International Journal of Cardiology 171 (2014) 199–208

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Bone marrow mesenchymal stromal cells rescue cardiac function in streptozotocin-induced diabetic rats Gustavo Monnerat-Cahli a, Mayra Trentin-Sonoda b, Bárbara Guerra a, Gabriel Manso a, Andrea C.F. Ferreira a, Diorney L.S.G. Silva a, Danielle C. Coutinho c, Marcela S. Carneiro-Ramos c, Deivid C. Rodrigues a, Mauricio C. Cabral-da-Silva a, Regina C.S. Goldenberg a, José H.M. Nascimento a, Antonio C. Campos de Carvalho a,d, Emiliano Medei a,⁎ a

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, Brasil c Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brasil d Instituto Nacional de Cardiologia, Rio de Janeiro, Brasil b

a r t i c l e

i n f o

Article history: Received 3 June 2013 Received in revised form 5 November 2013 Accepted 10 December 2013 Available online 18 December 2013 Keywords: Mesenchymal stromal cells Diabetes Cardiac electrophysiology Immunoregulation AMPK

a b s t r a c t Objectives: In the present study, we investigated whether MSC-transplantation can revert cardiac dysfunction in streptozotocin-induced diabetic rats and the immunoregulatory effects of MSC were examined. Background: Cardiac complications are one of the main causes of death in diabetes. Several studies have shown anti-diabetic effects of bone marrow mesenchymal stromal cells (MSC). Methods/results: The rats were divided in three groups: Non-diabetic, Diabetic and Diabetic-Treated with 5 × 106 MSC 4 weeks after establishment of diabetes. Four weeks after MSC-therapy, systemic metabolic parameters, immunological profile and cardiac function were assessed. MSC-transplantation was able to revert the hyperglycemia and body weight loss of the animals. In addition, after MSC-transplantation a decrease in corticosterone and IFN-γ sera levels without restoration of insulin and leptin plasma levels was observed. Also, MSC-therapy improved electrical remodeling, shortening QT and QTc in the ECG and action potential duration of left ventricular myocytes. No arrhythmic events were observed after MSC-transplantation. MSC-therapy rescued the cardiac beta-adrenergic sensitivity by increasing beta-1 adrenergic receptor expression. Both alpha and beta cardiac AMPK and p-AMPK returned to baseline values after MSC-therapy. However, total ERK1 and p-ERK1/2 were not different among groups. Conclusion: The results indicate that MSC-therapy was able to rescue cardiac impairment induced by diabetes, normalize cardiac AMPK subunit expression and activity, decrease corticosterone and glycemia and exert systemic immunoregulation. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia and, among other features, several cardiac complications [1]. In this context, diabetic cardiomyopathy is a major cause of morbidity and mortality [2–6]. Among the cardiac electrical alterations, QT and QTc interval, as well as QT dispersion are increased. Accordingly, these pro-arrhythmic electrocardiographic changes are caused by the prolongation of the action potential duration [2,3] as a consequence of cardiac electrical remodeling that occurs in this disease [2]. One of the

⁎ Corresponding author at: Instituto de Biofísica Carlos Chagas Filho-UFRJ, Av. Carlos Chagas Filho, 373-CCS-Bloco G, Rio de Janeiro, RJ 21941-902, Brasil. Tel./fax: + 55 21 25626555. E-mail address: [email protected] (E. Medei). 0167-5273/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2013.12.013

earliest signs of diabetic cardiomyopathy is mild left ventricular diastolic dysfunction, which is followed by systolic alterations with diminution of cardiac adrenergic sensitivity compromising the normal mechanical cardiac performance [7]. The sustained hyperglycemia is one of the most important factors contributing to cardiac impairment as a consequence of energy unbalance. AMP-activated protein kinase (AMPK) has recently emerged as a potential key factor in numerous cardiac complications and also in diabetes [8]. AMPK acts as an energy sensor and compensates for decreased energy use by upregulating sources of energy and downregulating processes of energy consumption that are not crucial to the cell. In addition, changes in its activity have been associated with diabetes-related hypoxia state [9]. Thus, AMPK activity and phosphorylation state can be either downregulated [10,11] or upregulated [12], in streptozotocininduced (STZ-induced) diabetic cardiomyopathy. While decreased AMPK activity was associated with inflammatory response [13] it has

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been demonstrated that the activation of AMPK was linked to an antiinflammatory response in diabetic rats [14]. Inflammation has also been demonstrated to play an important role on pathological processes in diabetes. Therefore, changes in the levels of key cytokines/chemokines, such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6 were described either in plasma and/or heart of streptozotocin-induced diabetes rats [13–16]. Since about 70% of pancreatic beta-cells are already destroyed by the time type-1 diabetes is diagnosed, efforts have been made to replace these cells or to improve function of the remaining pancreatic cells [17]. In this context, autologous bone marrow cell transplantation has emerged as a new option to treat type-1 diabetes in patients as described in pioneer work published by Voltarelli et al. [18]. In animal models, the small population of mononuclear bone marrow cells named “mesenchymal stromal cells” (MSC) also refereed as “mesenchymal stem cells” [19] was successfully used in STZ-induced diabetes either in rats [20–22] or mice [23,24]. The advantageous effects induced by MSC could be due of the potent immunomodulatory properties of these cells. Their immunoregulatory actions can modulate different types of cells, like antigen presenting and effector T and B cells, being applicable to several autoimmune disease and inflammatory processes [23,25–27]. In this context, it has been described that MSC-therapy ameliorated myocardial inflammation, reducing cytokine production and improving cardiac mechanical function on a rat model of myocarditis [25]. In fact, it was also demonstrated that MSC-therapy decreases the expression of proinflammatory cytokines improving cardiac function on an experimental model of inflammatory cardiomyopathy [27]. Additionally, it has been demonstrated that MSC-therapy can promote a cytokine profile shift from proinflammatory to anti-inflammatory in the pancreas of diabetic mice, unraveling an anti-diabetic effect induced by MSC-transplantation [23]. All of these beneficial effects observed with MSC-therapy on animal models are supporting nine clinical trials currently aiming to test the therapeutic effect of this cell population in patients with type-1 diabetes (www.clinicaltrials.gov). Cumulative evidence suggests that the anti-diabetic potential of MSC-therapy involves an immunoregulatory mechanism. While previous investigations have mainly focused on how MSC-therapy is able to reverse the hyperglycemic state of the streptozotocin-induced diabetes model (largely studying the pancreatic histoarchitecture and immunological pancreatic microenvironment), here we aimed to investigate whether MSC-therapy, modulating the systemic inflammatory state, is able to rescue the altered cardiac function in STZ-induced hyperglycemic rats. 2. Methods 2.1. Animals and experimental protocol Male Wistar rats (118.6 ± 8.0 g) were kept at constant temperature (23 °C) in a standard light/dark cycle (12 h/12 h) with free access to standard chow and water. The animals were divided in 3 groups: control non-diabetic (NORMAL), diabetic (DM) and diabetic treated with MSC (DM + MSC). Diabetes was induced by a single injection of streptozotocin (Sigma-Aldrich) (80 mg/kg i.v.) in 0.05 M citrate buffer (pH 4.5), in anesthetized (Isoflurane, Baker) animals. Animals from NORMAL group received the same volume of vehicle. Four weeks after establishment of diabetes (blood glucose higher than 300 mg/dL), diabetic rats were randomly separated in two groups: one that received a single transplantation of MSC (5 × 106) by systemic injection (retro-ocular plexus) (DM + MSC), and the second group, which receive only vehicle injection (DM). Four weeks after transplantation all animals were sacrificed with carbon dioxide asphyxiation followed by cervical dislocation. The protocols used in the present study were approved by the Animal Care and Use Committee at the Federal University of Rio de Janeiro (n°: 26). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. 2.2. Blood glucose and glucose tolerance test Blood glucose levels were determined using a glucose reagent strip and a standard automated glucometer (AccuChek Advantage II, Roche, Ireland). Briefly, animals were fasted overnight for 12 h and blood was obtained from the tip of the tail vein of the fully awake, non-anesthetized animal. After determination of the fasting blood glucose level, the

animals were loaded by gavage with 3.5 g glucose/kg body weight, and blood glucose levels were measured 15, 30, 60, 90 and 120 min later.

2.3. Serum insulin, leptin and corticosterone Serum insulin and corticosterone concentrations were measured using commercial radioimmunoassay kits, based on the presence of specific antibodies adhered to the internal surface of propylene tubes (ImmuChemTM Coated Tube Insulin 125I RIA kit) and double antibody (ImmuChemTM Double Antibody Corticosterone 125I RIA kit). Insulin (MP Biomedicals, LLC®, sensitivity of 5.5 μl U/ml) inter- and intra assay coefficients of variation varied from 5.0 to 8.9% and from 3.2 to 12.2%, respectively; Corticosterone (MP Biomedicals, LLC®, sensitivity of 25 ng/ml) inter and intra assay coefficients of variation varied from 6.5 to 7.2% and 4.4 to 10.3%, respectively (OH, USA). Serum leptin concentrations were measured using a specific radioimmunoassay (RIA) for rat leptin obtained from the Millipore Corporation (MA, U.S.A). Intra- and interassay coefficients of variation were 2.0–4.6% and 3.0–5.7% respectively, and the sensitivity was 0.639 ng/ml using 100 μl sample size. To obtain material to carry out these assays, the total amount of the blood extracted for serum isolation was about 1 ml.

2.4. Multipotent mesenchymal stem cell isolation and culture The total rat bone marrow was harvested by flushing the tibias and femurs with Dulbecco's modified Eagle medium (DMEM, Gibco-Invitrogen, USA) and the cell suspension centrifuged in Histopaque gradient at 400 ×g for 30 min (Histopaque 1.083 g/ml, 1:1, Sigma-Aldrich, USA). Mononuclear cells were collected from the histopaquemedium interface. Cells were washed in Phosphate-Buffered Saline (PBS) three times, counted in a hemocytometer, and checked for viability using 0.4% trypan blue. Cells were then plated at a density of 1.2 × 106 cells/cm2 and maintained at 37 °C in a 5% CO2 incubator for 1 week, during which medium was changed at least twice, washing away all floating hematopoietic cells. Culture medium used was DMEM supplemented with 20% fetal bovine serum (Gibco-Invitrogen, USA), 2 mM L-glutamine (Sigma-Aldrich), and antibiotics (100 U/ml penicillin G and 100 μg/ml streptomycin, Gibco). At approximately 80–90% confluence, adherent cells were detached from the culture flasks with 0.25% trypsin-EDTA (Sigma-Aldrich) replated at a density of 1.2 × 104 cells/cm2 and further propagated until passage 3.

2.5. ECG and action potential recording Electrocardiogram recording was carried out in conscious animals by noninvasive method. Electrodes were positioned in DI derivation and connected by flexible cables to a differential AC amplifier (model 1700, A-M Systems, USA), with signal low-pass filtered at 500 Hz and digitized at 1 kHz by a 16-bit A/D converter (Minidigi 1-D, Axon Instruments, USA) using Axoscope 9.0 software (Axon Instruments, USA). Data were stored in a PC for offline processing. To assess action potential recording, muscle strips were obtained and pinned to the bottom of a tissue bath in order to expose the endocardial side. The preparations were superfused with Tyrode's solution containing (in mM): 150.8 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 11.0 D-glucose, 10.0 HEPES (pH 7.4 adjusted with NaOH at 37.0 ± 0.5 °C) saturated with carbogen mixture (95% O2/5% CO2) at a flow of 5 ml/min (Gilson MINIPULS 3). The tissue was stimulated at four different basic cycle lengths. Transmembrane potential was recorded using glass microelectrodes (10–40 MΩ DC resistance) filled with 2.7 M KCl connected to a high input impedance microelectrode amplifier (MEZ7200, Nihon Kohden, Japan). Amplified signals were digitized (1440 Digidata A/D interface, Axon Instrument, Inc.) and stored in a computer for later analysis using software LabChart 7.3 (ADInstruments, Australia). The following parameters were analyzed: resting membrane potential (RMP), action potential amplitude (APA) and action potential duration at 90% (APD90), 50% (APD50) and 30% (APD30) repolarization. The Maximum rate of depolarization (Vmax) was calculated using five-point linear regression centered on the sample. The Maximum Negative Slope (Max. Neg. Slope) was calculated by the steepest downhill slope starting 5 ms after the peak using a linear regression during a window of 4 ms. The AP triangulation was calculated by subtracting APD40 from APD90. 2.6. Cardiac mechanical function In order to assess cardiac mechanical function the hearts were rapidly excised, cannulated by the aorta and perfused at constant flow 10 mL/min with Krebs–Henseleit buffer solution (KHB) containing (in mmol/L) NaCl 118.0, NaHCO3 25.0, KCl 4.7, KH2PO4 1.2, MgSO4.7H2O 1.2, CaCl2 1.25, and glucose 11.0. The KHB solution was saturated with carbogen gas (pH = 7.4) and held at 37.0 ± 0.5 °C. A small latex balloon, connected to a pressure transducer (MLT0380, ADInstruments, Australia), was inserted in the left ventricle, through the left atrium, and adjusted to an initial diastolic pressure of 10 mmHg. The transducer was connected to an amplifier (ML110, ADInstruments, Australia), to register the intraventricular pressures developed by the left ventricle (Left Ventricle developed pressure — LVDP). The pressure recordings were acquired at 1.0 kHz by an analogic-digital interface (PowerLab 400, ADInstruments, Australia) and stored in a computer for an offline analysis using LabChart 7.3 (ADInstruments, Australia). Next, a doseresponse curve with adrenergic agonist was performed (Isoproterenol: 0.1 nM, 0.3 nM, 0.8 nM, 1 nM, 10 nM, 100 nM; 500 nM; 1 μM).

G. Monnerat-Cahli et al. / International Journal of Cardiology 171 (2014) 199–208 2.7. RT-PCR and western blot The mRNA levels of the Beta1 Adrenergic receptor gene in the left ventricle heart tissue were quantified by qRT-PCR (qRT-PCR). GAPDH mRNA levels were used for normalization. The primer sequences were: Beta1 Adrenergic receptor forward: CTGCTACAACGA CCCCAAG, reverse: TCTTCACCTGTTTCTGGGC; and GAPDH forward: TGATTCTACCCACGGC AAGT, reverse: AGCATCACCCCATTTGATGT. To assess protein levels, total protein was extracted from the left ventricles and 70 μg of protein were resolved on SDS-PAGE (10% polyacrylamide) and then transferred onto nitrocellulose membranes. After 1 h of blocking with 5% milk in Tris-buffered saline, the membranes were probed with one of the following primary antibodies: AMPK-α (phosphorylated 1:250 and total 1:500, Cell Signaling Technology®, Inc. USA, catalog no. 9957) and AMPK-β1 (phosphorylated 1:1000 and total 1:2000, Cell Signaling Technology®, Inc., USA, catalog no. 9957) overnight. GAPDH was used as loading control for protein quantification (1:20,000). After final washes, the blots were then incubated with either IRDye Goat-Anti-Rabbit or GoatAnti-mouse IRDye® 680 CW secondary antibody (Rockland Immunochemicals® Gilbertsville, PA). Images of the blots were acquired with an infrared scanner from LICOR and analyzed using Odyssey software. ERK1 (1:500, Santa Cruz Biotechnologies catalog no. SC-93) and p-ERK1/2 (1:250, Santa Cruz Biotechnologies catalog no. sc-16982) followed the same protocol of incubations but with secondary antibodies conjugated to horseradish peroxidase (HRP) anti-rabbit (1:2500, Santa Cruz Biotechnologies catalog no. SC-2004) or anti-mouse (1:10000, Santa Cruz Biotechnologies catalog no SC-2005), developed by chemiluminescent reaction (ECL prime, GE) and detected by exposure of the blot to x-ray film (Kodak T-Mat S/RA X-ray Film). Optical density of the bands was quantified using Image J software (NIH, http://rsbweb.nih.gov/ij/). 2.8. Inflammatory plasma profile Blood was extracted and centrifuged at 3000 rpm for 15 min to isolate the serum. Luminex Performance Rat Cytokine Panel (R&D System) was used to measure levels of cytokines and the plates were read on Luminex® 100/200 (BioRad). 2.9. Statistical analysis Data are presented as mean ± SEM. Multiple comparisons between groups were performed using analysis of variance (ANOVA), followed by a Bonferroni or Newman-Keuls multiple comparison test. Data showing non Gaussian distribution (Kolgomorov-Smirnov test) were compared by Kruskal-Wallis test followed by Dunn's multiple comparison test. Values of P b 0.05 were considered statistically significant. All analysis were made using GraphPad Prism 5.0 (GraphPad Software, USA).

3. Results 3.1. MSC-transplantation improves glucose levels restoring corticosterone sera concentration High blood glucose and decreased levels of insulin and leptin are the hallmarks of STZ-induced diabetes in rats. As shown in Fig. 1 the animals used in this study reproduce this pattern, as well as a decrease on body weight, after a single dose of streptozotocin. The transplanted cells were systemically distributed and could be tracked for up to 7 days after transplantation (See Supplementary Fig. 1). Treatment with a single transplantation of MSC promoted an improvement in fasting serum glucose levels, leading to values similar to NORMAL rats (Fig. 1A). In addition MSC-transplantation reduced body weight loss (Fig. 1B) and restored corticosterone levels (Fig. 1C). However, the improvement in glucose control and the gain in body weight were not associated with an increase neither in leptin nor in insulin plasma levels (Fig. 1D, E). In fact, the DM + MSC rats were still intolerant to glucose as observed in Fig. 1F (Also in online Supplementary Table 1). Additionally, no difference was observed in the heart weight/body weight ratio among groups (NORMAL: 0.4 ± 0.01; DM: 0.5 ± 0.01; DM + MSC: 0.5 ± 0.04; P N 0.05). No difference in survival was observed among the studied groups; about more than 90% of rats survived in each group. 3.2. Systemic inflammatory profile after 4-weeks of MSC-therapy Among 9 cytokines studied, IFN-γ serum concentration detected in DM + MSC returned to values comparable to NORMAL rats. In addition, the plasma concentration of other six proinflammatory cytokines analyzed on DM + MSC group was not different when compared to the serum concentration of these inflammatory mediators on NORMAL group. Conversely, DM rats show a significant increment of these

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proinflamatory molecules when compared to serum concentration of the NORMAL group. Cytokine serum concentrations of both DM + MSC and DM did not show statistical difference, except for IFNγ. The serum concentration of the classical antiinflammatory cytokine IL-4 was similar among groups. The other antiinflammatory cytokine studied, IL-10, had higher concentration in serum of both DM and DM + MSC groups when compared to NORMAL rats. (See bars graphs on Fig. 2). 3.3. MSC-therapy reverses cardiac electrical remodeling Four-weeks after cell transplantation DM + MSC group presented an improvement in QT and corrected QT interval (QTc) in comparison to the DM group, but no differences in RR interval and QRS complex duration were observed among groups (Fig. 3) (Online Supplementary Table 2). In addition, no arrhythmic events were observed. Cardiac action potential analysis revealed that duration at 90% of repolarization was significantly prolonged in DM compared to NORMAL animals, and it was significantly shortened in DM + MSC rats when compared to DM animals (Fig. 4). These results were not rate dependent (Fig. 4B). Pro-arrhythmic markers such as triangulation and the maximal velocity of repolarization, which were increased in the DM group, were also improved after MSC-transplantation (Fig. 4C, D). No differences in other action potential parameters were observed among groups (Online Supplementary Table 3). 3.4. MSC-transplantation preserves heart mechanical function under adrenergic challenge No difference under basal conditions was observed on LVDP among groups (Fig. 5A, see inset bars graph). The hearts from the DM + MSC rats responded adequately to an adrenergic challenge, increasing LVDP (Fig. 5A, B). However, the DM group was not able to increase developed pressure under the same conditions (Fig. 5A; Online Supplementary Tables 4–6). Mechanistically, we demonstrated that MSC-therapy rescued mRNA levles of beta-1 adrenergic receptors in the left ventricle of diabetic rats (Fig. 5C). 3.5. MSC-therapy modulates AMPK but not ERK signaling pathways in the heart To assess the impact of the MSC-therapy upon signaling pathways activated in the heart, two protein kinases were investigated: (i) the expression and activity of AMPK and (ii) the expression of ERK1 and phospho-ERK1/2 (p-ERK1/2) were compared among the three groups. MSC-transplantation restored total AMPK α and β subunit expression, which were altered in DM hearts (Fig. 6A, B). In fact, MSCtherapy also rescued the levels of p-AMPK α and p-AMPK β subunits in hearts from diabetic animals, which are downregulated in nontreated diabetic rats (Fig. 6A, B). The levels of the other protein kinase studied, ERK1 and p-ERK1/2 were not different among groups (Fig. 6C). 4. Discussion Results from our observations further confirm that transplantation of MSC can reverse STZ-induced hyperglycemia. The novel finding reported here is that MSC improves cardiac function in diabetic rats. In accordance with our data, previous studies have demonstrated that MSC-transplantation in diabetic rats decreases hyperglycemia and promotes an increase in body weight of the diabetic animals [20–22,28]. However, previous to this work it was unclear whether this cell therapy could revert the one of the major causes of morbidity and mortality in diabetic patients, the diabetic cardiomyopathy [4]. In order to assess how MSC decreased sera glucose levels, the following hormones that play a key role in maintaining glycemic homeostasis

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Fig. 1. MSC-transplantation induced hyperglycemia reversion in diabetic rats: (A) Blood glucose throughout the protocol. (B) MSC-transplantation increases body weight. (C-E) Serum levels of corticosterone, leptin and insulin 4-weeks after MSC-transplantation. (F) Oral test to glucose tolerance 4-weeks after MSC-therapy. The results are expressed as mean ± SEM. *** P b 0.001 vs. NORMAL and DM + MSC; # P b 0.001 vs. NORMAL; ** P b 0.01 vs. NORMAL; P b 0.05 vs. DM. w: weeks. N = 8 to 10/group. White square = NORMAL; white triangle = DM; black circle = DM + MSC; white bars = NORMAL; gray bars = DM; black bars = DM + MSC.

were measured in serum from all groups studied: (i) corticosterone, (ii) insulin and (iii) leptin. Corticosterone is typically increased in diabetes [29,30]. It was reported that specific inactivation of the glucocorticoids receptors (GCCR) in the liver by the Cre/loxP system resulted in amelioration of STZ-induced hyperglycemia and caused hypoglycemia upon prolonged fasting [31]. Also, inflammation [32] high serum concentration of corticosterone was observed. Therefore, it has been described that the activation of the hypothalamic-hypophysis-adrenal axis by IL-6, IL-1ß, and TNF-α is an essential protective host response to inflammation [33]. This was supported by work of Fattori et al. showing that IL-6deficient-mice were not able to produce corticosterone after inflammation challenge [34]. Interestingly, our results showed that MSC transplanted animals show corticosterone serum levels similar to the

non-diabetic group and significantly lower values than DM group. This finding could be attributed, at least in part, to immunoregulatory properties that have been largely attributed to MSC [21]. In this context, here MSC-therapy reverted the diabetes-induced high serum concentration of IFN-γ an important proinflammatory marker. In fact, conversely to the data obtained when comparing the DM to the NORMAL group, the DM + MSC rats showed serum concentrations of all cytokines not to be different when compared to NORMAL rats. This novel and significant finding provides new insights into the mechanisms of MSC-induced normoglycemia. In agreement with this result, the body weight gain observed in the treated animals reflects the improvement of the metabolic state induced by MSC-therapy. The data are also in accordance with previous work transplanting MSC in STZ-induced diabetes in rats [21]. In contrast with these results, we did not find an enhancement neither

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Fig. 2. Systemic immunomodulation after 4-weeks of MSC-transplantation: Bars graphs summarize the serum immunomodulatory effect on both pro and anti-inflammatory cytokines studied. The results are expressed as mean ± SEM. * P b 0.05 vs. NORMAL; ** P b 0.01 vs. NORMAL; *** P b 0.001 vs. NORMAL; # P b vs. DM + MSC. N = 5 to 7 per group. White bars = NORMAL; gray bars = DM; black bars = DM + MSC.

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Fig. 3. MSC transplantation reverses cardiac electric remodeling: (A) Representative traces of electrocardiogram 4-weeks after transplantation shows QT interval shortening after MSC transplantation (bottom panel). (B-E) bar graphs summarize the ECG parameters measured 4-weeks after MSC transplantation. The results are expressed as mean ± SEM. ** P b 0.01 vs. NORMAL; # P b 0.05 vs. DM + MSC; N: NORMAL: 12; DM: 9; DM + MSC: 12. White bars = NORMAL; gray bars = DM; black bars = DM + MSC.

insulin nor leptin plasma levels after MSC-transplantation. In fact, even though the treated animals showed low glycemic values, the oral glucose tolerance test in this group showed that they were still intolerant to glucose, as also observed in the non-treated group. These data support the low sera insulin concentration observed in both MSC-treated and untreated diabetic groups. Among the few manuscripts showing the anti-diabetic effect of MSC on STZ-induced diabetic rat model, Boumaza et al. have shown a slight increment of sera insulin levels, but the authors reported that only 2 of 8 rats sustained sera insulin levels 21 days after MSC-transplantation [21]. Among the complications of diabetic cardiomyopathy, the electrical remodeling helps to create a cardiac microenvironment that evokes fatal ventricular arrhythmias [2,3]. Since previous publications have shown data that consistently support a beneficial effect of MSCtransplantation on the heart [35,36] we set out to study whether an anti-diabetic effect of MSC-transplantation could revert the STZinduced cardiac electrical remodeling in diabetic rats. In our study we

did not observe arrhythmic event in any of the animals studied. This data are in agreement with other studies [37]. A preserved mechanical function and beta-adrenergic sensitivity is crucial to maintain cardiovascular performance. While STZ-induced diabetes in rats did not impair basal cardiac contraction and relaxation performance for at least 2 months after diabetic induction, an early decrease in cardiac beta-adrenergic sensitivity was described [38]. Additionally, this has been shown not to be totally reestablished even under insulin treatment in the same animal model [39]. In the present study, the effects of cellular therapy with MSC on the preservation of the adrenergic sensitivity, increasing LVDP, led to a cardiac performance similar to NORMAL rats under adrenergic stimulus. Supporting this data, we demonstrated that treated diabetic rats rescue beta1-adrenergic receptor gene expression in the heart, while non-treated rats presented a downregulation of this transcript. The cardiac benefits promoted by MSC-therapy in STZ–induced diabetic rats could be also attributed to changes in various cardiac

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Fig. 4. MSC-therapy reverses cardiac action potential prolongation after 4-weeks: (A) Representative action potential traces from left ventricle endocardial tissue from all studied group. (B) The graphs summarize the action potential duration (APD) at 90% of repolarization under different basic cycle length (BCL) of stimulation (300, 500, 800 and 1000 ms). (C, D) The bar graphs summarize the triangulation duration and maximal negative slope of repolarization, respectively. The results are expressed as mean ± SEM. *** P b 0.001 vs. NORMAL and DM + MSC; ** P b 0.01 vs. NORMAL and DM + MSC; * P b 0.01 vs. NORMAL; # P b 0.05 vs. DM + MSC; N: NORMAL: 8; DM: 8; DM + MSC: 13. White square = NORMAL; white triangle = DM; black circle = DM + MSC; white bars = NORMAL; gray bars = DM; black bars = DM + MSC.

intracellular signaling networks, as a consequence of the improvement of the systemic metabolic profile. Here we have focused and studied two important kinases that play a vital role in STZ-induced diabetic cardiomyopathy, the AMPK and a member of the mitogen-activated protein kinase (MAPK) family, ERK. In this context, previous studies have reported a downregulation of cardiac p-AMPK-α in the same model used in the present work as a consequence of energy unbalance [11]. Our data shows that MSC transplantation can preserve AMPK protein expression and function. These results could be related to the improvement in glucose homeostasis induced by MSC-therapy, since similar results (increase in AMPK) were described after glycemic stabilization by long-term insulin treatment in the type-1 diabetes mice model [10]. Even though the actual information available about mechanical or electrophysiological changes as a

result of activation or inactivation of AMPK in such conditions is extremely limited, the authors speculate that the MSC-therapy, improving the metabolic balance - reflected by the AMPK result obtained here – has contributed to normalize the electrophysiological and mechanical cardiac function [10]. The expression and activation of the other kinase studied ERK, is usually associated with myocardial protection activating a “survival” pathway during stress conditions. In a diabetic rat model, Laviola et al. observed no change in heart p-ERK 6-weeks after STZ-induced diabetes. Conversely, it was demonstrated that after a longer period (20 weeks) of diabetes in rats, there is a downregulation of p-ERK in the heart [40]. In the present work we did not observe significant changes neither in total ERK1 nor in p-ERK1/2 in the diabetic hearts. In addition, MSCtherapy did not promote activation of this kinase.

Fig. 5. Heart mechanical function is conserved when challenged by adrenergic stimulus by restoration of beta-1 adrenergic receptor expression after MSC transplantation: (A) The graph shows the percentage (%) of LVDP increment as a function of isoproterenol concentration. The inset shows that there is no difference among groups under basal condition. (B) The bar graph shows LVDP measurement in 1 μM of ISO. (C) The expression of beta-1 adrenergic receptor mRNA in left ventricle tissue from the three groups. The results are expressed as mean ± SEM. ** P b 0.01 vs. NORMAL and DM + MSC; # P b 0.01 vs. NORMAL; * P b 0.05 vs. DM + MSC; N: NORMAL: 8; DM: 8; DM + MSC: 9. LVDP: left ventricular developed pressure; ISO: isoproterenol. White square = NORMAL; white triangle = DM; black circle = DM + MSC; white bars = NORMAL; gray bars = DM; black bars = DM + MSC.

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Fig. 6. The role of kinases on MSC-transplantation induced hyperglycemia reversion in diabetic rats heart: (A-C) top: representative immunoblotting of the protein of interest and the control load protein (GAPDH). Bottom: The bar graph summarizes the corresponding densitometric measurements of AMPK-α and p-AMPK-α (A). (B) AMPK-β 1 and p-AMPK- β. (C) Total ERK1, p-ERK1/2 and the ratio of p-ERK1/2/Total ERK1 in heart. The results are expressed as mean ± SEM. * P b 0.05 vs. NORMAL and DM + MSC; N: NORMAL: 8; DM: 7; DM + MSC: 8. White bars = NORMAL; gray bars = DM; black bars = DM + MSC.

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4.1. Clinical perspective and study limitations Cellular therapy using stem cells represents a potential strategy to treat several cardiovascular and metabolic diseases. In this context, bone marrow mesenchymal stromal cells emerge as a potential candidate for use in type-1 diabetes patients. This is supported by various preclinical studies demonstrating the anti-diabetic effect and the lack of arrhythmic events induced by MSC-transplantation [37]. These successful results have motivated the registration of at least 9 clinical trials in diabetes (http://www.clinicaltrials.gov) using cell-based therapies. The present work not only confirmed the anti-diabetic potential of MSC-therapy, but additionally, showed that this therapy is capable to reverse diabetes-induced cardiac alterations. While in the mice model of type-1 diabetes the mechanism by which MSC-transplantation improved sera glycemic values is well established, in rats it remains largely unknown. In mice insulin levels are increased after MSC-transplantation, as a consequence of the improvement in the inflammatory pancreatic microenvironment [23,24]. In STZinduced diabetic rats no evidence supports these data. In addition, our results showed that MSC-transplantation improved corticosterone, but not insulin and leptin sera concentration. This divergence between different animal models is part of the study limitation and requires careful examination before bringing this MSC-therapy to the clinical practice. Another limitation of the present work is the fact that we transplanted MSC from “non-diabetic” rats to diabetic ones. Autologous transplantation in humans may show different results because MSC isolated from diabetic animals have impaired paracrine potential [41]. However, it is well known that MSC show a low immunogenicity in allogeneic transplantation [42]. In summary, our results shown that MSC-transplantation: (i) can decrease hyperglycemia and increase body weight in STZ-induced diabetic rats, normalizing sera corticosterone concentration, but without normalizing insulin and leptin levels; (ii) induce a plasma immunomodulatory response, decreasing IFN-γ serum levels and partially reversing other proinflammatory cytokines studied; (iii) rescue cardiac electrical and mechanical function and ameliorate cardiac beta-adrenergic sensitivity; (iv) restore AMPK cardiac expression and activity to values comparable to non-diabetic rats. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2013.12.013. Acknowledgments This work was funded by the Brazilian National Research Council (CNPq, grants: 308168/2012-7 and 475218/2012-4), the Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ, grants: E-26/ 103.222/2011 and E-26/111.171/2011) and National Institutes of Science and Technology for Biology Structural and Bioimaging (grant: 573767/2008-4), Brazil. All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. References [1] Grundy SM, Benjamin IJ, Burke GL, et al. Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation 1999;100(10):1134–46. [2] Kahn JK, Sisson JC, Vinik AI. QT interval prolongation and sudden cardiac death in diabetic autonomic neuropathy. J Clin Endocrinol Metab 1987;64(4):751–4. [3] Magyar J, Rusznák Z, Szentesi P, Szûcs G, Kovács L. Action potentials and potassium currents in rat ventricular muscle during experimental diabetes. J Mol Cell Cardiol 1992;24(8):841–53. [4] Garcia MJ, McNamara PM, Gordon T, Kannel WB. Morbidity and mortality in diabetics in the Framingham population. Sixteen year follow-up study. Diabetes 1974;23(2):105–11. [5] Vinik AI, Ziegler D. Diabetic cardiovascular autonomic neuropathy. Circulation 2007;115(3):387–97. [6] An D, Rodrigues B. Role of changes in cardiac metabolism in development of diabetic cardiomyopathy. Am J Physiol Heart Circ Physiol 2006;291(4):H1489–506.

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