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Supplemental Digital Content is available in the text., Background: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. Methods: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. Results: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-blockers had a distinct cardiac ECM profile. Conclusions: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Aims We sought to investigate the correlation between speckle-tracking echocardiography (STE)-derived myocardial work (MW) and invasively measured contractility in a rat model of athlete's heart. We also assessed MW in elite athletes and explored its association with cardiopulmonary exercise test (CPET)-derived aerobic capacity. Methods and results Sixteen rats underwent a 12-week swim training program and were compared to controls (n = 16). STE was performed to assess global longitudinal strain (GLS), which was followed by invasive pressure-volume analysis to measure contractility [slope of end-systolic pressure–volume relationship (ESPVR)]. Global MW index (GMWI) was calculated from GLS curves and left ventricular (LV) pressure recordings. In the human investigations, 20 elite swimmers and 20 healthy sedentary controls were enrolled. GMWI was calculated through the simultaneous evaluation of GLS and non-invasively approximated LV pressure curves at rest. All subjects underwent CPET to determine peak oxygen uptake (VO2/kg). Exercised rats exhibited higher values of GLS, GMWI, and ESPVR than controls (−20.9 ± 1.7 vs. −17.6 ± 1.9%, 2745 ± 280 vs. 2119 ± 272 mmHg·%, 3.72 ± 0.72 vs. 2.61 ± 0.40 mmHg/μL, all PExercise < 0.001). GMWI correlated robustly with ESPVR (r = 0.764, P, Graphical Abstract ESPVR, slope of end-systolic pressure–volume relationship (i.e. the slope of the curve connecting the end-systolic points of the pressure–volume loops recorded during the transient occlusion of inferior vena cava); GLS, global longitudinal strain; GMWI, global myocardial work index; LV, left ventricular; VO2/kg, peak oxygen uptake.

Aims While global longitudinal strain (GLS) is considered to be a sensitive marker of left ventricular (LV) function, it is significantly influenced by loading conditions. We hypothesized that global myocardial work index (GMWI), a novel marker of LV function, may show better correlation with load‐independent markers of LV contractility in rat models of pressure‐induced or volume overload‐induced heart failure. Methods and results Male Wistar rats underwent either transverse aortic constriction (TAC; n = 12) or aortocaval fistula creation (ACF; n = 12), inducing LV pressure or volume overload, respectively. Sham procedures were performed to establish control groups (n = 12/12). Echocardiographic loops were obtained to determine GLS and GMWI. Pressure‐volume analysis with transient occlusion of the inferior caval vein was carried out to calculate preload recruitable stroke work (PRSW), a load‐independent ‘gold‐standard’ parameter of LV contractility. Myocardial samples were collected to assess interstitial and perivascular fibrosis area and also myocardial atrial‐type natriuretic peptide (ANP) and brain‐type natriuretic peptide (BNP) relative mRNA expression. Compared with controls, GLS was substantially lower in the TAC group (−7.0 ± 2.8 vs. −14.5 ± 2.5%; P

BACKGROUND Marfan syndrome (MFS) is a systemic connective tissue disorder belonging to a group of rare diseases. Several psychologically distressing factors can challenge life for MFS patients. The aim of the present study was, therefore, to assess the psychological and psychosocial aspects of MFS with the goal of identifying a means of improving disease management for patients. METHODS A total of 66 adult patients with MFS were enrolled into the study prospectively and were divided into operated (OP) and non-operated (NOP) subgroups. Multiple questionnaire tests were used to determine the mental and physical state of our patients. Demographic and surgical data were collected. The results of the tests were also compared to the Hungarostudy (HS) population (representing the average Hungarian population) by using a propensity-matched control. RESULTS OP group scores yielded more alcohol consumption (P

BackgroundClinical evidence suggests that the currently recommended approach to estimate the risk of aortic dissection in Marfan syndrome (MFS) is not reliable enough. Therefore, we investigated the possible role of visceral arterial tortuosity in the risk stratification.Methods and resultsSplenic and renal arteries of 37 MFS patients and 74 age and gender matched control subjects were segmented using CT angiography imaging. To measure tortuosity, distance metric (DM), sum of angles metric (SOAM), inflection count metric (ICM), and the ratio of ICM and SOAM (ICM/SOAM) were calculated. DM of the splenic, right and left renal artery was significantly higher in MFS patients than in controls (2.44 [1.92-2.80] vs. 1.75 [1.57-2.18]p p = 0.011; 1.40 [1.29-1.70] vs. 1.13 [1.09-1.23]p p < 0.001; 26.52 [20.69-30.24] vs. 19.95 [16.47-22.95]p p ConclusionTo our knowledge this is the first demonstration of increased arterial tortuosity in MFS on visceral arteries. Visceral arterial tortuosity, dominated by curves of lower frequency but higher amplitude according to the observed opposite tendency between the DM and SOAM metrics, could be a possible new predictor of serious manifestations of MFS.

Heart transplantation remains the definitive therapy of end-stage heart failure. Ischemia-reperfusion injury occurring during transplantation is a primary determinant of long-term outcome of heart transplantation and primary graft insufficiency. Modification of the nitric oxide/soluble guanylate cyclase/cyclic guanosine monophosphate signaling pathway appears to be one of the most promising among the pharmacological interventional options. We aimed at characterizing the cardio-protective effects of the soluble guanylate cyclase stimulator riociguat in a rat model of heterotopic heart transplantation. Donor Lewis rats were treated orally with either riociguat or placebo for two days (n = 9) in each transplanted group and (n = 7) in donor groups. Following explantation, hearts were heterotopically transplanted. After one hour reperfusion, left ventricular pressure-volume relations and coronary blood flow were recorded. Molecular biological measurements and histological examination were also completed. Left ventricular contractility (systolic pressure: 117 ± 13 vs. 48 ± 5 mmHg, p max: 2963 ± 221 vs. 1653 ± 159 mmHg/s, p min: −2014 ± 305 vs. −1063 ± 177 mmHg/s, p = 0.02; all at 120 µl of left ventricular volume), and alteration of coronary blood flow standardized to heart weight (2.55 ± 0.32 vs. 1.67 ± 0.22 ml/min/g, p = 0.03) were markedly increased following preconditioning with riociguat. Myocardial apoptosis markers were also significantly reduced in the riociguat pretreated group as well as the antioxidant markers were elevated. Pharmacological preconditioning with riociguat decreases ischemia-reperfusion injury and improves donor organ function in our animal model of heart transplantation. Therefore, riociguat might be a potential cardioprotective agent.

Background Biomechanical remodeling of coronary resistance arteries in physiological left ventricular hypertrophy has not yet been analyzed, and the possible sex differences are unknown. Methods Wistar rats were divided into four groups: male and female sedentary controls (MSe and FSe) and male and female animals undergoing a 12-week intensive swim training program (MEx and FEx). On the last day, the in vitro contractility, endothelium-dependent dilatation, and biomechanical properties of the intramural coronary resistance arteries were investigated by pressure microarteriography. Elastica and collagen remodeling were studied in histological sections. Results A similar outer radius and reduced inner radius resulted in an elevated wall to lumen ratio in the MEx and FEx animals compared to that in the sedentary controls. The wall elastic moduli increased in the MEx and FEx rats. Spontaneous and TxA2 agonist-induced tone was increased in the FEx animals, whereas endothelium-dependent relaxation became more effective in MEx rats. Arteries of FEx rats had stronger contraction, while arteries of MEx animals had improved dilation. Conclusions According to our results, the coronary arterioles adapted to an elevated load during long-term exercise, and this adaptation depended on sex. It is important to emphasize that in addition to differences, we also found many similarities between the sexes in the adaptive response to exercise. The observed sport adaptation in the coronary resistance arteries of rats may contribute to a better understanding of the physiological and pathological function of these arteries in active and retired athletes of different sexes.

Coenzyme A (CoA) is an essential cofactor required for intermediary metabolism. Perturbations in homeostasis of CoA have been implicated in various pathologies; however, whether CoA homeostasis is changed and the extent to which CoA levels contribute to ventricular function and remodeling during pressure overload has not been explored. In this study, we sought to assess changes in CoA biosynthetic pathway during pressure overload and determine the impact of limiting CoA on cardiac function. We limited cardiac CoA levels by deleting the rate-limiting enzyme in CoA biosynthesis, pantothenate kinase 1 (Pank1). We found that constitutive, cardiomyocyte-specific Pank1 deletion (cmPank1(−/−)) significantly reduced PANK1 mRNA, PANK1 protein, and CoA levels compared with Pank1-sufficient littermates (cmPank1(+/+)) but exerted no obvious deleterious impact on the mice at baseline. We then subjected both groups of mice to pressure overload-induced heart failure. Interestingly, there was more ventricular dilation in cmPank1(−/−) during the pressure overload. To explore potential mechanisms contributing to this phenotype, we performed transcriptomic profiling, which suggested a role for Pank1 in regulating fibrotic and metabolic processes during the pressure overload. Indeed, Pank1 deletion exacerbated cardiac fibrosis following pressure overload. Because we were interested in the possibility of early metabolic impacts in response to pressure overload, we performed untargeted metabolomics, which indicated significant changes to metabolites involved in fatty acid and ketone metabolism, among other pathways. Collectively, our study underscores the role of elevated CoA levels in supporting fatty acid and ketone body oxidation, which may be more important than CoA-driven, enzyme-independent acetylation in the failing heart. NEW & NOTEWORTHY Changes in CoA homeostasis have been implicated in a variety of metabolic diseases; however, the extent to which changes in CoA homeostasis impacts remodeling has not been explored. We show that limiting cardiac CoA levels via PANK deletion exacerbated ventricular remodeling during pressure overload. Our results suggest that metabolic alterations, rather than structural alterations, associated with Pank1 deletion may underlie the exacerbated cardiac phenotype during pressure overload.

Long-term exercise induces physiological cardiac adaptation, a condition referred to as athlete’s heart. Exercise tolerance is known to be associated with decreased cardiac passive stiffness. Passive stiffness of the heart muscle is determined by the giant elastic protein titin. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B. Titin-based passive stiffness may be controlled by altering the expression of the different isoforms or via post-translational modifications such as phosphorylation. Currently, there is very limited knowledge about titin’s role in cardiac adaptation during long-term exercise. Our aim was to determine the N2BA/N2B ratio and post-translational phosphorylation of titin in the left ventricle and to correlate the changes with the structure and transverse stiffness of cardiac sarcomeres in a rat model of an athlete’s heart. The athlete’s heart was induced by a 12-week-long swim-based training. In the exercised myocardium the N2BA/N2B ratio was significantly increased, Ser11878 of the PEVK domain was hypophosphorlyated, and the sarcomeric transverse elastic modulus was reduced. Thus, the reduced passive stiffness in the athlete’s heart is likely caused by a shift towards the expression of the longer cardiac titin isoform and a phosphorylation-induced softening of the PEVK domain which is manifested in a mechanical rearrangement locally, within the cardiac sarcomere.

BackgroundThe cardiovascular effects of training have been widely investigated; however, few studies have addressed sex differences in arteriolar adaptation. In the current study, we examined the adaptation of the gracilis arterioles of male and female rats in response to intensive training.MethodsWistar rats were divided into four groups: male exercise (ME) and female exercise (FE) animals that underwent a 12-week intensive swim-training program (5 days/week, 200 min/day); and male control (MC) and female control (FC) animals that were placed in water for 5 min daily. Exercise-induced cardiac hypertrophy was confirmed by echocardiography. Following the training, the gracilis muscle arterioles were prepared, and their biomechanical properties and functional reactivity were tested, using pressure arteriography. Collagen and smooth muscle remodeling were observed in the histological sections.ResultsLeft ventricular mass was elevated in both sexes in response to chronic training. In the gracilis arterioles, the inner radius and wall tension increased in female animals, and the wall thickness and elastic modulus were reduced in males. Myogenic tone was reduced in the ME group, whereas norepinephrine-induced vasoconstriction was elevated in the FE group. More pronounced collagen staining was observed in the ME group than in the MC group. Relative hypertrophy and tangential stress of the gracilis arterioles were higher in females than in males. The direct vasoconstriction induced by testosterone was lower in females and was reduced as an effect of exercise in males.ConclusionThe gracilis muscle arteriole was remodeled as a result of swim training, and this adaptation was sex dependent.

Vascular ischemia/reperfusion injury (IRI) contributes to graft failure and adverse clinical outcomes following coronary artery bypass grafting. Sodium-glucose-cotransporter (SGLT)-2-inhibitors have been shown to protect against myocardial IRI, irrespective of diabetes. We hypothesized that adding canagliflozin (CANA) (an SGLT-2-inhibitor) to saline protects vascular grafts from IRI. Aortic rings from non-diabetic rats were isolated and immediately mounted in organ bath chambers (control, n = 9–10 rats) or underwent cold ischemic preservation in saline, supplemented either with a DMSO vehicle (IR, n = 8–10 rats) or 50µM CANA (IR + CANA, n = 9–11 rats). Vascular function was measured, the expression of 88 genes using PCR-array was analyzed, and feature selection using machine learning was applied. Impaired maximal vasorelaxation to acetylcholine in the IR-group compared to controls was significantly ameliorated by CANA (IR 31.7 ± 3.2% vs. IR + CANA 51.9 ± 2.5%, p &lt, 0.05). IR altered the expression of 17 genes. Ccl2, Ccl3, Ccl4, CxCr4, Fos, Icam1, Il10, Il1a and Il1b have been found to have the highest interaction. Compared to controls, IR significantly upregulated the mRNA expressions of Il1a and Il6, which were reduced by 1.5- and 1.75-fold with CANA, respectively. CANA significantly prevented the upregulation of Cd40, downregulated NoxO1 gene expression, decreased ICAM-1 and nitrotyrosine, and increased PECAM-1 immunoreactivity. CANA alleviates endothelial dysfunction following IRI.

Sudden cardiac death among top athletes is very rare, however, it is 2-4 times more frequent than in the age-matched control population. In the present study, the electrophysiological consequences of long-term exercise training were investigated on Ca

Aim Here we aimed at investigating the relation between left ventricular (LV) contractility and myofilament function during the development and progression of pressure overload (PO)-induced LV myocardial hypertrophy (LVH). Methods Abdominal aortic banding (AB) was performed to induce PO in rats for 6, 12 and 18 weeks. Sham operated animals served as controls. Structural and molecular alterations were investigated by serial echocardiography, histology, quantitative real-time PCR and western blot. LV function was assessed by pressure-volume analysis. Force measurement was carried out in permeabilized cardiomyocytes. Results AB resulted in the development of pathological LVH as indicated by increased heart weight-to-tibial length ratio, LV mass index, cardiomyocyte diameter and fetal gene expression. These alterations were already present at early stage of LVH (AB-week6). Furthermore, at more advanced stages (AB-week12, AB-week18), myocardial fibrosis and chamber dilatation were also observed. From a hemodynamic point of view, the AB-wk6 group was associated with increased LV contractility, maintained ventriculo-arterial coupling (VAC) and preserved systolic function. In the same experimental group, increased myofilament Ca2+ sensitivity (pCa50) and hyperphosphorylation of cardiac troponin-I (cTnI) at Threonine-144 was detected. In contrast, in the AB-wk12 and AB-wk18 groups, the initial augmentation of LV contractility, as well as the increased myofilament Ca2+ sensitivity and cTnI (Threonine-144) hyperphosphorylation diminished, leading to impaired VAC and reduced systolic performance. Strong correlation was found between LV contractility parameters and myofilament Ca2+-sensitivity among the study groups. Conclusion Changes in myofilament Ca2+ sensitivity might underlie the alterations in LV contractility during the development and progression of PO-induced LVH.

Background Although exercise-induced cardiac hypertrophy has been intensively investigated, its development and regression dynamics have not been comprehensively described. In the current study, we aimed to characterize the effects of regular exercise training and detraining on left ventricular (LV) morphology and function. Methods Rats were divided into exercised (n = 12) and control (n = 12) groups. Exercised rats swam 200 min/day for 12 weeks. After completion of the training protocol, rats remained sedentary for 8 weeks (detraining period). Echocardiographic follow-up was performed regularly to obtain LV long- and short-axis recordings for speckle-tracking echocardiography analysis. Global longitudinal and circumferential strain and systolic strain rate were measured. LV pressure-volume analysis was performed using additional groups of rats to obtain haemodynamic data. Results Echocardiographic examinations showed the development of LV hypertrophy in the exercised group. These differences disappeared during the detraining period. Strain and strain rate values were all increased after the training period, whereas supernormal values rapidly reversed to the control level after training cessation. Load-independent haemodynamic indices, e.g., preload recruitable stroke work, confirmed the exercise-induced systolic improvement and complete regression after detraining. Conclusions and translational aspect Our results provide the first comprehensive data to describe the development and regression dynamics of morphological and functional aspects of physiological hypertrophy in detail. Speckle-tracking echocardiography has been proven to be feasible to follow-up changes induced by exercise training and detraining and might provide an early possibility to differentiate between physiological and pathological conditions.

OBJECTIVES Hypertension can impair structure and function of blood vessels. Experimental data describing the reverse remodeling process after a mechanical pressure unloading therapy in the vasculature is limited. We studied the influence of pressure unloading on both the structural and functional alterations of the aorta in a hypertensive rat model. METHODS Using isolated thoracic aortic rings in an in-vitro organ bath system, endothelium-dependent and endothelium-independent vasorelaxation were studied 6-weeks or 12-weeks after abdominal aortic banding (aortic banding-6-week or aortic banding-12-week), and 6-weeks after an aortic debanding procedure performed after the sixth experimental week of aortic banding (aortic banding + debanding-12-week). Age-matched rats were sham-operated (sham-6-week or sham-12-week). The aortic morphometry and histological fibrosis were studied, and the mRNA-expression of metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-2, and soluble guanylate cyclase subunits GUCY1a3 and GUCY1b3 were determined. RESULTS Aortic banding significantly increased systolic, diastolic, and pulse pressures. Structural changes (increased intima-media thickness and area normalized to body weight, aortic collagen content, higher MMP-2 and TIMP-2, and lower GUCY1a3 and GUCY1b3 mRNA-levels) and functional alterations (impaired endothelium-dependent and endothelium-independent vasorelaxation) have already taken place after 6 weeks of aortic banding. Pressure unloading, after established vascular changes, improved vascular function, resulted in reduced collagen content, and decreased both MMP-2 and TIMP-2 mRNA-expression. CONCLUSION Pressure-overload-induced vascular changes regressed due to mechanical unloading. Furthermore, debanding leads to a reductive tendency in fibrosis-associated gene expression and collagen accumulation. Collectively, the addition of drugs that target fibrosis to existing hypertensive treatment may present an attractive therapy against vascular remodeling.

In patients undergoing coronary artery bypass grafting (CABG), ischemia/reperfusion injury (IRI) is the main contributor to organ dysfunction. Aging-induced vascular damage may be further aggravated during CABG. Favorable effects of conditioned medium (CM) from mesenchymal stem cells (MSCs) have been suggested against IRI. We hypothesized that adding CM to saline protects vascular grafts from IRI in rats. We found that CM contains 28 factors involved in apoptosis, inflammation, and oxidative stress. Thoracic aortic rings from 15-month-old rats were explanted and immediately mounted in organ bath chambers (aged group) or underwent 24 h of cold ischemic preservation in saline-supplemented either with vehicle (aged-IR group) or CM (aged-IR+CM group), prior to mounting. Three-month-old rats were used as referent young animals. Aging was associated with an increase in intima-to-media thickness, an increase in collagen content, higher caspase-12 mRNA levels, and immunoreactivity compared to young rats. Impaired endothelium-dependent vasorelaxation to acetylcholine in the aged-IR group compared to the aged-aorta was improved by CM (aged 61 ± 2% vs. aged-IR 38 ± 2% vs. aged-IR+CM 50 ± 3%, p &lt, 0.05). In the aged-IR group, the already high mRNA levels of caspase-12 were decreased by CM. CM alleviates endothelial dysfunction following IRI in 15-month-old rats. The protective effect may be related to the inhibition of caspase-12 expression.

Background We aimed to identify sex differences in the network properties and to recognize the geometric alteration effects of long-term swim training in a rat model of exercise-induced left ventricular (LV) hypertrophy. Methods Thirty-eight Wistar rats were divided into four groups: male sedentary, female sedentary, male exercised and female exercised. After training sessions, LV morphology and function were checked by echocardiography. The geometry of the left coronary artery system was analysed on pressure-perfused, microsurgically prepared resistance artery networks using in situ video microscopy. All segments over > 80 μm in diameter were studied using divided 50-μm-long cylindrical ring units of the networks. Oxidative-nitrative (O-N) stress markers, adenosine A2A and estrogen receptor (ER) were investigated by immunohistochemistry. Results The LV mass index, ejection fraction and fractional shortening significantly increased in exercised animals. We found substantial sex differences in the coronary network in the control groups and in the swim-trained animals. Ring frequency spectra were significantly different between male and female animals in both the sedentary and trained groups. The thickness of the wall was higher in males as a result of training. There were elevations in the populations of 200- and 400-μm vessel units in males; the thinner ones developed farther and the thicker ones closer to the orifice. In females, a new population of 200- to 250-μm vessels appeared unusually close to the orifice. Conclusions Physical activity and LV hypertrophy were accompanied by a remodelling of coronary resistance artery network geometry that was different in both sexes.

Although regular exercise training is associated with cardiovascular benefits, the increased risk of atrial arrhythmias has been observed after vigorous exercise and has been related to oxidative stress. We aimed at investigating exercise-induced atrial remodeling in a rat model of an athlete’s heart and determining sex-specific differences. Age-matched young adult rats were divided into female exercised, female control, male exercised, and male control groups. After exercised animals completed a 12-week-long swim training protocol, echocardiography and in vivo cardiac electrophysiologic investigation were performed. Additionally, atrial histological and gene expression analyses were carried out. Post-mortem atrial weight data and histological examination confirmed marked atrial hypertrophy. We found increased atrial gene expression of antioxidant enzymes along with increased nitro-oxidative stress. No gene expression alteration was found regarding markers of pathological remodeling, apoptotic, proinflammatoric, and profibrotic processes. Exercise training was associated with a prolonged right atrial effective refractory period. We could not induce arrhythmias by programmed stimulation in any groups. We found decreased expression of potassium channels. Female gender was associated with lower profibrotic expression and collagen density. Long-term, balanced exercise training-induced atrial hypertrophy is not associated with harmful electrical remodeling, and no inflammatory or profibrotic response was observed in the atrium of exercised rats.

Background Brain death (BD) has been suggested to induce coronary endothelial dysfunction. Ischemia/reperfusion (IR) injury during heart transplantation may lead to further damage of the endothelium. Previous studies have shown protective effects of conditioned medium (CM) from bone marrow-derived mesenchymal stem cells (MSCs) against IR injury. We hypothesized that physiological saline-supplemented CM protects BD rats’ vascular grafts from IR injury. Methods The CM from rat MSCs, used for conservation purposes, indicates the presence of 23 factors involved in apoptosis, inflammation, and oxidative stress. BD was induced by an intracranial-balloon. Controls were subjected to a sham operation. After 5.5 h, arterial pressures were measured in vivo. Aortic rings from BD rats were harvested and immediately mounted in organ bath chambers (BD group, n = 7) or preserved for 24 h in 4 °C saline-supplemented either with a vehicle (BD-IR group, n = 8) or CM (BD-IR+CM group, n = 8), prior to mounting. Vascular function was measured in vitro. Furthermore, immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) have been performed. Results BD in donors was associated with significantly impaired hemodynamic parameters and higher immunoreactivity of aortic myeloperoxidase (MPO), nitrotyrosine, caspase-3, caspase-8, caspase-9, and caspase-12 compared to sham-operated rats. In organ bath experiments, impaired endothelium-dependent vasorelaxation to acetylcholine in the BD-IR group compared to BD rats was significantly improved by CM (maximum relaxation to acetylcholine: BD 81 ± 2% vs. BD-IR 50 ± 3% vs. BD-IR + CM 72 ± 2%, p Conclusions The preservation of BD rats’ vascular grafts with CM alleviates endothelial dysfunction following IR injury, in part, by reducing levels of inflammatory response and caspase-mediated apoptosis.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by the New National Excellence Programme (ÚNKP-19-3-I) of the Ministry for Innovation and Technology in Hungary, and the Artificial Intelligence Research Field Excellence Programme of the National Research, Development and Innovation Office of the Ministry of Innovation and Technology in Hungary. Background Global longitudinal strain (GLS) by speckle-tracking echocardiography (STE) is a sensitive parameter of left ventricular (LV) systolic function. Nevertheless, GLS is dependent on loading conditions. Through the analysis of pressure-strain loops, myocardial work was recently introduced and tested in different clinical scenarios. Myocardial work incorporates afterload, but still, it neglects changes in preload and LV geometry. Purpose Accordingly, our aim was to test our hypothesis that adding instantaneous LV size to myocardial work calculation can further mitigate the load-dependency of GLS, and therefore, a better correlation with intrinsic myocardial contractility can be achieved. Methods Volume overload-induced heart failure was established by an aortocaval fistula (ACF) in male Wistar rats (n = 12). Age-matched sham-operated animals served as controls (n = 12). STE was performed to assess GLS, which was immediately followed by invasive pressure-volume (P-V) analysis to assess LV pressure and to compute a gold-standard index of cardiac contractility (preload recruitable stroke work [PRSW]). Global myocardial work index (GMWI) was calculated from GLS and the invasively measured LV pressure. To compute GMWI indexed to LV area (GMWIA), the instantaneous power (calculated by multiplying the strain rate and the instantaneous LV pressure) was divided by the instantaneous LV area, and then it was integrated from mitral valve closure until mitral valve opening. Results LV ejection fraction did not differ significantly (ACF vs. controls: 59 ± 4 vs. 65 ± 9%, p = NS), whereas GLS (Figure 1A - representative animals) was slightly decreased in the ACF group (-13.2 ± 2.3 vs. -15.4 ± 1.9%, p Conclusions In the case of LV volume overload-induced heart failure, our pressure-area-strain loop-derived metric reflected LV contractility better than GLS and even GMWI. Therefore, the incorporation of instantaneous LV size into myocardial work calculation represents a promising clinical tool to assess and monitor intrinsic myocardial function independently of loading conditions. Abstract Figure 1

Funding Acknowledgements Type of funding sources: None. Speckle-tracking echocardiography (STE)-derived global longitudinal strain (GLS) is considered to be a sensitive marker of left ventricular (LV) function in a wide variety of cardiovascular diseases. Still, evidence suggests that GLS is significantly influenced by loading conditions. Myocardial work index (MWI) evaluates myocardial deformation in the context of afterload through the interpretation of strain in relation to instantaneous LV pressure. MWI may potentially overcome the limitations of mere strain calculation, and may better reflect cardiac contractility in hemodynamic overload states. Accordingly, our aim was to examine the relationship of GLS and MWI with load-independent markers of LV contractility in rat models of pressure- and volume overload-induced heart failure. Male Wistar rats underwent transverse aortic constriction (TAC; n = 12) to generate LV pressure overload, or aortocaval fistula (ACF; n = 12) was established to induce severe LV volume overload. In case of the control groups, sham procedures were performed (n = 12/12). Echocardiography loops were obtained to determine STE-derived GLS and global MWI. Pressure-volume analysis with transient occlusion of the inferior vena cava was carried out to calculate preload recruitable stroke work (PRSW), as a load-independent „gold-standard" parameter of LV contractility. GLS was mildly reduced in the ACF group (-13.2 ± 2.4 vs. -15.4 ± 2.0%, p GLS is significantly influenced by loading conditions, therefore, in case of severe pressure- or volume overload it may not be a reliable marker of LV contractility. In our rat model of pressure overload induced heart failure, contractility was maintained despite decreased GLS, while in the model of volume overload induced heart failure, GLS was maintained despite decreased contractility. MWI reflects contractility in hemodynamic overload states, therefore, it may be a more suitable marker of systolic function. Abstract Figure. Pressure-strain loops of the groups

Objectives: Peripheral arterial disease affects mainly the aorta and lower extremities and commonly results in tissue ischemia. Medical interventions to restore blood flow can induce reperfusion injury. We investigated the effects of both hypertension and ageing on vascular graft responsiveness under normoxia and ischemia/reperfusion (IR) conditions. Methods: Hemodynamic changes and aortic morphometry were studied in 18-month-old spontaneously hypertensive stroke prone (SHRSP, n=8) and age-matched normotensive Wistar (control, n=8) rats. Whereas thoracic aortic rings of the control- and SHRSP groups were immediately mounted in organ bath chambers (normoxia-group), aortic segments in the control-IR and SHRSP-IR groups underwent cold ischemic preservation, prior to mounting. Results: Significantly increased intima-media thickness, intima-media cross-sectional area, and lumen normalized to body weight were observed in SHRSP displaying a systolic blood pressure of 205±16 mmHg compared to controls 124±8 mmHg, p < 0.05. Phenylephrine-induced vasoconstriction was greater in aorta of SHRSP compared to controls. IR injury increased contractile responses to phenylephrine in both control and SHRSP compared to the corresponding groups, which was further increased in SHRSP-IR compared to control-IR (as percentage of KCl-contraction: SHRSP-IR 167±26% vs. control-IR 101±5%, p < 0.05). Compared to controls, vasoconstrictive response to high K+-induced depolarization was significantly reduced in SHRSP, control-IR, and SHRSP-IR. Additionally, KCl-induced contraction was reduced in SHRSP-IR compared to control-IR (SHRSP-IR 0.7±0.1g vs. control-IR 2.5±0.1g, p < 0.05). IR reduced maximum endothelium-dependent relaxation to acetylcholine in the normoxia control group, whereas it had no effect in the aortic rings from SHRSP. Conclusions: IR increases the susceptibility of aortic rings to vascular smooth muscle contractile dysfunction in 18-month-old hypertensive rats.

Introduction: Both sustained left ventricular (LV) pressure overload (PO) and volume overload (VO) induces LV remodeling and eventually development of heart failure (HF). Using rat models, the present study aimed to provide a detailed comparison of distinct aspects of LV function in PO- and VO-induced HF. Methods: PO and VO was induced by transverse aortic constriction (TAC, n=12) and aortocaval shunt (AV-shunt, n=12) creation respectively. Controls underwent corresponding sham operations (n=11). LV remodeling was characterized by echocardiography, histology, qRT PCR, and western blot. LV function was assessed by invasive pressure-volume (P-V) analysis. Results: Both sustained PO and VO resulted in the development of HF, as evidenced by increased LV BNP mRNA expression, pulmonary edema, and characteristic symptoms. While the extent of LV hypertrophy was comparable between the HF models, PO induced concentric while VO evoked eccentric LV remodeling. P-V analysis revealed impaired systolic function in both HF models. Accordingly, decreased ejection fraction and impaired ventriculo-arterial coupling (calculated as the ratio of arterial elastance/LV contractility [VAC]: 0.38±0.05 vs. 1.30±0.13, ShamTAC vs. TAC and 0.52±0.08 vs. 1.17±0.13, ShamAV-Shunt vs. AV-shunt; p0.05) underpinned the contractility-afterload mismatch, while in case of PO the increased afterload (arterial elastance: 0.77±0.07 vs. 2.64±0.28, ShamTAC vs. TAC and 0.80±0.07 vs. 0.54±0.05, ShamAV-Shunt vs. AV-shunt; p Conclusion: Systolic function was reduced in both HF models. However, different factors underpinned the impaired VAC in case of VO (reduced LV contractility) and PO (increased arterial elastance). Furthermore, although diastolic function deteriorated in both models, it occurred to a greater extent in case of PO.

Background Two-dimensional (2D) speckle tracking echocardiography (STE)-derived myocardial strain parameters are sensitive markers of left ventricular (LV) systolic function. Novel findings suggest that the contractile state of the myocardium, afterload and preload are major determinants of STE measurements. However, the hypothesis that longitudinal strain expresses the interaction between contractility and loading conditions rather than contractility alone in hemodynamic overload-induced heart failure (HF) has not been tested. Purpose This study aimed to explore the connection between longitudinal strain and contractility, afterload and preload in rat models of pressure overload (PO)- and volume overload (VO)-induced heart failure (HF). Methods Pressure overload (PO)-induced HF was evoked by transverse aortic constriction ([TAC], n=14). Volume overload (VO)-induced HF was established by an aortocaval fistula ([ACF], n=12). Age-matched sham operated animals served as controls. Pressure-volume analysis was carried out to compute cardiac contractility (slope of end-systolic pressure-volume relationship [ESPVR]), afterload (arterial elastance [Ea]) and ventriculo-arterial coupling ([VAC] = Ea/ESPVR). Preload was evaluated by meridional end-diastolic wall stress (σend-diastolic). STE was performed to assess global longitudinal strain (GLS). Results GLS was impaired in both PO-induced HF (−5.9±0.6 vs. −12.9±0.5%, TAC vs Sham, P Conclusion Both afterload and contractility define GLS. Hence, under conditions when both factors become altered, GLS reflects VAC. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): NVKP_16-1-2016-0017

Introduction In patients with chronic heart failure (CHF) left ventricular dysfunction results in elevated left atrial (LA) pressure, triggering pathological atrial remodelling and atrial fibrillation (AF). Nevertheless, it has been reported that some patients with CHF remain in sinus rhythm (SR) despite of the pathological structural alterations (e.g. dilation and fibrosis) of the LA. Of particular interest, data is scarce regarding the molecular explanation for the observed variability in AF development among CHF patients. Recent studies have indicated that alterations in microRNA (miRNA) expression might contribute to the pathogenesis of AF. However, the majority of previous studies focusing on miRNA expression compared healthy LA with SR to pathologically remodelled, dilated LA with AF. Consequently, whether dysregulation of miRNA expression directly contribute to AF and not only to pathological LA remodelling has not been tested before. Purpose The present study aimed to investigate miRNA expression in comparably remodelled LA from end-stage CHF patients with permanent AF (CHF-AF) or SR (CHF-SR). Methods LA samples were collected from male, non-diabetic, ischemic end-stage CHF patients undergoing heart transplantation (n=24). Patients were carefully selected to avoid any differences in age (55±2 vs. 54±2 years, CHF-AF vs. CHF-SR, n.s.), ejection fraction ([EF]: 22.5±1.8 vs. 23.3±2.5%, CHF-AF vs. CHF-SR, n.s.) LA diameters (longitudinal LA diameter: 56±4 vs. 48±5mm.; CHF-AF vs. CHF-SR, n.s.; horizontal LA diameter: 61±2 vs. 54±3, CHF-AF vs. CHF-SR, n.s.) and NYHA stage. As a molecular marker of atrial load, the mRNA expression of atrial natriuretic peptide (ANP) was measured with qRT-PCR. The extent of left atrial fibrosis was assessed on picrosirius red stained histological sections. Global LA miRNA expression profiling (including the measurement of 800 human miRNA) was carried out using a commercially available kit. Results LA mRNA expression of ANP was comparable between the AF-CHF and the SR-CHF groups, suggesting that atrial load occurred to the same level in the two experimental groups. Furthermore, no differences could be observed in the extent of atrial collagen content between the AF-CHF and the SR-CHF groups (collagen area: 20.3±1.3% vs. 23.9±3.1%, n.s.), providing evidence that fibrotic remodelling had occurred to a similar magnitude. The high-throughput miRNA measurement revealed no differences in atrial miRNA expression between the two study groups. Conclusion The present study provides evidence for the first time that AF is not associated with different LA miRNA expression in end-stage CHF patients with comparable level of LA dilatation, ANP expression (atrial load) and interstitial fibrosis. Based on these findings, the potential of miRNA-based therapeutic interventions might be limited in AF patients with ischemic end-stage CHF. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): NVKP_16-1-2016-0017

Background Regular, vigorous exercise induces morphological changes in the heart, including increased left ventricular (LV) volumes and mass. Although this represents a physiological adaptation to the increased cardiovascular demand, in a certain number of highly trained athletes, the LV remodelling is accompanied by decreased resting values of global longitudinal strain (GLS), exceeding even pathological limits. In this case, the differential diagnosis of overlapping pathological conditions represents a clinical challenge. Myocardial work (MW) evaluates myocardial deformation in the context of afterload; therefore, it may potentially overcome the limitations of GLS, and may better reflect the contractility of the athlete's heart. Purpose We sought to evaluate MW of elite athletes compared to sedentary volunteers. We also aimed to validate its robustness by investigating its relation to the invasively measured parameters of myocardial contractility in a rat model of the athlete's heart. Methods A total of 30 elite swimmers (19±4 years, 23±5 hours of training/week, 50% males) and 23 healthy sedentary controls (19±4 years, 40% males) were enrolled. Global MW index (GMWI) and constructive MW index (CMWI) were calculated using a vendor-specific module through the simultaneous evaluation of GLS by speckle-tracking echocardiography (STE) and the non-invasively approximated LV pressure curves (estimated from brachial cuff systolic pressure). In the rat model, LV hypertrophy was induced by swim training (n=18, 200 min/day for 12 weeks). An additional group of 17 untrained control rats were examined as well. STE was performed to assess GLS, which was followed by invasive pressure-volume (P-V) analysis to register LV pressure and to calculate cardiac contractility (slope of end-systolic P-V relationship [ESPVR]). GMWI and CMWI were assessed by evaluating the area of the LV pressure-strain loop. Results GLS was decreased in human athletes (athletes vs. controls: −18±2 vs. −19±1, p Conclusions Due to the adaptive LV remodelling, resting markers of LV systolic function may be decreased in elite athletes. Despite the reduced GLS, GMWI was preserved, and CMWI was supernormal in human athletes and the experimental model verified their correlation with contractility. Our findings indicate that MW indices in athletes may be better markers of systolic function compared to GLS. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office of Hungary

Introduction In diabetic patients, multiple cardiovascular outcome trials consistently showed the robust cardioprotective effects of the novel antidiabetic agents, sodium glucose cotransporter 2 (SGLT2) inhibitors. However, the DAPA-HF study using the SGLT2 inhibitor dapagliflozin have extended these observations onto non-diabetic patients with heart failure (HF), urging previous hypotheses regarding the cardioprotective effects of SGLT2 inhibitors to be revised. This is further complicated by the fact that SGLT2 is not expressed in the human myocardium neither under normal nor diseased states. Hence, it has been postulated that SGLT2 inhibitors might exert direct cardioprotection via non-specific inhibition of SGLT1, which is in turn highly expressed in the myocardium. Purpose Because literature data is scarce regarding the expression profile of myocardial SGLT1, we aimed to characterize left ventricular SGLT1 expression in humans with end-stage HF accordingly to HF aetiology and to investigate whether cardiac resynchronization therapy (CRT) affects SGLT1 expression. Methods From patients undergoing mitral valve replacement with otherwise no myocardial disease and preserved LV function, we collected control papillary muscles (Control, n=9). From patients with end-stage HF undergoing heart transplantation (n=72), we obtained LV anterior wall samples according to the following HF aetiology groups: hypertrophic cardiomyopathy (HCM, n=7); idiopathic dilated cardiomyopathy (DCM, n=12); ischaemic heart disease (IHD, n=14), IHD with type 2 diabetes mellitus (IHD+T2DM, n=11); and patients with CRT (CRT-DCM, n=9; CRT-IHD, n=9; CRT-IHD+T2DM, n=10). We measured LV SGLT1 expression on the gene and protein expression levels using qRT-PCR and western blotting, respectively. Echocardiography-derived LV end-diastolic diameter (LVEDD) and LV ejection fraction (LVEF) were registered prior to surgery. Results Compared to controls, LV SGLT1 mRNA and protein expressions were significantly upregulated in patients with DCM, IHD and IHD+T2DM (all P Conclusions Myocardial SGLT1 is upregulated in patients with HF (except HCM), and correlated strongly with parameters (LVEDD, LVEF) related to adverse LV remodelling. CRT was associated with reduced SGLT1 expression in DCM and IHD patients, but not in those with IHD+T2DM. Our results suggest that SGLT1 is upregulated in HF and might be implicated in adverse myocardial remodelling. Accordingly, whether SGLT2 inhibitors exert direct cardioprotection in HFrEF via non-specific inhibition of SGLT1 needs to be further elucidated. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Fund of Hungary, Higher Education Institutional Excellence Programme of the Ministry of Human Capacities of Hungary

Background Inflammation and cytokine release have been implicated in the pathogenesis of chronic heart failure (CHF). Of particular interest, Canakinumab, a monoclonal antibody against interleukin-1b (IL-1β), had provided benefit against cardiovascular events, suggesting that blockade of IL-1β secretion and signaling might be a promising new therapeutic target. Although, recent studies have provided evidence that inflammasome activation is the main contributor to IL-1β maturation, the role of inflammasome activation in CHF remains unknown. Objective Therefore, we aimed to assess inflammasome activation in myocardial samples from end-stage failing hearts. Methods Inflammasome activation was assessed by immunoblotting in left ventricular myocardial specimens harvested from patients with end-stage CHF. Furthermore, immunoblot measurements were also performed on translational animal models of CHF (e.g. rat models of permanent coronary artery ligation and transverse aortic constriction). Left ventricular monocyte and macrophage infiltration was detected by immunohistochemistry. To investigate the molecular background of inflammasome activation, a series of cell culture experiments were performed on AC16 human cardiomyocytes and THP-1 human monocytic cell lines. Results Out of the 4 major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human CHF while the NLRP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change. A similar expression pattern in AIM2 and NLRC4 was also noted in CHF animal models. Furthermore, robust infiltration of Iba1+ monocytes/macrophages was observed in human failing hearts as well as in different animal models of CHF. In vitro AIM2 inflammasome activation, as induced by transfection with double-stranded DNA [poly(deoxyadenylic-deoxythymidylic)] was reduced significantly by the pharmacological blockade of pannexin-1 channels. Conclusions AIM2 and NLRC4 inflammasome activation might contribute to chronic inflammation in CHF. Our findings suggest that pannexin-1 channels might be a promising novel target to reduce inflammasome activation. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): NVKP_16-1-2016-0017

Sudden cardiac death among top athletes is very rare, however, it is 2–4 times more frequent than in the age-matched control population. In the present study, the electrophysiological consequences of long-term exercise training were investigated on Ca2+ homeostasis and ventricular repolarization, together with the underlying alterations of ion channel expression, in a rat athlete's heart model. 12-week swimming exercise-trained and control Wistar rats were used. Electrophysiological data were obtained by using ECG, patch clamp and fluorescent optical measurements. Protein and mRNA levels were determined by the Western immunoblot and qRT-PCR techniques. Animals in the trained group exhibited significantly lower resting heart rate, higher incidence of extrasystoles and spontaneous Ca2+ release events. The Ca2+ content of the sarcoplasmic reticulum (SR) and the Ca2+ transient amplitude were significantly larger in the trained group. Intensive physical training is associated with elevated SR Ca2+ content, which could be an important part of physiological cardiac adaptation mechanism to training. However, it may also sensitize the heart for the development of spontaneous Ca2+ release and extrasystoles. Training-associated remodeling may promote elevated incidence of life threatening arrhythmias in top athletes.

Background The cardiac functional consequences of ischaemic stroke are still need to be elucidated, while according to ethical issues only non-inasive measurents were carried out in patients underwent transient cerebral ischaemia. Purpose We aimed at investigating left ventricular function using non-invasive and invasive modalities in a rat model of transient focal ischaemia. Methods Age-matched, young adult rats were used for this study. Serial left ventricular echocardiographic measurements and speckle-tracking analysis were performed in rats (n=9) underwent transient middle cerebral artery occlusion (MCAO) before, during and immediately after the induction of stroke, with a follow-up at 24, 48, 72 hours; 7, 11 and 14 days. In another experimental setting, 48 hours after stroke induction (MCAO group, n=9) we characterized left ventricular function by pressure-volume analysis, that was compared to sham-operated controls (Co group, n=9). Results Serial echocardiographic measurements showed impaired systolic function, that was most severe 48 hours after MCAO (global circumferential strain, GCS: −14.8±2.6% 48 hours after MCAO vs. −19.3±2.4% baseline, p Conclusions Our data suggests that MCAO is associated with reversible impairment of systolic function during echocardiographic measurements, however without alteration of intrinsic myocardial contractility. The tendency towards increased afterload might explain the observed alterations in rats underwent stroke. Funding Acknowledgement Type of funding source: None

Background When indicated, cardiac resynchronization therapy (biventricular pacing, CRT) decreases mortality in patients with heart failure (HF) and reduced ejection fraction, especially in those with non-ischemic cardiomyopathy. This is reflected by relatively rapid improvement of left ventricular (LV) end-diastolic diameter (LVEDD) and LV ejection fraction (LVEF) indicating reverse remodelling. These LV structural and functional improvements are accompanied by characteristic changes in LV gene expression profile. However, whether beneficial gene expression alterations related to biventricular pacing are sustained independently of structural and functional reverse remodelling is unclear. Purpose We aimed to compare LV fibrosis-related mRNA expression profile in end-stage HF patients with idiopathic dilated cardiomyopathy (DCM) who were not on CRT versus to those on CRT. Methods Left ventricular myocardial samples were harvested from end-stage HF patients undergoing heart transplantation (HTX). Inclusion criteria were negative family history of DCM, negative coronarography (i.e. non-ischemic), no relevant comorbidity (e.g. diabetes, hypertension) and no history of myocarditis. Accordingly, the following patient groups were included: 1.) DCM (n=12, 17% female, mean age [±standard deviation] 46.8±11.8 years) without CRT and 2.) CRT-DCM (n=12, 42% female, mean age 47.8±12.3 years) which comprised DCM patients on active CRT for mean 3.2±2.4 years until HTX. LV RNA was extracted and subjected to a commercially available mRNA expression panel interrogating 760 genes related to the development and regulation of fibrosis. Normalization to 10 housekeeping genes and batch corrections were conducted as per protocol. LV mRNA expression of atrial-natriuretic peptide (ANP) was quantified using qRT-PCR. Results Markers of reverse remodelling including LVEDD (73.4±8.3 mm vs 75.4±9.9 mm), LVEF (21.9±3.7% vs 18.5±6.8%) and LV ANP mRNA expression (arbitrary units: 1.05±1.80 vs 1.04±0.88) were comparable between DCM and CRT-DCM patients (all P>0.05), respectively. High-throughput mRNA expression screening revealed significant (all P Conclusions Independently of structural and functional reverse remodelling, CRT might be associated with slightly favourable LV expression profile of genes related to the regulation and development of fibrosis. This suggests that biventricular pacing might be beneficial on the molecular level beyond improvement of LV structure and function. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Fund of Hungary, Higher Education Institutional Excellence Programme of the Ministry of Human Capacities of Hungary

BackgroundMarfan syndrome (MFS) is a systemic connective tissue disorder with life-threatening manifestations affecting the ascending aorta. MFS is caused by dominant negative (DN) and haploinsufficient (HI) mutations of theFBN1gene. Our aim was to identify mutations of MFS patients with high detection rate and to investigate the use of a gene panel for patients with Marfanoid habitus. We also aimed to examine correlations between genotype and cardiovascular manifestations to predict “malignant” mutations.Methods136 individuals were enrolled. In the first phase, next-generation sequencing (NGS) and Sanger sequencing were performed for 57 patients to screen theFBN1gene, followed by multiplex ligation-dependent probe amplification (MLPA) in negative cases. For repeated negative results, NGS gene panel involving 9 genes was used. In the second phase, 79 patients were tested primarily with the same gene panel, negative samples were tested by MLPA.Results84 pathogenic mutations were detected, out of which 78 affectedFBN1, 6 non-FBN1mutations (2TGFB2, 1TGFBR2, 2TGFBR1, 1SMAD3) are associated with Loeys-Dietz syndrome (LDS). LDS patients had lower systemic score and they were younger, but their aortic involvement did not differ. MLPA detected 4 multi-exon deletions ofFBN1gene, which could not be identified by our first-step screening method. Aortic involvement (aortic dissection and/or dilation) did not differ significantly among HI and DN mutations (p = 0.061). Combined group of HI and DN mutations eliminating a disulphide-bonding cysteine (DN Cys) had significantly higher aortic involvement rate than DN mutations not eliminating a disulphide-bonding cysteine (DN non-Cys) (p p = 0.042 andp = 0.015, respectively).ConclusionsDue to the relevant number of mutations affecting genes other thanFBN1, preferred approach for testing individuals with Marfanoid habitus is using a gene panel rather than single-gene analysis, followed by MLPA for negative samples. DN Cys and HI mutations should be considered as risk factors for aortic involvement. Genetic testing for patients with Marfanoid features and a systemic score under 7 is recommended, as LDS patients may have lower scores, but they may have severe cardiovascular manifestations.

BACKGROUND Exercise training is associated with hypertrophy of left ventricle (LV). The aim of the present study is to evaluate sex differences in the adaptation of the coronary contractile function in physiological left ventricular hypertrophy induced by long-term swim training. METHODS Thirty-two Wistar rats were randomly divided into 4 groups: exercised male (ExM), exercised female (ExF), untrained control male (CoM), and untrained control female (CoF). The trained animals underwent a 12-week-long swim training program. After finishing the training program, LV morphology and function were checked by echocardiography. The spontaneous tone, thromboxane (TxA 2 ) agonist-induced vascular contractility and non-endothelial dilatation of the isolated intramural coronary resistance artery were examined by pressure microangiometry. The thromboxane receptor (TxA 2 R) protein expression in the wall of coronary arteries was examined using immunohistochemistry. RESULTS The LV mass index was significantly higher in the ExM and ExF groups, furthermore the LV mass index was significantly higher in female than in male animals. ExM animals had lower spontaneous tone than ExF. TxA 2 agonist-induced tone was raised only in ExF animals. The resistance coronary artery of exercised male animals had a significantly lower level of TxA 2 R positivity compared to exercised females. CONCLUSIONS Both sexes broaden their range of contractility following chronic swimming, but the vessel tone shifted toward contraction in exercised female rats, while these values shifted toward relaxation in males. These observations underline the significance of identifying potential gender differences in the chronic exercise-induced coronary vascular remodeling in human athletes.

Aims/hypothesis Large cardiovascular outcome trials demonstrated that the cardioprotective effects of sodium–glucose cotransporter 2 (SGLT2) inhibitors might reach beyond glucose-lowering action. In this meta-analysis, we sought to evaluate the potential infarct size-modulating effect of SGLT2 inhibitors in preclinical studies. Methods In this preregistered meta-analysis (PROSPERO: CRD42020189124), we included placebo-controlled, interventional studies of small and large animal models of myocardial ischaemia–reperfusion injury, testing the effect of SGLT2 inhibitor treatment on myocardial infarct size (percentage of area at risk or total area). Standardised mean differences (SMDs) were calculated and pooled using random-effects method. We evaluated heterogeneity by computing Τ2 and I2 values. Meta-regression was performed to explore prespecified subgroup differences according to experimental protocols and their contribution to heterogeneity was assessed (pseudo-R2 values). Results We identified ten eligible publications, reporting 16 independent controlled comparisons on a total of 224 animals. Treatment with SGLT2 inhibitor significantly reduced myocardial infarct size compared with placebo (SMD = −1.30 [95% CI −1.79, −0.81], p Τ2 = 0.58, I2 = 60%). SGLT2 inhibitors were only effective when administered to the intact organ system, but not to isolated hearts (p interaction R2 = 47%). While acute administration significantly reduced infarct size, chronic treatment was superior (p interaction R2 = 85%). The medications significantly reduced infarct size in both diabetic and non-diabetic animals, favouring the former (p interaction = 0.030, adjusted pseudo-R2 = 12%). Treatment was equally effective in rats and mice, as well as in a porcine model. Individual study quality scores were not related to effect estimates (p = 0.33). The overall effect estimate remained large even after adjusting for severe forms of publication bias. Conclusions/interpretation The glucose-lowering SGLT2 inhibitors reduce myocardial infarct size in animal models independent of diabetes. Future in vivo studies should focus on clinical translation by exploring whether SGLT2 inhibitors limit infarct size in animals with relevant comorbidities, on top of loading doses of antiplatelet agents. Mechanistic studies should elucidate the potential relationship between the infarct size-lowering effect of SGLT2 inhibitors and the intact organ system. Graphical abstract

BACKGROUND Maintenance of cell viability during cold storage is a key issue in organ transplantation. Methane (CH4) bioactivity has recently been recognized in ischemia/reperfusion conditions; we therefore hypothesized that cold storage in CH4-enriched preservation solution can provide an increased defense against organ dysfunction during experimental heart transplantation (HTX). METHODS The hearts of donor Lewis rats were stored for 60 minutes in cold histidine-tryptophan-ketoglutarate (Custodiol [CS]) or CH4-saturated CS solution (CS-CH4) (n = 12 each). Standard heterotopic HTX was performed, and 60 minutes later, the left ventricular (LV) pressure-volume relationships LV systolic pressure (LVSP), systolic pressure increment (dP/dtmax), diastolic pressure decrement, and coronary blood flow (CBF) were measured. Tissue samples were taken to detect proinflammatory parameters, structural damage (by light microscopy), endoplasmic reticulum (ER) stress, and apoptosis markers (CCAAT/enhancer binding protein [C/EBP] homologous protein, GRP78, glycogen synthase kinase-3β, very low-density lipoprotein receptor, caspase 3 and 9, B-cell lymphoma 2, and bcl-2-like protein 4), whereas mitochondrial functional changes were analyzed by high-resolution respirometry. RESULTS LVSP and dP/dtmax increased significantly at the largest pre-load volumes in CS-CH4 grafts as compared with the CS group (114.5 ± 16.6 mm Hg vs 82.8 ± 4.6 mm Hg and 3,133 ± 430 mm Hg/s vs 1,739 ± 169 mm Hg/s, respectively); the diastolic function and CBF (2.4 ± 0.4 ml/min/g vs 1.3 ± 0.3 ml/min/g) also improved. Mitochondrial oxidative phosphorylation capacity was more preserved (58.5 ± 9.4 pmol/s/ml vs 27.7 ± 6.6 pmol/s/ml), and cytochrome c release was reduced in CS-CH4 storage. Signs of HTX-caused myocardial damage, level of ER stress, and the transcription of proapoptotic proteins were significantly lower in CS-CH4 grafts. CONCLUSION The addition of CH4 during 1 hour of cold storage improved early in vitro graft function and reduced mitochondrial dysfunction and activation of inflammation. Evidence shows that CH4 reduced ER stress–linked proapoptotic signaling.

Rationale The beta-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a pro-adaptive response to cellular insults. To this end, increased protein O-GlcNAcylation improves short-term survival of cardiomyocytes subjected to acute injury. This observation has been repeated by multiple groups and in multiple models; however, whether increased protein O-GlcNAcylation plays a beneficial role in more chronic settings remains an open question. Objective Here, we queried whether increasing levels of cardiac protein O-GlcNAcylation would be beneficial during infarct-induced heart failure. Methods and results To achieve increased protein O-GlcNAcylation, we targeted Oga, the gene responsible for removing O-GlcNAc from proteins. Here, we generated mice with cardiomyocyte-restricted, tamoxifen-inducible haploinsufficient Oga gene. In the absence of infarction, we observed a slight reduction in ejection fraction in Oga deficient mice. Overall, Oga reduction had no major impact on ventricular function. In additional cohorts, mice of both sexes and both genotypes were subjected to infarct-induced heart failure and followed for up to four weeks, during which time cardiac function was assessed via echocardiography. Contrary to our prediction, the Oga deficient mice exhibited exacerbated—not improved—cardiac function at one week following infarction. When the observation was extended to 4 wk post-MI, this acute exacerbation was lost. Conclusions The present findings, coupled with our previous work, suggest that altering the ability of cardiomyocytes to either add or remove O-GlcNAc modifications to proteins exacerbates early infarct-induced heart failure. We speculate that more nuanced approaches to regulating O-GlcNAcylation are needed to understand its role—and, in particular, the possibility of cycling, in the pathophysiology of the failing heart.

The use of hearts with left-ventricular (LV) hypertrophy (LVH) could offer an opportunity to extend the donor pool for cardiac transplantation. We assessed the effects of LVH in 18-month-old spontaneously hypertensive stroke-prone (SHRSP) donor rats and following transplantation. In donors, cardiac function and structural alterations were assessed. Then, the hearts were transplanted into young normotensive-rats. We evaluated LV graft function 1 h after transplantation. The myocardial expression of 92 genes involved in apoptosis, inflammation, and oxidative-stress was profiled using PCR-array. Compared to controls, SHRSP-rats developed LVH, had increased LV systolic performance (slope of the end-diastolic pressure-volume (PV) relationship: 1.6±0.2 vs 0.8±0.1mmHg/μl,ppesratio after versus before transplantation: 32% vs 29%,p>0.05). Whereas nitrotyrosine immunoreactivity was higher, myeloperoxidase-positive cell infiltration was decreased in the SHRSP+transplanted compared to control+transplanted. Among the tested genes, LVH was associated with altered expression of 38 genes in donors, while transplantation of these hearts resulted in the change of four genes. Alterations in 18-month-old donor hearts, as a consequence of hypertension and LVH, were not associated with graft dysfunction in the early phase of reperfusion after transplantation.

Background and purpose Incidence and severity of obesity are increasing worldwide, however, efficient and safe pharmacological treatments are not yet available. Certain MAO inhibitors reduce body weight, although their effects on metabolic parameters have not been investigated. Here, we have assessed effects of a widely used, selective MAO-B inhibitor, selegiline, on metabolic parameters in a rat model of diet-induced obesity. Experimental approach Male Long-Evans rats were given control (CON) or a high-fat (20%), high-sucrose (15%) diet (HFS) for 25 weeks. From week 16, animals were injected s.c. with 0.25 mg·kg-1 selegiline (CON + S and HFS + S) or vehicle (CON, HFS) once daily. Whole body, subcutaneous and visceral fat was measured by CT, and glucose and insulin tolerance were tested. Expression of glucose transporters and chemokines was assessed by quantitative RT-PCR. Key results Selegiline decreased whole body fat, subcutaneous- and visceral adiposity, measured by CT and epididymal fat weight in the HFS group, compared with HFS placebo animals, without influencing body weight. Oral glucose tolerance and insulin tolerance tests showed impaired glucose homeostasis in HFS and HFS + S groups, although insulin levels in plasma and pancreas were unchanged. HFS induced expression of Srebp-1c, Glut1 and Ccl3 in adipose tissue, which were alleviated by selegiline. Conclusions and implications Selegiline reduced adiposity, changes in adipose tissue energy metabolism and adipose inflammation induced by HFS diet without affecting the increased body weight, impairment of glucose homeostasis, or behaviour. These results suggest that selegiline could mitigate harmful effects of visceral adiposity.

While heart transplantation (HTX) is the definitive therapy of heart failure, donor shortage is emerging. Pharmacological activation of soluble guanylate cyclase (sGC) and increased cGMP-signalling have been reported to have cardioprotective properties. Gemfibrozil has recently been shown to exert sGC activating effects in vitro. We aimed to investigate whether pharmacological preconditioning of donor hearts with gemfibrozil could protect against ischemia/reperfusion injury and preserve myocardial function in a heterotopic rat HTX model. Donor Lewis rats received p.o. gemfibrozil (150 mg/kg body weight) or vehicle for 2 days. The hearts were explanted, stored for 1 h in cold preservation solution, and heterotopically transplanted. 1 h after starting reperfusion, left ventricular (LV) pressure-volume relations and coronary blood flow (CBF) were assessed to evaluate early post-transplant graft function. After 1 h reperfusion, LV contractility, active relaxation and CBF were significantly (p

Decreased soluble guanylate cyclase activity and cGMP levels in diabetic kidneys were shown to influence the progression of nephropathy. The regulatory effects of soluble guanylate cyclase activators on renal signaling pathways are still unknown, we therefore investigated the renal molecular effects of the soluble guanylate cyclase activator cinaciguat in type-1 diabetic (T1DM) rats. Male adult Sprague-Dawley rats were divided into 2 groups after induction of T1DM with 60 mg/kg streptozotocin: DM, untreated (DM, n = 8) and 2) DM + cinaciguat (10 mg/kg per os daily, DM-Cin, n = 8). Non-diabetic untreated and cinaciguat treated rats served as controls (Co (n = 10) and Co-Cin (n = 10), respectively). Rats were treated for eight weeks, when renal functional and molecular analyses were performed. Cinaciguat attenuated the diabetes induced proteinuria, glomerulosclerosis and renal collagen-IV expression accompanied by 50% reduction of TIMP-1 expression. Cinaciguat treatment restored the glomerular cGMP content and soluble guanylate cyclase expression, and ameliorated the glomerular apoptosis (TUNEL positive cell number) and podocyte injury. These effects were accompanied by significantly reduced TGF-ß overexpression and ERK1/2 phosphorylation in cinaciguat treated diabetic kidneys. We conclude that the soluble guanylate cyclase activator cinaciguat ameliorated diabetes induced glomerular damage, apoptosis, podocyte injury and TIMP-1 overexpression by suppressing TGF-ß and ERK1/2 signaling.

In diabetes, retinal dysfunctions exist prior to clinically detectable vasculopathy, however the pathology behind these functional deficits is still not fully established. Previously, our group published a detailed study on the retinal histopathology of type 1 diabetic (T1D) rat model, where specific alterations were detected. Although the majority of human diabetic patients have type 2 diabetes (T2D), similar studies on T2D models are practically absent. To fill this gap, we examined Zucker Diabetic Fatty (ZDF) rats - a model for T2D - by immunohistochemistry at the age of 32 weeks. Glial reactivity was observed in all diabetic specimens, accompanied by an increase in the number of microglia cells. Prominent outer segment degeneration was detectable with changes in cone opsin expression pattern, without a decrease in the number of labelled elements. The immunoreactivity of AII amacrine cells was markedly decreased and changes were detectable in the number and staining of some other amacrine cell subtypes, while most other cells examined did not show any major alterations. Overall, the retinal histology of ZDF rats shows a surprising similarity to T1D rats indicating that despite the different evolution of the disease, the neuroretinal cells affected are the same in both subtypes of diabetes.

Background Marfan syndrome is a genetic disease, presenting with dysfunction of connective tissues leading to lesions in the cardiovascular and skeletal muscle system. Within these symptoms, the most typical is weakness of the connective tissue in the aorta, manifesting as aortic dilatation (aneurysm). This could, in turn, become annuloaortic ectasia, or life-threatening dissection. As a result, life-saving and preventative cardiac surgical interventions are frequent among Marfan syndrome patients. Aortic aneurysm could turn into annuloaortic ectasia or life-threatening dissection, thus life-saving and preventive cardiac surgical interventions are frequent among patients with Marfan syndrome. We hypothesized that patients with Marfan syndrome have different level of anxiety, depression and satisfaction with life compared to that of the non-clinical patient population. Methods Patients diagnosed with Marfan syndrome were divided into 3 groups: those scheduled for prophylactic surgery, those needing acute surgery, and those without need for surgery (n = 9, 19, 17, respectively). To examine the psychological features of the patients, Spielberger’s anxiety (STAI) test, Beck’s Depression questionnaire (BDI), the Berne Questionnaire of Subjective Well-being, and the Satisfaction with Life scale were applied. Results A significant difference was found in trait anxiety between healthy individuals and patients with Marfan syndrome after acute life-saving surgery (p 0.1). Finally, a significant, medium size effect was found between patient groups on the Joy in Living scale (F (2.39) = 3.51, p = 0.040, η2 = 0.15). Conclusions Involving psychiatric and mental-health care, in addition to existing surgical treatment interventions, is essential for more successful recovery of patients with Marfan syndrome.

Heart transplantation is the therapy of choice for end-stage heart failure. However, hemodynamic instability, which has been demonstrated in brain-dead donors (BDD), could also affect the posttransplant graft function. We tested the hypothesis that treatment of the BDD with the dopamine derivate n-octanoyl-dopamine (NOD) improves donor cardiac and graft function after transplantation. Donor rats were given a continuous intravenous infusion of either NOD (0.882 mg/kg/h, BDD+NOD, n = 6) or a physiological saline vehicle (BDD, n = 9) for 5 h after the induction of brain death by inflation of a subdural balloon catheter. Controls were sham-operated (n = 9). In BDD, decreased left-ventricular contractility (ejection fraction; maximum rate of rise of left-ventricular pressure; preload recruitable stroke work), relaxation (maximum rate of fall of left-ventricular pressure; Tau), and increased end-diastolic stiffness were significantly improved after the NOD treatment. Following the transplantation, the NOD-treatment of BDD improved impaired systolic function and ventricular relaxation. Additionally, after transplantation increased interleukin-6, tumor necrosis factor TNF-α, NF-kappaB-p65, and nuclear factor (NF)-kappaB-p105 gene expression, and increased caspase-3, TNF-α and NF-kappaB protein expression could be significantly downregulated by the NOD treatment compared to BDD. BDD postconditioning with NOD through downregulation of the pro-apoptotic factor caspase-3, pro-inflammatory cytokines, and NF-kappaB may protect the heart against the myocardial injuries associated with brain death and ischemia/reperfusion.

Heterotopic abdominal rat heart transplantation has been extensively used to investigate ischemic-reperfusion injury, immunological consequences during heart transplantations and also to study remodeling of the myocardium due to volume unloading. We provide a unique review on the latter and present a summary of the experimental studies on rat heart transplantation to illustrate changes that occur to the myocardium due to volume unloading. We divided the literature based on whether normal or failing rat heart models were used. This analysis may provide a basis to understand the physiological effects of mechanical circulatory support therapy.

Long-term exercise training is associated with characteristic cardiac adaptation, termed athlete's heart. Our research group previously characterized in vivo left ventricular (LV) function of exercise-induced cardiac hypertrophy in detail in a rat model; however, the effect of detraining on LV function is still unclear. We aimed at evaluating the reversibility of functional alterations of athlete's heart after detraining.Rats (n = 16) were divided into detrained exercised (DEx) and detrained control (DCo) groups. Trained rats swam 200 min·d for 12 wk, and control rats were taken into water for 5 min·d. After the training period, both groups remained sedentary for 8 wk. We performed echocardiography at weeks 12 and 20 to investigate the development and regression of exercise-induced structural changes. LV pressure-volume analysis was performed to calculate cardiac functional parameters. LV samples were harvested for histological examination.Echocardiography showed robust LV hypertrophy after completing the training protocol (LV mass index = 2.61 ± 0.08 DEx vs 2.04 ± 0.04 g·kg DCo, P0.05). This adaptation regressed after detraining (LV mass index = 2.01 ± 0.03 vs 1.97 ± 0.05 g·kg, n.s.), which was confirmed by postmortem measured heart weight and histological morphometry. After the 8-wk-long detraining period, a regression of the previously described exercise-induced cardiac functional alterations was observed (DEx vs DCo): stroke volume (SV; 144.8 ± 9.0 vs 143.9 ± 9.6 μL, P = 0.949), active relaxation (τ = 11.5 ± 0.3 vs 11.3 ± 0.4 ms, P = 0.760), contractility (preload recruitable stroke work = 69.5 ± 2.7 vs 70.9 ± 2.4 mm Hg, P = 0.709), and mechanoenergetic (mechanical efficiency = 68.7 ± 1.2 vs 69.4 ± 1.8, P = 0.742) enhancement reverted completely to control values. Myocardial stiffness remained unchanged; moreover, no fibrosis was observed after the detraining period.Functional consequences of exercise-induced physiological LV hypertrophy completely regressed after 8 wk of deconditioning.

OBJECTIVES Heart transplantation is the standard treatment in end-stage heart failure and at shortage of cardiac allografts is its major limiting factor. Striving to optimize the use of this limited resource, the aspect that long distance procurement may increase the available donor pool must be taken into consideration. As poly(ADP-ribose)polymerase (PARP)-activation has been identified as a key pathway of reperfusion injury, we assessed the hypothesis that its inhibition would allow an extension of cold preservation time and protect the graft against ischaemia/reperfusion injury. METHODS Hearts from donor rats were explanted, stored in a preservation solution (Custodiol) at 4 °C for 4 h or 8 h, and heterotopically transplanted. A vehicle or the PARP-inhibitor, INO-1001 (5 mg/kg), was administered during the reperfusion period. We evaluated post-transplant graft function with a Millar micromanometer at different left-ventricular volumes. Additionally, in organ bath experiments the effect of PARP-inhibition on endothelium-dependent and -independent vasorelaxation was evaluated after long-term cold ischaemic storage/warm reperfusion. RESULTS PARP-inhibition resulted in a better systolic functional recovery of grafts submitted to 4 h and 8 h ischaemia. Furthermore, INO-1001 decreased the left-ventricular end-diastolic pressure after 8 h of ischaemia. Coronary blood flow was significantly higher after PARP-inhibition in comparison to controls. Endothelium-dependent vasorelaxation was significantly better in the INO-1001-groups than in the vehicle-treated transplant groups. After 24-h hypothermic storage, treatment of aortic ring with INO-1001 during reoxygenation significantly improved endothelial dysfunction. CONCLUSIONS By inhibiting the PARP activation, INO-1001 can protect the graft and endothelium from the injury that is caused by prolonged cold myocardial ischaemia/reperfusion, thereby improving post-transplant graft function.