M. Campan

Ferritin as a reporter gene for in vivo tracking of stem cells by 1.5-T cardiac MRI in a rat model of myocardial infarction

The methods currently utilized to track stem cells by cardiac MRI are affected by important limitations, and new solutions are needed. We tested human ferritin heavy chain (hFTH) as a reporter gene for in vivo tracking of stem cells by cardiac MRI. Swine cardiac stem/progenitor cells were transduced with a lentiviral vector to overexpress hFTH and cultured to obtain cardiospheres (Cs). Myocardial infarction was induced in rats, and, after 45 min, the animals were subjected to intramyocardial injection of ∼200 hFTH-Cs or nontransduced Cs or saline solution in the border zone. By employing clinical standard 1.5-Tesla MRI scanner and a multiecho T2* gradient echo sequence, we localized iron-accumulating tissue only in hearts treated with hFTH-Cs. This signal was detectable at 1 wk after infarction, and its size did not change significantly after 4 wk (6.33 ± 3.05 vs. 4.41 ± 4.38 mm(2)). Cs transduction did not affect their cardioreparative potential, as indicated by the significantly better preserved left ventricular global and regional function and the 36% reduction in infarct size in both groups that received Cs compared with control infarcts. Prussian blue staining confirmed the presence of differentiated, iron-accumulating cells containing mitochondria of porcine origin. Cs-derived cells displayed CD31, α-smooth muscle, and α-sarcomeric actin antigens, indicating that the differentiation into endothelial, smooth muscle and cardiac muscle lineage was not affected by ferritin overexpression. In conclusion, hFTH can be used as a MRI reporter gene to track dividing/differentiating stem cells in the beating heart, while simultaneously monitoring cardiac morpho-functional changes.

Placental stem cells pre-treated with a hyaluronan mixed ester of butyric and retinoic acid to cure infarcted pig hearts: a multimodal study.

AIMS Pre-treating placenta-derived human mesenchymal stem cells (FMhMSCs) with a hyaluronan mixed ester of butyric and retinoic acid (HBR) potentiates their reparative capacity in rodent hearts. Our aim was to test FMhMSCs in a large-animal model by employing a novel combination of in vivo and ex vivo analyses. METHODS AND RESULTS Matched regional quantifications of myocardial function and viability were performed by magnetic resonance imaging (MRI) and positron emission tomography (PET) 4 weeks after myocardial infarction combined with intramyocardial injection of FMhMSCs (n = 7), or HBR-pre-treated FMhMSCs (HBR-FMhMSCs, n = 6), or saline solution (PBS, n = 7). Sham-operated pigs (n = 4) were used as control animals. Despite no differences in the ejection fraction and haemodynamics, regional MRI revealed, in pigs treated with HBR-FMhMSCs compared with the other infarcted groups, a 40% smaller infarct scar size and a significant improvement of the end-systolic wall thickening and circumferential shortening of the infarct border zone. Consistently, PET showed that myocardial perfusion and glucose uptake were, respectively, 35 and 23% higher in the border zone of pigs treated with HBR-FMhMSCs compared with the other infarcted groups. Histology supported in vivo imaging; the delivery of HBR-FMhMSCs significantly enhanced capillary density and decreased fibrous tissue by approximately 68%. Moreover, proteomic analysis of the border zone in the HBR-FMhMSCs group and the FMhMSCs group indicated, respectively, 45 and 30% phenotypic homology with healthy tissue, while this homology was only 26% in the border zone of the PBS group. CONCLUSION Our results support a more pronounced reparative potential of HBR-pre-treated FMhMSCs in a clinically relevant animal model of infarction and highlight the necessity of using combined diagnostic imaging to avoid underestimations of stem cell therapeutic effects in the heart.

Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression

Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-α, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions.

Severe Mechanical Dyssynchrony Causes Regional Hibernation-Like Changes in Pigs With Nonischemic Heart Failure

BACKGROUND Sustained left ventricular (LV) dyssynchrony can lead to heart failure (HF) in the absence of coronary artery stenosis. We tested whether myocardial hibernation underlies the LV functional impairment caused by high-frequency pacing, an established model of nonischemic dilated cardiomyopathy. METHODS AND RESULTS Regional LV contractile and perfusion reserve were assessed by magnetic resonance imaging, respectively, as end-systolic wall thickening (LVESWT) and myocardial perfusion reserve index (MPRI) at rest and during low-dose dobutamine stress (LDDS, 10 microg.kg.min intravenously for 10minutes) in failing minipigs (n=8). LV tissue was analyzed for glycogen deposits and other molecular hallmarks of hibernation. LDDS caused a marked increase in LVESWT (27+/-2.98 vs. 7.15+/-3 %, P < .05) and MPRI (2.1+/-0.5 vs. 1.3+/-0.3 P < .05) in the region that was activated first (pacing site) compared with the opposite region. Myocardial glycogen content was markedly increased in the pacing site (P < .05 vs. opposite region). In addition, gene expression of glycogen phosphorylase was reduced in pacing site compared with opposite regions (0.71+/-0.1 vs. 1.03+/-0.3, P < .05), whereas that of hexokinase type II was globally reduced by 83%. CONCLUSIONS The combination of high heart rate and sustained dyssynchronous LV contraction causes asymmetrical myocardial hibernation, in absence of coronary artery stenosis.

Sequencing and cardiac expression of natriuretic peptide receptors A and C in normal and heart failure pigs.

Pharmacological treatments able to activate natriuretic receptors (NPRs) and inhibit cardiac remodelling in heart failure (HF) patients, are currently under investigation. To better understand the therapeutic potential of the NPRs activation is necessary to dispose of experimental models devoid of confounding effects. The pig constitutes an animal model largely used but its genome is not completely sequenced. Aims of this study were to sequence NPR-A and NPR-C in Susscrofa and to evaluate ANP, BNP and NPRs mRNA expression in cardiac tissue of normal and HF minipigs in order to have a starting point for future studies devoted to check new potential drugs. Cardiac tissue was collected from adult male minipigs without (n=4) and with HF (n=5). Pig NPR-A (179bp) and NPR-C (203bp) mRNA were partially sequenced (GenBank n.: FJ518622, FJ518621). Compared to control, ANP and BNP gene expression resulted higher in all the cardiac chambers of HF heart. This increase is associated to a down-regulation of NPR-A and an up-regulation of NPR-C in HF. These sequences will provide a new tool to investigate the role of natriuretic peptides and of their receptors under physiological and pathological conditions and their response to therapeutic interventions.

Transthoracic Sensor for Noninvasive Assessment of Left Ventricular Contractility: Validation in A Minipig Model of Chronic Heart Failure

Background:  Invasively measured left ventricular (LV) dP/dt is the accepted standard for measuring acute and chronic directional changes in LV contractility. Recently, we developed a noninvasive force sensor based on an accelerometer positioned on the chest, which measures the vibrations generated by isovolumic myocardial contraction. The aim of this paper was to compare noninvasive (accelerometer) versus invasive (LV dP/dt) indices of myocardial contractility in a chronic minipig model of pacing‐induced heart failure (HF). Comparative assessment was performed both at rest and following dobutamine infusion.

Regional myocardial hybernation detected by low-dose dobutamine stress MRI in minipigs with prolonged left ventricular dyssynchronous contraction.

Purpose: Neuregulin-1 (NRG-1) is a paracrine growth factor released from endothelial cells in the heart. It stimulates ErbB2 and ErbB4 receptors on cardiomyocytes. Paracrine NRG-1 signalling in the myocardium is activated after exposure to anthracyclines and protects against the development of toxic cardiomyopathy. Conversely, inhibition of NRG-1 signalling after administration of trastuzumab, a monoclonal anti-ErbB2 antibody, exacerbates anthracycline-induced left ventricular (LV) dysfunction. The role of NRG-1 and ErbB activation in other forms of LV dysfunction is still incompletely understood. In this study, we hypothesized that myocardial NRG-1/ErbB signalling is activated during overdrive pacing-induced heart failure. Methods: Nineteen male beagle dogs with pacing-induced heart failure (HF) over 4 to 7 weeks underwent weekly echocardiograms and endomyocardial biopsies. Relative mRNA expression of target genes was measured with real-time RT-PCR. Phosphorylated ErbB2 and ErbB4 were measured by immunoprecipitation and Western blotting after sacrifice in 6 normal dogs, in 6 dogs with moderate HF (4 weeks of pacing) and in 7 dogs with overt congestive HF (7 weeks of pacing). Results: Rapid right ventricular pacing increased LV end-diastolic volume (64±4.3 ml/m2 at baseline and 113±7.2 ml/m2 at week 7, p<0.001) and decreased LV ejection fraction (67±1.6% at baseline and 37±2.6% at week 7, p<0.001). Over the same time course, ventricular NRG-1 mRNA expression increased 4-fold (p<0.01) and expression of a proteolytic enzyme promoting NRG-1 release, ADAM19, increased 2-fold (p<0.05). Left ventricular ErbB2 and ErbB4 receptors showed increasing degrees of overall and site-specific tyrosine phosphorylation over time. ErbB2 receptors underwent truncation exclusively in myocardium of dogs with moderate and severe HF, leading to the formation of a 95 kDa membrane-bound receptor remnant. Conclusions: Pacing-induced cardiomyopathy is accompanied by activation of myocardial NRG-1/ErbB signalling, as shown by increased NRG-1 expression and phosphorylation of ErbB2 and ErbB4 receptors in the LV. As a novel and intriguing observation, we describe the appearance of a truncated form of ErbB2 in the myocardium. In breast tumours, expression of this form is known to be associated with constitutive receptor activity and with poor cancer outcome. Whether, in analogy, ErbB2 truncation in the myocardium can be interpreted as a further manifestation of activated ErbB2 signalling remains to be confirmed, but may have interesting clinical applications.