filariae and force generators

I won’t lie; being the geeked-out loser that I am, I love reading about modern science. I’m often most impressed by advances in technology when I see the depths we’re able to probe into the origins of biological processes. Yet, the excitement I get from reading about novel therapeutics or mechanistic insights of the modern era is often dwarfed by how I feel when I read about seminal studies that laid the foundation for modern wizardry with barely a fraction of the tools. Two examples follow.

William Withering and the Foxglove
A terrific paper published last week in Science discusses the discovery and implementation of a new drug, omecamtiv mecarbil, for systolic heart failure. Basically, the authors started by revisiting the function of cardiac myocytes. Like any muscle fiber, cardiac muscle contracts via a cooperative actin-myosin system, for which myosin is the key. The myosin heavy chain has a head with two critical components: an ATP-hydrolyzing domain, and an actin-binding site. It also has a spring-like neck which allows for force transduction. The process by which myosin coordinates muscle contraction is actually quite interesting. In its “resting” state, myosin is bound to ATP; the ATP-bound myosin has very weak catalytic activity which will, over time, lead to slow hydrolysis and consumption of ATP. When a muscle is stimulated, release of acetylcholine at the neuromuscular endplate leads to activation of g-protein coupled receptors which, via increase of intracellular cAMP, leads to release of calcium from the sarcoplasmic reticulum. Calcium then binds to troponin C and tropomyosin, which release the actin-interacting region of the myosin head to bind actin. This event is critical, as actin-bound myosin has a higher rate of ATP hydrolysis; subsequenthydrolysis and release of inorganic phosphage by actin-bound myosin leads to the classic shortening of the myosin neck and the so-called “power stroke”, causing shortening of the sarcomere. In parallel, many of these shortenings lead to muscle contraction. Now, the key here is that this is a high-energy process (as anyone who exercises can tell you) but the majority of energy consumption comes not from the ATP hydrolysis by myosin but by the energy required to actively transport calcium back into the sarcoplasmic reticulum. The other key aspect of this process is that the rate-limiting step in contraction is the transition from weak (ATP-bound)actomyosin interaction to strong (ADP bound) actomyosin interaction → the rate of this step is limited by the rate of actin-bound ATP hydrolysis.

We’ll get back to this story in a second, but lets digress and talk briefly about heart failure. Systolic heart failure, in which the primary defect is in the contractility of the heart, is the dominant form of heart failure in the US (unlike in, for example, Africa, where diastolic heart failure appears to dominate). Improving contractility in heart failure takes two forms: optimizing conditions for cardiomyocyte contraction, and improving intrinsic cardiomyocyte function. By optimizing conditions for contraction we mean a) preload optimization to optimize starling forces (this is where beta-blockers are most useful) and b) afterload reduction to improve contractility (this is where ACE inhibitors and spironolactone appear to be most useful). There is a third aspect, diminishing pathologic cardiac fibrosis, in which both ACE inhibitors and spironolactone appear to be useful.

Improving intrinsic contractility is more difficult. The only drug that is thought to achieve this in an outpatient setting is digoxin. Which brings us to (drum roll) William Withering. Who was he? Well, he was born in 1741 in England, the son of a successful apothecary. He studied medicine at the University of Edinburgh where he had a particular interest in a syndrome known as “dropsy” (short for hydropsy, an old term for edema). He ultimately took up practice in the local village of Stratford, where, due to the interest of his new wife, an artists who specialized in botanical arrangements, he became interested in botany. In order to supplement his income, he moved in 1775 to Birmingham and became a member of the famous “Lunar Society” (a group of high-minded people who met at the monthly full moon and whose members included Charles Darwin’s grandfather, the discoverer of oxygen, the inventor of the steam engine, and Benjamin Franklin). Withering’s story becomes relevant to this scatterbrained post in 1775, when he becomes alerted to a familyremedy for dropsy: an herbal tea made from the leaves of the English foxglove plant, the active ingredient of which was digitalis.

It is important to note that Withering did not discover digitalis; indeed, it had been used since the 1200s for a variety of conditions. However,Withering’s unique skillset as a physician and botanist allowed him to study the effects of foxglove and report, in a landmark book, An Account of the Foxglove, a careful summary of 163 case studies over the course of a decade. In the book, he describes the preparation and administration of the drug, as well as a highly accurate description of digitalis toxicity (including overdiuresis, GI irritation, visual changes, and life threatening bradycardias and bradyarrhythmias). He then offers advice for therapeutic administration of digitalis, which, to this day, remains the only outpatient drug known to improve cardiac contractility.

And, that brings us back to the current day. Digoxin is obviously not the only drug that improves contractility; in the hospital (specifically, in the ICU), drugs known as inotropes are used to augment cardiac output. Chief among these drugs are dobutamine (a beta-1 receptor agonist) and milrinone (a phosphodiesterase 3 inhibitor). Both act ultimately by increasing intracellular levels of cyclic AMP, which increase calcium levels and therefore augment contractility. However, there are three major problems with use of these drugs.

• Both drugs act peripherally as well as centrally, causing peripheral vasodilation. In patients with cardiogenic shock, this can cause life-threatening hypotension
• Both drugs act by increasing intracellular calcium; because this is the primary avenue of ATP consumption in the cardiomyocyte, these drugs significantly increase myocardial oxygen consumption, which is a big problem in patients who have cardiogenic shock secondary to an ischemic event (like a myocardial infarction)
• Both drugs are highly arrhythmogenic for multiple reasons (they cause electrolyte abnormalities as well as tachycardia).

So these drugs are a problem (and the side effects are probably why use of these drugs has never had a proven mortality benefit). So we come back to our brilliant authors, who looked at the normal cycle of actomyosin contraction and realized that there was another area that could improve contractility. What if there were a drug which acted to speed up the rate-limiting step of contraction (ATP hydrolysis by the myosin head)? Better yet, what if this drug increased the rate of ATP hydrolysis by the myosin head only when it was bound to actin, therefore improving the efficiency of actomyosin interaction?

Well, that’s exactly what their drug, the inexplicably named omecamtiv mecarbil, does – it’s a cardiac myosin-specific ATPase agonist which preferentially acts when myosin is actin-bound. The drug clearly increases sarcomere shortening without changing the amount of intracellular calcium elevation (pictured); the net effect is a significant increase in stroke volume while sparing any effects on the heart rate and a net increase in cardiac output. So what are the problems with this drug? Well, there’s one main problem: because the drug potentiates ATP hydrolysis, it lengthens the amount of time that myosin interacts with actin. The net effect of this is that it increases the amount of time that the heart is in systole. Sounds good, but the problem is that coronary artery filling happens during diastole (when the coronary arteries aren’t compressed). So the dose is limited by myocardial ischemia from impaired diastolic filling. Still, this is an orally bioavailable drug which means that it may be valuable for patients with heart failure in both an inpatient and outpatient setting.

On to the next!

Worms.
So, filiariasis (infection by the thread-like filarial roundworms) is pretty gross. The key to their causing repulsive conditions like elephantiasis (where they obstruct the lymphatic system), disseminated rashes and urticaria, so-called “river blindness” (where one filiarial worm infects the eyes), and even serous cavity filariasis (where they occupy the peritoneal cavity) is their ability to disseminate while avoiding recognition by the immune response.

So scientists have been interested in how, exactly, they accomplish this stealth process for quite a long time. And in 1980, in a paper published in the New England Journal of Medicine, they pretty much figure it all out, with the use of some primitive tools. They isolated peripheral blood white blood cells from adult volunteers in Indonesia with recurrent lymphadenitis characteristic of filiariasis. They would then stimulate white blood cell isolates with either filarial antigens or, as a control, tuberculosis antigens, and compare the growth of stimulated to non-stimulated cultures (which they termed “stimulation index.”)

Their results, with these simple assays, were remarkable. To start, the authors found that peripheral cells from patients had no response to either TB or filarial antigens. However, when they depleted adherent cells from the peripheral cultures, responses to filarial antigens spiked (indicating that adherent cells were specifically suppressing anti-filarial responses).Additionally, if they delayed antigen exposure overnight before stimulating the peripheral cells, responses to filarial antigens also increased, suggesting that adherent cells are continuously suppressing anti-filarial responses in an antigen-dependent manner.

Finally, they found that supernatants from patients with filariasis suppressed both antifilarial and, to a lesser extent, anti-TB responses, suggesting that suppression by adherent cells occurred via secretion of soluble factors. All this, just by looking at cell counts!

In the years following, studies of the filarial nematode Acanthocheilnema vitae yielded a protein, ES-62, which was found to have numerous suppressive effects on the immune system. Notably, it was found to block macrophage production of Th1-type cytokines (sound familiar?) leading to preferential Th2 differentiation of T cells and suppressing T cell proliferation. It was also found to be clinically useful in the treatment of inflammatory arthritis and allergic asthma by suppressing macrophages and mast cells. In last week’s Nature Immunology, the authors found that recombinant ES-62 suppresses systemic inflammation induced by both TLR4 and TLR2 (2 innate immune receptors highly upregulated on macrophages and neutrophils during sepsis) by inducing ubiquitin-mediated degradation of TLR4 and the critical adaptor protein MyD88.

They then demonstrated that in a well-known mouse model of sepsis in which the cecum is ligated to serve as a nidus for infection, then punctured to release bacteria into the bloodstream, ES-62 can not only prevent sepsis but also treat it if administered within 6 hours of bacteremia while at the same time not interfering with clearance of bacteria from the peritoneum or bloodstream. Now don’t get me wrong; the ability to take those initial findings, isolate the protein responsible, and demonstrate its potential therapeutic efficacy is terrific. But the critical findings: that adherent peripheral blood cells produce a soluble factor in response to filarial antigens that effectively suppresses the adaptive immune response, were known over thirty years ago, and were discovered with little more than some volunteers, a centrifuge, an incubator, and a microscope.