Biology subjects

Ayagama, T.

Publications and source records attributed to Ayagama, T..

8 recordsLinked to original sources

bioRxiv2026

Molecular and Structural Basis of Cardiac Remodelling in Niemann-Pick Type C

Key Point SummaryNiemann-Pick disease type C (NPC) patients showed a high prevalence of ECG abnormalities, with additional echocardiographic evidence of altered left-ventricular structure and function. Npc1-/- mouse hearts exhibited age-associated glycosphingolipid accumulation accompanied by marked myocardial fibrosis and increased collagen deposition. Ex vivo electrophysiology revealed QT prolongation and atrioventricular conduction defects in Npc1-/- hearts, particularly under {beta}-adrenergic stress. Transcriptomic profiling identified inflammatory and fibrotic pathway activation consistent with the structural and electrophysiological abnormalities observed. Together, these findings demonstrate previously unrecognised cardiac involvement in NPC and support routine cardiac screening to improve clinical management. Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal storage disease caused by pathogenic variants in NPC1 or NPC2. Sudden death can occur due to seizures, but cardiac involvement has not been well defined. We performed 12-lead electrocardiograms (ECG) in 14 adult NPC patients (8 male, 6 female). Cardiac structure and function were examined in Npc1-/- adult mouse hearts, alongside wild-type controls. Glycosphingolipid accumulation was quantified by high-performance liquid chromatography, fibrosis and collagen deposition were quantified using Massons Trichrome (M&T) and Picrosirius Red (PR) staining. Whole-heart morphology, including chamber size and wall thickness, was assessed. Ex vivo ECG recordings assessed conduction abnormalities and arrhythmias. RNA-seq transcriptomics characterised molecular pathways altered in Npc1-/- hearts. 8/14 patients showed ECG abnormalities including abnormal QRS transitions (N=8), increased QRS amplitude (N=4), fascicular block (N=2), and abnormal T wave inversion (N=1). 13 patients also had transthoracic echocardiograms identifying mildly impaired LV systolic function (N=2) and increased wall thickness/LV mass (N=4). In Npc1-/- mice, age-related glycosphingolipid accumulation was associated with pronounced ventricular fibrotic remodelling. There was a significant increase in stained connective tissue area and connective tissue to cardiac tissue ratio in both M&T and PR staining. ECG from Langendorff-perfused Npc1-/- hearts showed QT prolongation and atrioventricular conduction abnormalities under isoprenaline stress. Transcriptomics revealed major changes in Npc1-/- hearts, consistent with histological fibrosis and linking NPC to inflammation-driven remodelling and arrhythmogenesis. These findings support routine cardiac screening in NPC patients and highlight the need for further studies to improve management and treatment.

physiology↗

bioRxiv2026

Proteomic Signatures of Mitochondrial Dysfunction Associated with Atrial Fibrillation in Goats

Atrial fibrillation (AF) increases energy demand in atrial myocytes, yet the mitochondrial mechanisms underlying this stress remain poorly defined. Using previously published proteomic data from left atrial tissue of AF and sham-operated goats, we performed organelle-specific bioinformatic analyses of the mitochondrial fraction. Over-representation and consensus pathway analyses consistently highlighted enrichment of oxidative phosphorylation (OXPHOS) subunits. Gene set enrichment and network analyses implicated Heat Shock Protein Family A Member 9 (HSPA9) as a potentially central regulatory hub coordinating the dysregulation of Complex I and III subunits, with 69% of regulatory relationships showing pathway concordance. These results indicate a coordinated, system-wide mitochondrial adaptation in AF, integrating energy production, proteostasis, and respiratory chain regulation.

molecular biology↗

bioRxiv2024

Lysosomal signalling pathways influence heart rhythm, and regulate atrial function

In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium (Ca2+) release to augment sarcoplasmic reticulum (SR) Ca2+ sequestration, producing larger Ca2+ transients. However, the role of lysosomal Ca2+ signals in pacemaker activity, a distinct Ca2+-operated function of the sinoatrial node (SAN), or in the atrial myocardium has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits the spontaneous beating rate response to beta-adrenergic stimulation in intact SAN. We found intracellular signaling microdomains between lysosomes and neighboring SR or mitochondria in mouse, and goat tissue. The spatial relationship between lysosomes and other Ca2+-handling organelles are altered in goat atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce cAMP in response to lysosomal signaling, adding a novel trigger for cyclic nucleotide signaling. Our findings support the hypothesis that lysosomal Ca2+ signaling contributes to regulation of cardiomyocyte cAMP levels and pacemaker activity.

physiology↗

bioRxiv2024

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the IP3 signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed Fluorescence resonance energy transfer (FRET) based cytosolic cAMP sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, addition of the alpha ()-1 agonist phenylephrine (PE, 3{micro}M) resulted in a bi-phasic FRET change (R1) 21.20 {+/-} 7.43% and (R2) 9.67 {+/-} 4.23% and addition of membrane permeant IP3 derivative, 2,3,6-tri-O-Butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20M) resulted in a peak of 20.31 {+/-} 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-Aminoethyl diphenylborinate (2-APB, 2.5M) or Xestospongin-C (0.3M) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3M) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs were not inhibited by 2-APB or Xestospongin-C. Finally, the localisation of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin C. This data support further investigation of the pro-arrhythmic nature and components of IP3 induced cAMP signalling to identify potential pharmacological targets. NEW & NOTEWORTHYThis study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in-part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the pro-arrhythmic nature of IP3-induced cAMP signaling.

physiology↗

bioRxiv2023

Compartmentalisation proteomics revealed endolysosomal protein network changes in a goat model of atrial fibrillation

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL help facilitate elimination of damaged membrane and cytosolic proteins, protein aggregates, membranous organelles and also play an important role in calcium signalling. Despite the importance of EL, their specific role in cardiovascular disease is not well understood. In particular, its unclear how EL contribute to atrial pathology over longer time frames. To shed light on this question, we conducted a comprehensive analysis that involved proteomics, transcriptomics, integrated analysis, electron tomography, western blotting, and enzyme assays. To identify the role of EL in atrial fibrillation (AF), we applied a recently published organelle protein isolation method. We used this method to study biopsies from AF goat model and analyse the EL-specific proteins and pathways involved in this condition. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates (TL), including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophago-vacuole formation, irregularly shaped mitochondria, and glycogen deposition, which provide insights into the ELs contribution to AF and related pathways and molecular mechanisms. Overall, our findings suggest that EL play an important role in the development of AF over longer time frames, and provide a more detailed understanding of the underlying molecular processes involved.

molecular biology↗

bioRxiv2022

Cross-talk between cAMP and Ca2+ signalling in cardiac pacemaker cells involves IP3-evoked Ca2+ release and stimulation of adenylyl cyclase 1

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. Here we investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sinoatrial node (SAN) cells to the -adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.46 % to 8.17% (P = 0.005) in the presence of 1 M ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.20 %, ST034307 = 16.32 %; P > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transient amplitude (CaT) generated by 10M PE in the presence and absence of 1M ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; P = 0.004), but did not result in an inhibition of CaT amplitude increase in response to PE in atrial cells. The results presented here demonstrate the involvement of AC1 in the downstream response of atrial pacemaker activity to -adrenoreceptor stimulation and IP3R calcium release.

pharmacology and toxicology↗

bioRxiv2021

Cardiac Atrial Compartmentalisation Proteomics: A Modified Density Gradient Method to Analyse Endo-lysosomal Proteins

The importance of lysosomes in cardiac physiology and pathology are well established, and evidence for roles in calcium signalling are emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endo-lysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/ml); and EL with negligible contamination from SR or Mito (1.04 g/ml), were analysed using Western Blot, enzyme activity assay and LC-MS/MS analysis (adapted discontinuous Percoll, and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and dataset of guinea-pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

molecular biology↗

bioRxiv2019

Functional cyclic AMP signalling is required for the actions of IP3 on the Ca2+ transient in cardiac atria and beating rate in sino-atrial node

Inositol trisphosphate (IP3) is a major Ca2+-mobilising second messenger and atrial IP3 receptor (IP3R) expression is greatly increased in atrial fibrillation (AF). Cardiac atrial and sino-atrial node (SAN) myocytes also express Ca2+-stimulated adenylyl cyclases (AC1 and AC8); however the pathways underlying atrial AC1 and AC8 activation are unknown. We investigated whether IP3 signalling in cardiac atria and SAN utilises ACs. Immunocytochemistry in isolated guinea pig atrial myocytes identified co-localisation of type 2 IP3Rs with AC8, while AC1 was located in close vicinity. UV photorelease of IP3 significantly enhanced Ca2+ transient amplitudes following stimulation of atrial myocytes (31 {+/-} 6 % increase 60 s post photorelease, n=16), an effect abolished by inhibitors of ACs (MDL-12,330) or PKA (H89). The maximum rate change observed in spontaneously-beating murine right atrial preparations exposed to phenylephrine (14.7 {+/-} 0.5 %, n=10) was significantly reduced by 2.5 mol/L 2-APB and abolished by a low dose of MDL-12,330. These observations are consistent with a functional interaction between IP3 and cAMP signalling involving Ca2+ stimulation of ACs in cardiac atria and the SAN. Structural evidence supports AC8 as the most likely effector. This signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly AF.

physiology↗