Biology subjects

Kirk, J. A.

Publications and source records attributed to Kirk, J. A..

13 recordsLinked to original sources

bioRxiv2026

Plakoglobin supports length-dependent force generation and cardiac resistance to fasting

Mechanical activity protects muscle from atrophy, but how contractile forces relate to anabolic signaling remains unclear. We previously showed that plakoglobin promotes insulin-PI3K-AKT-FoxO signaling and that its loss causes skeletal muscle atrophy. Here, super-resolution microscopy revealed that plakoglobin localizes with desmin at the sarcolemma and is lost from the membrane during fasting-induced skeletal muscle atrophy. In skinned muscle fibers, plakoglobin downregulation impaired stretch-induced increase in myofilament activation at long sarcomere length. During fasting, skeletal muscle lost sarcolemmal plakoglobin-containing complexes, insulin signaling, and mass and induced MuRF1 and MAFbx/Atrogin1. In contrast, cardiac muscle retained these complexes and anabolic signaling, and maintained its mass. Cardiac plakoglobin also resisted AAV-shJUP-mediated depletion despite viral delivery. Moreover, MEK inhibition disrupted this cardiac response, reduced AKT signaling, induced atrogenes, and decreased cardiac mass. Thus, plakoglobin supports force generation at increased sarcomere length, and preservation of plakoglobin-containing complexes and MEK-ERK signaling accompany cardiac resistance to fasting-induced atrophy.

cell biology↗

bioRxiv2026

A Novel Mechanism of Cardiomyopathy: Toxic Peptides Dysregulate Calcium Transport

A hallmark of dilated cardiomyopathy (DCM) is calcium mishandling, including reduced transport activity of the SERCA calcium pump in cardiac muscle cells. This has focused attention on SERCA as mechanism of disease and potential therapeutic target. Previously, diminished SERCA activity has been attributed to decreased protein expression, but recent studies suggest SERCA levels are unchanged in DCM. Thus, another mechanism must be responsible for the deficit. Since proteolysis is increased and proteosome function is impaired in DCM, we reasoned that accumulation of toxic protein fragments may contribute to SERCA dysfunction. In particular, previous studies showed diverse species of hydrophobic -helices can inhibit SERCA, so we hypothesized that SERCA may become congested with transmembrane peptides that mimic endogenous regulatory partners. We purified cell membranes from non-failing and DCM human ventricles and subjected them to mass spectrometry to identify protein species upregulated in DCM. Select candidates were screened for binding and inhibition of SERCA. Several small membrane proteins and membrane protein fragments bound avidly to SERCA and significantly reduced cellular calcium stores. The data suggest a novel pathophysiological mechanism in which transmembrane protein debris obstructs SERCA function and regulation, contributing to cardiac muscle dysfunction in heart failure.

physiology↗

bioRxiv2025

A novel two-component system controls vancomycin resistance in epidemic Clostridioides difficile

The glycopeptide antibiotic vancomycin is the frontline treatment for C. difficile infection in the UK. There have been only sporadic reports of resistance in the clinic but testing is rare so the true resistance landscape is unclear. We have previously shown that resistance can emerge rapidly in vitro via distinct but complementary pathways. Strain Bc2, characterised here, is an experimentally evolved derivative of C. difficile strain R20291 that displays a 16-fold increase in vancomycin MIC over its parent. Bc2 has point mutations in dacS, bclA3, CDR20291_0794, CDR20291_1871 and CDR20291_3124 (vnrS). By genetically engineering a wild-type vancomycin susceptible strain, we demonstrated that a combination of just two mutations, dacSc.798A>T and vnrSc.692G>T, both of which encode two-component system histidine kinases, was sufficient to recapitulate Bc2 resistance. We have previously shown that mutations in dacS can confer low level resistance via increases in the expression of a D,D-carboxypeptidase DacJ. dacSc.798A>T also led to increased transcription of dacJ and led to a modest increase in vancomycin MIC. Surprisingly vnrSc.692G>T led to overexpression of the vanG cluster, which encodes all of the enzymes needed for resistance via substitution of the terminal D-Ala on peptidoglycan lipid II precursors with D-Ser. Genomic analysis of a large collection of European C. difficile strains showed that a three gene cluster, which includes vnrS, vnrR (encoding the cognate response regulator) and an adjacent gene CDR20291_3123, is unique to the phylogenetic branch that contains strains belonging to epidemic ribotypes 027 and 176. Transcriptomic analysis of the wider VnrS regulon also revealed an additional previously unknown role in regulating the expression of the flagellum, an important virulence factor in C. difficile. Analysis of the response to vancomycin exposure also revealed that dacJ is one of a small set of genes that are upregulated shortly after antibiotic stress, even in the absence of mutations that typically lead to its overexpression. Together these data reveal a new synergistic route, needing only two point mutations, by which epidemic lineages of C. difficile can attain vancomycin resistance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/671617v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bba369org.highwire.dtl.DTLVardef@e562acorg.highwire.dtl.DTLVardef@1b112deorg.highwire.dtl.DTLVardef@4dcf8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Polycystin-2 is cardioprotective against myocardial infarction by regulating the calcium-mediated ER stress response.

BackgroundPatients with autosomal polycystic kidney disease (ADPKD) have an increased risk and worsened outcomes for acute myocardial infarction (AMI), but the mechanism behind this is unknown. Polycystin 2 (PC2), the protein encoded by one of the two main genes mutated in ADPKD, is a calcium-permeant channel ubiquitously expressed; however, its role in cardiomyocytes remains poorly understood. One hallmark of AMI is ER stress, which PC2 is known to regulate adaptively; however, whether PC2 regulates ER stress in the ischemic heart is unknown. ObjectiveThis study investigates the mechanism by which PC2 regulates ER stress in myocardial ischemia. Methods and ResultsPC2 protein was increased in human ischemic heart failure samples and murine myocardial infarct samples, and it was enriched at ER-mitochondrial contact sites. Induction of myocardial infarction (MI) in cardiomyocyte-specific PC2-KO mice led to cardiac dysfunction and reduced PERK expression compared to control MI mice. ER stress induced by tunicamycin in vitro blunted PERK phosphorylation and subsequent CHOP upregulation in PC2 KO cells. Tunicamycin-induced ER stress resulted in a PC2-dependent ER calcium leak and mitochondrial calcium transients, along with increased mitochondrial function, all of which were decreased in PC2 KO cells. Moreover, PC2 KO cells after ER stress exhibited decreased mitochondrial membrane potential and increased apoptosis. Isolated WT cardiomyocytes exhibited increased diastolic calcium after acute ER stress induction and increased mitochondrial uptake, neither of which was seen in PC2 KO cells. Re-expression of full-length PC2 in vitro restored both the calcium leak and PERK phosphorylation in PC2 KO cells under ER stress, but not a pathological mutant PC2 D511V, which impairs ion channel activity. ConclusionsPC2 is upregulated during ER stress, where it localizes at ER-mitochondrial contact sites and acts as an ER calcium leak channel, thereby restoring cellular homeostasis during the adaptive phase of ER stress. PC2 provides cardioprotection during ischemic events by preventing maladaptive ER stress, which contributes to cardiac dysfunction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/668747v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1e416d5org.highwire.dtl.DTLVardef@1971fd9org.highwire.dtl.DTLVardef@abeebcorg.highwire.dtl.DTLVardef@1c68f8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Titin cleavage impairs cardiac mechanical connectivity to drive diastolic failure and fibrosis

Titin, the largest human protein, serves as the elastic backbone of sarcomeres in muscle cells and imparts passive stiffness to cardiomyocytes. While proteolytic cleavage of elastic titin has been linked to cardiovascular disease, the isolated effects of titin stiffness loss in the living heart remain poorly understood. Here, we developed a knock-in mouse model allowing for the selective cleavage of cardiac titin springs in vivo. Using a time-resolved approach combining MRI, echocardiography, immunofluorescence, and molecular profiling, we demonstrate that titin cleavage does not dilate the heart but induces concentric remodeling, impaired diastolic function, and low-output heart failure. Mechanistically, compromised titin-based restoring forces trigger internal mechanical imbalance and dysconnectivity, which activate fibroblasts and promote extracellular matrix remodeling, revealing titins essential role in maintaining cardiac mechanical homeostasis.

physiology↗

bioRxiv2025

Calcium-Activated Sarcomere Contractility Drives Cardiomyocyte Maturation and the Response to External Mechanical Cues but is Dispensable for Sarcomere Formation

BackgroundUnderstanding the mechanisms of cardiomyocyte development is critical for fulfilling the potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although myocyte development is known to depend on internal and external mechanical cues, further investigation is required to understand the contributions of different signals and how they are integrated together to generate an adult cardiomyocyte. Here, we address this gap by examining the role of calcium-activated contractility in sarcomere formation and maturation and its influence on the iPSC-CM response to nanopatterns. MethodsWe generated iPSCs with homozygous D65A cardiac troponin C (cTnC) mutations. This mutation prevents calcium binding to site II of cTnC, resulting in tropomyosin blocking strong myosin binding to the thin filament and inhibiting sarcomere contraction. The iPSCs were differentiated into cardiomyocytes and matured in culture over 60 days. Cells were characterized via fluorescence imaging and calcium transient analysis. WT and mutant proteomes were examined via mass spectrometry throughout differentiation and maturation. We also replated partially matured cardiomyocytes onto nanopatterned surfaces to investigate how external mechanical signals affect maturation in contractile versus non-contractile cells. ResultsSurprisingly, we found that sarcomeres formed in the cTnC D65A cardiomyocytes, though these sarcomeres were underdeveloped and disorganized. Mutant cardiomyocytes also exhibited significant proteomic maturation defects and abnormal calcium transients. Plating D65A cardiomyocytes on nanopatterns improved structural and proteomic maturation. However, plating WT cardiomyocytes on nanopatterns led to a reduction in sarcomeric and oxidative phosphorylation protein content. ConclusionsCalcium-activated contractility is dispensable for sarcomerogenesis but critical for cardiomyocyte maturation. In non-contractile, mutant cardiomyocytes, nanopatterns enhance maturation, suggesting that external mechanical cues may partially compensate for defective contractility. However, nanopatterns did not facilitate WT maturation and may have hindered it. In addition to these novel findings, these large mass spectrometry datasets cataloging iPSC-CM maturation represent a useful resource for the cardiovascular community.

bioengineering↗

bioRxiv2023

One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse-genetics system

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of Infectious Bronchitis Virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

genetics↗

bioRxiv2022

An intact S-layer is advantageous to Clostridioides difficile within the host.

Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well known. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer revertants, following infection with the S-layer-null strain, FM2.5. Sequencing of the slpA gene revealed either correction of the original point mutation or modification of the sequence upstream of the mutation, which restored the reading frame, and translation of slpA. Selection of these strains was rapid, with up to 90% of isolates identified as revertants 24 h post infection. Two revertant isolates, RvA and RvB, showed modification of 3 and 13 amino acids respectively, compared to wild type sequence. Structural determination of SlpA from RvB revealed a different orientation of its domains, resulting in a reorganisation of the lattice assembly and changes in interacting interfaces which might result in functional differences. These revertants showed differing patterns of disease in vivo; RvA causing equivalent severity to R20291 and RvB an attenuated FM2.5-like phenotype. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates showed large changes in differentially expressed genes (DEGs) between R20291 and FM2.5 namely in TcdA/TcdB expression, in transcripts associated with sporulation and those linked to cell wall integrity, which may account for attenuation observed in vivo. In comparison, smaller differences were observed between RvA/R20291, and RvB/FM2.5 respectively, which correlated with observed disease severity in vivo. Cumulatively, these data highlight that the S-layer plays a role in C. difficile disease. Author SummaryThe S-layer of C. difficile is a paracrystalline array that covers the outer surface of the bacterial cell but its contribution to overall disease remains unclear. A previously described, spontaneous slpA-null mutant, FM2.5, with a point mutation in slpA offered an opportunity to study the role of the S-layer in vivo. Here, we confirm that this strain is less virulent in vivo despite effectively colonising the host and producing toxin. We also show in vivo selection for sequence modifications that restore slpA translation and produce an S-layer. While such modifications do not affect the overall 3D structure of individual SlpA (sub)domains, they can lead to altered orientation of the structural domains and subsequent S-layer assembly. Importantly, RNA-Seq analysis in vitro showed large differences in gene expression between FM2.5 and R20291. Detected differences in transcription of genes involved in toxin expression and sporulation suggests that the S-layer provides a selective survival advantage within the host, which contributes to disease severity.

microbiology↗

bioRxiv2022

The Clostridioides difficile S-Layer Protein A (SlpA) serves as a general phage receptor

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, but the lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but clear and unambiguous experimental evidence is lacking. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by phiCD38-2, phiCDIII and phiCD146, which normally infect the parental strain. Complementation assays with 12 different Slayer Cassette Types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by phiCD111, and SLCT-11 enabled infection by phiCD38-2 and phiCD146. Of note, expression of SLCTs 1, 6, 8, 9, 10 or 12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain, namely phiMMP02, phiMMP03, phiMMP04, phiCD506 and phiCD508. Adsorption assays showed that >50% adsorption was required for productive phage infection. Altogether, our data suggest that many phages use SlpA as their receptor and most importantly, morphologically distinct phages of the Siphoviridae and Myoviridae families target SlpA despite major differences in their tail structures. Our study therefore represents an important breakthrough in our understanding of the molecular interaction between C. difficile and its phages. IMPORTANCEPhage therapy represents an interesting alternative to treat Clostridioides difficile infections because contrary to antibiotics, most phages are highly species-specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage RBP binds to a specific SLCT, identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.

microbiology↗

bioRxiv2021

Myofilament Glycation in Diabetes Reduces Contractility by Inhibiting Tropomyosin Movement, is Rescued by cMyBPC Domains

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 M MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MGs functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosins blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosins position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF.

biophysics↗

bioRxiv2020

BAG3-dependent autophagy maintains sarcomere function in cardiomyocytes

The association between reduced myofilament force-generating capacity (Fmax) and heart failure (HF) is clear, however the underlying molecular mechanisms are poorly understood. Here, we show the Fmax decrease arises from impaired BAG3-mediated sarcomere turnover. Myofilament BAG3 decreased in human HF and predicted the extent of Fmax decrease. This relationship was confirmed using BAG3+/- mice, which had reduced Fmax and increased myofilament ubiquitination, suggesting impaired protein turnover. We show cardiac BAG3 operates via the chaperone-assisted selective autophagy complex (CASA), conserved from skeletal muscle, and confirm sarcomeric CASA localization is BAG3/proteotoxic stress-dependent. To determine if increasing BAG3 expression in HF would restore sarcomere proteostasis/Fmax, HF mice were treated with AAV9/BAG3. Gene therapy fully restored Fmax after four weeks and decreased ubiquitination. Using mass spectrometry, we identified several sarcomere proteins with increased ubiquitination in HF and four that decreased with AAV9/BAG3. Our findings indicate BAG3-mediated sarcomere turnover is required for myofilament functional maintenance.

physiology↗

bioRxiv2019

A cortex-specific PBP contributes to cephalosporin resistance in Clostridium difficile

Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridium difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We have identified and characterised a penicillin binding protein (PBP) that is required for cortex synthesis in C. difficile. Surprisingly this PBP was also found to contribute to cephalosporin resistance, indicating an additional role in the synthesis of vegetative cell wall. This is the first description of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.

microbiology↗

bioRxiv2018

Spatial organization of Clostridium difficile S-layer biogenesis

Surface layers (S-layers) are protective protein coats which form around all archaea and most bacterial cells. Clostridium difficile is a Gram-positive bacterium with an S-layer covering its peptidoglycan cell wall. The S-layer in C. difficile is constructed mainly of S-layer protein A (SlpA), which is a key virulence factor and an absolute requirement for disease. S-layer biogenesis is a complex multi-step process, disruption of which has severe consequences for the bacterium. We examined the subcellular localization of SlpA secretion and S-layer growth; observing formation of S-layer at specific sites that coincide with cell wall synthesis, while the secretion of SlpA from the cell is relatively delocalized. We conclude that this delocalized secretion of SlpA leads to a pool of precursor in the cell wall which is available to repair openings in the S-layer formed during cell growth or following damage.

microbiology↗