Maciej Zacharski

Development of porcine model of chronic tachycardia-induced cardiomyopathy

BACKGROUND There are few experimental models of heart failure (HF) in large animals, despite structural and functional similarities to human myocardium. We have developed a porcine model of chronic tachycardia-induced cardiomyopathy. METHODS Homogenous siblings of White Large breed swine (n=6) underwent continuous right ventricular (RV) pacing at 170 bpm; 2 subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were presented for euthanasia at subsequent stages: mild, moderate and end-stage HF. Left ventricle (LV) sections were analyzed histologically and relative ANP, BNP, phospholamban and sarcoplasmic reticulum calcium ATPase 2a transcript levels in LV were quantified by real time RT-PCR. RESULTS In the course of RV pacing, animals demonstrated reduced exercise capacity (time of running until being dyspnoeic: 6.6 ± 0.5 vs. 2.4 ± 1.4 min), LV dilatation (LVEDD: 4.9 ± 0.4 vs. 6.7 ± 0.4 cm), impaired LV systolic function (LVEF: 69 ± 8 vs. 32 ± 7 %), (all baseline vs. before euthanasia, all p<0.001). LV tissues from animals with moderate and end-stage HF demonstrated local foci of interstitial fibrosis, congestion, cardiomyocyte hypertrophy and atrophy, which was not detected in controls and mild HF animals. The up-regulation of ANP and BNP and a reduction in a ratio of sarcoplasmic reticulum calcium ATPase 2a and phospholamban in failing myocardium were observed as compared to controls. CONCLUSIONS In pigs, chronic RV pacing at relatively low rate can be used as an experimental model of HF, as it results in a gradual deterioration of exercise tolerance accompanied by myocardial remodeling confirmed at subcellular level.

Expression and Complex Formation of MMP9, MMP2, NGAL, and TIMP1 in Porcine Myocardium but Not in Skeletal Muscles in Male Pigs with Tachycardia-Induced Systolic Heart Failure

Matrix metalloproteinases (MMPs) are involved in the remodeling of extracellular matrix in various tissues. Their functioning could be related to the formation of complexes, containing MMP9, MMP2, tissue inhibitor of metalloproteinases type 1 (TIMP1), and neutrophil gelatinase-associated lipocalin (NGAL). Such complexes have not been investigated in either myocardial or skeletal muscles. We examined 20 male pigs with heart failure (HF), and 5 sham-operated animals. There were no differences in the mRNA expression of MMP9, MMP2, TIMP1, and NGAL between diseased and healthy animals, in either left ventricle (LV) myocardium or skeletal muscles. In LV from both diseased and healthy animals, in nonreducing and nondenaturing conditions, we demonstrated the presence of high molecular weight (HMW) complexes (130, 170, and 220 kDa) containing MMP9, TIMP1, and NGAL (also MMP2 in 220 kDa complex) without proteolytic activity, and a proteolytically active 115 kDa MMP9 form together with 72 and 68 kDa bands (proMMP2 and MMP2). Proteolytically active bands were also spontaneously released from HMW complexes. In skeletal muscles from both diseased and healthy animals, in nonreducing and nondenaturing conditions, we found no HMW complexes, and proteolytic activity was associated with the presence of 72 and 68 kDa bands (proMMP2 and MMP2).

Changes in parasympathetic system in medulla oblongata in male pigs in the course of tachycardia-induced cardiomyopathy

BACKGROUND Autonomic imbalance constituting a fundamental feature of heart failure (HF) has been assessed mainly at the periphery. Changes in the functioning of autonomic centers in the brain remain unclear. We investigated the molecular elements of parasympathetic system, i.e. α7 nicotinic acetylcholine receptor (α7nAChR) and enzymes metabolizing acetylcholine (acetylcholinesterase, AChE, choline acetyltransferase, ChAT) in medulla oblongata (MO) of male pigs with chronic tachycardia-induced cardiomyopathy. METHODS The mRNA levels of AChE, ChAT, α7nAChR and X-box binding protein 1 (spliced form, XBP1s) in MO were analyzed using qPCR, AChE and ChAT activities using spectrophotometry, proteasome activity using fluorometry, and the protein level of α7nAChR using Western blotting. RESULTS The development of systolic HF was accompanied by an increase in circulating catecholamines, a decrease in the AChE and α7nAChR mRNA in MO, an increase in AChE activity (all p<0.05), and no change in either the mRNA or activity of ChAT. Both circulating catecholamine levels and AChE activity were inversely related to systolic function of left myocardial ventricle (p<0.05). The level of α7nAChR protein in MO and its cytoplasmatic fraction were higher in pigs with moderate and severe HF as compared to the other animals (p<0.01). There was no difference in proteasome activity in MO between diseased and healthy animals, whereas the XBP1s mRNA decreased during HF progression (p<0.05). CONCLUSIONS Molecular elements of parasympathetic system are changed within the medulla oblongata during the progression of systolic non-ischemic heart failure in male pigs, indicating a functional link between MO and heart in HF.

Expression of sex steroid receptors and aromatase in adipose tissue in different body regions in men with coronary artery disease with and without ischemic systolic heart failure.

Abstract Background: The hormonal metabolism of adipose tissue differs across regions of fat. This issue has never been verified in male patients with coronary artery disease (CAD) with and without systolic heart failure (SHF). Methods: We examined 90 male patients with CAD with and without SHF and 42 healthy controls. Results: In patients with CAD with and without SHF, androgen receptor (AR) expression in adipose tissue of the lower leg was higher than AR expression of the thoracic wall and epicardial adipose tissue (EAT) (both p < .0001 for SHF patients and both p < .001 for patients without SHF). Expression of aromatase in adipose tissue of the lower leg among patients with CAD and SHF was higher than aromatase expression of the thoracic wall and EAT (p < .001 and p < .05, respectively), and in patients without SHF, it was higher only than aromatase expression of the thoracic wall (p < .05). There were no differences in expression of estrogen receptor (ER) between three regions of adipose tissue both in men with CAD with and without SHF. Conclusions: In male patients with CAD, site-related differences of adipose tissue in expression of AR and aromatase are present regardless of coexisting SHF with the highest hormonal activity within peripheral subcutaneous adipose tissue.

Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients’ outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such podoplanin-mediated effects can affect tube formation by endothelial cells and participate in their pathological properties in the tumor context. Our experimental data were supported by clinical studies. First, when IDC and DCIS were analyzed by immunohistochemistry according to the presence of podoplanin-expressing cells, the numbers of cancer-associated fibroblasts with high expression of this glycoprotein were significantly higher in IDC than in DCIS cases. Second, using immunofluorescence, the co-localization of PDPN-positive CAFs with blood vessels stained with antibody directed against CD34 was observed in tumor stroma of IDC samples.

Inducible NO synthase is constitutively expressed in porcine myocardium and its level decreases along with tachycardia-induced heart failure.

BACKGROUND The adverse effects of oxidative stress and the presence of proinflammatory factors in the heart have been widely demonstrated mainly on rodent models. However, larger clinical trials focusing on inflammation or oxidative stress in heart failure (HF) have not been carried out. This may be due to differences in the anatomy and physiology of the cardiovascular system between small rodents and large mammals. Thus, we investigated myocardial inflammatory factors, such as inducible NO synthase (iNOS) and oxidative stress indices in female pigs with chronic tachycardia-induced cardiomyopathy. METHODS Homogenous female siblings of Large White breed swine (n=15) underwent continuous right ventricular (RV) pacing at 170bpm, whereas five sham-operated subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were euthanized at subsequent stages of the disease: mild, moderate and severe HF. Left ventricle (LV) sections were examined with electron microscopy. The relative expression of iNOS in LV was determined by quantitative PCR. The protein level of iNOS was determined by Western blotting and immunohistochemistry. The level of the S-nitrosylated (S-NO) protein in LV was determined after S-NO moieties were substituted by biotin, followed by a colorimetrical detection with streptavidin. Malondialdehyde (MDA), a marker of lipid peroxidation, was evaluated in the LV and serum using thiobarbituric acid. The aconitase activity (based on measurement of the concomitant formation of NADPH from NADP(+)), a marker of oxidative stress, was analyzed in mitochondrial and cytosolic LV fractions. The concentration of interleukin-1β (IL-1β) was measured in LV homogenates using enzyme-linked immunosorbent assay. RESULTS RV pacing resulted in an impairment of LV systolic function, LV dilatation and neurohormonal activation. The electron microscopy revealed abnormalities within the cardiomyocytes of failing hearts, i.e. swollen mitochondria and myofibril derangement. iNOS was expressed in the control LV myocardium. The development of HF was accompanied by a decrease in iNOS mRNA (P<.05), which was also reflected at a protein level, and a decrease in the protein S-nitrosylation (P<.05). Both iNOS mRNA and S-NO relative moiety levels were inversely related to the dilatation of the LV (P<.05). There was no difference in the concentration of MDA in the LV and serum. Similarly, no differences in the concentration of IL-1β LV were found between diseased and healthy animals. Aconitase activity was decreased only in the LV mitochondrial fraction of pigs with severe HF. CONCLUSIONS iNOS was shown to be constitutively expressed within porcine LV. Its level decreases during the progression of systolic nonischemic HF in the pig model. Thus, it can be assumed that an up-regulation of proinflammatory factors is not involved in porcine tachycardia-induced cardiomyopathy and that the impact of oxidative stress may be restricted to the mitochondria in this HF model.

Anti-inflammatory properties and expression in selected organs of canine interleukin-1β splice variant 1.

The IL-1β gene can be also be spliced with the intron 4 retention; the result is a IL-1β splice variant 1 (IL-1βsv1), which was significantly up-regulated in failing myocardium of dogs suffering from chronic degenerative valvular disease (CDVD). Expression of IL-1βsv1 was assessed, at both RNA and protein levels, in organs affected by heart failure, namely, kidneys, liver, and lungs from 35 dogs suffering chronic degenerative valvular disease (CDVD) and in 20 disease free control dogs. IL-1βsv1 RNA was detected in the dogs from both groups. In the CDVD group, the highest RNA and protein IL-1βsv1 levels were observed in lungs, followed, in that order, by the liver and kidneys. IL-1βsv1 protein was found in the cytoplasm of hepatocytes and IL-1βsv1-overexpressing DH82 cells. In lungs, IL-1βsv1 was localized in the cytoplasm and in the nuclei of bronchiolar epithelial and smooth-muscle cells. Cytoplasmic and nuclear IL-1βsv1 expression was observed in macrophages, and a strong nuclear signal was detected in epithelial cells of the alveolar sacs. Following lipopolysaccharide (LPS) stimulation, overexpression of IL-1βsv1 in DH82 cells decreased the pro-inflammatory response. Our results indicate that IL-1βsv1 is constitutively expressed in both normal tissues and in tissues from cases of heart failure. The presence of IL-1βsv1 in tissues exposed to invading agents and its anti-inflammatory activity in DH82 cells may point to its immunomodulatory role in vivo.

P-R interval in porcine model of chronic tachycardia-induced cardiomyopathy

Abstract The aim of the study was to assess the atrioventricular conduction in the model of porcine pacing induced tachycardiomyopathy. Fifty-one swine were examined: 27 were paced and 24 served as a control group. Every 4 weeks, the animals were anaesthetised for 1 h and an ECG Holter was performed. Thirty minutes after the onset of anaesthesia, P-R and R-R intervals were measured. Each result was assigned to the subgroup according to the animal’s weight and the presence or absence of previous pacing. P-R interval was longer in animals after at least 4 weeks of rapid ventricular stimulation than in adjusted group of the animals according to the body mass. Multivariate analysis has showed that longer P-R interval was related to male gender, higher body mass, slower heart rate, and history of previous pacing. Chronic ventricular pacing led to the slowing of atrioventricular conduction. The presence of differences in the duration of R-R intervals between groups was only found in swine weighing 120-139 kg. The R-R interval was shorter in paced animals, whereas PR interval was longer in that group, indicating that PR prolongation is related to electrical or structural remodelling of the cardiac conductive tissue but not increased sympathetic nervous system activity, which is expected to produce corresponding changes in PR and R-R intervals.