Cellular Hypertrophy Research Articles

Abstract 14660: Truncating Variants in MYBPC3 Cause Ubiquitin Proteasome System Dysfunction Related to Hypertrophic Cardiomyopathy in Human Induced Pluripotent Stem Cell Model

Introduction: Hypertrophic cardiomyopathy (HCM) is the most common inherited monogenic cardiomyopathy. Cardiac myosin binding protein C ( MYBPC3 ) is the most frequently mutated gene in this disease, and most pathogenic variants in MYBPC3 are truncating variants including splice site variants. The ubiquitin proteasome system (UPS), a major intracellular protein degradation system, plays an important role in the heart. Although impairment of UPS is reportedly caused by truncated cMyBP-C, the underlying molecular mechanism causing HCM is largely unknown. Hypothesis: Truncating variants in MYBPC3 lead to impaired UPS activity in human iPS cell-derived cardiomyocytes (hiPSC-CMs) and are associated with HCM phenotypes. Methods: Patient derived iPSC clones were generated from HCM patients with MYBPC3 variants (IVS11-9G>A and IVS27+1G>A). Corrected isogenic control of IVS27+1G>A was generated by base editing. To evaluate UPS activity, we employed ubiquitin fusion degradation reporter, Ub-G76V-GFP, which provides fluorescence readout of UPS activity. BNP expression of hiPSC-CMs were evaluated by antibody labeling and fluorescence-activated cell sorting (FACS). Contractility and Ca 2+ transient were measured from single biopatterned hiPSC-CMs. Results: Both MYBPC3 variant lines exhibited UPS impairment as reflected by increased GFP fluorescence compared to isogenic and healthy controls (Fig. A). IVS27+1G>A showed higher expression of BNP (Fig. B and C). Enhanced contractility and Ca 2+ handling abnormalities were observed in MYBPC3 variant iPSC-CM lines as compared to control (Fig. D and E). Conclusions: Impairment of UPS activity may be associated with a marker of cellular hypertrophy, and contractility and Ca 2+ handling abnormalities in hiPSC-CMs with MYBPC3 truncating variants. These findings support prior reports suggesting that decreased UPS activity contribute to the pathophysiological mechanism of HCM caused by truncating MYBPC3 variants.

Repeated Administration of Cisplatin Transforms Kidney Fibroblasts through G2/M Arrest and Cellular Senescence.

Cisplatin is a potent chemotherapeutic used for the treatment of many types of cancer, but it has nephrotoxic side effects leading to acute kidney injury and subsequently chronic kidney disease (CKD). Previous work has focused on acute kidney tubular injury induced by cisplatin, whereas the chronic sequelae post-injury has not been well-explored. In the present study, we established a kidney fibroblast model of CKD induced by repeated administration of cisplatin (RAC) as a clinically relevant model. In NRK-49F rat kidney fibroblasts, RAC upregulated α-smooth muscle actin (α-SMA) and fibronectin proteins, suggesting that RAC induces kidney fibroblast-to-myofibroblast transformation. RAC also enhanced cell size, including the cell attachment surface area, nuclear area, and cell volume. Furthermore, RAC induced p21 expression and senescence-associated β-galactosidase activity, suggesting that kidney fibroblasts exposed to RAC develop a senescent phenotype. Inhibition of p21 reduced cellular senescence, hypertrophy, and myofibroblast transformation induced by RAC. Intriguingly, after RAC, kidney fibroblasts were arrested at the G2/M phase. Repeated treatment with paclitaxel as an inducer of G2/M arrest upregulated p21, α-SMA, and fibronectin in the kidney fibroblasts. Taken together, these data suggest that RAC transforms kidney fibroblasts into myofibroblasts through G2/M arrest and cellular senescence.

Toxicological effects of the textile industrial effluent to an Indian freshwater fish, Clarias batrachus

In this study, the histological effects of textile industrial effluent in the freshwater fish, Clarias batrachus was studied after 10, 20, and 30 days period of exposure. The 24, 48, 72, and 96 hours LC50 values were found to be 58.476, 53.231, 47.253, and 37.429% respectively. During the experiment, the treated fishes showed abnormal behaviors like erratic swimming, hyper-excitation, rapid opercular movement, and thick mucus covering which indicated the toxicity of the effluent. The observed changes in the gills were epithelial hyperplasia with lamellar fusion, necrosis, vacuolar degeneration, and atrophy of primary and secondary gill lamellae. On the other hand, the liver showed the formation of the number of vacuoles, enlargement of nuclei of some cells, degeneration of cytoplasm in hepatocytes, atrophy, a rupture in blood vessels, and disposition of hepatic cords. The kidney also showed abnormalities like degeneration of proximal and distal convoluted tubule, vacuolation of renal interstitial tissue, necrosis, cellular hypertrophy, and granular cytoplasm.

Aberrant regulation of autophagy disturbs fibrotic liver regeneration after partial hepatectomy.

Reports indicate that autophagy is essential for maintaining hepatocyte proliferative capacity during liver regeneration. However, the role of autophagy in fibrotic liver regeneration is incompletely elucidated. We investigated the deregulation of autophagic activities in liver regeneration after partial hepatectomy using a CCl4-induced fibrosis mouse model. The baseline autophagic activity was significantly increased in the fibrotic liver. After 50% partial hepatectomy (PHx), liver regeneration was remarkably decreased, accompanied by increased hepatocyte size and binuclearity ratio. Moreover, the expression of autophagy-related proteins was functionally deregulated and resulted in a reduction in the number of autophagosome and autophagosome-lysosome fusions. We further showed upregulation of autophagy activities through verapamil administration, improved hepatocyte proliferation capacity, and restricted cellular hypertrophy and binuclearity ratio. In conclusion, we demonstrated that the impairment of liver regeneration is associated with aberrant autophagy in fibrotic liver and that enhancing autophagy with verapamil may partially restore the impaired liver regeneration following PHx.

Senotherapeutics and Their Molecular Mechanism for Improving Aging

Aging is defined as physiological dysfunction of the body and a key risk factor for human diseases. During the aging process, cellular senescence occurs in response to various extrinsic and intrinsic factors such as radiation-induced DNA damage, the activation of oncogenes, and oxidative stress. These senescent cells accumulate in many tissues and exhibit diverse phenotypes, such as resistance to apoptosis, production of senescence-associated secretory phenotype, cellular flattening, and cellular hypertrophy. They also induce abnormal dysfunction of the microenvironment and damage neighboring cells, eventually causing harmful effects in the development of various chronic diseases such as diabetes, cancer, and neurodegenerative diseases. Thus, pharmacological interventions targeting senescent cells, called senotherapeutics, have been extensively studied. These senotherapeutics provide a novel strategy for extending the health span and improving age-related diseases. In this review, we discuss the current progress in understanding the molecular mechanisms of senotherapeutics and provide insights for developing senotherapeutics.

Innovative histological and histochemical characterization of tongue biopsies from patients with burning mouth syndrome.

The present pilot study aims to perform an innovative histological and histochemical characterization of samples from patients with burning mouth syndrome (BMS) to correlate these findings with the clinical scenario. To carry out this objective, the study samples were stained with the hematoxylin-eosin stain and later, an histochemical study was carried out to determine the composition of the extracellular matrix (ECM) using the stains of Alcian Blue, Picrosirius, Reticulin from Gomori and Verhoeff. The results of this study revealed histological patterns compatible with cellular hypertrophy in different layers of the epithelium as well as a greater keratinization in BMS cases. On the other hand, a lower amount of proteoglycans and a greater amount of collagen fibers were observed compared to the control. In addition, older patients had fewer reticular fibers and younger patients had fewer elastic fibers compared to the control. In conclusion, the present study shows the existence of a correlation between the histological patterns, age and symptoms of patients with BMS. Therefore, it is necessary to develop synergistic studies in order to assess and implement new classification systems that could improve the therapeutic approach of patients with BMS.

Deficiency of pituitary adenylate cyclase-activating peptide and its receptor PAC1 confers interstitial cardiac fibrosis in a murine model of metabolic stress

Abstract Background Fibrosis is a hallmark of cardiac remodeling and is present in a variety of cardiac diseases. In particular, in the highly prevalent heterogeneous group of cardiac dysfunctions with preserved left ventricular ejection fraction (LVEF), interstitial fibrosis has been identified as a pivotal pathophysiological factor. Current evidence suggests that comorbidities, mainly from the metabolic spectrum, promote chronic systemic low-grade inflammation with secondary affection of the heart, leading to fibrosis and thus impairing cardiac structure and function. In contrast to cardiac dysfunction with reduced LVEF, which is predominantly of ischemic origin, effective antifibrotic treatment options are very limited emphasizing the urgent need for new therapeutic targets. In this context, the recent report of an antifibrotic effect of the pleiotropic neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) in irradiation-induced myocardial damage [1] renders it an intriguing new candidate. Purpose The aim of this study is to elucidate the role of PACAP and of PAC1 receptor in cardiac fibrosis in a murine model of metabolic stress. Methods PACAP−/− and PAC1−/− C57/Bl6 J mice were crossbred with an ApoE−/− strain and metabolic stress was induced by feeding a cholesterol-enriched diet (CED) for 10 weeks. Standard chow (SD) served as control diet. Tissue samples of the ventricles were processed for FFPE histological analysis with hematoxylin/eosin, Giemsa and picrosirius red stains and subjected to morphometric quantification of cardiomyocyte cross diameters and fibrosis using a software-based image analysis approach. Results After 10 weeks of CED, a statistically significant higher extent of myocardial fibrosis was detected in PACAP−/−/ApoE−/− and PAC1−/−/ApoE−/− compared to ApoE−/− mice (Fig. 1). The pattern of fibrous deposition was consistent with that of reactive interstitial fibrosis and affected both ventricles uniformly. Morphometric analysis revealed no macroscopic or cellular hypertrophy in the PACAP−/−/ApoE−/− and PAC1−/−/ApoE−/− mice compared with ApoE−/− mice after CED. All morphological changes were exclusively present after feeding CED and were not detectable with SD feeding. Conclusions This study provides novel evidence for a significant involvement of PACAP and its receptor PAC1 in the development of metabolically induced cardiac fibrosis. The antifibrotic effect of PACAP, which can be inferred in this context, is mediated to a significant extent via the PAC1 receptor, and only becomes apparent under metabolic stress conditions. The absence of macroscopic and cellular cardiac hypertrophy in PACAP deficient or PAC1 deficient mice as demonstrated here precludes relevant hemodynamic effects of PACAP and PAC1 receptor, respectively. According to the results of our pilot study, PACAP analogues or PAC1 receptor agonists may offer a therapeutic potential to attenuate metabolically triggered cardiac fibrosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This research was partly supported by the Von Behring-Röntgen-Stiftung Schloss 1 D-35037 Marburg, Germany.

Acute exposure of minimally oxLDL elicits survival responses by downregulating the mediators of NLRP3 inflammasome in cultured RAW 264.7 macrophages.

Lipid burden in macrophages driven by oxidized low-density lipoprotein (oxLDL) accelerates the foam cell formation and the activation of sterile inflammatory responses aggravating the atherosclerosis. However, there is limited information on the mediators and the pathways involved in the possible survival responses, especially at the initial phase, by lipid burden in macrophage cells on encountering oxLDL. The present study was designed to assess the expression status of major mediators involved in the NLRP3 inflammasome pathway of sterile inflammation and the cellular responses in oxLDL-challenged cultured RAW 264.7 macrophage cells. OxLDL-treated RAW 264.7 macrophage cells displayed a decreased expression of the key sterile inflammatory mediators, TLR4, TLR2, ASC, NLRP3 and IL-18 at protein and transcript levels; however, they displayed increased level of IL-1β, RAGE and TREM1 at protein level. Biological responses including lipid uptake, lipid peroxidation, cellular hypertrophy, mitochondrial density and mitochondrial membrane potential were significantly increased in oxLDL-treated macrophages. Moreover, superoxide production was significantly decreased in the oxLDL-treated macrophages compared to the control. Overall, the findings revealed the expression status of key sterile mediators and the macrophage response during the initial phase of oxLDL exposure tend towards the prevention of inflammation. Further understanding would open novel translational opportunities in the management of atherosclerosis.

Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification.

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provokedthe osteocyte differentiation of osteoblast precursor cells under a three-dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self-organized scaffold-free spheroid model reconstructed by pre-chondrocyte cells. Within 2-day cultivation in the absence of the chemically induced chondrogenesis supplements, the chondrocyte marker was greatly expressed in the inner region of the spheroid, whereas the hypertrophic chondrocyte marker was strongly detected in the surface region of the spheroid. Notably, we found out that the gene expression levels of osteocyte markers were also greatly upregulated compared to the conventional 2D monolayer. Moreover, after long-term cultivation for 28 days, it induced morphological changes in the spheroid, such as cellular hypertrophy and death. In this study, in order to recapitulate the initial stage of the endochondral ossification, we highlighted the potentials of the 3D culture method to drive the hypertrophic chondrocyte differentiation of the pre-chondrocyte cells.

Abstract P2055: Secondary Structure And Antisense Oligonucleotides Regulate Cardiac Messenger RNA Translation And Modulates Cardiomyocyte Hypertrophy

Translation of upstream open reading frames (uORFs) typically abrogates translation for the main ORF (mORF) and regulates protein expression. uORFs are present in nearly 50% of messenger RNA (mRNA) transcripts, but their impact and regulation on biological processes are understudied. Mining of ribosome profiling data from next-generation sequencing analysis of human and mouse hearts reveals that most translated uORFs reside within mRNAs of transcription factors, which play essential roles in cardiomyocyte (CM) development and growth. We show that the uORF start codon synergizes with an immediate downstream double-stranded RNA (dsRNA) element to activate uORF and inhibit mORF translation. Biochemical and genetic evidence support that this mechanism is utilized by mRNAs encoding multiple transcription factors such as GATA4 (GATA binding protein 4), encoding a master transcription regulator of CM differentiation and pathological hypertrophy. Intriguingly, a trans-acting regulatory factor, DEAD-box RNA helicase DDX3X, is involved in unwinding the dsRNA structure and promoting mORF translation. We develop two types of antisense oligonucleotides (ASOs) that mimic the unwinding or stabilizing of the dsRNA secondary structure, thereby suppressing or enhancing the translation of this uORF, respectively. Genetic or chemical inactivation of this GATA4 uORF using CRISPR-Cas9 or ASOs in human embryonic stem cells lead to enhanced differentiation into CMs and cellular hypertrophy. At the organismal level, short-term treatment of isoproterenol and surgery-induced cardiac hypertrophy mouse models with uORF-enhancing ASOs reduces GATA4 mRNA translation and antagonizes cardiac hypertrophy and remodeling. As a summary, this uORF-dsRNA element is a new tunable regulator of cardiac transcription factor mRNA translation and can be targeted to alter cellular and organismal phenotypes. Moreover, mechanism-based translation-manipulating ASOs can serve as promising biotechnological tools to regulate gene expression in vitro and in vivo and can be adapted to create novel RNA-based therapeutics.

Abstract GS112: An Epigenome-wide Association Study Paired With Cell-type-specific Data Identifies Key Regulators Of Heart Failure

Background: Heart Failure (HF) is driven by the interactions of multiple genetic, epigenetic, and environmental factors both within and between the cell types of the heart. Our lab uses the Hybrid Mouse Diversity Panel (HMDP), a large cohort of inbred mouse strains, to perform systems genetics analyses of isoproterenol-induced cardiac hypertrophy and failure by leveraging the benefits of a curated model organism population to identify key drivers of phenotype. We are now expanding this study into the DNA methylome, which has been convincingly linked to cardiac-associated phenotypic changes in prior studies. Methods and Results: Using Reduced Representational Bisulfite Sequencing, we profiled left ventricular tissue samples from 88 HMDP strains subjected to isoproterenol challenge (30 mg/kg/day for 21 days) and matched control animals. We identified nearly 170,000 CpGs whose methylation status varies across the HMDP and 179 significant associations (FDR of 5%) between CpG methylation and phenotypic variation using the epigenome-wide association study algorithm MACAU, including 37 associations linking CpG methylation in unchallenged hearts to 19 post-challenge phenotypes. To identify high-confidence candidate genes, we combined our loci with data from the Wellcome Trust Mouse Genomes Resource, transcriptomic and metabolomic data from the HMDP, Hi-C data, and cell-type-specific gene expression and methylation data from healthy and failing mouse hearts. We are systematically querying the resulting 78 candidates using in silico and in vitro approaches. These genes include Mospd3 , which is associated with right ventricular hypertrophy and whose knockdown in vitro results in reduced cardiomyocyte hypertrophy and changes to hypertrophy-related gene expression. Also observed were Atp9a , whose expression levels are associated with significant global DNA methylation changes between control and ISO-treated animals and whose knockdown likewise causes a reduction in observed cellular hypertrophy, and Mdga1 , whose promoter methylation status is linked to changes in 5% of expressed genes of the heart during heart failure. Further analysis and in vivo study of these loci will further our understanding of the role of DNA methylation in heart failure.

Abstract P2011: The Role Of A Novel Alpha-crystallin B Chain Variant In Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder affecting 1 in 500 people in the general population. Characterized by asymmetric left ventricular hypertrophy, cardiomyocyte disarray, cardiac fibrosis and increased risk of sudden cardiac death, HCM is in fact a highly complex disease with heterogenous clinical presentation, onset and complication. While mutations in the sarcomere gene can account for a substantial proportion of familial cases of HCM, up to 70% of HCM patients do not carry such sarcomere variants and the causal mutations for their diseases remain elusive. Recently, we identified a novel variant of the alpha-crystallin B chain (CRYAB R123W ) in a pair of monozygotic twins who developed concordant HCM phenotypes that manifested over a nearly identical time course. Yet, how CRYAB R123W promotes HCM phenotype remains unclear. Here, we generated mice carrying the Cryab R123W -knockin allele and demonstrated that hearts from these animals exhibit increased elastance, reduced end diastolic volume and increased E/E’ consistent with increased systolic and reduced diastolic function. Upon transverse aortic constriction, mice carrying the Cryab R123W allele developed pathological left ventricular hypertrophy with substantial cardiac fibrosis and progressively reduced ejection fraction. In contrast to another well-characterized CRYAB variant (R120G) which induced Desmin aggregation, no evidence of protein aggregation was observed in hearts expressing CRYAB R123W despite its potent effect on driving cellular hypertrophy. Unexpectedly, CRYAB R123W appears to enhance calcium signaling by promoting nuclear localization of NFAT through direct interaction with calcineurin. Mice also were more susceptible to ventricular tachycardia with programmed stimulation. Single nuclei RNA-sequencing of mouse hearts is underway and preliminarily reveals reduction in oxidative phosphorylation, consistent with known effects of Cryab on mitochondrial function. Thus, our data establishes the Cryab R123W allele as a novel genetic model of HCM and unveiled additional sarcomere-independent mechanisms of cardiomyocyte hypertrophy.

Host Cytoskeleton Gene Expression Is Correlated with the Formation of Ascovirus Reproductive Viral Vesicles.

Ascoviruses are large DNA viruses that primarily infect lepidopteran larvae. They differ markedly from other plant or animal viruses by initiating replication in the nucleus, then inducing nuclear lysis followed by extensive cellular hypertrophy and subsequent cleavage of the entire enlarged cell into numerous viral vesicles. Most progeny virions are assembled in these vesicles as they circulate in the hemolymph. Here, we report transcriptome studies of host cytoskeletal genes in larvae infected with ascoviruses from 6 h to 21 days post-infection (dpi). We focused on the cabbage looper, Trichoplusia ni, infected with the Trichoplusia ni ascovirus (TnAV), along with supporting studies on the fall armyworm, Spodoptera frugiperda, infected with the Spodoptera frugiperda ascovirus (SfAV). In T. ni, many cytoskeleton genes were upregulated at 48 hours post-infection (hpi), including 29 tubulins, 21 actins, 21 dyneins, and 13 kinesins. Mitochondrial genes were upregulated as much as two-fold at 48 hpi and were expressed at levels comparable to controls in both T. ni and S. frugiperda, even after 21 dpi, when several cytoskeleton genes remained upregulated. Our studies suggest a temporal correlation between increases in the expression of certain host cytoskeletal genes and viral vesicle formation. However, these results need confirmation through functional genetic studies of proteins encoded by these genes.

Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

Histopathological aberrations and oxidative stress responses in Nile tilapia Oreochromis niloticus as influenced by dietary florfenicol and its metabolites

The intensification of aquacultural practices demands the regulated use of antibiotics to contain diseases. This study investigated the accrual and depletion of florfenicol (FFC) and its metabolite florfenicol amine (FFA) in Oreochromis niloticus juveniles following dietary administration of 15 and 45 mg FFC/kg biomass/day for 10 days, the oxidative stress responses and histopathological alterations in the kidney and liver tissues. The therapeutic dose caused no mortalities but reduced the feed intake significantly. The induction of oxidative stress in the liver and kidney tissues upon FFC-dosing was revealed by the increase in malondialdehyde, ferric reducing antioxidant power, total nitric oxide, and decrease in glutathione-S-transferase on day 10. The dietary FFC caused degeneration of renal tubular epithelium, inflammation, mineralization, hydropic swelling and necrosis in the kidney and glycogen-type vacuolation, cytoplasmic degeneration, cellular hypertrophy, and nuclear abnormalities in the liver signifying its nephrotoxic and hepatotoxic potential. The elimination of FFC was rapid in the liver and kidney but it persevered in muscle for 3 weeks. The depletion of FFA was faster in the liver and muscle, while it persevered in the kidney, signifying the probable reaction between free radicals and FFC metabolites. The withdrawal period was computed to be 6 days. Within 3 weeks of termination of dosing, the changes in biomarkers and histoarchitecture were recuperated. Though the dietary FFC induced oxidative stress and alterations in the kidney and liver histoarchitecture, our findings suggested that FFC was well tolerated by O. niloticus and is practically safe at the therapeutic dose.

AKT-mTOR signaling-mediated rescue of PRKAG2 R302Q mutant-induced familial hypertrophic cardiomyopathy by treatment with β-adrenergic receptor (β-AR) blocker metoprolol.

Protein kinase AMP-activated non-catalytic subunit gamma 2 gene (PRKAG2) cardiac syndrome, caused by mutations in PRKAG2, often shows myocardial hypertrophy and abnormal glycogen deposition in cardiomyocytes. However, it remains incurable due to a lack of a management guideline for treatment. We constructed a fluorescently labeled adenovirus carrying the wild-type or R302Q mutant of the PRKAG2 gene, infected neonatal rat cardiomyocytes (NRCMs) and H9C2 cell lines, and then analyzed changes in AMP-activated protein kinase (AMPK) activity, cell hypertrophy, glycogen storage, and cell proliferation when presence or absence of metoprolol or protein kinase A (PKA) inhibition H89, and then analyzed the changes in AKT-mTOR signal transduction activity. Overexpression of PRKAG2 R302Q in primary cardiomyocytes increased the activity of AMPK, induced cellular hypertrophy and glycogen storage, and promoted the phosphorylation levels of AKT-mTOR signaling pathway. Application of either β1-adrenergic receptor (β1-AR) blocker metoprolol or PKA inhibitor H89 to the cardiomyocytes rescued the hypertrophic cardiomyopathy (HCM)-like phenotypes induced by PRKAG2 R302Q, including suppression of both AKT-mTOR phosphorylation and AMPK activity. The current study not only determined the mechanism regulating HCM induced by PRKAG2 R302Q mutant, but also demonstrated a therapeutic strategy using β1-AR blocker to treat the patients with PRKAG2 cardiac syndrome.

Ameliorative Role of Fluconazole Against Abdominal Aortic Constriction-Induced Cardiac Hypertrophy in Rats.

Cytochrome P450 1B1 (CYP1B1) is known to be involved in the pathogenesis of several cardiovascular diseases, including cardiac hypertrophy and heart failure, through the formation of cardiotoxic metabolites named as mid-chain hydroxyeicosatetraenoic acids (HETEs). Recently, we have demonstrated that fluconazole decreases the level of mid-chain HETEs in human liver microsomes, inhibits human recombinant CYP1B1 activity, and protects against angiotensin II-induced cellular hypertrophy in H9c2 cells. Therefore, the overall purpose of this study was to elucidate the potential cardioprotective effect of fluconazole against cardiac hypertrophy induced by abdominal aortic constriction (AAC) in rats. Male Sprague-Dawley rats were randomly assigned into 4 groups such as sham control rats, fluconazole-treated (20 mg/kg daily for 4 weeks, intraperitoneal) sham rats, AAC rats, and fluconazole-treated (20 mg/kg) AAC rats. Baseline and 5 weeks post-AAC echocardiography were performed. Gene and protein expressions were measured using real-time PCR and Western blot analysis, respectively. The level of mid-chain HETEs was determined using liquid chromatography-mass spectrometry. Echocardiography results showed that fluconazole significantly prevented AAC-induced left ventricular hypertrophy because it ameliorated the AAC-mediated increase in left ventricular mass and wall measurements. In addition, fluconazole significantly prevented the AAC-mediated increase of hypertrophic markers. The antihypertrophic effect of fluconazole was associated with a significant inhibition of CYP1B1, CYP2C23, and 12-LOX and a reduction in the formation rate of mid-chain HETEs. This study demonstrates that fluconazole protects against left ventricular hypertrophy, and it highlights the potential repurposing of fluconazole as a mid-chain HETEs forming enzymes' inhibitor for the protection against cardiac hypertrophy.

Role of exchange protein directly activated by cAMP in cardiac aging

The aging of the heart is characterized by the presence of fibrosis, hypertrophy, inflammation, apoptosis, and senescence. It has been shown that an increase of cAMP in the aging heart aggravates age-related pathologies, but the players involved in this process have not been determined yet. In this sense, cAMP exerts its function through different effectors among which the Exchange protein directly activated by cAMP 1 (EPAC1) is involved in cardiac remodeling leading to heart failure making this protein a potential target to modulate cardiac aging. This project aims to determine the potential role of EPAC1 in cardiac aging and its possible mechanism of action. In rat neonatal cardiomyocytes we used EPAC's specific activator 8-CPT and inhibitor CE3F4 and evaluated Beta-galactosidase activity and DNA damage to determine senescence activation. For in vivo studies we used 6 month-old and 24 month-old WT or KO EPAC1 mice. To analyse diastolic dysfunction, we performed cardiac echography before and after an acute injection of Isoproterenol. We also evaluated cardiomyocyte cross-sectional areas by immunostaining and the presence of inflammation and senescence markers by qPCR. Finally, we used human samples of young, old patients and HFpEF patients and measured the levels of expression of EPAC1 by qPCR. We found that activating EPAC1 induced both beta-galactosidase activation and DNA damage and that both processes could be reversed by CE3F4. We also found that Doxorubicin-induced senescence was decreased by blocking EPAC1 activation. In vivo, we showed that EPAC1 protein expression was increased in old mice. In addition, we found that after Isoproterenol stress, isovolumic relaxation time (IVRT) was unchanged in old EPAC1 KO mice compared to WT. Immunostaining analysis demonstrated that cardiomyocyte hypertrophy was decreased in Old KO mice compared to WT. Interestingly, we found that senescence and inflammation markers were increased in the WT but not in the EPAC1 KO mice. Finally, mRNA expression of EPAC1 was upregulated in HFpEF patients compared to young subjects. Our findings show that EPAC1 promotes the detrimental process of cardiac aging by increasing senescence, inflammation, and cellular hypertrophy highlighting its potential as a therapeutic target for cardiovascular diseases related to aging.

The Impacts of Ultra-Slim Plus and Slim Smart on the Morphology of Kidneys and Renal Function of Albino Rats as Weight Reducing Drugs

Aims and objectives: Current study was designed to investigate the effects of two weight reducing drugs i.e. Slim Smart and Ultra-Slim Plus on kidneys and renal function. Experimental Study: This study was conducted in the Experimental Institute (Animal House) of The University of Lahore, Anatomy departments and Biochemistry department from November 2021 to May 2022. A total 25 adult male albino rat were collected and divided them into three different groups. Plain distilled water, Slim Smart and Ultra Slim plus were used in glass bottles. Under an anatomical microscope, the TA index was used to quantify interstitial fibrosis, glomerular hyalinization, vascular alterations, and tubular atrophy. Cell infiltration was computed as a percentage of inflammatory cells / HPF, and the cortical medulla ratio (C / M) was also calculated. SPSS version 20 was used to gather and analyze the raw data. One-way ANOVA was used to compare groups for quantitative variables such as renal cell infiltration. Group (mean ± standard) error was used for this analysis. Post-tacky tests were also used for cortical medullary ratios and (p< 0.05) was considered important. Results: Sections of Albino Rate kidney stained with H&E and PAS and examined histologically revealed mesangial cellular hypertrophy, thickened vascular walls, and lymphocyte infiltration. The results of group-A and group-C were significant but in group-B much inflammation was seen. A slightly significant changes (p< 0.05) were seen in serum creatinine and blood urea levels in different groups. Conclusion: Slim Smart and Ultra Slim Plus induce mesangial hyper cellularity and lymphocyte infiltration, as well as thickening of blood vessel walls. These alterations are consistent with interstitial nephritis, thus individuals with renal problems should use these medications with care. Keywords: Slim Smart, Ultra Slim Plus, Mesangial hyper cellularity, Lymphocyte infiltration, Nephrotoxicity

F-box and WD repeat-containing protein 7 ameliorates angiotensin II-induced myocardial hypertrophic injury via the mTOR-mediated autophagy pathway.

Myocardial hypertrophy is a common heart disease that is closely associated with heart failure. The expression of F-box and WD repeat-containing protein 7 (FBW7) is significantly downregulated in angiotensin (Ang) II-induced cardiac fibroblasts, suggesting that it may possess an important function in cardiac development. The present study attempted to explore the role of FBW7 in Ang II-induced myocardial hypertrophic injury and its associated mechanism of action. Reverse transcription-quantitative PCR and western blotting were used to determine the expression levels of FBW7 in Ang II-induced H9C2 cells. The expression levels of autophagy-related and mTOR signaling pathway-related proteins were detected using western blotting. Cell viability was assessed using the Cell Counting Kit-8 assay. The apoptosis rate of H9C2 cells was detected using TUNEL assay and western blotting. Cellular hypertrophy and fibrosis were assessed using phalloidin staining and western blotting. Levels of inflammatory factors were examined using ELISA and western blotting, whereas levels of oxidative stress-related markers were detected by corresponding kits. The results indicated that FBW7 expression was downregulated in Ang II-induced H9C2 cells. FBW7 upregulation enhanced the expression levels of autophagy-related proteins and activated mTOR-mediated cellular autophagy. FBW7 overexpression promoted the cell viability, inhibited Ang II-induced apoptosis, cellular hypertrophy and fibrosis in H9C2 cells via the autophagic pathway, as well as inflammation and oxidative stress. Overall, the data indicated that FBW7 overexpression ameliorated Ang II-induced hypertrophic myocardial injury via the mTOR-mediated autophagic pathway.