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Background Barth syndrome (BTHS) is a rare X‐linked mitochondrial disorder caused by mutations in the TAFAZZIN gene, which disrupts cardiolipin (CL) remodelling and mitochondrial function. While cardiac manifestations of BTHS are well characterized in male patients, the mechanisms underlying skeletal muscle weakness and fatigability are poorly understood. Methods We investigated neuromuscular and mitochondrial alterations in a novel murine model (TazPM) carrying a patient‐derived D75H point mutation knocked into the Tafazzin locus. This mutation preserves protein abundance but abolishes enzymatic activity. Skeletal muscle function was assessed via weightlifting and hanging tests. Muscle fibre composition and neuromuscular junction (NMJ) integrity were evaluated using immunofluorescence, western blotting and in vivo electrophysiology. Mitochondrial morphology was examined by transmission electron microscopy, and bioenergetics were quantified using ultra‐performance liquid chromatography. Stress signalling was assessed by western blotting. Results Male TazPM mice exhibited seven‐fold elevated total monolysocardiolipin and five‐fold reduced mature CL levels, confirming deficient transacylase activity. These mice exhibited lower muscle strength and endurance, 32% smaller muscle fibres of all types and a shift towards fast‐twitch type 2B fibres, which are more susceptible to fatigue. Electrophysiological analysis revealed a 60% reduction in motor unit number and an increase in average single motor unit potential, indicating motor neuron remodelling. NMJ protein analysis showed decreased MUSK and DOK7 and increased CHRNA1, suggesting impaired NMJ integrity. Despite mitochondrial structural abnormalities and reduced expression of key mitochondrial proteins (NDUFB8, MCU, TMEM65), resting ATP, phosphocreatine and adenine nucleotide ratios were unchanged in both glycolytic and oxidative muscles. However, stress signalling pathways were markedly activated, including phosphorylation of eIF2α, increased CHOP, DELE1, p53 expression and altered Wnt/β‐catenin signalling components. Conclusions Whole‐body deficiency of tafazzin enzymatic activity, as occurs in BTHS, is sufficient to result in widespread neuromuscular remodelling, including fibre size/type shifts, motor unit loss, NMJ dysregulation and stress pathway activation, without overt energetic failure at rest. These findings suggest that myopathy in BTHS arises not solely from mitochondrial ATP insufficiency but rather from cumulative structural and signalling adaptations.

Fibroblasts, which are the most numerous cell type in the heart, interact with cardiomyocytes in vitro and affect their function; however, they are considered to play a secondary role in cardiac hypertrophy and failure. Here we have shown that cardiac fibroblasts are essential for the protective and hypertrophic myocardial responses to pressure overload in vivo in mice. Haploinsufficiency of the transcription factor-encoding gene Krüppel-like factor 5 (Klf5) suppressed cardiac fibrosis and hypertrophy elicited by moderate-intensity pressure overload, whereas cardiomyocyte-specific Klf5 deletion did not alter the hypertrophic responses. By contrast, cardiac fibroblast-specific Klf5 deletion ameliorated cardiac hypertrophy and fibrosis, indicating that KLF5 in fibroblasts is important for the response to pressure overload and that cardiac fibroblasts are required for cardiomyocyte hypertrophy. High-intensity pressure overload caused severe heart failure and early death in mice with Klf5-null fibroblasts. KLF5 transactivated Igf1 in cardiac fibroblasts, and IGF-1 subsequently acted in a paracrine fashion to induce hypertrophic responses in cardiomyocytes. Igf1 induction was essential for cardioprotective responses, as administration of a peptide inhibitor of IGF-1 severely exacerbated heart failure induced by high-intensity pressure overload. Thus, cardiac fibroblasts play a pivotal role in the myocardial adaptive response to pressure overload, and this role is partly controlled by KLF5. Modulation of cardiac fibroblast function may provide a novel strategy for treating heart failure, with KLF5 serving as an attractive target.

Background Chinese urban residents consume more salt from meals prepared outside home than in the past. The purpose of this study is to understand Chinese consumer demand for salt reduction as expressed through their orders on meal delivery apps (MDAs), restaurants’ willingness to promote salt reduction, and the extent to which restaurants comply with reduced salt requests. Methods We analyzed consumer comments extracted from 718 restaurants on a Chinese MDA called ELEME for orders made in the July-December 2020 timeframe. A self-designed questionnaire was distributed to the restaurant managers to assess restaurants’ attitude towards salt reduction upon signing up for the study, and laboratory validation was conducted to test whether dishes ordered with reduced salt requests by consumers actually contained less salt. Results A total of 25,982 (0.7%) orders out of 3,630,798 orders contained consumer comments. Of the consumer comments, 40.6% (10,549) were about requests for less salt in dishes. Totally 91.5% of 421 surveyed restaurants showed a willingness to respond to consumers’ reduced salt requests. The median sodium content measured in the reduced-salt dishes by the laboratory was significantly lower than that in their regular salt counterparts ( P  < 0.05). Conclusions We observed substantial consumer demand for salt reduction while ordering meals on the MDA and that restaurants did, in response, reduce the sodium content in the meals they provided. As meals delivered via MDAs comprise an increasing proportion of outside foods consumed, there is an opportunity for public health experts and policy makers to work with MDAs and restaurants to promote healthier food selections. Trial registration ChiCTR2100047729.

Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele (TazPM) that phenocopies BTHS clinical traits. As TazPM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile TazPM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile TazPM hearts. However, adult TazPM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult TazPM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.

Barth syndrome (BTHS) is a rare, infantile-onset, X-linked mitochondriopathy exhibiting a variable presentation of failure to thrive, growth insufficiency, skeletal myopathy, neutropenia, and heart anomalies due to mitochondrial dysfunction secondary to inherited TAFAZZIN transacetylase mutations. Although not reported in BTHS patients, male infertility is observed in several Tafazzin (Taz) mouse alleles and in a Drosophila mutant. Herein, we examined the male infertility phenotype in a BTHS-patient-derived D75H point-mutant knockin mouse (TazPM) allele that expresses a mutant protein lacking transacetylase activity. Neonatal and adult TazPM testes were hypoplastic, and their epididymis lacked sperm. Histology and biomarker analysis revealed TazPM spermatogenesis is arrested prior to sexual maturation due to an inability to undergo meiosis and the generation of haploid spermatids. Moreover, TazPM testicular mitochondria were found to be structurally abnormal, and there was an elevation of p53-dependent apoptosis within TazPM seminiferous tubules. Immunoblot analysis revealed that TazPM gamete genome integrity was compromised, and both histone γ-H2Ax and Nucleoside diphosphate kinase-5 protein expression were absent in juvenile TazPM testes when compared to controls. We demonstrate that Taz-mediated transacetylase activity is required within mitochondria for normal spermatogenesis, and its absence results in meiotic arrest. We hypothesize that elevated TazPM spermatogonial apoptosis causes azoospermia and complete infertility.

Background & Aims: Liver fibrosis is a pathological healing process resulting from hepatic stellate cell (HSC) activation and the generation of myofibroblasts from activated HSCs. The precise underlying mechanisms of liver fibrogenesis are still largely vague due to lack of understanding the functional heterogeneity of activated HSCs during liver injury. Approach and Results: In this study, to define the mechanism of HSC activation, we performed the transcriptomic analysis at single-cell resolution (scRNA-seq) on HSCs in mice treated with carbon tetrachloride (CCl4). By employing LRAT-Cre:Rosa26mT/mG mice, we were able to isolate an activated GFP-positive HSC lineage derived cell population by fluorescence-activated cell sorter (FACS). A total of 8 HSC subpopulations were identified based on an unsupervised analysis. Each HSC cluster displayed a unique transcriptomic profile, despite all clusters expressing common mouse HSC marker genes. We demonstrated that one of the HSC subpopulations expressed high levels of mitosis regulatory genes, velocity, and monocle analysis indicated that these HSCs are at transitioning and proliferating phases at the beginning of HSCs activation and will eventually give rise to several other HSC subtypes. We also demonstrated cell clusters representing HSC-derived mature myofibroblast populations that express myofibroblasts hallmark genes with unique contractile properties. Most importantly, we found a novel HSC cluster that is likely to be critical in liver regeneration, immune reaction, and vascular remodeling, in which the unique profiles of genes such as Rgs5, Angptl6, and Meg3 are highly expressed. Lastly, we demonstrated that the heterogeneity of HSCs in the injured mouse livers is closely similar to that of cirrhotic human livers. Conclusions: Collectively, our scRNA-seq data provided insight into the landscape of activated HSC populations and the dynamic transitional pathway from HSC to myofibroblasts in response to liver injury.

Testicular development starts in utero and maturation continues postnatally, requiring a cascade of gene activation and differentiation into different cell types, with each cell type having its own specific function. As we had previously reported that the Capping protein inhibiting regulator of actin (Cracd) gene was expressed in the adult mouse testis, herein we examine when and where the β-catenin associated Cracd is initially expressed during postnatal testis development. Significantly, Cracd mRNA is present in both the immature postnatal and adult testis in round spermatid cells, with highest level of expression occurring during the first wave of meiosis and spermatogenesis. In the juvenile testes, Cracd is initially expressed within the innermost region but as maturation occurs, Cracd mRNA switches to a more peripheral location. Thereafter, Cracd is downregulated to maintenance levels in the haploid male germ cell lineage. As Cracd mRNA was expressed within developing round spermatids, we tested its effectiveness as a biomarker of non-obstructive azoospermia using transgenic knockout mice models. Meaningfully, Cracd expression was absent in Deleted in azoospermia like (Dazl) null testis, which exhibit a dramatic germ cell loss. Moreover, Cracd was abnormally regulated and ectopically mis-expressed in Polypyrimidine tract binding protein-2 (Ptbp2) conditional germ cell restricted knockout testis, which exhibit a block during spermatid differentiation and a reduction in the number of late stage spermatocytes coincident with reduced β-catenin expression. Combined, these data suggest that Cracd is a useful first wave of spermatogenesis biomarker of azoospermia phenotypes, even prior to an overt phenotype being evident.

Autonomic innervation is an essential component of cardiovascular regulation that is first established from the neural crest (NC) lineage in utero and continues developing postnatally. Although in vitro studies have indicated that SH2-containing protein tyrosine phosphatase 2 (SHP-2) is a signaling factor critical for regulating sympathetic neuron differentiation, this has yet to be shown in the complex in vivo environment of cardiac autonomic innervation. Targeting SHP-2 within postmigratory NC lineages resulted in a fully penetrant mouse model of diminished sympathetic cardiac innervation and concomitant bradycardia. Immunohistochemistry of the sympathetic nerve marker tyrosine hydroxylase revealed a progressive loss of adrenergic ganglionic neurons and reduction of cardiac sympathetic axon density in Shp2 cKOs. Molecularly, Shp2 cKOs exhibit lineage-specific suppression of activated phospo-ERK1/2 signaling but not of other downstream targets of SHP-2 such as pAKT. Genetic restoration of the phosphorylated-extracellular signal-regulated kinase (pERK) deficiency via lineage-specific expression of constitutively active MEK1 was sufficient to rescue the sympathetic innervation deficit and its physiological consequences. These data indicate that SHP-2 signaling specifically through pERK in postmigratory NC lineages is essential for development and maintenance of sympathetic cardiac innervation postnatally.

Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a complex genetic disorder with a high predisposition of numerous skeletal dysplasias including short stature, osteoporosis, kyphoscoliosis, and fracture non-union (pseudoarthrosis). We have developed murine models that phenocopy many of the skeletal dysplasias observed in NF1 patients, including reduced bone mass and fracture non-union. We also show that the development of these skeletal manifestations requires an Nf1 haploinsufficient background in addition to nullizygous loss of Nf1 in mesenchymal stem/progenitor cells (MSCs) and/or their progenies. This is replicated in two animal models of NF1, PeriCre(+);Nf1(flox/-) and Col2.3Cre(+);Nf1(flox/-) mice. Adoptive transfer experiments demonstrate a critical role of the Nf1+/- marrow microenvironment in the impaired fracture healing in both models and adoptive transfer of WT bone marrow cells improves fracture healing in these mice. To our knowledge, this is the first demonstration of a non-cell autonomous mechanism in non-malignant NF1 manifestations. Collectively, these data provide evidence of a combinatory effect between nullizygous loss of Nf1 in osteoblast progenitors and haploinsufficiency in hematopoietic cells in the development of non-malignant NF1 manifestations.