Possible retinotoxicity of long-term vardenafil treatment.

Phosphodiesterase (PDE) inhibitors - such as vardenafil - are used primarily for treating erectile dysfunction via increasing cyclic guanosine monophosphate (cGMP) levels. Recent studies have also demonstrated their significant cardioprotective effects in several diseases, including diabetes, upon long-term, continuous application. However, PDE inhibitors are not specific for PDE5 and also inhibit the retinal isoform. A sustained rise in cGMP in photoreceptors is known to be toxic; therefore, we hypothesized that long-term vardenafil treatment might result in retinotoxicity. The hypothesis was tested in a clinically relevant animal model of type 2 diabetes mellitus. Histological experiments were performed on lean and diabetic Zucker Diabetic Fatty rats. Half of the animals were treated with vardenafil for six months, and the retinal effects were evaluated. Vardenafil treatment alleviated rod outer segment degeneration but decreased rod numbers in some positions and induced changes in the interphotoreceptor matrix, even in control animals. Vardenafil treatment decreased total retinal thickness in the control and diabetic groups and reduced the number of nuclei in the outer nuclear layer. Müller cell activation was detectable even in the vardenafil-treated control animals, and vardenafil did not improve gliosis in the diabetic group. Vardenafil-treated animals showed complex retinal alterations with improvements in some parameters while deterioration in others. Our results point towards the retinotoxicity of vardenafil, even without diabetes, which raises doubts about the retinal safety of long-term continuous vardenafil administration. This effect needs to be considered when approving PDE inhibitors for alternative indications.

mRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions.

A small fraction of people vaccinated with mRNA-lipid nanoparticle (mRNA-LNP)-based COVID-19 vaccines display acute or subacute inflammatory symptoms whose mechanism has not been clarified to date. To better understand the molecular mechanism of these adverse events (AEs), here, we analyzed in vitro the vaccine-induced induction and interrelations of the following two major inflammatory processes: complement (C) activation and release of proinflammatory cytokines. Incubation of Pfizer-BioNTech's Comirnaty and Moderna's Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1ß < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1ß, and TNF-α, suggesting C-dependence of these cytokines' induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines. Thus, the pharmacological attenuation of inflammatory AEs may not be achieved via monotherapy with the tested C inhibitors; efficacy may require combination therapy with different C inhibitors and/or other anti-inflammatory agents.

Recipient Pericardial Apolipoprotein Levels Might Be an Indicator of Worse Outcomes after Orthotopic Heart Transplantation.

BACKGROUND: End-stage heart failure (ESHF) leads to hypoperfusion and edema formation throughout the body and is accompanied by neurohormonal and immunological alterations. Orthotopic heart transplantation (HTX) has been used as a beneficial option for ESHF. Due to the shortage of donor hearts, the ideal matching and timing of donors and recipients has become more important. PURPOSE: In this study, our aim was to explore the relationship between the clinical outcomes of HTX and the cytokine and apolipoprotein profiles of the recipient pericardial fluid obtained at heart transplantation after opening the pericardial sac. MATERIALS AND METHODS: The clinical data and the interleukin, adipokine, and lipoprotein levels in the pericardial fluid of twenty HTX recipients were investigated. Outcome variables included primer graft dysfunction (PGD), the need for post-transplantation mechanical cardiac support (MCS), International Society for Heart and Lung Transplantation grade ≥2R rejection, and mortality. Recipient risk scores were also investigated. RESULTS: Leptin levels were significantly lower in patients with PGD than in those without PGD (median: 6.36 (IQR: 5.55-6.62) versus 7.54 (IQR = 6.71-10.44); p = 0.029). Higher ApoCII levels (median: 14.91 (IQR: 11.55-21.30) versus 10.31 (IQR = 10.02-13.07); p = 0.042) and ApoCIII levels (median: 60.32 (IQR: 43.00-81.66) versus 22.84 (IQR = 15.84-33.39); p = 0.005) were found in patients (n = 5) who died in the first 5 years after HTX. In patients who exhibited rejection (n = 4) in the first month after transplantation, the levels of adiponectin (median: 74.48 (IQR: 35.51-131.70) versus 29.96 (IQR: 19.86-42.28); p = 0.039), ApoCII (median: 20.11 (IQR: 13.06-23.54) versus 10.32 (IQR: 10.02-12.84); p = 0.007), and ApoCIII (median: 70.97 (IQR: 34.72-82.22) versus 26.33 (IQR: 17.18-40.17); p = 0.029) were higher than in the nonrejection group. Moreover, the pericardial thyroxine (T4) levels (median: 3.96 (IQR: 3.49-4.46) versus 4.69 (IQR: 4.23-5.77); p = 0.022) were lower in patients with rejection than in patients who did not develop rejection. CONCLUSION: Our results indicate that apolipoproteins can facilitate the monitoring of rejection and could be a useful tool in the forecasting of early and late complications.

Alpha-1-Antitrypsin Protects Vascular Grafts of Brain-Dead Rats Against Ischemia/Reperfusion Injury.

INTRODUCTION: Endothelial dysfunction is a potential side effect of brain death (BD). Ischemia/reperfusion (IR) injury during heart transplantation may lead to further endothelial damage. Protective effects of alpha-1-antitrypsin (AAT), a human neutrophil serine protease inhibitor, have been demonstrated against IR injury. We hypothesized that AAT protects brain-dead rats' vascular grafts from IR injury. METHODS: Donor rats were subjected to BD by inflation of a subdural balloon. After 5.5 h, aortic rings were immediately mounted in organ baths (BD, n = 6 rats) or preserved in saline, supplemented either with vehicle (BD-IR, n = 8 rats) or AAT (BD-IR + AAT, n = 14 rats) for 24 h. During organ bath experiment, rings from both IR groups were exposed to hypochlorite to simulate warm reperfusion-associated endothelial injury. Endothelial function was measured ex vivo. Immunohistochemical staining for caspases was carried out and DNA-strand breaks were evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Data are presented as median (interquartile range). RESULTS: AAT improved IR-induced decreased maximum endothelium-dependent vasorelaxation to acetylcholine in the BD-IR + AAT aortas compared to the BD-IR group (BD: 83 (9-28) % versus BD-IR: 49 (39-60) % versus BD-IR + AAT: 64 (24-42) %, P < 0.05). Additionally, an increase in the rings' sensitivity to acetylcholine was noted after AAT (pD2-value: BD-IR + AAT: 7.35 (7.06-7.89) versus BD-IR: 6.96 (6.65-7.21), P < 0.05). Caspase-3, -8, -9, and -12 immunoreactivity and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were significantly decreased by AAT. CONCLUSIONS: AAT alleviates endothelial dysfunction, prevents increased caspase-3, -8, -9, and -12 levels, and decreases apoptotic DNA breakage due to BD and IR injury. This suggests that AAT treatment may be therapeutically beneficial to reduce IR-induced vascular damage.

Zymosan Particle-Induced Hemodynamic, Cytokine and Blood Cell Changes in Pigs: An Innate Immune Stimulation Model with Relevance to Cytokine Storm Syndrome and Severe COVID-19.

Hemodynamic disturbance, a rise in neutrophil-to-lymphocyte ratio (NLR) and release of inflammatory cytokines into blood, is a bad prognostic indicator in severe COVID-19 and other diseases involving cytokine storm syndrome (CSS). The purpose of this study was to explore if zymosan, a known stimulator of the innate immune system, could reproduce these changes in pigs. Pigs were instrumented for hemodynamic analysis and, after i.v. administration of zymosan, serial blood samples were taken to measure blood cell changes, cytokine gene transcription in PBMC and blood levels of inflammatory cytokines, using qPCR and ELISA. Zymosan bolus (0.1 mg/kg) elicited transient hemodynamic disturbance within minutes without detectable cytokine or blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This was followed by a transient granulopenia and then, up to 6 h, major granulocytosis, resulting in a 3-4-fold increase in NLR. These changes were paralleled by massive transcription and/or rise in IL-6, TNF-alpha, CCL-2, CXCL-10, and IL-1RA in blood. There was significant correlation between lymphopenia and IL-6 gene expression. We conclude that the presented model may enable mechanistic studies on late-stage COVID-19 and CSS, as well as streamlined drug testing against these conditions.

Donor Pericardial Interleukin and Apolipoprotein Levels May Predict the Outcome after Human Orthotopic Heart Transplantation.

The proinflammatory cascade that is activated at the time of brain death plays a crucial role in organ procurement. Our aim of this study was to explore the relationship between the clinical outcome of orthotopic heart transplantation, as well as cytokine and apolipoprotein profiles of the pericardial fluid obtained at donation. Interleukin, adipokine and lipoprotein levels in the pericardial fluid, as well as clinical data of twenty donors after brain death, were investigated. Outcome variables included primary graft dysfunction, the need for posttransplantation mechanical cardiac support and International Society for Heart and Lung Transplantation grade ≥ 2R rejection. Hormone management and donor risk scores were also investigated. Lower levels of IL-6 were observed in primary graft dysfunction (median: 36.72 [IQR: 19.47-62.90] versus 183.67 [41.21-452.56]; p = 0.029) and in the need for mechanical cardiac support (44.12 [20.12-85.70] versus 247.13 [38.51-510.38]; p = 0.043). Rejection was associated with lower ApoAII (p = 0.021), ApoB100 (p = 0.032) and ApoM levels (p = 0.025). Lower adipsin levels were detected in those patients receiving desmopressin (p = 0.037); moreover, lower leptin levels were found in those patients receiving glucocorticoid therapy (p = 0.045), and higher T3 levels were found in those patients treated with L-thyroxine (p = 0.047) compared to those patients not receiving these hormone replacement therapies. IL-5 levels were significantly associated with UNOS-D score (p = 0.004), Heart Donor Score (HDS) and Adapted HDS (p < 0.001). The monitoring of immunological and metabolic changes in donors after brain death may help in the prediction of potential complications after heart transplantation, thus potentially optimizing donor heart allocation.

Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

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 ß1, 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 ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-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 ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Sex similarities and differences in the reverse and anti-remodeling effect of pressure unloading therapy in a rat model of aortic banding and debanding.

Investigating the effect of sex on pressure unloading therapy in a clinical scenario is limited by several nonstandardized factors. Hence, we sought to study sex-related similarities and differences under laboratory conditions. Pressure overload was induced in male and female rats by aortic banding (AB) for 6 and 12 wk. Age-matched sham-operated animals served as controls. Pressure unloading was performed by aortic debanding at week 6. Different aspects of myocardial remodeling were characterized by echocardiography, pressure-volume analysis, histology, qRT-PCR, and explorative proteomics. Hypertrophy, increased fetal gene expression, interstitial fibrosis, and prolonged active relaxation were noted in the AB groups at week 6 in both sexes. However, decompensation of systolic function and further deterioration of diastolic function only occurred in male AB rats at week 12. AB induced similar proteomic alterations in both sexes at week 6, whereas characteristic differences were found at week 12. After debanding, regression of hypertrophy and recovery of diastolic function took place to a similar extent in both sexes. Nevertheless, fibrosis, transcription of ß-myosin-to-α-myosin heavy chain ratio, and myocardial proteomic alterations were reduced to a greater degree in females than in males. Debanding exposed anti-remodeling properties in both sexes and prevented the functional decline in males. Female sex is associated with greater reversibility of fibrosis, fetal gene expression, and proteomic alterations. Nevertheless, pressure unloading exposes a more pronounced anti-remodeling effect on the functional level in males, which is attributed to the more progressive functional deterioration in AB animals.NEW & NOTEWORTHY The present study is the first to assess the role of sex on pressure unloading-induced reverse and anti-remodeling in a rat model of aortic banding and debanding. Our data indicate that female sex is associated with a greater reversibility of fibrosis, fetal gene expression, and proteomic alterations compared with males. Nevertheless, pressure unloading exposes more anti-remodeling effect on the functional level in males, which is attributed to the more rapid functional deterioration in aortic-banded animals.

Sex differences in rat renal arterial responses following exercise training.

During aerobic exercise, hemodynamic alterations occur. Although blood flow in skeletal muscle arteries increases, it decreases in visceral vessels because of mesenterial vasoconstriction. However, maintaining renal blood flow during intensive sport is also a priority. Our aim was to investigate the changes of vascular reactivity and histology of isolated renal artery of male and female rats in response to swim training. Wistar rats were distributed into four groups: male sedentary (MSed), male trained (MTr), female sedentary (FSed), and female trained (FTr). Trained animals underwent a 12-wk-long intensive swimming program. Vascular function of isolated renal artery segments was examined by wire myography. Phenylephrine-induced contraction was lower in FSed than in MSed animals, and it was decreased by training in male but not in female animals. Inhibition of cyclooxygenases by indomethacin reduced contraction in both sedentary groups, and in MTr but not in FTr animals. Inhibition of nitric oxide production increased contraction in both trained groups. Acetylcholine induced relaxation was similar in all experimental groups showing predominant NO-dependency. Elastin and smooth muscle cell actin density was reduced in female rats after aerobic training. This study shows that, as a result of a 12-wk-long training, there are sex differences in renal arterial responses following exercise training. Swimming moderates renal artery vasoconstriction in male animals, whereas it depresses elastic fiber and smooth muscle actin density in females.NEW & NOTEWORTHY We provided the first detailed analysis of the adaptation of the renal artery after aerobic training in male and female rats. As a result of a 12-wk-long training program, the pharmacological responses of renal arteries changed only in male animals. In phenylephrine-induced contraction, cyclooxygenase-mediated vasoconstriction mechanisms lost their significance in female rats, whereas NO-dependent relaxation became a significant contraction reducing factor in both sexes. Early structural changes, such as reduced elastin and smooth muscle cell actin evolves in females.

Evaluation of muscle-specific and metabolism regulating microRNAs in a chronic swimming rat model.

Making benefit from the epigenetic effects of environmental factors such as physical activity may result in a considerable improvement in the prevention of chronic civilization diseases. In our chronic swimming rat model, the expression levels of such microRNAs were characterized, that are involved in skeletal muscle differentiation, hypertrophy and fine-tuning of metabolism, which processes are influenced by chronic endurance training, contributing to the metabolic adaptation of skeletal muscle during physical activity. After chronic swimming, the level of miR-128a increased significantly in EDL muscles, which may influence metabolic adaptation and stress response as well. In SOL, the expression level of miR-15b and miR-451 decreased significantly after chronic swimming, which changes are opposite to their previously described increment in insulin resistant skeletal muscle. MiR-451 also targets PGC-1α mRNA, whiches expression level significantly increased in SOL muscles, resulting in enhanced biogenesis and oxidative capacity of mitochondria. In summary, the microRNA expression changes that were observed during our experiments suggest that chronic swim training contributes to a beneficial metabolic profile of skeletal muscle.

Poor Preoperative Nutritional Status, but Not Hormone Levels, Are Associated With Mortality After Cardiac Surgery.

OBJECTIVES: The authors' aim was to examine the preoperative hormone and nutritional status in patients undergoing elective cardiac surgery. DESIGN AND SETTINGS: The authors' research was a single-center, prospective, observational study (ClinicalTrials.gov: NCT03736499). PARTICIPANTS & INTERVENTIONS: The authors examined 252 patients who underwent elective cardiac surgery. Preoperative thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), prolactin, and testosterone levels were collected and analyzed after the surgery. The Geriatric Nutritional Risk Index (GNRI), Controlling Nutritional Status (CONUT), and Prognostic Nutritional Index (PNI) were all calculated as a sum and groups. Frailty was calculated based on the modified Frailty Index-11. The primary outcome was overall mortality. MEASUREMENTS AND MAIN RESULTS: The mean age of the patients was 64.23 years (standard deviation: 11.07 years). Thirty-three patients (13.01%) died during the median follow-up time of 20.48 months (interquartile range: 18.90-22.98 months). Thyroid hormones were examined as continuous variables and also in 3 groups based on low, normal, and high hormone levels. Continuous TSH (p = 0.230), continuous fT3 (p = 0.492), and continuous fT4 (p = 0.657) were not significantly associated with total mortality. After adjustment for the European System for Cardiac Operative Risk Evaluation II and postoperative complications, the following nutritional scores were associated with total mortality: GNRI < 91 (adjusted hazard ratio [AHR]: 4.384; 95% confidence interval [CI]: 1.866-10.303, p = 0.001), the higher CONUT group (AHR: 1.736; 95% CI: 1.736-2.866, p = 0.031), and a PNI < 48 points (AHR: 3.465; 95% CI: 1.735-6.918, p < 0.001). The modified Frailty Index-11 was not associated with mortality. CONCLUSIONS: Before cardiac surgery, nutritional status should be assessed because the findings may help to decrease mortality. The hormone levels were not associated with mortality.

Improvement of Left Ventricular Graft Function Using an Iron-Chelator-Supplemented Bretschneider Solution in a Canine Model of Orthotopic Heart Transplantation.

Demand for organs is increasing while the number of donors remains constant. Nevertheless, not all organs are utilized due to the limited time window for heart transplantation (HTX). Therefore, we aimed to evaluate whether an iron-chelator-supplemented Bretschneider solution could protect the graft in a clinically relevant canine model of HTX with prolonged ischemic storage. HTX was performed in foxhounds. The ischemic time was standardized to 4 h, 8 h, 12 h or 16 h, depending on the experimental group. Left ventricular (LV) and vascular function were measured. Additionally, the myocardial high energy phosphate and iron content and the in-vitro myocyte force were evaluated. Iron chelator supplementation proved superior at a routine preservation time of 4 h, as well as for prolonged times of 8 h and longer. The supplementation groups recovered quickly compared to their controls. The LV function was preserved and coronary blood flow increased. This was also confirmed by in vitro myocyte force and vasorelaxation experiments. Additionally, the biochemical results showed significantly higher adenosine triphosphate content in the supplementation groups. The iron chelator LK614 played an important role in this mechanism by reducing the chelatable iron content. This study shows that an iron-chelator-supplemented Bretschneider solution effectively prevents myocardial/endothelial damage during short- as well as long-term conservation.

CD3+/CD56+ NKT-like Cells Show Imbalanced Control Immediately after Exercise in Delayed-Onset Muscle Soreness.

The purpose of the study was to carry out an immunophenotypical characterization with a special focus on natural killer cells of junior swimmers from the Hungarian National Swim Team before and after an intensive acute exercise. Nineteen swimmers, ten females and nine males, completed the exercise protocol. Sixteen swimmers experienced delayed-onset muscle soreness. Most of our findings substantiated earlier results, such as the increase in the percentage of the CD3-/CD56+ natural killer cells and the CD3-/CD56dim+ NK cells, and the decrease in the percentage of CD3+ T cells among lymphocytes after the exercise protocol. The drop of natural killer cell activity back to the pre-exercise level was in line with earlier findings. Interestingly, the percentage of CD3+/CD56+ NKT-like cells did not change significantly in those three swimmers who did not report delayed-onset muscle soreness. On the contrary, the percentage of CD3+/CD56+ NKT-like cells among lymphocytes increased in fourteen and decreased in two swimmers reporting delayed-onset muscle soreness. This study for the first time demonstrated a link between the delayed-onset muscle soreness and the imbalanced control of CD3+/CD56+ NKT-like cells among lymphocytes. However, validation of this association in a larger sample size study will be necessary.

Sodium-glucose cotransporter 2 inhibitors reduce myocardial infarct size in preclinical animal models of myocardial ischaemia-reperfusion injury: a meta-analysis.

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 < 0.00001), referring to a 33% [95% CI 20%, 47%] difference. Heterogeneity was moderate (Τ2 = 0.58, I2 = 60%). SGLT2 inhibitors were only effective when administered to the intact organ system, but not to isolated hearts (p interaction <0.001, adjusted pseudo-R2 = 47%). While acute administration significantly reduced infarct size, chronic treatment was superior (p interaction <0.001, adjusted pseudo-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.

Cardiac PANK1 deletion exacerbates ventricular dysfunction during pressure overload.

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.

Omecamtiv mecarbil evokes diastolic dysfunction and leads to periodic electromechanical alternans.

Omecamtiv mecarbil (OM) is a promising novel drug for improving cardiac contractility. We tested the therapeutic range of OM and identified previously unrecognized side effects. The Ca2+ sensitivity of isometric force production (pCa50) and force at low Ca2+ levels increased with OM concentration in human permeabilized cardiomyocytes. OM (1 µM) slowed the kinetics of contractions and relaxations and evoked an oscillation between normal and reduced intracellular Ca2+ transients, action potential lengths and contractions in isolated canine cardiomyocytes. Echocardiographic studies and left ventricular pressure-volume analyses demonstrated concentration-dependent improvements in cardiac systolic function at OM concentrations of 600-1200 µg/kg in rats. Administration of OM at a concentration of 1200 µg/kg was associated with hypotension, while doses of 600-1200 µg/kg were associated with the following aspects of diastolic dysfunction: decreases in E/A ratio and the maximal rate of diastolic pressure decrement (dP/dtmin) and increases in isovolumic relaxation time, left atrial diameter, the isovolumic relaxation constant Tau, left ventricular end-diastolic pressure and the slope of the end-diastolic pressure-volume relationship. Moreover, OM 1200 µg/kg frequently evoked transient electromechanical alternans in the rat in vivo in which normal systoles were followed by smaller contractions (and T-wave amplitudes) without major differences on the QRS complexes. Besides improving systolic function, OM evoked diastolic dysfunction and pulsus alternans. The narrow therapeutic window for OM may necessitate the monitoring of additional clinical safety parameters in clinical application.

Sex-related differences of early cardiac functional and proteomic alterations in a rat model of myocardial ischemia.

BACKGROUND: Reduced cardiovascular risk in premenopausal women has been the focus of research in recent decades. Previous hypothesis-driven experiments have highlighted the role of sex hormones on distinct inflammatory responses, mitochondrial proteins, extracellular remodeling and estrogen-mediated cardioprotective signaling pathways related to post-ischemic recovery, which were associated with better cardiac functional outcomes in females. We aimed to investigate the early, sex-specific functional and proteomic changes following myocardial ischemia in an unbiased approach. METHODS: Ischemia was induced in male (M-Isch) and female (F-Isch) rats with sc. injection of isoproterenol (85 mg/kg) daily for 2 days, while controls (M-Co, F-Co) received sc. saline solution. At 48 h after the first injection pressure-volume analysis was carried out to assess left ventricular function. FFPE tissue slides were scanned and analyzed digitally, while myocardial proteins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using isobaric labeling. Concentrations of circulating steroid hormones were measured with LC-MS/MS. Feature selection (PLS and PLS-DA) was used to examine associations among functional, proteomic and hormonal datasets. RESULTS: Induction of ischemia resulted in 38% vs 17% mortality in M-Isch and F-Isch respectively. The extent of ischemic damage to surviving rats was comparable between the sexes. Systolic dysfunction was more pronounced in males, while females developed a more severe impairment of diastolic function. 2224 proteins were quantified, with 520 showing sex-specific differential regulation. Our analysis identified transcriptional, cytoskeletal, contractile, and mitochondrial proteins, molecular chaperones and the extracellular matrix as sources of disparity between the sexes. Bioinformatics highlighted possible associations of estrogens and their metabolites with early functional and proteomic alterations. CONCLUSIONS: Our study has highlighted sex-specific alterations in systolic and diastolic function shortly after ischemia, and provided a comprehensive look at the underlying proteomic changes and the influence of estrogens and their metabolites. According to our bioinformatic analysis, inflammatory, mitochondrial, chaperone, cytoskeletal, extracellular and matricellular proteins are major sources of intersex disparity, and may be promising targets for early sex-specific pharmacologic interventions.

Exercise-induced alterations of myocardial sarcomere dynamics are associated with hypophosphorylation of cardiac troponin I.

Although the knowledge of sports cardiology advanced significantly in the recent years, the molecular mechanisms by which exercise training augments cardiac performance is poorly understood. Here we aimed at determining left ventricular (LV) myocardial sarcomeric protein modifications in a rat model of exercise training and detraining. Young male Wistar rats were divided into exercised (Ex) and control (Co) groups. Trained rats swam 200 min/day for 12 weeks. Detrained (DEx) and control (DCo) rats remained sedentary for 8 weeks after completion of the 12-week-long protocol. Ca2+-regulated active force production (Fa⁢c⁢t⁢i⁢v⁢e), its Ca2+-sensitivity (pCa50) and Ca2+-independent passive tension (Fp⁢a⁢s⁢s⁢i⁢v⁢e) were determined in isolated permeabilized cardiomyocytes and phosphorylation levels of sarcomeric proteins were assayed by biochemical methods. Means of maximal Ca2+-activated isometric force (Fm⁢a⁢x) and pCa50 values were higher (p < 0.05) in the Ex group (28.0 ± 1.4 kN/m2 and 5.91 ± 0.03, respectively, mean ± SEM) than those in the Co group (15.8 ± 0.8 kN/m2 and 5.81 ± 0.03, respectively). Fp⁢a⁢s⁢s⁢i⁢v⁢e did not differ between these two groups. The level of cardiac troponin I (cTnI) phosphorylation decreased upon exercise (from 1.00 ± 0.02 to 0.66 ± 0.06, p < 0.05; in relative units). Site specific phosphorylation assays revealed cTnI hypophosphorylations at the protein kinase A (PKA)-specific Ser-22/23 sites and at the protein kinase C (PKC)-specific Thr-143 site. Mechanical and biochemical parameters of the DEx and DCo groups did not differ from each other following the detraining period. Exercise-induced hypertrophy is associated with reversible increases in Ca2+-dependent force production and its Ca2+-sensitivity in LV cardiomyocytes, which can be associated with changes in cTnI phosphorylation.

Increased Expression of N2BA Titin Corresponds to More Compliant Myofibrils in Athlete's Heart.

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.