BACKGROUND AND AIMS:Advanced cancer may resemble a heart failure (HF)-like phenotype marked by cardiac wasting, dyspnoea, congestion, and/or physical dysfunction. The trial evaluated safety and efficacy of HF therapy among patients with advanced cancer receiving specialized palliative care to improve patients' self-care ability. METHODS:Patients with stage 4 solid tumours with a life expectancy of 1-6 months receiving specialized palliative care were enrolled. Patients were required to meet at least two cardiovascular risk criteria and at least one criterion for functional limitation. Participants were randomized 1:1 to receive optimized HF therapy (up to four drugs: sacubitril/valsartan, empagliflozin, ivabradine, ferric carboxymaltose) or placebo in a double-blind setting. The primary hierarchical endpoint included: (i) days alive and able to wash oneself, (ii) ability to walk 4 m, and (iii) self-reported patient global assessment (PGA) of subjective well-being, during the 30-day placebo-controlled phase. RESULTS:In five centres, 93 patients were randomized. The primary endpoint did not differ between groups (win ratio 0.95, 95% confidence interval [CI] 0.57-1.58; P = .83). Overall, mortality was 32% at 30 days (not different between groups). In patients alive at 30 days, HF therapy reduced N-terminal pro-B-type natriuretic peptide levels by 41% (P = .040), increased left ventricular ejection fraction by 2.9% (P = .036), and improved PGA scores (odds ratio 0.22, 95% CI 0.06-0.75; P = .016). CONCLUSIONS:In a population with advanced cancer receiving specialized palliative care and high early mortality, optimized HF therapy did not improve patients' self-care ability. Among survivors at 30 days, improvements in quality of life measures and cardiac biomarkers suggest potential benefit of individualized HF therapy, which is hypothesis generating and needs validation.

Neutrophils are key in acute and chronic cardiovascular diseases (CVD) by mediating inflammation during myocardial infarction, stroke, and heart failure. Reactive oxygen species (ROS) generated by neutrophil NADPH oxidase 2 (NOX2) have been linked to CVD pathology by inducing tissue injury via oxidative stress. There is, however, only limited data investigating the impact of neutrophil-derived ROS on clinical outcome in patients with CVD. This study measured neutrophil oxidative burst capacity (NOBC) in patients with CVD and evaluated its association with major adverse cardiac events (MACE) and infection. In a population of 201 (median age 69 years (interquartile range (IQR) = 61–80 years); 33.4

BACKGROUND:Amyloid transthyretin cardiomyopathy (ATTR-CM) results from extracellular deposition of misfolded transthyretin (TTR), causing progressive heart failure. Naturally-occurring antibodies (nAbs) targeting misfolded proteins exist in neurodegenerative disease, but their presence in ATTR-CM is unknown. The objective of this study is to determine whether nAbs against TTR (nAbsTTR) exist in humans and whether they are influenced by disease or its treatment. METHODS:Serum from healthy donors, umbilical cord blood (UCB), and patients with ATTR-CM - both untreated and receiving TTR-stabilizing therapy - was analyzed for nAbsTTR using immunoassays, blotting, and binding studies. Functional activity was evaluated in a fibril formation assay. RESULTS:nAbsTTR binding both native and amyloid TTR (ATTR) with high affinity (KD 30 nM/7 nM) were detected in healthy serum and UCB. NAbsTTR levels were significantly altered in ATTR-CM compared to controls: nAbsTTR (IgG) were higher while nAbsTTR (IgM) were lower. nAbsTTR of both subtypes significantly increased by 22% (p ≤ 0.05) in patients receiving TTR-stabilizing therapy. In vitro, nAbsTTR suppressed TTR fibril aggregation. CONCLUSIONS:Naturally-occurring TTR-targeting antibodies are present from birth, modulated by disease and therapy, and inhibit fibril formation. These findings reveal an unrecognized immune mechanism with potential relevance for ATTR-CM pathogenesis and treatment.

Abstract Cardiosphere‐derived cells (CDCs) are a promising in vitro model for studying myocardial ischaemia–reperfusion (I–R) injury and testing potential therapeutic interventions. This study investigated the suitability of CDCs as a model for myocardial infarction (MI) and the effects of prolyl hydroxylase inhibitor (PHI) administration during reperfusion. CDCs were generated from neonatal mouse hearts and characterized by immunofluorescence, revealing a heterogeneous mixture of cardiomyocytes, smooth muscle cells, endothelial cells, and stem cells. The CDCs were subjected to oxygen–glucose deprivation (OGD) followed by reperfusion with or without the PHI dimethyloxalylglycine (DMOG). Cell viability, hypoxia‐inducible factor 1‐alpha (HIF‐1α) accumulation, and gene expression were analysed. The results showed that DMOG administration during reperfusion reduced lactate dehydrogenase release, indicating decreased cell death. HIF‐1α protein levels increased during OGD and were further stabilized by DMOG during reperfusion. The expression of HIF‐1α target genes, such as vascular endothelial growth factor (Vegfa), and genes involved in regeneration and cardiac function, including connective tissue growth factor (Ctgf), cyclin D2 (Ccnd2), and beta‐1 adrenergic receptor (Adrb1), was modulated by OGD and DMOG treatment. Comparisons with an in vivo mouse I–R injury model revealed similarities in gene expression patterns. In conclusion, CDCs serve as an effective in vitro model for studying I–R injury, closely resembling the in vivo situation. Furthermore, PHI administration during reperfusion reduces cell death and modulates the expression of genes involved in cardioprotection and regeneration, highlighting the potential of PHIs as a therapeutic strategy for I–R injury.

Recent advances in mitochondrial network dynamic and signalling highlight mitochondria as key therapeutic targets across diverse diseases. Yet, high drug development failure rates reflect an incomplete understanding of upstream molecular regulators of mitochondrial fate. Here, we address this gap by reverse engineering of the BH3-only protein BNIP3. Structural modelling and sequence-function analyses of its N-terminus identify a critical functional domain and amino acid hotspots that directly activate BCL-2 executioner proteins, triggering mitochondrial cell death. Leveraging these insights, we develop a BNIP3 antagonist peptide (B-017) that disrupts interactions between BNIP3 and BCL-2 executioner proteins, preserving mitochondrial integrity. B-017 demonstrates target specificity, a favourable safety profile, and robust suppression of cell death signalling in human cells. In clinically relevant animal models, it reduces tissue damage in the heart, brain, and liver. Together, these findings position B-017 as a promising therapeutic candidate targeting mitochondrial dysfunction.

Background: Inorganic nitrate is abundant in leafy green vegetables and has been shown to exert positive cardiovascular effects through nitric oxide-related pathways. The enteral microbiome is an emerging key player in cardiovascular diseases and depends on dietary habits. Whether dietary inorganic nitrate impacts on the microbiome and atherosclerosis-associated microbiome-dependent metabolites like short chain fatty acids (SCFA) and trimethylamine N-oxide (TMAO) is unknown. Methods: In a double-blind randomized controlled trial, 30 healthy volunteers were included who either received dietary nitrate (0.12 mmol/kg bodyweight) or placebo (equimolar amounts of sodium chloride) for 30 days. The microbiome metabolites TMAO and SCFA were analyzed. The enteral microbiome was analyzed by 16S-rRNA sequencing at baseline and follow-up. Results: Systolic blood pressure decreased after nitrate supplementation (baseline 124.73 mmHg vs. follow up 120 mmHg, p < 0.05) with no change in controls. Dietary nitrate supplementation increased TMAO levels (nitrate baseline 349.28 μ/L vs. nitrate follow-up 481.15 μ/L, p < 0.05), while SCFA levels remained unchanged. The relative abundance of Akkermansia and taxa of Clostridiales were higher in individuals with high compared to normal TMAO levels after nitrate supplementation, while Shannon diversity, richness and evenness did not differ between both groups. Conclusions: Our results indicate that dietary nitrate supplementation is associated with alterations to the enteral microbiome with an impact on proatherogenic metabolites. Further work is warranted to investigate the causal relationship between dietary nutrients, the microbiome and downstream metabolites.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the industrialized world. The gut microbiome influences CVD, through atherogenic metabolites like trimethylamine N-oxide (TMAO) or protective effects through short-chain fatty acids (SCFA) production. The specific alterations in the gut microbiome and downstream metabolites in acute coronary syndrome (ACS) and chronic coronary syndrome (CCS) remain unclear. We enrolled ACS patients within 24 h of clinical presentation with a follow-up of 28 days, using CCS patients as controls. Gut microbiome composition, downstream metabolites, and cardiovascular function were assessed at both baseline and follow-up. Microbiome-derived metabolites were analyzed and gut microbiome samples were characterized by 16S rRNA gene analysis. We enrolled 40 patients, with 20 patients each in the ACS and CCS group. Alpha diversity of the microbiome did not differ throughout the follow-up. After ACS gut microbiome composition changed during the follow-up period with increased levels of Butyricicoccus and Butyricoccaceae, a pattern not observed in the CCS cohort. Downstream analysis of microbiome-derived metabolites SCFA revealed increased serum levels of butanoic acid, while TMAO levels remained unchanged. This small prospective observational non-randomized study, suggests that ACS may trigger an enrichment of butanoic acid-producing bacteria in the gut microbiome, accompanied by an increase in serum butanoic acid levels over 28 days. No significant changes in TMAO were observed. These insights could help develop approaches to reduce the burden of CVD. As a small pilot study, these findings require validation in larger ACS cohorts. Trial registration NCT, NCT05456802, Registered 30 June 2022, https://clinicaltrials.gov/study/NCT05122689

Complex cellular interactions determine functional and structural tissue remodeling during reperfused acute myocardial infarction (repAMI). These processes show distinct spatial distribution as the injured heart muscle is segmented into different areas (damage area, area at risk (AAR), and remote area). Three-dimensional (3D) visualization of these areas is essential for the analysis of various interactions between resident cardiac cells and infiltrating immune cells, enabling the identification of possible treatment targets. Here, a protocol is described for simultaneous and automatable 3D visualization and quantification of the cardiac damage area, AAR, and infiltrating immune cells (e.g., neutrophils) after repAMI. This includes intravital antibody-mediated staining of cardiac damage area (CD31neg) and neutrophil infiltration (Ly6G+) following ex vivo visualization of AAR by retrograde antibody perfusion and further non-toxic tissue clearing for light sheet fluorescence microscopy (LSFM) imaging. This technique allows the spatial analysis of target cells, e.g., infiltrating immune cells and damaged areas in an intact mouse heart after repAMI. Traditional histology and immunohistochemistry can be performed after non-toxic tissue clearing and image acquisition with computer-assisted post-processing. This allows multiplexing information gain within the same mouse heart, strengthening data robustness and being especially significant in a highly complex injury like repAMI.

Myoglobin (Mb) plays an important role at rest and during exercise as a reservoir of oxygen and has been suggested to regulate NO center dot bioavailability under hypoxic/acidic conditions. However, its ultimate role during exercise is still a subject of debate. We aimed to study the effect of Mb deficiency on maximal oxygen uptake ((V)over dot(O2max)) and exercise performance in myoglobin knockout mice (Mb(-/-)) when compared to control mice (Mb(+/+)). Furthermore, we also studied NO center dot bioavailability, assessed as nitrite (NO2-) and nitrate (NO3-) in the heart, locomotory muscle and in plasma, at rest and during exercise at exhaustion both in Mb(-/-) and in Mb(+/+) mice. The mice performed maximal running incremental exercise on a treadmill with whole-body gas exchange measurements. The Mb(-/-) mice had lower body mass, heart and hind limb muscle mass (P < 0.001). Mb(-/-) mice had significantly reduced maximal running performance (P < 0.001). (V)over dot(O2max) expressed in ml min(-1) in Mb(-/-) mice was 37% lower than in Mb(+/+) mice (P < 0.001) and 13% lower when expressed in ml min(-1) kg body mass(-1) (P = 0.001). Additionally, Mb(-/-) mice had significantly lower plasma, heart and locomotory muscle NO2- levels at rest. During exercise NO2- increased significantly in the heart and locomotory muscles of Mb(-/-) and Mb(+/+) mice, whereas no significant changes in NO2- were found in plasma. Our study showed that, contrary to recent suggestions, Mb deficiency significantly impairs (V)over dot(O2max) and maximal running performance in mice.

OBJECTIVE:To evaluate wolfram as a photon and beta absorber in the management of uveal melanoma with radiotherapy, examining its potential ocular adverse effects and physiologic tolerance using an in vivo rabbit ocular model. METHODS:A method of manufacturing implants from mixtures of wolfram and silicone was developed. Their shielding effect on the radiation of sources used in ocular brachytherapy was investigated by dosimetric measurement in an eye phantom as well as numerical simulations. Different wolfram implantation techniques, such as extraocular fixation of a wolfram-silicone implant (n = 1), vitrectomy with silicone oil and intravitreal injection of a wolfram-silicone oil suspension (n = 2), and concurrent attachment of a wolfram implant onto the sclera (n = 2), were tested to investigate the long-term effects of wolfram. A vitrectomy with silicone oil without wolfram implantation was carried out in 2 rabbits (n = 2), constituting the control group. The eyes were enucleated after 3 months for histologic analysis. RESULTS:Wolfram-silicone mixtures have been dosimetrically proven to be very effective radiation absorbers for use in ocular brachytherapy. Severe complications, such as endophthalmitis, secondary glaucoma, cornea decompensation, and vessel occlusion, were not documented in the tested rabbit eyes after the application of wolfram. Histologic examination of the bulbi after enucleation showed epiretinal gliosis without further pathologic findings in all eyes after vitrectomy. CONCLUSIONS:The results of this study show that wolfram and wolfram-silicone implants constitute a promising candidate as potential radiation shielding substrates.

Reliability of power supply for current implantable electronic devices is a critical issue for longevity and for reducing the risk of device failure. Energy harvesting is an emerging technology, representing a strategy for establishing autonomous power supply by utilizing biomechanical movements in human body. Here, a novel "Twistron energy cell harvester" (TECH), consisting of coiled carbon nanotube yarn that converts mechanical energy of the beating heart into electrical energy, is presented. The performance of TECH is evaluated in an in vitro artificial heartbeat system which simulates the deformation pattern of the cardiac surface, reaching a maximum peak power of 1.42 W kg-1 and average power of 0.39 W kg-1 at 60 beats per minute. In vivo implantation of TECH onto the left ventricular surface in a porcine model continuously generates electrical energy from cardiac contraction. The generated electrical energy is used for direct pacing of the heart as documented by extensive electrophysiology mapping. Implanted modified carbon nanotubes are applicable as a source for harvesting biomechanical energy from cardiac motion for power supply or cardiac pacing.

Immune checkpoint inhibitor (ICI) therapy represents a ground-breaking paradigm in cancer treatment, harnessing the immune system to combat malignancies by targeting checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). The use of ICI therapy generates distinctive immune-related adverse events (irAEs) including cardiovascular toxicity, necessitating targeted research efforts. This comprehensive review explores preclinical models dedicated to ICI-mediated cardiovascular complications including myocarditis. Tailored preclinical models of ICI-mediated myocardial toxicities highlight the key role of CD8+ T cells, emphasizing the profound impact of immune checkpoints on maintaining cardiac integrity. Cytokines and macrophages were identified as possible driving factors in disease progression, and at the same time, initial data on possible cardiac antigens responsible are emerging. The implications of contributing factors including thoracic radiation, autoimmune disorder, and the presence of cancer itself are increasingly understood. Besides myocarditis, mouse models unveiled an accelerated progression of atherosclerosis, adding another layer for a thorough understanding of the diverse processes involving cardiovascular immune checkpoint signalling. This review aims to discuss current preclinical models of ICI cardiotoxicity and their potential for improving enhanced risk assessment and diagnostics, offering potential targets for innovative cardioprotective strategies. Lessons from ICI therapy can drive novel approaches in cardiovascular research, extending insights to areas such as myocardial infarction and heart failure.

Uveal melanoma (UM) is the most common primary cancer of the eye in adults. A new systemic therapy is needed to reduce the high metastasis and mortality rate. As β-blockers are known to have anti-tumor effects on various cancer entities, this study focuses on investigating the effect of β1-selective blockers atenolol, celiprolol, bisoprolol, metoprolol, esmolol, betaxolol, and in particular, nebivolol on UM. The study was performed on 3D tumor spheroids as well as 2D cell cultures, testing tumor viability, morphological changes, long-term survival, and apoptosis. Flow cytometry revealed the presence of all three β-adrenoceptors with a dominance of β2-receptors on cell surfaces. Among the blockers tested, solely nebivolol concentration-dependently decreased viability and altered 3D tumor spheroid structure. Nebivolol blocked the repopulation of cells spreading from 3D tumor spheroids, indicating a tumor control potential at a concentration of ≥20 µM. Mechanistically, nebivolol induced ATP depletion and caspase-3/7 activity, indicating that mitochondria-dependent signaling is involved. D-nebivolol or nebivolol combined with the β2-antagonist ICI 118.551 displayed the highest anti-tumor effects, suggesting a contribution of both β1- and β2-receptors. Thus, the present study reveals the tumor control potential of nebivolol in UM, which may offer a perspective for co-adjuvant therapy to reduce recurrence or metastasis.

CD47 is a cell surface protein controlling phagocytotic activity of innate immune cells. CD47 blockade was investigated as an immune checkpoint therapy in cancer treatment, enhancing phagocytosis of tumor cells by macrophages. Anti-CD47 treatment also reduced injury size during reperfused acute myocardial infarction (repAMI) by enhancing phagocytotic acitivity of macrophages. Little is known about the impact of CD47 blockade on neutrophils, representing the main portion of early infiltrating immune cells after repAMI. Therefore, we performed 45 min of cardiac ischemia followed by 24 h of reperfusion, observing a decreased cardiac injury size measured by triphenyl tetrazolium chloride (TTC) Evan’s blue staining. We were able to detect this effect with an innovative three-dimensional method based on light sheet fluorescence microscopy (LSFM). This further allowed us a simultaneous analysis of neutrophil infiltration, showing an unaltered amount of injury-associated neutrophils with reduced cardiac injury volume from repAMI. This observation suggests modulated phagocytosis of cell debris by neutrophils. Therefore, we performed flow cytometry analysis, revealing an increased phagocytotic activity of neutrophils in vitro. These findings highlight that CD47 blockade also enhances phagocytosis of cardiac cell debris by neutrophils, which might be an additional protective effect of anti-CD47 treatment after repAMI.

The cardiac bioavailability of peptide drugs that inhibit harmful intracellular protein–protein interactions in cardiovascular diseases remains a challenging task in drug development. This study investigates whether a non-specific cell-targeted peptide drug is available in a timely manner at its intended biological destination, the heart, using a combined stepwise nuclear molecular imaging approach. An octapeptide (heart8P) was covalently coupled with the trans-activator of transcription (TAT) protein transduction domain residues 48–59 of human immunodeficiency virus-1 (TAT-heart8P) for efficient internalization into mammalian cells. The pharmacokinetics of TAT-heart8P were evaluated in dogs and rats. The cellular internalization of TAT-heart8P-Cy(5.5) was examined on cardiomyocytes. The real-time cardiac delivery of 68Ga-NODAGA-TAT-heart8P was tested in mice under physiological and pathological conditions. Pharmacokinetic studies of TAT-heart8P in dogs and rats revealed a fast blood clearance, high tissue distribution, and high extraction by the liver. TAT-heart-8P-Cy(5.5) was rapidly internalized in mouse and human cardiomyocytes. Correspondingly, organ uptake of hydrophilic 68Ga-NODAGA-TAT-heart8P occurred rapidly after injection with an initial cardiac bioavailability already 10 min post-injection. The saturable cardiac uptake was revailed by the pre-injection of the unlabeled compound. The cardiac uptake of 68Ga-NODAGA-TAT-heart8P did not change in a model of cell membrane toxicity. This study provides a sequential stepwise workflow to evaluate the cardiac delivery of a hydrophilic, non-specific cell-targeting peptide. 68Ga-NODAGA-TAT-heart8P showed rapid accumulation in the target tissue early after injection. The implementation of PET/CT radionuclide-based imaging methodology as a means to assess effective and temporal cardiac uptake represents a useful and critical application in drug development and pharmacological research and can be extended to the evaluation of comparable drug candidates.

Cancer survival rates have increased significantly because of improvements in therapy regimes and novel immunomodulatory drugs. Recently, combination therapies of anthracyclines and immune checkpoint inhibitors (ICIs) have been proposed to maximize neoplastic cell removal. However, it has been speculated that a priori anthracycline exposure may prone the heart vulnerable to increased toxicity from subsequent ICI therapy, such as an anti-programmed cell death protein 1 (PD1) inhibitor. Here, we used a high-dose anthracycline mouse model to characterize the role of the PD1 immune checkpoint signaling pathway in cardiac tissue using flow cytometry and immunostaining. Anthracycline treatment led to decreased heart function, increased concentration of markers of cell death after six days and a change in heart cell population composition with fewer cardiomyocytes. At the same time point, the number of PD1 ligand (PDL1)-positive immune cells and endothelial cells in the heart decreased significantly. The results suggest that PD1/PDL1 signaling is affected after anthracycline treatment, which may contribute to an increased susceptibility to immune-related adverse events of subsequent anti-PD1/PDL1 cancer therapy.

AbstractBackgroundRecruitment and activation of brown adipose tissue (BAT) results in increased energy expenditure (EE) via thermogenesis and represents an intriguing therapeutic approach to combat obesity and treat associated diseases. Thermogenesis requires an increased and efficient supply of energy substrates and oxygen to the BAT. The hemoprotein myoglobin (MB) is primarily expressed in heart and skeletal muscle fibres, where it facilitates oxygen storage and flux to the mitochondria during exercise. In the last years, further contributions of MB have been assigned to the scavenging of reactive oxygen species (ROS), the regulation of cellular nitric oxide (NO) levels and also lipid binding. There is a substantial expression of MB in BAT, which is induced during brown adipocyte differentiation and BAT activation. This suggests MB as a previously unrecognized player in BAT contributing to thermogenesis.Methods and ResultsThis study analyzed the consequences of MB expression in BAT on mitochondrial function and thermogenesis in vitro and in vivo. Using MB overexpressing, knockdown or knockout adipocytes, we show that expression levels of MB control brown adipocyte mitochondrial respiratory capacity and acute response to adrenergic stimulation, signalling and lipolysis. Overexpression in white adipocytes also increases their metabolic activity. Mutation of lipid interacting residues in MB abolished these beneficial effects of MB. In vivo, whole‐body MB knockout resulted in impaired thermoregulation and cold‐ as well as drug‐induced BAT activation in mice. In humans, MB is differentially expressed in subcutaneous (SC) and visceral (VIS) adipose tissue (AT) depots, differentially regulated by the state of obesity and higher expressed in AT samples that exhibit higher thermogenic potential.ConclusionsThese data demonstrate for the first time a functional relevance of MBs lipid binding properties and establish MB as an important regulatory element of thermogenic capacity in brown and likely beige adipocytes.

The programmed cell death protein 1 (PD1) immune checkpoint prevents inflammatory tissue damage by inhibiting immune reactions. Understanding the relevance of cardiac PD1 signaling may provide new insights into the inflammatory events under baseline conditions and disease. Here, we demonstrate distinct immunological changes upon PD1 deficiency in healthy hearts and during reperfused acute myocardial infarction (repAMI). In PD1-deficient mice, upregulated inflammatory cytokines were identified under baseline conditions including cardiac interleukins and extracellular signal-related kinase 1/2 (ERK1/2). A murine in vivo repAMI model to determine inflammatory changes in the early phase showed downregulation of the ligand PDL1, paralleled by an endothelial injury, indicated by loss of the CD31 signal. Immunofluorescence imaging showed decreased PDL1 expression specifically in the infarct zone, highlighting an involvement in PDL1 in myocardial injury response. Pharmacological depletion of PD1 prior to repAMI did not alter the area of infarction but led to increased numbers of CD8(+) T cells in treated mice. We conclude that PD1/PDL1 signaling plays a significant role in healthy hearts and repAMI, emphasizing the relevance of adaptive immunity during myocardial injury. The findings highlight the risk for adverse outcomes from acute myocardial infarction in the growing group of patients receiving immune checkpoint inhibitor therapy.

Skeletal muscles are an important reservoir of nitric oxide (NO•) stored in the form of nitrite [NO2−] and nitrate [NO3−] (NOx). Nitrite, which can be reduced to NO• under hypoxic and acidotic conditions, is considered a physiologically relevant, direct source of bioactive NO•. The aim of the present study was to determine the basal levels of NOx in striated muscles (including rat heart and locomotory muscles) with varied contents of tissue nitrite reductases, such as myoglobin and mitochondrial electron transport chain proteins (ETC-proteins). Muscle NOx was determined using a high-performance liquid chromatography-based method. Muscle proteins were evaluated using western-immunoblotting. We found that oxidative muscles with a higher content of ETC-proteins and myoglobin (such as the heart and slow-twitch locomotory muscles) have lower [NO2−] compared to fast-twitch muscles with a lower content of those proteins. The muscle type had no observed effect on the [NO3−]. Our results demonstrated that fast-twitch muscles possess greater potential to generate NO• via nitrite reduction than slow-twitch muscles and the heart. This property might be of special importance for fast skeletal muscles during strenuous exercise and/or hypoxia since it might support muscle blood flow via additional NO• provision (acidic/hypoxic vasodilation) and delay muscle fatigue.