Biology subjects

Lal, H.

Publications and source records attributed to Lal, H..

6 recordsLinked to original sources

bioRxiv2025

3D human skeletal muscle organoids reveal distinct effects of high-dose dihydronicotinamide riboside on muscle development

The evaluation of NAD+-boosting compounds in human skeletal muscle is hindered by limitations of traditional 2D cultures and animal models. Human-relevant, three-dimensional (3D) engineered skeletal muscle organoids offer a promising platform to assess the biological effects of metabolic modulation. Here we engineered 3D human skeletal muscle organoids to investigate the impact of dihydronicotinamide riboside (NRH), a potent NAD+ precursor. Sustained exposure to high NRH concentrations (500 {micro}M) enhanced early differentiation markers, including increased myotube fusion and fast-twitch fiber area, but concurrently induced structural defects such as disrupted sarcomeric organization, enlarged acetylcholine receptor clusters, and impaired acetylcholine-stimulated calcium signaling. These results reveal that excessive and sustained NAD+ elevation can uncouple rapid differentiation from proper maturation in muscle tissue. Our results highlight the importance of dose and duration optimization for NAD+-boosting compounds and establish 3D engineered muscle organoids as a valuable non-animal platform for mechanistic toxicology and preclinical safety assessment.

bioengineering↗

bioRxiv2023

PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

Adaptation to hypothermic stress is important for skeletal muscle cells, but a comprehensive knowledge of molecular mediators is lacking. We show that adaptation to mild hypothermia (320C) improves the ability of skeletal muscle myoblasts to differentiate into myotubes in vitro. We performed proteomic analysis of mouse myoblasts exposed to mild hypothermia for various time points and identified dynamic changes in mitochondrial metabolism and proteostasis. This revealed that RBM3, an RNA-binding protein, increases progressively with acute and chronic exposure to hypothermic stress, and is necessary for the enhanced differentiation upon hypothermic adaptation. We also demonstrate that overexpression of RBM3 at physiological temperatures is sufficient to (i) enhance mitochondrial metabolism as judged by a decrease in the AMPK energy-sensing pathway, (ii) increase levels of proteins associated with translation and increase levels of 4E-BP1 phosphorylation, (iii) increase stem cell markers (MyoD1, PAX7), and improve differentiation of myoblasts from both young and aged mice.

biochemistry↗

bioRxiv2021

Cardiac fibroblast GSK-3α mediates adverse myocardial fibrosis via IL-11 and ERK pathway

BackgroundHeart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK-3{beta} as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK-3 in myocardial fibrosis is unknown. Methods and ResultsHerein, we employed two entirely novel conditional fibroblast-specific and tamoxifen-inducible mouse models to define the role of CF-GSK-3 in fibroblast activation and myocardial fibrosis. Specifically, GSK-3 was deleted from cardiac fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or periostin-promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury, and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In both settings, GSK-3 deletion restricted fibrotic remodeling and improved cardiac function. To investigate underlying mechanisms, we examined the effect of GSK-3 deletion on myofibroblast transformation and pro-fibrotic TGF{beta}1-SMAD3 signaling in vitro. A significant reduction in cell migration, collagen gel contraction, and -SMA expression in TGF{beta}1 treated GSK-3 KO MEFs confirmed that GSK-3 is required for myofibroblast transformation. Surprisingly, GSK-3 deletion did not affect SMAD3 activation, indicating the pro-fibrotic role of GSK-3 is SMAD3 independent. To further delineate the underlying mechanism, total proteins were isolated from CFs of WT and KO animals at 4 weeks post-injury, and kinome profiling was performed by utilizing PamStation(R)12 high throughput microarray platform. The kinome analysis identified the downregulation of RAF family kinase activity in GSK3-KO-CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. Importantly, flow cytometric analysis of CFs confirmed a significant decrease in pERK levels in KO mice. Additionally, our in vitro studies demonstrated that GSK-3 deletion prevented TGF{beta}1 induced ERK activation thereby validating our findings from kinome analysis. Interestingly, IL-11, a fibroblast specific downstream effector of TGF{beta}1, was very low in GSK-3 KO MEFs as compared to WT and ERK inhibition further reduced IL-11 expression in them. All these results indicate that GSK-3 mediates pro-fibrotic response in the injured heart through IL-11 and ERK pathway. ConclusionCF-GSK-3 plays a causal role in myocardial fibrosis that could be therapeutically targeted for future clinical applications.

molecular biology↗

bioRxiv2020

Repurposing Nintedanib for Pathological Cardiac Remodeling and Dysfunction

BackgroundHeart Failure (HF) is the leading cause of death worldwide. Myocardial fibrosis, one of the clinical manifestations implicated in almost every form of heart disease, contributes significantly to HF development. However, there is no approved drug specifically designed to target cardiac fibrosis. Nintedanib (NTB) is an FDA approved tyrosine kinase inhibitor for idiopathic pulmonary fibrosis (IPF) and chronic fibrosing interstitial lung diseases (ILD). The favorable clinical outcome of NTB in IPF patients is well established. Furthermore, NTB is well tolerated in IPF patients irrespective of cardiovascular comorbidities. However, there is a lack of direct evidence to support the therapeutic efficacy and safety of NTB in cardiac diseases. Methods and ResultsWe examined the effects of NTB treatment on cardiac fibrosis and dysfunction using a murine model of HF. Specifically, 10 weeks old C57BL/6J male mice were subjected to Transverse Aortic Constriction (TAC) surgery. NTB was administered once daily by oral gavage (50mg/kg) till 16 weeks post-TAC. Cardiac function was monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 16 weeks post-TAC. In the control group, systolic dysfunction started developing from 4 weeks post-surgery and progressed till 16 weeks. However, NTB treatment prevented TAC-induced cardiac functional decline. In another experiment, NTB treatment was stopped at 8 weeks, and animals were followed till 16 weeks post-TAC. Surprisingly, NTBs beneficial effect on cardiac function was maintained even after treatment interruption. NTB treatment remarkably reduced cardiac fibrosis as confirmed by Massons trichome staining and decreased expression of collagen genes (COL1A1, COL3A1). Compared to TAC group, NTB treated mice showed lower HW/TL ratio and cardiomyocyte cross-sectional area. Our in vitro studies demonstrated that NTB prevents myofibroblast transformation, TGF{beta}1-induced SMAD3 phosphorylation, and production of fibrogenic proteins (Fibronectin-1). However, NTB significantly altered vital signaling pathways in both, isolated fibroblast and cardiomyocytes, suggesting that its biological effect and underlying cardiac protection mechanisms are not limited to fibroblast and fibrosis alone. ConclusionOur findings provide a proof of concept for repurposing NTB to combat adverse myocardial fibrosis and encourage the need for further validation in large animal models and subsequent clinical development for HF patients.

pathology↗

bioRxiv2019

CADHERIN-11 BLOCKADE REDUCES INFLAMMATION DRIVEN FIBROTIC REMODELING AND IMPROVES OUTCOMES AFTER MYOCARDIAL INFARCTION

BackgroundOver one million Americans experience myocardial infarction (MI) every year, and the resulting scar and subsequent cardiac fibrosis contribute to heart failure and death. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not yet been studied in the context of cardiac remodeling after MI. We hypothesized that targeting CDH11 function after MI would reduce inflammation-driven fibrotic remodeling and infarct expansion to improve functional outcomes in mice. MethodsMI was induced by ligation of the left anterior descending artery in transgenic mice with reduced or ablated CDH11, wild type mice receiving bone marrow transplants from Cdh11 transgenic animals, and wild type mice treated with a functional blocking antibody against CDH11 (SYN0012). Cardiac function was measured by echocardiography, expression of cell populations was quantified by flow cytometry, and tissue remodeling by altered histological assessment and transcription of inflammatory and pro-angiogenic genes by qPCR. Co-culture was used to assess interactions between cardiac fibroblasts and macrophages. ResultsMI increased transcription of Cdh11 in non-cardiomyocyte cells. Mice with deletion of Cdh11 and wild type mice receiving bone marrow transplants from Cdh11 transgenic animals had improved cardiac function and dimensions after MI. Animals given SYN0012 had improved cardiac function, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 also reduced the number of bone marrow-derived myeloid cells and increased pro-angiogenic cells in the heart three days after MI, consistent with a decrease in transcription and expression of IL-6 in the infarct region. Cardiac fibroblast and macrophage interactions led to an increase in IL-6 secretion that was reduced with SYN0012 treatment in vitro. ConclusionsOur findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI, and that targeting CDH11 with a blocking antibody improves cardiac function after MI. This improvement is likely mediated by altered recruitment of bone marrow-derived cells, thereby limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.

molecular biology↗

bioRxiv2019

IL-10-producing B cells are enriched in murine pericardial adipose tissues and ameliorate the outcome of acute myocardial infarction

Acute myocardial infarction (MI) provokes an inflammatory response in the heart that removes damaged tissues to facilitate repair. However, exaggerated and/or persistent inflammation compromises healing, which may be counteracted by regulatory immune mechanisms. A key regulatory factor in an inflammatory response is the anti-inflammatory cytokine IL-10, which can be produced by a number of immune cells including subsets of B lymphocytes. Here, we investigated IL-10-producing B cells in pericardial adipose tissues (PATs) and their role in the healing process following acute MI in mice. We found abundant IL-10-producing B cells in PATs under homeostatic conditions, with the majority of them bearing cell surface CD5 (CD5+ B cells). These cells were detected early in life, maintained a steady presence during adulthood, and resided in fat-associated lymphoid clusters (FALCs). The cytokine IL-33 was preferentially expressed in PATs under homeostatic conditions and contributed to enrichment of IL-10-producing CD5+ B cells in PATs. CD5+ B cells expanded in PATs following MI, and accumulated in the infarcted heart during the resolution of MI-induced inflammation. B cell-specific deletion of IL-10 worsened cardiac function after MI, exacerbated myocardial injury, and delayed resolution of inflammation. These findings reveal a significant contribution of IL-10-producing B cells to the anti-inflammatory mechanism that terminates MI-induced inflammation, and identify these cells as novel therapeutic targets to improve the outcome of MI. Significance StatementMyocardial infarction (MI) remains a leading cause of mortality and morbidity worldwide. Although it is now recognized that a balanced and timely terminated pro-inflammatory response following acute MI is essential in promoting tissue repair, the underlying regulatory mechanisms are poorly defined. In this report, we show that IL-10-producing B cells in mice 1) are enriched in pericardial adipose tissues (PATs) and influenced by cytokine IL-33 under homeostatic conditions; 2) expand in PATs following MI and accumulate in the infarcted heart during the resolution of MI-induced inflammation; and 3) facilitate resolution of inflammation and reduce myocardial injury to preserve cardiac function after MI. These findings identify IL-10-producing B cells as novel therapeutic targets to improve the outcome of MI.

immunology↗