The neonatal heart experiences rapid metabolic growth after birth to meet increasing energetic and biosynthetic demands. How mitochondrial cofactor availability limits this transition remains unclear. Here, we demonstrate that mitochondrial S-adenosylmethionine (mitoSAM) import through SLC25A26 becomes limiting shortly after birth and specifically restricts protein lipoylation, although other mitoSAM-dependent processes are partially preserved. Loss of Slc25a26 impaired lipoylation-dependent flux through pyruvate and α-ketoglutarate dehydrogenases, restricting tricarboxylic acid cycle carbon entry and depleting aspartate and nucleotide pools. Conversely, mitochondrial gene expression remained intact, and respiratory chain enzyme activities showed partial impairment, indicating that lipoylation is the most mitoSAM-sensitive pathway during postnatal heart adaptation. These metabolic limitations were linked to sustained cardiomyocyte cell-cycle activity, delayed structural maturation, and early cardiomyopathy. Supplementing with medium-chain triglycerides during the suckling-to-weaning transition partially stabilized metabolism and prolonged survival. Overall, our findings identify a stage-specific metabolic vulnerability in the postnatal heart characterized by hierarchical mitoSAM utilization within the mitochondria.

Mitochondrial DNA (mtDNA) mutations accumulate with age, but their mechanistic contribution to aging remains unclear. The classical mtDNA mutator mouse expresses a proofreading-deficient mtDNA polymerase (POLG D257A ) and accumulates mtDNA mutations across all tissues leading to premature aging. However, this model cannot resolve whether the aging phenotype results from systemic dysfunction or cell-intrinsic effects of somatic mtDNA mutations. To overcome this limitation, we generated Polg iMut mice allowing spatial and temporal control of POLG D257A expression. We demonstrate here that mtDNA mutations induced in cardiomyocytes cause progressive contractile dysfunction and respiratory chain deficiency in the heart without accompanying systemic pathology. Proteomic analyses link cardiac mosaic respiratory chain dysfunction to a progressive immune response, characterized by up-regulation of antigen-processing proteins and immune cell infiltration. In contrast, longevity-associated pathways are suppressed and uncoupled from mitochondrial and immune alterations, indicating distinct regulatory mechanisms. These findings demonstrate that mtDNA mutations can drive cardiac dysfunction and reveal a mechanistic link between mitochondrial dysfunction, immune responses, and aging.

Heart failure (HF) remains a major global health burden despite advances in pharmacological and device-based therapies. Current guideline-directed medical therapy (GDMT) slows disease progression through neurohormonal modulation but does not address the fundamental deficit in contractile function, particularly in patients with advanced HF with reduced ejection fraction (HFrEF). Conventional inotropes improve contractility but worsen outcomes. Recent advances have identified novel therapeutic approaches that enhance cardiac contractility without the deleterious effects of conventional inotropes. This review examines pathophysiologic mechanisms compromising contractility in HFrEF and therapeutic strategies that enhance contractility, spanning both traditional calcium-dependent inotropes and emerging classes of small molecules. Their mechanisms of action, along with supporting preclinical and clinical data, are discussed to highlight their future therapeutic potential. These novel strategies reflect a paradigm shift from indirect neurohormonal modulation toward direct myocardial modulation, potentially reshaping the therapeutic landscape for both acute and chronic HF with targeted, efficacious, and safer interventions.

Acyl ghrelin increases cardiac output (CO) and contractility in patients with heart failure with reduced ejection fraction (HFrEF). In healthy humans, acyl ghrelin increases gastric emptying rate (GER) and hunger, a potential add-on benefit for HFrEF patients suffering from cachexia with symptoms of delayed gastric emptying and loss of appetite. The aim of this study was to determine if post-prandial acyl ghrelin infusion increases GER and hunger in HFrEF patients compared to CO. HFrEF patients (n = 29) arrived fasted and received a 500-kcal breakfast followed by 1.5 g paracetamol. They next received placebo (vehicle, n = 15) or acyl ghrelin infusion (0.1 µg/kg/min, n = 14) for 120 min. Blood was tapped for plasma at 0, 30, 60, 120, and 150 min into the meal. Hunger scores and CO were recorded. Plasma paracetamol was measured to assess GER. Paracetamol concentration peaked at 30 min in 8 of 14 (57

The neonatal heart undergoes a rapid metabolic transition from fetal glycolysis to oxidative phosphorylation, requiring coordinated metabolic remodeling. Mechanisms driving this transition remain unclear. Here, we demonstrate that sufficient mitochondrial S-adenosylmethionine (mitoSAM), imported via the solute carrier Slc25a26 , is essential for this shift by sustaining the lipoylation of 2-oxoacid dehydrogenases, critical for TCA cycle activation. Proteomic and metabolomic profiling revealed that reduced mitoSAM availability impaired lipoylation, blocking TCA cycle function and restricting nucleotide synthesis, while mitochondrial gene expression and respiratory capacity remained largely intact. In vivo EdU labeling showed persistent cardiomyocyte proliferation imposing further strain on nucleotide pools. Supplementation with medium-chain triglycerides during the suckling-to-weaning transition restored metabolic function and normalized cardiac growth and morphology. Our data reveal a critical developmental window in which mitoSAM-dependent lipoylation ensures heart maturation.

OBJECTIVES:Dermatomyositis (DM) is characterised by systemic inflammation, debilitating muscle weakness, cutaneous lesions, and increased mortality. An upregulation of type I interferon (IFN)-stimulated genes is observed in patients with DM. However, it remains unclear whether type I IFNs cause muscle weakness in DM. This study aimed to investigate the role of IFN-α/β receptor signalling in muscle weakness induced by factors in DM serum. METHODS:In ex vivo experiments, flexor digitorum brevis muscles were isolated from healthy mice and incubated 24 hours with 10% healthy serum or serum from patients with DM (n = 9). To modulate IFN signalling, an antibody against the type I IFN receptor α/β subunit 1 (IFNAR1) or the Janus kinase-signal transducer and activator of transcription inhibitor ruxolitinib was used. RNA sequencing, followed by bioinformatics analysis, was conducted to identify differentially expressed genes and affected pathways related to IFN signalling. RESULTS:Incubation with serum from patients with DM, but not that from healthy controls, caused significant muscle weakness manifested by a reduction in muscle force. Bioinformatic analyses revealed downregulation of type I IFN-inducible genes with IFNAR1 antibody. Pathway analysis showed enrichment of several IFN-related pathways. Inhibition of type I IFN signalling with either an IFNAR1 antibody or ruxolitinib abolished DM serum-induced effects. CONCLUSIONS:Factors in serum from patients with DM can activate the type I IFN signalling pathway in skeletal muscles, which constitutes an important causal factor for muscle weakness. Our data support a mechanistic model where blood-borne factors contribute to muscle disease phenotypes and underscore the therapeutic possibilities of pharmacological interventions targeting the IFNAR1 signalling pathway.

Heart failure with reduced ejection fraction (HFrEF) is characterized by impaired cardiac contractility. AC01, a small-molecule ghrelin receptor agonist, enhances contractility in cardiomyocytes. This study evaluated the in vivo hemodynamic effects of AC01 in a mouse HFrEF model and in cynomolgus monkeys. In HFrEF mice, intravenous AC01 significantly increased cardiac output, stroke volume, and ejection fraction versus vehicle, without any apparent detriment to diastolic function. Pressure-volume loop analysis demonstrated load-independent inotropic effects. In monkeys, oral AC01 increased cardiac output and stroke volume while reducing heart rate, without lowering central aortic pressure. These effects were sustained over 14 days of oral dosing. Additionally, AC01 improved autonomic balance by increasing parasympathetic and decreasing sympathetic activity. Overall, AC01 produced rapid, consistent, and sustained improvements in systolic function across species, supporting its potential as a novel, load-independent inotropic therapy for heart failure.

BACKGROUND:To investigate efficacy, safety, and tolerance of high-intensity interval training (HIIT) vs. clinical standard low-moderate intensity home-based exercise (CON) to improve aerobic capacity, muscle endurance, and mitochondrial function in patients with recent onset, idiopathic inflammatory myopathies (IIM). METHODS:Twenty-three patients with recent onset IIM were randomised into HIIT or CON groups. Both groups underwent 12 weeks of exercise training. The HIIT did 3 sessions/week, always supervised during the first three weeks. Then, training was supervised 1-3 session per week based on an individual assessment of the participants' preference and ability to perform HIIT in the clinic. The CON received one supervised session and then exercised five days per week at home, following clinical standard. Primary outcome was maximal exercise test (VO2peak l/min and ml/kg x min, peak power (Watt), time-to-exhaustion TTE min/sec), with secondary outcomes mitochondrial protein expression in muscle. Safety was assessed by disease activity (serum levels of muscle enzymes, muscle strength (MMT8), Physician Global Assessment, pain, and fatigue (VAS, 0-100). FINDINGS:HIIT resulted in a 16% increase in VO2peak L/min, significantly higher than the 1.8% change in CON (95% CI 0.1; 0.47). Peak power and TTE improved significantly more in HIIT, 18% and 23%, respectively, compared to CON, 8% and 12% (95% CI 3.9; 30.8 and 00:06; 03:18, respectively). Muscle biopsies (HIIT n = 7, CON n = 6) showed increases (p < 0.05) in central mitochondrial protein expression in HIIT but not CON, suggesting enhanced mitochondrial function. Both groups maintained stable serum muscle enzymes indicating no increase in disease activity from the intervention. Muscle disease activity remained low and unchanged in both groups (95% CI -1.2; 1.0), physician global activity and MMT8 significantly improved within CON (95% CI -1.7; -0.26 and 0.1; 3.9, respectively) but not in the HIIT group. INTERPRETATION:HIIT is an effective and safe exercise intervention to improve aerobic fitness, muscle endurance, and mitochondrial function in patients with recent onset IIM. This approach should be considered an adjuvant treatment in managing IIM, potentially health-enhancing for these patients. FUNDING:Swedish Research Council, the Swedish Rheumatism Association, Stockholm County Research Grant (ALF), King Gustaf V 80-year Foundation, the Swedish Heart and Lung Foundation, Promobilia Foundation, and Stig Thune Foundation.

Catalase, a canonical antioxidant enzyme, has been shown to prevent maladaptive oxidative damage and protect against aging-associated deterioration of cellular functions. In cardiac tissue, catalase protects cardiac myocytes against a variety of pathological changes by maintaining normal contractile function. Here we elucidated how catalase can protect cardiomyocyte excitation-contraction (EC) coupling we focused on exploring the subcellular location and specific effects of catalase in cardiomyocytes. To determine the effects of catalase on cardiomyocyte EC-coupling, we used adenoviral overexpression and pharmacological inhibition of catalase with 3AT in cultured adult mouse ventricular myocytes combined with detailed characterization of Ca2+ signaling. We found that endogenous catalase co-localizes with RyRs in the subspace between the junctional sarcoplasmic reticulum (SR) and t-tubules. When virally overexpressed, catalase increases the ROS scavenging capacity of myocytes and enhances Ca2+-release by increasing synchronization of local Ca2+-release. To improve Ca2+-release, catalase acts to prevent membrane peroxidation and prevents degradation of t-tubule structures. Both the functional and structural effects of catalase were reversed by catalase inhibition with 3AT. The effect of catalase on Ca2+-release was more pronounced in cardiomyocytes isolated from transverse aortic constriction-operated, hypertrophied mouse hearts. Overall, our data suggest that catalase located to the subspace has a specific role in stabilizing and protecting Ca2+-release structures from degradation in conditions associated with increased oxidative stress.

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric soft tissue sarcoma with limited therapeutic options, particularly for cases resistant to conventional treatments. The SUMOylation pathway, which plays a key role in regulating the cell cycle, apoptosis, and transcription, has emerged as a potential therapeutic target in RMS. Elevated levels of SUMO1 and SUMO2/3 conjugates in RMS cell lines, compared to normal human skeletal muscle cells, underscore the association between upregulated SUMOylation and aggressive cancer phenotypes. Understanding these molecular underpinnings is critical for the development of innovative and effective treatments. The investigation encompassed transcriptomic and protein analyses to profile SUMOylation pathway components across alveolar and embryonal RMS subtypes, aiming to identify heterogeneity that could guide personalized therapy approaches. TAK-981, a small molecule that selectively inhibits the SUMOylation of target proteins, was evaluated in combination with chemotherapeutic agents for additive or synergistic effects. Additionally, its impact on radiosensitivity and key signaling pathways, such as AKT, ERK and CAV1 phosphorylation, was assessed to elucidate its mechanism of action. Transcriptomic and proteomic analyses revealed distinct expression profiles of SUMOylation pathway components across RMS subtypes, highlighting heterogeneity that could guide personalized therapeutic strategies. Notably, SAE1 protein was overexpressed in RMS tissues and cells, positioning it as a potential biomarker for this cancer. Its activity was effectively counteracted by TAK-981, a SUMO inhibitor that demonstrated significant therapeutic potential by suppressing RMS cell proliferation and migration, and enhancing the cytotoxic effects of chemotherapeutic agents actinomycin D and doxorubicin. However, TAK-981 did not increase radiosensitivity, suggesting its selective action through chemical inhibition mechanisms. Mechanistically, TAK-981 reduced phosphorylation of key signaling proteins, including AKT, ERK and CAV1, which are critical for RMS cell survival. The findings of this study establish TAK-981 as a promising therapeutic agent for RMS. The results also provide foundational insights into the role of SUMOylation associated with the new biomarker SAE1 in RMS and its subtypes, paving the way for the development of personalized treatment strategies that leverage SUMO pathway inhibition.

AIMS:Impaired cardiac function, reduced nitric oxide (NO) bioavailability, and inflammation are key contributors to the pathogenesis and progression of heart failure with reduced ejection fraction (HFrEF). This study aimed to investigate whether dietary inorganic nitrate supplementation can attenuate cardiac dysfunction and adverse remodeling in HFrEF by enhancing NO signaling. METHODS:Two mouse models of HFrEF, induced by myocardial infarction (MI) or transverse aortic constriction (TAC), were treated with dietary nitrate or a control diet for 4-6 weeks, initiating the treatment on day 3 after myocardial injury. Echocardiography and pressure volume (PV) loop analysis were employed to assess cardiac function and hemodynamics. Histology staining was performed to assess the degree of cardiac fibrosis. Myograph experiments were conducted to assess aortic vasorelaxation. Biomarkers related to hypertrophy, fibrosis, and inflammation were analyzed in cardiac tissues through Q-PCR analysis and immunofluorescence staining. RESULTS:In HFrEF mice, long-term inorganic nitrate treatment increased systolic and diastolic function, enhanced vascular relaxation, and reduced both replacement and reactive fibrosis. In the nitrate group, cardiac gene expression showed downregulation of hypertrophy-, fibrosis-, and inflammation-related markers, alongside upregulation of anti-inflammatory markers associated with M1-to-M2 macrophage polarization. Immunofluorescence confirmed reduced fibrosis and increased anti-inflammatory protein biomarkers associated with increased serum nitrate and cardiac cGMP levels. CONCLUSIONS:Early initiation of dietary nitrate supplementation after myocardial injury enhances cardiac and vascular function, reduces fibrosis and inflammation, and holds promise as a cardioprotective strategy to reduce the progression of HFrEF through NO-signaling.

Abstract Background Acyl ghrelin (ghrelin) increases cardiac output (CO) and contractility in patients with heart failure with reduced ejection fraction (HFrEF). In healthy humans, ghrelin increases gastric emptying rate (GER) and hunger, a potential add-on benefit for HFrEF patients suffering from cachexia with symptoms of delayed gastric emptying and loss of appetite. Aim Determine if post-prandial ghrelin infusion increases GER and hunger in HFrEF patients; compare to CO. Methods This study was a component of a double-blind placebo-controlled trial of acyl ghrelin vs. placebo in HFrEF. Patients (n=29) arrived fasted and received a 500 kcal breakfast and 1.5 g paracetamol. They next received placebo (vehicle, n=15) or ghrelin infusion (0.1 µg/kg/min, n=14) for 120 min. Blood was tapped for plasma at 0, 30, 60, 120 and 150 min into the meal. Plasma concentration of paracetamol was measured to assess GER of a liquid bolus. Hunger scores and CO were recorded. Mann-Whitney rank sum test was used for statistics. Results Paracetamol peaked at 30 min in 8 of 14 (57%) in the ghrelin treatment group versus 1 of 15 (7%) in the placebo group (P=0.004, Fig 1). Remaining patients peaked at later time points. There were no anomalous peaks at 0 min baseline or 2880 min (2 days). The ghrelin treatment group data was segregated into rapid (peak at 30 min, n=8) and slow (peak >30 min, n=6) GER subgroups. The rapid subgroup had a ghrelin treatment effect on CO (one-way repeat measures) (P<0.001, Fig 2). Baseline (t=0) CO for the rapid GER subgroup was 4.06 ±1.41 L/min (median ±SD) and 3.95 ±0.62 L/min for the slow GER subgroup (P=0.31). Data points shown are median ± SEM, each patient normalized to own baseline (100%); * P<0.05, ** P<0.01, *** P<0.001, pairwise comparison to baseline. Hunger gradually increased. The greatest increase in hunger was at 150 min, at which time median fold increase in hunger relative baseline was 1.6 (placebo), 1.7 (ghrelin, slow GER) and 2.3 (ghrelin, rapid GER). These hunger differences between groups did not reach significance. However, this trend was consistent with ghrelin induction of hunger, especially in those with potent GER and CO responses. Conclusions Ghrelin increases GER, and potentially hunger, in HFrEF patients in addition to increasing CO. Some HFrEF patients may benefit from this add-on effect.Fig 1.Time of paracetamol peak.Fig 2.Cardiac output.

Sprint interval training (SIT) is a time-efficient type of endurance training that involves large type 2 muscle fibre recruitment. Effective antioxidant supplementation may mitigate positive training adaptations by limiting the oxidant challenge. Our aim was to test whether SIT affects type 2 more than type 1 muscle fibres, and whether the muscular training response is mitigated by antioxidant treatment. Young men performed three weekly SIT sessions (4-6 × 30 s all-out cycling) for 3 weeks while treated with antioxidants (vitamin C, 1 g day-1; vitamin E, 235 mg day-1) or placebo. Vastus lateralis biopsies were taken to measure (i) activation of genes for reactive oxygen/nitrogen species (ROS) sensors and inflammatory mediators with quantitative RT-PCR and (ii) fibre type-specific proteome adaptations using MS-based proteomics. Vitamin treatment decreased the upregulation of genes for ROS sensors and inflammatory regulators during the first SIT session. The 3 weeks of SIT caused generally larger proteome adaptations in type 2 than in type 1 fibres, and this included larger increases in abundance of proteins involved in mitochondrial energy production. Vitamin treatment blunted the SIT-induced proteome adaptations, whereas it did not affect the training-induced improvement in maximal cycling performance. In conclusion, (i) the large type 2 fibre recruitment and resulting proteome adaptations are instrumental to the effectiveness of SIT and (ii) antioxidant supplementation counteracts positive muscular adaptations to SIT, which would blunt any improvement in submaximal endurance performance, whereas it does not affect the improvement in maximal cycling performance, where O2 delivery to muscle would be limiting. KEY POINTS: Sprint interval training (SIT) is a time-efficient type of endurance training that involves large recruitment of fast-twitch muscle fibres. Treatment with antioxidants may mitigate the positive effects of endurance training. Fibre type-specific proteomics performed on muscle biopsies obtained from young men before and after 3 weeks of SIT showed larger training effects in fast- than in slow-twitch fibres. Antioxidant treatment in the form of vitamin C and E pills counteracted the positive muscular adaptations to the 3 weeks of SIT. These results increase our understanding of why SIT is an effective endurance training regime and provide further evidence against the common belief that antioxidant supplements are beneficial in a physical exercise context.

Digital sequencing uses unique molecular identifiers (UMIs) to correct for polymerase induced errors and amplification biases. Here, we design 19 different structured UMIs to minimize the capacity of primers to form non-specific PCR products during library construction using SiMSen-Seq, a PCR-based digital sequencing approach with flexible multiplexing capabilities suitable for tumor-informed mutation analysis. All structured UMI designs demonstrate enhanced assay performance compared with an unstructured reference UMI. The best performing structured UMI design shows significant improvements in all tested aspects of assay and sequencing performance with the ability to reliable detect low variant allele frequencies.

Sprint interval training (SIT) is a time-efficient type of endurance training that involves large type 2 muscle fibre recruitment. Effective antioxidant supplementation may mitigate positive training adaptations by limiting the oxidant challenge. Our aim was to test whether SIT affects type 2 more than type 1 muscle fibres, and whether the training response is mitigated by antioxidant treatment. Young men performed three SIT sessions (6 x 30 s all-out cycling) per week for three weeks while treated with antioxidants (vitamin C, 1 g/day; vitamin E, 235 mg/day) or placebo. Vastus lateralis biopsies were taken to measure (i) activation of genes for reactive oxygen/nitrogen species (ROS) sensors and inflammatory mediators with quantitative RT PCR and (ii) fibre type-specific proteome adaptations using mass spectrometry-based proteomics. Vitamin treatment decreased the upregulation of genes for ROS sensors and inflammatory regulators during the first SIT session. The three weeks of SIT caused generally larger proteome adaptations in type 2 than in type 1 fibres, and this included larger increases in abundance of proteins involved in mitochondrial energy production. Vitamin treatment blunted the SIT-induced proteome adaptations, whereas it did not affect the training-induced improvement in maximal cycling performance. In conclusion, (i) the large type 2 fibre recruitment and resulting proteome adaptations are instrumental to the effectiveness of SIT, and (ii) antioxidant supplementation counteracts positive muscular adaptations to SIT, which would blunt any improvement in submaximal endurance performance, whereas it does not affect the improvement in maximal cycling performance, where O2 delivery to muscle would be limiting. ### Competing Interest Statement The authors have declared no competing interest.

Angiosarcoma is a rare and aggressive type of soft-tissue sarcoma with high propensity to metastasize. For patients with metastatic angiosarcoma, prognosis is dismal and treatment options are limited. To improve the outcomes, identifying patients with poor treatment response at an earlier stage is imperative, enabling alternative therapy. Consequently, there is a need for improved methods and biomarkers for treatment monitoring. Quantification of circulating tumor-DNA (ctDNA) is a promising approach for patient-specific monitoring of treatment response. In this case report, we demonstrate that quantification of ctDNA using SiMSen-Seq was successfully utilized to monitor a patient with metastatic angiosarcoma. By quantifying ctDNA levels using 25 patient-specific mutations in blood plasma throughout surgery and palliative chemotherapy, we predicted the outcome and monitored the clinical response to treatment. This was accomplished despite the additional complexity of the patient having a synchronous breast cancer. The levels of ctDNA showed a superior correlation to the clinical outcome compared with the radiological evaluations. Our data propose a promising approach for personalized biomarker analysis to monitor treatment in angiosarcomas, with potential applicability to other cancers and for patients with synchronous malignancies.