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Mutations in PRPH2 are a relatively common cause of sight-robbing inherited retinal degenerations (IRDs). Peripherin-2 (PRPH2) is a photoreceptor-specific tetraspanin protein that structures the disk rim membranes of rod and cone outer segment (OS) organelles, and is required for OS morphogenesis. PRPH2 is noteworthy for its broad spectrum of disease phenotypes; both inter- and intra-familial heterogeneity have been widely observed and this variability in disease expression and penetrance confounds efforts to understand genotype-phenotype correlations and pathophysiology. Here we report the generation and initial characterization of a gene-edited animal model for PRPH2 disease associated with a nonsense mutation (c.1095:C>A, p.Y285X), which is predicted to truncate the peripherin-2 C-terminal domain. Young (P21) Prph2Y285X/WT mice developed near-normal photoreceptor numbers; however, OS membrane architecture was disrupted, OS protein levels were reduced, and in vivo and ex vivo electroretinography (ERG) analyses found that rod and cone photoreceptor function were each severely reduced. Interestingly, ERG studies also revealed that rod-mediated downstream signaling (b-waves) were functionally compensated in the young animals. This resiliency in retinal function was retained at P90, by which time substantial IRD-related photoreceptor loss had occurred. Altogether, the current studies validate a new mouse model for investigating PRPH2 disease pathophysiology, and demonstrate that rod and cone photoreceptor function and structure are each directly and substantially impaired by the Y285X mutation. They also reveal that Prph2 mutations can induce a functional compensation that resembles homeostatic plasticity, which can stabilize rod-derived signaling, and potentially dampen retinal dysfunction during some PRPH2-associated IRDs., (© The Author(s) 2024. Published by Oxford University Press.)

Synapse-associated protein 97 (SAP97) is a scaffolding protein crucial for the functional expression of several cardiac ion channels and therefore proper cardiac excitability. Alterations in the functional expression of SAP97 can modify the ionic currents underlying the cardiac action potential and consequently confer susceptibility for arrhythmogenesis. In this study, we generated a murine model for inducible, cardiac-targeted Sap97 ablation to investigate arrhythmia susceptibility and the underlying molecular mechanisms. Furthermore, we sought to identify human SAP97 ( DLG1 ) variants that were associated with inherited arrhythmogenic disease. The murine model of cardiac-specific Sap97 ablation demonstrated several ECG abnormalities, pronounced action potential prolongation subject to high incidence of arrhythmogenic afterdepolarizations and notable alterations in the activity of the main cardiac ion channels. However, no DLG1 mutations were found in 40 unrelated cases of genetically elusive long QT syndrome (LQTS). Instead, we provide the first evidence implicating a gain of function in human DLG1 mutation resulting in an increase in Kv4.3 current ( I to ) as a novel, potentially pathogenic substrate for Brugada syndrome (BrS). In conclusion, DLG1 joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. Dysfunction in these critical components of cardiac excitability can potentially result in fatal cardiac disease. NEW & NOTEWORTHY The gene encoding SAP97 ( DLG1 ) joins a growing list of genes encoding ion channel-interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1- encoded SAP97's candidacy as a minor Brugada syndrome susceptibility gene.

Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchain-forming P/rds generated abundant high-curvature OS membranes, which were improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions., Competing Interests: The authors declare no competing interest.

Peripherin-2/rds is required for biogenesis of vertebrate photoreceptor outer segment organelles. Its localization at the high-curvature rim domains of outer segment disk membranes suggests that it may act to shape these structures; however, the molecular function of this protein is not yet resolved. Here, we apply biochemical, biophysical, and imaging techniques to elucidate the role(s) played by the protein's intrinsically disordered C-terminal domain and an incipient amphipathic α-helix contained within it. We investigated a deletion mutant lacking only this α-helix in stable cell lines and Xenopus laevis photoreceptors. We also studied a soluble form of the full-length ∼7-kDa cytoplasmic C terminus in cultured cells and purified from Escherichia coli The α-helical motif was not required for protein biosynthesis, tetrameric subunit assembly, tetramer polymerization, localization at disk rims, interaction with GARP2, or the generation of membrane curvature. Interestingly, however, loss of the helical motif up-regulated membrane curvature generation in cellulo , introducing the possibility that it may regulate this activity in photoreceptors. Furthermore, the incipient α-helix (within the purified soluble C terminus) partitioned into membranes only when its acidic residues were neutralized by protonation. This suggests that within the context of full-length peripherin-2/rds, partitioning would most likely occur at a bilayer interfacial region, potentially adjacent to the protein's transmembrane domains. In sum, this study significantly strengthens the evidence that peripherin-2/rds functions directly to shape the high-curvature rim domains of the outer segment disk and suggests that the protein's C terminus may modulate membrane curvature-generating activity present in other protein domains., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims: Pulmonary vein ganglia (PVG) are targets for atrial fibrillation ablation. However, the functional relevance of PVG to the normal heart rhythm remains unclear. Our aim was to investigate whether PVG can modulate sinoatrial node (SAN) function., Methods and Results: Forty-nine C57BL and seven Connexin40+/EGFP mice were studied. We used tyrosine-hydroxylase (TH) and choline-acetyltransferase immunofluorescence labelling to characterize adrenergic and cholinergic neural elements. PVG projected postganglionic nerves to the SAN, which entered the SAN as an extensive, mesh-like neural network. PVG neurones were adrenergic, cholinergic, and biphenotypic. Histochemical characterization of two human embryonic hearts showed similarities between mouse and human neuroanatomy: direct neural communications between PVG and SAN. In Langendorff perfused mouse hearts, PVG were stimulated using 200-2000 ms trains of pulses (300 μs, 400 µA, 200 Hz). PVG stimulation caused an initial heart rate (HR) slowing (36 ± 9%) followed by acceleration. PVG stimulation in the presence of propranolol caused HR slowing (43 ± 13%) that was sustained over 20 beats. PVG stimulation with atropine progressively increased HR. Time-course effects were enhanced with 1000 and 2000 ms trains (P < 0.05 vs. 200 ms). In optical mapping, PVG stimulation shifted the origin of SAN discharges. In five paroxysmal AF patients undergoing pulmonary vein ablation, application of radiofrequency energy to the PVG area during sinus rhythm produced a decrease in HR similar to that observed in isolated mouse hearts., Conclusion: PVG have functional and anatomical biphenotypic characteristics. They can have significant effects on the electrophysiological control of the SAN.

Background: Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse., Objective: To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs., Methods: Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data., Results: APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs., Conclusions: Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs., (Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Histone H3 lysine 4 (H3K4me) methyltransferases and their cofactors are essential for embryonic development and the establishment of gene expression patterns in a cell-specific and heritable manner. However, the importance of such epigenetic marks in maintaining gene expression in adults and in initiating human disease is unclear. Here, we addressed this question using a mouse model in which we could inducibly ablate PAX interacting (with transcription-activation domain) protein 1 (PTIP), a key component of the H3K4me complex, in cardiac cells. Reducing H3K4me3 marks in differentiated cardiomyocytes was sufficient to alter gene expression profiles. One gene regulated by H3K4me3 was Kv channel-interacting protein 2 (Kcnip2), which regulates a cardiac repolarization current that is downregulated in heart failure and functions in arrhythmogenesis. This regulation led to a decreased sodium current and action potential upstroke velocity and significantly prolonged action potential duration (APD). The prolonged APD augmented intracellular calcium and in vivo systolic heart function. Treatment with isoproterenol and caffeine in this mouse model resulted in the generation of premature ventricular beats, a harbinger of lethal ventricular arrhythmias. These results suggest that the maintenance of H3K4me3 marks is necessary for the stability of a transcriptional program in differentiated cells and point to an essential function for H3K4me3 epigenetic marks in cellular homeostasis.

Cardiac calsequestrin (CSQ) is synthesized on rough endoplasmic reticulum (ER), but concentrates within the junctional sarcoplasmic reticulum (SR) lumen where it becomes part of the Ca(2+)-release protein complex. To investigate CSQ trafficking through biosynthetic/secretory compartments of adult cardiomyocytes, CSQ-DsRed was overexpressed in cultured cells and examined using confocal fluorescence microscopy. By 48h of adenovirus treatment, CSQ-DsRed fluorescence had specifically accumulated in perinuclear cisternae, where it co-localized with markers of rough ER. From rough ER, CSQ-DsRed appeared to traffic directly to junctional SR along a transverse (Z-line) pathway along which sec 23-positive (ER-exit) sites were enriched. In contrast to DsRed direct fluorescence that presumably reflected DsRed tetramer formation, both anti-DsRed and anti-CSQ immunofluorescence did not detect the perinuclear CSQ-DsRed protein, but labeled only junctional SR puncta. These putative CSQ-DsRed monomers, but not the fluorescent tetramers, were observed to traffic anterogradely over the course of a 48h overexpression from rough ER towards the cell periphery. We propose a new model of CSQ and junctional SR protein traffic in the adult cardiomyocyte, wherein CSQ traffics from perinuclear cisternae, along contiguous ER/SR lumens in cardiomyocytes as a mobile monomer, but is retained in junctional SR as a polymer., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: Inherited arrhythmias can be caused by mutations in the cardiac ryanodine receptor (RyR2). The cellular source of these arrhythmias is unknown. Isolated RyR2(R4496C) mouse ventricular myocytes display arrhythmogenic activity related to spontaneous Ca(2+) release during diastole. On the other hand, recent whole-heart epicardial and endocardial optical mapping data demonstrate that ventricular arrhythmias in the RyR2(R4496C) mouse model of catecholaminergic polymorphic ventricular tachycardia (CPVT) originate in the His-Purkinje system, suggesting that Purkinje cells, and not ventricular myocytes, may be the cellular source of arrhythmogenic activity. The relative effect of the RyR2(R4496C) mutation on calcium homeostasis in ventricular myocytes versus Purkinje cells is unknown., Objective: This study sought to determine which cardiac cell type is more severely affected, in terms of calcium handling, by expression of the RyR2(R4496C) mutant channel: the ventricular myocytes or the Purkinje cells., Methods and Results: To discriminate Purkinje cells from ventricular myocytes, we crossed the RyR2(R4496C) mouse model of CPVT with the Cx40(EGFP/+) transgenic mouse. This genetic cross yields Purkinje cells that express eGFP, and therefore fluoresce green when excited by the appropriate wavelength; ventricular myocytes, which do not express connexin 40, are not green. Intracellular calcium was measured in each cell type using calcium-sensitive probes. Purkinje cells of the RyR2(R4496C) mouse model of CPVT show an approximately 2x greater rate (P < .05) and approximately 2x to 3x greater amplitude (P < .000001) of spontaneous calcium release events than ventricular myocytes isolated from the same heart., Conclusion: These results demonstrate that focally activated arrhythmias originate in the specialized electrical conducting cells of the His-Purkinje system in the RyR2(R4496C) mouse model of CPVT., (Copyright 2010 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle calsequestrin (skelCSQ) and cardiac calsequestrin (cardCSQ) are resident proteins of the ER/SR, but mechanisms by which CSQ is retained inside membrane lumens remain speculative. A structural model that predicts linear CSQ polymers has been developed that might explain CSQ concentration and localization inside junctional SR lumens, however little evidence exists for polymer formation in intact cells or for its effects on subcellular localization. We previously showed that cardCSQ is efficiently retained within the ER, but its retention is lost under conditions expected to disrupt its polymerization. In the present study, we found unexpectedly that skelCSQ shows no co-localization with cardCSQ in COS cells or in rat neonatal heart cells, but instead concentrates in a membrane compartment (ERGIC) that is just distal to that of cardCSQ. Consistent with this difference in immunofluorescent localization, the structures of CSQ ((316)Asn-linked) glycans showed two types of pre-Golgi processing. Despite the difference in subcellular distribution of individual wild-type forms of CSQ, however, pairs of different CSQ molecules (for example, different isoforms or different fluorescent fusion proteins) consistently co-localized, suggesting that separate forms of CSQ polymerize in different parts of the same secretory pathway, while different CSQ pairs localize together through heteropolymerization.

In junctional sarcoplasmic reticulum, binding to cardiac triadin-1 provides a mechanism by which the Ca(2+)-release channel/ryanodine receptor may link with calsequestrin to regulate Ca(2+) release. Calsequestrin and triadin-1 both contain N-linked glycans, but about half of triadin-1 in the heart remains unglycosylated. To investigate mechanisms for this incomplete glycosylation, we overexpressed triadin-1 as a series of glycoform variants in non-muscle cell lines and neonatal heart cells using plasmid and adenoviral vectors. We showed that the characteristic incomplete glycosylation stemmed from properties of the glycosylation sequence that are conserved among triadin splice variants, including the close proximity of Asn(75) to the sarcoplasmic reticulum inner membrane. Although triadin-1 appeared by SDS-PAGE analysis as a 35/40-kDa doublet in all cells, variations occurred in the relative levels of the two glycoforms depending on the cell type and whether overexpression involved a plasmid or adenoviral vector. Treatment of triadin-1 with the proteasome inhibitor MG-132 led to striking changes in the relative levels of triadin-1 that indicated active breakdown of unglycosylated, but not glycosylated, triadin-1. Besides substantial increases in the relative levels of unglycosylated triadin-1, proteasome inhibition led to an accumulation of two new modified forms of triadin-1 that were seen with triadin-1 only when it is not glycosylated on Asn(75). Effects of tunicamycin and endoglycosidase H confirmed that these novel isoforms represent two alternative N-linked glycosylation sites, indicating that an alternative topology occurs infrequently leading to yet other glycoforms with short half-lives.

The mitogen-activated protein kinase (MAPk) signaling pathway, which plays a critical role in the proliferation of mammalian cells, is frequently up-regulated in human tumors and may contribute to the transformed phenotype. Since a major limitation of current cancer chemotherapy is prevalent resistance to cytotoxic drugs, this study determined whether alterations in growth factor signaling through MAPk may contribute to this phenomenon in human neuroblastoma cell lines. Drug-resistant SKNSH cell lines were established by long-term incubation with increasing concentrations to 10(-6) M doxorubicin (SKNSH rDOX6) or MDL 28842 (SKNSH rMDL6). The expression of epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF)-induced EGFR tyrosine phosphorylation were lower in drug-resistant SKNSH cells than their wild-type counterparts. In SKNSH rDOX6 cells, decreased activation and reduced nuclear translocation of MAPk in response to EGF, or lysophosphatidic acid (LPA), or phorbol 12-myristate 13-acetate (PMA), were observed. In SKNSH rMDL6 cells, although MAPk could be activated to wild-type levels by ligand stimulation, the translocation of active MAPk to the nucleus was also reduced. These results suggest that resistance to cytotoxic drugs in human neuroblastoma cell lines is associated with a decrease in growth factor signaling through the MAPk pathway.