Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.

BACKGROUND: Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes. METHODS: Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. RESULTS: We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. CONCLUSIONS: These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

Hypoxia-induced stabilization of HIF2A promotes cardiomyocyte proliferation by attenuating DNA damage.

Introduction: Gradual exposure to a chronic hypoxic environment leads to cardiomyocyte proliferation and improved cardiac function in mouse models through a reduction in oxidative DNA damage. However, the upstream transcriptional events that link chronic hypoxia to DNA damage have remained obscure. Aim: We sought to determine whether hypoxia signaling mediated by the hypoxia-inducible factor 1 or 2 (HIF1A or HIF2A) underlies the proliferation phenotype that is induced by chronic hypoxia. Methods and Results: We used genetic loss-of-function models using cardiomyocyte-specific HIF1A and HIF2A gene deletions in chronic hypoxia. We additionally characterized a cardiomyocyte-specific HIF2A overexpression mouse model in normoxia during aging and upon injury. We performed transcriptional profiling with RNA-sequencing on cardiac tissue, from which we verified candidates at the protein level. We find that HIF2A - rather than HIF1A - mediates hypoxia-induced cardiomyocyte proliferation. Ectopic, oxygen-insensitive HIF2A expression in cardiomyocytes reveals the cell-autonomous role of HIF2A in cardiomyocyte proliferation. HIF2A overexpression in cardiomyocytes elicits cardiac regeneration and improvement in systolic function after myocardial infarction in adult mice. RNA-sequencing reveals that ectopic HIF2A expression attenuates DNA damage pathways, which was confirmed with immunoblot and immunofluorescence. Conclusion: Our study provides mechanistic insights about a new approach to induce cardiomyocyte renewal and mitigate cardiac injury in the adult mammalian heart. In light of evidence that DNA damage accrues in cardiomyocytes with aging, these findings may help to usher in a new therapeutic approach to overcome such age-related changes and achieve regeneration.

Networks that Govern Cardiomyocyte Proliferation to Facilitate Repair of the Injured Mammalian Heart.

Cardiovascular diseases are the number one cause of death worldwide and in the United States (US). Cardiovascular diseases frequently progress to end-stage heart failure, and curative therapies are extremely limited. Intense interest has focused on deciphering the cascades and networks that govern cardiomyocyte proliferation and regeneration of the injured heart. For example, studies have shown that lower organisms such as the adult newt and adult zebrafish have the capacity to completely regenerate their injured heart with restoration of function. Similarly, the neonatal mouse and pig are also able to completely regenerate injured myocardium due to cardiomyocyte proliferation from preexisting cardiomyocytes. Using these animal models and transcriptome analyses, efforts have focused on the definition of factors and signaling pathways that can reactivate and induce cardiomyocyte proliferation in the adult mammalian injured heart. These studies and discoveries have the potential to define novel therapies to promote cardiomyocyte proliferation and repair of the injured, mammalian heart.

Metabolic Control of Cardiomyocyte Cell Cycle.

Current therapies for heart failure aim to prevent the deleterious remodeling that occurs after MI injury, but currently no therapies are available to replace lost cardiomyocytes. Several organisms now being studied are capable of regenerating their myocardium by the proliferation of existing cardiomyocytes. In this review, we summarize the main metabolic pathways of the mammalian heart and how modulation of these metabolic pathways through genetic and pharmacological approaches influences cardiomyocyte proliferation and heart regeneration.

FDA approved drugs with antiviral activity against SARS-CoV-2: From structure-based repurposing to host-specific mechanisms.

The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5'-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.

The Use of Mebendazole in COVID-19 Patients: An Observational Retrospective Single Center Study.

Background: An in-silico screen identified mebendazole with potential antiviral activity that could be a repurposed drug against SARS-CoV-2. Mebendazole is a well-tolerated and cheap antihelminthic agent that is readily available worldwide and thus could be a therapeutic tool in the fight against COVID-19. Methods: This is an observational retrospective study of PCR-confirmed COVID-19 patients who received mebendazole with the intention-to-treat. The study included an inpatient cohort (157 inpatients) and an outpatient cohort (185 outpatients). Of the 157 inpatients and 185 outpatients, 68 (43.3%) and 94 (50.8%) received mebendazole, respectively. Patients who presented within the same timeframe but did not receive mebendazole were used as controls. Patients received standard-of-care treatment including remdesivir, dexamethasone, and anticoagulants as deemed necessary by the treating physician. The following clinical outcomes were evaluated: for the inpatient cohort, length of stay (LOS) at the hospital, need for ventilation (combined invasive and noninvasive), and mortality; for the outpatient cohort, time to symptom resolution, need for hospitalization, and mortality. Results: For the inpatient cohort, the median age did not differ between the treatment and control groups; 62 (56, 67) vs. 62 (56, 68), P, and there was a comparable proportion of males in both groups; 43 (63%) vs. 55 (62%), P=0.85. The hospital LOS was 3.5 days shorter in the treatment group compared to the control group (P < 0.001). There were fewer patients who required invasive or noninvasive ventilation in the treatment group, 2 (2.9%) vs. 7 (7.9%), and the mortality rate is lower in the treatment group, 3 (4.4%) vs. 8 (9.0%), though the differences did not reach statistical significance. For the outpatient cohort, the median age was lower in the treatment group compared with the control group; 40 (34, 48) vs. 48 (41, 54), P < 0.001. There was a comparable proportion of males between both groups; 50 (53%) vs. 52 (57%), P=0.59. Patients in the treatment group were 3.3 days closer to symptom resolution (P < 0.001). There were numerically fewer patients requiring hospitalization in the treatment group compared with the control group, 3 (3.2%) vs. 6 (6.6%), though this did not reach statistical significance (P=0.33). Conclusion: In this retrospective observational study, the use of mebendazole in COVID-19 patients was associated with shorter hospitalizations in the inpatient cohort and shorter durations of symptom resolution in the outpatient cohort. The findings from this small observational study are hypothesis-generating and preclude drawing conclusions about clinical efficacy. Further studies are needed to examine the role of mebendazole in the treatment of COVID-19 patients.

Atovaquone for treatment of COVID-19: A prospective randomized, double-blind, placebo-controlled clinical trial.

Background: An in silico screen was performed to identify FDA approved drugs that inhibit SARS-CoV-2 main protease (Mpro), followed by in vitro viral replication assays, and in vivo pharmacokinetic studies in mice. These studies identified atovaquone as a promising candidate for inhibiting viral replication. Methods: A 2-center, randomized, double-blind, placebo-controlled trial was performed among patients hospitalized with COVID-19 infection. Enrolled patients were randomized 2:1 to atovaquone 1500 mg BID versus matched placebo. Patients received standard of care treatment including remdesivir, dexamethasone, or convalescent plasma as deemed necessary by the treating team. Saliva was collected at baseline and twice per day for up to 10 days for RNA extraction for SARS-CoV-2 viral load measurement by quantitative reverse-transcriptase PCR. The primary outcome was the between group difference in log-transformed viral load (copies/mL) using a generalized linear mixed-effect models of repeated measures from all samples. Results: Of the 61 patients enrolled; 41 received atovaquone and 19 received placebo. Overall, the population was predominately male (63%) and Hispanic (70%), with a mean age of 51 years, enrolled a mean of 5 days from symptom onset. The log10 viral load was 5.25 copies/mL vs. 4.79 copies/mL at baseline in the atovaquone vs. placebo group. Change in viral load did not differ over time between the atovaquone plus standard of care arm versus the placebo plus standard of care arm. Pharmacokinetic (PK) studies of atovaquone plasma concentration demonstrated a wide variation in atovaquone levels, with an inverse correlation between BMI and atovaquone levels, (Rho -0.45, p = 0.02). In post hoc analysis, an inverse correlation was observed between atovaquone levels and viral load (Rho -0.54, p = 0.005). Conclusion: In this prospective, randomized, placebo-controlled trial, atovaquone did not demonstrate evidence of enhanced SARS-CoV-2 viral clearance compared with placebo. However, based on the observed inverse correlation between atovaquone levels and viral load, additional PK-guided studies may be warranted to examine the antiviral effect of atovaquone in COVID-19 patients.

Parasitological, Molecular, and Epidemiological Investigation of Cryptosporidium Infection Among Cattle and Buffalo Calves From Assiut Governorate, Upper Egypt: Current Status and Zoonotic Implications.

Details about the epidemiological patterns and real contributions of different reservoir animals in maintaining the transmission cycle of Cryptosporidium spp. in Upper Egypt remain lacking. This study was designed to investigate the occurrence of Cryptosporidium spp. in cattle and buffalo (n = 608) from Upper Egypt. The parasite for the resulting positive samples by fecal examination was molecularly identified using nested PCR targeting the small subunit rRNA. Moreover, several explanatory variables, including animals' age, sex, condition, seasonal variations, were examined to describe the epidemiological pattern of the disease. Interestingly, the fecal examination revealed that 33.55% (204/608) of the animals under study were infected with Cryptosporidium, including 38.27% among cattle and 28.16% among buffalo. The parasite was molecularly identified using nested PCR, and their amplicons were identified in almost all fecal samples using microscopy (202/204). According to age as an individual variable factor, the infection rates of Cryptosporidium spp. in cattle calves with ages of <1, 1-3, and >3 months were 39.13, 34.04, and 54.54%, respectively. Meanwhile, in buffalo calves, the occurrence rates were 28.57, 27.27, and 29.41%, respectively. Regarding sex, female cattle calves were more susceptible to Cryptosporidium infection (51.28%) than males (26.19%) (p < 0.05), whereas male buffalo calves had a higher infection rate (32.25%) than females (25%). According to seasonal variations, the infection rates of Cryptosporidium spp. in cattle calves during spring, summer, autumn, and winter were 42.11, 30.43, 30, and 52.63%, respectively. In contrast, lower infection rates of 30, 21.42, 23.52, and 35% were reported in buffalo calves during spring, summer, autumn, and winter, respectively. The rate of infection was 45.16% in diarrheic cattle calves and 15.78% in non-diarrheic ones (p < 0.05). Meanwhile, the infection rate was 33.96% in diarrheic buffalo calves and 11.11% in non-diarrheic ones (p < 0.05). This study reported a higher occurrence of Cryptosporidium infection among the animals under study and revealed that buffalos and cattle can contribute to maintaining the transmission cycle of this zoonotic parasite in Upper Egypt.

Compartmentalized metabolism supports midgestation mammalian development.

Mammalian embryogenesis requires rapid growth and proper metabolic regulation1. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development2,3. Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle4,5. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.

NBS1-CtIP-mediated DNA end resection suppresses cGAS binding to micronuclei.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor-in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor-functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head-associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1's interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.

Extracellular vesicle-based interorgan transport of mitochondria from energetically stressed adipocytes.

Adipocytes undergo intense energetic stress in obesity resulting in loss of mitochondrial mass and function. We have found that adipocytes respond to mitochondrial stress by rapidly and robustly releasing small extracellular vesicles (sEVs). These sEVs contain respiration-competent, but oxidatively damaged mitochondrial particles, which enter circulation and are taken up by cardiomyocytes, where they trigger a burst of ROS. The result is compensatory antioxidant signaling in the heart that protects cardiomyocytes from acute oxidative stress, consistent with a preconditioning paradigm. As such, a single injection of sEVs from energetically stressed adipocytes limits cardiac ischemia/reperfusion injury in mice. This study provides the first description of functional mitochondrial transfer between tissues and the first vertebrate example of "inter-organ mitohormesis." Thus, these seemingly toxic adipocyte sEVs may provide a physiological avenue of potent cardio-protection against the inevitable lipotoxic or ischemic stresses elicited by obesity.

Mitochondrial fatty acid utilization increases chromatin oxidative stress in cardiomyocytes.

The inability of adult mammalian cardiomyocytes to proliferate underpins the development of heart failure following myocardial injury. Although the newborn mammalian heart can spontaneously regenerate for a short period of time after birth, this ability is lost within the first week after birth in mice, partly due to increased mitochondrial reactive oxygen species (ROS) production which results in oxidative DNA damage and activation of DNA damage response. This increase in ROS levels coincides with a postnatal switch from anaerobic glycolysis to fatty acid (FA) oxidation by cardiac mitochondria. However, to date, a direct link between mitochondrial substrate utilization and oxidative DNA damage is lacking. Here, we generated ROS-sensitive fluorescent sensors targeted to different subnuclear compartments (chromatin, heterochromatin, telomeres, and nuclear lamin) in neonatal rat ventricular cardiomyocytes, which allowed us to determine the spatial localization of ROS in cardiomyocyte nuclei upon manipulation of mitochondrial respiration. Our results demonstrate that FA utilization by the mitochondria induces a significant increase in ROS detection at the chromatin level compared to other nuclear compartments. These results indicate that mitochondrial metabolic perturbations directly alter the nuclear redox status and that the chromatin appears to be particularly sensitive to the prooxidant effect of FA utilization by the mitochondria.

Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain.

Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.

Experimental Hypoxia as a Model for Cardiac Regeneration in Mice.

Experimental hypoxia has been used for decades to examine the adaptive response to low-oxygen environments. Various models have been studied, including flies, worms, fish, rodents, and humans. Our lab has recently used this technology to examine the effect of environmental hypoxia on mammalian heart regeneration. In this chapter, we describe studies of systemic hypoxia in mice. We found that systemic hypoxia can blunt oxidative DNA damage and induce cardiomyocyte proliferation. While our primary interests are focused on cardiovascular research, these hypoxia protocols are applicable to any other organ system.

Mitochondrial Substrate Utilization Regulates Cardiomyocyte Cell Cycle Progression.

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.

A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.