Intercalated disk structure, tissue heterogeneity and ion channel distribution modulate conduction and local calcium influx

The Journal of Physiology · 2026-02-24 · 1 citations

Abstract The intercalated disk (ID) is the specialized cellular structure that connects cardiomyocytes. Electrogenic proteins are known to be preferentially located at the ID, such as the voltage‐gated sodium channel (NaV1.5), inward‐rectifying potassium channel (Kir2.1), sodium‐potassium ATPase (NKA) and the L‐type calcium channel (CaV1.2). Experimental evidence shows that modifying ID properties alters conduction, and that perturbed ID structures are found in patients with cardiac arrhythmias. In our previous work, we have shown that chamber‐specific ID structures and changes in intermembrane distance lead to changes in tissue‐level conduction velocity. Here, we expand our model to include the dynamics of multiple ions within the extracellular cleft as well as representations of multiple ionic currents and gap junctions (GJs) within the ID. First, we observe that ionic fluxes at the cleft critically alter local ionic currents: Na + depletion in the cleft leads to a compensatory influx of Ca 2+ , which in turn drives a significant increase in ID calcium current. Furthermore, we find that concentrated Na + channel or GJ clusters lead to slowed conduction at the tissue level. Finally, tissue‐scale heterogeneities in ID structure lead to conduction block or spatially heterogeneous conduction velocity, suggesting a newly identified mechanism for cardiac re‐entry. Our results show that local ion channel clustering can regulate cardiac conduction. Moreover, the interplay between ion channel localization and ion concentration dynamics suggests a novel mechanism to enhance robustness of local calcium currents within the ID. image Key points Intercalated disk and extracellular cleft structure have been previously shown to modulate cardiac conduction and regulate local sodium currents. In this study, we find that cleft sodium depletion drives cleft calcium influx within the extracellular cleft space and increases local intercalated disk calcium current. Enhanced sodium channel or gap junction clustering tends to slow conduction. Tissue heterogeneity in intercalated disk disruption and channel clustering can lead to localized conduction block.

Author Reply to Peer Reviews of Astrocytic connexin43 phosphorylation contributes to seizure susceptibility after mild Traumatic Brain Injury

2025-08-02

Abstract 4368688: PERM1 Enhances Cardiac Contractility via Sarcomeric Metabolic Integration and Downregulation of MYBPC3

Circulation · 2025-11-03

Background: Heart failure with reduced ejection fraction (HFrEF) affects over 3 million adults in the United States and is associated with high morbidity and mortality, with five-year survival rates below 50%. HFrEF is characterized by impaired myocardial contractility and energy metabolism, with disrupted coupling between sarcomeric force production and energy transduction, known as mechano-energetics. Our previous study demonstrated that adeno-associated virus (AAV)-mediated gene delivery of PERM1, a striated muscle-specific regulator of mitochondrial bioenergetics, enhances cardiac contractility in mice, underscoring its therapeutic potential in HFrEF. However, the mechanisms by which PERM1 modulates myocardial contractility remain largely unknown. Hypothesis: We hypothesized that PERM1 enhances cardiac contractility via a non-canonical mechanism by acting as a signaling nexus that links metabolic regulation to sarcomeric function. Methods and Results: Bioinformatic analysis of mass spectrometry-based screening identified myosin-binding protein C3 (MYBPC3), a cardiac-specific regulator that limits actin-myosin cross-bridge formation, as a PERM1-interacting protein. Co-immunoprecipitation confirmed interactions of PERM1 with both MYBPC3 and creatine kinase B (CKB), a stress-responsive isoform essential for ATP delivery to the sarcomere. Super-resolution stochastic optical reconstruction microscopy (STORM) revealed complexing of CKB with troponin C in cardiomyocytes from AAV-PERM1-treated hearts, which was markedly reduced in PERM1-null hearts (Figure 1). Furthermore, MYBPC3 expression was significantly decreased in AAV–PERM1–treated hearts (74.2% reduction vs. AAV-GFP controls, p<0.01). Conclusions: These findings suggest that PERM1 enhances cardiac contractility by downregulating MYBPC3 to promote actin-myosin interactions and by anchoring CKB to the sarcomere to couple energy metabolism with contractile function. Collectively, our data uncover a novel role for PERM1 in regulating myocardial contractility through direct sarcomeric metabolic integration.

Optimized enrichment of murine blood–brain barrier vessels with a critical focus on network hierarchy in post-collection analysis

Scientific Reports · 2025-05-06 · 1 citations

Cerebrovascular networks contain a unique region of interconnected capillaries known as the blood-brain barrier (BBB). Positioned between upstream arteries and downstream veins, these microvessels have unique structural features, such as the absence of vascular smooth muscle cells (vSMCs) and a relatively thin basement membrane, to facilitate highly efficient yet selective exchange between the circulation and the brain interstitium. This vital role in neurological health and function has garnered significant attention from the scientific community and inspired methodology for enriching BBB capillaries. Extensive characterization of the isolates from such protocols is essential for framing the results of follow-on experiments and analyses, providing the most accurate interpretation and assignment of BBB properties. Seeking to aid in these efforts, here we visually screened output samples using fluorescent labels and found considerable reduction of non-vascular cells following density gradient centrifugation (DGC) and subsequent filtration. Comparatively, this protocol enriched brain capillaries, though larger diameter vessels associated with vSMCs could not be fully excluded. Protein analysis further underscored the enrichment of vascular markers following DGC, with filtration preserving BBB-associated markers and reducing - though not fully removing - arterial/venous contributions. Transcriptional profiling followed similar trends of DGC plus filtration generating isolates with less non-vascular and non-capillary material included. Considering vascular network hierarchy inspired a more comprehensive assessment of the material yielded from brain microvasculature isolation protocols. This approach is important for providing an accurate representation of the cerebrovascular segments being used for data collection and assigning BBB properties specifically to capillaries relative to other regions of the brain vasculature.

Cytoplasmic connexin43-microtubule interactions promote glioblastoma stem-like cell maintenance and tumorigenicity

Cell Death and Disease · 2025-05-16 · 11 citations

Glioblastoma (GBM) is the most common primary tumor of the central nervous system. One major challenge in GBM treatment is the resistance to chemotherapy and radiotherapy observed in subpopulations of cancer cells, including GBM stem-like cells (GSCs). These cells have the capacity to self-renew and differentiate and as such, GSCs participate in tumor recurrence following treatment. The gap junction protein connexin43 (Cx43) has complex roles in oncogenesis and we have previously demonstrated an association between Cx43 and GBM chemotherapy resistance. Here, we report, for the first time, increased direct interaction between non-junctional Cx43 and microtubules in the cytoplasm of GSCs. We hypothesize that non-junctional Cx43/microtubule complexing is critical for GSC maintenance and survival and sought to specifically disrupt this interaction while maintaining other Cx43 functions, such as gap junction formation. Using a Cx43 mimetic peptide of the carboxyl terminal tubulin-binding domain of Cx43 (JM2), we successfully disrupted Cx43 interaction with microtubules in GSCs. Importantly, administration of JM2 significantly decreased GSC survival in vitro, and limited GSC-derived and GBM patient-derived xenograft tumor growth in vivo. Together, these results identify JM2 as a novel peptide drug to ablate GSCs in GBM treatment.

Abstract Th0063: Targeting connexin 43 cyclin E complexes reduces neointima formation

Arteriosclerosis Thrombosis and Vascular Biology · 2025-04-01

The gap junction protein connexin 43 (Cx43) has been associated with human pathological vascular smooth muscle cell (SMC) proliferation and neointima formation. We previously identified that mitogen-activated protein kinase (MAPK) phosphorylation of Cx43 results in binding with the cell cycle protein cyclin E, facilitating neointima formation in mice. However, the specific nature of these interactions and their relevance to human disease has never been described. We aimed to define protein interaction sites for Cx43 and cyclin E and develop novel compounds to disrupt their effects on SMC. Using an ex vivo human saphenous vein model of neointima formation, we identified increased MAPK-phosphorylated Cx43 and cyclin E-Cx43 interactions in human explant tissues. We used peptide arrays to define a cyclin E-Cx43 binding region and generated ‘CycliCx’, a stearate-linked phospho-mimetic peptide. CycliCx inhibits the effects of PDGF-β treatment on human SMC, altering Cx43 trafficking and interactions with cyclin E and reducing SMC proliferation and migration. RNAseq analysis identified that CycliCx significantly inhibits PDGF-β-induced proliferative pathways in SMC and limits proliferation. CycliCx limits neointima formation in mice in vivo and in ex vivo human saphenous vein explants. Our data provide the strongest evidence to date, showing mechanistic regulation of SMC proliferation by the Cx43 protein and identifying a significant potential therapeutic strategy for preventing neointimal formation.

An inducible genetic model of chronic hypoxic signaling in cardiomyocytes precipitates severe cardiomyopathy and remodeling

bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-15

Abstract Cardiovascular disease remains the leading cause of death globally, underscoring the need for physiologically relevant models to investigate mechanisms of heart failure and arrhythmia. Chronic activation of hypoxic signaling pathways, particularly via the hypoxia-inducible factor (HIF) axis, is a key contributor to cardiac remodeling under stress. A major regulator of HIF signaling is the von Hippel-Lindau tumor suppressor (VHL) which, under normoxic conditions, targets HIFα for proteasomal degradation. Loss of VHL results in HIFα accumulation and persistent hypoxic signaling, but constitutive cardiomyocyte-specific Vhl knockout models are confounded by developmental effects and early mortality. Here, we develop and characterize an inducible, cardiomyocyte-specific Vhl knockout mouse model as a non-invasive and temporally controlled system to study chronic hypoxic stress and its contribution to cardiac remodeling and disease. VhlLoxP/LoxP ;αMHC-MerCreMer +/− mice were administered tamoxifen to induce Vhl deletion in adult cardiomyocytes. Within 5–7 days post-induction, mice displayed reduced ejection fraction, increased cardiac diameter, and elevated expression of cardiac stress markers. Transcriptomic and protein analyses revealed downregulation of key genes involved in cardiac structure and electrophysiology, including Gja1 (Cx43), Cdh2 (N-cadherin), Cacna1c (Ca V 1.2), and Kcnq1 . Importantly, these changes preceded overt cardiac remodeling, as confirmed in an abbreviated tamoxifen protocol. This inducible Vhl knockout model recapitulates hallmark features of dilated cardiomyopathy and highlights a subset of cardiac structural and ion channel genes as sensitive early responders to chronic hypoxic stress. This platform enables mechanistic dissection of disease onset and progression in ischemic heart disease and serves as a well-controlled and reproducible model for evaluating novel therapeutic strategies.

Abstract Wed061: An Inducible Genetic Model of Chronic Cardiomyocyte-specific Hypoxic Signaling Elicits Rapid Cardiac Remodeling

Circulation Research · 2025-08-01

Background: Cardiovascular disease is the leading cause of death in the United States, with a continuing need to understand molecular mechanisms of heart failure and arrhythmia. A potential method for reproducibly modeling heart disease in vivo involves genetic manipulation of the hypoxia inducible factor (HIF) pathway. During normoxia, Von Hippel-Lindau tumor suppressor (VHL, gene name Vhl ) targets HIFα for degradation. VHL loss mimics hypoxic stress through preventing HIFα degradation, leading to transcriptional activation/repression of responsive genes. While constitutive cardiomyocyte-specific Vhl knockout ( Vhl -/- ) mice display cardiac abnormalities, lack of temporal control introduces developmental effects. An inducible model would be favorable for more controlled insult to model chronic cardiac hypoxic stress in vivo in adult animals. We aimed to establish an inducible cardiomyocyte-specific Vhl -/- mouse line to model chronic cardiac hypoxic stress in vivo . Methods: Vhl-LoxP/LoxP ;αMHC-MerCreMer +/- mice were induced with tamoxifen administration over 3-5 day timecourses. Cardiac function and structure were assessed via echocardiography (n = 6-8). Cardiac transcript and protein expression were investigated through RNAseq (n = 3), RT-qPCR (n = 5-8), and western blot (n = 5-8). Results: As early as 5 days post induction, Vhl -/- mice display decreased ejection fraction and increased cardiac diameter. At day 8, Vhl -/- hearts demonstrate increased mRNA levels of cardiac stress markers and reduced mRNA levels of critical cardiac structural and electrophysiological genes including: Cdh2 (N-cadherin), Cacna1c (Ca V 1.2), and Kcnq1 (KQT member 1), together with severely reduced mRNA and protein levels of the gap junction protein connexin43. To determine whether these molecular changes precede cardiac remodeling, we harvested Vhl -/- hearts at day 3 and found altered expression of key cardiac structural and functional genes levels by RNAseq. Conclusions: We find that cardiomyocyte-specific chronic hypoxic signaling precipitates cardiac remodeling analogous to dilated cardiomyopathy. At the molecular level, expression of genes crucial for cardiac structure and electrophysiology are particularly sensitive to chronic hypoxic signaling.

Viral Infection and Connexin Dysfunction in the Heart

Current Cardiology Reports · 2025-03-27 · 2 citations

PURPOSE OF REVIEW: Gap junctions, comprising connexin proteins, enable the direct intercellular electrical coupling of cardiomyocytes, and disruption of this process is arrhythmogenic. In addition, gap junctions effect metabolic coupling and of relevance to this review, propagate host antiviral immune responses. Accordingly, connexins have emerged as viral targets during infection. This review summarizes current knowledge regarding contributions of inflammation vs virally encoded factors in driving alterations to cardiac gap junction function. RECENT FINDINGS: In addition to host immune-mediated effects on cardiac electrophysiology and gap junctions in myocarditis, there is now increasing appreciation for virally encoded factors targeting connexin function in acute/active infection. We now know diverse viral species have independently evolved to directly target connexin function during infection. Understanding both the direct and indirect effects of viral infection on cardiac gap junctions is critical to inform treatment strategies and development of novel therapeutics for acute infection as a distinct disease process from chronic myocarditis.

Novel Pannexin 1 isoform is increased in cancer

bioRxiv (Cold Spring Harbor Laboratory) · 2024-09-09

ABSTRACT Pannexin 1 (PANX1) is upregulated in many cancers, where its channel activity and signalling promote tumorigenic properties. Here, we report that potential internal translation start sites exist in mouse and human PANX1 which have implications in trafficking and protein interaction. Using mouse PANX1 constructs for each internal methionine (M) we saw that the shorter PANX1 isoforms were glycosylated, able to traffic to the cell surface and PANX1-M37 formed channels which could be activated by C-terminus cleavage or α1-adrenoceptor stimulation. Furthermore, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer, and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, and using Expasy PeptideCutter we did not find any evidence of hPANX1 cleavage sites which would produce a 25 kDa fragment, suggesting a potential alternative translation initiation site as the source of hPANX1-25K. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at multiple sites including the canonical N255 and novel N338 and N394 residues, and can interact with both β-catenin and full length hPANX1. Using cell surface biotinylation and immunocytochemistry, we also determined hPANX1-25K exhibits a predominantly intracellular localization. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and patient-matched normal skin, indicating that it may be differentially regulated in normal and cancer cells.