A surprising discovery of live mitochondria in human blood

Written by: Abby Stahl

Original Article: Al Amir Dache et al. The FASEB Journal 2020
The Gist of It
You may have heard that mitochondria are the “powerhouse” of the cell, but what is DNA from these tiny organelles doing circulating in the bloodstream?  That was the question a team of scientists at the Institut Regional du Cancer de Montpellier in France asked in response to several studies showing that DNA from mitochondria could be found in circulation.
Mitochondria are important producers of cellular energy that evolved millions of years ago through a symbiotic relationship between primitive eukaryotic cells (our ancestors) and free-living prokaryotes (bacteria). Mitochondria have retained many of their bacterial features today, including having their own DNA. The fact that mitochondrial DNA is in the bloodstream has been known by doctors for years, and its presence can even be used to help diagnose diseases and conditions such as diabetes, cancer, and heart disease. Surprisingly, this team of researchers found that there are 50,000 times more copies of mitochondrial DNA in the bloodstream than nuclear DNA, or the genomic DNA at the “core” of the cell. However, the purpose of this mitochondrial cell-free DNA (McfDNA) was still unknown.
By collecting blood plasma from healthy volunteers, the scientists found that McfDNA was longer and more stable than nuclear cell-free DNA (NcfDNA), suggesting the McfDNA might be protected or encapsulated by some larger structure. The researchers went on to find that spinning the plasma at a high speed caused sedimentation of most of the McfDNA. Spinning at high speed can also cause intact mitochondria to be spun down. In fact, using a dye that labels live mitochondria, they found an abundance of the organelles within the centrifuged blood plasma. More powerful electron microscopy confirmed that these mitochondria existed without a cell around them, and metabolic studies showed that these mitochondria were functional at consuming oxygen, an important step in energy production.
These research studies are the first to report the presence of intact and functional mitochondria in blood from healthy individuals. The scientists estimate that there are between 200,000 and 3.7 million cell-free, intact mitochondria per milliliter of blood plasma and that these mitochondria might have crucial biological roles still to be discovered!
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Blood serum collected from healthy patients was found to contain live mitochondria capable of converting oxygen (O2) to energy (ATP) and encapsulating stable, circulating, cell-free mitochondrial DNA.

The Nitty Gritty
In this study, the authors began by comparing the stability of DNA found in the blood of healthy individuals. They hypothesized that NcfDNA would be more stable than McfDNA due to the presence of supporting histone proteins that mitochondrial DNA lacks. To their surprise, the McfDNA had a greater DNA Integrity Index, meaning the McfDNA had a mean size greater than 300 base pairs when assessed from 13 blood samples. Sequencing of cell-free DNA from 80 additional individuals showed that McfDNA contained two populations of DNA – small fragments between 30–300 base pairs (approximately 20%) and large pieces greater than 1000 base pairs (approximately 80%); these populations could not be sequenced due to limits of sequencing technology.
To further determine the structure of this stable McfDNA, the researchers centrifuged blood samples at increasing speeds to sediment different components. Interestingly, the McfDNA sedimented at 16,000 × g, whereas NcfDNA was retained in the blood plasma, suggesting McfDNA was larger and denser. Importantly, the researchers also verified these findings in cells that had been cultured in media to confirm that the McfDNA was not being released by platelets during the experimental handling of blood samples, showing an abundance of McfDNA pelleted at 16,000 × g in cell cultures. In all cases, the McfDNA was too large to pass through a 0.22 mm filter, suggesting it might be retained within mitochondria organelles themselves, which are typically 0.4–1.5 mm in size and are known to sediment at 7,000–20,000 × g .
To confirm these suspicions, the scientists used MitoTracker Green dye to visualize live mitochondria and found them in serum samples and cell media supernatants. Western blot analysis also confirmed the presence of membrane transport proteins, TOM22 and TIM23, in these McfDNA fractions. Finally, the researchers used electron microscopy and identified structures with a double membrane and similar size to mitochondria in the samples, although they noted some morphological differences.
After determining that mitochondria were present and surrounding the McfDNA in blood and cell samples, the scientists asked whether these mitochondria were metabolically functional and able to produce energy, a process known as respiration. Using a Seahorse XF flux analyzer, the researchers found that the isolated mitochondria consumed oxygen and were sensitive to inhibition and activation by known regulators of respiration.
These studies show that human blood contains a previously unknown and significant population of mitochondria that can be useful for diagnostics; however, the physiological roles of these cell-free organelles (if any) remain a mystery for now.
Original Research Article: Al Amir Dache Z, A., et al. “Blood contains circulating cell‐free respiratory competent mitochondria.” FASEB J (2020). doi: 10.1096/fj.201901917RR. [Epub ahead of print].

Delivering Drugs with Sound, Part 2: Treating muscles to treat the brain

Written by: Ken Estrellas

Original Article: Li et al. Molecular Therapy Methods & Clinical Development 2020
The Gist of It
You may recall that in my last post, I presented a study that used ultrasound waves, high-frequency sound waves typically undetectable by human hearing, to deliver drugs to cartilage. A recent study published by Li and colleagues extended this concept to a different area of the body – the brain – and applied it to a significant clinical issue that my fellow contributor Abel Cortinas wrote about in the final post of 2019: Alzheimer’s disease (AD). As Abel mentioned, a protein called amyloid beta (Aβ) clumps up in the brain of patients with AD, forming plaques associated with the disease. Previous studies have shown that there is less of an enzyme called neprilysin in the brains of patients with AD; also, getting rid of neprilysin increases the amount of Aβ made. Consequently, increasing the amount of neprilysin might help reduce the presence of these clumps, potentially relieving the symptoms of AD. However, increasing levels of neprilysin in the brain can be difficult from a practical standpoint, since administering the enzyme itself to the brain or using viral gene therapy to produce the enzyme both come with a significant risk of complications. In this study, the authors take an interesting approach: using ultrasound to inject modified genetic material known as plasmids that can lead to the production of neprilysin. However, instead of injecting this gene therapy directly into the brain, the leg was selected as an injection site instead. Since muscles contain a large number of blood vessels, the injected plasmids are able to circulate throughout the body, including the brain. The authors tested this approach in mice with a form of AD and observed that mice treated with ultrasound injections of plasmid had both more neprilysin and less Aβ protein in the brain one month after injection. These treated mice were also better able to navigate a complex water maze than untreated AD mice, suggesting that cognitive decline (a symptom of AD) had slowed. Importantly, the treated mice showed no signs of damage to the injected muscles or systemic damage in the kidneys. Overall, this study presents an alternative approach to the treatment of AD that appears promising and deserves further investigation.
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Ultrasound was used to deliver a gene therapy to muscle, with the effects observed in the brain

The Nitty Gritty
In this study, Li et al. explored the potential of ultrasound-assisted administration of 11,090 base pair plasmids to hindlimb muscles to more effectively overexpress neprilysin in the brain tissue of APP/PS1 mice, a model of AD, decreasing local concentrations of Aβ protein. The plasmids, which mediated overexpression of human neprilysin (hNEP) under the control of a cytomegalovirus (CMV) promoter, were delivered via microbubbles into the gastrocnemius muscles of N = 20 APP/PS1 mice with the simultaneous administration of 1.7 MHz, 1.0 W/cm2 ultrasound irradiation for one minute. One month after injection, increased levels of neprilysin were observed in the brains and skeletal muscles of hNEP-treated APP/PS1 mice compared with levels in untreated APP/PS1 mice, as determined by western blot. Significantly decreased levels of Aβ were detected in the brains of treated mice via western blot after one month compared with the levels in untreated mice, and this was visually observed via immunohistochemistry. A Morris Water Maze was used to compare potential functional improvements in treated vs untreated mice; treated mice showed a significantly decreased time to find a hidden platform in the shallow water maze (latency) and traveled a significantly shorter distance to do so (distance) after one month of treatment, whereas wild type C57/BL control mice and untreated APP/PS1 mice did not demonstrate any differences. Histological assessment of skeletal muscle, lung, and kidney tissue from treated mice using hematoxylin and eosin (H&E) staining revealed no signs of significant damage after one month. These results appear to present an initially promising strategy to overexpress neprilysin specifically in brain tissue without the need to cross the blood-brain barrier with recombinant enzyme or the complications associated with virally mediated gene therapy. Nonetheless, the authors acknowledge the need to assess the half-life of overexpressed hNEP, and the potential for long-term functional improvements – or declines – remain unknown.
Original Research Article: Li, Y., et al. “Expression of neprilysin in skeletal muscle by ultrasound-mediated gene transfer (sonoporation) reduces amyloid burden for Alzheimer’s disease.” Mol Ther Methods Clin Dev (2020) [Epub ahead of print].

Malaria: When fighting infection in your body doesn’t actually fight infection for the community

Written By: Rebecca Tweedell

Original Article: Joyner et al. PLoS Pathogens 2019
The Gist of It
Malaria is a devastating disease resulting in more than 228 million cases worldwide and over 400,000 deaths in 2018, mostly in children under the age of 5. Due to the major global public health threat of this disease, there has been intense effort to study it. We know that malaria is caused by the Plasmodium species of parasites. These parasites have a complicated lifecycle, which is one of the reasons it is so challenging to study them. A key feature of some Plasmodium parasites’ lifecycle is that they can form hypnozoites, dormant forms that act like spores, hiding silently in the liver until they reactivate later on to cause what is known as a relapse, or a second infection after the initial “primary” infection is already over. A recent study from scientists at the Malaria Host-Pathogen Interaction Center at Emory University explored how the immune system responds to these relapses. In a rhesus macaque model of infection, they found that, in contrast to primary infections, relapses did not cause the typical symptoms of fever and inflammation. Also, during relapses, the number of parasites in the blood did not get as high as it did during the primary infection. This is at least partly because memory B cells were activated; memory B cells are important immune cells that “remember” a previous infection the body has had, allowing it to fight the pathogen more successfully the second time around. The fact that this immune response led to the absence of clinical symptoms and a lower number of parasites in the blood during relapse seem like a promising result, but upon further exploration of the parasites that were in the blood, the researchers made a worrying finding. While the total number of parasites was lower during relapse, the percentage of these parasites that were gametocytes (the form of the parasite that can be picked up by a mosquito to allow transmission of the pathogen) was actually much higher than in primary infections. This means that although the immune response is doing a good job of limiting the infection, it is not able to stop the production of gametocytes and prevent the further transmission of the parasite. So while your body may be fighting its own infection successfully, this may not translate to a reduction in the number of infections in the community overall. This is an important point to know as we continue to search for strategies to fight this pathogen and prevent its deadly spread.
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During infection with Plasmodium, memory B cells formed as a result of the primary infection can keep parasite numbers low during relapse, but gametocytes are still around and make up a larger percentage of the parasites, meaning the parasite can still be transmitted to other people.

The Nitty Gritty
Joyner et al. established a model of primary infection and relapse using P. cynomolgi in rhesus macaques. The macaques were inoculated with sporozoites to initiate the primary infection. After peak parasitemia in the blood was reached, a single sub-curative dose of artemether was given to reduce but not eliminate the parasites (to prevent the development of severe disease), and then the parasitemia was allowed to rebound. Then the macaques were treated with a curative artemether regimen to clear all blood stage parasites. Subsequently, they were monitored for the occurrence of relapse, which would be due to the emergence of hypnozoites based on this infection and treatment scheme. They observed that relapses were not as clinically severe as primary infections, with anemia, fevers, and thrombocytopenia absent during relapse. Transcriptional analysis of whole blood samples revealed that clusters of genes associated with B cells, T cells, cell signaling, and antigen presentation were upregulated during relapse. By analyzing the peripheral blood mononuclear cells with flow cytometry, they found that during relapse the number of unswitched and switched memory B cells increased. They analyzed the functionality of the antibodies produced by allowing opsonization of Plasmodium-infected red blood cells (iRBCs) with heat-inactivated plasma and testing for phagocytosis by THP-1 cells. They found that the plasma collected during relapse increased the percentage of phagocytosed iRBCs compared with the plasma from naïve animals and compared with the plasma collected during the peak of parasitemia during the primary infection. Finally, the researchers analyzed the effects of the immune response on the parasites during relapse. They found that the overall number of parasites in the blood during relapse never reached the level achieved during primary infection. When they further parsed the data to look at the number of gametocytes specifically, the overall number was also reduced during relapse compared with primary infection. However, the percentage of parasites that were gametocytes was significantly higher during relapse. This suggests that while a strong adaptive immune response is formed that can limit the clinical manifestations of disease, this response will not actually prevent gametocyte formation and subsequent transmission.
Original Research Article: Joyner, C.J., et al. “Humoral immunity prevents clinical malaria during Plasmodium relapses without eliminating gametocytes.” PLoS Pathog 15.9 (2019): e1007974.

How are babies kept safe from mom’s immune system?

Written By: Kaitlyn Sadtler

Original Article: Salvany-Celades et al. Cell Reports 2019
The Gist of It
Why is it that our body rejects transplant organs (even from relatives) but mothers’ bodies are able to safely carry a developing fetus? Nutrients and oxygen travel from the mother to the fetus to support its growth, but normally the mother’s immune cells do not reject this foreign being inside of their own body. Certain kinds of immune cells – known as regulatory T cells (Tregs) – do just as their name suggests, they regulate immune responses. These cells are found at the interface between the fetal and maternal tissue, within the placenta. The placenta is composed of cells from both the mother and the fetus, with blood from both being separated by only a few cells. This allows the oxygen and nutrients from the mother to transfer to the developing fetus, but also provides a site where maternal and fetal cells directly interact. The Tregs here receive signals from fetal cells known as extravillous trophoblasts (“extra” = outside of, “villous” = the fetal blood vessels, and “trophoblasts” = cells from the embryo derived from the same cells that make up the amniotic sac) and also maternal decidual macrophages (“decidua” = lining of pregnant uterus). These cells communicate with the Tregs to prevent immune rejection of the fetus. Salvany-Celades and colleagues described three distinct types of Tregs that are found in this interface. These three cell types can inhibit the activity of both activated helper T cells, which can normally produce proinflammatory cytokines, and cytotoxic T cells, which normally kill cells that are infected by viruses (or donor cells in transplants). As we learn more about the way that these immune cells are working in an optimal setting, we are more aware of signs to look for to identify when something is going wrong; importantly, this also allows us to develop new treatments to help prevent damaging immune responses against developing fetuses.
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Three distinct types of regulatory T cells help train mom’s immune system to prevent it from reacting against the developing fetus.

The Nitty Gritty
Salvany-Celades et al. identified three subtypes of regulatory T cells in the decidual tissues of the maternal-fetal interface in first trimester decidua. These three are divided based on their expression of surface markers: (1) CD25hiFoxP3+, (2) PD1hiIL-10+, and (3) TIGIT+FoxP3dim. These cells were sorted and then tested for their ability to prevent CD4+ and CD8+ effector T cell activation. All three cell types reduced the proliferation of CD4+ effector T cells as determined by CFSE labeling and co-culture and stimulation with anti-CD3 anti-CD28 beads. The first class of Tregs (CD25hi) also inhibited CD8+ T cell proliferation, whereas the other two classes did not. Furthermore, when these classes were isolated from term placenta, they did not have as great of a regulatory capacity as first-term Tregs. The PD1hi Tregs were dependent upon IL-10 signaling, as determined by IL-10R antibody blocking, but this was not required for CD25hi Tregs. When co-cultured with peripheral CD4+ T cells, extravillous trophoblasts (EVTs) and decidual macrophages were able to increase the proportion of FoxP3+ and HELIOS+ Tregs. EVTs and decidual macrophages were also able to induce Tregs in a transwell system, but to a lesser extent than with direct co-culture, suggesting both soluble signals and cell-cell contact are important in the generation of iTregs (induced Tregs) by EVTs and decidual macrophages. Blocking HLA-C on EVTs decreased their ability to promote the differentiation of PD1hi Tregs through cell-cell contact. Overall, this work describes the interaction of three types of Tregs within decidual tissue of first-trimester pregnancies that are educated by both EVTs and decidual macrophages to promote regulation and prevent pathogenic immune responses in pregnancy.
Original Research Article: Salvany-Celades, M., et al. “Three types of functional regulatory T cells control T cell responses at the human maternal-fetal interface.Cell Rep 27.9 (2019): 2537–2547.

Evaluating Alzheimer’s disease through a better understanding of GAD67

Written By: Abel B. Cortinas

Original Article: Wang et al. Molecular Neurodegeneration 2017
The Gist of It:
Alzheimer’s disease (AD) is a complex, irreversible, and usually age-related brain disorder that slowly destroys one’s memory and thinking skills. Although it is difficult to accurately measure the exact number of people affected, the US National Institute on Aging estimates that approximately 5.5 million Americans have Alzheimer’s dementia, with most of them age 65 and older. There are currently five prescription drugs approved by the US Food and Drug Administration to treat the cognitive symptoms (that is, memory loss, confusion, and problems with thinking and reasoning) of AD. Unfortunately, there is no cure or disease-modifying therapy to treat the actual cause of AD, in large part because of the complexity of the disease itself. And, although there are currently over 130 drugs in clinical trials for the treatment of AD, the failure rate of all drugs over the past 15 years in AD research is daunting (greater than 99%!). Researchers at both Pennsylvania State University and South China Normal University studied a specific protein, GAD67, known to be involved in signaling pathways in the brain and linked to a number of other neurological disorders (like epilepsy and schizophrenia). Using mice that developed AD but also had less of the GAD67 protein, researchers revealed three key findings. First, reducing the amount of the Gad67 gene (which is responsible for making the GAD67 protein) present led to a significant decrease in the amount of a different protein called amyloid beta. Amyloid beta is notoriously known for clumping up and forming plaques in the brains of patients with AD. Second, having less of the GAD67 protein resulted in less inflammation in the brain as well as lower amounts of an important signaling molecule in the brain called gamma amino-butyric acid, or GABA for short. Lastly, these mice partially recovered their sense of smell and memory association, a condition known as the olfactory (that is, the sense of smell) sensory deficit, which is a reported symptom in patients with AD. Together, these findings suggest that the Gad67 gene and protein play an important role in the development and progression of AD. This result provides a potential strategy for treatment through inhibition or gene silencing / editing.
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THE SELECTIVE KNOCKDOWN OF THE Gad67 GENE IN MICE POTENTIALLY PLAYS AN IMPORTANT ROLE IN MODULATING THE EFFECTS OF ALZHEIMER’S DISEASE, A DISEASE THAT CURRENTLY HAS NO APPROVED THERAPIES FOR TREATMENT

The Nitty Gritty:
Wang et al. created an Alzheimer’s disease (AD) mouse model with Gad67 haploinsufficiency, referred to as AD+GG+ mice, by crossing GAD67-GFP knock-in mice with 5xFAD mice that had five human familial AD mutations in amyloid precursor protein and presenilin-1 genes. The researchers found that Gad67 haploinsufficiency resulted in significantly lower amounts of amyloid beta (Aβ) burden in the AD+GG+ bigenic mice compared with amounts in 5xFAD mice. Aβ42 immunostaining at the frontal cortex as well as quantitative detection using an Aβ ELISA protocol in the olfactory bulb and the piriform cortex demonstrated downregulated Aβ production in Gad67 haploinsufficient 5xFAD mice. Quantitative analysis of Aβ42 in the hippocampus demonstrated a slight reduction in the amount of Aβ in AD+GG+ mice, but the reduction in the hippocampus was not as significant as in the cortical areas. Further immunostaining with thioflavin-s verified that Aβ deposits were reduced in both the frontal cortex and piriform cortex of the bigenic mice. The effect of Gad67 haploinsufficiency on both GABA production and electrophysiological tonic GABA currents in astrocytes was also studied. Immunostaining revealed significantly less astrocytic GABA in the Gad67 haploinsufficient bigenic mice compared with normal 5xFAD mice. Quantitative analyses performed in the frontal cortex, piriform cortex, and hippocampal CA1 regions demonstrated a uniform reduction in astrocytic GABA. Contrastingly, neuronal GABA was not significantly changed across all groups of mice. Subsequently, whole-cell patch-clamp recordings were performed on the layer IV-VI cortical neurons to determine whether reduced GABA content resulted in reduced tonic GABA currents. They found that Gad67 haploinsufficiency resulted in a reduction of the abnormal GABA tonic currents in cortical neurons of 5xFAD mice. Next, microglia reactivity was assessed. Due to prior research suggesting that microglia activity is regulated by GABA production from astrocytes, immunoreactivity was measured after exposure to the proinflammatory reagent nitric oxide synthase (iNOS). In Gad67 haploinsufficient mice, the iNOS immunoreactivity was significantly reduced compared with immunoreactivity in non-AD mice in the frontal cortex and piriform cortex, as assessed by immunostaining and subsequent quantitative analysis. Lastly, Wang et al. investigated whether Gad67 haploinsufficiency rescued olfactory deficits in 5xFAD mice. Odor habituation and cross dis-habituation behavior tests were performed on all groups. The results indicated that Gad67 haploinsufficiency rescued olfactory deficits in 5xFAD mice. Taken together, Gad67 gene deficiency provided reasonable evidence to support the targeting of both the gene and its resulting protein as a potentially promising strategy to combat AD.
Original research article: Wang, Y., et al. “Gad67 haploinsufficiency reduces amyloid pathology and rescues olfactory memory deficits in a mouse model of Alzheimer’s disease. 12.1 (2017): 73.

A Healthy Gut Leads to a Healthy Heart

Written by: Sayan Roychowdhury

Original article: Battson et al. American Journal of Physiology Heart and Circulatory Physiology 2019
The Gist of it:
Many people have heard that the bacteria in our gut play an important role in digestion, but did you know they can also affect your weight and cardiovascular health? Although some bacteria are associated with disease, others are vital for your immune system, heart function, and weight control. And understanding this link can be extremely important – as of 2018, almost 30% of the world’s population is considered obese; within the United States, this number is up to 40%. In overweight people, cardiovascular disease (CVD) is more than twice as likely to lead to death than it is in healthy-weight people. This increased risk is caused by stiffer arteries and restricted blood flow to the heart. Finding ways to manage the risk of CVD is crucial, especially for obese patients.
Mice that are obese have vastly different microbiomes, the population of microbes living inside their gut, than mice that are a healthy weight. This knowledge begs the question, is it possible for obese mice to have a decreased risk of dying from CVD if they have a healthy gut microbiome? A group of scientists from Colorado State University think so. Their recent paper explores the connection between the gut microbiome and physical changes to the heart and nearby arteries. These researchers exchanged the gut microbes between obese mice and healthy-weight mice and studied their cardiovascular pathology at the end of an 8-week period. They discovered that when obese mice were given gut microbes from healthy-weight mice, it reduced their cardiovascular pathology. Healthy-weight mice transplanted with the obese gut microbes displayed the opposite effects and had increased pathology. These results are exciting because they show that altering the gut microbiome could be an important strategy to combat the risk of CVD in people, in addition to diet and weight loss.
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Swapping microbes from the guts of healthy-weight and obese mice leads to changes in their cardiovascular pathology.

The Nitty Gritty:
Cecal contents were collected from two sets of mice, obese leptin-deficient (Ob) and control (Con) C57BL/6J, and used as donor samples for cecal transplants. The endogenous gut microbiota in recipient mice was suppressed prior to the transplants. Four experimental groups were created: 1) control mice given control microbiota (Con + Con), 2) control mice given obese microbiota (Con + Ob), 3) obese mice given control microbiota (Ob + Con), and 4) obese mice given obese microbiota (Ob + Ob). All groups were confirmed to exhibit the transplanted microbiota by analyzing fecal matter collected from the colon. To measure arterial stiffness, an aortic pulse wave velocity was calculated using the distance between the two probes divided by the time between the ECG R wave and the tail end of the Doppler signal. The arterial stiffness was significantly higher in mice from the Con + Ob group compared with Con + Con mice, while the stiffness in mice from the Ob + Con group was slightly lower than in mice from the Ob + Ob group. To determine the size of myocardial infarcts, no-flow global ischemia was first induced in the excised hearts, and then the hearts were reperfused for 2 hours. Finally, the left ventricle (LV) was isolated, weighed, and stained to distinguish living from infarcted tissue. LV mass was shown to be increased in both of the groups transplanted with the Ob microbiota. Infarct size was up to 20% larger in hearts from the Con + Ob group compared with hearts from the Con + Con groups, while the size in Ob + Ob hearts was significantly larger than in Ob + Con hearts. In summary, this work provides evidence that a change in gut microbiota composition can lead to modified cardiovascular pathology, independent of weight and diet.
Original research article: Battson, M.L., et al. “Gut microbiota regulates cardiac ischemic tolerance and aortic stiffness in obesity.” Am J Physiol Heart Circ Physiol 317.6 (2019): H1210–H1220.

 

 

Treating Heart Failure by “Vagus Nerve Stimulation (VNS)” implanted devices

Written By: Sravya Kotaru

Original article: Ouyang et.al. Gene 2019
The Gist of it:
The human heart pumps blood in two steps – first, blood fills into the two lower chambers of the heart called ventricles; then, the heart muscle contracts and pushes the blood out of the ventricles to other organs through blood vessels. Chronic systolic heart failure happens when the heart is not able to push enough blood out when it contracts. The main cause is an imbalance between signals that activate the heart pump and those that relax it. This results in an over-active and tired heart. Medications commonly prescribed for this type of heart failure aim to reduce the signals that activate the heart and bring back the balance. A new treatment focuses on the other side of the balance by electrically stimulating a nerve that typically relaxes the heart, called the vagus nerve. This treatment uses a device implanted under the skin near the collar bone that sends electrical signals to the vagus nerve in the neck, where it connects the brain to the heart. Scientists found that when the vagus nerve is stimulated, the heart makes increased amounts of a special type of RNA called microRNA-183-3p. MicroRNAs, unlike the more well-known mRNAs, do not contain the information to make proteins. Instead, they block certain mRNAs from making their specific proteins. MicroRNA-183-3p reduces the production of a protein called BNIP3L in the heart. In an over-active heart, BNIP3L increases the removal of mitochondria, the power plants of the heart that generate energy for its function; this removal makes the heart tire out. VNS treatment reduces the amount of BNIP3L in the heart and reverses its over-activating effects, thus protecting the heart from failure. This research has not only explained how VNS treatment is effective at the molecular level but has also opened up the possibility of developing new drugs that can mimic the effects of VNS to treat heart failure.
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Vagus nerve stimulation treats chronic systolic heart failure and increases blood output from the heart by increasing the amount of microRNA-183-3p in the heart, which in turn reduces the amount of BNIP3L protein. This restores energy generation in the heart by limiting the BNIP3L-mediated removal of the energy powerhouses, called mitochondria, thus protecting the heart from tiring out and failing.

The Nitty Gritty:
Chronic systolic heart failure (CSHF) is a complex disorder characterized by insufficient blood circulation to the lungs and other organs. A recent therapeutic method that has proven to be effective in patients with CSHF is vagus nerve stimulation (VNS), which involves electrically stimulating the parasympathetic cranial nerve X (or vagus nerve) that connects the brain to the heart, throat, and abdomen. Using an animal model that reproduced CSHF mitigation by VNS treatment in rats, Ouyang et al. investigated the molecular mechanisms of VNS action. The authors observed that VNS treatment increased the left ventricle ejection fraction (LVEF, i.e. output from systolic contraction) and decreased the left ventricular end-systolic and end-diastolic volumes (LVESF and LVEDF). VNS treatment also decreased the amount of B-type natriuretic peptide (BNP) hormone, indicating lower blood volume and heart wall extension at the end of each contraction. They observed that VNS-treated rats had higher amounts of the microRNA (miR)-183-3p and the anti-autophagy marker Bcl-2 and lower amounts of the pro-autophagy markers LC3 II/I and Beclin-1. They further observed that miR-183-3p directly silences the expression of the Bcl-2–interacting protein 3-like (BNIP3L) protein, which is known to initiate autophagy by recruiting the LC3 phagosome components. Moreover, the VNS-mediated increase in the amount of miR-183-3p led to a decrease in the amount of phosphorylation and thus inactivation of the mTOR and Akt signaling molecules. In summary, the authors discovered that VNS alleviates CSHF via the upregulation of miR-183-3p in cardiac cells and through the subsequent suppression of BNIP3L-initiated autophagy in an mTOR/Akt pathway-dependent manner.
Original Research Article: Ouyang, S., et.al. “MicroRNA-183-3p up-regulated by vagus nerve stimulation mitigates chronic systolic heart failure via the reduction of BNIP3L-mediated autophagy.” Gene (2019). doi: 10.1016/j.gene.2019.144136 [Epub ahead of print].