A systematic review on the biochemical threshold of mitochondrial genetic variants.

Mitochondrial DNA (mtDNA) variants cause a range of diseases from severe pediatric syndromes to aging-related conditions. The percentage of mtDNA copies carrying a pathogenic variant, variant allele frequency (VAF), must reach a threshold before a biochemical defect occurs, termed the biochemical threshold. Whether the often-cited biochemical threshold of >60% VAF is similar across mtDNA variants and cell types is unclear. In our systematic review, we sought to identify the biochemical threshold of mtDNA variants in relation to VAF by human tissue/cell type. We used controlled vocabulary terms to identify articles measuring oxidative phosphorylation (OXPHOS) complex activities in relation to VAF. We identified 76 eligible publications, describing 69, 12, 16, and 49 cases for complexes I, III, IV, and V, respectively. Few studies evaluated OXPHOS activities in diverse tissue types, likely reflective of clinical access. A number of cases with similar VAFs for the same pathogenic variant had varying degrees of residual activity of the affected complex, alluding to the presence of modifying variants. Tissues and cells with VAFs <60% associated with low complex activities were described, suggesting the possibility of a biochemical threshold of <60%. Using Kendall rank correlation tests, the VAF of the m.8993T > G variant correlated with complex V activity in skeletal muscle (τ = -0.58, P = 0.01, n = 13); however, no correlation was observed in fibroblasts (P = 0.7, n = 9). Our systematic review highlights the need to investigate the biochemical threshold over a wider range of VAFs in disease-relevant cell types to better define the biochemical threshold for specific mtDNA variants.

Renal denervation restores biomechanics of carotid arteries in a rat model of hypertension.

The prevalence of hypertension increases with aging and is associated with increased arterial stiffness. Resistant hypertension is presented when drug treatments fail to regulate a sustained increased blood pressure. Given that the mechanisms between the sympathetic nervous system and the kidney play an important role in blood regulation, renal denervation (RDN) has emerged as a therapeutic potential in resistant hypertension. In this study, we investigated the effects of RDN on the biomechanical response and microstructure of elastic arteries. Common carotid arteries (CCA) excised from 3-month, 8-month, and 8-month denervated rats were subjected to biaxial extension-inflation test. Our results showed that hypertension developed in the 8-month-old rats. The sustained elevated blood pressure resulted in arterial remodeling which was manifested as a significant stress increase in both axial and circumferential directions after 8 months. RDN had a favorable impact on CCAs with a restoration of stresses in values similar to control arteries at 3 months. After biomechanical testing, arteries were imaged under a multi-photon microscope to identify microstructural changes in extracellular matrix (ECM). Quantification of multi-photon images showed no significant alterations of the main ECM components, elastic and collagen fibers, indicating that arteries remained intact after RDN. Regardless of the experimental group, our microstructural analysis of the multi-photon images revealed that reorientation of the collagen fibers might be the main microstructural mechanism taking place during pressurization with their straightening happening during axial stretching.

Teamwork makes the dream work: functional collaborations between families, scientists, and healthcare providers to drive progress in the treatment of Leigh Syndrome.

BACKGROUND: Leigh syndrome, an inherited neurometabolic disorder, is estimated to be the most common pediatric manifestation of mitochondrial disease. No treatments are currently available for Leigh syndrome due to many hurdles in drug discovery efforts. Leigh syndrome causal variants span over 110 different genes and likely lead to both unique and shared biochemical alterations, often resulting in overlapping phenotypic features. The mechanisms by which pathogenic variants in mitochondrial genes alter cellular phenotype to promote disease remain poorly understood. The rarity of cases of specific causal variants creates barriers to drug discovery and adequately sized clinical trials. BODY: To address the current challenges in drug discovery and facilitate communication between researchers, healthcare providers, patients, and families, the Boston University integrative Cardiovascular Metabolism and Pathophysiology (iCAMP) Lab and Cure Mito Foundation hosted a Leigh Syndrome Symposium. This symposium brought together expert scientists and providers to highlight the current successes in drug discovery and novel models of mitochondrial disease, and to connect patients to providers and scientists to foster community and communication. CONCLUSION: In this symposium review, we describe the research presented, the hurdles ahead, and strategies to better connect the Leigh syndrome community members to advance treatments for Leigh syndrome.

Renal denervation restores biomechanics of carotid arteries in a rat model of hypertension.

The prevalence of hypertension increases with aging and is associated with increased arterial stiffness. Resistant hypertension is presented when drug treatments fail to regulate a sustained increased blood pressure. Given that the mechanisms between the sympathetic nervous system and the kidney play an important role in blood regulation, renal denervation (RDN) has emerged as a therapeutic potential in resistant hypertension. In this study, we investigated the effects of RDN on the biomechanical response and microstructure of elastic arteries. Common carotid arteries (CCA) were excised from 3-, 8- and 8-month-old denervated rats, and subjected to biaxial extension-inflation test. Our results showed that hypertension developed in the 8-month-old rats. The sustained elevated blood pressure resulted in arterial remodeling which was manifested as a significant stress increase in both axial and circumferential directions after 8 months. RDN had a favorable impact on CCAs with a restoration of stresses in values similar to control arteries at 3 months. After biomechanical testing, arteries were imaged under a multi-photon microscope to identify microstructural changes in extracellular matrix (ECM). Quantification of multi-photon images showed no significant alterations of the main ECM components, elastic and collagen fibers, indicating that arteries remained intact after RDN. Regardless of the experimental group, our microstructural analysis of the multi-photon images revealed that reorientation of the collagen fibers might be the main microstructural mechanism taking place during pressurization with their straightening happening during axial stretching.

Proposed mechanisms of hypertension and risk of adverse cardiovascular outcomes in LGBT communities.

Hypertension (HTN), a highly prevalent public issue affecting one in two adults in the United States, has recently been shown to differentially burden individuals belonging to marginalized communities, such as the lesbian, gay, bisexual, and transgender (LGBT) communities. The minority stress theory posits that a unique combination of marginalization-related psychosocial stressors and coping behaviors may underlie the increased burden of diseases like HTN in LGBT populations. Uncontrolled or poorly managed HTN often leads to the development of adverse cardiovascular outcomes, such as heart failure (HF). Despite our understanding of minority stress theory and demonstrated associations between LGBT identities and HTN, the mechanisms whereby psychosocial stress drives HTN in LGBT populations remain unclear. This mini-review discusses the physiological systems governing blood pressure and the epidemiology of HTN across different subgroups of LGBT people. In addition, we propose mechanisms demonstrated in the general population whereby psychological stress has been implicated in elevating blood pressure that may be occurring in LGBT populations. Finally, we discuss the limitations of current studies and methodological frameworks to make suggestions for study designs to better delineate the mechanisms of psychosocial stress-related HTN in LGBT communities.

The Mighty NUMT: Mitochondrial DNA Flexing Its Code in the Nuclear Genome.

Nuclear-mitochondrial DNA segments (NUMTs) are mitochondrial DNA (mtDNA) fragments that have been inserted into the nuclear genome. Some NUMTs are common within the human population but most NUMTs are rare and specific to individuals. NUMTs range in size from 24 base pairs to encompassing nearly the entire mtDNA and are found throughout the nuclear genome. Emerging evidence suggests that the formation of NUMTs is an ongoing process in humans. NUMTs contaminate sequencing results of the mtDNA by introducing false positive variants, particularly heteroplasmic variants present at a low variant allele frequency (VAF). In our review, we discuss the prevalence of NUMTs in the human population, the potential mechanisms of de novo NUMT insertion via DNA repair mechanisms, and provide an overview of the existing approaches for minimizing NUMT contamination. Apart from filtering known NUMTs, both wet lab-based and computational methods can be used to minimize the contamination of NUMTs in analyses of human mtDNA. Current approaches include: (1) isolating mitochondria to enrich for mtDNA; (2) applying basic local alignment to identify NUMTs for subsequent filtering; (3) bioinformatic pipelines for NUMT detection; (4) k-mer-based NUMT detection; and (5) filtering candidate false positive variants by mtDNA copy number, VAF, or sequence quality score. Multiple approaches must be applied in order to effectively identify NUMTs in samples. Although next-generation sequencing is revolutionizing our understanding of heteroplasmic mtDNA, it also raises new challenges with the high prevalence and individual-specific NUMTs that need to be handled with care in studies of mitochondrial genetics.

Importance of survey demographic questions to foster inclusion in medicine and research and reduce health inequities for LGBTQIA2S+ individuals.

A clear, inclusive, and accurate approach to the collection of demographic information in clinical research and medical practice is critical to understanding the healthcare needs of the specific population. Inclusive demography constitutes appropriate and accurate characterization of an individual's sexual orientation and gender identity (SOGI) data. Appropriate demography fosters sense of inclusion and belonging for those belonging to medically marginalized communities such as the lesbian, gay, bisexual, transgender, queer, intersex, asexual, and Indigenous Two-Spirit (LGBTQIA2S+) communities and improves health outcomes. Acquiring inclusive demographics in healthcare research is needed for the following critical reasons. First, LGBTQIA2S+ individuals experience undue psychological harm when their identities are not appropriately captured in survey data, promoting further alienation of the LGBTQIA2S+ community in medicine and research. Second, LGBTQIA2S+ populations are disproportionately burdened by several major cardiovascular and cardiovascular-associated diseases, including hypertension and diabetes. Failure to include these populations, and accurately characterize their participation, in research leads to failure to identify associations between identities and disease, resulting in worse health outcomes. Furthermore, this lack of precision in current data for sex, gender, and sexual orientation may lead to inaccurate data for all populations, not just the LGBTQIA2S+ community. Finally, there are currently major political and social threats and attacks on the LGBTQIA2S+ community and, in particular, on transgender and gender-diverse individuals. Proper medical inclusion and advocacy for the LGBTQIA2S+ community by the medical community may help protect the community from further undue harm through creating sense of belonging and reductions in marginalization-related health inequities.

Systematic dissection, preservation, and multiomics in whole human and bovine hearts.

OBJECTIVES: We sought to develop a rigorous, systematic protocol for the dissection and preservation of human hearts for biobanking that expands previous success in postmortem transcriptomics to multiomics from paired tissue. BACKGROUND: Existing cardiac biobanks consist largely of biopsy tissue or explanted hearts in select diseases and are insufficient for correlating whole organ phenotype with clinical data. METHODS: We demonstrate optimal conditions for multiomics interrogation (ribonucleic acid (RNA) sequencing, untargeted metabolomics) in hearts by evaluating the effect of technical variables (storage solution, temperature) and simulated postmortem interval (PMI) on RNA and metabolite stability. We used bovine (n=3) and human (n=2) hearts fixed in PAXgene or snap-frozen with liquid nitrogen. RESULTS: Using a paired Wald test, only two of the genes assessed were differentially expressed between left ventricular samples from bovine hearts stored in PAXgene at 0 and 12 hours PMI (FDR q<0.05). We obtained similar findings in human left ventricular samples, suggesting stability of RNA transcripts at PMIs up to 12 hours. Different library preparation methods (mRNA poly-A capture vs. rRNA depletion) resulted in similar quality metrics with both library preparations achieving >95% of reads properly aligning to the reference genomes across all PMIs for bovine and human hearts. PMI had no effect on RNA Integrity Number or quantity of RNA recovered at the time points evaluated. Of the metabolites identified (855 total) using untargeted metabolomics of human left ventricular tissue, 503 metabolites remained stable across PMIs (0, 4, 8, 12 hours). Most metabolic pathways retained several stable metabolites. CONCLUSIONS: Our data demonstrate a technically rigorous, reproducible protocol that will enhance cardiac biobanking practices and facilitate novel insights into human CVD. CONDENSED ABSTRACT: Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Current biobanking practices insufficiently capture both the diverse array of phenotypes present in CVDs and the spatial heterogeneity across cardiac tissue sites. We have developed a rigorous and systematic protocol for the dissection and preservation of human cardiac biospecimens to enhance the availability of whole organ tissue for multiple applications. When combined with longitudinal clinical phenotyping, our protocol will enable multiomics in hearts to deepen our understanding of CVDs.

Central Gαi2 Protein Mediated Neuro-Hormonal Control of Blood Pressure and Salt Sensitivity.

Hypertension, a major public health issue, is estimated to contribute to 10% of all deaths worldwide. Further, the salt sensitivity of blood pressure is a critical risk factor for the development of hypertension. The hypothalamic paraventricular nucleus (PVN) coordinates neuro-hormonal responses to alterations in plasma sodium and osmolality and multiple G Protein-Coupled Receptors (GPCRs) are involved in fluid and electrolyte homeostasis. In acute animal studies, our laboratory has shown that central Gαi/o subunit protein signal transduction mediates hypotensive and bradycardic responses and that Gz/q, proteins mediate the release of arginine vasopressin (AVP) and subsequent aquaretic responses to acute pharmacological stimuli. Extending these studies, our laboratory has shown that central Gαi2 proteins selectively mediate the hypotensive, sympathoinhibitory and natriuretic responses to acute pharmacological activation of GPCRs and in response to acute physiological challenges to fluid and electrolyte balance. In addition, following chronically elevated dietary sodium intake, salt resistant rats demonstrate site-specific and subunit-specific upregulation of Gαi2 proteins in the PVN, resulting in sympathoinhibition and normotension. In contrast, chronic dietary sodium intake in salt sensitive animals, which fail to upregulate PVN Gαi2 proteins, results in the absence of dietary sodium-evoked sympathoinhibition and salt sensitive hypertension. Using in situ hybridization, we observed that Gαi2 expressing neurons in parvocellular division of the PVN strongly (85%) colocalize with GABAergic neurons. Our data suggest that central Gαi2 protein-dependent responses to an acute isotonic volume expansion (VE) and elevated dietary sodium intake are mediated by the peripheral sensory afferent renal nerves and do not depend on the anteroventral third ventricle (AV3V) sodium sensitive region or the actions of central angiotensin II type 1 receptors. Our translational human genomic studies have identified three G protein subunit alpha I2 (GNAI2) single nucleotide polymorphisms (SNPs) as potential biomarkers in individuals with salt sensitivity and essential hypertension. Collectively, PVN Gαi2 proteins-gated pathways appear to be highly conserved in salt resistance to counter the effects of acute and chronic challenges to fluid and electrolyte homeostasis on blood pressure via a renal sympathetic nerve-dependent mechanism.

Sensory Afferent Renal Nerve Activated Gαi2 Subunit Proteins Mediate the Natriuretic, Sympathoinhibitory and Normotensive Responses to Peripheral Sodium Challenges.

We have previously reported that brain Gαi2 subunit proteins are required to maintain sodium homeostasis and are endogenously upregulated in the hypothalamic paraventricular nucleus (PVN) in response to increased dietary salt intake to maintain a salt resistant phenotype in rats. However, the origin of the signal that drives the endogenous activation and up-regulation of PVN Gαi2 subunit protein signal transduction pathways is unknown. By central oligodeoxynucleotide (ODN) administration we show that the pressor responses to central acute administration and central infusion of sodium chloride occur independently of brain Gαi2 protein pathways. In response to an acute volume expansion, we demonstrate, via the use of selective afferent renal denervation (ADNX) and anteroventral third ventricle (AV3V) lesions, that the sensory afferent renal nerves, but not the sodium sensitive AV3V region, are mechanistically involved in Gαi2 protein mediated natriuresis to an acute volume expansion [peak natriuresis (µeq/min) sham AV3V: 43 ± 4 vs. AV3V 45 ± 4 vs. AV3V + Gαi2 ODN 25 ± 4, p < 0.05; sham ADNX: 43 ± 4 vs. ADNX 23 ± 6, AV3V + Gαi2 ODN 25 ± 3, p < 0.05]. Furthermore, in response to chronically elevated dietary sodium intake, endogenous up-regulation of PVN specific Gαi2 proteins does not involve the AV3V region and is mediated by the sensory afferent renal nerves to counter the development of the salt sensitivity of blood pressure (MAP [mmHg] 4% NaCl; Sham ADNX 124 ± 4 vs. ADNX 145 ± 4, p < 0.05; Sham AV3V 125 ± 4 vs. AV3V 121 ± 5). Additionally, the development of the salt sensitivity of blood pressure following central ODN-mediated Gαi2 protein down-regulation occurs independently of the actions of the brain angiotensin II type 1 receptor. Collectively, our data suggest that in response to alterations in whole body sodium the peripheral sensory afferent renal nerves, but not the central AV3V sodium sensitive region, evoke the up-regulation and activation of PVN Gαi2 protein gated pathways to maintain a salt resistant phenotype. As such, both the sensory afferent renal nerves and PVN Gαi2 protein gated pathways, represent potential targets for the treatment of the salt sensitivity of blood pressure.

Gaining Insight into Mitochondrial Genetic Variation and Downstream Pathophysiology: What Can i(PSCs) Do?

Mitochondria are specialized organelles involved in energy production that have retained their own genome throughout evolutionary history. The mitochondrial genome (mtDNA) is maternally inherited and requires coordinated regulation with nuclear genes to produce functional enzyme complexes that drive energy production. Each mitochondrion contains 5-10 copies of mtDNA and consequently, each cell has several hundreds to thousands of mtDNAs. Due to the presence of multiple copies of mtDNA in a mitochondrion, mtDNAs with different variants may co-exist, a condition called heteroplasmy. Heteroplasmic variants can be clonally expanded, even in post-mitotic cells, as replication of mtDNA is not tied to the cell-division cycle. Heteroplasmic variants can also segregate during germ cell formation, underlying the inheritance of some mitochondrial mutations. Moreover, the uneven segregation of heteroplasmic variants is thought to underlie the heterogeneity of mitochondrial variation across adult tissues and resultant differences in the clinical presentation of mitochondrial disease. Until recently, however, the mechanisms mediating the relation between mitochondrial genetic variation and disease remained a mystery, largely due to difficulties in modeling human mitochondrial genetic variation and diseases. The advent of induced pluripotent stem cells (iPSCs) and targeted gene editing of the nuclear, and more recently mitochondrial, genomes now provides the ability to dissect how genetic variation in mitochondrial genes alter cellular function across a variety of human tissue types. This review will examine the origins of mitochondrial heteroplasmic variation and propagation, and the tools used to model mitochondrial genetic diseases. Additionally, we discuss how iPSC technologies represent an opportunity to advance our understanding of human mitochondrial genetics in disease.

Sympathetic Regulation of the NCC (Sodium Chloride Cotransporter) in Dahl Salt-Sensitive Hypertension.

Increased sympathoexcitation and renal sodium retention during high salt intake are hallmarks of the salt sensitivity of blood pressure. The mechanism(s) by which excessive sympathetic nervous system release of norepinephrine influences renal sodium reabsorption is unclear. However, studies demonstrate that norepinephrine can stimulate the activity of the NCC (sodium chloride cotransporter) and promote the development of SSH (salt-sensitive hypertension). The adrenergic signaling pathways governing NCC activity remain a significant source of controversy with opposing studies suggesting a central role of upstream α1- and ß-adrenoceptors in the canonical regulatory pathway involving WNKs (with-no-lysine kinases), SPAK (STE20/SPS1-related proline alanine-rich kinase), and OxSR1 (oxidative stress response 1). In our previous study, α1-adrenoceptor antagonism in norepinephrine-infused male Sprague-Dawley rats prevented the development of norepinephrine-evoked SSH in part by suppressing NCC activity and expression. In these studies, we used selective adrenoceptor antagonism in male Dahl salt-sensitive rats to test the hypothesis that norepinephrine-mediated activation of the NCC in Dahl SSH occurs via an α1-adrenoceptor dependent pathway. A high-salt diet evoked significant increases in NCC activity, expression, and phosphorylation in Dahl salt-sensitive rats that developed SSH. Increases were associated with a dysfunctional WNK1/4 dynamic and a failure to suppress SPAK/OxSR1 activity. α1-adrenoceptor antagonism initiated before high-salt intake or following the establishment of SSH attenuated blood pressure in part by suppressing NCC activity, expression, and phosphorylation. Collectively, our findings support the existence of a norepinephrine-activated α1-adrenoceptor gated pathway that relies on WNK/SPAK/OxSR1 signaling to regulate NCC activity in SSH.

Hypothalamic Paraventricular Nucleus Gαi2 (Guanine Nucleotide-Binding Protein Alpha Inhibiting Activity Polypeptide 2) Protein-Mediated Neural Control of the Kidney and the Salt Sensitivity of Blood Pressure.

We have previously reported that in salt-resistant rat phenotypes brain, Gαi2 (guanine nucleotide-binding protein alpha inhibiting activity polypeptide 2) proteins are required to maintain blood pressure and sodium balance. However, the impact of hypothalamic paraventricular nucleus (PVN) Gαi2 proteins on the salt sensitivity of blood pressure is unknown. Here, by the bilateral PVN administration of a targeted Gαi2 oligodeoxynucleotide, we show that PVN-specific Gαi2 proteins are required to facilitate the full natriuretic response to an acute volume expansion (peak natriuresis [µeq/min] scrambled (SCR) oligodeoxynucleotide 41±3 versus Gαi2 oligodeoxynucleotide 18±4; P<0.05) via a renal nerve-dependent mechanism. Furthermore, in response to chronically elevated dietary sodium intake, PVN-specific Gαi2 proteins are essential to counter renal nerve-dependent salt-sensitive hypertension (mean arterial pressure [mm Hg] 8% NaCl; SCR oligodeoxynucleotide 128±2 versus Gαi2 oligodeoxynucleotide 147±3; P<0.05). This protective pathway involves activation of PVN Gαi2 signaling pathways, which mediate sympathoinhibition to the blood vessels and kidneys (renal norepinephrine [pg/mg] 8% NaCl; SCR oligodeoxynucleotide 375±39 versus Gαi2 oligodeoxynucleotide 850±27; P<0.05) and suppression of the activity of the sodium chloride cotransporter assessed as peak natriuresis to hydrochlorothiazide. Additionally, central oligodeoxynucleotide-mediated Gαi2 protein downregulation prevented PVN parvocellular neuron activation, assessed by FosB immunohistochemistry, in response to increased dietary salt intake. In our analysis of the UK BioBank data set, it was observed that 2 GNAI2 single nucleotide polymorphism (SNP) (rs2298952, P=0.041; rs4547694, P=0.017) significantly correlate with essential hypertension. Collectively, our data suggest that selective targeting and activation of PVN Gαi2 proteins is a novel therapeutic approach for the treatment of salt-sensitive hypertension.

Inhibition of microglial activation in rats attenuates paraventricular nucleus inflammation in Gαi2 protein-dependent, salt-sensitive hypertension.

NEW FINDINGS: • What is the central question of this study? We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi2 protein-dependent, salt-sensitive hypertension. • What is the main finding and its importance? The main finding is that PVN-specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi2 protein-dependent, salt-sensitive hypertension in Sprague-Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension. ABSTRACT: The central mechanisms underlying salt-sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt-resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi2 protein-dependent, salt-sensitive hypertension. Male Sprague-Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal-salt (NS; 0.6% NaCl) or high-salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co-infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro-inflammatory cytokines TNFα, IL-1ß and IL-6 and the anti-inflammatory cytokine IL-10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt-sensitive hypertension, increased plasma noradrenaline, PVN pro-inflammatory cytokine mRNA upregulation, anti-inflammatory cytokine mRNA downregulation and PVN-specific microglial activation in rats receiving a targeted Gαi2 but not scrambled ODN. Minocycline co-infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi2 ODN-infused animals on HS. Our data suggest that central Gαi2 protein prevents microglial-mediated PVN inflammation and the development of salt-sensitive hypertension.

Tumour-derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour-bearing mice.

BACKGROUND: Cancer cachexia is a metabolic wasting syndrome that is strongly associated with a poor prognosis. The initiating factors causing fat and muscle loss are largely unknown. Previously, we found that leukaemia inhibitory factor (LIF) secreted by C26 colon carcinoma cells was responsible for atrophy in treated myotubes. In the present study, we tested whether C26 tumour-derived LIF is required for cancer cachexia in mice by knockout of Lif in C26 cells. METHODS: A C26 Lif null tumour cell line was made using CRISPR-Cas9. Measurements of cachexia were compared in mice inoculated with C26 vs. C26Lif-/- tumour cells, and atrophy was compared in myotubes treated with medium from C26 vs. C26Lif-/- tumour cells. Levels of 25 cytokines/chemokines were compared in serum of mice bearing C26 vs. C26Lif-/- tumours and in the medium from these tumour cell lines. RESULTS: At study endpoint, C26 mice showed outward signs of sickness while mice with C26Lif-/- tumours appeared healthy. Mice with C26Lif-/- tumours showed a 55-75% amelioration of body weight loss, muscle loss, fat loss, and splenomegaly compared with mice with C26 tumours (P < 0.05). The heart was not affected by LIF levels because the loss of cardiac mass was the same in C26 and C26Lif-/- tumour-bearing mice. LIF levels in mouse serum was entirely dependent on secretion from the tumour cells. Serum levels of interleukin-6 and G-CSF were increased by 79-fold and 68-fold, respectively, in C26 mice but only by five-fold and two-fold, respectively, in C26Lif-/- mice, suggesting that interleukin-6 and G-CSF increases are dependent on tumour-derived LIF. CONCLUSIONS: This study shows the first use of CRISPR-Cas9 knockout of a candidate cachexia factor in tumour cells. The results provide direct evidence for LIF as a major cachexia initiating factor for the C26 tumour in vivo. Tumour-derived LIF was also a regulator of multiple cytokines in C26 tumour cells and in C26 tumour-bearing mice. The identification of tumour-derived factors such as LIF that initiate the cachectic process is immediately applicable to the development of therapeutics to treat cachexia. This is a proof of principle for studies that when carried out in human cells, will make possible an understanding of the factors causing cachexia in a patient-specific manner.