathological analyses provide further experimental evidence that stem cell grafting can improve myocardial performance and survival during postinfarction heart remodelling and offer background to the novel concept that genetically manipulated grafted cells can effectively be used for local delivery of therapeutic cardiotropic molecules [10,33,34]. The current findings also indicate that RLX can be an effective agent in protecting the post-infarcted heart from adverse remodelling, in keeping with the emerging concept that this hormone, due to its collagen-remodelling, pro-angiogenic and vasodilatatory properties [23][24][25], can play a major cardioprotective role [17,21,22,34].
This study confirms and extends our previous findings obtained by transplantation of RLX-producing C2C12 cells in postinfarcted swine, examined 1 month after cell injection [15]. The current rat experimental model provides similar evidence that improved ECM turnover and neo-angiogenesis are a prominent feature of the functional benefits afforded by C2C12 cells and RLX, even upon long-term cardiac remodelling (  scar [17], thereby improving diastolic compliance and ventricular contractile dynamics. When exogenous RLX was combined with grafting of C2C12/RLX cells, we observed a further beneficial effect, consistent with a statistically significant increase in the overall volume of viable myocardium. This effect appears to depend, at least in part, to the protection against myocardial cell apoptosis. Interestingly, this fits well with the recent observation that RLX antagonizes apoptosis in cultured rat cardiomyocytes [35]. Considering the effects of exogenously given RLX observed in animal models of acute myocardial infarction [36,37], we suggest that local secretion of RLX by C2C12/RLX cells could exert paracrine effects on cardiac host tissue, favouring tissue perfusion and reducing local inflammation and sclerosis. By these mechanisms, RLX may promote survival and functional recovery of the stunned, peri-necrotic myocardium. It is even possible that, besides RLX, C2C12 cells could locally release other cardiotropic substances, as suggested by the observation that transplantation of C2C12/GFP also increases myocardial viability and cardiac functional performance. In fact, stem cells have been reported to release pro-survival and pro-angiogenic molecules when engrafted in the post-ischaemic heart [38,39]. This is consistent with the recent 'paracrine' hypothesis to explain the functional benefits of stem cell therapy in the post-infarcted, dysfunctional heart [14,15,33,34,40].
The current findings also indicate that the retrograde coronary venous route for cell delivery is safe, as the mortality upon re-operation was similar in the treated and untreated animals, and adequate for the injected C2C12 cells to engraft into the host myocardium, where they could survive for at least 2 months after delivery. The relatively higher amounts of C2C12/RLX cells found at termination in the post-infarction scar as compared with C2C12/GFP cells suggest that RLX may promote cell proliferation or, most likely, may favour cell engraftment and/or survival by re-modelling and improving the hostile milieu of the scar tissue [34]. Interestingly, C2C12 cells were localized mainly in the perivascular space, where they retain their original features of poorly differentiated precursors with no morphological sign of muscle-like differentiation. The inability of the C2C12 myoblasts to execute a myogenic differentiation programme in the cardiac scar has been previously highlighted in a similar study in the swine in vivo [15] and can be conceivably explained by the lack of appropriate micro-environmental signals in a non-physiological host tissue [41]. This finding offers additional support to the concept that transplanted myoblasts can improve the function of post-infarcted heart by indirect mechanisms, independent of de novo regeneration of contractile tissue [7,8,14,34], and that RLX may be a candidate therapeutic to enhance efficacy of recovery.
From a practical point of view, the present study further underscores the therapeutic value of stem cell transplantation in postinfarction cardiac dysfunction, especially when the grafted cells are used as vehicle for the local release of cardiotropic substances capable of improving adverse heart remodelling [42]. The current findings also point to the option of using delayed rather than immediate cell therapy following acute myocardial infarction to achieve better efficacy, in keeping with previous reports [43].