removal-an-internal-translational-start-site-from-mrna-while

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily ...

echocardiography-recording-in-awake-miniature-pigs

Echocardiography Recording in Awake Miniature Pigs

Echocardiography uses ultrasonic waves to non-invasively assess cardiac structure and function and is the standard of care for cardiac assessment and ...

Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)(s878), that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblin(s634) revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)(s878) line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS.

Genetic and physiologic dissection of the vertebrate cardiac conduction system.

The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function. 

3-OST-7 regulates BMP-dependent cardiac contraction.

Correlating Cardiac Origin Neurohormonal Stress Levels With Heart Failure Outcomes.

The cardiomyopathy of Duchenne muscular dystrophy (DMD) is an important cause of morbidity and mortality in affected males with this dreaded muscle disease. Previous studies have implicated changes in expression and subcellular localization of connexin-43 (Cx43), the major ventricular gap junction protein, in DMD cardiomyopathy. In this issue of the JCI, Himelman et al. explore how hypophosphorylation of Cx43 at a triplet of serine residues (S325/S328/S330) in the regulatory C-terminus contributes to multiple features of the cardiomyopathy phenotype. Using a mouse model of DMD cardiomyopathy in which phosphomimetic glutamic acids are substituted for serines at these residues in Cx43, Himelman et al. observed reduced gap junction remodeling and lateralization of Cx43 immunosignals, protection against isoproterenol-induced arrhythmias, and improved Ca2+ homeostasis. This study contributes to the understanding of pathologic Cx43 remodeling and encourages further research into developing strategic interventions to mitigate cardiac dysfunction and arrhythmias in DMD patients.

A role for connexin-43 in Duchenne muscular dystrophy cardiomyopathy.

Connexin 43 (Cx43) is a gap junction protein that assembles at the cell border to form intercellular gap junction (GJ) channels which allow for cell-cell communication by facilitating the rapid transmission of ions and other small molecules between adjacent cells. Non-canonical roles of Cx43, and specifically its C-terminal domain, have been identified in the regulation of Cx43 trafficking, mitochondrial preconditioning, cell proliferation, and tumor formation, yet the mechanisms are still being explored. It was recently identified that up to six truncated isoforms of Cx43 are endogenously produced via alternative translation from internal start codons in addition to full length Cx43, all from the same mRNA produced by the gene . GJA1-11k, the 11kDa alternatively translated isoform of Cx43, does not have a known role in the formation of gap junction channels, and little is known about its function. Here, we report that over expressed GJA1-11k, unlike the other five truncated isoforms, preferentially localizes to the nucleus in HEK293FT cells and suppresses cell growth by limiting cell cycle progression from the G/G phase to the S phase. Furthermore, these functions are independent of the channel-forming full-length Cx43 isoform. Understanding the apparently unique role of GJA1-11k and its generation in cell cycle regulation may uncover a new target for affecting cell growth in multiple disease models.

An Alternatively Translated Connexin 43 Isoform, GJA1-11k, Localizes to the Nucleus and Can Inhibit Cell Cycle Progression.

Connexin-43 (Cx43) gap junctions provide intercellular coupling, which ensures rapid action potential propagation and synchronized heart contraction. Alterations in Cx43 localization and reductions in gap junction coupling occur in failing hearts, contributing to ventricular arrhythmias and sudden cardiac death. Recent reports have found that an internally translated Cx43 isoform, GJA1-20k, is an auxiliary subunit for the trafficking of Cx43 in heterologous expression systems. Here, we have created a mouse model by using CRISPR technology to mutate a single internal translation initiation site in Cx43 (M213L mutation), which generates full-length Cx43, but not GJA1-20k. We found that GJA1M213L/M213L mice had severely abnormal electrocardiograms despite preserved contractile function, reduced total Cx43, and reduced gap junctions, and they died suddenly at 2 to 4 weeks of age. Heterozygous GJA1M213L/WT mice survived to adulthood with increased ventricular ectopy. Biochemical experiments indicated that cytoplasmic Cx43 had a half-life that was 50% shorter than membrane-associated Cx43. Without GJA1-20k, poorly trafficked Cx43 was degraded. The data support that GJA1-20k, an endogenous entity translated independently of Cx43, is critical for Cx43 gap junction trafficking, maintenance of Cx43 protein, and normal electrical function of the mammalian heart.

Auxiliary trafficking subunit GJA1-20k protects connexin-43 from degradation and limits ventricular arrhythmias.

Heart failure is an important, and growing, cause of morbidity and mortality. Half of patients with heart failure have preserved ejection fraction, for whom therapeutic options are limited. Here we report that cardiac bridging integrator 1 gene therapy to maintain subcellular membrane compartments within cardiomyocytes can stabilize intracellular distribution of calcium-handling machinery, preserving diastolic function in hearts stressed by chronic beta agonist stimulation and pressure overload. This study identifies that maintenance of intracellular architecture and, in particular, membrane microdomains at t-tubules, is important in the setting of sympathetic stress. Stabilization of membrane microdomains may be a pathway for future therapeutic development.

In Mice Subjected to Chronic Stress, Exogenous cBIN1 Preserves Calcium-Handling Machinery and Cardiac Function.

Cardiac Bridging Integrator 1 (cBIN1) is a membrane deformation protein that generates calcium microdomains at cardiomyocyte t-tubules, whose transcription is reduced in heart failure, and is released into blood. cBIN1 score (CS), an inverse index of plasma cBIN1, measures cellular myocardial remodeling. In patients with heart failure with preserved ejection fraction (HFpEF), CS diagnoses ambulatory heart failure and prognosticates hospitalization. The performance of CS has not been tested in patients with heart failure with reduced ejection fraction (HFrEF).

cBIN1 Score (CS) Identifies Ambulatory HFrEF Patients and Predicts Cardiovascular Events.

: Cardiac bridging integrator 1 (cBIN1) organizes transverse tubule (t-tubule) membrane calcium handling microdomains required for normal beat-to-beat contractility. cBIN1 is transcriptionally reduced in heart failure (HF). We recently found that cBIN1 pretreatment can limit HF development in stressed mice. Here, we aim to explore whether cBIN1 replacement therapy can improve myocardial function in continuously stressed hearts with pre-existing HF. : Adult male mice were subjected to sham or transverse aortic constriction (TAC) surgery at the age of 8-10 weeks old. Adeno-associated virus 9 (AAV9) transducing cBIN1-V5 or GFP-V5 (3 × 10 vg) was administered through retro-orbital injection at 5 weeks post-TAC. Mice were followed by echocardiography to monitor cardiac function until 20 weeks after TAC. Overall survival, heart and lung weight (LW), and HF incidence were determined. In a second set of animals in which AAV9-cBIN1 pretreatment prevents HF, we recorded cardiac pressure-volume (PV) loops and obtained myocardial immunofluorescence imaging. : The overall Kaplan-Meir survival of AAV9-cBIN1 mice was 77.8%, indicating a significant partial rescue between AAV9-GFP (58.8%) and sham (100%) treated mice. In mice with ejection fraction (EF) ≥30% prior to AAV9 injection at 5 weeks post-TAC, AAV9-cBIN1 significantly increased survival to 93.3%, compared to 62.5% survival for AAV9-GFP treated mice. The effect of exogenous cBIN1 was to attenuate TAC-induced left ventricular (LV) dilation and prevent further HF development. Recovery of EF also occurs in AAV9-cBIN1-treated mice. We found that EF increases to a peak at 6-8 weeks post-viral injection. Furthermore, PV loop analysis identified that AAV9-cBIN1 increases both systolic and diastolic function of the post-TAC hearts. At the myocyte level, AAV9-cBIN1 normalizes cBIN1 expression, t-tubule membrane intensity, and intracellular distribution of Cav1.2 and ryanodine receptors (RyRs). : In mice with pre-existing HF, exogenous cBIN1 can normalize t-tubule calcium handling microdomains, limit HF progression, rescue cardiac function, and improve survival.

Exogenous Cardiac Bridging Integrator 1 Benefits Mouse Hearts With Pre-existing Pressure Overload-Induced Heart Failure.

SARS-CoV-2 is responsible for the 2020 global COVID-19 pandemic. In patients with COVID-19, multiple cardiovascular (CV) manifestations have been reported. SARS coronavirus 2 infection can lead to inflammatory CV disease first via takeover of the angiotensin-converting enzyme-2 enzyme as a cell receptor as well as the macrophage activation syndrome in severe illness. We review the CV manifestations of COVID-19 and therapeutics under investigation. We discuss the potential long-term CV sequelae after recovery from COVID-19 and the gaps in knowledge including the pathophysiological links between acute cardiac injury, myocardial inflammation and chronic cardiomyopathy. Future investigational efforts could result in significant diagnostic and therapeutic advances potentially impacting the broader field of chronic heart failure and cardiac recovery.

SARS-CoV-2 as an inflammatory cardiovascular disease: current knowledge and future challenges.

Huntington's disease skeletal muscle has altered T-tubules.

Coronary microvascular dysfunction (CMD) is associated with heart failure with preserved ejection fraction (HFpEF); however, pathophysiology is not well described.

Association of coronary microvascular dysfunction and cardiac bridge integrator 1, a cardiomyocyte dysfunction biomarker.

The Connexin43 gap junction gene  has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k.

Connexin 43 (Cx43) is the primary gap junction protein of mammalian heart ventricles and is encoded by the gene  which has a single coding exon and therefore cannot be spliced. We previously identified that  mRNA undergoes endogenous internal translation initiated at one of several internal AUG (M) start codons, generating N-terminal truncated protein isoforms that retain the C-terminus distal to the start site. GJA1-20k, whose translation initiates at mRNA M213, is usually the most abundant isoform in cells and greatly increases after ischemic and metabolic stress. GJA1-20k consists of a small segment of the last transmembrane domain and the complete C-terminus tail of Cx43, with a total size of about 20 kDa. The original role identified for GJA1-20k is as an essential subunit that facilitates the trafficking of full-length Cx43 hexameric hemichannels to cell-cell contacts, generating traditional gap junctions between adjacent cells facilitating, in cardiac muscle, efficient spread of electrical excitation. GJA1-20k deficient mice (generated by a M213L substitution in ) suffer poor electrical coupling between cardiomycytes and arrhythmogenic sudden death two to 4 weeks after their birth. We recently identified that exogenous GJA1-20k expression also mimics the effect of ischemic preconditioning in mouse heart. Furthermore, GJA1-20k localizes to the mitochondrial outer membrane and induces a protective and DRP1 independent form of mitochondrial fission, preserving ATP production and generating less reactive oxygen species (ROS) under metabolic stress, providing powerful protection of myocardium to ischemic insult. In this manuscript, we focus on the detailed roles of GJA1-20k in mitochondria, and its interaction with the actin cytoskeleton.

GJA1-20k and Mitochondrial Dynamics.

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)..

Live-cell imaging and analysis of actin-mediated mitochondrial fission.

Internal translation is a form of post-translation modification as it produces different proteins from one mRNA molecule by beginning translation at a methionine coding triplet downstream of the first methionine. Internal translation can eliminate domains of proteins that otherwise restrict movement or activity, thereby creating profound functional diversity. Connexin43 (Cx43), encoded by the gene  is the main gap junction protein necessary for propagating action potentials between adjacent cardiomyocytes.  can be internally translated to produce a peptide 20 kD in length named GJA1-20k. This review focuses on the role of GJA1-20k in maintaining cardiac electrical rhythm as well as in ischemic preconditioning (IPC). Connexin43 is the only ion channel we are aware that has been reported to be subject to internal translation. We expect many other ion channels also undergo internal translation. The exploration of post-translational modification of ion channels, and in particular of internal translation, has the potential to greatly increase our understanding of both canonical and non-canonical ion channel biology.

Internal translation of Gja1 (Connexin43) to produce GJA1-20k: Implications for arrhythmia and ischemic-preconditioning.

GJA1-20k Rescues Cx43 Localization and Arrhythmias in Arrhythmogenic Cardiomyopathy.

Preclinical large animal models of chronic heart failure (HF) are crucial to both understanding pathological remodeling and translating fundamental discoveries into novel therapeutics for HF. Canine models of ischemic cardiomyopathy are historically limited by either high early mortality or failure to develop chronic heart failure. Twenty-nine healthy adult dogs (30 ± 4 kg, 15/29 male) underwent thoracotomy followed by one of three types of left anterior descending (LAD) coronary artery ligation procedures:  ( = 4) (simple LAD: proximal and distal LAD ligation);  ( = 14) (simple LAD plus lateral wall including ligation of the distal first diagonal and proximal first obtuse marginal); and  ( = 11) (total LAD devascularization or TLD: simple LAD plus ligation of proximal LAD branches to both the right and left ventricles). Dogs were followed until chronic severe HF developed defined as left ventricular ejection fraction (LVEF) < 40% and NH-terminal-prohormone B-type natriuretic peptide (NT-proBNP) > 900 pmol/L. Overall early survival (48-h postligation) in 29 dogs was 83% and the survival rate at postligation 5 wk was 69%.  and  had 100% and 71% early survival, respectively, yet only a 50% success rate of developing chronic HF.  had excellent survival at postligation 48 h (91%) and a 100% success in the development of chronic ischemic HF. The TLD approach, which limits full LAD and collateral flow to its perfusion bed, provides excellent early survival and reliable development of chronic ischemic HF in canine hearts. The novel total left anterior descending devascularization (TLD) approach in a canine ischemic heart failure model limits collateral flow in the ischemic zone and provides excellent early survival and repeatable development of chronic ischemic heart failure in the canine heart. This work provides a consistent large animal model for investigating heart failure mechanisms and testing novel therapeutics.

ArticleTotal : 2	Year
Publication title Cited by 4	2022
Publication title Cited by 4	2023

Article	Year
Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS biology\| PubMed ID: 18479184	2008
3-OST-7 regulates BMP-dependent cardiac contraction. PLoS biology\| PubMed ID: 24311987	2013
Correlating Cardiac Origin Neurohormonal Stress Levels With Heart Failure Outcomes. JAMA cardiology\| PubMed ID: 31876897	2020
A role for connexin-43 in Duchenne muscular dystrophy cardiomyopathy. The Journal of clinical investigation\| PubMed ID: 32091412	2020
An Alternatively Translated Connexin 43 Isoform, GJA1-11k, Localizes to the Nucleus and Can Inhibit Cell Cycle Progression. Biomolecules\| PubMed ID: 32244859	2020
Auxiliary trafficking subunit GJA1-20k protects connexin-43 from degradation and limits ventricular arrhythmias. The Journal of clinical investigation\| PubMed ID: 32525845	2020
In Mice Subjected to Chronic Stress, Exogenous cBIN1 Preserves Calcium-Handling Machinery and Cardiac Function. JACC. Basic to translational science\| PubMed ID: 32613144	2020
cBIN1 Score (CS) Identifies Ambulatory HFrEF Patients and Predicts Cardiovascular Events. Frontiers in physiology\| PubMed ID: 32670075	2020
Exogenous Cardiac Bridging Integrator 1 Benefits Mouse Hearts With Pre-existing Pressure Overload-Induced Heart Failure. Frontiers in physiology\| PubMed ID: 32670093	2020
SARS-CoV-2 as an inflammatory cardiovascular disease: current knowledge and future challenges. Future cardiology\| PubMed ID: 33739142	2021
Huntington's disease skeletal muscle has altered T-tubules. The Journal of general physiology\| PubMed ID: 33978682	2021
Association of coronary microvascular dysfunction and cardiac bridge integrator 1, a cardiomyocyte dysfunction biomarker. Clinical cardiology\| PubMed ID: 34528718	2021
Protective mitochondrial fission induced by stress-responsive protein GJA1-20k. eLife\| PubMed ID: 34608863	2021
GJA1-20k and Mitochondrial Dynamics. Frontiers in physiology\| PubMed ID: 35399255	2022
Live-cell imaging and analysis of actin-mediated mitochondrial fission. STAR protocols\| PubMed ID: 36542522	2022
Internal translation of Gja1 (Connexin43) to produce GJA1-20k: Implications for arrhythmia and ischemic-preconditioning. Frontiers in physiology\| PubMed ID: 36569758	2022
GJA1-20k Rescues Cx43 Localization and Arrhythmias in Arrhythmogenic Cardiomyopathy. Circulation research\| PubMed ID: 36927183	2023
A canine model of chronic ischemic heart failure. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 36961487	2023

Robin M. Shaw

Biography

JoVE Journal Publications

Other Publications