G-CSF/SCF reduces inducible arrhythmias in the infarcted heart potentially via increased connexin43 expression and arteriogenesis

Granulocyte colony-stimulating factor (G-CSF), alone or in combination with stem cell factor (SCF), can improve hemodynamic cardiac function after myocardial infarction. Apart from impairing the pump function, myocardial infarction causes an enhanced vulnerability to ventricular arrhythmias. Therefore, we investigated the electrophysiological effects of G-CSF/SCF and the underlying cellular events in a murine infarction model. G-CSF/SCF improved cardiac output after myocardial infarction. Although G-CSF/SCF led to a twofold increased, potentially proarrhythmic homing of bone marrow (BM)-derived cells to the area of infarction, <1% of these cells adopted a cardial phenotype. Inducibility of ventricular tachycardias during programmed stimulation was reduced 5 wk after G-CSF/SCF treatment. G-CSF/SCF increased cardiomyocyte diameter, arteriogenesis, and expression of connexin43 in the border zone of the infarction. An enhanced expression of the G-CSF receptor demonstrated in cardiomyocytes and other cell types of the infarcted myocardium indicates a sensitization of the heart to direct influences of this cytokine. In addition to paracrine effects potentially caused by the increased homing of BM-derived cells, these might contribute to the therapeutic effects of G-CSF.

Myocardial infarction is responsible for about one third of heart failure cases (1)(2)(3). It is defi ned as the loss of myocardial tissue that is replaced by scar tissue (4,5). The formation of scar tissue is not only one of the most important factors compromising the pump function of the failing heart, but it is also an important risk factor for ventricular arrhythmias because it constitutes anatomical conduction barriers that cause electrical reentrant circuits. Ventricular tachyarrhythmias are the main cause for sudden death in heart failure patients (4)(5)(6). Therefore, new approaches for the therapy of heart failure should not only improve hemodynamic function, but they should also aim at a reduction of the vulnerability to arrhythmias. Initial preclinical and clinical trials have demonstrated that the transfer of BM-derived stem and precursor cells into the infarcted myocardium can improve left ventricular systolic function (7)(8)(9). Similar results were obtained by the application of the cytokine granulocyte colony-stimulating factor (G-CSF), alone or in combination with stem cell factor (SCF), which is known to mobilize BM-derived cells (10)(11)(12)(13)(14). The publications of Orlic et al. (10,15,16) suggest that the benefi cial eff ects of these therapies are due to a signifi cant degree of structural regeneration of the infarcted hearts by transdiff erentiation of the immigrated BM-derived cells to cardiomyocytes. In contrast, others (17)(18)(19) were unable to reproduce these results. However, recent data by Gnecchi et al. (20) indicate that paracrine (humoral) factors secreted by BM-derived cells promote cardiomyocyte survival. Recently, a new aspect was added by the work of Harada et al. (14), who identifi ed direct eff ects of G-CSF on car diomyocytes in vitro.
However, the mere increase of BM-derived cells in the infarcted heart by cytokine treatment as well as their potential transdiff erentiation into noncardiomyocytes or even cardiomyocytes might carry a risk for pro-arrhythmia. The transfer of skeletal myoblasts into failing hearts has been shown to provoke ventricular tachycardias in patients (21), and certain types of cardiomyocytes derived from in vitrodiff erentiated embryonic stem cells exhibited prolonged action potential durations, afterdepolarizations, and a potential for arrhythmogenesis (22). On the other hand, reducing the infarct size has the potential to reduce the risk of ventricular arrhythmias after myocardial infarction. Therefore, the aim of this study was to systematically evaluate the midterm electrophysiological eff ects of G-CSF/SCF treatment on hearts subjected to myocardial infarction, and to reveal cellular changes potentially underlying the observed physiological alterations.

G-CSF/SCF promotes the homing of enhanced green fl uorescent protein (EGFP) + cells in the infarcted left ventricle of mice
The homing of BM-derived cells to the infarcted myocardium on the one hand might contribute to the benefi cial effects of G-CSF/SCF by paracrine mechanisms. On the other hand, it might constitute the cellular basis for an enhanced susceptibility to arrhythmia. Because of these notions, the homing of BM-derived cells was comparatively analyzed in G-CSF/SCF-treated and untreated infarcted hearts.
EGFP-transgenic donor mice were used for BM transplantation (BMT) into irradiated recipient animals before these were subjected to infarction and G-CSF/SCF treatment. As expected, ligation of the left anterior descending coronary artery (LAD) caused anterior myocardial infarction as evident by replacement of myocardium with fi brotic scar tissue 5 wk after the infarction (Fig. 1 A). Morphometric analysis of the hearts of 15 BMT animals (4 G-CSF/SCF treated and 11 untreated) killed 5 wk after induction of myocardial infarction identifi ed EGFP + (BM-derived) cells predominantly in the area of myocardial infarction and its border zone (Fig. 1 B). Furthermore, G-CSF/SCF led to a twofold increase of homing of EGFP + cells to the infarcted area and the border zone of myocardial infarction (P < 0.05 for G-CSF/SCF vs. controls). In agreement with this fi nding, G-CSF/SCF led to a threefold rise in white blood cell concentration in the peripheral blood, accompanied by a doubling of the neutrophil proportion in splenectomized mice (not depicted). In 10 BMT animals not subjected to infarction (fi ve G-CSF/SCF treated and fi ve untreated), the accumulation of EGFP + cells 5 wk after G-CSF/SCF treatment was 4-6-fold lower than in the non-infarcted myocardial tissue of animals with myocardial infarction (Fig. 1 B).
98-99% of EGFP + cells in the myocardium were white blood cells as evident by coimmunolocalization of CD45 is characterized by thinning of the left ventricular wall and replacement of myocardium with fi brotic tissue (green) and infl ammatory cells. A subendocardial layer of myocardium survived after myocardial infarction. LV, left ventricle; RV, right ventricle; S, septum. (B) Quantitative analysis of EGFP + cells in the infarcted and non-infarcted regions as well as in the border zone of G-CSF/SCF-treated and untreated hearts 5 wk after induction of myocardial infarction. Also, percentages of EGFP + cells in the hearts of G-CSF/ SCF-treated and untreated EGFP BM chimeras not subjected to infarction are indicated. Asterisks above bars indicate statistically signifi cant differences of percentages of EGFP + cells between the border zone or area of infarction and non-infarcted areas of the same animals for controls or G-CSF/SCF mice (P < 0.05). Asterisks above parentheses indicate significant differences between the border zone or area of infarction of controls and G-CSF/SCF group. (C) Colocalization (right) of EGFP fl uorescence (left) and CD45 immunofl uorescence (middle) indicates that the majority of BM-derived cells are white blood cells. (D) Green fl uorescent cardiomyocytes (*) in the scar tissue of a G-CSF/SCF animal (D1) and the noninfarcted area of a non-G-CSF/SCF animal (D2) 5 wk after induction of myocardial infarction in recipients of EGFP-expressing BM. Intact cardiomyocytes are labeled by immunofl uorescence for troponin T using a red fl uorochrome (Alexa Fluor 594). The green color represents the autofl uorescence of EGFP + (BM-derived) cells. ARTICLE ( Fig. 1 C), regardless of location (infarcted vs. non-infarcted area), and protocol type (G-CSF/SCF vs. control). In >100 sections from the hearts of G-CSF/SCF and control animals, only one troponin T + /EGFP + cell with central nucleus, myofi laments, and cross-striation ( Fig. 1 D) was identifi ed in an area of infarction. In the non-infarcted areas, troponin T + /EGFP + cells were casually found (up to fi ve cells per cross section).

G-CSF receptor (G-CSFR) is expressed in several cell types of the heart and is up-regulated by infarction
Because G-CSF and SCF were administered systemically, expression of the corresponding receptors was analyzed in the heart to fi nd evidence for potential direct eff ects on myocardial tissue. G-CSFR expression was evaluated by immunofl uorescence microscopy 1 d and 5 wk after induction of myocardial infarction as well as by quantitative real-time RT-PCR at six time points after infarction. 1 d after myocardial infarction, prominent G-CSFR expression in the non-infarcted area was predominantly detected in small interstitial and vascular cells (Fig. 2 A, 1), whereas in the infarcted area, cardiomyocytes also exhibited weak G-CSFR immunofl uorescence (Fig. 2 A, 2). In contrast, 5 wk after induction of myocardial infarction, cardiomyocytes exhibited marked G-CSFR expression both in the non-infarcted area and in the border zone ( Fig. 2 A, 3 and 4). Cardiomyocytes, immunopositive for G-CSFR, display a bright red corona, indicating membrane localization of the receptor. Animals investigated 1 d after myocardial infarction were chimeras with EGFP-expressing BM (see Materials and methods). Because the small interstitial cells, immunopositive for G-CSFR, were not EGFP + , these cells were not of hematopoietic origin but were resident myocardial cells. In contrast to G-CSFR, the receptor for SCF (c-kit) was not detectable by immunofl uorescence 5 wk after myocardial infarction (not depicted).
Real-time RT-PCR analysis of myocardial tissue of untreated animals revealed an up-regulation of G-CSF and G-CSFR mRNA levels within the fi rst 24 h after induction of myocardial infarction, which was more pronounced in the aff ected tissue than in the non-infarcted area (Fig. 2 B).

G-CSF/SCF improves hemodynamic function
Myocardial infarction induced by LAD ligation led to regional wall motion abnormalities of the anterior wall, reduced fractional shortening, and typical electrocardiogram (ECG) changes seen after anterior myocardial infarction in the mouse (Fig. 3, A and B). Infarction size was relatively homogeneous throughout the experiments and did not differ between G-CSF/SCF hearts and controls, neither in pathological measurements (Table I) nor in vivo, assessed by the akinetic area of the left ventricle on echocardiography (G-CSF/SCF treated 5.4 ± 0.8 mm 2 vs. controls 6.7 ± 1.1 mm 2 ; P = 0.39). Also, left ventricular contractility did not diff er between G-CSF/SCF-treated mice and controls.
It is worthy to note that fractional shortening showed a trend toward better function, and septal wall width bordering the area of myocardial infarction showed a trend toward greater thickness in G-CSF/SCF mice. Despite the lack in infarct size reduction, cardiac output was significantly increased in G-CSF/SCF mice compared with controls ( Fig. 3 C).

G-CSF/SCF does not provoke afterdepolarizations and spontaneous ventricular tachycardias in the intact heart
Based on reports of proarrhythmic eff ects provoked by the transplantation of skeletal myoblasts (21), the proarrhythmic risk of the increased homing of BM-derived cells was assessed by observing spontaneous ventricular rhythm after atrioventricular nodal block (23)(24)(25). This protocol did not provoke more spontaneous arrhythmias in G-CSF/SCFtreated hearts compared with controls (7 out of 25 G-CSF/ SCF-treated hearts with spontaneous arrhythmias vs. 7 out of 26 controls; P = NS). Furthermore, ventricular action potential durations were shorter in G-CSF/SCF mouse hearts when pacing rates were high, whereas action potential   (Table II).

G-CSF/SCF reduces inducibility of ventricular arrhythmias and increases connexin43 expression
Ventricular arrhythmias after myocardial infarction are often a result of conduction slowing in the border zone of the infarcted myocardium and associated with decreased intercellular connections in this region (26). Programmed stimulation, the best assay to provoke such reentrant arrhythmias, provoked less ventricular tachycardias in G-CSF/SCF-treated mice when either a single premature extra stimulus (Fig. 4 A, top) or two consecutive premature stimuli (Fig. 4 A, bottom) were applied (Fig. 4 B). As expected, a reduction of con-nexin43 expression (90% reduction) was found in the border zone of the infarction (Fig. 4 C and Videos S1-S4, available at http://www.jem.org/cgi/content/full/jem.20051151/DC1), consistent with conduction slowing, the hallmark of inducible ventricular arrhythmias after myocardial infarction. G-CSF/SCF-treated hearts, in contrast, had markedly higher connexin43 levels in the border zone of the infarction when compared with untreated controls (25% of normal con-nexin43 levels; Fig. 4 C and Videos S1-S4).

G-CSF/SCF increases arteriogenesis and cardiomyocyte hypertrophy in the border zone of infarction
G-CSF/SCF enhanced arterialization in the border zone of myocardial infarction as indicated by an increased number of vessels per fi eld of view (Fig. 5 A). However, concordant with increased arteriogenesis, the mean vessel diameter was not aff ected. Cardiomyocyte hypertrophy was more pronounced in the border zone of the infarction after G-CSF/ SCF treatment (Fig. 5 B).

DISCUSSION
Main fi ndings G-CSF/SCF treatment increased the homing of BM-derived cells (mainly white blood cells) into the infarcted myocardium twofold. In addition, up-regulation of G-CSFR expression in the infarcted myocardium suggests a sensitization for direct eff ects of the cytokine. G-CSF/SCF increased cardiac output regardless of infarction size and integration of BM-derived cells into the myocardium. G-CSF/SCF reduced the inducibility of ventricular arrhythmias. This antiarrhythmic eff ect can be attributed to an increased expression of connexin43 in cardiomyocytes in the border zone of the infarction. Although provocation of afterdepolarizations was a specifi c aim of this study, there was no tendency for spontaneous arrhythmias or afterdepolarizations after G-CSF/ SCF treatment. The observed physiological eff ects of cytokine treatment were associated with cardiomyocyte hypertrophy and an enhanced arterialization in the border zone of the infarction.  19), because a signifi cant degree of transdiff erentiation was not observed, this allows us to speculate that the observed therapeutic eff ects may be due to the secretion of stimulatory factors by white blood cells. However, the debate as to how G-CSF/SCF-based cardiac regenerative therapies improve cardiac function (10,(15)(16), and whether transdiff erentiation of BM-derived cells is the underlying mechanism of myocardial regeneration (27,28), has recently been extended by Harada et al. (14). Their results indicate a signifi cant survival-promoting eff ect of G-CSF treatment on cardiomyocytes and endothelial cells that prevented left ventricular remodeling after myocardial infarction, suggesting that G-CSF may directly act on cardiomyocytes and endothelial cells. However, in contrast to Orlic et al. (10) and our work, they did not use splenectomized mice for their experiments. Diff erences in neutrophil accumulation in the blood between splenectomized and nonsplenectomized mice might explain why they were unable to identify diff erences between the homing of BM-derived cells in G-CSF-treated and control hearts. Although the threefold rise in white blood cell count in peripheral blood measured in our G-CSF/SCF-treated  animals is in the range observed in nonsplenectomized animals (29), the boost in the proportion of neutrophils is less pronounced in nonsplenectomized animals (not depicted). Nevertheless, Adachi et al. (30), who also did not use splenectomized animals for their experiments, reported an increased infi ltration of BM-derived side population cells into the G-SCF-treated infarcted heart. Together with recent data (20) about the secretion by BM-derived mesenchymal stem cells of so far unknown paracrine factor(s), capable of reducing the infarct size in a rat model of permanent coronary artery occlusion, their results demonstrate that the question of the relative contribution of direct and paracrine eff ects to the therapeutic action of G-CSF is not definitely answered.

ARTICLE
Our results regarding G-CSFR expression are in agreement with Harada et al. (14) as well as Schneider et al. (31) and suggest that the infarcted myocardium is sensitized for direct eff ects of G-CSF by an increased expression of the G-CSFR. Together with the parallel increase of local G-CSF expression after myocardial infarction (Fig. 2 B), these data suggest the existence of an endogenous mechanism of G-CSF signaling activation potentially mediating cardioprotective eff ects. Similar results were obtained in a model of brain infarction (31). However, in view of the twofold-increased homing of BM-derived cells, they also do not allow to favor one of the explanations (paracrine vs. direct eff ects) but leave the alternative open that both mechanisms act in parallel. Nevertheless, the recent observation of a rise of G-CSF concentration in the blood accompanied by an increase of pe-ripheral CD34 + cells (endothelial progenitor cells) after acute myocardial infarction in humans (32) allows us to speculate that myocardial damage by itself leads to the activation of an endogenous BM mobilization mechanism, possibly enhancing the recruitment of BM-derived cells from the blood.

Improvement of cardiac output
Concordant with results from some (33), but not all (14), groups, we found no change in infarct size after G-CSF/SCF treatment. In addition, we did not fi nd evidence for the generation of new myocardium after G-CSF/SCF treatment. Cardiomyocyte hypertrophy in the border zone of the infarction was, however, increased in G-CSF/SCF-treated hearts. Increased binding of G-CSF to its receptor on cardiomyocytes activates the Akt (34,35) as well as the Jak/STAT Figure 5. Infl uence of G-CSF/SCF on arterialization and cardiomyocyte diameter. (A) Increased arterialization of the area of infarction, including the border zone, in G-CSF/SCF-treated hearts. Left: In situ identifi cation of arterial vessels, visualized by α-smooth muscle actin staining (red fl uorescence) in the non-infarcted (left panels) and infarcted areas plus the border zone (right panels) of untreated (top panels) and G-CSF/ SCF-treated hearts (bottom panels). Right: Quantitative analysis of arterialization. *, statistical signifi cance of difference (P < 0.05); #, a p-value determined by performing a nonparametric test (Wilcoxon signed rank test) because a normality test failed. Performing the corresponding para-metric test would lead to P < 0.05. (B) G-CSF/SCF increases cardiomyocyte diameter in the border zone of the infarction. Left: In situ detection of cardiomyocyte diameter in the non-infarcted areas (left panels) and in the border zone of infarction (right panels) of untreated (top panels) and G-CSF/SCF-treated hearts (bottom panels). Red fl uorescence, cardiomyocyte boundaries; DAPI staining, nuclei; green autofl uorescence, cardiomyocytes. Right: Quantitative analysis. There was no difference in normal myocardium between controls and G-CSF/SCF-treated hearts in normal myocardium, but cardiomyocyte diameter was increased in G-CSF/SCFtreated hearts in the border zone of the infarction.
pathway (14,36), which are both known to impart hypertrophic signaling in cardiomyocytes. For the Akt pathway, it has also been demonstrated that its transgene-mediated, constitutive activation in mouse cardiomyocytes improves their contractile function in vitro as well as in vivo (37).
The increased vascularization in the border zone of the infarction (Fig. 5 A) might have also contributed to the improved cardiac output by enabling increased supply of oxygen and nutrients. Angiogenesis might be at least in part induced by direct eff ects of G-CSF, indicated by distinct immunolocalization of G-CSFR in vessel wall cells 1 d after induction of myocardial infarction. This fi nding is in accordance with Harada et al. (14), who detected G-CSFR expression in endothelial cells, and with Chen et al. (38), who demonstrated that G-CSF induces proliferation of vascular smooth muscle cells. Activation of the Akt as well as the Jak/STAT pathway by the binding of G-CSF to its receptor also might have been responsible for the enhanced induction of arteriogenesis in the G-CSF/SCF-treated hearts as suggested by work of Iwanaga et al. (39) and Harada et al. (14).

Lack of proarrhythmic effects of G-CSF/SCF
Despite the homing of a large number of BM-derived cells into the border zone of myocardial infarction (up to 30% of all nucleated cells), a dedicated protocol to provoke afterdepolarizations and ventricular arrhythmias did not provoke afterdepolarizations or spontaneous ventricular tachycardias in G-CSF/SCF-treated hearts. The lack of diff erentiation into myocytes of the majority of BM-derived cells, observed by us and others (17)(18)(19)40), and consideration of the electrotonic attenuation of abnormal electrical behavior of a single cell when it is integrated into the myocardial syncytium, can explain that we did not fi nd afterdepolarizations in the intact heart.
Reduced inducibility of ventricular arrhythmias G-CSF/SCF-treated hearts had less inducible ventricular arrhythmias during programmed ventricular stimulation than controls (Fig. 4 B). Slow conduction due to decreased intercellular coupling in the border zone of a healed myocardial infarction is the pivotal factor for reentrant ventricular tachycardias in this setting (26,41,42). Reentry due to slow zigzag conduction in the border zone of an infarction is mainly caused by a dramatic decrease in expression of gap junctional proteins, specifi cally in connexin43 (26,43). Indeed, a reduction of connexin43 expression by ‫%09ف‬ is suffi cient to enhance inducibility of ventricular tachycardias in transgenic mice (44). A comparable (90%) reduction of connexin43 expression was found in cardiomyocytes in the border zone of the infarction in the absence of G-CSF/SCF treatment (Fig.  4 C). G-CSF/SCF partially reversed the down-regulation of connexin43 expression (Fig. 4 C) by increasing connexin43 levels in the border zone of the infarction to ‫%52ف‬ of normal expression. This level of connexin expression is sufficient to maintain almost normal conduction velocities (44) and can explain why G-CSF/SCF-treated hearts were pro-tected against induction of ventricular tachyarrhythmias in this study.
This study examined the midterm electrophysiological eff ects of G-CSF/SCF into the infarcted myocardium. Arrhythmic events, including sudden arrhythmic death, may occur late after myocardial infarction, and current revascularization therapies during acute myocardial infarction usually limit infarct size in the clinical setting. The G-CSF/SCF treatment protocol was chosen here in agreement with the pioneering work by Orlic et al. (10) to allow for comparison of our data with those published by this group. Further studies are needed to establish a protocol for BM mobilization that is clinically applicable, i.e., where treatment begins during the acute phase of a myocardial infarction.
In summary, our data suggest that G-CSF/SCF has benefi cial functional hemodynamic, electrophysiological, and microstructural eff ects in the absence of formation of new myocardium from BM-derived cells.

MATERIALS AND METHODS
Animals. 114 mice, which were 58 CD1 outbred mice (Charles River Deutschland GmbH; body weight 23-30 g at the day of myocardial infarction) and 56 female C57BL/6 inbred mice (Charles River Deutschland GmbH; body weight 20-26 g at the day of BMT), were studied. C57BL/6 mice served as hosts for BM transplantation from EGFP-expressing transgenic donor mice (also C57BL/6 background; reference 45). These mice were used to analyze myocardial homing of BM-derived cells. All experiments were performed in accordance with the German Law on the Care and Use of Laboratory Animals and were approved by the local Institutional Review Board.

Transplantation of EGFP-expressing BM-derived cells and splenectomy.
To investigate the eff ect of G-CSF/SCF on mobilization and myocardial homing of BM-derived cells, 34 recipient mice underwent total body irradiation at a dose of 11 Gy (0.98 Gy/min), and intravenous infusion of BM (2 × 10 7 mononuclear cells) was harvested from EGFP-expressing mice the next day. The procedural details have been described previously (46). Blood cell chimerism determined 8 wk after BMT by FACS analysis showed >95% donor cells (EGFP + ) in all transplanted mice. Myocardial homing of BM-derived cells was studied 10 wk after BMT.
With the exception of eight C57BL/6 animals only used for the analysis of BM mobilization in nonsplenectomized mice, all experimental animals were splenectomized according to a published protocol (10) at least 2 wk before BMT or G-CSF/SCF treatment in non-BMT animals.

ARTICLE
White blood cell analysis. After 4 d of G-CSF/SCF treatment, 200 µl blood was obtained from the tail vein of 31 animals (15 controls and 16 treated with G-CSF/SCF). Automatic full blood count measurements using the KK-21 hematology analyzer (Sysmex GmbH) were performed. After Pappenheim staining of blood smears, the proportion of lymphocytes, neutrophils, and monocytes in at least 100 white blood cell counts was calculated.
Echocardiography and surface ECG recordings. Sedated mice (ketamine/xylazine) underwent Doppler echocardiographic studies 5 wk after infarction immediately before the electrophysiological study according to published procedures (47). For additional details, see the supplemental Materials and methods.
Electrophysiological study in isolated hearts. The heart was excised under general anesthesia, and the aorta was cannulated and retrogradely perfused using 37°C Krebs-Henseleit buff er (in mmol/liters; NaCl 118, NaHCO3 24.88, KH2PO 41.18, glucose 5.55, Na-pyruvate 2, MgSO4 0.83, CaCl2 1.8, KCl 4.7) equilibrated with a 95% oxygen/5% carbon dioxide gas mixture. The heart was mounted on a vertical Langendorff apparatus (Hugo Sachs Electronic-Harvard Apparatus GmbH) and constantly perfused at 100 ± 5 mmHg perfusion pressure, corresponding to coronary fl ow rates of 4 ± 1 ml/min. An octapolar mouse electrophysiologic catheter (CIBER MOUSE; NuMED) was placed in the right atrium and ventricle for atrial and ventricular pacing. A tissue bath ECG was recorded from Ag-AgCl electrodes immersed in superfused sponges fl anking the isolated heart. Three murine monophasic action potentials (MAPs) were continuously and simultaneously recorded from the right and left ventricular epicardium (23)(24)(25). One MAP was positioned within the border zone of the infarct to record afterdepolarizations. All recordings were obtained simultaneously throughout the experimental protocols. ECG signals were amplifi ed and fi ltered by an ECG amplifi er at a bandwidth of 0.1 to 300 Hz (Hugo Sachs Electronic-Harvard Apparatus GmbH). MAP signals were amplifi ed using dedicated MAP amplifi ers (Boston Scientifi c Inc.). Additional details of the experimental setup have been described previously (23)(24)(25).
After placing all catheters in a stable position, atrial pacing was performed at diff erent pacing cycle lengths to measure steady-state action potential durations. To provoke ventricular arrhythmias initiated by afterdepolarizations, the atrioventricular node was ablated and the spontaneous ventricular rhythm was observed for 10 min (23,24). Thereafter, to test the propensity to reentry, programmed ventricular stimulation was performed using up to two premature stimuli (S2 and S3) at diff erent pacing cycle lengths (80-140 ms; reference 48). All signals were digitized at 1 or 2 KHz and stored on digital media for offl ine analysis.
Histology and immunofl uorescence. After completion of the electro-physiologi25cal procedure, hearts were either fi xed in Bouin's solution and stained using Goldner's Trichrom staining, or fi xed in 3.7% formaldehyde, followed by dehydration in 10% sucrose solution, O.C.T. embedding, and cryoconservation for fl uorescent detection of EGFP-expressing cells or immunofl uorescent detection of various antigens according to an established protocol (49). The following primary antibodies were used: rat anti-mouse CD45 as a marker for blood cells (AB3088; Abcam Limited), monoclonal anti-troponin T as a marker of diff erentiated cardiomyocytes (cardiac isoform AB-1, clone 13-11; Lab Vision), rabbit anti-rat connexin43 polyclonal antibodies for the detection of gap junctions (71-0700; Zytomed GmbH), monoclonal rat anti-reticular fi broblast antibodies (clone ER-TR7; Novus Biologicals Inc.), rabbit smooth muscle anti-actin antibody for the detection of arteries and arterioles (RB-9010; Lab Vision Corporation), rabbit c-kit (SCF receptor) antibody (sc-168; Santa Cruz Biotechnology, Inc.), and rabbit anti-G-CSFR antibody (sc-694; Santa Cruz Biotechnology, Inc.). For additional information see the supplemental Materials and methods.
Sections were examined using a confocal fl uorescence microscope (Axioplan 2 and LSM 510 Meta; Carl Zeiss MicroImaging, Inc.). Images were digitized and transferred to a personal computer. For quantifi cation of diff er-ent cell populations, the number of EGFP + cells and of EGFP + cells coexpressing the immunohistological marker (anti-CD45 or anti-troponin T antibody) were counted in three randomly selected fi elds of view at the same magnifi cation (400) in the infarcted area, the border zone, and in the noninfarcted area of the left ventricle. For quantitative analysis of connexin expression, the number of connexin43 + spots and the proportion of the connexin43 + area in relation to the nonfi brotic myocardial area of the respective fi eld of view were determined on digitized images taken at high magnifi cation (400) from at least two nonconsecutive slides per mouse heart (four control and fi ve G-CSF/SCF-treated animals) using Adobe Photoshop CS (Adobe Systems GmbH) and ImageJ 1.32 imaging software (National Institute of Mental Health). A minimum of eight images from the border zone and the non-infarcted area was analyzed in each heart.
Quantitative analysis of the arterial vessel area (smooth muscle anti-actin + area of vessels and enclosed lumina) was analyzed as described for the measurement of the connexin43 + area (see above). In addition, the number of arterial vessels per fi eld of view as well as the diameter of each vessel per fi eld of view were determined.
Histological sections immunostained for fi broblasts/connective tissue were also used for short axis diameter measurement of cardiomyocytes at the nuclear level (100 cells per region and animal) using published methods (50) adapted for immunofl uorescence; i.e., DAPI staining was used for the detection of nuclei and cardiomyocytes were identifi ed based on their autofl uorescence.

RNA isolation and real-time RT-PCR.
Infarcted hearts of male CD1 mice at diff erent time points after surgery (30 min, n = 2; 1 h, n = 3; 4 h, n = 3; 24 h, n = 2; 1wk, n = 2; 6 wk, n = 3) were explanted and the area of infarction, including its border zone, was separated from the non-infarcted myocardial tissue. Ventricular tissues of seven explanted hearts of untreated male CD1 mice served as controls.
Isolation of RNA from mouse heart tissue was performed using the RNeasy Fibrous Tissue Mini kit (QIAGEN) according to the manufacturer's protocol. Reverse transcription was performed using the ImProm-IITM Reverse Transcription System (Promega) according to the manufacturer's protocol. cDNA samples reverse transcribed from RNAs were analyzed by quantitative RT-PCR using primers (see the supplemental Materials and methods) purchased from MWG-BIOTECH AG. Quantitative RT-PCR was performed using SYBR Green Reaction Mix (Eurogentec) on an ABI PRISM 7900HT Detection System (Applied Biosystems). Each sample was run in duplicate. The expression of each gene within the diff erent tissue samples was quantifi ed relative to cyclophilin A expression according to the Sequence Detector User Bulletin 2 (Applied Biosystems).
Statistics. All procedures and analyses (functional and histological) were performed by a blinded experimenter with respect to treatment group (G-CSF/SCF vs. control). Depending on the existence of equal variances and normal distribution of data, treatment groups were compared using an unpaired t test and one-way ANOVA, respectively, or the corresponding nonparametric test procedures (Mann-Whitney U test and Kruskal-Wallis test). A chi-square test and log rank test were used to compare nominal parameters (e.g., inducibility of arrhythmias or mortality).