Metabolic-associated steatohepatitis (MASH) involves hepatocyte damage that cannot be explained solely by lipid accumulation. Here, to discover injury-specific pathways, we focused on a gene of uncertain function, EF-Hand Domain Family Member D1 (EFHD1), identified in human genome-wide association studies of liver injury but not liver fat. We show that EFHD1, a Ca2+-dependent actin crosslinker, stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS), detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release. During MASH, EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence. This structural failure promotes mitochondrial double-stranded RNA escape and activation of a maladaptive antiviral PKR-dependent stress response, a causal relationship also supported by Mendelian randomization in humans. Consequently, inhibiting EFHD1 in human and mouse models blunts hepatocyte damage. These findings identify EFHD1 as a Ca2+-dependent ERMCS stabilizer, reveal a hepatocyte-intrinsic injury pathway, and suggest EFHD1 inhibition as a therapeutic strategy.

Metabolic-associated steatohepatitis (MASH) involves hepatocyte damage that cannot be explained solely by lipid accumulation. Here, to discover injury-specific pathways, we focused on a gene of uncertain function, EF-Hand Domain Family Member D1 (EFHD1), identified in human genome-wide association studies of liver injury but not liver fat. We show that EFHD1, a Ca2+-dependent actin crosslinker, stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS), detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release. During MASH, EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence. This structural failure promotes mitochondrial double-stranded RNA escape and activation of a maladaptive antiviral PKR-associated stress response, a causal relationship also supported by Mendelian randomization in humans. Consequently, inhibiting EFHD1 in human and mouse models blunts hepatocyte damage. These findings identify EFHD1 as a Ca2+-dependent ERMCS stabilizer, reveal a hepatocyte-intrinsic injury pathway, and suggest EFHD1 inhibition as a therapeutic strategy.

Background: Arrhythmogenic cardiomyopathy (ACM) arises from mutations in desmosomal genes, such as desmoglein (DSG) and desmoplakin (DSP), which converge into a common pathophysiological phenotype at the cellular level, whose hallmarks include disruption of Connexin43 (Cx43) trafficking and membrane localization and suppression of the Wnt/β-catenin signaling pathway. These cellular changes in turn contribute to cardiomyocyte loss, fibrofatty infiltration, and decreased cellular coupling, leading to heart failure, arrhythmias, and sudden cardiac death. GJA1-20k, an internally translated isoform of Cx43, has previously shown therapeutic potential in a DSG model of ACM by preserving Cx43 trafficking and cell-cell coupling. Objective: To investigate the role of GJA1-20k in protecting against a DSP model of ACM by evaluating its effect on Cx43 trafficking and Wnt/β-catenin signaling. Methods: A DSP mutant mouse model of ACM ( Dsp -/- ) received retroorbital injections of AAV9 vectors expressing either GJA1-20k-GFP or GST-GFP at 1×10 12 vg/kg. Echocardiographic measurements were recorded at 4-week intervals until the endpoint was reached, wherein hearts were excised and underwent further processing for histological and biochemical assays. Mechanistic pathways identified were validated in vitro using cell lines. Results: Dsp -/- mice that received GST developed heart failure associated with pathological fibrosis and cardiac remodeling. However, GJA1-20k treated Dsp -/- mice had preserved heart function and absent fibrotic infiltration. At the cardiomyocyte level, GST-treated Dsp -/- mice had significantly reduced Cx43 localization to the intercalated discs and reduced intercalated disc and nuclear localization of β-catenin compared to control mice. This was accompanied by an increase in β-catenin phosphorylation and degradation. However, upon GJA1-20k administration, Dsp -/- mice had restored Cx43 trafficking, increased β-catenin nuclear translocation and activity, and reduced β-catenin degradation. Conclusion: GJA1-20k offers therapeutic potential against ACM by preventing pathological changes in Cx43 localization and Wnt/β-catenin signaling. Moreover, this work introduces the novel concept of next generation gene therapy, whereby targeting common pathological cellular phenotypes downstream of the causative mutation serves as a viable low dose therapeutic approach to diseases of different genetic origin such as ACM.

Introduction: GJA1-20k, an N-terminus truncated isoform of the gap junction protein Connexin 43, is stress responsive and mimics ischemia preconditioning, reducing ischemia-reperfusion (IR) injury. GJA1-20k localizes to the outer membrane of mitochondria, stabilizes actin, and mediates an actin mediated mitochondrial protection. The mechanism is not known how GJA1-20k and actin work towards mitochondrial protection. Methods: Cell-free TIRF imaging was performed to study direct interactions between actin and purified GJA1-20k. High-resolution confocal microscopy was applied to visualize GJA1-20k and actin organization around mitochondria in intact HEK cells and cell-free suspensions wherein rhodamine-labeled actin, purified GJA1-20k, and isolated mitochondria were incubated together. Results: Cell-free TIRF imaging establishes that GJA1-20k directly binds to actin , forming both actin clusters and stabilized actin filaments. As expected, live-cell imaging reveals that GJA1-20k is enriched at the mitochondrial outer membrane. The direct binding of GJA1-20k and actin results in a rich collection of actin around mitochondria, as evidenced in both live-cell imaging and cell-free suspensions containing only purified actin, GJA1-20k, and isolated mitochondria. 3D reconstruction reveals that mitochondrial localization of GJA1-20k causes formation of dense actin sheets enveloping mitochondria, which we call “mitochondrial actin cages” which appear to limit the ability of mitochondria to swell under stress conditions. Conclusions: GJA1-20k induced actin cages could be critical to GJA1-20k mediated protection against IR injury. The observed actin around mitochondria could prevent pathological mitochondrial swelling, preserving mitochondrial integrity and function in oxidative stress conditions.

Background: Arrhythmogenic Cardiomyopathy (ACM) is an insidious hereditary heart disease that results in structural and electrical cardiac abnormalities leading to arrhythmogenesis and heart failure. ACM is characterized by cardiomyocyte loss, decreased cellular coupling, and fibrofatty replacement. The disease arises from mutations in desmosomal genes, such as plakophilin (PKP), desmoglein (DSG), and desmoplakin (DSP). The pathogenesis of this disease is linked to dysfunction at the level of several cellular processes and pathways, including Wnt/β-catenin signaling. Recent evidence has shown that GJA1-20k, a truncated isoform of Connexin-43 (Cx43) generated by internal translation, has a therapeutic role in preserving Cx43 trafficking and cell-cell coupling in ACM of DSG origin. This study aims to investigate the role of GJA1-20k in protecting against ACM of DSP origin by evaluating its effect in cell models on Cx43 trafficking and Wnt/β-catenin signaling. Methods: DSP knockdown was performed on HEK cells and mouse neonatal cardiomyocytes. The role of GJA1-20k was determined using imaging, biochemical, and molecular biology techniques. Results: Protein levels of Cx43 and β-catenin were decreased on Western blot (WB) upon DSP knockdown. These findings were corroborated through imaging wherein the signal intensity for Cx43 at cellular junctions and β-catenin at the membrane was significantly decreased compared to the control group. β-catenin signal intensity was also decreased within the nucleus. Upon administration of GJA1-20k, protein levels of Cx43 were normalized on WB. Moreover, the ratio of phosphorylated β-catenin (inactive) to total β-catenin was decreased. Imaging studies identified a significant increase in Cx43 and β-catenin at the membrane in ACM models treated with GJA1-20k. This was also accompanied by an increase in β-catenin signal intensity within the nucleus. A TOP-flash luciferase assay was performed and revealed a significant increase in β-catenin transcriptional activity in the presence of GJA1-20k despite DSP knockdown. Conclusions: These results suggest that GJA1-20k is able to rescue Cx43 trafficking and recover Wnt/β-catenin signaling in cells and cardiomyocytes lacking DSP. The ability of GJA1-20k to restore dysfunctional genetic pathways involved in arrhythmogenesis and fibro-adipogenesis make it an attractive potential candidate for targeted therapy against ACM.

Background: The diagnosis and treatment of autoimmune optic neuritis (ON) has improved with the accessibility and reliability of aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) antibody testing, yet autoantibody-negative ON remains common. This study describes the demographic, clinical, and outcome data in patients with isolated ON across the pediatric and adult cohort. Methods: A retrospective chart review of University of Utah Health patients with the International Classification of Diseases (ICD) code of ICD-9 377.30 (ON unspecified), ICD-9 377.39 (other ON), or ICD-10 H46 (ON) and at least 2 ophthalmologic evaluations were conducted between February 2011 and July 2023. Only isolated cases of ON without other brain or spinal demyelinating lesions were evaluated. Differences in demographic and clinical characteristics between AQP4, MOG, and Other-ON were determined. Results: Of the 98 patients (15 children and 83 adults), 9 (9.2%) were positive for AQP4-IgG and 35 (35.7%) tested positive for MOG-IgG. Fifty-four were classified into Other-ON, of which 7 (13.0%) had recurrence or new demyelinating lesions during a median follow-up of 12.5 months—2 were ultimately diagnosed with recurrent isolated ON (RION), 1 with chronic relapsing inflammatory ON (CRION), 2 with multiple sclerosis, 1 with collapsin response-mediator protein (CRMP)-5-ON, and 1 with seronegative neuromyelitis optica spectrum disorder. Four patients were treated with long-term immunosuppressive therapy. No patients with RION or CRION had preceding infections; they had first recurrences of ON within 2 months. At presentation, AQP4-ON (75%) and MOG-ON (48.8%) had more severe vision loss (visual acuity <20/200) than Other-ON (23.2%, P = 0.01). At the 1-month follow-up, 93.0% of patients with MOG-ON and 89.3% of patients with Other-ON demonstrated a visual acuity ≥20/40, compared with only 50% of patients with AQP4-ON (P < 0.01). By the last follow-up, 37.5% of the AQP4-ON still exhibited visual acuity <20/40, including 25% who experienced severe vision loss (visual acuity <20/200). By contrast, over 95% of patients with MOG-ON and Other-ON maintained a visual acuity of ≥20/40. In our cohort, over a quarter of pediatric cases presented with simultaneous bilateral ON, 40% had a preceding infection, and 44.4% initially presented with a visual acuity <20/200. Two pediatric cases had recurrence, and both were MOG-ON. By their last follow-up, all pediatric cases had achieved a visual acuity of 20/40 or better. In addition, pediatric cases were more likely to exhibit disc edema compared with adult cases (100% vs 64%, P < 0.01). Conclusions: Despite recent advances in identification and availability of testing for AQP4-IgG and MOG-IgG, over half of patients who presented with isolated ON remained with an “idiopathic” diagnostic label. As more than 1 in 10 patients with AQP4-IgG and MOG-IgG negative ON experienced recurrence or develop new demyelinating lesions, clinicians should provide anticipatory guidance and closely monitor for potential long-term outcomes. In addition, it is crucial to re-evaluate the diagnosis in cases of poor recovery, ON recurrence, and the emergence of new neurological symptoms, as ON can often be the initial presentation of other conditions.

While membrane proteins such as ion channels continuously turn over and require replacement, the mechanisms of specificity of efficient channel delivery to appropriate membrane subdomains remain poorly understood. GJA1-20k is a truncated Connexin43 (Cx43) isoform arising from translation initiating at an internal start codon within the same parent GJA1 mRNA and is requisite for full-length Cx43 trafficking to cell borders. GJA1-20k does not have a full transmembrane domain, and it is not known how GJA1-20k enables forward delivery of Cx43 hemichannels. Here, we report that a RPEL-like domain at the C terminus of GJA1-20k binds directly to actin and induces an actin phenotype similar to that of an actin-capping protein. Furthermore, GJA1-20k organizes actin within the cytoplasm to physically outline a forward delivery pathway for microtubule-based trafficking of Cx43 channels to follow. In conclusion, we find that the postal address of membrane-bound Cx43 channel delivery is defined by a separate protein encoded by the same mRNA of the channel itself.

Current approaches, such as fixed-cell imaging or single-snapshot imaging, are insufficient to capture cytoskeleton-mediated mitochondrial fission. Here, we present a protocol to capture actin-mediated mitochondrial fission using high -resolution time-lapse imaging. We describe steps starting from cell preparation and mitochondria labeling through to live-cell imaging and final analysis. This approach is also applicable for analysis of multiple cytoskeleton-mediated organelle events such as vesicle trafficking, membrane fusion, and endocytic events in live cells. For complete details on the use and execution of this protocol, please refer to Shimura et al. (2021).1

Introduction: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a promising treatment for non-compressible torso hemorrhage (NCTH). However, REBOA complications, specifically ischemia-reperfusion injury, significantly limit its therapeutic benefits and broad acceptance. GJA1-20k is a stress response protein that protects mitochondrial function. Given the central role of deranged cellular metabolism after shock, we hypothesized that GJA1-20k would mitigate REBOA-associated ischemia-reperfusion injury as shown by lowering serum lactate concentrations in a pig model of hemorrhagic shock and REBOA. Methods: Twelve Yorkshire pigs were anesthetized and subjected to hemorrhagic shock by removing 25% of the estimated blood volume, followed by 45 minutes of complete supra-coeliac REBOA. Twenty-five minutes into the occlusion period, animals received a 1-hr intravenous infusion of 0.9% saline (placebo) or GJA1-20k (0.01 mg/kg) and were transfused with autologous blood. After the balloon catheter was deflated and removed, pigs received critical care and algorithmic resuscitation with IV fluids and norepinephrine to maintain a mean arterial pressure greater than 65 mmHg. Results: While there was no difference in serum lactate concentration at the end of the study (Control: 5.6 [3.0-6.7] mmol/L, GJA1-20k: 3.5 [3.0-3.8] mmol/L, p=0.1), the GJA1-20k treatment group had a reduction in resuscitation fluid requirements (Control: 62.5 [50-90] mL/kg, GKA1-20k: 22.5 [0-50] mL/kg, p=0.03). Importantly, with GJA1-20k treatment, 2 animals required no fluid boluses, and another only required 10 mL/kg of IV fluids. GJA1-20k group was showed a reduction in final serum creatinine concentration (Control: 2.8 +/-0.3 mg/dL, GJA1-20k: 2.4 +/- 0.3 mg/dL, p=0.03). Conclusions: While there was no significant difference in serum lactate concentration between groups, GJA1-20k significantly reduced fluid resuscitation requirements and renal injury following REBOA in a pig model of hemorrhagic shock. Mitochondrial protection is a potential target for mitigating REBOA-associated ischemia-reperfusion injury after hemorrhagic shock.

Dynamin-Related Protein 1 (DRP1) is a major mediator of mitochondrial fission and its mutation results with hyper-fused mitochondria and, in mice, a dilated cardiomyopathy. GJA1-20k is the most abundant N-terminus truncated isoform of Connexin 43 (Cx43) formed by internal translation and was originally identified as a trafficking subunit of Cx43. It has been recently identified that GJA1-20k is located on the mitochondrial membrane and plays a role in mediating ischemia preconditioning by promoting a protective mitochondrial fission. Interestingly, GJA1-20k mediated fission occurs even under DRP1 inhibition in vitro. Here, we hypothesize that GJA1-20k is downstream of DRP1 and that exogenous GJA1-20k administration rescues hyper-fusion of mitochondria in DRP1-suppressed mouse hearts. Using an AAV9-mediated DRP1 dominant negative mutant (Drp1-K38A) to suppress DRP1, we confirmed that the Drp1-K38A mouse hearts have an increase in cardiac mitochondrial size (158.9 ± 3.7% of control) and decrease in cardiac function (ejection fraction is 43.1 ± 1.5% versus 64.0 ± 4.1% in control animals), as assayed by electron microscopy and in vivo echocardiography, respectively. However, the administration of exogenous GJA1-20k via AAV9 transduction normalizes mitochondrial size (net 14.1 ± 3.3% decrease from control group) and rescues cardiac function (ejection fraction 61.6 ± 2.7%), suggesting that the progression to the cardiomyopathy is prevented by GJA1-20k administration. In summary, GJA1-20k functions downstream of DRP1 and can rescue the effect DRP1 suppression on mitochondrial size and cardiac function.

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell-cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell-cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell-cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell-cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell-cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block - both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

GJA1-20k is an internally translated, N-terminus truncated isoform of the gap junction protein Connexin 43 (Cx43). We have identified essential roles of GJA1-20k for Cx43 trafficking to cell membrane, actin stabilization, and regulation of mitochondrial dynamics. Recently, we found that GJA1-20k works with actin to induce a “protective” mitochondrial fission, protecting cells and hearts against ischemia-reperfusion (I/R) injury. This GJA1-20k mediated mitochondrial fission does not require the canonical fission mediator, Dynamin-Related Protein 1 (DRP1) pathway. At present, however, little is known how GJA1-20k occurs at mitochondrial membrane. Our in silico analysis indicates that the N-terminus of GJA1-20k contains a potent binding site to Heat Shock Protein 70 (HSP70). Since HSP70 is a well known mitochondrial transporter, we hypothesized that HSP70 interacts with GJA1-20k to support its translocation to mitochondria. To explore the involvement of HSP70, in HeLa cells we knocked down HSP70 in the presence of exogenous GJA1-20k. An absence of HSP70 inhibited GJA1-20k localization to mitochondrial membrane. In fact, in the absence of HSP70, most GJA1-20k was aggregated, suggesting a failure to distribute. Given that the expression of both HSP70 and GJA1-20k are upregulated during I/R injury, these data indicate that both stress-response proteins cooperate to localize GJA1-20k to mitochondrial membrane, initiating protective mitochondrial fission.