Aim: Glutaraldehyde (GDA) is an active ingredient in many instrument disinfectants and is effective against a broad spectrum of microorganisms. In the past, the virus-inactivating properties of these products were mainly claimed based on quantitative suspension tests with different test viruses. Recently, however, a European Norm EN 17111:2018 has been published which allows examination of instrument disinfectants in a surface carrier test, simulating practical conditions. Therefore, it is of interest to evaluate GDA for the ability to inactivate the viruses used in this European Norm as test viruses. Methods: The virucidal efficacy of GDA as the active ingredient in instrument disinfectants was evaluated with 4 different test viruses in a method simulating practical conditions (EN 17111:2018).Results: With a fixed exposure time of five minutes at 20°C, 100 ppm GDA were necessary to inactivate vaccinia virus, classifying it as a limited spectrum virucidal activity for pre-cleaning products. For adenovirus, 125 ppm GDA were required, whereas for murine norovirus as a surrogate for human norovirus, 4,000 ppm GDA were required for a significant reduction of viral titres. Both non-enveloped viruses must be tested to prove virucidal activity in EN 17111:2018. But even 4,000 ppm were not enough to yield a 4 log10 reduction of the murine parvovirus at 20°C. This virus is only required as a test virus using this method if temperatures ≥40°C are used. Conclusion: GDA, as the active ingredient of many instrument disinfectants, shows virucidal efficacy at 20°C. The necessary concentrations are strongly dependent on the stability of the test viruses. Due to the high stability of murine norovirus, GDA levels of 4,000 ppm were required to inactivate this virus within the 5-minute exposure time. Glutaraldehyde (GDA) has long been the active ingredient in many high-level instrument disinfectants for semi-critical devices or instruments. An aqueous 2% alkaline GDA solution is one of the most common disinfectants for endoscope decontamination in hospitals.Picornaviridae family in a quantitative suspension test describing the virucidal activity under various test conditions , .The murine parvovirus, minute virus of mice (MVM), strain Crawford (ATCC VR 1346) was included in this study, which should only be used for temperatures ≥40°C in EN 17111:2018. MVM (passage 4) was obtained from the Paul Ehrlich Institute, Langen, Germany. All details of this quantitative carrier test are described in the EN 17111:2018. The surface-sandblasted frosted glass carriers were prepared as follows. One volume of 0.3% BSA solution was mixed with nine volumes of test virus suspension. In brief, 50 µL of this virus inoculum were pipetted onto the inoculation square of the carrier, followed by drying. The glass carrier with the dried virus inoculum was placed in a 30-mL cylindrical screw tube containing 10 mL of the GDA solution. Due to existing data with GDA-based instrument disinfectants, different concentrations were tested against the 4 test viruses. At the end of the exposure time, the carriers were transferred into a second screw tube with MEM and glass beads, followed by mixing for 60 s. Infectivity was stopped by immediate serial dilution (1:10 dilutions) with ice-cold MEM. Next, 100 µL of each dilution were placed in eight wells of a sterile polystyrene flat-bottomed 96-well microtitre plate containing 100 µL cell suspension. Titre reduction is shown as the difference between the virus titre after exposure time with different GDA concentrations and the virus titre with hard water. In parallel, tests were run without any virus as control for cytotoxicity.10TCID50/mL with a 95% confidence interval. A reduction of infectivity of ≥4 log10 steps  is regarded in the EN 17111:2018 as evidence of virucidal efficacy [The virus titres were determined using the method of Spearman  and Kaerefficacy .The virucidal efficacy of GDA as the active ingredient in instrument disinfectants was evaluated with 4 different test viruses in a method simulating practical conditions. The results are given in Figure 1 By performing EN 17111:2018, the virus-inactivating properties of instrument disinfectants can be tested with a method simulating practical conditions . MVA, AdFor GDA as the fixed reference in EN 17111:2018, the chemical and physical parameters are described in detail in the European Norm. However, no data for the concentration used as the reference are available to date. Therefore, our data should additionally help determine exact reference concentrations. As expected, MVA – representing all enveloped viruses – was the most fragile virus. 100 ppm was enough for viral inactivation, whereas 50 ppm GDA failed. Interestingly, a great difference regarding the stability of both non-enveloped viruses was observed. MNV  required much higher concentrations than did AdV (125 ppm), demonstrating a distinctively greater stability towards GDA in the test simulating practical conditions.10 steps with AdV, whereas MNV inactivation required 1,000 ppm PAA at 20°C with five minutes’ exposure time [In a previous test with practical conditions testing peracetic acid (PAA), MNV was also more stable than AdV. 400 ppm PAA at 20°C was enough for a reduction of 4 log10 reduction of virus titres in the surface test, whereas in our study on instrument disinfectants, 4,000 ppm GDA failed to produce such an inactivation (RF=3.06±0.29). This means that results from the instrument test EN 17111:2018 cannot be transferred to surface disinfectants, which must be tested in a different way.Some surface disinfectants used in the healthcare setting are also based on GDA. Therefore, it might be interesting to compare results of instrument tests where carriers are immersed as shown here to results of carrier tests overlying the virus suspension. With a test based on stainless steel discs for surface disinfectants, it was shown that concentrations between 125 and 500 ppm GDA were necessary for AdV inactivation within a 5-minute exposure time , whereasIn summary, our data will help to evaluate GDA-based instrument disinfectants often used for manual cleaning for their efficacy to inactivate enveloped and non-enveloped viruses, which are now described as test viruses in the recently published EN 17111:2018 . FurtherJS works as a consultant for Dr. Brill + Partner GmbH Institute for Hygiene and Microbiology. The other authors declare that they have no competing interests.FB, JoeS, ES and JS formulated the study questions and designed the study. BB, DP and BB were responsible performing all experimental data. DT was responsible for data evaluation. All authors read and approved the final manuscript.


Akebia Saponin D (ASD), a triterpenoid saponin, was shown to have protective effects in certain neuronal cells. The purpose of the present study was to investigate the possibility of ASD to prevent tumor necrosis factor (TNF)-induced axonal loss and the ASD modulation of the biologic process of autophagy in optic nerves. Rats were given intravitreal administration of TNF, simultaneous administration of 2, 20, or 200 pmol ASD and TNF, or ASD alone. LC3-II and p62 expression, which is a marker of autophagic flux, and phosphorylated p38 (p-p38) expression in optic nerves were examined by immunoblot analysis. Morphometric analysis revealed a significant ameliorated effect of ASD against TNF-induced optic nerve damage. p62 was significantly increased in the optic nerve in TNF-treated eyes, but this increase was totally prevented by ASD. The ASD alone injection showed significant reduction of p62 levels compared with the PBS-treated control eyes. LC3-II was significantly increased by ASD treatment in the TNF-injected eyes. p-p38 was significantly increased in the optic nerve in TNF-treated eyes, but this increase was completely prevented by ASD. The protective effects of ASD may be associated with enhanced autophagy activation and inhibition of p-p38. Autophagy, a cellular process which includes the degradation of cytoplasmic and axoplasmic components, has been linked to the pathophysiology of certain human diseases, such as neurodegenerative diseases –4. SeverDipsacus asper Wall. A previous report showed that ASD increased autophagic flux in the mice hepatic steatosis model [Akebia saponin D (ASD) is extracted from herbal medicine is model . In neuris model , and ASDis model . In addiis model . Among Mis model . Notablyis model , suggestExperiments were performed on 8-week-old male Wistar rats. All studies were conducted according to the Association for Research in Vision and Ophthalmology (ARVO) statement on the Use of Animals in Ophthalmic and Vision Research and were approved by the Ethics Committee of the Institute of Experimental Animals of St. Marianna University Graduate School of Medicine. The animals were maintained under controlled conditions, with temperature at 23 ± 1 °C, humidity at 55 ± 5%, and light from 06:00 to 18:00.Intravitreal injection was carried out as described previously . In anesOne and two weeks after administration, optic nerves (4 mm in length) were isolated, homogenized, and centrifuged at 15,000 × g for 15 min at 4 °C as described previously . Superna2 each; total area of 7232.3 μm2 per eye) were acquired and quantified using Aphelion image-processing software. The number of axons per eye was averaged and presented as the number/mm2.Quantification of each axon was conducted with optic nerves from 32 rats as described previously , 19. TheThe eyes were enucleated 1 week after injection and eye caps were made. The eye cap samples were immersed in 4% paraformaldehyde. Paraffinized sections were made longitudinally through the optic disc. The primary antibodies were anti-p-p38 antibody  and anti-neurofilament-L antibody . The secondary antibodies used were FITC-labeled and rhodamine-labeled antibodies. The samples on slides were mounted in DAPI-containing medium .Data are presented as mean ± S.E.M. Differences among groups in axon data and immunoblot data were analyzed using one-way ANOVA, following by Dunnett’s post hoc test. A probability value of less than 0.05 was considered to represent a statistically significant difference.Consistent with our previous study , compareConsistent with our previous study , the expThere was no significant change in the LC3-II level in optic nerve 2 weeks after TNF injection. The ASD injection significantly increased LC3-II level as compared with TNF alone (Fig. A previous study reported that p-p38 exists in RGCs , but no Immunohistochemical study found abundant p-p38 immunoreactivity in TNF-treated group and that some of these immunoreactivities were colocalized with neurofilament immunoreactivity in optic nerve (Fig. Our previous study showed that induction of autophagy is involved in axonal protection against TNF- induced degeneration in the optic nerve , 21. AltIn conclusion, these results suggested that the axonal protection of ASD may be involved in autophagy activation by inhibition of p-p38.


Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes. Heart failure with preserved ejection fraction (HFpEF) is a global, major health issue for which no effective therapies are available. Here, the authors discover that the interplay between two transcription factors, Xbp1s and FoxO1, is critical for metabolic adaptation and lipid handling in HFpEF-stressed cardiomyocytes. In striking contrast with the other major form of HF—HF with reduced ejection fraction, HFrEF—the incidence and prevalence of HFpEF have increased steadily over the last decade. As of today, more than two-thirds of patients with HF over 65 years of age suffer from the clinical syndrome of HFpEF3. Despite the enormity of the problem, efficacious treatment options for these millions of individuals are lacking4.Heart failure with preserved ejection fraction (HFpEF) is now the most prevalent form of HF worldwide4. However, the vast majority of them present with obesity and metabolic syndrome8. Obesity increases the risk of HFpEF over HFrEF and is associated with worsening of functional parameters in HFpEF6. Indeed, elements of metabolic alteration have been identified as crucial pathophysiological drivers of HFpEF in both preclinical models and clinical studies10. Despite the well-recognized association between obesity and HFpEF, mechanistic elements governing the excess lipid accumulation in cardiomyocytes—i.e. cardiac lipid overload—are poorly understood.Limited understanding of pathophysiological mechanisms underlying HFpEF is a prominent reason for the failure of clinical trials thus far. HFpEF is a heterogeneous syndrome and not all patients with HFpEF harbor the same predisposing conditions11. ER stress triggers the unfolded protein response (UPR), a multidimensional, adaptive signaling pathway capable of mitigating stress under many conditions. Despite the name, stresses other than unfolded proteins can activate the UPR. Current evidence points to a clear association between the UPR and hallmarks of metabolic syndrome: obesity and dyslipidemia12. Indeed, either prolonged dysregulation of lipid synthesis or failure of UPR activation to resolve insults can disrupt the homeostatic ER environment and promote disease development14.Disruption of endoplasmic reticulum (ER) homeostasis—also known as ER stress—is a common feature in disease-stressed cardiomyocytes9. Across the spectrum of cardiovascular diseases, suppression of the IRE1α/Xbp1s arm of the UPR is an alteration unique to HFpEF. Indeed, restoring Xbp1s in HFpEF cardiomyocytes substantially ameliorates the syndrome in mice10. Looking forward, how disruption of the UPR contributes to lipotoxic alterations in cardiomyocytes and cardiometabolic disease is poorly characterized, and mechanisms whereby Xbp1s ameliorates the HFpEF phenotype remain elusive. Hence, we set out to define and manipulate mechanisms downstream of Xbp1s in cardiometabolic HFpEF and decipher its cardioprotective actions.In mammals, the UPR involves three ER transmembrane protein sensors: IRE1α (inositol-requiring kinase 1α), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6). Differential roles of the individual UPR transducers have been proposed. The IRE1α pathway is the most highly conserved branch of the UPR. Upon ER stress, IRE1α initiates the unconventional splicing of mRNA encoding Xbp1 (X-box–binding protein 1) to generate spliced Xbp1 (Xbp1s), a powerful transcription factor involved in many cellular functions essential to stress responsiveness. We described a cardioprotective role of Xbp1s in HFpEF, providing evidence of IRE1α/Xbp1s dysregulation in cardiomyocyte function in both experimental and clinical HFpEFHere, we show that myocardial lipid accumulation and cardiac metabolic alterations in HFpEF are coupled with increases in the abundance and activity of FoxO1 (Forkhead box protein O1), a conserved transcription factor involved in cell metabolism. FoxO1 governs lipid accumulation in cardiomyocytes, and overexpression of Xbp1s triggers FoxO1 ubiquitination and proteasomal degradation. Using gain- and loss-of-function approaches we demonstrate that genetic deletion of FoxO1, as well as Xbp1s overexpression, in cardiomyocytes each ameliorates the HFpEF phenotype and reduces cardiac steatosis. Finally, we identify the E3 ubiquitin ligase STUB1 (STIP1 homology and U-Box-containing protein 1) as a novel and direct transcriptional target of Xbp1s implicated in Xbp1s-dependent degradation of FoxO1 in cardiomyocytes. These findings link UPR alterations with metabolic dysregulation in HFpEF, unveiling the Xbp1s-FoxO1 axis as a critical mechanism in the pathogenesis of lipid abnormalities in cardiomyocytes.10. As expected, cardiac tissue samples from HFpEF mice manifest a significant increase in Oil Red O staining for neutral lipids  in the presence or absence of the proteasome inhibitor MG132. In NRVMs, Xbp1s overexpression increased FoxO1 polyubiquitination  to recapitulate the numerous features of clinical HFpEF9. Indeed, single hits, either HFD or l-NAME alone, do not elicit HFpEF in mice9. To evaluate FoxO1 activation in the setting of these different stresses, we assessed its nuclear localization. Nuclear fractionation of left ventricular (LV) samples revealed no increase in nuclear FoxO1 in animals treated with l-NAME alone  and endothelial dysfunction-driven hypertension /oleate mono-unsaturated fatty acid (FA) complex. Twenty-four and 48 hours of treatment were sufficient to induce lipid accumulation . FoxO1-cKO mice and littermate controls (F/F) were exposed to HFpEF or control diet for seven weeks Fig. . Both sy18. Surveying the gene promoter regions of the ubiquitin ligases known to be involved in FoxO1 degradation, we identified a conserved UPR element (UPRE) nucleotide sequence—the prototypical binding site of Xbp1s—in the promoter region of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-Box-containing protein 1)  Fig. . To purs 1) Fig. . A simil 1) Fig. . Chromat 1) Fig. . In addi 1) Fig. , confirmTo corroborate our findings, we infected NRVMs with AdXBP1s or treated them with TUN, observing significant increases in STUB1 mRNA Fig.  and protOur data suggest that Xbp1s activates STUB1 transcription, promoting the proteolytic degradation of FoxO1. To examine this more directly, we measured the impact of STUB1 on the half-life of FoxO1 protein. NRVMs were treated with cycloheximide to block protein translation, and steady-state levels of FoxO1 over the course of 8 h were measured. Loss of STUB1 led to a significant decrease in the early rate of decay of FoxO1 and increased the protein’s half-life in this assay from ~4 h to almost 8 h. Fig. . In suppTo delineate the role of STUB1 in Xbp1s-dependent control of FoxO1 in cardiomyocytes, we evaluated FoxO1 mRNA and protein levels in STUB1-depleted NRVMs in the setting of Xbp1s overexpression. STUB1 knockdown in cardiomyocytes did not affect FoxO1 transcript levels under basal conditions or after Xbp1s overexpression Fig. . Strikin20. Whereas infection of NRVMs with AdXBP1s resulted in increases in SKP2 mRNA 9, reduces myocardial lipid accumulation; (2) FoxO1 promotes lipid accumulation in HFpEF cardiomyocytes; (3) Xbp1s promotes FoxO1 protein degradation; (4) cardiomyocyte-specific deletion of FoxO1 ameliorates the HFpEF phenotype and diminishes cardiac steatosis; (5) the E3 ubiquitin ligase STUB1, a direct transcriptional target of Xbp1s, is also downregulated in HFpEF and governs FoxO1 stability in cardiomyocytes. In aggregate, we unveil the role of FoxO1 as a driver of lipid accumulation in cardiomyocytes, functioning under the control of its molecular brake, Xbp1s, and emerging as a critical contributor to HFpEF pathogenesis. We propose a novel pathophysiological model of HFpEF in which suppression of the Xbp1s arm of the UPR blunts STUB1 expression, leading to the stabilization and overactivation of FoxO1 in cardiomyocytes with consequent cardiomyocyte steatosis and associated detrimental sequelae  Xbp1s, which is decreased in HFpEFlae Fig. .Fig. 6Pr22. Indeed, metabolic disorder-related HFpEF is the most prevalent phenotype of HFpEF in the community23. Prior work has described fatty infiltration of cardiomyocytes in failing hearts, causally linking cardiac lipid overload with systolic and diastolic dysfunction26. Thus, HFpEF can be considered the most common clinical manifestation of lipotoxic cardiomyopathy.Obesity and diabetes significantly increase the risk of atherosclerotic coronary artery disease and myocardial infarction, which almost inevitably impairs systolic function leading to HFrEF. However, obese people are at markedly increased risk of HFpEF independent of the occurrence of ischemic cardiac injury10 and STUB1, coupled with activation of FoxO1, suggesting a feedback circuit whereby Xbp1s acts to increase transcription of STUB1 to blunt the persistent activation of FoxO1 observed under HFD conditions. Conversely, in HFpEF hearts, downregulation of Xbp1s leads to suppression of STUB1 transcription, further increasing FoxO1 activation and increasing cardiomyocyte lipid accumulation. Therefore, as obesity alone does not elicit HFpEF, the mechanistic underpinnings of obesity-induced cardiomyopathy are different from those in HFpEF, supporting a model in which FoxO1 overactivation in HFpEF leads to the detrimental functional consequences of cardiomyocyte lipid accumulation.It is important to note that HFpEF, both clinically and mechanistically, does not completely overlap with obesity, and that obesity is one—the most common—of the many comorbidities shaping the complexity of the pathophysiological mechanisms of HFpEF. Accordingly, in obese (HFD-fed) mice, we observed activation, rather than suppression, of Xbp1s28. Conversely, impaired FA oxidation is a hallmark of metabolic dysfunction in HF. In patients with HFrEF without metabolic abnormalities  only a minimal increase in myocardial triglyceride content has been observed25. Conversely, in obese/diabetic patients with HFrEF, significant accumulation of intramyocardial lipid has been reported25. These data suggest that impaired FA oxidation alone is insufficient to elicit significant myocardial steatosis, and cardiac lipid overload is largely a function of increased FA uptake/biogenesis. Indeed, although increased FA oxidation has long been considered a culprit contributing to cardiac dysfunction in obese models, recent data suggest that increased FA oxidation does not cause cardiac dysfunction in mice30. Taken together, these observations suggest that cardiac lipotoxicity is not attributable to increased fatty-acid oxidation per se, but rather to an imbalance of fatty-acid supply, storage, and use. Consistent with this, we have observed an increase in cardiac lipogenic signature coupled with reduction in mitochondrial FA oxidation in cardiometabolic HFpEF. Similarly, in two models of reduced cardiac lipid accumulation in HFpEF (Xbp1 overexpression and FoxO1 deletion) we did not observe an increase in FA oxidation in cardiomyocytes, suggesting that the prevalent mechanisms of lipid accumulation in HFpEF cardiomyocytes are activation of FA uptake/biogenesis pathways.The precise mechanisms whereby lipids induce cardiac dysfunction are unclear. In principle, pathological lipid accumulation in cardiomyocytes can result from increased FA uptake/biogenesis, decreased FA oxidation, or a combination of the two. In the hearts of obese humans and rodents, an increase, rather than a decrease, in FA oxidation has been reported32. We have previously demonstrated, in both clinical and experimental HFpEF, inflammation-dependent suppression of the IRE1α/Xbp1s arm of the UPR9. Restoration of Xbp1s levels in failing cardiomyocytes results in diminished diastolic dysfunction and amelioration of the global HFpEF phenotype9. Here, we have unveiled proteasomal degradation of FoxO1 as a major mechanism of Xbp1s-dependent cardioprotective effects in HFpEF.Dysregulation of the UPR pathway has emerged as a hallmark of cardiac alterations in HFpEF33. For example, in hepatocytes, Xbp1s regulates lipogenesis35, a function that is independent of its classical role in ER stress and critically involves FoxO115. With respect to cardiac biology, FoxO1 participates in remodeling, autophagy, apoptosis, responses to oxidative stress, and regulation of metabolism37. In addition, FoxO1 has emerged as an important target of insulin and other growth factors in the myocardium16, and we have reported that FoxO1 is persistently activated in cardiac tissue from murine models of long-term diabetes17. Despite this, upstream regulators of FoxO1-dependent regulation of lipid homeostasis in cardiomyocytes remain largely unknown, and a role of FoxO1 in HFpEF has not been reported previously.The interplay between the UPR and lipid biology is complex. FA can activate the UPR, and specific branches of the UPR regulate lipid metabolism38. However, whereas we observed STUB1-dependence of FoxO1 protein degradation upon Xbp1s activation, we recognize that this may not be the only mechanism involved in FoxO1 degradation upon metabolic stress in cardiomyocytes; other mechanisms may play a role. STUB1, also known as carboxyl terminus of Hsc70-interacting protein (ChIP), is a dual function co-chaperone/ubiquitin ligase that is highly expressed in the heart and vasculature39. Interestingly, a cardioprotective role of STUB1 involving, at least in part, the modulation of lipid accumulation, has been observed in two models of HFrEF, suggesting a central role of STUB1 in cardiomyocyte homeostasis41. Direct targeting of transcription factors for therapeutic purposes has proven challenging42. Therefore, the identification of negative upstream regulators of FoxO1 such as STUB1 might result in valuable therapeutic targets to diminish FoxO1 activation and its negative metabolic consequences. Indeed, strategies aiming to enhance STUB1 functionality for therapeutic applications in cardiovascular disease have been proposed recently43, supporting the potential feasibility of targeting STUB1 for clinical gain.Xbp1s is a powerful transcription factor acting through a variety of transcriptional targets involved in many fundamental cellular responses to stress. We report here that the E3 ubiquitin ligase STUB1 is a direct downstream target of Xbp1s. STUB1 is a major FoxO1 ubiquitin ligase driving its proteasomal degradation37. Together, these data suggest that FoxO1 plays different roles in cardiac pathology depending on the specific stress conditions.We have demonstrated that FoxO1 suppression in HFpEF is beneficial. Interestingly, these beneficial effects occur in the absence of a significant reduction in cardiac hypertrophy. By contrast, we recently described a pro-hypertrophic role for FoxO1 acting through cardiomyocyte thyroid hormone metabolism44 and insulin resistance worsens HF, a feedback regulatory circuit contributing to HF susceptibility and progression. We and others have positioned FoxO1 as a pivotal element in the control of insulin signaling in the heart. Forced expression of FoxO1 in cardiomyocytes culminates in reduced insulin sensitivity and impaired glucose metabolism16. In HFpEF hearts, we observe increased activity of PDK4, which will repress glucose oxidation45. Indeed, an increase in PDK4 levels is a common feature of hearts subjected to metabolic stress. Interestingly, in cardiometabolic HFpEF the increase in PDK4 is independent of FoxO1.Another major metabolic abnormality observed in HF is systemic insulin resistance. HF causes insulin resistance47. Despite this, debate regarding the functional consequences of persistent diabetes or obesity on cardiac function persists, with some reporting little or no effect49 and others reporting evolution toward HFrEF50. Here, we have provided the clinical context—the syndrome of HFpEF—in which FoxO1 overactivation promotes cardiac lipotoxicity. We observed impaired cardiac mitochondrial FA oxidation in HFpEF. Interestingly, in FoxO1-cKO mice subjected to HFpEF, we observed a trend toward amelioration of mitochondrial fatty-acid oxidation in the absence of changes in glucose oxidation. Collectively, these results imply that a major disruption of energetic substrate utilization occurs in HFpEF, and the downregulation of FoxO1 tends to mitigate this energetic disturbance, acting through a decrease in HFpEF-induced lipotoxicity.Beyond glucose metabolism, less well-known is the role of FoxO1 in regulating cardiac lipid homeostasis. FoxO1 nuclear compartmentalization and enhancement of its transcriptional activity, together with increases in FA uptake, have been observed in models of long-term diabetes and obesity10, whereas some evidence suggests a predilection for HFpEF in women51. Despite this, sexual dimorphism in HFpEF pathophysiology is an underdeveloped area of investigation. Therefore, elucidation of sex-neutral signaling pathways involved in HFpEF pathogenesis is warranted.It is important to note that cardiometabolic alterations in HFpEF, and their underlying pathophysiological mechanisms, exhibit a certain degree of sex dependence. Female mice seem to be protected from experimental HFpEFHere, we have unveiled an Xbp1s-FoxO1 signaling circuit in diseased cardiomyocytes, thereby defining events downstream of the UPR in heart that govern metabolic events. We have identified a biological axis, Xbp1s-STUB1-FoxO1, in the regulation of cardiac steatosis in HFpEF, providing a novel pathophysiological model for cardiometabolic HFpEF. The field of experimental HFpEF is rapidly moving from a simplistic view in which HFpEF equals diastolic dysfunction to a view of HFpEF as a complex syndrome in which metabolic alterations contribute importantly to its pathogenesis. As such, delineation of alterations in lipid metabolism and their consequences on cardiac function is of paramount importance in deciphering mechanisms underlying this prevalent and devastating syndrome.52. Cardiomyocyte-specific FoxO1 knockout (cKO) animals, mice harboring a floxed FoxO1 allele were crossed with α-MHC-MerCreMer transgenic mice. FoxO1-cKO and corresponding control floxed or α-MHC-MerCreMer mice were maintained on an FVB genetic background. Male adult (10/12-week-old) mice were used in the experiments. Mice were maintained on a 12-hour light/dark cycle from 6 AM to 6 PM, 65–75 °F, 40–50% humidity and had unrestricted access to food  and water. N[w]-nitro-l-arginine methyl ester  was supplied in the drinking water or embedded in the chow . HFpEF was induced by feeding mice with HFD + L-NAME diet from 5 to 15 weeks10. Pregnant Sprague-Dawley rats were purchased (Charles River Laboratories) and were used for the sole purpose of harvesting primary cardiomyocytes from 1–2-day old rat pups.All experiments involving animals conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center. All studies were in compliance with all ethical regulations. C57BL/6 N mice were used for wild-type (WT) studies. Tetracycline responsive elements (TRE)-Xbp1s mice were crossed with mice harboring a tetracycline transactivator (tTA) transcription factor driven by α-myosin heavy chain promoter (αMHC-tTA) to generate mice with cardiomyocyte-specific inducible overexpression of Xbp1s (Xbp1s TG)10. All parameters were measured at least 3 times, and averages are presented.Transthoracic echocardiography was performed using a VisualSonics Vevo 2100 system equipped with MS400 transducer . Indices of systolic function were obtained in conscious, gently restrained mice from M-mode scans at midventricular level. Indices of diastolic function were obtained in anesthetized mice from apical four-chamber view using pulsed-wave and tissue Doppler imaging. Anesthesia was induced by 5% isoflurane and, during echocardiogram acquisition, isoflurane was reduced to 1.0–1.5% and adjusted to maintain a heart rate >400 beats per min10 until exhaustion. Exhaustion was defined as the inability of the mouse to return to running within 10 s of direct contact with an electric-stimulus grid.Exercise tests were performed as depicted in the figures using the following protocol. After acclimatization, mice ran uphill (20°) on a treadmill apparatus (Columbus Instruments) according to a ramping speed protocol53. Cardiac sections were visualized with a Leica DM2000 upright photomicroscope. Total triglyceride content was determined biochemically from myocardial tissue collected immediately after euthanasia. Colorimetric quantification of total triglycerides was performed using Triglycerides Reagent—Thermo Fisher Scientific according to the manufacturer’s instructions54.Hearts were harvested and flash frozen in an embedding medium containing a 3:1 mixture of Tissue Freezing Medium  and gum tragacanth . 0.3% Oil Red O  staining was performed on 5 μm-thick sections according to standard procedures9. Hearts were dissected and immediately digested by retrograde perfusion with perfusion buffer  for 1 min followed by buffer containing liberase TM  and trypsin (0.025%) for 14 min. The left ventricle was dissected and minced in a perfusion medium supplemented with 10% dialyzed fetal bovine serum. After filtration, AMVMs were allowed to sediment by gravity and non-cardiomyocytes were discarded. Calcium reintroduction was performed stepwise from 0 to 1.8 mM in six steps. AMVMs were stained with LipidTOX deep red  for 1 h according to the manufacturer’s instructions. Images were obtained using a Zeiss LSM 880 upright confocal fluorescence microscope. NRVMs were isolated from 1 to 2-day-old Sprague-Dawley rats9. NRVMs were cultured in Dulbecco’s modified Eagle’s medium/M199 (3:1) containing 3% fetal bovine serum and antibiotics before performing the experiments. NRVMs were treated in serum-free medium with BSA/oleic acid complexes  at the concentration of 200 μM for 3 h, 6 h, 24 h or 48 h. Free fatty-acid BSA  was used as control. Neutral lipids were visualized by Oil O Red staining as described above. For gene knockdown, NRVMs were transfected with three sequence-independent, STUB1-, FoxO1-, and siSKP2  using Lipofectamine RNAiMax (Invitrogen) in Opti-MEM (Gibco). After 6 h, cells were washed twice with growth medium and culture overnight in growth medium. For adenovirus-mediated protein overexpression, cells were transduced for 6 h with AdXBP1s52, AdcaFoxO137, and AdSTUB1 adenoviruses with increasing multiplicity of infection (MOI) of virus, and cells were harvested 24-hour post-infection. A GFP adenovirus (AdGFP) construct was used as control at the same MOI. Human STUB1 cDNA was obtained from UT Southwestern Center for Human Genetics and subcloned into an adenovirus expression vector using Adeno-X Adenoviral System 3 (Takara) following the manufacturer’s instructions. Adeno-X 293 cell line was purchased from Clontech . When appropriate, NRVMs and HEK293 cells were treated with MG132 , bafilomycin A1 , cell-permeable protease inhibitor cocktail , TUN , MK-3946 , cycloheximide .AMVMs were isolated from littermates of each experimental group as followsTo evaluate cell survival, LDH release was quantified using the CytoTox96 cytotoxicity kit (Promega) according to the manufacturer’s instructions. Each experiment was performed in three biological replicates each time in triplicate. LDH release was calculated as follows: (medium LDH)/(medium LDH + intracellular LDH).55); Lamin A/C ; PDK4 (previously developed56), OXPHOS cocktail . 10,000-fold dilution of GAPDH . Uncropped scans of immunoblot images are available in the Supplementary Information.Whole protein extracts from frozen mouse hearts, AMVMs and NRVMs were prepared by lysis in ice-cold modified radio immunoprecipitation assay (RIPA) buffer , 2 mM EDTA) containing protease and phosphatase inhibitors. Nuclear and cytosolic extracts were prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) following the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4–20% gradient gels (Bio-Rad) and transferred to nitrocellulose membranes. An Odyssey scanner (LI-COR version 3.0) was used as detection system. The separation between the nuclear and cytosolic fractions was verified by blotting for the cytosolic protein GAPDH and the nuclear membrane protein Lamin A/C. Tandem ubiquitin binding entities for the detection of ubiquitinated protein were used according to the manufacturer’s instructions (Life Sensors). Proteins were detected with a 1000-fold dilution of the following primary antibodies: FoxO1 ; FoxO3 ; Xbp1s ; GFP ; STUB1 ; Ubiquitin ; LC3 . A total of 500 ng RNA was used for reverse transcription using iScript reagent (Bio-Rad). qPCR reactions were performed in triplicate with SYBR master mix (Bio-Rad). The 257. NRVM cell numbers were counted using a hemocytometer. In brief, 1 × 104 cardiomyocytes were incubated in a solution containing KMES 60 mM, MgCl 223 mM, KH2PO4 10 mM, Hepes 20 mM, taurine 20 mM, mannitol 110 mM, EGTA 0.5 mM, and DTT 0.3 mM (pH 7.1) at 4 °C, and permeabilized with saponin (50 ng/mL) for 5 min before substrates were added. Isolated mitochondria from hearts (0.25 mg/mL) were incubated with the following buffer: 210 mM MOPS, 70 mM mannitol,10 mM sucrose, 5 mM KH2PO4, pH = 7.458. To measure complex I mediated OCR, glutamate (10 mM) and malate (5 mM) were added. State3 respiration was activated by adding ADP (2.5 mM), whereas state 4 respiration was measured after adding oligomycin (1 μM). For complex II-mediated OCR, 5 mM succinate was added. For the measuring of palmitoyl/carnitine-mediated OCR, 25 μM palmitoyl-carnitine was added to each experiment.NRMVs were transduced with AdGFP and AdXbp1s as described above. Cardiac mitochondria were isolated by ultracentrifugation in ice-cold isolation buffer . Oxygen consumption was measured using a fluorescence-based oxygen sensor  connected to a phase measurement system from the same company. The sensor was calibrated according to the manufacturer’s instructions37. The luciferase vector was transfected into HEK293 cells using Fugene HD (Roche) and into NRVMs using Lipofectamine 3000 (Thermo-Fisher). Luciferase activity was measured using a dual-luciferase kit (Promega) and normalized to renilla activity. Quantitative analyses of ChIP assays were conducted to assess Xbp1s occupancy at the STUB1 promoter using the Zymo-Spin ChIP kit according to the manufacturer’s instructions (Epigenetics). In brief, AdGFP- and AdXbp1s-infected NRVMs were exposed to 1% formaldehyde with gentle shaking . After quenching the cross-linking reaction with glycine, cells were washed twice with PBS, and then homogenized in nuclear extraction buffer. The homogenate was centrifuged  and the supernatant was discarded. The nuclear pellet was resuspended in nuclear lysis buffer, sonicated, and centrifuged  to prepare chromatin solution. Chromatin solution was diluted 10-fold and incubated with control (IgG) and Xbp1s antibody overnight at 4 °C with rotation. DNA purified from chromatin solutions was used as input. Purified DNA with or without immunoprecipitation was used to determine relative Xbp1s occupancy by comparing the Ct value of 10-fold diluted input DNA with undiluted immunoprecipitated DNA samples in control (IgG) and Xbp1s antibody. Primers used were : GGCGGAGCCTTGGTCTGA, CGATCCGCCACCGGAACT).Transcriptional assays using pDR4, a luciferase vector harboring two STUB1-UPREs in the presence of AdXbp1s or AdGFP control were performed in HEK293  and NRVMsp < 0.05 was considered statistically significant. All experiments were performed with at least three biological replicates. Statistical analyses were conducted using GraphPad Prism software 8.0. No statistical analysis was used to predetermine sample sizes; estimates were made based on our previous experience, experimental approach, availability, and feasibility required to obtain statistically significant results. Experimental animals were randomly assigned to each experimental/control group. Investigators were blinded to the genotypes of the individual animals during the experiments.Results are presented as mean ± SEMM. Differences were analyzed by two-tailed unpaired Kolmogorov–Smirnov test for experiments with two groups and one-way or two-way analysis of variance plus Sidak’s or Tukey’s post hoc test for multiple comparisons as appropriate in experiments including ≥3 groups. A minimum value of Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Breed management and conservation are based on documents (pedigree) that record the relationships among animals although they are not always complete and error-free. Thanks to the DNA analysis, it is possible to check the correct genealogy and breed history. Our study focuses on the Italian Maremmano horse breed, whose Studbook was established in 1980. Starting from a selection of 74 maternal lines, we analyzed the mitochondrial DNA (which is transmitted to offspring only by mother) of 92 samples and verified the pedigree data of 12 dam lines by matching genealogical and mitochondrial information. We found some mistakes in three maternal lines, as the samples belonging to the same lineage showed different mitochondrial DNAs, thus suggesting that the information recorded in the Studbook is wrong and the samples do not descend from the same dam founder. With this research, we confirm the utility of combining genealogical and historical information with molecular techniques in order to prevent errors in pedigree data and the loss of the genetic diversity of local breeds.The Maremmano horse is considered one of the most important Italian warmblood breeds which originated from an ancient population. In 1980, the National Association of Maremmano Breeders established the first Studbook that recorded 440 dams and four sire founders. In this study, we selected the most significant maternal lines in terms of offspring  and analyzed their mitochondrial DNA control regions. We found a high variability, reflecting the importance of this local breed as a genetic resource to be preserved. Through multiple sampling, we then verified the pedigree information for 12 dam lines by matching genealogical data with mitochondrial haplotypes. A complete concordance was demonstrated in nine lineages, while for the other we highlighted a different number of haplotypes for each line , thus suggesting that the information recorded in the Studbook could be wrong and the samples do not descend from the same maternal founder. Our combined analysis provides the opportunity to confirm the ancestry of animals and could be employed to prevent errors in pedigree data also for other breeds and species. The set of known parent-offspring relationships in a population is called pedigree, which is often graphically displayed as a family tree diagram and largely employed to derive the relationships among individuals. Over the decades, pedigree has been used in breeding programs as a predictive tool for the unique and reliable identification of individual organisms in breeding management and conservation ,2,3. PedEach animal should get a unique identification number at birth, and its parents should be known without any doubt. Extensive pedigree records should trace the whole breed history since its establishment; unfortunately, no pedigree is complete and error-free as records are scarce because of different factors:(1) cut-off dates for recording the genealogies ;(2) animals of unknown origins are included in the Studbooks ;(3) exchanges of young animals, parents, or semen at mating, and uncontrolled mating;(4) wrong registration and administrative mistakes;(5) fraud.In this scenario, the development of molecular techniques has a key role in supporting the traditional pedigree which is based on relationships among animals. A combination of both pedigree and molecular data has been demonstrated to be optimal ,8,9,10. The present study focused on the Maremmano horse, which is considered one of the most important Italian horse breeds for leisure and sports. This warmblood horse descends from an ancient local population spread in Maremma, an Italian territory along the Tyrrhenian coast of Southwestern Tuscany and Northern Latium, located about 100–200 km northwest of Rome. During the Etruscan period and over the centuries, the Maremmano horse was influenced by various genetic types introduced by people that dominated this territory . Although a significant proportion of Maremmano horses descends from a strongly limited number of male lines , it is In this complex scenario, our research aims to match the pedigree data available for the Maremmano breed with mitochondrial haplotypes in order to assess its genetic diversity and verify the reliability of current genealogies, as phenotypic traits and genealogical data are insufficient to ascertain the horse history and the real pedigree.Genealogical information recorded in the Maremmano Studbook was considered in order to choose all principal dam lines and select samples with a known pedigree up to four consecutive generations of maternal ancestors. Generation intervals (GI) were calculated as the difference between the average age of offspring and parents. Also pedigree completeness (PEC) was calculated including maternal and paternal lines. The genetic distance between horses was evaluated by calculating the average relatedness (AR) coefficient  and only animals with at least 20 descendants were selected for this study. Pedigree data were obtained from the National Horse Breeders Association of Maremmano Breed (ANAM). These data are based on old documents  which are often incomplete and recorded up to the subject arbitrarily considered the breed-founder. For this reason, the data were verified by referring to the archives of the Sports Horse Research Center (University of Perugia), that were created thanks to the access to the main national Studbook databases for scientific purposes and allowed the cross-checking of genealogical information which are integrated, where it is possible, with molecular genetic techniques.A total of 92 Maremmano horses  belonging to 74 female lines from different Italian regions  were analyzed .Among them, due to the sampling complexity as these horses are bred in the wild, we were able to verify the pedigree information through the mtDNA analysis of 12 of the most important maternal lineages in terms of offspring, by analyzing two or more samples for each dam line, for a total of 30 samples . They belonged to the four main male lines Aiace (N = 12), Ingres (N = 2), Otello (N = 13), and Ussero (N = 3) and were representative of all the four Italian regions mentioned above.For each animal, the pedigree reported in the ANAM Studbook was checked for possible mistakes and arranged for the analysis by using the Pedigree Viewer v.6.3 software , which iBy using a script in Fortran 95 software, it was possible to calculate the percentage of Thoroughbred blood for each animal. The algorithm computes the Thoroughbred blood percentage reported in the pedigree through logical considerations: the blood percentage of the animals identified as Thoroughbred is 100%. Each horse, whether male or female, transfers to the offspring half of its own coefficient so that the product resulting from mating two Thoroughbred has 100% Thoroughbred blood percentage, which is inherited half from the sire and half from the dam. Therefore, an individual horse who has just one Thoroughbred parent has 50% Thoroughbred blood whereas an individual horse with one Thoroughbred grandparent has 25% Thoroughbred blood.The software computed the Thoroughbred blood percentage in Maremmano horses asThe average per year of birth of these Thoroughbred blood coefficients was reported in a plot in order to analyze the trend of the Maremmano breeding program. Sire and dam ancestors’ lineages of the pedigree were reconstructed by using Fortran 95 software, which allowed us to trace backwards the maternal and paternal lineages of each individual pedigree till the ancestors without genealogical information which are supposed to be the breed founders. A preliminary selection of the animals was carried out by employing this software in order to perform a molecular analysis of Y chromosome  and mitochondrial DNA. After specific logical tests, the program returns an output file containing the name of each individual with the relative male and female ancestors back to the founder and indicating the number of generations that separates them. For each male and female founder, the total number of individuals belonging to its lineage, as well as the number of living and still in activity animals was computed.After the pedigree analysis, only animals with at least 20 descendants were selected for this study, for a total of 92 individuals belonging to 74 dam lines. Among them, the pedigree information was verified for 30 samples belonging to 12 of the most important Maremmano dam lines in terms of offspring , Nizza I, Pomposina, and Velia), by analyzing 27 mtDNA control-region sequences from Cardinali et al. [® Automated Nucleic Acid Extractor, following the provided protocol. All experimental procedures were reviewed and approved by the Animal Research Ethics Committee of the University of Perugia (Prot. N.2017_01).For these three sequences, DNA was extracted from 1.2 mL of peripheral blood samples by automated extraction through the MagCorewww.fluxusengineering.com).Mitochondrial DNA control-region was amplified between sites 15,364 and 563 and Sanger-sequenced as already reported by Cardinali and colleagues . FragmenThe pedigree file, including 16,588 horses  born from 1920 to 2019 (30 September 2019) provided by ANAM Studbook, was verified. In the first year of ANAM official activity 203 foals were registered and this number did not change in the following two years. Then, the number increased to 540 in 1991, reaching its maximum in 1997 . Subsequently, the number of foals decreased to 370 in 2000 and remained almost stable until 2012 when it decreased to 216 because of the economic crisis. The number registered in 2019 was practically the same as the number registered when the Studbook first started .The average ± SD Thoroughbred blood percentage in the whole population was 19.33% ± 2.65: 19.81% ± 2.06 in males and 18.92% ± 2.71 in females . It has to be pointed out that the percentage of Thoroughbred blood in pedigree has increased 0.19% per year since 1980; however, the increase more than doubled to 0.51% in the first 10 years of the ANAM activity and remained stable in the following years.The average inbreeding (F) in the whole population was 2.90% and ranged from a minimum of 1.29% (1981) to a maximum of 6.16% (2014); this value increases on average 0.10% per year although it decreased to 4.64% in 2017, due to the execution of a Breeders Association inbreeding containment program started in 2012 .The average inbreeding increases to 4.54% in the inbred animals while this value reaches 3.92% in the collected sample.The mean of the AR coefficient in the whole population was 5.17 % and ranged from a minimum of 0.01% to a maximum of 14.62%.The average generation interval was 10.65 years; the greater pathway was observed between the Maremmano sire and foal (11.64 years), while the lower (9.65 years) was between the mare and foal. The average PEC was 99.34% including maternal and paternal lines.The sire lineage, identified by the “home made” software for the whole Maremmano population, confirmed four main lines , and smaller lines with a restricted living progeny. As for the dam lineages, a selection based on not related lines with at least 20 descendants and living progeny was carried out. The use of this procedure permitted to identify 74 maternal lineages, among which there are the most famous dam founders: Arpa 3, a mare born in 1974, whose progeny is part of the unit of Tenuta Presidenziale di Castelporziano mares [From the individuals belonging to the 74 maternal lineages, 92 animals were both tested and sampled as described in the Material and Methods section, on which a mitochondrial DNA analysis was carried out. The 92 individuals showed an average AR coefficient of 3.57 with a minimum of 0.01% and a maximum of 12.23%. Among the 610 bp analyzed, 75 polymorphic sites were found, all of them represented by single nucleotide polymorphism (SNP) with an average nucleotide diversity of (π) 0.02063 .Fifty-one different haplotypes were identified (Nh), with an average haplotype diversity index (Hd) of 0.981 , showingA total of 16  haplogroups were identified, with the higher frequencies for L, G, B, and M  .After the comparison between the genealogical data from the pedigree and the molecular data from the mtDNA analysis, a total of 12 dam lines were checked, as it was possible to confirm if two or more samples belonged to the same maternal line . In our Knowledge of relationships among horses is essential for the genetic management of breeds and represents one of the principal tools employed to optimize the conservation strategies. It is traditionally retrieved from pedigree, which is essentially never complete or error-free. With the development of molecular techniques, traditional relationship coefficients based on pedigree information are gradually replaced by those calculated from molecular data. In this framework, the present study combined the available pedigree data from the Maremmano breed with mitochondrial DNA haplotypes, in order to assess its genetic diversity and verify the reliability of some genealogies. This uniparental molecular marker can be applied to historical issues of maternity in absence of biological material from putative dams or even from offspring . The mtDOur study confirms the completeness of genealogical information and the traditional importance that breeders gave to the female lines; these two factors had a key role in preserving the genetic diversity of the Maremmano horse. Thanks to accurate genealogical analysis, 16,588 Maremmano horses registered in the studbook were better identified, and male and female ancestors were traced. From the reconstruction of sire and mare lineages, it was confirmed that all Maremmano horses derive from 4 males and 440 dam founders, of which 332 mares were born between 1960 and 1975 [Among them, the most significant maternal lines were selected for a total of 74 lineages. When combined with genealogical and historical information, mtDNA data proved to be helpful in evaluating maternal inheritance and in identifying founding mares. Furthermore, mtDNA surveys contributed to the current understanding of the domestication process ,20,21,22For these reasons, we collected blood samples from 92 Maremmano horses belonging to the 74 dam lines which were considered the most representative of this local breed . Among them, a molecular concordance with studbook records was demonstrated through multiple sampling in nine lines , and Velia), while genealogical mistakes emerged for three dam lines , probably due to wrong data registration. Our findings confirm the importance of the pedigree reliability for the analysis of genetic structure, that could be applied to other local breeds and species, as previously done ,6.The analysis of Maremmano mtDNA control regions revealed a high mitochondrial variability within the breed, testified by 75 mutated sites that allowed to identify 51 haplotypes and 16 different haplogroups, thus confirming its remarkable genetic diversity ,27. AmonThe development of molecular techniques gradually sustains traditional pedigree-based analysis; a studbook is not always error-free therefore mtDNA analysis can provide genetic information about the maternal lines. Our study showed that the combination of both sources not only could be optimal but could also enable a phylogenetic reconstruction of domestic horse origins. The mtDNA analysis provides breeders with the opportunity to confirm the ancestry of their horses and could be used to prevent possible frauds in falsifying the genealogical data. Furthermore, through the analysis of the entire mitogenome and the comparison with other breeds, it will be possible to characterize all the different haplogroups existing in the Maremmano breed and incorporate this scientific approach in the selection plans, in order to provide important support to preserve its genetic diversity.In conclusion, the present research confirms the power of mitochondrial DNA surveys for testing pedigree authenticity and improving the monitoring and management of livestock populations and demonstrates the utility of mtDNA analysis in describing the maternal legacy among samples by excluding those maternally related or without certain origins. This research can be considered as a preliminary study for further analyses focused on verifying the reliability of pedigree data for the remaining Maremmano dam lines and can be also extended to other local breeds and species.


Applying a quality-by-design approach, the influence of critical formulation variables, i.e., the L5 content and the L5:siRNA ratio (w/w), on critical quality attributes (CQAs) was investigated systematically using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). The CQAs were identified based on the quality target product profile and included size, polydispersity index, zeta potential, encapsulation efficiency and loading for achieving efficient and safe TNF-α gene silencing in activated RAW 264.7 cells. Formulations inducing efficient gene silencing and low cytotoxicity were identified, and the optimal formulations displayed L5 contents of 15 and 20% (w/w), respectively, and an L5:siRNA weight ratio of 15:1. All tested formulations within the OOS mediated efficient and sequence-specific TNF-α gene silencing in RAW 264.7 cells at TNF-α-siRNA concentrations, which were significantly lower than the concentrations required of non-encapsulated TNF-α-siRNA, highlighting the benefit of the delivery system. The results also demonstrate that increasing the loading of siRNA into the delivery system does not necessarily imply enhanced gene silencing. This opens new avenues for further exploitation of LPNs as a robust platform technology for delivering TNF-α siRNA to macrophages, e.g., in the management of COPD.RNA interference (RNAi) has an unprecedented potential as a therapeutic strategy for reversibly silencing the expression of any gene. Therapeutic delivery of the RNAi mediator, i.e., small interfering RNA (siRNA), can be used to address diseases characterized by gene overexpression, for example inflammatory conditions like chronic obstructive pulmonary disease (COPD). Macrophages play a key role in COPD pathogenesis and are recruited to the airways and lung parenchyma, where they release proinflammatory cytokines, e.g., tumor necrosis factor-alpha (TNF-α). Hence, targeting TNF-α with siRNA is a promising therapeutic approach for COPD management. However, a safe and effective delivery system is required for delivery of TNF-α siRNA into the cytosol of hard-to-transfect macrophages. The purpose of this study was to optimize the intracellular delivery of TNF-α siRNA to the lipopolysaccharide-activated murine macrophage cell line RAW 264.7 using lipidoid-polymer hybrid nanoparticles (LPNs) composed of the lipid-like transfection agent lipidoid 5 (L Caenorhabditis elegans more than two decades ago  is a regulatory pathway in eukaryotic cells in which gene expression is inhibited at the messenger RNA (mRNA) level by sequence-specific double-stranded RNA, for example small interfering RNA (siRNA) . More than 210 million people are affected by COPD, and by 2030, COPD is expected to be the fourth largest cause of death worldwide . The agonistic effect was also confirmed in silico. Interestingly, this agonistic effect was abrogated when L5 was formulated as LPNs loaded with siRNA. However, no systematic studies have been performed to date to investigate the effect of the L5 content and the L5:TNF-α siRNA weight ratio on the in vitro safety and gene silencing effect of LPNs. Hence, we used a systematic quality-by-design (QbD) approach implementing risk assessment and design of experiments (DoE) for optimizing the loading of therapeutically relevant TNF-α siRNA in LPNs. The QbD approach and DoE are valuable tools as maximal information is provided from the least number of experiments. The QbD process involves the following steps: (i) identification of the quality target product profile (QTPP) i.e., the product attributes that are critical to stability, safety and efficacy; (ii) identification of the critical quality attributes (CQA); (iii) identification of critical process parameters (CPP), and (iv) layout and implementation of DoE to establish the relationship between the CQAs and the CPPs. This information is used to define a process design space i.e., the optimal operating space (OOS) that will result in an end product of the desired QTPP  and negative control siRNA were generously provided by GlaxoSmithKline  as dried, purified and desalted duplexes (w: 20 kDa) was obtained from Wako Pure Chemical Industries . L5 was synthesized, purified, and characterized as previously reported  method, essentially as reported previously  was varied from 15 to 25% (w/w), and the L5:TNF-α siRNA ratio ranged from 5.0:1 to 15.0:1 (w/w). The final batch size per formulation was 15 mg. The physicochemical properties  of the LPNs were determined as described previously  values and physicochemical properties, respectively, using an I-optimal design with two critical independent variables, i.e., L5 content and L5:TNF-α siRNA weight ratio (5 content ranged from 15 to 25% (w/w), while the latter ranged from 5.0:1 to 15.0:1 ] to the independent variables were measured. Critical responses were identified and subjected to model fitting by using analysis of variance (ANOVA), and the best model fit was selected based on statistical parameters, i.e., the p-value, the R2, the difference between adjusted and predicted R2, and the adequate precision. The results were further optimized numerically and graphically to construct desirability and overlay plots, respectively. Statistical optimization was performed using Design Expert .Initially, the effect of Lht ratio . The L5 o 15.0:1 . Seven r2-95% atmospheric air incubator at 37°C, the growth medium was renewed every second day, and the cells were subcultured twice a week by detaching them from the culture flask  using a cell scraper. Cells were seeded in 6-well tissue culture plates (Sigma Aldrich) at a density of 1.0 × 106 cells/well. Subsequently, nanoparticle suspensions were added to each well resulting in final siRNA concentrations of 2.8, 5.6, 11.3, 27.9, and 55.8 nM, respectively, in duplicates, followed by incubation for 21 h. To each well, 5 ng/mL  lipopolysaccharide  was added, and the cells were subsequently incubated for additional 3 h. After 24 h, the cells were lysed with 350 μL NucleoSpin cell lysis buffer , and total RNA was isolated and purified using the NucleoSpin RNA Plus kit (Macherey-Nagel). Total RNA was checked for purity and quantified by UV-Vis spectroscopy . Purified RNA was reverse transcribed using the iScript cDNA synthesis kit . The real-time polymerase chain reaction (PCR) or quantitative PCR (qPCR) was performed in duplicate for the reference housekeeping genes [β-actin (ACTB) and β-glucuronidase (GUSB)] and in triplicate for the TNF-α gene using a LightCycler® 480  and the SYBR I Green® Master Mix (Roche) as reported previously  analysis, followed by quantification relative to LPS-treated cells using the comparative ΔΔCP method . The cells were maintained in Dulbecco's Modified Eagle's Medium with high (4.5 g/L) glucose  supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine , and 10% (v/v) fetal bovine serum . The cells were grown in a 5% CO Pfaffl, .2-95% atmospheric air. At 26 h, the cell culture medium containing MTT was removed from the wells, and the plate was thoroughly air-dried overnight in the dark before adding 200 μL dimethyl sulfoxide to solubilize formazan MTT crystals. The cell viability was determined based on the amount of formazan crystals formed in cells incubated with the test formulations vs. the control cells by measuring the absorbance at 570 nm using a microplate reader .RAW 264.7 cells were seeded in a 96-well plate at a density of 5,000 cells/well in a final volume of 180 μL and allowed to adhere by incubation for 4 h. Subsequently, the cells were incubated for 24 h with 20 μL test formulations in quadruplicates at eight different TNF-α siRNA concentrations (1.38–222.8 nM). The final volume in the well was 200 μL. At 24 h, the cell culture medium was withdrawn from each well. Adherent cells were washed with PBS, followed by the addition of 180 μL cell culture medium and 20 μL freshly prepared 12 mM methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Sigma Aldrich). The plates were incubated for 2 h at 37°C in 5% COpost-hoc multiple comparison test. A p-value ≤ 0.05 was considered statistically significant.Data were analyzed using GraphPad Prism  and represented as mean values ± standard deviation (SD). Statistically significant differences were assessed by one-way analysis of variance (ANOVA) followed by a Tukey's z-average, (ii) PDI, (iii) zeta potential, (iv) encapsulation efficiency, (v) loading of siRNA, (vi) in vitro transfection efficiency, and (vii) in vitro cell viability.The QTPP of the TNF-α siRNA-loaded LPNs was defined  to ensur5 content , on the physicochemical properties of the LPNs, the other variable, i.e., the L5:TNF-α siRNA ratio, was kept constant at four different levels   . Signifi5 content constant and varying the L5:TNF-α siRNA ratio. The z-average and PDI of the LPNs remained almost constant at all tested L5:TNF-α siRNA ratios (5:TNF-α siRNA ratio was decreased (corresponding to a higher amount of siRNA without increasing the L5 content). Hence, the L5 content and the L5:TNF-α siRNA ratio influence the zeta potential and the siRNA loading. The encapsulation efficiency was neither affected by the L5 content nor the L5:TNF-α siRNA ratio, and no significant differences were observed. However, the responses were within the QTPP (>60%). Based on these results, the L5 content relative to the final batch weight  and the L5:TNF-α siRNA ratio  were selected for inclusion in a systematic DoE.The physicochemical properties of the formulations were explored further by keeping the LA ratios . In cont5 content and the L5:TNF-α siRNA ratio affected the gene silencing. At an siRNA concentration of 5.6 nM for LPNs having 15% L5 content, the TNF-α mRNA expression was ~60%. In contrast, the expression was at least 80% with the remaining LPNs. At the highest tested siRNA concentration (55.8 nM), the TNF-α mRNA expression generally decreased as the L5 content increased  and the L5:TNF-α siRNA ratio  as the L5 content increased from 15 to 25% , the R2 was only −0.3 and 0.27, respectively, hence these responses were excluded from the analysis. For all modeled responses (R2 was in reasonable agreement with the predicted R2 (the difference was <0.2), while the adequate precision was higher than four. In addition, the lack of fit was not significant for all modeled responses. Formulations displaying an L5:TNF-α siRNA wt. ratio of 5.0:1 were excluded from the mathematical modeling because the IC50 values for gene silencing fit the model equations linearly from 7.5:1 to 15.0:1 ratio.The obtained responses  were fitesponses , the adj50 could not be determined due to negligible effects if the formulations on cell viability) and the prediction model analyzed using ANOVA . The IC50 values of TNF-α siRNA-loaded LPNs with L5 content of 15 and 20% having an L5:TNF-α siRNA ratio of 15.0:1 relative to non-encapsulated siRNA were 4.5 ± 1.2 and 3.2 ± 0.6-fold times lower, respectively  constant. This was followed by assessment of all formulations for their effect on TNF-α gene silencing and cell viability in activated RAW 264.7 macrophages.Adopting a QbD approach ensures the quality of medicines by employing statistical, analytical, and risk management methodologies in the design, development, and manufacturing of medicines  by keeping the L5:TNF-α siRNA ratio 15.0:1, two formulations were prepared, and it was found that the z-averages were 159.1 and 154.8 nm with PDIs of 0.216 and 0.272, respectively (p < 0.01). One explanation could be that as the amount of L5 is increased, the PLGA content decreases, which means a lower concentration of the latter in the organic phase during the preparation process and consequently a reduction in particle size. This may be attributed to a decrease in solvent viscosity, increase in diffusion coefficient, and the decrease in PLGA content in the droplets of the organic phase resulting in smaller particles after solvent evaporation , in agreement with previous reports  siRNA-loaded LPNs as we have reported previously  as the L5 content increased from 15 to 25%, highlighting the role of L5 in electrostatic interactions with siRNA. The relatively lower encapsulation efficiency could be due to the high pH (7.5) during the emulsification process as L5 might not be fully protonated . No statistically significant differences were observed with respect to TNF-α siRNA loading between the two formulations. Similar results were obtained for LPNs loaded with an antisense oligonucleotide (ASO) (Thanki et al., 5 is not complete and that more siRNA could be complexed. However, in general, excess siRNA may not necessarily translate into higher gene silencing because the cationic character of the lipid decreases, eventually resulting in poor transport across the cell membrane and thus poor transfection (Fröhlich, 5 content and the L5:TNF-α siRNA ratio affect the physicochemical properties of the LPNs, hence they were included in the systematic QbD-based optimization of the LPNs.When keeping the LIn vitro studies using the MTT assay exhibit that the LPNs, in general, do not have a cytotoxic potential at the measured concentration ranges. The toxicity is predominantly induced by the positively charged lipid component of the nanoparticulate carrier. However, the use of synthetic lipids or lipidoids help overcome these delivery challenges (Akinc et al., Nucleic acid delivery has traditionally been hindered by the toxicity associated with their delivery systems. In the past years, novel carrier systems and analogs have been developed to overcome this problem (Love et al., 5 component. It has been reported that certain lipidoids. e.g., DLinDMA, at a concentration of 1 μg/mL of siRNA induce a significant increase in apoptotic macrophages (Basha et al., In the present study, we identified the effect of the independent variables on the cell viability where all the tested formulations had comparable siRNA concentrations. It was found that non-encapsulated TNF-α siRNA and scrambled siRNA did not affect the cell viability at the tested concentrations. Thus, the toxicity of the formulations is most likely caused by the L5 content and L5:TNF-α siRNA ratios were tested at five concentrations of encapsulated TNF-α siRNA. All formulations, except the ones displaying an L5:TNF-α siRNA ratio of 7.5:1, exhibited IC50 values 2.6- to 4.6-fold lower than the LPNs loaded with negative control siRNA and the non-loaded TNF-α siRNA. These results show that using LPNs as a delivery system is clearly advantageous and the silencing is due to the specific sequence of TNF-α siRNA. This may be due to the fact that non-loaded TNF-α siRNA cannot cross the cell membrane (De Paula et al., 5:TNF-α siRNA ratio was increased from 7.5:1 to 15.0:1 at 15, 20, and 25% L5 content. Interestingly, LPNs with a 5.0:1 ratio silenced TNF-α gene expression more efficiently than 7.5:1. We do not fully understand the reason for these results, which needs to be addressed in future studies. Although the IC50 values are quite different for every formulation, it is evident that at the highest tested concentration of siRNA, the gene expression of TNF-α in LPS-activated macrophages treated with LPNs containing 15, 20, and 25% L5 and having an L5:TNF-α siRNA ratio of 15.0:1 was only 19, 12, and 6%, respectively. Similar observations have been documented previously where increasing the lipidoid content resulted in more efficient silencing of Factor VII mRNA (Akinc et al., 50 values are highly variable and depend on many factors, including the composition of the formulation (Thanki et al., 5 having an L5: TNF-α siRNA ratio of 15.0:1 were found to be most optimal for gene silencing in macrophages, as determined from their IC50 values (To study the effect of the independent variables on the TNF-α mRNA expression, 12 formulations with different L0 values .in vitro gene silencing (Dormenval et al., in vivo (Jansen et al., z-average, PDI, zeta potential, siRNA loading, and encapsulation efficiencies are almost identical with the latter, which may be due to comparable nucleotide length and similar modification patterns (5 content and the L5:TNF-α siRNA weight ratio as shown from the coefficients (in vitro and in vivo after pulmonary administration, which demonstrated that the sustained release of siRNA from LPNs correlates well with cell uptake and in vivo biodistribution (Thanki et al., in vitro and in vivo release kinetics, which is highly comparable to our previously reported findings (Thanki et al., To date, several different lipid carriers have been studied for siRNA delivery. It is generally challenging to compare silencing and non-specific effects obtained in different studies due to highly variable experimental conditions, e.g., cell type, LPS concentration, incubation time, and presence of serum proteins. These tend to destabilize cationic nanoparticles and thus invalidate the observations, since the actual amount of siRNA that reaches the cytosol may vary (Buyens et al., patterns , whereasficients . RecentlThe OOS was constructed based on the results obtained from the above experiments and on the criteria set in the QTPP. Ten formulations displaying a desirability score from 0.671 to 0.796 were identified. The desirability values are between 0 and 1, the latter being the most desirable. However, this term may be misinterpreted because the desirability function is fully dependent on how close the upper and lower limits are set in the QTPP. The goal of optimization is how well the set conditions meet the goals and not about gaining the highest desirability (Desirability Function, in vitro gene silencing effect and safety. We show that both the L5 content and the L5:TNF-α siRNA ratio significantly affect the zeta potential, siRNA loading and TNF-α gene silencing. It was also found that the responses to the independent variables were complex, but in general there seemed to be an optimal range for loading siRNA into LPNs. QbD with statistical analysis facilitated the modeling of these responses with a high coefficient of determination, which can be useful in predicting responses based on user input of the independent variables. The LPN formulations loaded with TNF-α siRNA showed a 1.3- to 4.6-fold reduction in the effective dose for in vitro gene silencing as compared to the dose required for gene silencing by non-encapsulated TNF-α siRNA, highlighting the importance of a delivery system. It was also shown that the zeta potential is not the only determinant of toxicity of a formulation and that reducing the positive surface charge does not necessarily imply reduced gene silencing in every scenario. We further show that higher siRNA loading does not necessarily correlate with higher gene silencing in vitro. Finally, an OOS was identified based on graphical optimization, and point formulations displaying L5 contents of 15 and 20% and L5:TNF-α siRNA weight ratio of 15.0:1 were identified that satisfied all the criteria set in the QTPP and had a balanced toxicity/efficacy profile. These results demonstrate the importance of systematic formulation design for loading therapeutic TNF-α siRNA in LPNs with optimal gene silencing and safety and support our ongoing efforts for TNF-α siRNA-mediated therapeutic management of COPD in preclinical animal models.In conclusion, we demonstrate the application of a systematic QbD approach for loading therapeutic TNF-α siRNA into LPNs with optimal The original contributions presented in the study are included in the article/AL, AT, KT, and CF designed the study. AL, KT, and AW performed the laboratory work. AL, AT, KT, and CF interpreted the data. AL, AT, and CF drafted the manuscript. AL, AT, AW, KT, HF, and CF provided scientific input throughout the study period and drafting of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TNF-α perfusion did not significantly change thresholds for compound action potential (CAP) responses, which reflect cochlear nerve activity, or distortion product otoacoustic emissions, which reflect outer hair cell integrity. However, intracochlear TNF-α perfusion reduced CAP amplitudes and increased the number of inner hair cell synapses without paired post-synaptic terminals, suggesting a pattern of synaptic degeneration that resembles that observed in primary cochlear neuropathy. Additionally, etanercept, a TNF-α blocker, protected against TNF-α-induced synaptopathy when administered systemically prior to intracochlear TNF-α perfusion. Findings motivate further investigation into the harmful effects of chronically elevated intracochlear levels of TNF-α, and the potential for etanercept to counter these effects.Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that plays a prominent role in the nervous system, mediating a range of physiologic and pathologic functions. In the auditory system, elevated levels of TNF-α have been implicated in several types of sensorineural hearing loss, including sensorineural hearing loss induced by vestibular schwannoma, a potentially fatal intracranial tumor that originates from the eighth cranial nerve; however, the mechanisms underlying the tumor's deleterious effects on hearing are not well-understood. Here, we investigated the effect of acute elevations of TNF-α in the inner ear on cochlear function and morphology by perfusing the cochlea with TNF-α  Hearing loss is the most common sensory deficit in the world, affecting 466 million people today, and projected to affect 900 million by 2050 . The mosAlthough SNHL is typically caused by aging, overexposure to noise or ototoxic drugs, or genetic mutation, an additional potentially life-threatening cause of SNHL is vestibular schwannoma (VS), a non-malignant tumor arising from Schwann cells of the vestibular portion of the eighth cranial nerve, and extending into the cerebellopontine angle of the brain. A striking 95% of VS patients develop SNHL; additional symptoms can include tinnitus, balance difficulties, and facial paralysis . The conThese reports motivate the hypothesis that VS-induced SNHL is caused, at least in part, by VS-secreted factors that directly damage cochlear hair cells and neurons. Indeed, our laboratory has previously shown that human VSs grouped based on patient hearing ability tend to have different gene expression profiles, suggesting molecular contributions to VS-induced SNHL . FocusinTNF-α is a pro-inflammatory cytokine with key functions in diverse cellular processes, including regulation of the pro-inflammatory responses and maintenance of cellular homeostasis. While the expression of TNF-α is typically undetectable in the normal cochlea , it can in vivo intracochlear TNF-α perfusion on cochlear structure and function in guinea pigs. These experiments have implications for understanding the mechanisms of VS-induced SNHL in addition to other forms of SNHL, including ISSNHL, AIED, and noise-induced hearing loss. We perfused TNF-α via the round window, which opens into the perilymph-filled scala tympani, because (a) VS-secreted TNF-α is likely to reach the cochlea via the fundus of the internal auditory canal, which is continuous with the cochlea's perilymph-filled lumina, (b) perilymph bathes the vast majority of cochlear cells, and (c) the round window is surgically accessible in a minimally invasive fashion, using an approach that is similar to the approach routinely used in humans for cochlear implantation. We show that acute in vivo TNF-α perfusion leads to an increase in the number of synaptic ribbons without paired post-synaptic receptors, suggesting synaptic degeneration. We also demonstrate the efficacy of etanercept, a TNF-α-inhibitor, for protecting against TNF-α-induced synaptic damage when administered prior to TNF-α. Results suggest a TNF-α-induced pathologic profile that is similar to what is observed in primary cochlear neuropathy.Here, we investigate the effects of The experimental timeline is outlined in n = 5), TNF-α , and prevention . Animals were anesthetized through intraperitoneal injections of pentobarbital sodium , and intramuscular injections of fentanyl (0.2 mg/kg) and haloperidol (10 mg/kg) as described previously  were randomly assigned to one of three experimental groups: control , or TNF-α  according to the condition being tested. The TNF-α concentration selected for the present experiments is higher than what we previously reported for human VS secretions , followed by intracardiac perfusion with 4% paraformaldehyde (PFA). Inner ears were immediately extracted and prepared for histological assessment as described below.Hearing was assessed via compound action potentials (CAPs), a measure of the synchronous firing of a population of auditory nerve fibers, and distortion product otoacoustic emissions, a proxy for outer hair cell function, over the course of each experiment. Specifically, hearing was tested just prior to cochleostomy placement, after positioning of the microcannula, and every 2 h after the first intracochlear perfusion until the experiment's termination .All sound stimuli were delivered via a custom acoustic assembly comprising two dynamic drivers as sound sources and a miniature electret microphone to measure the sound pressure level (SPL) at the animal's ear , 32. Res2 onset envelope; 16/s) delivered at SPLs from 10 to 85 dB SPL proceeding in 5 dB steps. The electrode response was amplified , filtered (0.3–3 kHz bandpass), and averaged (128 samples at each frequency-level combination). Threshold was defined as the lowest stimulus level at which a repeatable wave could be identified in the response waveform. The minimum input tone pip SPL that could generate a CAP amplitude of at least 0.4 μV was taken to be the CAP threshold of hearing; this value was empirically determined to be the voltage at which CAP amplitudes began to rise linearly with increasing SPL in our system. If the CAP amplitude was <0.4 μV at the maximum tone-pip SPL of 85 dB, the threshold was set to 85 dB.CAPs were recorded in response to tone-pip stimuli  corresponding to the frequency 2f1–f2 are robust and are frequently measured to assess outer hair cell motility, which is required to generate the distortion products.These DPOAEs were recorded as response amplitude vs. primary level functions ; f2/f1 = 1.2 in the ear canal was amplified and digitally sampled at a rate of 200 kHz. Fourier analysis was used to determine the magnitudes of the DPOAE responses at f1, f2, and 2f1–f2. In a DPOAE magnitude spectrum, for a given frequency f, the noise floor was calculated as the average of the response magnitudes at the two frequencies above f and the two frequencies below f. The minimum input SPL required to generate a 2f1–f2 response with magnitude greater than the noise floor at 2f1–f2 was taken to be the DPOAE threshold.Immediately following euthanasia and intracardiac perfusion with 4% PFA, inner ears were extracted and dissected to remove extraneous bulla and tissue. Both the round and oval windows were opened and PFA was perfused through the scala tympani and scala vestibuli to ensure thorough fixation of the organ of Corti. Cochleae remained in 4% PFA for 2 h on a shaker and were subsequently placed in 0.12 M EDTA for 2 weeks to decalcify the otic capsule for whole mount preparation.Histologic sections of the organ of Corti were prepared as previously described . In brieWhole mounts were imaged using a Leica TCS SP8 laser-scanning confocal microscope. Preliminary low magnification (10X) images were acquired and subsequently arranged from base to apex and labeled according to frequency region  using a custom ImageJ plug-in . In eachHigh magnification z-stacks were uploaded to Amira 3D Software for Life Sciences (Thermo Fisher Scientific) for automated identification and segmentation of ribbons. Specifically, custom software  was usedH-tests were applied using IBM SPSS and Microsoft Excel software.Differences in DPOAE and CAP thresholds before and after intracochlear perfusion were assessed by two-way ANOVA. Error bars in 2  VS tumor secretions contain high levels of TNF-α , (b) VS in vivo experiments follow up on our previous ex vivo investigations into the effects of applying both recombinant TNF-α and human VS secretions to murine cochlear explants (ex vivo studies, while secretions from specific VS tumors caused inner hair cell damage, outer hair cell damage, and nerve fiber disorganization, application of recombinant TNF-α caused nerve fiber disorganization and a decrease in neurite counts without evidence of any hair cell damage. The present in vivo studies were also conducted using recombinant TNF-α, and the observed synaptic degeneration corroborates the findings of nerve fiber disorganization in the previous ex vivo studies. Thus, it is likely that the VS secretion-induced hair cell damage observed in our previous ex vivo studies occurred due to a longer or different method of exposure, and/or was mediated by a different ototoxic secreted factor or some other mechanism altogether. Relevantly, it is known from studies conducted in the central nervous system that overexpression of TNF-α can cause acute synaptic degeneration via glutamate-mediated cytotoxicity. Specifically, excess TNF-α production has been shown to lead to neuronal death (2+-permeable AMPA receptors that is caused by selective downregulation of the GluA2 subunit of the AMPA receptors in these neurons. Returning to the peripheral auditory system, it is widely accepted that glutamate is the afferent neurotransmitter responsible for communication between inner hair cells and spiral ganglion neuron terminals in the cochlea (The present explants . In the al death  mediatedal death , and it al death . In partal death  reported cochlea  and that cochlea . Becausein vivo study on TNF-α-induced intracochlear cytotoxicity, Keithley et al. (in vivo, and they observed only minimal differences between these groups in inner and outer hair cell counts in vitro. These findings are consistent with our present results; specifically, we also did not observe significant differences in CAP thresholds between the TNF-α and control groups, and did not observe significant differences between these groups in hair cell counts. Keithley et al. did not comment on CAP amplitudes or synaptic integrity in their report. While we cannot directly compare Keithley et al.'s results with ours, because we measured tone-evoked CAPs, both studies nonetheless have important parallels, and reach similar conclusions. Taken together, our current in vivo results and previously published ex vivo results on application of human VS secretions to mouse cochlear explants suggest that in cases of VS-induced SNHL where inner and outer hair cell death are observed, the hair cell degeneration cannot be explained by acute TNF-α-mediated cytotoxicity alone.In a previous y et al.  investigy et al. , 55. WhiOur finding that administration of etanercept, a TNF-α-blocker, protected the inner ear from TNF-α induced structural and functional transformation is consistent with a large body of literature showing that blocking TNF-α in several contexts of SNHL can prevent subsequent SNHL –59. One in vivo and in vitro studies assessing TNF-α's ototoxic effect on the organ of Corti, animals or cochlear explants, respectively, were kept alive for several days during or after TNF-α treatment before histological processing and assessment (in vitro studies showed significant deterioration over the course of several days of incubation in TNF-α-doped culture medium, and significant differences between 2, 4, and 8 days specifically (An important limitation of the present study is the acute nature of the experimental design; specifically, hearing was assessed up to 6 h post-TNF-α perfusion, when animals were immediately sacrificed and ears extracted for histology. By contrast, in previous sessment , 53. Theifically . The acuin vivo chronic intracochlear exposure to TNF-α and assessment of hearing and cochlear morphology at several sequential time points, in addition to further exploring etanercept's ability to protect against TNF-α-induced SNHL, and rescue hearing function post-exposure to TNF-α. Given that the brainstem is also located near the cerebellopontine angle, future in vitro and in vivo brain studies should investigate the brainstem's potential vulnerability to high levels of TNF-α in VS secretions.In summary, although we did not observe any direct effects of TNF-α on CAP or DPOAE thresholds or on hair cell counts, our experiments did reveal that TNF-α specifically caused a decrease in CAP amplitudes accompanied by an increase in the number of unpaired or orphaned ribbon synapses within 6 h of exposure. This finding suggests a pathologic profile that is similar to what is observed in primary cochlear neuropathy—in the latter disease, reductions in ABR Wave I amplitudes and synaptic degeneration are observed with minimal changes in ABR Wave I and DPOAE thresholds and minimal loss of inner and outer hair cells in mice , 40 and The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Massachusetts Eye and Ear Institutional Animal Care and Use Committee.KS conceived of the project and supervised all the work. KS and MS designed experiments. MS and SK performed electrophysiological measurements. SK and RL performed confocal immunohistochemistry and synaptic counting. SK, RL, and LL performed cochlear microdissections. SK, JI, MS, RL, and KS analyzed data. JI and KS wrote the manuscript. All authors edited the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heart failure (HF) is the most rapidly growing cardiovascular health burden worldwide. HF can be classified into three groups based on the percentage of the ejection fraction (EF): heart failure with reduced EF (HFrEF), heart failure with mid-range—also called mildly reduced EF— (HFmrEF), and heart failure with preserved ejection fraction (HFpEF). HFmrEF can progress into either HFrEF or HFpEF, but its phenotype is dominated by coronary artery disease, as in HFrEF. HFrEF and HFpEF present with differences in both the development and progression of the disease secondary to changes at the cellular and molecular level. While recent medical advances have resulted in efficient and specific treatments for HFrEF, these treatments lack efficacy for HFpEF management. These differential response rates, coupled to increasing rates of HF, highlight the significant need to understand the unique pathogenesis of HFrEF and HFpEF. In this review, we summarize the differences in pathological development of HFrEF and HFpEF, focussing on disease-specific aspects of inflammation and endothelial function, cardiomyocyte hypertrophy and death, alterations in the giant spring titin, and fibrosis. We highlight the areas of difference between the two diseases with the aim of guiding research efforts for novel therapeutics in HFrEF and HFpEF.  Heart failure (HF) is the most prominent cause of hospitalization globally, with 3.6 million newly diagnosed patients annually, resulting in a socioeconomic burden of billions of euros per year . Heart fHFpEF is preceded by chronic comorbidities, such as hypertension, type 2 diabetes mellitus (T2DM), obesity, and renal insufficiency, whereas HFrEF is often preceded by the acute or chronic loss of cardiomyocytes due to ischemia, a genetic mutation, myocarditis, or valvular disease ,6. This Despite the fact that acute cardiovascular events associated with HFrEF and HFpEF share many risk factors , some coInappropriate or exaggerated inflammation is a key component underlying damage to the heart in both HFrEF and HFpEF, but has different roles in the onset and propagation. Depending on the trigger, the inflammatory response can be classified into three groups: sterile-, metabolic risk-, and non-sterile-induced inflammation .Sterile inflammation occurs due to post-ischemic or toxic necrosis, massive trauma, haemorrhage, and/or resuscitation, such as in the HFrEF condition MI  Figure . During In the meantime, the activation of adrenergic signalling alerts the bone marrow to reduce the production of hematopoietic stem cell (HSC) retention factors . ConsequWhile the inflammatory response in HFrEF is the result of cardiomyocyte damage, rather than the systemic comorbidities, inflammation in HFpEF is the result of a cluster of extra-cardiac metabolic and inflammatory risk factors, including obesity, diabetes, anaemia, hypertension, chronic obstructive pulmonary disease (COPD), auto-immune diseases, and renal insufficiency  Figure . Obese aThe “systemic microvascular paradigm” for HFpEF development proposes that low-grade chronic systemic inflammation initiates detrimental microvascular changes . In lineNon-sterile inflammation can be induced by a viral infection. Viral myocarditis (VM), for example, is characterized by a cardiac infection caused by a normally benign virus, triggering an exaggerated immune response in immune-susceptible persons. The initial acute phase is characterized by increased circulating and cardiac neutrophils and monocytes, while in later phases, increased circulating T-cells, specifically cytotoxic T-cells, and B-cells can be detected ,21. ThisIn short, regardless of the cause of HFrEF, inflammation results in cardiac remodelling driven by cardiomyocyte damage or loss, which causes cardiomyocyte autophagy, apoptosis, and necrosis ,28,29,30Endothelial cells represent the majority of non-cardiomyocyte cells (60%). Structural and/or functional abnormalities will therefore strongly impact cardiac function . EndotheIncreased oxidative stress, imbalanced nitric oxide (NO) bioavailability, or neurohormonal activation and vasoconstriction in response to reduced cardiac output (CO) are central mechanisms causing endothelial dysfunction ,112,113.In addition to an impaired NO bioavailability, there is an association between endothelial dysfunction and reduced endogenous production of estrogens after menopause . Endogenpassive) in cardiac biopsies of HFrEF patients compared to HFpEF patients I to activate cross-bridge formation between myofilament proteins. For example, patients with end-stage HFrEF have elevated myocardial [Na+]I [+]I may increase the Ca2+ influx via reverse mode NCX and limit the Ca2+ extrusion via forward mode NCX, leading to elevated levels of sub-sarcolemmal [Ca2+]I [2+ removal [2+ levels are increased in patients with HFpEF, indicating reduced Ca2+ removal, as observed in HFrEF. However, unlike HFrEF, this deficit in Ca2+ removal in HFpEF is not dependent on the Na+ gradient [Cardiac relaxation also depends on a decreased level of intracellular calcium , and increased perivascular and interstitial fibrosis. In contrast, in HFrEF, titin isoform switching is less consistent, and more flexible isoforms even present in end-stage non-ischemic dilated cardiomyopathy, therefore left ventricular stiffness ranges from being unaffected, to increased, or even reduced.Despite the fact that HFrEF and HFpEF share many risk factors and co-morbidities, there are substantial differences in systemic inflammation, cardiac remodelling , and stiffness . HFrEF, mostly occurs in male patients and is the consequence of cardiomyocyte loss. In contrast, HFpEF is often diagnosed in elderly female patients, who suffer from (a cluster of) non-cardiac comorbidities, such as hypertension, T2DM, stroke, anaemia, pulmonary disease, liver disease, sleep apnoea, gout, and cancer. HFpEF is characterized by low-grade chronic systemic inflammation and capillary dysfunction, with consequential low-grade cardiac inflammation. However, as most studies are conducted on models of established HFpEF, studies during the disease onset are required to elucidate the common denominator underlying the associated complications for HFpEF. In HFrEF, systemic and cardiac inflammation are secondary to the causes of cardiomyocyte loss. Whereas in HFpEF endothelial dysfunction mainly precedes its progression, in HFrEF endothelial dysfunction may rather be the consequence. Depending on the stimulus, perivascular (metabolic risks in HFpEF), interstitial , or replacement  fibrosis occurs and is differentially managed between the two conditions. The increased left ventricular stiffness in HFpEF is caused by reduced Ca