Analysis of the Model of Atherosclerosis Formation in Pig Hearts as a Result of Impaired Activity of DNA Repair Enzymes.

Excessive consumption of food rich in saturated fatty acids and carbohydrates can lead to metabolic disturbances and cardiovascular disease. Hyperlipidemia is a significant risk factor for acute cardiac events due to its association with oxidative stress. This leads to arterial wall remodeling, including an increase in the thickness of the intima media complex (IMT), and endothelial dysfunction leading to plaque formation. The decreased nitric oxide synthesis and accumulation of lipids in the wall result in a reduction in the vasodilating potential of the vessel. This study aimed to establish a clear relationship between markers of endothelial dysfunction and the activity of repair enzymes in cardiac tissue from a pig model of early atherosclerosis. The study was conducted on 28 female Polish Landrace pigs, weighing 40 kg (approximately 3.5 months old), which were divided into three groups. The control group (n = 11) was fed a standard, commercial, balanced diet (BDG) for 12 months. The second group (n = 9) was fed an unbalanced, high-calorie Western-type diet (UDG). The third group (n = 8) was fed a Western-type diet for nine months and then switched to a standard, balanced diet (regression group, RG). Control examinations, including blood and urine sampling, were conducted every three months under identical conditions with food restriction for 12 h and water restriction for four hours before general anesthesia. The study analyzed markers of oxidative stress formed during lipid peroxidation processes, including etheno DNA adducts, ADMA, and NEFA. These markers play a crucial role in reactive oxygen species analysis in ischemia-reperfusion and atherosclerosis in mammalian tissue. Essential genes involved in oxidative-stress-induced DNA demethylation like OGG1 (8-oxoguanine DNA glycosylase), MPG (N-Methylpurine DNA Glycosylase), TDG (Thymine-DNA glycosylase), APEX (apurinic/apirymidinic endodeoxyribonuclease 1), PTGS2 (prostaglandin-endoperoxide synthase 2), and ALOX (Arachidonate Lipoxygenase) were measured using the Real-Time RT-PCR method. The data suggest that high oxidative stress, as indicated by TBARS levels, is associated with high levels of DNA repair enzymes and depends on the expression of genes involved in the repair pathway. In all analyzed groups of heart tissue homogenates, the highest enzyme activity and gene expression values were observed for the OGG1 protein recognizing the modified 8oxoG. Conclusion: With the long-term use of an unbalanced diet, the levels of all DNA repair genes are increased, especially (significantly) Apex, Alox, and Ptgs, which strongly supports the hypothesis that an unbalanced diet induces oxidative stress that deregulates DNA repair mechanisms and may contribute to genome instability and tissue damage.

Effect of low-dose hydrocortisone and inhaled nitric oxide on inflammatory mediators and local pulmonary metalloproteinases activity in LPS-induced sepsis in piglets.

Hospital mortality in sepsis varies between 30-45%. It has been shown that administration of inhaled nitric oxide (iNO) and intravenous corticosteroid in a porcine endotoxemia model attenuated the systemic inflammatory response. We explored the anti-inflammatory effect of a double-treatment strategy (iNO + low-dose steroid) on the lungs in a long-term porcine endotoxic shock model. As metalloproteinases (MMPs) are involved in the initiation of multiple organ dysfunction in septic shock, we evaluated the influence of this combination therapy on MMP2 and MMP9 activity and proIL-1ß maturation. A shock-like condition was established in 23 animals by continuous infusion of E. coli lipopolysaccharide (LPS) for 10 h. Then the animals were observed for 10 h. Twelve pigs received iNO and hydrocortisone (iNO treatment started 3 h after the initial LPS infusion and continued until the end of the experiment). Eleven pigs were controls. Pigs treated with iNO and hydrocortisone displayed less inflammatory infiltrates in the lungs than the controls and a lower level of IL-1ß. The proMMP2 was significantly decreased in the iNO and hydrocortisone group. The amount of an active MMP9 (~ 60 kDa) was decreased in the iNO and hydrocortisone group. Total gelatinolytic activity was lower in the iNO and hydrocortisone group. Reduced MMP activity was accompanied by a 2.5-fold decrease of the active IL-1ß form (17 kDa) in the pulmonary tissue of iNO combined with hydrocortisone exposed pigs. We demonstrated that in a porcine endotoxemia model the NO inhalation combined with intravenous hydrocortisone led to the attenuation of the inflammatory cascade induced by bacterial LPS. The decrease in pulmonary MMPs activities was accompanied by reduced proIL-1ß processing.

Assessment of Myocardial Diastolic Dysfunction as a Result of Myocardial Infarction and Extracellular Matrix Regulation Disorders in the Context of Mesenchymal Stem Cell Therapy.

The decline in cardiac contractility due to damage or loss of cardiomyocytes is intensified by changes in the extracellular matrix leading to heart remodeling. An excessive matrix response in the ischemic cardiomyopathy may contribute to the elevated fibrotic compartment and diastolic dysfunction. Fibroproliferation is a defense response aimed at quickly closing the damaged area and maintaining tissue integrity. Balance in this process is of paramount importance, as the reduced post-infarction response causes scar thinning and more pronounced left ventricular remodeling, while excessive fibrosis leads to impairment of heart function. Under normal conditions, migration of progenitor cells to the lesion site occurs. These cells have the potential to differentiate into myocytes in vitro, but the changed micro-environment in the heart after infarction does not allow such differentiation. Stem cell transplantation affects the extracellular matrix remodeling and thus may facilitate the improvement of left ventricular function. Studies show that mesenchymal stem cell therapy after infarct reduces fibrosis. However, the authors did not specify whether they meant the reduction of scarring as a result of regeneration or changes in the matrix. Research is also necessary to rule out long-term negative effects of post-acute infarct stem cell therapy.

Hemosiderin Accumulation in Liver Decreases Iron Availability in Tachycardia-Induced Porcine Congestive Heart Failure Model.

Despite advances in the management of iron deficiency in heart failure (HF), the mechanisms underlying the effects of treatment remain to be established. Iron distribution and metabolism in HF pathogenesis need to be clarified. We used a porcine tachycardia-induced cardiomyopathy model to find out how HF development influences hepatic and myocardial iron storing, focusing on ferritin, the main iron storage protein. We found that cumulative liver congestion (due to the decrease of heart function) overwhelms its capacity to recycle iron from erythrocytes. As a consequence, iron is trapped in the liver as poorly mobilized hemosiderin. What is more, the ferritin-bound Fe3+ (reflecting bioavailable iron stores), and assembled ferritin (reflecting ability to store iron) are decreased in HF progression in the liver. We demonstrate that while HF pigs show iron deficiency indices, erythropoiesis is enhanced. Renin-angiotensin-aldosterone system activation and hepatic hepcidin suppression might indicate stress erythropoiesisinduced in HF. Furthermore, assembled ferritin increases but ferritin-bound Fe3+ is reduced in myocardium, indicating that a failing heart increases the iron storage reserve but iron deficiency leads to a drop in myocardial iron stores. Together, HF in pigs leads to down-regulated iron bioavailability and reduced hepatic iron storage making iron unavailable for systemic/cardiac needs.

Primary Human Cardiomyocytes and Cardiofibroblasts Treated with Sera from Myocarditis Patients Exhibit an Increased Iron Demand and Complex Changes in the Gene Expression.

Cardiac fibroblasts and cardiomyocytes are the main cells involved in the pathophysiology of myocarditis (MCD). These cells are especially sensitive to changes in iron homeostasis, which is extremely important for the optimal maintenance of crucial cellular processes. However, the exact role of iron status in the pathophysiology of MCD remains unknown. We cultured primary human cardiomyocytes (hCM) and cardiofibroblasts (hCF) with sera from acute MCD patients and healthy controls to mimic the effects of systemic inflammation on these cells. Next, we performed an initial small-scale (n = 3 per group) RNA sequencing experiment to investigate the global cellular response to the exposure on sera. In both cell lines, transcriptomic data analysis revealed many alterations in gene expression, which are related to disturbed canonical pathways and the progression of cardiac diseases. Moreover, hCM exhibited changes in the iron homeostasis pathway. To further investigate these alterations in sera-treated cells, we performed a larger-scale (n = 10 for controls, n = 18 for MCD) follow-up study and evaluated the expression of genes involved in iron metabolism. In both cell lines, we demonstrated an increased expression of transferrin receptor 1 (TFR1) and ferritin in MCD serum-treated cells as compared to controls, suggesting increased iron demand. Furthermore, we related TFR1 expression with the clinical profile of patients and showed that greater iron demand in sera-treated cells was associated with higher inflammation score (interleukin 6 (IL-6), C-reactive protein (CRP)) and advanced neurohormonal activation (NT-proBNP) in patients. Collectively, our data suggest that the malfunctioning of cardiomyocytes and cardiofibroblasts in the course of MCD might be related to alterations in the iron homeostasis.

Catabolic/Anabolic Imbalance Is Accompanied by Changes of Left Ventricular Steroid Nuclear Receptor Expression in Tachycardia-Induced Systolic Heart Failure in Male Pigs.

BACKGROUND: Steroid hormones play an important role in heart failure (HF) pathogenesis, and clinical data have revealed disordered steroidogenesis in male patients with HF. However, there is still a lack of studies on steroid hormones and their receptors during HF progression. Therefore, a porcine model of tachycardia-induced cardiomyopathy corresponding to HF was used to assess steroid hormone concentrations in serum and their nuclear receptor levels in heart tissue during the consecutive stages of HF. METHODS AND RESULTS: Male pigs underwent right ventricular pacing and developed a clinical picture of mild, moderate, or severe HF. Serum concentrations of dehydroepiandrosterone, testosterone, dihydrotestosterone, estradiol, aldosterone, and cortisol were assessed by enzyme-linked immunosorbent assay. Androgen receptor, estrogen receptor alpha, mineralocorticoid receptor, and glucocorticoid receptor messenger RNA levels in the left ventricle were determined by qPCR.The androgen level decreased in moderate and severe HF animals, while the corticosteroid level increased. The estradiol concentration remained stable. The quantitative real-time polymerase chain reaction revealed the downregulation of androgen receptor in consecutive stages of HF and increased expression of mineralocorticoid receptor messenger RNA under these conditions. CONCLUSIONS: In the HF pig model, deteriorated catabolic/anabolic balance, manifested by upregulation of aldosterone and cortisol and downregulation of androgen signaling on the ligand level, was augmented by changes in steroid hormone receptor expression in the heart tissue.

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-1ß (IL-1ß) 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-1ß 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.

Sex differences in porcine left ventricular myocardial remodeling due to right ventricular pacing.

BACKGROUND: Although sex differences in heart failure (HF) prevalence and severity have been recognized, its molecular mechanisms are poorly understood. We used a tachycardia-induced cardiomyopathy model to determine the sex specific remodeling pattern in male and female adult pigs. METHODS: We compared the echocardiographic and molecular measures of myocardial remodeling in 19 male and 12 female pigs with chronic symptomatic systolic HF due to right ventricle (RV) pacing (170 bpm) and 6 male and 5 female sham-operated controls. Males achieved subsequent HF stages earlier than females. RESULTS: The progression of symptomatic HF was associated with the reduction of the left ventricle (LV) ejection fraction in both sexes (all p < 0.05). A significant LV dilatation occurred only in males (p < 0.001). The HF development was accompanied by an increased pro-hypertrophic factor GATA4 and TGF-ß1 messenger RNA (mRNA) expression in the LV only in male pigs (all p < 0.01). The total gelatinolytic activity in LV was higher in males than females (irrespective of HF, p < 0.05), and the HF progression was associated with a reduced total gelatinolytic activity (p < 0.05) in the LV only in males. No differences in LV myocardial collagen content were found between HF groups and sexes. Cardiomyocyte cross-sectional diameter was significantly smaller in male hearts as compared to female (p < 0.05). CONCLUSIONS: Male and female porcine hearts respond differently to RV pacing. Males, most likely due to a higher extracellular matrix turnover, demonstrated a significant LV dilatation, followed by a strong induction of pro-hypertrophic program, and an earlier development of symptomatic HF.

Normal electrocardiographic and echocardiographic (M-mode and two-dimensional) values in Polish Landrace pigs.

BACKGROUND: Swine are recognized animal models of human cardiovascular diseases. Normal values of cardiac morphology and function have been published for swine but for smaller number of pigs and not for swine whose weights ranged up 100 kg. In order to improve the value of results of an investigation on cardiac morphology and function in swine when such data are extrapolated to humans, the aim of this study was to document electrocardiographic and echocardiographic measures of cardiac morphology and function in swine. The study comprised 170 single and repeated measurements that were made in 132 healthy domestic swine (Sus domesticus) whose weights ranged between 20-160 kg and were used as controls in three different experiments. All electrocardiographic and echocardiographic measurements in all swine were done under general anaesthesia. RESULTS: Statistically significant correlations were found between body weight and heart rate (HR), the duration of the P-wave, the duration of the QRS interval, the duration of the QT interval, and the corrected QT ratio (QTc). Since body weight was positively correlated with age, statistically significant correlations were also found between age and HR, the duration of the P-wave, the duration of the QRS interval, the duration of the QT interval, and the QTc. We found that the thickness of the left ventricular wall and the internal diameter of the left ventricle increased with age and body weight. We also found positive trends between body weight and ejection fraction and body weight and fractional shortening. We also found a positive relationship between age, body weight, and the ratio of the left ventricular internal diameter to its wall thickness, as well as the relative left atrial size. CONCLUSION: Many electro- and echocardiographic measures of cardiac morphology and function of healthy swine are related to their body weight. When the electro- and echocardiographic measures of domestic swine and humans are compared, the most comparable electrocardiographic values are those that were determined in swine whose body weights are not greater than 70 kg. In contrast, the most comparable echocardiographic measures are those that were determined in swine with a body weight of 40-110 kg.

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 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).

Effects of tamoxifen on estrogen receptor-α level in immune cells and humoral specific response after immunization of C3H/He male mice with syngeneic testicular germ cells (TGC).

Estrogens and estrogen receptors (ERs) are potent regulators of the immune response. Disruption of ERα or modulation of its function by selective ligands during experimental autoimmune conditions changes the course of disease by influencing specific humoral and cellular responses. However, it is not known whether fluctuation in the ERα level and the variable accessibility to its ligands in immune cells influence the development of specific immune responses against auto-antigens. This study was designed to evaluate the expression level of ERα in splenic immune cells and the specific humoral immune response in male C3H/He/W mice immunized with syngeneic testicular germ cells (TGC) in the presence of tamoxifen. Levels of ERα protein in immune cell subpopulations of immunized mice (assessed by flow cytometry) increased in MHCII(+)CD86(+), MHCII(+)CD86(- ), F4/80(+)MHCII(+), immature macrophages (F4/80(+)/MHCII(- )), and CD3(+)CD4(+) T cells. Addition of tamoxifen decreased the level of ERα in MHCII(+)CD86(+), MHCII(+)CD86(- ), F4/80(+)MHCII(+), immature macrophages (F4/80(+)/MHCII(- )), and the CD19(+)CD3(- ) cell subpopulation of immunized mice. Therefore, immunization with syngeneic antigen and tamoxifen treatment evoked cell-type specific changes in the level of ERα. Irrespective of tamoxifen treatment the humoral response in immunized animals toward TGCs was similar, suggesting that modulation of the level of ERα in immune cells is not directly related to specific auto-antibody production.

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.

Increased expression of interleukin-1beta and its novel splice variant in canine hearts with volume overload.

Volume overload frequently caused in dogs by chronic degenerative valvular disease (CDVD), eventually leads to cardiac failure. Experimental and clinical evidences demonstrate that increased interleukin-1beta serum level in patients with heart insufficiency correlates with the severity of failure irrespective of its etiology. Very little is known about the IL-1beta expression in failing vs. non-failing myocardium. IL-1beta transcript level was determined in the CDVD dogs (n=17) and control animals (n=9) without cardiac insufficiency by real-time PCR. IL-1beta transcript level in failing hearts was higher than in the control. In both groups the highest IL-1beta level was detected in the left ventricles. Although IL-1beta is a major pro-inflammatory cytokine most of the CDVD dogs displayed no inflammatory infiltrates into the myocardium. Massive fibrosis was observed in the control group, unlike the failing hearts, in which cardiomyocyte hypertrophy and atrophy dominated. The alternative IL-1beta transcript identified here (IL-1betasv1) was significantly elevated in the failing myocardium compared with the control group. Increased IL-1beta expression seems to be associated with mechanical heart overload. Its endogenous origin, and certain histopathological findings attributed to IL-1beta indicate its importance in cardiac hypertrophy and failure. The lack of some typical IL-1beta actions, i.e. inflammatory, pyrogenic and fibrotic, may suggest a different role of this cytokine in myocardium. It appears that the canine IL-1beta gene can be transcribed in two ways in heart tissue, with the IL-1betasv1 form present mainly in failing hearts.