Sarcomeres are the fundamental functional units of skeletal muscle, essential for both force generation and metabolic homeostasis. While sarcomere degradation has been extensively studied, the mechanisms that preserve its integrity remain poorly defined. Here, we identify HECTD1 as an E3 ubiquitin ligase required for sarcomere maintenance and mitochondrial integrity. We show that HECTD1 ubiquitylates and stabilizes the chaperones KLHL40/41, which protect thin-filament components from misfolding and degradation. Consequently, reducing Hectd1 expression in myotubes coordinately decreases the levels of multiple sarcomere proteins. Skeletal muscle–specific Hectd1 knockout mice ( Hectd1 mKO) show severe sarcomere and mitochondrial disorganization and dysfunction, progressive muscle weakness, exercise and glucose intolerance, and unresolved tissue remodeling. Importantly, human iPSC-derived myotubes carrying a patient-associated HECTD1 mutation, recapitulate key molecular features of the Hectd1 mKO. These findings establish HECTD1 as a central regulator linking sarcomere proteostasis to mitochondrial function and identify its dysfunction as a cause of myopathy with mitochondriopathy.

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 diseases are clinically and genetically heterogeneous, often complicating diagnosis. Here, we describe four unrelated individuals with suspected mitochondrial disease who shared similar neuroimaging features, including bilateral symmetrical supra- and infratentorial white-matter abnormalities, together with variable movement disorders and intellectual impairment. Whole-genome sequencing identified the same homozygous MRPS22 variant (c.798_799delinsTA) in all four patients. MRPS22 encodes a component of the mitochondrial small ribosomal subunit (mtSSU). Functional studies in patient-derived fibroblasts showed impaired mitoribosome assembly and reduced de novo mitochondrial translation. Despite largely preserved steady-state levels of OXPHOS proteins, respiratory chain analysis identified a mild, isolated complex I deficiency. Proteomic profiling revealed reduced levels of mitochondrial ribosomal proteins and dysregulation of mitochondrial translation pathways. In line with the proteomic findings, RNA sequencing of fibroblasts from three patients revealed a distinct transcriptional signature compared with controls, with mitochondrial translation emerging as the most affected pathway. Mitochondrial-encoded transcripts were decreased, whereas nuclear-encoded mitochondrial genes were generally increased. Structural modelling suggested that the variant disrupts key interactions important for mitoribosome stability. While previously reported MRPS22 variants have been associated with severe, often prenatal-onset disease, the individuals described here exhibited a milder phenotype, thereby expanding the clinical spectrum of MRPS22-related disorders. Together, these findings support the pathogenicity of this variant and highlight the value of integrated genomic and functional analyses in diagnosing mitochondrial disease.

Abstract Neurological disorders are a major cause of death and disability worldwide. The brain’s energy metabolism is essential to its proper function, yet the mechanisms driving neuroenergetic dysfunction remain poorly understood. A key challenge is the limited availability of human-relevant models that can reproduce the complexity of brain physiology. An Organ-on-Chip (OoC) system was developed to mimic the neurovascular unit metabolic coupling by incorporating human isogenic iPSC-derived endothelial-like cells, pericyte-like cells, astrocytes, and a cerebral organoid, representing the main cellular components of the NVU. The novel, customized microfluidic platform enables research on neurovascular coupling by interconnecting a blood-brain barrier-on-a-chip model with a 3D brain parenchymal compartment to mimic physiological conditions. Graphical abstract

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.

Abstract Background As clinical genetics evolves towards the broader field of clinical genomics, the diagnostic approach to rare diseases is undergoing a paradigm shift. This transformation has significantly impacted rare disease diagnostics, increasingly done through gene panels, whole exome and whole genome sequencing. To advance beyond genomics into precision medicine and encompass the breadth of relevant clinical scenarios, a true systems shift is required that challenges conventional barriers and enables the formation of cross-disciplinary, integrated environments. Methods The Genomic Medicine Center Karolinska Rare Diseases (GMCK-RD) has, for the past 10 years, brought together healthcare and academia to enable large-scale genome sequencing in a clinical diagnostics context. Within GMCK-RD, experts from various medical disciplines collaborate closely with clinical geneticists, bioinformaticians, and researchers to integrate genome sequencing into healthcare. Results In total, 15 644 individuals with suspected rare diseases were analyzed using clinical genome sequencing, including pediatric (48%), adult (48%) and fetal (4%) samples. The overall diagnostic yield was 22.6%, providing a diagnosis for 3 538 individuals with variants in 1 570 genes. Moreover, a rare disease analysis tool suite developed and validated in house includes a bioinformatic pipeline allowing for comprehensive data analysis covering a wide range of genetic variants including SNVs, INDELs, repeat expansions, uniparental disomies, balanced and unbalanced structural variants as well as insertions of mobile elements. Results are visualized and interpreted in custom-developed decision support systems functioning as an interpretation portal as well as a knowledge-base to capture the interpretation efforts made in a structured format allowing future secondary use. Conclusions Altogether, GMCK-RD has shifted healthcare in our region towards precision diagnostics. We emphasize the need to transition from traditional clinical genetic diagnostics to a broader clinical genomics approach. Beyond this shift, we advocate integrating genomics with specialized clinical and laboratory medicine, a concept pioneered for inborn errors of metabolism (IEM) with stepwise spread to additional disease groups. In this model, a multidisciplinary unit combines screening, targeted diagnostics, individualized treatment, and long-term patient follow-up. Here we provide a road map and guide for inspiration for centers aiming to implement genome sequencing in rare disease diagnostics.

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.

Polyadenylation is a conserved post-transcriptional RNA modification with fundamentally different consequences for RNA fate across biological systems. In bacteria, chloroplasts, and plant mitochondria, adenylation is generally associated with RNA turnover and degradation, whereas its role in metazoan mitochondria remains incompletely understood. In metazoa, polyadenylation is best known for generating complete UAA stop codons in a subset of mitochondrial mRNAs. However, this explanation does not fully account for the evolutionary conservation of the modification, its diverse RNA substrates, or the broad phenotypic consequences of disrupted polyadenylation. In this review, we re-examine RNA adenylation and propose that, in metazoan mitochondria, polyadenylation primarily establishes a permissive 3' end state that governs RNA maturation, stability, translational competence, and decay. This perspective provides a unifying explanation for the diverse functions attributed to mitochondrial polyadenylation.

S-adenosylmethionine (SAM) is the principal methyl donor in cells and is essential for mitochondrial gene expression, influencing RNA modifications, translation, and ribosome biogenesis. Using direct long-read RNA sequencing in mouse tissues and embryonic fibroblasts, we show that processing of the mitochondrial ribosomal gene cluster fails in the absence of mitochondrial SAM, leading to an accumulation of unprocessed precursors. Proteomic analysis of ribosome fractions revealed these precursors associated with processing and assembly factors, indicating stalled biogenesis. Structural analysis by cryo-electron microscopy demonstrated that SAM-dependent methylation is required for peptidyl transferase centre formation during mitoribosome assembly. Our findings identify a critical role for SAM in coordinating mitoribosomal RNA processing and large subunit maturation, linking cellular methylation potential to mitochondrial translation capacity.

We present an optimised luminometric method for measuring muscle mitochondrial ATP production rate (MAPR), adapted to a 96-well microplate format. The enhanced assay enables quantification of ATP production from 12 or more substrate combinations within 15 min, using only 10 μL of isolated mitochondria. The method demonstrates high accuracy and precision, with a validated measurement range of 0.3-70 nmol/min/L. To support clinical interpretation, a reference dataset was established from 92 individuals aged seven months to 79 years. All these individuals were referred for muscle biopsy but were subsequently deemed unlikely to have a mitochondrial disorder following comprehensive clinical evaluation. An overview of the current version of our assays for oxidative phosphorylation (OXPHOS) enzymes is also provided. As proof of concept, we present three patients carrying pathogenic variants in mitochondrial DNA (ATP6 and MT-TL1) and the nuclear PDHA1 gene. All exhibited decreased MAPR with one or more substrates, along with additional clinical, biochemical, and morphological features consistent with mitochondrial disease. Furthermore, we illustrate the age-dependent development of MAPR in muscle across the human lifespan, demonstrating a 60-80 % higher maximal capacity for oxidative ATP production in adults compared with young children. In contrast, MAPR supported by fatty acid-derived substrates remains unchanged over the same period. In conclusion, the improved MAPR assay offers a robust and efficient tool for assessing mitochondrial function in both clinical diagnostics and research. Its high-throughput format and reliable performance make it particularly well-suited for the investigation of suspected mitochondrial disorders.

Sweden has one neonatal screening laboratory and two centers conducting diagnostic workup for inborn errors of metabolism (IEM). Next-generation sequencing (NGS) has been gradually introduced as a confirmatory diagnostic test in the Swedish newborn screening program. Here, we describe the use of NGS in the diagnostic workup of IEM in screening-detected babies in Sweden between 2015 and 2023. During this period, 1,023,344 newborn children were screened, and 81 of 290 IEM cases were genetically confirmed using NGS. Planned improvements to the program are to perform genetic validation directly on the initial dried blood spot (DBS). As whole-genome sequencing (WGS) is superior in detecting causative genetic variants compared to Sanger sequencing, targeted NGS, and whole-exome sequencing (WES), it will likely become the method of choice more broadly in the future. A strong focus is to consolidate the nationally coordinated DBS newborn screening program, with all its individual components, including screening, targeted diagnostics, individualized treatment, and follow-up. This challenges the current regionalized organization of Swedish healthcare, which hinders close national collaboration between experts and sharing of data, as well as equal access to advanced treatments for identified patients, regardless of their place of birth.

AbstractAberration of mitochondrial function is a shared feature of many human pathologies, characterised by changes in metabolic flux, cellular energetics, morphology, composition, and dynamics of the mitochondrial network. While some of these changes serve as compensatory mechanisms to maintain cellular homeostasis, their chronic activation can permanently affect cellular metabolism and signalling, ultimately impairing cell function. Here, we use a Drosophila melanogaster model expressing a proofreading-deficient mtDNA polymerase (POLγexo-) in a genetic screen to find genes that mitigate the harmful accumulation of mtDNA mutations. We identify critical pathways associated with nutrient sensing, insulin signalling, mitochondrial protein import, and autophagy that can rescue the lethal phenotype of the POLγexo- flies. Rescued flies, hemizygous for dilp1, atg2, tim14 or melted, normalise their autophagic flux and proteasome function and adapt their metabolism. Mutation frequencies remain high with the exception of melted-rescued flies, suggesting that melted may act early in development. Treating POLγexo- larvae with the autophagy activator rapamycin aggravates their lethal phenotype, highlighting that excessive autophagy can significantly contribute to the pathophysiology of mitochondrial diseases. Moreover, we show that the nucleation process of autophagy is a critical target for intervention.

BACKGROUND. Mitochondrial diseases belong to the group of inborn errors of metabolism (IEM), with a prevalence of 1 in 2,000-5,000 individuals. They are the most common form of IEM, but, despite advances in next-generation sequencing technologies, almost half of the patients are left genetically undiagnosed. METHODS. We investigated a cohort of 61 patients with defined mitochondrial disease to improve diagnostics, identify biomarkers, and correlate metabolic pathways to specific disease groups. Clinical presentations were structured using human phenotype ontology terms, and mass spectrometry-based proteomics was performed on primary fibroblasts. Additionally, we integrated 6 patients carrying variants of uncertain significance (VUS) to test proteomics as a diagnostic expansion. RESULTS. Proteomic profiles from patient samples could be classified according to their biochemical and genetic characteristics, with the expression of 5 proteins (GPX4, MORF4L1, MOXD1, MSRA, and TMED9) correlating with the disease cohort, thus acting as putative biomarkers. Pathway analysis showed a deregulation of inflammatory and mitochondrial stress responses. This included the upregulation of glycosphingolipid metabolism and mitochondrial protein import, as well as the downregulation of arachidonic acid metabolism. Furthermore, we could assign pathogenicity to a VUS in MRPS23 by demonstrating the loss of associated mitochondrial ribosome subunits. CONCLUSION. We established mass spectrometry-based proteomics on patient fibroblasts as a viable and versatile tool for diagnosing patients with mitochondrial disease. FUNDING. The NovoNordisk Foundation, Knut and Alice Wallenberg Foundation, Wellcome Centre for Mitochondrial Research, UK Medical Research Council, and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children.

Human mitochondria harbour a circular, polyploid genome (mtDNA) encoding 11 messenger RNAs (mRNAs), two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). Mitochondrial transcription produces long, polycistronic transcripts that span almost the entire length of the genome, and hence contain all three types of RNAs. The primary transcripts then undergo a number of processing and maturation steps, which constitute key regulatory points of mitochondrial gene expression. The first step of mitochondrial RNA processing consists of the separation of primary transcripts into individual, functional RNA molecules and can occur by two distinct pathways. Both are carried out by dedicated molecular machineries that substantially differ from RNA processing enzymes found elsewhere. As a result, the underlying molecular mechanisms remain poorly understood. Over the last years, genetic, biochemical and structural studies have identified key players involved in both RNA processing pathways and provided the first insights into the underlying mechanisms. Here, we review our current understanding of RNA processing in mammalian mitochondria and provide an outlook on open questions in the field.

Pyruvate dehydrogenase complex deficiency (PDCD) is a defect of aerobic carbohydrate metabolism that causes neurological disorders with varying degrees of severity. We report the clinical, biochemical, and molecular findings in patients with primary and secondary PDCD caused by novel atypical genetic variants. Whole-genome sequencing (WGS) identified the synonymous variants c.447A>G, p.(Lys149=) and c.570C>T, p.(Cys190=) in pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), the deep intronic variants c.1023+2267G>A and c.1023+2302A>G in pyruvate dehydrogenase complex component X (PDHX), and c.185+15054G>A in thiamine pyrophosphokinase (TPK1). Analysis by Sanger and RNA sequencing of cDNA from patient blood and/or cultured fibroblasts showed that the synonymous variants in PDHA1 lead to aberrant splicing and skipping of exons 5 and 5-6 in one of the patients and transcripts lacking exon 6 in the other. The deep intronic variants in PDHX and TPK1 lead to insertion of intronic sequence in the corresponding transcripts. The splice defects in PDHA1 were more pronounced in cultured fibroblasts than in blood. Our findings expand the spectrum of pathogenic variants causing PDCD and highlight the importance of atypical variants leading to aberrant splicing. The severity of the splice defects and resulting biochemical dysfunction varied between tissues, stressing the importance of performing biochemical and transcript analysis in affected tissues. The two males with hemizygous synonymous PDHA1 variants have a mild phenotype and higher PDH enzyme activity than expected, which is consistent with aberrant but leaky splicing with a proportion of the transcripts remaining correctly spliced.

Loss-of-function variants in the PRKN gene encoding the ubiquitin E3 ligase PARKIN cause autosomal recessive early-onset Parkinson’s disease (PD). Extensive in vitro and in vivo studies have reported that PARKIN is involved in multiple pathways of mitochondrial quality control, including mitochondrial degradation and biogenesis. However, these findings are surrounded by substantial controversy due to conflicting experimental data. In addition, the existing PARKIN-deficient mouse models have failed to faithfully recapitulate PD phenotypes. Therefore, we have investigated the mitochondrial role of PARKIN during ageing and in response to stress by employing a series of conditional Parkin knockout mice. We report that PARKIN loss does not affect oxidative phosphorylation (OXPHOS) capacity and mitochondrial DNA (mtDNA) levels in the brain, heart, and skeletal muscle of aged mice. We also demonstrate that PARKIN deficiency does not exacerbate the brain defects and the pro-inflammatory phenotype observed in mice carrying high levels of mtDNA mutations. To rule out compensatory mechanisms activated during embryonic development of Parkin -deficient mice, we generated a mouse model where loss of PARKIN was induced in adult dopaminergic (DA) neurons. Surprisingly, also these mice did not show motor impairment or neurodegeneration, and no major transcriptional changes were found in isolated midbrain DA neurons. Finally, we report a patient with compound heterozygous PRKN pathogenic variants that lacks PARKIN and has developed PD. The PARKIN deficiency did not impair OXPHOS activities or induce mitochondrial pathology in skeletal muscle from the patient. Altogether, our results argue that PARKIN is dispensable for OXPHOS function in adult mammalian tissues.

Neurological conditions conquer the world; they are the leading cause of disability and the second leading cause of death worldwide, and they appear all around the world in every age group, gender, nationality, and socioeconomic class. Despite the growing evidence of an immense impact of perturbations in neuroenergetics on overall brain function, only little is known about the underlying mechanisms. Especially human insights are sparse, owing to a shortage of physiologically relevant model systems. With this perspective, we aim to explore the key steps and considerations involved in developing an advanced human in vitro model for studying neuroenergetics. We discuss biological and technological strategies to meet the requirements of a predictive model, aiming at providing a guide and inspiration for future in vitro models of neuroenergetics.

The pyruvate dehydrogenase complex (PDC) is responsible for the conversion of pyruvate into acetyl-CoA, which is used for energy conversion in cells. PDC activity is regulated by phosphorylation via kinases and phosphatases (PDK/PDP). Variants in all subunits of the PDC and in PDK3 have been reported, with varying phenotypes including lactic acidosis, neurodevelopmental delay, peripheral neuropathy, or seizures. Here, we report a de novo heterozygous missense variant in PDK1 (c.1139G > A; p.G380D) in a girl with developmental delay and early onset severe epilepsy. To investigate the role of PDK1G380D in energy metabolism and neuronal development, we used a zebrafish model. In zebrafish embryos we show a reduced number of cells with mitochondria with membrane potential, reduced movements, and a delay in neuronal development. Furthermore, we observe a reduction in the phosphorylation of PDH-E1α by PDKG380D, which suggests a disruption in the regulation of PDC activity. Finally, in patient fibroblasts, a mild reduction in the ratio of phosphorylated PDH over total PDH-E1α was detected. In summary, our findings support the notion that this aberrant PDK1 activity is the cause of clinical symptoms in the patient.