A canine model of chronic ischemic heart failure.

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: group 1 (n = 4) (simple LAD: proximal and distal LAD ligation); group 2 (n = 14) (simple LAD plus lateral wall including ligation of the distal first diagonal and proximal first obtuse marginal); and group 3 (n = 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 NH2-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%. Groups 1 and 2 had 100% and 71% early survival, respectively, yet only a 50% success rate of developing chronic HF. Group 3 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.NEW & NOTEWORTHY 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.

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

The Connexin43 gap junction gene GJA1 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.

Cell-Type-Specific Immune Dysregulation in Severely Ill COVID-19 Patients.

Recent studies have demonstrated immunologic dysfunction in severely ill coronavirus disease 2019 (COVID-19) patients. We use single-cell RNA sequencing (scRNA-seq) to analyze the transcriptome of peripheral blood mononuclear cells (PBMCs) from healthy (n = 3) and COVID-19 patients with moderate disease (n = 5), acute respiratory distress syndrome (ARDS, n = 6), or recovering from ARDS (n = 6). Our data reveal transcriptomic profiles indicative of defective antigen presentation and interferon (IFN) responsiveness in monocytes from ARDS patients, which contrasts with higher responsiveness to IFN signaling in lymphocytes. Furthermore, genes involved in cytotoxic activity are suppressed in both natural killer (NK) and CD8 T lymphocytes, and B cell activation is deficient, which is consistent with delayed viral clearance in severely ill COVID-19 patients. Our study demonstrates that COVID-19 patients with ARDS have a state of immune imbalance in which dysregulation of both innate and adaptive immune responses may be contributing to a more severe disease course.

Cell type-specific immune dysregulation in severely ill COVID-19 patients.

Coronavirus disease 2019 (COVID-19) has quickly become the most serious pandemic since the 1918 flu pandemic. In extreme situations, patients develop a dysregulated inflammatory lung injury called acute respiratory distress syndrome (ARDS) that causes progressive respiratory failure requiring mechanical ventilatory support. Recent studies have demonstrated immunologic dysfunction in severely ill COVID-19 patients. To further delineate the dysregulated immune response driving more severe clinical course from SARS-CoV-2 infection, we used single-cell RNA sequencing (scRNAseq) to analyze the transcriptome of peripheral blood mononuclear cells (PBMC) from hospitalized COVID-19 patients having mild disease (n = 5), developing ARDS (n = 6), and recovering from ARDS (n = 6). Our data demonstrated an overwhelming inflammatory response with select immunodeficiencies within various immune populations in ARDS patients. Specifically, their monocytes had defects in antigen presentation and deficiencies in interferon responsiveness that contrasted the higher interferon signals in lymphocytes. Furthermore, cytotoxic activity was suppressed in both NK and CD8 lymphocytes whereas B cell activation was deficient, which is consistent with the delayed viral clearance in severely ill COVID-19 patients. Finally, we identified altered signaling pathways in the severe group that suggests immunosenescence and immunometabolic changes could be contributing to the dysfunctional immune response. Our study demonstrates that COVID-19 patients with ARDS have an immunologically distinct response when compared to those with a more innocuous disease course and show a state of immune imbalance in which deficiencies in both the innate and adaptive immune response may be contributing to a more severe disease course in COVID-19.

Prognostic value of point-of-care ultrasound during cardiac arrest: a systematic review.

BACKGROUND: Despite significant improvements in cardiopulmonary resuscitation, sudden cardiac arrest is one of the leading causes of mortality in the United States. Ultrasound is a widely available tool that can be used to evaluate the presence of cardiac wall motion during cardiac arrest. Several clinical studies have evaluated the use of ultrasound to visualize cardiac motion as a predictor of mortality in cardiac arrest patients. However, there are limited data summarizing the prognostic value of point of care ultrasound evaluation during resuscitation. We performed a systematic literature review of the existing evidence examining the clinical utility of point-of-care ultrasound evaluation of cardiac wall motion as a predictor of cardiac resuscitation outcomes. METHODS/RESULTS: We performed a systematic PubMed search of clinical studies up to July 23, 2019 evaluating point-of-care sonographic cardiac motion as a predictor of mortality following cardiac resuscitation. We included studies written in English that reviewed short-term outcomes and included adult populations. Fifteen clinical studies met inclusion criteria for assessing cardiac wall motion with point-of-care ultrasound and outcomes following cardiac resuscitation. Fourteen of the fifteen studies showed a statistically significant correlation between the presence of cardiac motion on ultrasound and short-term survival. This was most evident in patients with ventricular fibrillation or ventricular tachycardia as a presenting rhythm. Absence of cardiac motion non-survival. The data were pooled and the overall pooled odds ratio for return of spontaneous circulation in the presence of cardiac motion during CPR was 12.4 +/1 2.7 (p <  0.001). CONCLUSION: Evaluation of cardiac motion on transthoracic echocardiogram is a valuable tool in the prediction of short-term cardiac resuscitation outcomes. Given the safety and availability of ultrasound in the emergency department, it is reasonable to apply point-of-care ultrasound to cardiopulmonary resuscitation as long as its use does not interrupt resuscitation.

Interaction of α Carboxyl Terminus 1 Peptide With the Connexin 43 Carboxyl Terminus Preserves Left Ventricular Function After Ischemia-Reperfusion Injury.

Background α Carboxyl terminus 1 (αCT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that αCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cε phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of αCT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study αCT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cε phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an α-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that αCT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of αCT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of αCT1 (αCT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of αCT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.

Diabetes Increases Cryoinjury Size with Associated Effects on Cx43 Gap Junction Function and Phosphorylation in the Mouse Heart.

Diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack. The poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing, response to injury and infection. Studies in rodents have implicated a role for the gap junction protein connexin 43 (Cx43) in regulating the injury response in diabetic skin wounds. In this study, we sought to determine whether diabetes alters Cx43 molecular interactions or intracellular communication in the cryoinjured STZ type I diabetic mouse heart. We found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration. Consistent with these findings, we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice. This decrease in coupling is associated with a concomitant increase in phosphorylation of Cx43 at serine 368, a residue known to decrease channel conductance. Taken together, our results suggest that insulin regulates both gap junction-mediated intercellular communication and injury propagation in the mouse heart.

Your Father and Grandfather's Atrial Fibrillation: A Review of the Genetics of the Most Common Pathologic Cardiac Dysrhythmia.

Atrial fibrillation (AF) remains the most common pathologic dysrhythmia in humans with a prevalence of 1-2% of the total population and as high as 10% of the elderly. AF is an independent risk marker for cardiovascular mortality and morbidity, and given the increasing age of the population, represents an increasing burden of disease. Although age and hypertension are known risk factors for development of AF, the study of families with early onset AF revealed mutations in genes coding for ion channels and other proteins involved in electrotonic coupling as likely culprits for the pathology in select cases. Recent investigations using Genome-Wide Association Studies have revealed several single nucleotide polymorphisms (SNPs) that appear to be associated with AF and have highlighted new genes in the proximity of the SNPs that may potentially contribute to the development of the dysrhythmia. Here we review the genetics of AF and discuss how application of GWAS and next generation sequencing have advanced our knowledge of AF and further investigations may yield novel therapeutic targets for the disease.

Extracorporeal Membrane Oxygenation Support for Hypokalemia-induced Cardiac Arrest: A Case Report and Review of the Literature.

BACKGROUND: Hypokalemia is a reversible cause of cardiac arrest in patients presenting to the emergency department (ED). Extracorporeal membrane oxygenation (ECMO) is an established technology for cardiopulmonary support with emerging roles in resuscitation. Here, we review the literature of hypokalemic-induced cardiac arrests and discuss one such case successfully managed with ECMO. CASE REPORT: A 23-year-old Central American man who presented to a community ED under federal custody with several days of nausea and vomiting was found to have a serum potassium level of 1.5 mEq/L. Repeat serum potassium level was 1.1 mEq/L upon arrival to our facility. Within 2 h of arrival, despite electrolyte repletion, he suffered cardiac arrest. Advanced cardiac life support was performed for 45 min. ECMO was initiated while active chest compressions were performed. After aggressive potassium repletion, return of spontaneous circulation was achieved and ECMO was eventually discontinued. Further investigation ultimately confirmed the presence of a potassium-wasting nephropathy, for which the patient had been treated with chronic potassium supplementation prior to entering federal custody. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: ECMO is a well-established modality for cardiopulmonary support, with an emerging role for patients in undifferentiated cardiac arrest presenting to the ED. There is a growing interest in the utility of ECMO in these circumstances. This report highlights hypokalemia as an important cause of cardiac arrest, reviews the treatment and causes of hypokalemia, and demonstrates a potential role for ECMO as a critical temporizing measure to provide time for potassium repletion.

Cryoinjury models of the adult and neonatal mouse heart for studies of scarring and regeneration.

A major limitation in studies of the injured heart is animal-to-animal variability in wound size resulting from commonly used techniques such as left anterior descending coronary artery ligation. This variability can make standard errors sufficiently large that mean separation between treatment and control groups can be difficult without replicating numbers (n) of animals in groups by excessive amounts. Here, we describe the materials and protocol necessary for delivering a standardized non-transmural cryoinjury to the left ventricle of an adult mouse heart that may in part obviate the issue of injury variance between animals. As reported previously, this cryoinjury model generates a necrotic wound to the ventricle of consistent size and shape that resolves into a scar of uniform size, shape, and organization. The cryo-model also provides an extended injury border zone that exhibits classic markers of remodeling found in surviving cardiac tissue at the edge of a myocardial infarction, including connexin43 (Cx43) lateralization. In a further extension of the method, we describe how we have adapted the model to deliver a cryoinjury to the apex of the heart of neonatal mice-a modification that may be useful for studies of myocardial regeneration in mammals.

The connexin43 carboxyl terminus and cardiac gap junction organization.

The precise spatial order of gap junctions at intercalated disks in adult ventricular myocardium is thought vital for maintaining cardiac synchrony. Breakdown or remodeling of this order is a hallmark of arrhythmic disease of the heart. The principal component of gap junction channels between ventricular cardiomyocytes is connexin43 (Cx43). Protein-protein interactions and modifications of the carboxyl-terminus of Cx43 are key determinants of gap junction function, size, distribution and organization during normal development and in disease processes. Here, we review data on the role of proteins interacting with the Cx43 carboxyl-terminus in the regulation of cardiac gap junction organization, with particular emphasis on Zonula Occludens-1. The rapid progress in this area suggests that in coming years we are likely to develop a fuller understanding of the molecular mechanisms causing pathologic remodeling of gap junctions. With these advances come the promise of novel approach to the treatment of arrhythmia and the prevention of sudden cardiac death. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury.

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.

A peptide mimetic of the connexin43 carboxyl terminus reduces gap junction remodeling and induced arrhythmia following ventricular injury.

RATIONALE: Remodeling of connexin (Cx)43 gap junctions (GJs) is linked to ventricular arrhythmia. OBJECTIVES: A peptide mimetic of the carboxyl terminal (CT) of Cx43, incorporating a postsynaptic density-95/disks-large/ZO-1 (PDZ)-binding domain, reduces Cx43/ZO-1 interaction and GJ size remodeling in vitro. Here, we determined: (1) whether the Cx43-CT mimetic αCT1 altered GJ remodeling following left ventricular (LV) injury in vivo; (2) whether αCT1 affected arrhythmic propensity; and (3) the mechanism of αCT1 effects on arrhythmogenicity and GJ remodeling. METHODS AND RESULTS: A cryoinjury model generating a reproducible wound and injury border zone (IBZ) in the LV was used. Adherent methylcellulose patches formulated to locally release αCT1 (< 48 hours) were placed on cryoinjuries. Relative to controls, Cx43/ZO-1 colocalization in the IBZ was reduced by αCT1 by 24 hours after injury. Programmed electric stimulation ex vivo and optical mapping of voltage transients indicated that peptide-treated hearts showed reduced inducible arrhythmias and increased ventricular depolarization rates 7 to 9 days after injury. At 24 hours and 1 week after injury, αCT1-treated hearts maintained Cx43 in intercalated disks (IDs) in the IBZ, whereas by 1 week after injury, controls demonstrated Cx43 remodeling from IDs to lateralized distributions. Over a postinjury time course of 1 week, αCT1-treated IBZs showed increased Cx43 phosphorylation at serine368 (Cx43-pS368) relative to control tissues. In biochemical assays, αCT1 promoted phosphorylation of serine368 by protein kinase (PK)C-ε in a dose-dependent manner that was modulated by, but did not require ZO-1 PDZ2. CONCLUSIONS: αCT1 increases Cx43-pS368 in vitro in a PKC-ε-dependent manner and in the IBZ in vivo acutely following ventricular injury. αCT1-mediated increase in Cx43-pS368 phosphorylation may contribute to reductions in inducible-arrhythmia following injury.

ZO-1 determines adherens and gap junction localization at intercalated disks.

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.

Translational lessons from scarless healing of cutaneous wounds and regenerative repair of the myocardium.

Regenerative healing is the process by which injured tissues are restored to their original structure and function. Many species are capable of healing in this manner. However, in mammals the healing response in most tissues is marked by fibroblast proliferation and scar tissue deposition. While scarring contributes to efficient resolution of mammalian wounds and restoration of at least partial structural and functional support, the final result of scar formation can be more deleterious than the initial insult. This is especially true in the heart, which is sensitive to electrical heterogeneities and altered mechanical properties produced by scarring. Several therapeutic modalities promoting regeneration in skin wounds have been developed that modulate various aspects of the healing process. Targets include cytokine stimulation, control of fibroblast activation, modulation of gap junctions, and stem cell differentiation. Here, we review and compare mechanisms of injury, repair, and scarring in the skin and heart and discuss the promise and caveats of future therapies that may translate to improving repair of myocardial tissues.

Novel therapies for scar reduction and regenerative healing of skin wounds.

Fibrotic scars deposited during skin wound healing can cause disfiguration and loss of dermal function. Scar differentiation involves inputs from multiple cell types in a predictable and overlapping sequence of cellular events that includes inflammation, migration/proliferation and extracellular matrix deposition. Research into the molecular mechanisms underpinning these processes in embryonic and adult wounds has contributed to the development of a growing number of novel therapeutic approaches for improving scar appearance. This review discusses some of these emerging strategies for shifting the balance of healing from scarring to regeneration in the context of non-pathological wounds. Particular focus is given to potential therapies based on transforming growth factor (TGF)-beta signaling and recent unexpected findings involving targeting of gap junctional connexins. Lessons learned in promoting scarless healing of cutaneous injuries might provide a basis for regenerative healing in other scenarios, such as spinal cord rupture or myocardial infarction.