Biology subjects

Huang, H.-L.

Publications and source records attributed to Huang, H.-L..

7 recordsLinked to original sources

bioRxiv2026

Beyond Glycogen Storage: AMPKγ2 Regulates Cardiac Hypertrophy and Electrophysiology via Myosin Interaction

IntroductionVariants in PRKAG2 cause hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2-related hypertrophy with increased glycogen storage, many HCM phenotypes remain unexplained. We aimed to uncover how PRKAG2 variants induce myocyte hypertrophy and electrical changes during early cardiac development. MethodsWe generated transgenic zebrafish expressing wild-type (TgWT) or pathogenic variant (TgR299Q) Prkag2 cDNA under a myocardium-specific promoter, and examined cardiac electrophysiology, contractile function, and cytoarchitecture during cardiogenesis and in adult hearts. ResultsTgR299Q fish showed hypertrophic cardiomyocytes and progressive contractile abnormalities, recapitulating human HCM phenotypes. Cardiomyocyte glycogen was elevated in adult but not embryonic hearts. Despite the absence of glycogen accumulation at 6-day post-fertilization, TgR299Q hearts showed electrical abnormalities, including reduced conduction velocity and prolonged action potential and Ca2+ transient durations. We observed decreased AMPK phosphorylation in the TgR299Q hearts. However, AMPK activation did not rescue the electrophysiological abnormalities in TgR299Q. Proximity ligation assays and co-immunoprecipitation identified a physical interaction between AMPK{gamma}2 and myosin, enhanced by the R299Q variant and accompanied by increased AMPK{gamma}2 localization to the myofilament. Na/Ca{superscript 2} exchanger (NCX) inhibition increased Ca2+ duration and diastolic Ca2+ in TgWT but not TgR299Q hearts, indicating reduced free cytosolic Ca2+ for NCX-mediated extrusion in TgR299Q. These findings suggest that enhanced AMPK{gamma}2-myosin interaction may promote myofilament Ca{superscript 2} retention, thereby prolonging Ca{superscript 2} transient duration and APD in the mutant. Notably, the myosin inhibitor mavacamten reduced AMPK{gamma}2-myosin interaction in TgR299Q hearts, and both mavacamten and vmhcl knockdown rescued the early electrophysiological abnormalities. ConclusionsThe PRKAG2 variant altered cardiac excitability, contractility, and Ca2+ handling during cardiogenesis, independent of glycogen accumulation. Enhanced interactions between AMPK{gamma}2 and myosin contributed to these early changes. Our study revealed a novel link between cellular energy sensing and contractile machinery, with therapeutic potential for modulating contractile function in cardiomyopathies.

physiology↗

bioRxiv2025

Structural and Functional Mechanisms Underlying Activation Gate Dynamics and IFM Motif Accessibility in Human Nav1.5

Voltage-gated sodium channels are vital for regulating excitability in muscle and nerve cells, and their dysregulation is linked to a range of diseases. However, therapeutic targeting of Nav channels remains challenging due to a limited understanding of their gating mechanisms. Here, we present a cryo-EM structure of human Nav1.5 in an intermediate open state, stabilized by interactions between the N-terminal domain and the S6I segment. This structure reveals a previously uncharacterized Na+ binding site adjacent to the conserved inactivation (IFM) motif. Molecular dynamics simulations demonstrate that monovalent cations stably occupy this site, while electrophysiological recordings demonstrate that ion binding modulates IFM motif docking and fast inactivation kinetics. Our findings reveal that IFM accessibility is dynamically regulated in this intermediate state, challenging the canonical hinged-lid model of fast inactivation. Collectively, our study provides a revised structural framework for Nav1.5 gating mechanisms, suggesting an alternative pathway for ion accessibility that may inform better mechanistic and therapeutic strategies for treating Nav1.5-related cardiac arrhythmias.

biophysics↗

bioRxiv2022

Structural Mechanisms of Actin Isoforms

Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle changes in their design principles determine the interaction with myosin motors and actin-binding proteins. The functional diversity is further increased by posttranslational modifications (PTMs). Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal -actin (3.37[A]), cardiac -actin (3.07[A]), {beta}-actin (2.99[A]), and {gamma}-actin (3.38[A]) in the Mg2+{middle dot}ADP state with their native PTMs. The structures revealed isoform-specific conformations of the N-terminus that shifts closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Retropropagated structural changes further show that myosin binding modulates actin filament structure. Further, our structures enabled us to reveal the location of disease-causing mutations and to analyze them with respect to known locations of PTMs. Collectively, the previously unknown structures of single-isotype, posttranslationally modified bare cardiac -actin, {beta}-actin, and {gamma}-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms.

biochemistry↗

bioRxiv2022

Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration

Cardiovascular disease is the leading cause of death worldwide due to the inability of adult heart to regenerate after injury. N6-methyladenosine (m6A) methylation catalyzed by the enzyme methyltransferase-like 3 (Mettl3) plays important roles in various physiological and pathological bioprocesses. However, the role of m6A in heart regeneration remains largely unclear. To study m6A function in heart regeneration, we modulated Mettl3 expression in vitro and in vivo. Knockdown of Mettl3 significantly increased the proliferation of cardiomyocytes and accelerated heart regeneration following heart injury in neonatal and adult mice. However, Mettl3 overexpression decreased cardiomyocyte proliferation and suppressed heart regeneration in postnatal mice. Conjoint analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq identified Fgf16 as a downstream target of Mettl3-mediated m6A modification during postnatal heart regeneration. RIP-qPCR and luciferase reporter assays revealed that Mettl3 negatively regulates Fgf16 mRNA expression in an m6A-Ythdf2-dependent manner. The silencing of Fgf16 suppressed the proliferation of cardiomyocytes. However, the overexpression of {Delta}Fgf16, in which the m6A consensus sequence was mutated, significantly increased cardiomyocyte proliferation and accelerated heart regeneration in postnatal mice compared with wild-type Fgf16. Our data demonstrate that Mettl3 post-transcriptionally reduces Fgf16 mRNA levels through an m6A-Ythdf2-dependen pathway, thereby controlling cardiomyocyte proliferation and heart regeneration.

cell biology↗

bioRxiv2020

DNA extraction and sequencing of the Mawangdui ancient cadaver protected by formalin

Mawangdui ancient Cadaver is the first wet corpse found in the world, which is famous for being immortal for over two thousands of years. After being unearthed, the female corpse was immersed in the formalin protective solution for more than 40 years. We used magnetic bead method and formalin fixed paraffing (FFPE) method to extract the DNA of the female corpse, respectively. PCR amplification, sanger sequencing, library building, high throughput sequencing (testing) and data processing were carried out on the DNA samples, and about 0.5% of the whole genome coverage sequencing data was obtained. Comparing the results of DNA trough two extraction and sequencing methods. We found that the FFPE and high throughput sequencing methods is better than others for DNA extraction of the ancient samples which were preserved in formalin, providing a guidance for dealing with formalin preserved ancient samples in the future.

molecular biology↗

bioRxiv2020

Non-canonical regulation of glycogenolysis and the Warburg phenotype by soluble adenylyl cyclase

Cyclic AMP is produced in cells by two very different types of adenylyl cyclases: the canonical transmembrane adenylyl cyclases (tmACs, ADCY1[~]9) and the evolutionarily more conserved soluble adenylyl cyclase (sAC, ADCY10). While the role and regulation of tmACs is well documented, much less is known of sAC in cellular metabolism. We demonstrate here that sAC is an acute regulator of glycolysis, oxidative phosphorylation and glycogen metabolism, tuning their relative bioenergetic contributions. Suppression of sAC activity leads to aerobic glycolysis, enhanced glycogenolysis, decreased oxidative phosphorylation, and an elevated cytosolic NADH/NAD+ ratio, resembling the Warburg phenotype. Importantly, we found that glycogen metabolism is regulated in opposite directions by cAMP depending on its location of synthesis and downstream effectors. While the canonical tmAC-cAMP-PKA axis promotes glycogenolysis, we identify a novel sAC-cAMP-Epac1 axis that suppresses glycogenolysis. These data suggest that sAC is an autonomous bioenergetic sensor that suppresses aerobic glycolysis and glycogenolysis when ATP levels suffice. When the ATP level falls, diminished sAC activity induces glycogenolysis and aerobic glycolysis to maintain energy homeostasis.

biochemistry↗

bioRxiv2019

Mutant Zp1 results in Zona Pellucida Lacking and Female Infertility in Rats and Humans

Zona pellucida (ZP) plays a vital role in reproductive processes including oogenesis, fertilization and preimplantation development of embryo. The ZP of humans is composed of four glycoproteins (ZP1-ZP4), same as rats ZP. Our previous research reported a first case of human infertility due to ZP1 mutation, but the mechanism was unclear. Here we developed a genome editing in vivo rat model and a co-transfected in vitro cell model to investigate the pathogenic effect. In rat homozygous for the homologous mutation, ZP were absent in all of collected eggs. Further the growing and fully grown oocytes in the mutant ovaries completely lack a ZP but with detectable intracellular ZP1 protein. After mating with male rats, none of the mutant female rats got pregnant. Moreover, the co-transfected cell experiments and the ovarian experiments showed that the truncated ZP1 sequestered intracellularly ZP3 and ZP4 to impede their release outside, resulting in an intracellular accumulation of ZP1, ZP3 and ZP4, leading to absence of ZP in mutant oocytes. Our results clearly establish the causal role of ZP1 mutation on ZP defects and female infertility.\n\nSummary statementRat model mirrored completely the phenotypes observed in humans, infertility and abnormal eggs that lack a zona pellucida, through the negative effects of ZP1 mutation.

genetics↗