Do the left half and right halves of the diaphragm undergo the same displacement during breathing?

Do the left half and right halves of the diaphragm undergo the same displacement during breathing?

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Do the left half and right halves of the diaphragm of a normal person move exactly the same distance between inhaling and exhaling?

A short answer is that the displacement of the right and left part of the diaphragm during breathing may not be the same.


Thoracic diaphragm (Wikipedia):

In humans, the diaphragm is slightly asymmetric-its right half is higher up (superior) to the left half, since the large liver rests beneath the right half of the diaphragm. There is also a theory that the diaphragm is lower on the other side due to the presence of the heart.

Genetic specification of left-right asymmetry in the diaphragm muscles and their motor innervation (PubMed):

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry.


Excursion (displacement during breathing) of the right and left part of the diaphragm may or may not be the same, which may differ from case to case.

Manual evaluation of the diaphragm muscle (PubMed):

In most cases, the diaphragm shows a symmetrical respiratory excursion of ~2-10 cm…

Imaging of the Diaphragm: Anatomy and Function (RadioGraphics):

The excursion may be somewhat asymmetric and there may be a slight delay or lag on one side, typically the right.


The ideal thoracoscopic pleurodesis method for preventing recurrence of spontaneous pneumothorax remains controversial. This study was conducted to compare the patterns, effects, and thoracic volume changes achieved using a variety of thoracoscopic procedures in rabbits.

Materials and methods

Thirty-six New Zealand White rabbits were randomly assigned to undergo the following thoracoscopic procedures in the left hemithorax: (a) parietal pleural abrasion (b) minocycline instillation (c) combination of abrasion and minocycline or (d) examination alone. The rabbits were euthanatized 30 days after the operation to determine pleurodesis score, area of greatest adhesion, thoracic volume change, and histopathological findings.


Grossly, pleural abrasion produced moderate localized apical pleural symphysis with no obvious thoracic volume change. Minocycline instillation induced moderate generalized pleurodesis with a significant decrease in thoracic volume. The combination of abrasion and minocycline instillation produced the greatest generalized pleurodesis as well as a significant decrease in thoracic volume. On microscopic examination, the combination procedure produced the greatest inflammation and fibrosis of the visceral and parietal pleura. Increased intensity of pleurodesis score as well as pleural inflammation and fibrosis is associated with decreased thoracic volume.


Thoracoscopic pleurodesis achieved using pleural abrasion and minocycline instillation induced different patterns of pleurodesis, and a combination of each method generated a synergy and produced a better pleurodesis. However, as the generalization and intensity of the pleurodesis were inversely associated with thoracic volume, the optimal method should be determined on an individual basis according to the clinical situation.

25) The Phases of Lateral Deviation

Let’s analyze the process that leads the body to develop a common scoliosis. Let’s observe, thereby, in detail the five phases that bring the skull to sink, the physiological curvature to change and the spine to undergo torsion.

The first phase to analyze is obviously Phase 0. In this phase we concentrate on the skull, as it is from here that the process originates that leads to scoliosis. In fact, as we have repeated at other points, this is a descending process, which begins with the skull and has repercussions as far down as the soles of the feet.

In Phase 0, shown in picture 60, we take as an example an ideal human skeleton. Let’s start from a hypothetical position of perfect symmetry.

The blue line that divides the skeleton in half ends perpendicular to the floor. This line that divides the skeleton into two perfect halves, two mirror images, is called the vertical line.

This physical state of perfection is not met with in nature, except in rare cases. All human beings (the professional model, the athlete, the farmer, the office worker, etc.) are imbalanced, mostly to the one side or the other. Some more, some less.

Certainly the degree of asymmetry differs from case to case. There are people who because of their asymmetry remain disabled, people who all in all succeed in living a dignified life, and finally there are those who succeed in becoming life-long athletes. It all depends on the extent of the asymmetry.

Assuming that the image portrayed in picture 60 is a perfect skeleton, extremely rare in nature, we begin to make it conform to what is usually seen in life by actually reducing the dental height, until preventing the skull from governing itself. In picture 61 a reduction of dental height is carried out on the left dental arch.

The first thing that shows up is a loss of symmetry of the dental arches that leads as a result to asymmetrical work on the part of the masseter and temporal muscles (levator muscles of the jaw).

With the removal of dental height on the left dental arch the skull loses its support on the left side. Conversely, the support of the skull remains unchanged on the right side.

As a result the masseters shorten themselves, forcing a contact at that point precisely due to the lack of a reaction force (Newton’s Third Law) on the part of the teeth. As we have already said, it should be the teeth which counterbalance the force exerted by the masseter and temporal muscles. We see how the skeleton, no longer perfect, begins to take on the aspect of a common asymmetrical skeleton.

In Phase 1 the skull, forced to sink down, conforms to the asymmetrical condition which, in descending fashion, is destined to provoke chain reactions in the rest of the skeleton, which takes on an asymmetrical appearance. Let’s look at this in detail.

Let’s begin by saying that the skull is run through by a yellow line. This yellow line divides the skull into two equal halves. This line allows us to see the change in inclination of the skull in respect to the jaw (orange line) and to the vertical line (blue line).

Given the lack of dental height on the left side, the skull begins to give way somewhat on the left side as it is being pulled downward by the masseter and temporal muscles.

Falling to the left, the skull alters its inclination in respect to the axes of reference. In picture 61 the axes of reference are the light-blue vertical line and the orange horizontal line (the line of the jaw).

With an inclination to the left the skull begins sinking truly in this direction. As it sinks the musculature on the right extends, pulling the right shoulder towards it, which begins to rise.

As a result, the entire right side of the body (left in the picture 61) raises itself, causing the pelvis to rotate in a clockwise direction. The rotation of the pelvis draws the leg up and changes the arch support of the right foot.

Symptoms in Phase 1 are very mild. Muscle tension is not excessive, for which reason psychological tension is also limited.

With progression of the fall of the skull we pass to Phase 2.

During Phase 2 we start to observe the first changes that take place in other parts of the skeleton. The descending nature of this phenomenon starts to become apparent.

In Phase 2, due to the effect of the masseter and temporal muscles, the skull continues its clockwise rotation, changing its inclination in respect to the blue vertical line and comes closer to the jaw (to the left), there where it lacks support.

Due to the alteration of the inclination of the skull, which moves from right to left, the right shoulder is drawn upward.

The skull pulls the right shoulder towards itself because it is kept at that point through the participation of the rhomboid muscles and those of the neck.

The entire right side begins to tense up and as a result increases the clockwise rotation of the pelvis.

The first significant difference in Phase 2 in regard to Phase1 is to be found in the inclination of the jaw, which tends to come nearer to the skull there where it lacks dental height. The entire jaw lifts itself momentarily only in this phase as it is pulled upward by a skull which is trying to remain straight on its vertical axis.

We can say that Phase 2 is an aggravation of Phase1. The significant difference remains the change in inclination of the jaw, which defers to the skull.

Due to the change in inclination of the jaw, the supra- and infrahyoid muscles take on asymmetrical muscular burdens. These muscular burdens provoke a series of symptoms precisely in this area due to an unphysiological circulation of the blood.

The problems seen here affect various areas: the tonsils, the throat, the oral cavity, the thyroid, speech formation, swallowing, etc.

The left side of the body becomes taut and contracts in spasms, creating suffering which the individual interprets as being psychological, precisely for the reason that he cannot identify the real cause. As the sinking of the skull progresses, the intervertebral compressions increase until they press on the blood vessels.

Finally, as a last result, the right side of the pelvis lifts, altering also the arch support of the feet. Often posturologists treat this with insoles, but it is clear that this is merely a “band aid” for a problem that has its origin elsewhere. As written previously, whoever finds himself in this phase feels vaguely unwell in such a way that is not easy to define, and has psychosomatic problems.

Phase 3 is the penultimate one in terms of an aggravation of the overall asymmetry of the body. In this phase the body begins to undergo serious changes that alter it dramatically.

In Phase 3 we see right away two notable differences in regard to the previous phase:

1) The skull has continued its rotation towards the left shoulder while the jaw has not returned to the line of the vertical axis.

2) The jaw changes its inclination once again, returning parallel to the ground. With the change in inclination of the jaw, also the skull continues its rotation due to its sinking towards the left side (on the right side of the picture).

As well, during this phase, the skull is pulled downwards by the muscles of the back. This brings on the phenomenon of scoliosis.

How is scoliosis brought about, in this phase?

In Phase 2 the muscles of the right side of the body are in spasm, in order to keep the skull from falling to the left. At this point the central part of the back begins to curve to the left, creating a scoliosis. That happens because the muscles of the left side of the body contract and in this way pull the spine with them. At this point the center of mass of the skull returns to its axis and reduces the muscular tension on the right side of the body. It is this phenomenon that generates scoliosis.

This process takes place because scoliosis is in fact a compensatory mechanism that serves to bring the center of gravity of the skull back to the vertical line (blue line) with a resulting reduction in muscular strain.

As we have said previously, with the addition of scoliosis the center of mass of the skull returns to its axis, and the right shoulder sinks. The sinking of the right shoulder relaxes the tension in the musculature of the right side. At the same time, due to the shortening of the muscles on the left side the pelvis begins a counterclockwise rotation.

In this condition, the three yellow lines of shoulder, pelvis and feet turn out to be almost parallel.

This example demonstrates very well how the center of equilibrium always tends to keep the skull on its vertical axis which passes through the center of gravity.

Due to this involuntary, unaware and unconscious activity, the body is trying, either physically or psychologically, to keep the skull on its vertical axis.

This process has been described in Phases 6 and 7 of the displacement seen in profile. Thanks to continuing strain, in this phase we can have serious psychological symptoms (anxiety and panic attacks) and the rise of physical problems (herniated discs and gastrointestinal problems) due to the considerable amount of compressions that have come to be created in the body.

We have arrived at the last phase of our frontal displacement. At this stage there is an aggravation of the previous phase that accentuates the compensations that are created.

As can be seen in picture 64 the skull continues to collapse to the left. It is just for this reason that the body finds a new way to compensate by means of the right shoulder that is pulled downward.

The lowering of the right shoulder depends on two simultaneous and interlocking phenomena:

1) The musculature of the right side tenses up like crazy

2) The skull, in order to return to its axis, increases the curvature of a spine already sharply arched towards the left.

Both of these phenomena contribute to the lowering of the right shoulder. At this point the pelvis also continues its clockwise rotation, pulling the muscles of the left side down.

Thus we have a higher left hip, which pulls the left leg with it, making it shorter than the right one.

The rib cage is forced into a spasm, encaged by the muscles. It is affected by the displacement of the spine and of the elevation of the left shoulder.

In this condition the rib cage compresses inexorably everything that there is within it the heart, the lungs, the liver, the stomach and the diaphragm.

Due to these compressions, a number of internal organs can be subject to particular symptoms, such as shortness of breath, panic attacks, and gastrointestinal problems.

The muscles of the neck at this point are tense and painful. They work asymmetrically due to their varying lengths. They too are forced to adapt to the compensatory situation.

In the area of the throat, on the other hand, symptoms and common pathologies are generated such as recurring sore throats, thyroid problems, problems with swallowing, speech problems, etc.

Regarding the pelvis and the legs, in such a condition it becomes very difficult to achieve any notable results in sports. Thus, as a result many people tend to abandon the athletic activities they are very fond of.

In fact, people with a serious asymmetry are victims of frequent accidents, joint problems, constant pain, etc.

In this last phase the organism is definitely suffering. It presents with a multitude of symptoms, either physical or psychological, that often are attributed to stress.

It is interesting to see how the human body “curls up” on itself. This “curling up” can be devastating for the health, over time.

Luckily, my own frontal situation was still in a Phase 3, but if I hadn’t taken action in a timely fashion I would

have reached the phase just described, Phase 4.

The picture 65, on the side, portrays me at the start of the straightening process. Although my chief problem was a notable lack of profile form caused by a huge lack of vertical dimension in my teeth in the premolar and molar areas, I was also not in good shape from a frontal point of view. In fact, as indicated previously, I was in a Phase 3.

My belly falls outwards due to the compressions of the diaphragm the spinal column shows a distinct deviation to the left.

Even though I was very thin, I seemed to have a “beer belly” due to a severe lumbar lordosis that pushed it outwards. The lumbar lordosis, apart from pushing the viscera of the abdominal cavity outwards, pressed also on the diaphragm, impeding its proper function.

In picture 65 a curvature of the spine to the left is seen (dotted red line). It can be seen the considerable asymmetry of the lower abdominal muscles in the area of the pelvis. How the left abdominal area has come to be much more protruding.

In this condition I had considerable problems with breathing as well as considerable gastrointestinal problems.

In picture 66, given in evidence, the compressions are affecting normal inhalation in the thoracic area. Such an asymmetry generates the symptoms described above, so much is clear.

In any case, we are not all imbalanced in the same way, and it not a given that a serious skeletal asymmetry brings a serious symptomatic with it.

Often it happens that one sees crookedly-made people or those with a facial asymmetry who however do not have any symptoms. Then, there are people that we see are relatively balanced but who present with a definitive symptomatic. Let’s see if we can understand the reason.

The main reason lies in the unpredictability of the muscular compensation. In some individuals the compressions can affect the arteries, in others the esophagus, the stomach or the diaphragm. When it is the stomach that is compressed, the symptoms present in the form of gastrointestinal problems, rather than in muscular complaints, as in the case of compressions of the cervical spine. For this reason, due to asymmetry some individuals may suffer more than others.

In addition it must be said that not all those in Phase 4 present with an imbalance apparent to the naked eye. My case was not one of those.


Pregnant women experience numerous adjustments in their endocrine system that help support the developing fetus. The fetal-placental unit secretes steroid hormones and proteins that alter the function of various maternal endocrine glands. Sometimes, the changes in certain hormone levels and their effects on their target organs can lead to gestational diabetes and gestational hypertension.

Estrogen, progesterone, and human chorionic gonadotropin (hCG) levels throughout pregnancy.

Estrogen, progesterone, and 17α-hydroxyprogesterone (17α-OHP) levels during pregnancy in women. [1] The dashed vertical lines separate the trimesters. Determinations were via radioimmunoassay. [1]

Levels of sex hormones and SHBG during pregnancy in women. [2] The dashed vertical lines separate the trimesters. Determinations were via radioimmunoassay. [2]

Fetal-placental unit Edit

Levels of progesterone and estrogen rise continually throughout pregnancy, suppressing the hypothalamic axis and subsequently the menstrual cycle. The progesterone is first produced by the corpus luteum and then by the placenta in the second trimester. Women also experience increased human chorionic gonadotropin (β-hCG), which is produced by the placenta.

Pancreatic Insulin Edit

The placenta also produces human placental lactogen (hPL), which stimulates maternal lipolysis and fatty acid metabolism. As a result, this conserves blood glucose for use by the fetus. It can also decrease maternal tissue sensitivity to insulin, resulting in gestational diabetes. [3]

Pituitary gland Edit

The pituitary gland grows by about one-third as a result of hyperplasia of the lactrotrophs in response to the high plasma estrogen. [4] Prolactin, which is produced by the lactrotrophs increases progressively throughout pregnancy. Prolactin mediates a change in the structure of the breast mammary glands from ductal to lobular-alveolar and stimulates milk production.

Parathyroid Edit

Fetal skeletal formation and then later lactation challenges the maternal body to maintain their calcium levels. [5] The fetal skeleton requires approximately 30 grams of calcium by the end of pregnancy. [4] The mother's body adapts by increasing parathyroid hormone, leading to an increase in calcium uptake within the gut as well as increased calcium reabsorption by the kidneys. Maternal total serum calcium decreases due to maternal hypoalbuminemia, but the ionized calcium levels are maintained. [4]

Adrenal glands Edit

Total cortisol increases to three times of non-pregnant levels by the third trimester. [4] The increased estrogen in pregnancy leads to increase corticosteroid-binding globulin production and in response the adrenal gland produces more cortisol. [4] The net effect is an increase of free cortisol. This contributes to insulin resistance of pregnancy and possibly striae. [4] Despite the increase in cortisol, the pregnant mom does not exhibit Cushing syndrome or symptoms of high cortisol. One theory is that high progesterone levels act as an antagonist to the cortisol.

The adrenal gland also produces more aldosterone, leading to an eight-fold increase in aldosterone. [4] Women do not show signs of hyperaldosterone, such as hypokalemia, hypernatremia, or high blood pressure.

The adrenal gland also produces more androgens, such as testosterone, but this is buffered by estrogen's increase in sex-hormone binding globulin (SHBG). [4] SHBG binds avidly to testosterone and to a lesser degree DHEA. [4]

Thyroid Edit

The thyroid enlarges and may be more easily felt during the first trimester. The increase in kidney clearance during pregnancy causes more iodide to be excreted and causes relative iodine deficiency and as a result an increase in thyroid size. Estrogen-stimulated increase in thyroid-binding globulin (TBG) leads to an increase in total thyroxine (T4), but free thyroxine (T4) and triiodothyronine (T3) remain normal. [4]

Endocrine function tests in pregnancy Edit

Effect of pregnancy on endocrine function tests. [4]
Hormone Test Result
FSH, LH GnRH stimulation Unresponsive from third gestation until several weeks postpartum
Growth Hormone Insulin tolerance test Response increases during first half of pregnancy and then normalizes until several weeks postpartum
TSH TRH stimulation Response unchanged
Pancreatic Insulin Glucose tolerance test Peak glucose increases, and glucose concentration remains elevated for longer
Adrenal Cortisol ACTH infusion Exaggerated cortisol and aldosterone responses
Metyrapone Diminished response
Mineralocorticoids ACTH infusion No deoxycorticosterone response
Dexamethasone No deoxycorticosterone response

A woman's breasts change during pregnancy to prepare them for breastfeeding a baby. Normal changes include:

  • Tenderness of the nipple or breast
  • An increase in breast size over the course of the pregnancy
  • Changes in the color or size of the nipples and areola
  • More pronounced appearance of Montgomery's tubercles (bumps on the areola)

From about the 16th week of pregnancy the breasts are able to begin to produce milk. It’s not unusual for small amounts of straw-coloured fluid called colostrum to leak from the nipples. Breast lumps also sometimes develop during pregnancy but these are generally benign cysts or fibroadenoma which are not cause for concern. If the nipples begin to leak any blood tinged fluid a woman should consult her doctor. [6]

A woman's breasts grow during pregnancy, usually 1 to 2 cup sizes [ citation needed ] and potentially several cup sizes. A woman who wore a C cup bra prior to her pregnancy may need to buy an F cup or larger bra while nursing. [7] A woman's torso also grows and her bra band size may increase one or two sizes. [8] [9] An average of 80% of women wear the wrong bra size, [10] and mothers who are preparing to nurse can benefit from a professional bra fitting from a lactation consultant. [9]

Once the baby is born, after the initial stage of breastfeeding with colostrum, the mother will experience her breasts filling with milk (sometimes referred to as “the milk coming in”). This can happen up to about 50–73 hours after birth. Once full lactation begins, the woman's breasts swell significantly and can feel achy, lumpy and heavy (which is referred to as engorgement). Her breasts may increase in size again by an additional 1 or 2 cup sizes, but individual breast size may vary depending on how much the infant nurses from each breast. [8] [9] A regular pattern of nursing is generally established after 8–12 weeks, and a woman's breasts will usually reduce in size, but may remain about 1 cup size larger than prior to her pregnancy. [8] Changes in breast size during pregnancy may be related to the sex of the infant, as mothers of female infants have greater changes in breast size than mothers of male infants. [11]

Many people and even medical professionals mistakenly think that breastfeeding causes the breasts to sag (referred to as ptosis). [12] [13] [14] As a result, some new parents are reluctant to nurse their infants. In February 2009, Cheryl Cole told British Vogue that she hesitated to breastfeed because of the effect it might have on her breasts. "I want to breastfeed," she said, "but I’ve seen what it can do, so I may have to reconsider." [15] In actuality, breastfeeding is not considered to be a major contributor to ptosis of the breasts. In fact, the biggest factors affecting ptosis are cigarette smoking, a woman's body mass index (BMI), her number of pregnancies, her breast cup size before pregnancy, and age. [16] [17]

Breast size does not determine the amount of milk a woman will produce or whether she will be able to successfully breastfeed her baby. [18] Larger breast size pre pregnancy is a sign there are more fatty cells within the breast, which do not affect milk production. A more important indicator is breast changes during the course of pregnancy. If a woman does not experience any nipple or breast changes during pregnancy this is an indication she may have a rare condition such as breast hypoplasia which could lead to more difficulty breastfeeding. Women whose breasts are simply smaller but who have experienced some breast changes are likely to have a successful breastfeeding experience.

The heart adapts to the increased cardiac demand that occurs during pregnancy in many ways.

  • Cardiac output (Lit./Min.): 6.26
  • Stoke Volume (Ml.): 75
  • Heart Rate (Per min.): 85
  • Blood Pressure: Unaffected

Cardiac output increases throughout early pregnancy, and peaks in the third trimester, usually to 30-50% above baseline. [5] Estrogen mediates this rise in cardiac output by increasing the pre-load and stroke volume, mainly via a higher overall blood volume (which increases by 40–50%). [19] The heart rate increases, but generally not above 100 beats/ minute. Total systematic vascular resistance decreases by 20% secondary to the vasodilatory effect of progesterone. Overall, the systolic and diastolic blood pressure drops 10–15 mm Hg in the first trimester and then returns to baseline in the second half of pregnancy. [5] All of these cardiovascular adaptations can lead to common complaints, such as palpitations, decreased exercise tolerance, and dizziness. [5] However, there is no evidence that exercise leads to any risk to baby, even during the late stages of pregnancy. [20]

Uterine enlargement beyond 20 weeks' size can compress the inferior vena cava, which can markedly decrease the return of blood into the heart or preload. As a result, healthy pregnancy patients in a supine position or prolonged standing can experience symptoms of hypotension. [21]

Chapter 20 - Pulmonary Function in Aging Humans

In this chapter, the impact of aging upon the respiratory system and how these changes may affect the regulation of blood gases are explored. While researching the chemical and mechanical sensors that initiate deviations from the inherent ventilatory rhythm it was found that the ventilatory response to hypoxic and hypercapnic challenges appears to be less sensitive in most, but not all, older individuals. It is possible that chemoreceptor sensitivity decreases with aging, but the available human evidence is more consistent with a reduced inspiratory muscle drive accompanying altered central nervous system function. However, animal studies have provided evidence of the carotid bodies becoming atrophied in older rats. Thus, it is possible that age-dependent reductions in ventilatory sensitivity can accompany changes to both chemoreceptor and medullary functions. In addition, the respiratory system obtains feedback from mechanical and metabolic sensors. Aging clearly affects mechanoreceptor feedback, with elderly individuals being less able to distinguish changes in elastic and resistive respiratory loads. Further, during exercise, particularly in habitually sedentary older people, there may be a significant expiratory flow limitation, with the tidal volume falling inside the closing capacity. In combination with changes in gas exchange, one finds that older individuals generally require greater minute ventilation when performing the same absolute work. Aging does not challenge the respiratory system to the point at which either the ventilatory or the gas exchange mechanisms may fail. Indeed, most changes occur very gradually and without significant adverse health implications, even for the most elderly of people.



  • Root of the penis (radix): It is the attached part, consisting of the bulb of penis in the middle and the crus of penis, one on either side of the bulb. It lies within the superficial perineal pouch.
  • Body of the penis (corpus): It has two surfaces: dorsal (posterosuperior in the erect penis), and ventral or urethral (facing downwards and backwards in the flaccid penis). The ventral surface is marked by a groove in a lateral direction. of the penis consists of the shaft skin, the foreskin, and the preputial mucosa on the inside of the foreskin and covering the glans penis. The epithelium is not attached to the underlying shaft so it is free to glide to and fro. [5]


The human penis is made up of three columns of tissue: two corpora cavernosa lie next to each other on the dorsal side and one corpus spongiosum lies between them on the ventral side. [6]

The enlarged and bulbous-shaped end of the corpus spongiosum forms the glans penis with two specific types of sinusoids, which supports the foreskin, or prepuce, a loose fold of skin that in adults can retract to expose the glans. [7] The area on the underside of the penis, where the foreskin is attached, is called the frenum, or frenulum. The rounded base of the glans is called the corona. The perineal raphe is the noticeable line along the underside of the penis.

The urethra, which is the last part of the urinary tract, traverses the corpus spongiosum, and its opening, known as the meatus / m iː ˈ eɪ t ə s / , lies on the tip of the glans penis. It is a passage both for urine and for the ejaculation of semen. Sperm are produced in the testes and stored in the attached epididymis. During ejaculation, sperm are propelled up the vas deferens, two ducts that pass over and behind the bladder. Fluids are added by the seminal vesicles and the vas deferens turns into the ejaculatory ducts, which join the urethra inside the prostate gland. The prostate as well as the bulbourethral glands add further secretions, and the semen is expelled through the penis.

The raphe is the visible ridge between the lateral halves of the penis, found on the ventral or underside of the penis, running from the meatus (opening of the urethra) across the scrotum to the perineum (area between scrotum and anus). [8]

The human penis differs from those of most other mammals, as it has no baculum (or erectile bone) and instead relies entirely on engorgement with blood to reach its erect state. A distal ligament buttresses the glans penis and plays an integral role to the penile fibroskeleton, and the structure is called "os analog," a term coined by Geng Long Hsu in the Encyclopedia of Reproduction. [9] It is a remnant of baculum evolved likely due to change in mating practice. [10]

The human penis cannot be withdrawn into the groin, and it is larger than average in the animal kingdom in proportion to body mass. The human penis is reciprocating from a cotton soft to a bony rigidity resulting from penile arterial flow varied between 2-3 to 60-80 mL/Min implies the most ideal milieu to apply Pascal's law in the entire human body the overall structure is unique. [9]

Penile measurements vary, with studies that rely on self-measurement reporting a significantly higher average size than those which rely on measurements taken by health professionals. As of 2015 [update] , a systematic review of 15,521 men (and the best research to date on the topic, as the subjects were measured by health professionals) concluded that the average length of an erect human penis is 13.12 cm (5.17 inches) long, while the average circumference of an erect human penis is 11.66 cm (4.59 inches). [3] [4]

Among all primates, the human penis is the largest in girth, but is comparable to the chimpanzee penis and the penises of certain other primates in length. [11] Penis size is affected by genetics, but also by environmental factors such as fertility medications [12] and chemical/pollution exposure. [13] [14] [15] The longest officially documented human penis was found by physician Robert Latou Dickinson. It was 34.3 cm (13.5 in) long and 15.9 cm (6.26 in) around. [16]

Normal variations

    are raised bumps of somewhat paler color around the base (sulcus) of the glans which typically develop in men aged 20 to 40. As of 1999, different studies had produced estimates of incidence ranging from 8 to 48 percent of all men. [17] They may be mistaken for warts, but are not harmful or infectious and do not require treatment. [18] are small, raised, yellowish-white spots 1–2 mm in diameter that may appear on the penis, which again are common and not infectious.
  • Sebaceous prominences are raised bumps similar to Fordyce's spots on the shaft of the penis, located at the sebaceous glands and are normal. is an inability to retract the foreskin fully. It is normal and harmless in infancy and pre-pubescence, occurring in about 8% of boys at age 10. According to the British Medical Association, treatment (topical steroid cream and/or manual stretching) does not need to be considered until age 19.
  • Curvature: few penises are completely straight, with curves commonly seen in all directions (up, down, left, right). Sometimes the curve is very prominent but it rarely inhibits sexual intercourse. Curvature as great as 30° is considered normal and medical treatment is rarely considered unless the angle exceeds 45°. Changes to the curvature of a penis may be caused by Peyronie's disease.

Differences between female and male organs

In the developing fetus, the genital tubercle develops into the glans of the penis in males and into the clitoral glans in females they are homologous. The urogenital fold develops into the skin around the shaft of the penis and the urethra in males and into the labia minora in females. [1] The corpora cavernosa are homologous to the body of the clitoris the corpus spongiosum is homologous to the vestibular bulbs beneath the labia minora the scrotum, homologous to the labia majora and the foreskin, homologous to the clitoral hood. [1] [19] The raphe does not exist in females, because there, the two halves are not connected.

Growth in puberty

On entering puberty, the penis, scrotum and testicles will enlarge toward maturity. During the process, pubic hair grows above and around the penis. A large-scale study assessing penis size in thousands of 17- to 19-year-old males found no difference in average penis size between 17-year-olds and 19-year-olds. From this, it can be concluded that penile growth is typically complete not later than age 17, and possibly earlier. [20]


In males the expulsion of urine from the body is done through the penis. The urethra drains the bladder through the prostate gland where it is joined by the ejaculatory duct, and then onward to the penis. At the root of the penis (the proximal end of the corpus spongiosum) lies the external sphincter muscle. This is a small sphincter of striated muscle tissue and is in healthy males under voluntary control. Relaxing the urethra sphincter allows the urine in the upper urethra to enter the penis properly and thus empty the urinary bladder.

Physiologically, urination involves coordination between the central, autonomic, and somatic nervous systems. In infants, some elderly individuals, and those with neurological injury, urination may occur as an involuntary reflex. Brain centers that regulate urination include the pontine micturition center, periaqueductal gray, and the cerebral cortex. [21] During erection, these centers block the relaxation of the sphincter muscles, so as to act as a physiological separation of the excretory and reproductive function of the penis, and preventing urine from entering the upper portion of the urethra during ejaculation. [22]

Voiding position

The distal section of the urethra allows a human male to direct the stream of urine by holding the penis. This flexibility allows the male to choose the posture in which to urinate. In cultures where more than a minimum of clothing is worn, the penis allows the male to urinate while standing without removing much of the clothing. It is customary for some boys and men to urinate in seated or crouched positions. The preferred position may be influenced by cultural or religious beliefs. [23] Research on the medical superiority of either position exists, but the data are heterogenic. A meta-analysis [24] summarizing the evidence found no superior position for young, healthy males. For elderly males with LUTS, however, the sitting position when compared to the standing position is differentiated by the following:

  • the post void residual volume (PVR, ml) was significantly decreased
  • the maximum urinary flow (Qmax, ml/s) was increased
  • the voiding time (VT, s) was decreased

This urodynamic profile is related to a lower risk of urologic complications, such as cystitis and bladder stones.


An erection is the stiffening and rising of the penis, which occurs during sexual arousal, though it can also happen in non-sexual situations. Spontaneous erections frequently occur during adolescence due to friction with clothing, a full bladder or large intestine, hormone fluctuations, nervousness, and undressing in a nonsexual situation. It is also normal for erections to occur during sleep and upon waking. (See nocturnal penile tumescence.) The primary physiological mechanism that brings about erection is the autonomic dilation of arteries supplying blood to the penis, which allows more blood to fill the three spongy erectile tissue chambers in the penis, causing it to lengthen and stiffen. The now-engorged erectile tissue presses against and constricts the veins that carry blood away from the penis. More blood enters than leaves the penis until an equilibrium is reached where an equal volume of blood flows into the dilated arteries and out of the constricted veins a constant erectile size is achieved at this equilibrium. The scrotum will usually tighten during erection.

Erection facilitates sexual intercourse though it is not essential for various other sexual activities.

Erection angle

Although many erect penises point upwards (see illustration), it is common and normal for the erect penis to point nearly vertically upwards or nearly vertically downwards or even horizontally straight forward, all depending on the tension of the suspensory ligament that holds it in position.

The following table shows how common various erection angles are for a standing male, out of a sample of 1,564 males aged 20 through 69. In the table, zero degrees is pointing straight up against the abdomen, 90 degrees is horizontal and pointing straight forward, while 180 degrees would be pointing straight down to the feet. An upward pointing angle is most common. [25]

Occurrence of erection angles
angle (°)
from vertically upwards
of males
0–30 4.9
30–60 29.6
60–85 30.9
85–95 9.9
95–120 19.8
120–180 4.9


Ejaculation is the ejection of semen from the penis. It is usually accompanied by orgasm. A series of muscular contractions delivers semen, containing male gametes known as sperm cells or spermatozoa, from the penis. Ejaculation usually happens as the result of sexual stimulation, but it can be due to prostatic disease in rare cases. Ejaculation may occur spontaneously during sleep (known as a nocturnal emission or wet dream). Anejaculation is the condition of being unable to ejaculate.

Ejaculation has two phases: emission and ejaculation proper. The emission phase of the ejaculatory reflex is under control of the sympathetic nervous system, while the ejaculatory phase is under control of a spinal reflex at the level of the spinal nerves S2–4 via the pudendal nerve. A refractory period succeeds the ejaculation, and sexual stimulation precedes it. [26]

The human penis has been argued to have several evolutionary adaptations. The purpose of these adaptations is to maximise reproductive success and minimise sperm competition. Sperm competition is where the sperm of two males simultaneously resides within the reproductive tract of a female and they compete to fertilise the egg. [27] If sperm competition results in the rival male's sperm fertilising the egg, cuckoldry could occur. This is the process whereby males unwittingly invest their resources into offspring of another male and, evolutionarily speaking, should be avoided. [28]

The most researched human penis adaptations are testis and penis size, ejaculate adjustment and semen displacement. [29]

Testis and penis size

Evolution has caused sexually selected adaptations to occur in penis and testis size in order to maximise reproductive success and minimise sperm competition. [30] [31]

Sperm competition has caused the human penis to evolve in length and size for sperm retention and displacement. [31] To achieve this, the penis must be of sufficient length to reach any rival sperm and to maximally fill the vagina. [31] In order to ensure that the female retains the male's sperm, the adaptations in length of the human penis have occurred so that the ejaculate is placed close to the female cervix. [32] This is achieved when complete penetration occurs and the penis pushes against the cervix. [33] These adaptations have occurred in order to release and retain sperm to the highest point of the vaginal tract. As a result, this adaptation also leaves the sperm less vulnerable to sperm displacement and semen loss. Another reason for this adaptation is that, due to the nature of the human posture, gravity creates vulnerability for semen loss. Therefore, a long penis, which places the ejaculate deep in the vaginal tract, could reduce the loss of semen. [34]

Another evolutionary theory of penis size is female mate choice and its associations with social judgements in modern-day society. [31] [35] A study which illustrates female mate choice as an influence on penis size presented females with life-size, rotatable, computer generated males. These varied in height, body shape and flaccid penis size, with these aspects being examples of masculinity. [31] Female ratings of attractiveness for each male revealed that larger penises were associated with higher attractiveness ratings. [31] These relations between penis size and attractiveness have therefore led to frequently emphasized associations between masculinity and penis size in popular media. [35] This has led to a social bias existing around penis size with larger penises being preferred and having higher social status. This is reflected in the association between believed sexual prowess and penis size and the social judgement of penis size in relation to 'manhood'. [35]

Like the penis, sperm competition has caused the human testicles to evolve in size through sexual selection. [30] This means that large testicles are an example of a sexually selected adaptation. The human testicles are moderately sized when compared to other animals such as gorillas and chimpanzees, placing somewhere midway. [36] Large testicles are advantageous in sperm competition due to their ability to produce a bigger ejaculation. [37] Research has shown that a positive correlation exists between the number of sperm ejaculated and testis size. [37] Larger testes have also been shown to predict higher sperm quality, including a larger number of motile sperm and higher sperm motility. [30]

Research has also demonstrated that evolutionary adaptations of testis size are dependent on the breeding system in which the species resides. [38] Single-male breeding systems—or monogamous societies—tend to show smaller testis size than do multi-male breeding systems or extra-pair copulation (EPC) societies. Human males live largely in monogamous societies like gorillas, and therefore testis size is smaller in comparison to primates in multi-male breeding systems, such as chimpanzees. The reason for the differentiation in testis size is that in order to succeed reproductively in a multi-male breeding system, males must possess the ability to produce several fully fertilising ejaculations one after another. [30] This, however, is not the case in monogamous societies, where a reduction in fertilising ejaculations has no effect on reproductive success. [30] This is reflected in humans, as the sperm count in ejaculations is decreased if copulation occurs more than three to five times in a week. [39]

Ejaculate adjustment

One of the primary ways in which a male's ejaculate has evolved to overcome sperm competition is through the speed at which it travels. Ejaculates can travel up to 30–60 centimetres at a time which, when combined with its placement at the highest point of the vaginal tract, acts to increase a male's chances that an egg will be fertilised by his sperm (as opposed to a potential rival male's sperm), thus maximising his paternal certainty. [34]

In addition, males can—and do—adjust their ejaculates in response to sperm competition and according to the likely cost-benefits of mating with a particular female. [40] Research has focused primarily on two fundamental ways in which males go about achieving this: adjusting ejaculate size and adjusting ejaculate quality.

The number of sperm in any given ejaculate varies from one ejaculate to another. [41] This variation is hypothesised to be a male's attempt to eliminate, if not reduce, his sperm competition. A male will alter the number of sperm he inseminates into a female according to his perceived level of sperm competition, [29] inseminating a higher number of sperm if he suspects a greater level of competition from other males.

In support of ejaculate adjustment, research has shown that a male typically increases the amount he inseminates sperm into his partner after they have been separated for a period of time. [42] This is largely due to the fact that the less time a couple is able to spend together, the chances the female will be inseminated by another male increases, [43] hence greater sperm competition. Increasing the number of sperm a male inseminates into a female acts to get rid of any rival male's sperm that may be stored within the female, as a result of her potential extra-pair copulations (EPCs) during this separation. Through increasing the amount he inseminates his partner following separation, a male increases his chances of paternal certainty. This increase in the number of sperm a male produces in response to sperm competition is not observed for masturbatory ejaculates. [29]


Males also adjust their ejaculates in response to sperm competition in terms of quality. Research has demonstrated, for example, that simply viewing a sexually explicit image of a female and two males (i.e. high sperm competition) can cause males to produce a greater amount of motile sperm than when viewing a sexually explicit image depicting exclusively three females (i.e. low sperm competition). [44] Much like increasing the number, increasing the quality of sperm that a male inseminates into a female enhances his paternal certainty when the threat of sperm competition is high.

Female phenotypic quality

A female's phenotypic quality is a key determinant of a male's ejaculate investment. [45] Research has shown that males produce larger ejaculates containing better, more motile sperm when mating with a higher quality female. [40] This is largely to reduce a male's sperm competition, since more attractive females are likely to be approached and subsequently inseminated by more males than are less attractive females. Increasing investment in females with high quality phenotypic traits therefore acts to offset the ejaculate investment of others. [45] In addition, female attractiveness has been shown to be an indicator of reproductive quality, with greater value in higher quality females. [46] It is therefore beneficial for males to increase their ejaculate size and quality when mating with more attractive females, since this is likely to maximise their reproductive success also. Through assessing a female's phenotypic quality, males can judge whether or not to invest (or invest more) in a particular female, which will influence their subsequent ejaculate adjustment.

Semen displacement

The shape of the human penis is thought to have evolved as a result of sperm competition. [47] Semen displacement is an adaptation of the shape of the penis to draw foreign semen away from the cervix. This means that in the event of a rival male's sperm residing within the reproductive tract of a female, the human penis is able to displace the rival sperm, replacing it with his own. [48]

Semen displacement has two main benefits for a male. Firstly, by displacing a rival male's sperm, the risk of the rival sperm fertilising the egg is reduced, thus minimising the risk of sperm competition. [49] Secondly, the male replaces the rival's sperm with his own, therefore increasing his own chance of fertilising the egg and successfully reproducing with the female. However, males have to ensure they do not displace their own sperm. It is thought that the relatively quick loss of erection after ejaculation, penile hypersensitivity following ejaculation, and the shallower, slower thrusting of the male after ejaculation, prevents this from occurring. [48]

The coronal ridge is the part of the human penis thought to have evolved to allow for semen displacement. Research has studied how much semen is displaced by differently shaped artificial genitals. [49] This research showed that, when combined with thrusting, the coronal ridge of the penis is able to remove the seminal fluid of a rival male from within the female reproductive tract. It does this by forcing the semen under the frenulum of the coronal ridge, causing it to collect behind the coronal ridge shaft. [49] When model penises without a coronal ridge were used, less than half the artificial sperm was displaced, compared to penises with a coronal ridge. [49]

The presence of a coronal ridge alone, however, is not sufficient for effective semen displacement. It must be combined with adequate thrusting to be successful. It has been shown that the deeper the thrusting, the larger the semen displacement. No semen displacement occurs with shallow thrusting. [49] Some have therefore termed thrusting as a semen displacement behaviour. [50]

The behaviours associated with semen displacement, namely thrusting (number of thrusts and depth of thrusts), and duration of sexual intercourse, [50] have been shown to vary according to whether a male perceives the risk of partner infidelity to be high or not. Males and females report greater semen displacement behaviours following allegations of infidelity. In particular, following allegations of infidelity, males and females report deeper and quicker thrusting during sexual intercourse. [49]

Circumcision has been suggested to affect semen displacement. Circumcision causes the coronal ridge to be more pronounced, and it has been hypothesised that this could enhance semen displacement. [34] This is supported by females' reports of sexual intercourse with circumcised males. Females report that their vaginal secretions diminish as intercourse with a circumcised male progresses, and that circumcised males thrust more deeply. [51] It has therefore been suggested that the more pronounced coronal ridge, combined with the deeper thrusting, causes the vaginal secretions of the female to be displaced in the same way as rival sperm can be. [34]



The 3 isoforms of nonmuscle myosin (NM) II (NMII-A, NMII-B, and NMII-C) play various roles during mouse embryonic development. Previous work, using knockout and hypomorphic mice, showed that Myh10 encoding myosin heavy chain II-B is critical for cardiac and brain development. Ablating or decreasing NMII-B by 80% results in cardiac (ventricular septal defect, double outlet of the right ventricle) and brain defects but not midline fusion defects. Neither NMII-A nor II-C seems to play roles in early myocardial development.

Methods and Results—

We had previously generated point mutant knock-in mice and now report novel findings as a result of expressing motor-deficient NMII-B at wild-type levels. Homozygous mice die at embryonic day 14.5 in cardiac failure, exhibiting abnormalities not seen in NMII-B null and hypomorphic mice: a failure in midline fusion resulting in a cleft palate, ectopia cordis, and a large omphalocele. Fusion of the sternum and endocardial cushions is impaired in the mutant mice associated with a failure in apoptosis of the mesenchymal cells. Failure to disassemble myocyte cell–cell adhesions during cardiac outflow tract development contributes to impaired outflow tract myocardialization and displacement of the aorta to the right ventricle.


Expression of motor-impaired NMII-B disrupts normal ventral body wall closure because of a dominant-negative effect. This is not because of the loss of NMII-B function but rather a gain-of-function resulting from prolonged cross-linking of NMII-B to actin filaments, thereby interfering with the dynamics of actomyosin cytoskeletal structure. Furthermore, impaired NMII-B motor activity inhibits outflow tract myocardialization, leading to mislocalization of the aorta.


Nonmuscle myosin (NM) II plays important roles in various cellular processes, including cell migration, cell morphology, cytokinesis, and cell–cell adhesion. 1 Three different NMII heavy chains (NMHCs) are expressed and encoded by 3 different genes: Myh9 2,3 Myh10, 2 and Myh14. 4,5 The protein products are referred to as NMHCII-A, NMHCII-B, and NMHCII-C, respectively, and mutations in NMHCII-A 6,7 and NMHCII-C 8,9 cause several human syndromes. To study how a mutation in NMII-B could affect pathophysiological processes in vivo, we mutated Arg709 to Cys in the motor domain of NMHCII-B in mice (B R709C /B R709C ). This amino acid and the surrounding residues are conserved in all myosin II family members, including skeletal, cardiac, and smooth muscle myosin.

Clinical Perspective on p 265

To understand the effect of R709C mutation on NMII-B activity, we previously characterized a baculovirus-expressed heavy meromyosin (HMM) fragment, R709C-HMMII-B, which contains the NMII enzymatic and actin-binding domains. 10 R709C-HMMII-B showed 2 important changes in biological properties compared with wild-type HMMII-B: a 70% decrease in its actin-activated MgATPase activity and a complete loss in its ability to propel actin filaments in an in vitro motility assay. Furthermore, R709C-HMMII-B displayed an increased affinity for actin and spent a prolonged period bound to actin filaments during cross-bridge cycling. 11

As a part of generating B R709C /B R709C mice using homologous recombination, we inserted the neomycin cassette for selection of the mutant embryonic stem cells into the Myh10 intron, 5′ of exon 16, thus initially producing hypomorphic mice (B R709CN /B R709CN ) that expressed a decreased (20%) amount of the mutant NMII-B. These mice developed cardiac and brain abnormalities similar to NMII-B null (B − /B − ) mice, although the onset of the abnormalities was delayed compared with the knockouts. 12,13 Somewhat surprisingly, when we removed the cassette encoding neomycin resistance thereby increasing the expression of mutant NMII to wild-type levels, the hydrocephalus and defects in myocyte cytokinesis were rescued, although the abnormalities in neuronal cell migration were not. 11,14 We interpreted these results as showing that NMII has 2 distinct functions in vivo: a structural–scaffolding function that is important for cell–cell adhesion and relies on the ability of NMII to form bipolar filaments that cross-link actin. This would explain the ability of the mutant NMII-B and other isoforms to rescue the defect in cell adhesion in the neuroepithelial cells lining the spinal canal, which causes the hydrocephalus. 14 In contrast, the inability of the mutant NMII-B to rescue the neuronal migration defects was thought to reflect the defect in the mutant NMII-B motor activity as measured by the decreased MgATPase activity and the loss of in vitro motility, a property unique to each isoform.

In the present report, we characterize the novel abnormalities found in B R709C /B R709C and B + /B R709C mice, which differ significantly from B − /B − and hypomorphic mice. These include a major defect in midline fusion resulting in a cleft palate (homozygotes only), ectopia cordis (homozygotes only), and an omphalocele containing the liver and intestines, and diaphragmatic herniation and structural cardiac abnormalities (homozygotes only), defects similar to those first described in humans by Cantrell et al. 15

Materials and Methods

NMHCII-B Mutant Mice

B − /B − , B R709CN /B R709CN , and B R709C /B R709C , B a* /B a* mice were generated as previously described 12,16,17 and are available through the Mutant Mouse Regional Resource Centers (Nos. 16991, 16142, 15983, and 16998). B − /B − , B R709CN /B R709CN , and B a* /B a* mice are maintained in a mixed background of 129/Sv and C57BL/6. All procedures were conducted using an approved animal protocol in accordance with National Heart, Lung, and Blood Institute Animal Care and Use Committee.

Histology and Immunofluorescence Staining

Staining was performed as described. 12 Primary antibodies (Table I in the Data Supplement) for immunostaining were incubated at 4°C overnight after antigen retrieval in 10 mmol/L citrate buffer (pH 6). The confocal images were collected using a Zeiss LSM 510-META. In all cases, when possible, comparison was made among littermates. For each genotype, we analyzed ≥5 mice.


The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using the In Situ Cell Death Detection Fluorescein Kit, following the manufacturer’s instructions (Roche Applied Science).

Statistical Analyses

Data are expressed as mean±SD. The Student t test was performed to compare 2 means. A 1-way ANOVA was used to compare ≥3 means at a time. Data passed normality test for statistical analysis.


Defects in Ventral Wall Closure in B R709C /B R709C and B + /B R709C Mice

The B R709C /B R709C mice died during embryonic development between embryonic day (E) 14.5 and E16.5. As shown in Figure 1B, E14.5 B R709C /B R709C mice developed generalized edema (white arrow), indicating a major failure in cardiac function. Figure Ib in the Data Supplement shows evidence for massive congestive failure in the liver of B R709C /B R709C mice indicated by the presence of sinusoidal dilatation, focal hemorrhages, and congestion (compare with wild-type Figure 1A Figure Ia in the Data Supplement). Measurements of cardiac function using in utero echocardiography at E14.5 showed a marked decrease in fractional shortening and heart rate and an increase in the left ventricular dimension in systole in B R709C /B R709C embryos (Table II in the Data Supplement). B R709C /B R709C embryos develop an umbilical hernia (Figure 1B, orange arrow), indicating a failure in ventral body wall closure. Figure 1C and 1D shows sagittal sections of E13.5 embryos from B + /B R709C and B R709C /B R709C embryos. In both cases, the liver is abnormally herniated outside the body (arrows). Approximately 50% of B + /B R709C mice and all B R709C /B R709C mice develop an omphalocele. Furthermore, ≈50% of B R709C /B R709C mice develop ectopia cordis, with the heart protruding outside the thoracic chamber (Figure 1F, black arrow). Ectopia cordis was not seen in B + /B R709C mice, suggesting that the severity of the defects in ventral body wall closure is dependent on the dosage of mutant NMII-B.

Figure 1. Congestive heart failure and midline fusion defects in B R709C /B R709C mice. Representative images of wild-type (B + /B + A) and B R709C /B R709C (B) mice at embryonic day (E) 14.5, showing generalized edema (white arrow) and an umbilical hernia (orange arrow) in a B R709C /B R709C mouse. Hematoxylin and eosin (H&E)–stained sagittal sections of E13.5 embryos show a herniated liver in B + /B R709C (C, arrow) and B R709C /B R709C (D, arrow) mice. E and F, H&E-stained cross sections of E14.5 embryos show ectopia cordis in a B R709C /B R709C mouse (F, black arrow). A similar section from a B + /B + mouse is shown in E. In 50% of B R709C /B R709C mice, the 2 halves of the lower sternum are widely separated (F, green arrows compared with E, green arrow), allowing the heart to protrude outside the thoracic chamber. G and H, H&E-stained cross sections of E14.5 embryos show a cleft palate in a B R709C /B R709C mouse (H, arrows). In the B + /B + section (G), the 2 palatal shelves contact each other (arrow). Scale bars (AF), 1 mm G and H, 500 μm.

B R709C /B R709C embryos also develop cleft palates. At E14.5, the left and right palatal shelves of B + /B + mice are positioned in a horizontal plane above the tongue and are joined (Figure 1G, arrow). In contrast, the B R709C /B R709C palatal shelves are much shorter, positioned vertically, and flank the tongue with a major gap between them (Figure 1H, arrows). This defect does not seem to be because of a delay in the development of B R709C /B R709C embryos. Those few B R709C /B R709C embryos that survive to E16.5 still show cleft palates (Figure II in the Data Supplement).

Congenital Diaphragmatic Hernia in Heterozygous and Homozygous NMII-B Mutant Mice

B R709C /B R709C and B + /B R709C mice show abnormal development of the diaphragm, which leads to herniation of the liver into the thoracic cavity. Figure 2A, 2B, and 2C shows sagittal sections of the developing diaphragm of B + /B + , B + /B R709C , and B R709C /B R709C mice at E13.5 stained with antibodies to NMHCII-A (green) and striated muscle myosin (MF20, red). The skeletal muscle cells of B + /B + mice are uniformly distributed throughout the entire dorsal area of the diaphragm (A, red yellow and white boxes, enlarged in D and G). However in both B + /B R709C (B) and B R709C /B R709C (C) mice, skeletal muscle cells accumulate abnormally in the central region (white boxes in B and C, enlarged in H and I). This results in significantly fewer muscles cells at the most lateral region of the diaphragm (yellow boxes in B and C, enlarged in E and F) consistent with a defect in migration of the skeletal muscle cells. To quantify the distribution of muscle cells, we divided the diaphragm into 3 equal segments—ventral, middle, and lateral—and calculated the percentages of muscle cells for each segment from 3 mice of each genotype. These values are 38.5±1.3%, 30.3±1.8%, and 31.1±1.2% for ventral, middle, and lateral segments, respectively, in the B + /B + diaphragm 56.2±1.3%, 38.1± 0.5%, and 5.8±1.7%, respectively, in the B + /B R709C diaphragm and 57.9±1.5%, 41.7±1.2%, and 0.43±0.3%, respectively, in the B R709C /B R709C diaphragm. Statistical analysis shows a significant increase in muscle cells in the ventral segments and a significant reduction in the lateral segments of B + /B R709C and B R709C /B R709C compared with the B + /B + diaphragm (ANOVA, P<0.05). The absence of muscle cells makes this part of the diaphragm vulnerable to herniation. Because the ventral body wall is wide open in B R709C /B R709C mice, the diaphragmatic hernia is more easily observed in B + /B R709C mice (Figure IIIb and IIIc in the Data Supplement). The amuscular lateral region of the diaphragm of an E19.5 B + /B R709C mouse becomes abnormally thin permitting the liver to protrude into the thoracic cavity (Figure IIIb in the Data Supplement, black arrow). Figure IIIa in the Data Supplement shows an equivalent section from a control E19.5 B + /B + embryo. Figure IIIc in the Data Supplement shows a 2-month-old B + /B R709C mouse, which did not develop an obvious omphalocele but still developed a severe diaphragmatic hernia that permitted the intestines to protrude into the thoracic cavity. The lack of muscle cells in the lateral regions of the mutant diaphragms is not associated with increased apoptosis, because no obvious apoptotic cells were observed in the developing diaphragms from the 3 genotypes.

Figure 2. Defects in diaphragm development in B + /B R709C and B R709C /B R709C embryos. A to I, Immunofluorescence confocal images of embryonic day (E) 13.5 mouse sagittal sections near the middle of the torso stained for nonmuscle myosin heavy chain II-A (NMHCII-A green) and striated muscle myosin (MF20, red) show loss of skeletal muscle cells in the lateral-most region of the diaphragm in B + /B R709C and B R709C /B R709C embryos (B and C, yellow boxes enlarged in E and F). In the B + /B + embryo, skeletal muscle cells are numerous in this region (A, yellow box enlarged in D). Skeletal muscle cells accumulate near the midline of the B + /B R709C and B R709C /B R709C diaphragm (B and C, white boxes enlarged in H and I) compared with the B + /B + diaphragm (A, white box enlarged in G). 4',6-diamidino-2-phenylindole (blue) stains nuclei. Scale bars (AC), 200 μm D to I, 50 μm.

Importantly, none of the defects described above with respect to midline fusion are seen in B − /B − or B R709CN /B R709CN mice. 12,16 The midline fusion defects are unlikely to be because of background strain differences in these mouse lines. Both B + /B R709C and B − /B R709C , but not B + /B − offspring from B + /B − and B + /B R709C crosses, developed defects in ventral body wall closure. We next studied the cellular mechanisms underlying these defects.

Impaired Apoptosis in the Fusing Sternum of B R709C /B R709C Mice

Ectopia cordis is usually associated with defects in sternal fusion. 18 In B + /B + mice at E14.5, the fusing halves of the lower sternum are aligned side by side (Figure 1E, green arrow). In B R709C /B R709C mice, they are widely separated (Figure 1F, green arrows Figure 3D, inset). To understand the cellular mechanisms underlying this defect, we examined apoptotic activity in the fusing sternum of E14.5 B + /B + and B R709C /B R709C mice. Most of the B + /B + sternal mesenchymal cells are undergoing apoptosis manifested by nuclear condensation and fragmentation (Figure 3A, arrows). In contrast, few apoptotic cells were found in the same area in B R709C /B R709C mice (Figure 3B). TUNEL assays confirmed the apoptosis in B + /B + sternums (Figure 3C, green), which was decreased in B R709C /B R709C mice (Figure 3D, green). The percentage of apoptotic mesenchymal cells in B + /B + and B R709C /B R709C sternums was 14.4±7.7% and 1.4±0.8% (P<0.001, t test), respectively (n=5 mice for each genotype). Previous studies from cultured cells have shown that NMII is required for the final stages of apoptosis. 19–21 We next examined a step in the upstream pathway, activation of caspase-3, using immunostaining for activated caspase-3. In B + /B + mice, a significant number of mesenchymal cells (46.2±7.2%) in the fusing sternum were positive for activated caspase-3 (Figure 3E, red), whereas few mesenchymal cells (5.4±2.3% n=5 mice each genotype P<0.001, t test) stained positively for activated caspase-3 in the B R709C /B R709C sternum (Figure 3F, red). We then examined these same cells for expression of p53, the signaling molecule that initiates apoptosis. There were no major difference in p53 expression in B R709C /B R709C sternal mesenchymal cells compared with B + /B + mesenchymal cells (Figure 3E and 3F, green). The relative average fluorescence intensities of p53 staining from B + /B + and B R709C /B R709C sternums were 52.8±1.5% and 61.9±5.3% (n=3 mice each P>0.05, t test). These results indicate that NMII-B functions upstream of caspase-3 but downstream of p53 in regulating mesenchymal cell apoptosis of the fusing sternum. The requirement for enzymatic NMII activity in apoptosis has been reported in various cell types in culture. We further asked which enzymes are responsible for activation of NMII during sternal fusion. We examined the expression of myosin light chain kinase (MLCK) and Rho kinase (ROCK1) in the fusing sternum and found that ROCK1, but not MLCK, is expressed (Figure IV in the Data Supplement). Thus, ROCK1-mediated NMII activation is most likely involved in normal sternum fusion, although further investigation is required to test this hypothesis.

Figure 3. Impaired apoptosis in the fusing lower sternum of B R709C /B R709C embryos. A and B, Hematoxylin and eosin (H&E)–stained mesenchymal cells in the middle of the embryonic day (E) 14.5 fusing sternum show an extensive accumulation of apoptotic cells with condensed and fragmented chromosomes in B + /B + mice (A, green arrows). Few apoptotic cells are seen in B R709C /B R709C mice (B). Confocal images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays show apoptotic cells near the midline in the fusing sternum of B + /B + mice (C, green), which are not seen in B R709C /B R709C mice (D). The insets (H&E images) indicate areas shown in C and D. Confocal images of the sternal area stained with antibodies for activated caspase-3 (red) and p53 (green) from E14.5 mouse embryos show a decrease in caspase-3–positive cells in B R709C /B R709C mice (F, red) compared with B + /B + mice (E, red). No difference in p53 staining was seen between B + /B + and B R709C /B R709C mice (E and F, green). G to I, Confocal images of E14.5 mouse embryos stained with antibodies for nonmuscle myosin heavy chain (NMHC) II-A (G, red), II-B (H, red), and II-C (I) show that both NMHCII-A and NMHCII-B, but not NMHCII-C, are expressed in the fusing sternum. 4',6-diamidino-2-phenylindole (blue) stains nuclei. Scale bars (A and B), 25 μm C to I, 50 μm.

Sternal Mesenchymal Cells Express both NMII-A and NMII-B But Not NMII-C

It has previously been reported that dorsal wall closure in Drosophila (corresponding to mammalian ventral wall closure) requires zipper, the sole Drosophila NMII isoform. 22 However, mice and humans express 3 different isoforms of NMII. We, therefore, examined the expression patterns of NMII-A, NMII-B, and NMII-C in the developing mouse sternum. Figure 3G to 3I shows immunofluorescence confocal images for NMII-A, NMII-B, and NMII-C from an E14.5 wild-type mouse embryo. Both NMII-A (G) and II-B (H), but not II-C (I), were detected in mesenchymal cells of the developing sternums. Previous reports have demonstrated that ablation of NMII-B did not impair ventral body wall closure in mice, 16 indicating that expression of NMII-A alone is sufficient to support ventral body wall closure. Importantly, despite normal expression of NMII-A, expression of R709C-NMII-B leads to defects in ventral body wall closure. Because these defects did not occur in B − /B − mice, they are unlikely the result of loss of NMII-B function. This raises the possibility that in B R709C /B R709C mice, the mutant NMII-B isoform is interfering with the normal function of NMII-A during sternal fusion.

Failure in Fusion and Remodeling of the Atrioventricular Cushions in B R709C /B R709C Mouse Hearts

B R709C /B R709C mouse hearts show defects in fusion and remodeling of the atrioventricular endocardial cushions, which are not seen in B − /B − or wild-type hearts. Figure 4A to 4I shows the developing atrioventricular valves of B + /B + , B R709C /B R709C , and B − /B − mouse hearts from E11.5 to E14.5. At E11.5, before the fusion of the superior and inferior cushions, no differences in shape, size, and positioning of the cushions were found among B + /B + (A), B R709C /B R709C (B) and B − /B − (C) hearts, suggesting a normal endothelial–mesenchymal transition in developing B R709C /B R709C and B − /B − hearts. At E12.5 the B + /B + atrioventricular cushions have fused and started to elongate (D). In contrast, the B R709C /B R709C cushions have not fused and show no sign of elongation (E). By E14.5, B + /B + atrioventricular cushions have developed into elongated, mature mitral, and tricuspid valves (G). The superior and inferior cushions of B R709C /B R709C mice are still not fused or remodeled (H), suggesting that the defects in B R709C /B R709C atrioventricular cushions are not simply because of a delay in development. In B − /B − hearts, the atrioventricular cushions were fused normally at E12.5 (Figure 4F). However, the maturation into cardiac valves is delayed at E14.5 (Figure 4I) at the time B − /B − mice start to die.

Figure 4. Defects in fusion and remodeling of the atrioventricular cushions in B R709C /B R709C mouse hearts. A to I, Hematoxylin and eosin–stained heart sections of B + /B + , B R709C /B R709C , and B − /B − embryos show developmental progression of atrioventricular (AV) cushions from embryonic day (E) 11.5 to E14.5. E11.5 AV cushions show no differences in size, morphology, and positioning among B + /B + (A), B R709C /B R709C (B), and B − /B − (C) hearts. B + /B + AV cushions fuse and start to elongate at E12.5 (D) and acquire mature mitral (MV) and tricuspid (TV) valve leaflets by E14.5 (G). B R709C /B R709C cushions remain unfused and show no sign of maturation at E12.5 (E) and E14.5 (H). The fusion of AV cushions in B − /B − hearts appears normal at E12.5 (F) however, further maturation into cardiac valves is delayed at E14.5 (I) compared with the B + /B + mouse (E). J and K, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay shows defective apoptosis in developing B R709C /B R709C cushions. Apoptotic cells are readily seen in B + /B + cushions (J, green), but few apoptotic cells are found in B R709C /B R709C cushions (K). Scale bars (AI), 40 μm J and K, 25 μm. IC indicates inferior AV cushion and SC, superior AV cushion.

Similar to its role in sternal formation, apoptosis also plays an important role in the development of the endocardial atrioventricular cushions. We next investigated whether apoptosis was impaired in the developing B R709C /B R709C endocardial cushions using TUNEL assays. At E12.5, fusion of the superior and inferior cushions in B + /B + hearts was accompanied by significant numbers of apoptotic mesenchymal cells (Figure 4J, green 11.2±3.0%). However, few apoptotic cells were detected in B R709C /B R709C cushions at E12.5, which fail in fusion (Figure 4K 1.1±0.9% n=5 mice each genotype P<0.001, t test). Note that at E11.5 no obvious apoptotic cells were detected in either B + /B + or B R709C /B R709C atrioventricular cushions (Figure Va and Vb in the Data Supplement). Furthermore, we did not observe obvious apoptotic cells in B R709C /B R709C cushions at E14.5 (Figure Vd in the Data Supplement), suggesting that the defect in apoptosis is not because of a developmental delay in the B R709C /B R709C mouse heart. These results emphasize the role of NMII in apoptosis. Similar to the developing sternum, mesenchymal cells in developing atrioventricular cushions express NMII-A (Figure VIa in the Data Supplement, green) and NMII-B (Figure VIb in the Data Supplement, green) but no detectable II-C (Figure VIc in the Data Supplement). These results are consistent with the hypothesis that mutant NMII-B interferes with the normal function of NMII-A in B R709C /B R709C cardiac atrioventricular cushion development. This is also consistent with our findings that NMII-B/II-C double-ablated mice showed no defect in ventral wall closure or atrioventricular cushion fusion. 23

Defects in Outflow Tract Myocardialization in B R709C /B R709C and B − /B − Mouse Hearts

Both B − /B − 16 and B R709C /B R709C hearts showed defects in outflow tract (OFT) alignment, with both the aorta and the pulmonary artery emanating from the right ventricle (double outlet of the right ventricle [DORV] Figure 5A–5C 10 of 10 B R709C /B R709C mice). During heart development, the OFT cardiac cushions are initially composed of cardiac jelly. After invasion by endocardial cells and cardiac neural crest cells, the cushions expand, fuse, and consequently form a mesenchymal outlet septum. The mesenchyme of the proximal region of the outlet septum is then replaced by cardiac myocytes during a process called myocardialization. 24 In mice, the invasion by cardiac myocytes occurs between E10.5 and E13.5. Figure 5D and 5E shows hematoxylin and eosin–stained images of developing hearts at E11.5, illustrating the invasion by the cardiac myocytes of the B + /B + cardiac cushion (Figure 5D, arrow) but absence of invasion in B R709C /B R709C mice (Figure 5E, arrow). During this process, the cardiac myocytes from the outer layer of myocardium in the OFT lose their tight cell–cell adhesions, become polarized, and migrate into the adjacent cushions. Inhibition of myocardialization leads to abnormal alignment of the OFT. 24 Therefore, we examined myocardialization in the OFTs of wild-type and B R709C /B R709C mice using immunofluorescence confocal microscopy with antibodies to MF20 to delineate the cardiac myocytes and antibodies to NMHCII-B to identify both cardiac myocytes and cardiac nonmyocytes. Figure 5F shows that at E11.5, the B + /B + myocardial cells bordering the OFT are polarized, with extended lamellipodia- and filopodia-like structures protruding into the adjacent mesenchymal cells of the cushions (Figure 5F, arrows Figure VIIa in the Data Supplement). However, in B R709C /B R709C mice there is a discrete boundary between the OFT myocardium and the cushion because the cardiac myocytes remain compact with no sign of invasion (Figure 5G Figure VIIb in the Data Supplement).

Figure 5. Defective myocardialization of developing outflow tract (OFT) in B R709C /B R709C mouse hearts. A to C, Serial hematoxylin and eosin (H&E)–stained heart sections from an embryonic day (E) 14.5 B R709C /B R709C embryo show abnormal configuration of the great arteries with double outlet of the right ventricle. D and E, H&E-stained sections of E11.5 mouse hearts show that the cardiac myocytes in the developing OFT are invading the underlying cardiac cushions (CC) in the B + /B + mouse heart (D, arrow) but not in the B R709C /B R709C heart (E, arrow). F and G, Immunofluorescence confocal microscope images of the OFT from E11.5 mouse hearts stained with antibodies for nonmuscle myosin heavy chain II-B (NMHCII-B green) and MF20 (red, marker for sarcomeric myosin indicating cardiac myocytes) show that the B + /B + cardiac myocytes are invading the cardiac cushion (F, arrows) but the B R709C /B R709C myocytes are not (G). H and I, Immunofluorescence images of the developing OFT from E11.5 mouse hearts stained with antibodies for N-cadherin (green) show that in the B + /B + OFT there is no obvious enrichment of N-cadherin at the boundaries between cardiac myocytes (H). In contrast, in the B R709C /B R709C myocytes, N-cadherin is enriched at the cell–cell boundaries (I, arrows). 4',6-diamidino-2-phenylindole (blue) stains nuclei. Scale bars (AE, 200 μm) F and G, 50 μm H and I, 10 μm. AO indicates aorta PA, pulmonary artery and RV, right ventricle.

Next, we looked for the cause of the failure of migration in the B R709C /B R709C myocytes. Because phosphorylation of the regulatory MLC (MLC20) is required for activation of NMII, we used antibodies to the 2 most likely kinases that are known to phosphorylate MLC20, ROCK1 and MLCK, to see whether they were present in the OFT and whether their pattern of expression was altered in the B R709C /B R709C OFT. Figure VIIa and VIIb in the Data Supplement shows that ROCK1 is present in the cardiac myocytes in the OFT of both B + /B + and B R709C /B R709C mice. This kinase is not detected in the ventricular myocytes (Figure VIIc and VIId in the Data Supplement). In contrast, MLCK was detected in the OFT and ventricular myocytes of both the normal and mutant mice (Figure VIII in the Data Supplement). Figure VIIe and VIIf in the Data Supplement shows that MLC20 is phosphorylated in both wild-type and B R709C /B R709C cardiac myocytes. This makes it unlikely that the failure in migration is because of a lack of MLC20 phosphorylation or alteration in the ROCK1 or MLCK expression and suggests that the defect in myocyte migration entails an intrinsic kinetic property of the mutant NMII-B.

We sought a mechanism related to the kinetic properties of the mutant and wild-type NMII to explain the defect in migration in B R709C /B R709C cardiac myocytes. Previous work has shown that the disassembly of cell–cell adhesion junctions requires NMII activity. 25,26 Examination of the cell–cell adhesion boundaries in the myocytes of the OFT by immunofluorescence confocal microscopy showed marked differences between B + /B + and B R709C /B R709C cardiac myocytes. Figure 5I (arrows) shows that the cell adhesion molecule N-cadherin (the only classical cadherin expressed in the myocardium) is concentrated at the cell–cell boundary of the cardiac myocytes in the B R709C /B R709C OFT. This indicates that the B R709C /B R709C cardiac myocytes retain tight cell–cell adhesions. In contrast, there is no cortical concentration of N-cadherin in the actively migrating B + /B + cardiac myocytes at E11.5 (Figure 5H). These results suggest that the mutation R709C, which decreases NMII-B MgATPase activity and increases the time NMII-B spends bound to actin, inhibits the disassembly of cell–cell adhesions in the cardiac myocytes. This results in a failure in myocardialization, thereby contributing to the development of DORV.

The aorta of B − /B − hearts is also abnormally localized to the right ventricle as previously reported. 16 Figure 6 shows that similar to the B R709C /B R709C OFT, the B − /B − OFT shows defects in myocardialization (Figure 6C) and disassembly of cardiac myocyte cell–cell adhesions (Figure 6D) compared with a B + /B + littermate (Figure 6A and 6B). Because normal alignment of the aorta is impaired in B R709C /B R709C and B − /B − hearts, these findings suggest a requirement for wild-type NMII-B enzymatic motor activity in these processes during normal mouse heart development. This is also consistent with our finding that genetic replacement of NMII-B with NMII-A does not rescue the DORV 17 because of the defects in OFT myocardialization (Figure IX in the Data Supplement).

Figure 6. Defective myocardialization of the developing outflow tract (OFT) in B − /B − mouse hearts. A to D, Immunofluorescence confocal microscope images of embryonic day (E) 11.5 mouse cardiac outflow tracts stained with antibodies for desmin (A and C, red, a marker for cardiac myocytes) or N-cadherin (AD, green). N-cadherin localization shows that the cardiac myocytes are invading the underlying cardiac cushions in the B + /B + mouse heart (A, red) but not in the B − /B − heart (C, red) causing a defect in OFT myocardialization in B − /B − mouse hearts. Staining of the cardiac intercellular adhesion molecule N-cadherin shows that in the B + /B + OFT there is no obvious localization of N-cadherin at the boundaries between cardiac myocytes (A and B, green). In the B − /B − OFT, N-cadherin is localized at the cell–cell boundaries (C and D, green), indicating a failure in disassembly of cardiac myocyte cell–cell adhesions. Scale bars, 10 μm.


The Table summarizes the phenotypes observed from our 3 NMII-B genetically altered mouse models. The findings from these mutant mice have resulted in 2 hypotheses with respect to the mechanism underlying the NMII-B mutation. The first is that the novel gain-of-function defects found in these mice are because of the interference of R709C-NMII-B with the normal function of a second NMII, NMII-A. The second is that the mechanism underlying these defects arises from the 2 different functions of the NMII-B molecule: the motor function and the structural function.

Table. Phenotypes of Nonmuscle Myosin II-B Knockout and R709C Mutant Mice

We have analyzed ≥10 mice for each genotype. The phenotypes seen in mutant mice are 100% penetrant except as indicated.

Evidence that the mutant NMII-B is interfering with the normal function of NMII-A during ventral wall closure or endocardial cushion fusion is as follows: hypomorphic B R709CN /B R709CN mice, mice ablated for NMII-B or NMII-C, or mice doubly ablated for NMII-B and NMII-C show normal closure of the ventral body wall and normal endocardial cushion fusion. Therefore, expression of NMII-A alone is sufficient for ventral body wall development. In addition, these defects are also observed in the B R709C /B R709C homozygous mice, which do not contain any normal NMII-B, so interference with the normal isoform of NMII-B is not possible. Furthermore, the 2 affected tissues do express significant amounts of NMII-A (but not NMII-C). This raises the novel possibility that the mutant NMII-B is interfering with NMII-A. In vitro motility studies using baculovirus-expressed NMII-A HMM and mutant NMII-B HMM showed that the presence of the R709C-NMII-B HMM markedly slowed the movement of NMII-A HMM. 10 Mechanistically, prolonged binding of R709C-NMII-B to actin filaments 11 could affect the dynamics of actomyosin stress fibers, 27 which, in general, contain both NMII-A and NMII-B. 28 It is of interest that there have been several reports implicating a mutation in NMII-A 29–31 but not NMII-C 32 in the generation of a cleft palate in humans, consistent with our hypothesis that the mutant NMII-B is interfering with NMII-A. Of course, we cannot rule out interference with a NM of a different class.

Previous work has shown that NMII-A–ablated mice die at E6.5 and the heterozygous II-A mice are entirely normal, 33 so we cannot directly test our hypothesis. However, in support of this mechanism is our finding of a significant gene dose-dependent effect: All B R709C /B R709C mutant mice are severely affected, showing abnormalities including cleft palate, ectopia cordis (50%), and omphalocele. Approximately one half of B + /B R709C mice, expressing 50% mutant NMII-B compared with wild-type mice, are born with an omphalocele only, and hypomorphic mice (B R709CN /B R709CN ) expressing only 20% mutant myosin display neither defect.

The requirement for NMII function in ventral body wall closure is also supported by results from ROCK-ablated mice, which show a failure in body wall closure associated with a deficiency in the formation of actomyosin bundles in the umbilical ring. 34 In a chick model for defective ventral body wall closure, this abnormality was attributed to reduced myosin activity because of decreased ROCK expression. 35 In both cases, the defects are similar to our B + /B R709C mice but much milder compared with B R709C /B R709C mice. This is most likely because of a partial inactivation of NMII function in ROCK-ablated mice because other kinases are also capable of activating NM-II. 1

Our second hypothesis is that the defects we observed in B R709C /B R709C mice arise from 2 distinct, although not unrelated functions of myosin: NMII can use either its enzymatic motor domain to translocate actin filaments or NMII can act more as a structural protein to cross-link actin filaments. Both of these functions require the binding of myosin to actin however, translocation of actin requires a particular isoform-specific, actin-activated MgATPase activity and duty cycle (amount of time the myosin head stays strongly bound to actin) to perform a normal functional role in motile processes such as cell migration. These functions of NMII are more sensitive to mutations that alter kinetic properties and cannot be rescued by other NMII isoforms with different motor and kinetic properties. 17 An example of this is generation of the DORV in the B R709C /B R709C mice in which the mutant NMII-B cannot dissociate the cell–cell adhesion complex nor can it participate in migration of the myocytes into the cardiac cushion. We postulate that the result of this inability to migrate normally into the cardiac cushion is mislocalization of the aortic root to the right ventricle. Of note is a report by Phillips et al 36 attributing the development of a DORV to abnormalities in NMII function. Another example of defective migration is found in the skeletal muscle cells of the developing diaphragm R709C-NMII-B mice resulting in diaphragmatic herniation. An additional consequence of the NMII-B motor mutation is the apparent failure of the cardiac and sternal mesenchymal cells to undergo a normal apoptotic program. The loss of apoptosis results in abnormal valve formation and a defect in sternal fusion, 2 novel defects not seen in NMII-B–ablated or hypomorphic mice.

Mice either ablated for NMII-B or expressing R709C-NMII-B, either in reduced (20%) or wild-type amounts, develop abnormalities in myocardialization during cardiac OFT development, leading to misalignment of the aorta with the right ventricle. Our results indicate that both the expression level and the normal enzymatic activity of NMII-B are essential for normal OFT myocardialization. MLCK and ROCK are 2 of the major kinases that activate NMII activity. Specific expression of ROCK1 in OFT cardiac myocytes during myocardialization suggests that ROCK1 is the major upstream kinase activating NMII-B. This is supported by findings from loop-tail (Lp) mice where abnormal OFT myocardialization is associated with disruption of noncanonical Wnt/planar cell polarity–mediated RhoA/ROCK1 signaling. 36 Decreased expression of ROCK1 in the proximal OFT cardiac myocytes was also described in connexin 43 knockout mice that developed DORV with abnormal OFT myocardialization. 37 Because no changes in ROCK1 expression and NMII activation (MLC20 phosphorylation) were observed in the B R709C /B R709C OFT, we attribute the defects in myocardialization directly to the impaired R709C-NMII-B enzymatic activity. Our results further point to the importance of NMII-B–mediated disruption of cardiac myocyte cell–cell contacts during OFT myocardialization. Cardiac myocytes lose their epithelial context and migrate into the adjacent mesenchymal cushions during myocardialization. Expression of R709C-NMII-B in mice prevents OFT cardiac myocytes from detaching from surrounding cells, and the retention of cell–cell adhesions thereby inhibits their migration into the cushion tissue. Loss of the NMII enzymatic activity and prolonged binding of the mutant NMII to actin contribute to the inability of R709C-NMII-B to disrupt cell–cell adhesions. This is consistent with the requirement for NMII activity to perturb pre-existing epithelial cell–cell adhesions in culture. 38,39 All of the above evidence is consistent with a Wnt/RhoA/ROCK1/NMII-B pathway in regulating myocardialization during OFT development. Abnormal OFT alignment is one of the most common congenital heart defects. Defects in OFT myocardialization seem to be the common end pathway leading to this abnormality.

During revision of this article, Dr Wendy Chung's group reported a patient carrying a nonsense mutation resulting in a premature stop codon in the MYH10 transcript. 40 Among various abnormalities, the patient developed a congenital diaphragmatic hernia, which is one of the defects of pentalogy of Cantrell and is seen in our NMII-B mutant mice reported here. Our present plans call for testing our hypothesis by searching for possible mutations in NMII-B and related proteins in patients with the diagnosis of pentalogy of Cantrell.


We thank Dr Mary Anne Conti for her significant contributions to this article. Dr Sachiyo Kawamoto and members of the Laboratory of Molecular Cardiology also provided critical comments on the article. We also thank Dr Kazuyo Takeda for echocardiography. Drs Chengyu Liu and Yubin Du (National Heart, Lung, and Blood Institute [NHLBI] Transgenic Core) and Drs Christian A. Combs and Daniela Malide (NHLBI Light Microscopy Core) provided outstanding service and advice. Antoine Smith and Dalton Saunders provided excellent technical assistance.

Sources of Funding

This research was supported by the Division of Intramural Research , National Heart, Lung, and Blood Institute .


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Middeldorp ME, Pathak RK, Meredith M, Mehta AB, Elliott AD, Mahajan R, Twomey D, Gallagher C, Hendriks JML, Linz D, et al.


Purpose: To determine the clinical impact of the Varian Real-Time Position Monitor (RPM) respiratory gating system for treatment of liver tumors.

Methods and Materials: Ten patients with liver tumors were selected for evaluation of this passive system, which tracks motion of reflective markers mounted on the abdomen with an infrared-sensitive camera. At simulation, a fluoroscopic movie, breathing trace, and CT scans synchronized at end-expiration (E-E) and end-inspiration were acquired in treatment position using the RPM system. Organs and gross tumor volume were contoured on each CT. Each organ’s positional change between two scan sets was quantified by calculation of the center of volume shift and an “index coefficient,” defined as the volume common to the two versions of the organ to the volume included in at least one (intersection/union). Treatment dose was determined by use of normal tissue complication probability calculations and dose-volume histograms. Gated portal images were obtained to monitor gating reproducibility with treatment.

Results: Eight patients received 177 treatments with RPM gating. Average superior-to-inferior (SI) diaphragm motion on initial fluoroscopy was reduced from 22.7 mm without gating to 5.1 mm with gating. Comparing end-inspiration to E-E CT scans, average SI movement of the right diaphragm was 11.5 mm vs. 2.2 mm for two E-E CT scans. For all organs, average E-I SI organ motion was 12.8 mm vs. 2.0 mm for E-E studies. Index coefficients were closer to 1.0 for E-E than end-inspiration scans, indicating gating reproducibility. The average SI displacement of diaphragm apex on gated portal images compared with DRR was 2.3 mm. Treatment was prolonged less than 10 minutes with gating. The reproducible decrease in organ motion with gating enabled reduction in gross tumor volume-to-planning target volume margin from 2 to 1 cm. This allowed for calculated dose increases of 7%–27% (median: 21.3%) in 6 patients and enabled treatment in 2.

Conclusion: Gating of radiotherapy for liver tumors enables safe margin reduction on tumor volume, which, in turn, may allow for dose escalation.


In summary, here we developed a novel network-based representation of the musculoskeletal system, constructed a mathematical modeling framework to predict recovery, and validated that prediction with data acquired from athletic injuries. Moreover, we directly linked the network structure of the musculoskeletal system to the organization of cortical architecture, suggesting an evolutionary pressure for optimal network control of the body. We compared the structure, function, and control of the human musculoskeletal system to a null system in which small groups of closely related muscles are rewired with each other. Our results suggest that the structure, function, and control of the musculoskeletal system are emergent from the highly detailed, small-scale organization, and when this small-scale organization is destroyed, so are the emergent features. Our work directly motivates future studies to test whether faster recovery may be attained by not only focusing rehabilitation on the primary muscle injured but also directing efforts towards muscles that the primary muscle impacts. Furthermore, our work supports the development of a predictive framework to determine the extent of musculoskeletal repercussions from insults to the primary motor cortex. An important step in the network science of clinical medicine [87], our results inform the attenuation of secondary injury and the hastening of recovery.

Watch the video: Διαφραγματικη Αναπνοη 159 (July 2022).


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