Pathological Remodeling Research Articles

Cardiac contractility modulation: an effective treatment strategy for heart failure beyond reduced left ventricular ejection fraction?

Heart failure (HF) with preserved ejection fraction (HFpEF) causes a progressive limitation of functional capacity, poor quality of life (QoL) and increased mortality, yet unlike HF with reduced ejection fraction (HFrEF) there are no effective device-based therapies. Both HFrEF and HFpEF are associated with dysregulations in myocardial cellular calcium homeostasis and modifications in calcium-handling proteins, leading to abnormal myocardial contractility and pathological remodelling. Cardiac contractility modulation (CCM) therapy, based on a pacemaker-like implanted device, applies extracellular electrical stimulation to myocytes during the absolute refractory period of the action potential, which leads to an increase in cytosolic peak calcium concentrations and thereby the force of isometric contraction promoting positive inotropism. Subgroup analysis of CCM trials in HFrEF has demonstrated particular benefits in patients with LVEF between 35% and 45%, suggesting its potential effectiveness also in patients with higher LVEF values. Available evidence on CCM in HFpEF is still preliminary, but improvements in terms of symptoms and QoL have been observed. Future large, dedicated, prospective studies are needed to evaluate the safety and efficacy of this therapy in patients with HFpEF.

Cardiac pericytes mediate the remodeling response to myocardial infarction.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-β signaling. Deleting TGF-β receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Advanced myocardial deformation echocardiography for the athlete's heart evaluation: Functional and mechanistic analysis

Echocardiographic assessment of the athlete's heart remains challenging because of a phenotypic overlap between reactive physiological adaptation and pathological remodelling. The lower range of normal values of left ventricle systolic ejection fraction (LVEF) and global longitudinal strain (GLS) in athletes makes it difficult to differentiate changes related to adaptive remodelling or indicative of early cardiomyopathy. This study sought to identify echocardiographic phenotype of endurance athletes using 2D speckle tracking imaging with a multi-layer approach and to define predictive factors of subtle LV systolic dysfunction. Healthy male athletes who underwent a pre-participation medical evaluation at the University Hospital of Nancy between 2013 and 2020 were included. Clinical and echocardiographic data were compared with healthy men from the STANISLAS cohort. Subtle LV dysfunction was defined as a GLS < 17.5%. A total of 191 athletes and 161 control subjects were studied. Athletes demonstrated lower LVEF (57.9% vs. 62.6%; P < 0.01) and lower GLS (17.8% vs. 21.1%; P < 0.01). No significant differences were found between athletes with and without subtle LV dysfunction regarding clinical characteristics, structural echocardiographic features, and exercise capacity. Athletes with subtle LV dysfunction exhibited a lower endocardial GLS (18.8% vs. 22.7%; P < 0.02), a lower epicardial GLS (14.0% vs. 16.6%; P < 0.01) and a greater endocardial/epicardial GLS ratio (1.36 vs. 1.32; P < 0.01). No significant difference was found regarding mechanical dispersion (P = 0.46). The endurance athlete's heart is characterized by a specific myocardial deformation pattern with a greater endocardial/epicardial GLS ratio. Subtle LV dysfunction seems mainly driven by a decreased epicardial GLS and not related to exercise capacity, structural remodelling or mechanical dispersion (Fig. 1).

Autonomic control of ventricular function in health and disease: current state of the art.

Cardiac autonomic dysfunction is one of the main pillars of cardiovascular pathophysiology. The purpose of this review is to provide an overview of the current state of the art on the pathological remodeling that occurs within the autonomic nervous system with cardiac injury and available neuromodulatory therapies for autonomic dysfunction in heart failure. Data from peer-reviewed publications on autonomic function in health and after cardiac injury are reviewed. The role of and evidence behind various neuromodulatory therapies both in preclinical investigation and in-use in clinical practice are summarized. A harmonic interplay between the heart and the autonomic nervous system exists at multiple levels of the neuraxis. This interplay becomes disrupted in the setting of cardiovascular disease, resulting in pathological changes at multiple levels, from subcellular cardiac signaling of neurotransmitters to extra-cardiac, extra-thoracic remodeling. The subsequent detrimental cycle of sympathovagal imbalance, characterized by sympathoexcitation and parasympathetic withdrawal, predisposes to ventricular arrhythmias, progression of heart failure, and cardiac mortality. Knowledge on the etiology and pathophysiology of this condition has increased exponentially over the past few decades, resulting in a number of different neuromodulatory approaches. However, significant knowledge gaps in both sympathetic and parasympathetic interactions and causal factors that mediate progressive sympathoexcitation and parasympathetic dysfunction remain. Although our understanding of autonomic imbalance in cardiovascular diseases has significantly increased, specific, pivotal mediators of this imbalance and the recognition and implementation of available autonomic parameters and neuromodulatory therapies are still lagging.

The Chemistry and Biology of Collagen Hybridization.

Collagen provides mechanical and biological support for virtually all human tissues in the extracellular matrix (ECM). Its defining molecular structure, the triple-helix, could be damaged and denatured in disease and injuries. To probe collagen damage, the concept of collagen hybridization has been proposed, revised, and validated through a series of investigations reported as early as 1973: a collagen-mimicking peptide strand may form a hybrid triple-helix with the denatured chains of natural collagen but not the intact triple-helical collagen proteins, enabling assessment of proteolytic degradation or mechanical disruption to collagen within a tissue-of-interest. Here we describe the concept and development of collagen hybridization, summarize the decades of chemical investigations on rules underlying the collagen triple-helix folding, and discuss the growing biomedical evidence on collagen denaturation as a previously overlooked ECM signature for an array of conditions involving pathological tissue remodeling and mechanical injuries. Finally, we propose a series of emerging questions regarding the chemical and biological nature of collagen denaturation and highlight the diagnostic and therapeutic opportunities from its targeting.

FGF21-FGFR1 controls mitochondrial homeostasis in cardiomyocytes by modulating the degradation of OPA1

Fibroblast growth factor 21 (FGF21) is a pleiotropic hormone secreted primarily by the liver and is considered a major regulator of energy homeostasis. Recent research has revealed that FGF21 could play an important role in cardiac pathological remodeling effects and prevention of cardiomyopathy; however, the underlying mechanism remains largely unknown. This study aimed to determine the mechanism underlying the cardioprotective effects of FGF21. We engineered FGF21 knock out mice and subsequently elucidated the effects of FGF21 and its downstream mediators using western blotting, qRT-PCR, and mitochondrial morphological and functional analyses. FGF21 knockout mice showed cardiac dysfunction, accompanied by a decline in global longitudinal strain (GLS) and ejection fraction (EF), independent of metabolic disorders. Mitochondrial quality, quantity, and function were abnormal, accompanied by decreased levels of optic atrophy-1 (OPA1) in FGF21 KO mice. In contrast to FGF21 knockout, cardiac-specific overexpression of FGF21 alleviated the cardiac dysfunction caused by FGF21 deficiency. In an in vitro study, FGF21 siRNA deteriorated mitochondrial dynamics and impaired function induced by cobalt chloride (CoCl2). Both recombinant FGF21 and adenovirus-mediated FGF21 overexpression could alleviate CoCl2-induced mitochondrial impairment by restoring mitochondrial dynamics. FGF21 was essential for maintaining mitochondrial dynamics and function of the cardiomyocytes. As a regulator of cardiomyocyte mitochondrial homeostasis under oxidative stress, FGF21 could be an important new target for therapeutic options for patients with heart failure.

Effects of norepinephrine‑induced activation of rat vascular adventitial fibroblasts on proliferation and migration of BMSCs involved in vascular remodeling.

Vascular remodeling caused by vascular injury such as hypertension and atherosclerosis is a complex process involving a variety of cells and factors, and the mechanism is unclear. A vascular injury model was simulated by adding norepinephrine (NE) to culture medium of vascular adventitial fibroblasts (AFs). NE induced activation and proliferation of AFs. To investigate the association between the AFs activation and bone marrow mesenchymal stem cells (BMSCs) differentiation in vascular remodeling. BMSCs were cultured with supernatant of the AFs culture medium. BMSC differentiation and migration were observed by immunostaining and Transwell assay, respectively, while cell proliferation was measured using the Cell Counting Kit-8. Expression levels of smooth muscle actin (α-SMA), TGF-β1 and SMAD3 were measured using western blot assay. The results indicated that compared with those in the control group, in which BMSCs were cultured in normal medium, expression levels of α-SMA, TGF-β1 and SMAD3 in BMSCs cultured in medium supplemented with supernatant of AFs, increased significantly (all P<0.05). Activated AFs induced the differentiation of BMSCs into vascular smooth muscle-like cells and promoted proliferation and migration. AFs activated by NE may induce BMSCs to participate in vascular remodeling. These findings may help design and develop new approaches and therapeutic strategies for vascular injury to prevent pathological remodeling.

Pathological ventricular remodelling and purinergic signaling: Emerging roles of the ATP-gated P2X4 receptor

Pathological ventricular remodelling is an abnormal healing process following myocardial infarction (MI), which mechanisms remain debated. Several hypotheses have been proposed to explain the links between inflammatory response secondary to MI and the evolution towards pathological remodelling, including the involvement of ATP and its cellular receptors: the purinergic receptors. Our working hypothesis is that there is a maintenance/resurgence of a tissue inflammatory response in MI patients with pathological remodelling that would hinder the healing process and may be associated with a specific purinergic profile. The objective of the project is to study the impact of ischemia-reperfusion on cardiac fibroblast's’ profiles and to identify an associated purinergic signature. First, we studied the effect of ischaemia (5 h) and reperfusion (24, 48 h) on human primary cardiac fibroblasts (HCF) phenotype by studying the expression of markers associated with the activated state: periostin and the differentiated state: smooth muscle alpha-actin (αSMA) by Western blot. Simultaneously, the expression level of P2 purinergic receptors (P2R) was quantified by RT-qPCR and WB over time. In a second step, we chose to investigate the involvement of P2X4 in the activation and differentiation of cardiac fibroblasts. Therefore, we blocked its activity during reperfusion with its specific antagonist 5-BDBD (5 μM). In addition, wound healing studies were conducted with and without P2X4's antagonist to investigate its involvement in the migratory capacity of cardiac fibroblasts. Ischaemia-reperfusion (IR) appears to induce an initial increase in periostin expression at 24 h followed by a decrease at 48 h as compared to normoxia. This may be related to a more important secretion of periostin at 48 h, as suggested by its increase in supernatants. Concerning αSMA, IR induces a decrease in its expression in a sustained manner over time. Regarding P2X4, there is a decrease in its mRNA and protein levels. The use of its antagonist in normoxia and IR decreases αSMA expression but does not affect periostin expression. Similarly, its blockade decreases the basal migratory capacity of fibroblasts. IR seems to prevent fibroblast differentiation by maintaining them into an activated state. P2X4 could potentially be involved by decreasing fibroblast differentiation, thus impacting αSMA expression. Yet, other purinergic receptors whose expression levels vary following MI might also be involved.

PE-454609-1 MYOCARDIAL WORK IN VENTRICULAR PREEXCITATION

Ventricular preexcitation alters the electromechanical activation sequence and may induce left ventricular (LV) contractile discoordination and pathologic remodelling in selected cases. To evaluate LV myocardial work before and after accessory pathway (AP) ablation in patients with WPW preexcitation and to compare them to normal controls. Mechanical activation mapping and myocardial work calculation was performed in 11 pediatric patients with the WPW syndrome/pattern and left sided or paraseptal APs undergoing catheter ablation (successful in 10/11) and 8 healthy age-matched controls using speckle tracking echocardiography and the GE ECHOPAC version 204.57.0.745 software with the integrated AFI module. An 18-segmental echocardiographic LV model was used. Global LV myocardial work efficiency (GWE) was calculated as the ratio of global constructive work to the sum of global constructive and global wasted work (GWW). The time from delta wave onset to R wave peak in lead V6 (delta to R) was used to quantify the degree of preexcitation. Before ablation GWW was higher (164 vs 102 mmHg%, P=0.015) and GWE lower (median 91 vs 94%, P=0.010) in patients vs controls. After successful ablation GWW decreased from 164 to 93 mmHg% (P=0.006) and GWE increased from 91.1 to 94.0 % (P=0.006). GWE tended to correlate negatively with the delta to R interval (R=0.59, P=0.056). Before ablation, segmental WE increased with distance from ventricular AP insertion from median 69.5 % at AP location to 98.0 % in distant segments (P<0.001). After ablation, WE was equally distributed among LV segments (Figure). Mechanical activation mapping localized APs with a median (IQR) segmental distance of 0 (0-1) segments from the EP mapped location. Ventricular preexcitation induces significant LV myocardial work inefficiency in segments adjacent to AP which correlates with the degree of preexcitation and is normalized after ablation. The amount of wasted work in asymptomatic preexcitation may be considered when assessing the potential for pathologic LV remodelling or discussing the indication for prophylactic ablation. Mechanical activation mapping may be used to localize manifest APs.

Role of mitochondrial dynamics in abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is an asymptomatic pathology representing 75% of aortic aneurysms and occurring mainly in men over 65 years old, with a high mortality rate (80%) due to aortic rupture. Currently, only surgical management are used, and no pharmacological treatment exists. A potential therapeutic track would be the regulation of mitochondrial functions, already involved in neurological, ophthalmic, and cardiac pathologies. However, mitochondrial dynamics regulation, especially important within smooth muscle cells, is still untapped in abdominal aortic aneurysm. Our aim is to understand the role of mitochondrial dynamics in abdominal aortic aneurysm. We used a murine model of 6 months old of hypertensive Ldlr-/- male mice (angiotensin II, 1 mg/kg/day) associated with an hypercholesterolemic diet (HCD) for 28 days. Moreover, 6 months old Opa1+/- hypertensive male mice were used to study aortic remodeling. Aortic samples were studied by histomorphometric analyzes, RT-qPCR and Western blot. Ldlr-/- mice suffering from an abdominal aortic aneurysm showed a significant increase in mitochondrial fission (FIS1, DRP1) and mitophagy (PARKIN, PINK1). In addition, our results demonstrated strong pathological adventitial remodeling in supra-renal abdominal aorta in Opa1+/- hypertensive mice with a significant increase of Mmp2/Timp2 ratio in gene expression involved in aneurysm remodeling. Those results were associated with a significant decrease in smooth muscle cell contraction gene expression, inducing phenotypic cell switch, and pathological arterial remodeling. Mitochondrial dynamics dysfunction associated with hypertensive stress are involved in abdominal aortic aneurysm development.

Indoxyl-sulfate activation of the AhR-NFκB pathway promotes IL-6 secretion and subsequent osteogenic differentiation in human valvular interstitial cells

Calcific aortic valve disease (CAVD) results from fibro-calcific degeneration of the aortic valve leaflets, causing major cardiovascular complications. In chronic kidney disease (CKD) patients, this pathological remodelling is more frequent, appears earlier and progresses faster leading to a mortality rate three times higher than in the general population. The uremic toxin Indoxyl-sulfate (IS), a powerful predictor of cardiovascular mortality in CKD patients, is a strong promoter of ectopic calcification, which role in CAVD has never been studied. This work aimed to evaluate whether IS influences primary human valvular interstitial cells (hVICs) mineralization in-vitro. Primary hVICs isolated from aortic tricuspid valves collected from CAVD patients were exposed to increasing concentrations of IS (IS normal [Isn]: 0.5 Mg/mL, IS uremic [Isu]: 37 Mg/mL or IS maximum [Ismax]: 233 Mg/mL) in the presence of an osteogenic medium (OM) containing 2.4 mmol/L calcium and 2.5 mmol/L phosphate. HVICs osteogenic transition was assessed by qRT-PCR following Bmp2 and Runx2 mRNA. Their mineralization was quantified by the o-cresolphthalein method. Inflammation was assessed following NFκB activation by western blot and IL-1β, IL-6 and TNF-α secretion by ELISA. A siRNA approach allowed determining the signaling pathway involved. IS increased OM-induced hVICs osteogenic transition and calcification concentration-dependently, an effect blocked after transfection with siRNA targeting IS receptor AhR. Exposure to [ISu] or [ISmax] promoted P65 phosphorylation, the blockade of which inhibited IS-induced mineralization, suggesting a key role for inflammation in this process. HVICs did not secrete TNF-α, produced few IL-1β but secreted high amounts of IL-6 in response to IS, which was blocked by silencing AhR or P65. The use of an antibody neutralizing IL-6 abolished IS-induced procalcific effects. IS promotes hVICs mineralization through AhR-dependent activation of NFκB pathway and subsequent release of IL-6. The fact that IL-6 neutralization blocks IS procalcific effects is of particular interest since interventional studies evaluating the cardiovascular impact of anti-IL-6 antibodies in CKD are underway.

Exercise stress cardiac magnetic resonance imaging in the assessment of induced cardiovascular responses in cardiac patients: a scoping review protocol.

This scoping review will describe cardiac magnetic resonance imaging testing protocols used in combination with exercise (Ex-CMR) to assess cardiovascular responses. The review will document the advantages and limitations of these protocols in cardiac patients. Ex-CMR characterizes the heart, differentiating between normal and pathological cardiac remodeling with considerable accuracy. However, there is no review detailing existing Ex-CMR protocols. This is particularly important since not all Ex-CMR protocols seem to induce enough stress to effectively characterize cardiac remodeling, hence the need for a review to report on the current evidence. This review will consider studies that use Ex-CMR testing protocols to assess cardiovascular responses, revealing cardiac remodeling in patients whose age at the time of the study was ≥ 18 years. The review will be conducted in accordance with the JBI methodology for scoping reviews and reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR). The following databases will be searched: PubMed, Embase, ISI Web of Science, OpenGrey, Grey Matters, and OAlster. Articles in English and French will be included and there will be no limitation set for the date of publication. Data will be extracted from papers included in the scoping review by 2 independent reviewers and will be classified in summary tables. Open Science Framework https://osf.io/hvn75/?view_only=f6cf8fc2112e498d89c39639dbce70d1 .

Pressure overload induces ISG15 to facilitate adverse ventricular remodeling and promote heart failure.

Inflammation promotes adverse ventricular remodeling, a common antecedent of heart failure. Here, we set out to determine how inflammatory cells affect cardiomyocytes in the remodeling heart. Pathogenic cardiac macrophages induced an IFN response in cardiomyocytes, characterized by upregulation of the ubiquitin-like protein IFN-stimulated gene 15 (ISG15), which posttranslationally modifies its targets through a process termed ISGylation. Cardiac ISG15 is controlled by type I IFN signaling, and ISG15 or ISGylation is upregulated in mice with transverse aortic constriction or infused with angiotensin II; rats with uninephrectomy and DOCA-salt, or pulmonary artery banding; cardiomyocytes exposed to IFNs or CD4+ T cell-conditioned medium; and ventricular tissue of humans with nonischemic cardiomyopathy. By nanoscale liquid chromatography-tandem mass spectrometry, we identified the myofibrillar protein filamin-C as an ISGylation target. ISG15 deficiency preserved cardiac function in mice with transverse aortic constriction and led to improved recovery of mouse hearts ex vivo. Metabolomics revealed that ISG15 regulates cardiac amino acid metabolism, whereas ISG15 deficiency prevented misfolded filamin-C accumulation and induced cardiomyocyte autophagy. In sum, ISG15 upregulation is a feature of pathological ventricular remodeling, and protein ISGylation is an inflammation-induced posttranslational modification that may contribute to heart failure development by altering cardiomyocyte protein turnover.

T-Cell Mineralocorticoid Receptor Deficiency Attenuates Pathologic Ventricular Remodelling After Myocardial Infarction.

Mineralocorticoid receptor (MR) antagonists have been widely used to treat heart failure (HF). Studies have shown that MR in T cells plays important roles in hypertension and myocardial hypertrophy. However, the function of T-cell MR in myocardial infarction (MI) has not been elucidated. In this study, we used T-cell MR knockout (TMRKO) mouse to investigate the effects of T-cell MR deficiency on MI and to explore the underlying mechanisms. Echocardiography and tissue staining were used to assess cardiac function, fibrosis, and myocardial apoptosis after MI. Flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect immune cell filtration and inflammation. T-cell MR deficiency significantly improved cardiac function, promoted myocardial repair, and inhibited myocardial apoptosis, fibrosis, and inflammation after MI. Luminex assays revealed that TMRKO mice had significantly lower levels of interferon-gamma (IFN-γ) and interleukin-6 (IL-6) in serum and infarcted myocardium than littermate control mice. In cultured splenic T cells, MR deficiency suppressed IL-6 expression, whereas MR overexpression enhanced IL-6 expression. Chromatin immunoprecipitation (ChIP) assay demonstrated that MR bound to the MR response element on the promoter of IL-6 gene. Finally, T-cell MR deficiency significantly suppressed accumulation of macrophages in infarcted myocardium and differentiation of proinflammatory macrophages, thereby alleviating the consequences of MI. T-cell MR deficiency improved pathologic ventricular remodelling after MI, likely through inhibition of accumulation and differentiation of proinflammatory macrophages. At the molecular level, MR may work through IFN-γ and IL-6 in T cells to exert functions in MI.

Sex, age and menopause differentially impact gut barrier function and proteomic profile

Cardiovascular disease (CVD) and metabolic syndrome (MetS) depends on sex, age and hormonal status and is worsened by comprised gut barrier function. Previous work establishes that loss of estrogen and age comprises intestinal barrier function permeability exacerbating systemic inflammation, increasing gut oxidative stress, and elevating pro-inflammatory cytokines. We hypothesize that loss of estrogen during menopause underlies sex- and age-dependent pathological remodeling in gut barrier function leading to increased risk for CVD and MetS. C57BL-6 male and female mice were fed a high-fat-diet (HFD, 45% fat); menopause was induced via 21 days of 4-vinylcycohexane diepoxide (VCD) intraperitoneal injections (controls were injected with sesame oil) in adult and aged female mice. Diestrus was reached 14+days after the end of VCD injections and menopause was determined by cytology. Upon sacrifice, plasma was collected, and intestines were flash frozen. LPS assays were performed on plasma samples of premenopausal (n=8), menopausal (n=10), aged menopausal (n=6), and male mice (n=11). Male mice, and aged menopausal mice had significantly higher circulating endotoxin levels than premenopausal mice, while menopausal mice did not significantly differ from pre-menopause. These results indicate that age and sex impact barrier leakiness. Further, these results highlight that barrier integrity in menopause is distinctly different from aged menopause mice.Quantitative proteomics of ileum samples in premenopausal (n=5), menopausal (n=5), aged menopausal (n=5), and male mice (n=5) identified 7664 proteins with &gt;1 unique peptide of which 134 proteins were significant (p&lt;0.05) with a max fold change ˂1.27 across all groups. Protein expression of all groups was assessed using principal component analysis. Aged menopausal mice cluster closer to premenopausal mice than menopausal mice. These results indicate that changes in the ileum during menopause are different than those observed in aging. Compared to premenopausal mice, menopausal mice have significantly increased in proteins involved in the Go-biological processes including an enrichment in proteins involved in the innate immune system (6.3 fold enrichment, FE, -log10p=0.00006), phagocytosis recognition (19.82 FE, -log10p=1.14), defense response to bacterium (10.066 FE, -log10p=9.56), and B cell receptor signaling pathways (20.311 FE, -log10p=9.33). In contrast, male mice compared to premenopausal mice show a significant decrease in proteins involved in phagocytosis recognition (22.57 FE, -log10p=4.28), defense response to bacterium (12.898 FE, -log10p=2.76), and B cell receptor signaling pathway (20.56 FE, -log10p=8.12). These results indicate an increase in immune activity in menopausal female mice and a decrease in male mice compared to premenopausal female mice. In summary, these data demonstrate the change within the gut during menopause is dissimilar to those seen in male and aging mice. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elevated bile acids are associated with left ventricular structural changes in biliary atresia.

In children with biliary atresia (BA), pathologic structural changes within the heart, which define cirrhotic cardiomyopathy, are associated with adverse perioperative outcomes. Despite their clinical relevance, little is known about the pathogenesis and triggers of pathologic remodeling. Bile acid excess causes cardiomyopathy in experimental cirrhosis, but its role in BA is poorly understood. Echocardiographic parameters of left ventricular (LV) geometry [LV mass (LVM), LVM indexed to height, left atrial volume indexed to BSA (LAVI), and LV internal diameter (LVID)] were correlated with circulating serum bile acid concentrations in 40 children (52% female) with BA listed for transplantation. A receiver-operating characteristic curve was generated to determine optimal threshold values of bile acids to detect pathologic changes in LV geometry using Youden index. Paraffin-embedded human heart tissue was separately analyzed by immunohistochemistry for the presence of bile acid-sensing Takeda G-protein-coupled membrane receptor type 5. In the cohort, 52% (21/40) of children had abnormal LV geometry; the optimal bile acid concentration to detect this abnormality with 70% sensitivity and 64% specificity was 152µmol/L (C-statistics=0.68). Children with bile acid concentrations >152µmol/L had ∼8-fold increased odds of detecting abnormalities in LVM, LVM index, left atrial volume index, and LV internal diameter. Serum bile acids positively correlated with LVM, LVM index, and LV internal diameter. Separately, Takeda G-protein-coupled membrane receptor type 5 protein was detected in myocardial vasculature and cardiomyocytes on immunohistochemistry. This association highlights the unique role of bile acids as one of the targetable potential triggers for myocardial structural changes in BA.

BS-452758-1 SUPPRESSION-REPLACEMENT GENE THERAPY REVERSE REMODELS THE INTRACELLULAR CALCIUM HANDLING DISEASE PHENOTYPE OF TNNT2-MEDIATED HYPERTROPHIC CARDIOMYOPATHY

Hypertrophic cardiomyopathy (HCM), characterized by otherwise unexplained left ventricular hypertrophy, has a prevalence of 1:500. Pathogenic variants in TNNT2-encoded cardiac troponin T causes a thin filament subtype of sarcomeric HCM. A key cellular feature of the pathological remodeling in TNNT2-mediated HCM involves dysregulation of intracellular calcium handling in ventricular cardiomyocytes (CMs). To develop a TNNT2 “suppression-replacement (SupRep)” gene therapy to reverse the intracellular calcium perturbations associated with thin filament, sarcomeric HCM using induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) derived from a patient with TNNT2-R288P-mediated HCM. Custom-designed TNNT2 targeting shRNAs were tested for knockdown (KD) efficiency using TSA201 cells and RT-qPCR. A dual-component SupRep TNNT2 gene therapy was created by cloning into a single construct a custom-designed TNNT2 shRNA that produced greatest KD (suppression) and a “shRNA-immune” (shIMM) TNNT2 cDNA (replacement). Patient-specific TNNT2-R288P and CRISPR/Cas9 variant-corrected isogenic control (IC) iPSC-CMs were generated from a patient with HCM. The intracellular calcium indicator, Fluo-4, was used to detect intracellular free calcium. Our lead TNNT2-shRNA had an 80% KD efficiency of TNNT2. TNNT2-R288P iPSC-CMs treated with TNNT2-SupRep showed a similar KD efficiency. Compared to the IC iPSC-CMs, the TNNT2-R288P iPSC-CMs showed increased irregular events (30.4% vs. 10.9%, p<0.0001), prolonged calcium transient duration 90 (CTD90, 1.01 ± 0.03 s vs. 0.89 ± 0.02 s, p<0.0001) and prolonged peak to 90% decay time (0.57 ± 0.02 s vs. 0.49 ± 0.01 s, p=0.0012). Compared to untreated cells, transfection with TNNT2-SupRep gene therapy, the TNNT2-R288P iPSC-CMs showed a decrease in irregular events (30.4% vs. 14%, p<0.0001), shortened CTD90 (1.01 ± 0.03 s vs. 0.91 ± 0.01 s, p=0.003), and shortened peak to 90% decay time (0.57 ± 0.02 s vs. 0.50 ± 0.02 s, p=0.02). There was no significant difference in irregular events, CTD90, or peak to 90% decay time between TNNT2-R288P iPSC-CMs treated with TNNT2-SupRep and IC iPSC-CMs. Here, we provide the first proof-of-principle SupRep gene therapy for TNNT2-mediated HCM. TNNT2-SupRep gene therapy almost fully restored the impaired intracellular calcium handling properties of TNNT2-R288P patient-derived iPSC-CMs back to the normal state achieved by the CRISPR/Cas9 gene-edited cure.

Raman microspectroscopy identifies fibrotic tissues in collagen-related disorders via deconvoluted collagen type I spectra.

Fibrosis is a consequence of the pathological remodeling of extracellular matrix (ECM) structures in the connective tissue of an organ. It is often caused by chronic inflammation, which over time, progressively leads to an excess deposition of collagen type I (COL I) that replaces healthy tissue structures, in many cases leaving a stiff scar. Increasing fibrosis can lead to organ failure and death; therefore, developing methods that potentially allow real-time monitoring of early onset or progression of fibrosis are highly valuable. In this study, the ECM structures of diseased and healthy human tissue from multiple organs were investigated for the presence of fibrosis using routine histology and marker-independent Raman microspectroscopy and Raman imaging. Spectral deconvolution of COL I Raman spectra allowed the discrimination of fibrotic and non-fibrotic COL I fibers. Statistically significant differences were identified in the amide I region of the spectral subpeak at 1608cm-1, which was deemed to be representative for structural changes in COL I fibers in all examined fibrotic tissues. Raman spectroscopy-based methods in combination with this newly discovered spectroscopic biomarker potentially offer a diagnostic approach to non-invasively track and monitor the progression of fibrosis. STATEMENT OF SIGNIFICANCE: Current diagnosis of fibrosis still relies on histopathological examination with invasive biopsy procedures. Although, several non-invasive imaging techniques such as positron emission tomography, single-photon emission computed tomography and second harmonic generation are gradually employed in preclinical or clinical studies, these techniques are limited in spatial resolution and the morphological interpretation highly relies on individual experience and knowledge. In this study, we propose a non-destructive technique, Raman microspectroscopy, to discriminate fibrotic changes of collagen type I based on a molecular biomarker. The changes of the secondary structure of collagen type I can be identified by spectral deconvolution, which potentially can provide an automatic diagnosis for fibrotic tissues in the clinical applicaion.

Targeting Fibrosis to Improve Cancer Outcomes

Abstract Scleroderma (SSc) is an idiopathic autoimmune connective tissue disease. The three hallmarks of SSc include 1) vasculopathy resulting in 2) progressive fibrosis or thickening of the skin, lungs, and internal organs and 3) aberrant immune infiltration. Fibrotic pulmonary involvement is the leading cause of death in SSc patients. Recent data from John Hopkins Scleroderma Center have shown a significantly increased risk of lung cancer in SSc patients. However, the mechanisms linking the two diseases are not well understood. Moreover, due to the close temporal relationship between SSc and lung cancer diagnosis, it is unclear whether lung fibrosis precedes tumorigenesis or conversely lung tumorigenesis precedes fibrosis. It has become evident that certain fibrotic diseases can progress towards cancer. Seemingly, non-alcoholic steatohepatitis or HBV/HCV infection-induced liver cirrhosis can progress to hepatocellular carcinoma. Vice versa, many solid tumors are fibrotic, and desmoplasia is referred to the pathological remodeling observed in the tumor microenvironment. There seems to be a tight link between fibrosis and cancer although the directionality of this progression is unknown. Moreover, several fibrotic processes involving the ß-catenin/Wnt, YAP/TAZ and hedgehog pathways in addition to fibrogenic growth factors (TGFß, VEGF, FGF) have been known to drive cancer. We are developing a murine model to study the mechanisms driving fibrosis to cancer progression in the lungs.

Homeobox A9 is a novel mediator of vascular smooth muscle cell phenotypic switching and proliferation by regulating methyl-CpG binding protein 2

Aberrant proliferation and phenotypic switching of vascular smooth muscle cells (VSMCs) are considered to be the main pathological processes of atherosclerotic plaque formation. Methyl-CpG binding protein 2 (MECP2) affects cell differentiation via modulating VSMC-specific gene expression and acts as a driver for the development of atherosclerosis (AS). Here, we aimed to elucidate the role of homeobox A9 (HOXA9) on aberrant VSMCs upon injury or AS, and whether HOXA9-mediated VSMC injury was associated with MECP2. Adeno-associated virus serotype 8-mediated knockdown of HOXA9 rescued aortic pathological injury of apolipoprotein E-deficient (ApoE−/−) mice fed a high-fat diet (HFD), characterized by the reduction of lipid accumulation and foam cell formation. Further in vitro evidence suggested that proliferation and migration of primary mouse VSMCs (mVSMCs) stimulated by oxidized low-density lipoprotein (ox-LDL) were inhibited after HOXA9 silencing. In addition, HOXA9 silencing blocked VSMC phenotypic switching from contractile to a pathological synthetic state. HOXA9 overexpression caused opposite alterations in ox-LDL-stimulated mVSMCs. Mechanistically, MECP2 was transcriptionally activated by HOXA9. Forced expression of MECP2 impaired the anti-proliferation, anti-migration, and phenotypic switching abilities of HOXA9 silencing in VSMCs upon ox-LDL stimulation. Collectively, our findings reveal that the role of HOXA9 in pathological vascular remodeling may attribute to transcriptional regulation of MECP2.