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Small Cell Lung Cancer (SCLC)

15% of all Lung Cancers. Most aggressive Lung Cancer type. Poor 5-year survival rate of 6% vs.16% for Lung Cancer overall. Responsive to initial chemotherapy. Targeted therapies not currently recommended


Non-Small Cell Lung Cancer (NSCLC)

85% of all Lung Cancers. Various molecular aberrations identified. Standard treatment is platinum-based chemotherapy. Approved targeted treatment options available. Heterogeneous, with 3 major histologic subtypes: adenocarcinoma, squamous, and large cell carcinoma.


molecular targets for therapy in lung cancer

Epidermal Growth Factor Receptor (mutations): Drugs: Erlotinib, Gefitinib, Afatinib. Anaplastic Lymphoma Kinase (ALK) (gene rearrangements): Drugs: Crizotinib, Ceritinib



1. Tyrosine Kinase Inhibitors (EGFRTKIs). These attack the internal domain and inhibit. 2. Monoclonal Antibodies. These attach the extracellular domain of EGFR. Patients with EGFR Mutations have superior effect on EGFRTKIs compared to chemotherapy.


Anaplastic lymphoma kinase (ALK)

Is a receptor tyrosine kinase. The EML4-ALK fusion gene is responsible for approximately 3-5% of non-small-cell lung cancer(NSCLC). The vast majority of cases are adenocarcinomas. The standard test used to detect this gene in tumor samples is fluorescence in situ hybridization (FISH) by a US FDA approved kit. Recently Roche Ventana has got the approval of Chinese FDA and European FDA to test mutation by IHC. Other techniques like reverse-transcriptase PCR (RT-PCR) can also be used to detect lung cancers with an ALK gene fusion but not recommended. ALK lung cancers are found in patients of all ages, although on average these patients may be somewhat younger. ALK lung cancers are more common in light cigarette smokers or nonsmokers, but a significant number of patients with this disease are current or former cigarette smokers. EML4-ALK-rearrangement in NSCLC is exclusive and not found in EGFR- or KRAS-mutated tumors. BIOMARKERS: Fluorescent in situ Hybridzation ALK-Protein Expression, Fusion gene demonstrated by PCR. ALK fusion gene results in formation of cytoplasmic chimeric proteins with constitutive kinase activity. Rarer fusion partners for ALK such as KIF5B and TFG have also been reported in NSCLC. The ALK fusion gene appears to be a distinct NSCLC molecular subset susceptible to targeted inhibition


Regulation of T-cell Function by Cell Surface Markers

the immune response is regulated by an array of molecules. This is achieved in part by the regulation of T cell activation, which requires two signals: activation through the t-cell receptor (TCR) by recognition of antigen presented by MHC on antigen-presenting cells (APCs). Then, the ligation of costimulatory and coinhibitory molecules expressed on T cells and APCs.


Programmed death-ligand 1 (PD-L1)

Is highly expressed in lung cancer. transmembrane protein that has been speculated to play a major role in suppressing the immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis. Normally the immune system reacts to foreign antigens where there is some accumulation in the lymph nodes or spleen which triggers a proliferation of antigen-specific CD8+ T cell. The formation of PD-1 receptor / PD-L1 complex transmits an inhibitory signal which reduces the proliferation of these CD8+ T cells at the lymph nodes and supplementary to that PD-1 is also able to control the accumulation of foreign antigen specific T cells in the lymph nodes through apoptosis which is further mediated by a lower regulation of the gene Bcl-2. Engagement of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. It appears that upregulation of PD-L1 may allow cancers to evade the host immune system. PD-L1 Is Broadly Expressed in Human Cancer. Cell membrane localization is induced by IFN alpha, beta, and gamma; LPS; GMCF, VEGF, IL-4, 10. The hope is to treat with PD1 or PDL1 antibodies.



Nivolumab acts as an immunomodulator by blocking ligand activation of the programmed cell death 1 (PD-1) receptor on activated T cells.


tunica intima

comprises the inner layer of the ves- sel and contains a layer of endothelial cells in intimate contact with the blood, in addition (in larger arteries) to a layer of elastic and loose collagenous tissues containing intimal cells.


tunica media

a layer in the middle of the vessel, which may be comprised of multiple layers of elastic laminae, smooth muscle cells, or collagen.


tunica adventitia

The outer supporting layer is the tunica adventitia and is comprised of collagenous tissue. In larger vessels, the adventitia contains blood vessels (the vasa vasorum, (vessels of vessels)) that actually supply oxygen and nutrients to the adventitia and outer part of the media, as they cannot diffuse to these regions from the blood inside the vessel.



Arteries are quite thick-walled as compared to veins, and have approximately the same wall thickness as the lumen of the vessel itself. The largest arteries have a thick media with multiple elastic layers, but the elastic layers gradually decrease proceeding from the heart to the arterioles. Smooth muscle is found in the media from the aorta to the arterioles. The elastic layers are crucial to permit expansion of the vessels after systolic contraction of the heart, dampening the systolic blood pressure. Smooth muscle, particularly in the arterioles, permits control of blood flow to capillary beds.


Elastic arteries

The aorta and larger arteries branching from it are referred to as elastic arteries. The intima has a thin layer of endothelial cells and an under- lying layer of collagen/elastin-rich fibers and contains fibroblasts and myointimal cells which have similar structural features to smooth muscle cells. The media contains multiple elastic lay- ers (about 30-40 in the aorta, with some collagenous fibers and smooth muscle cells sandwiched in between. Their adventitia contains vasa vasorum.


myointimal cells

the smooth muscle cells of the intima of a blood vessel


vasa vasorum

a network of small blood vessels that supply the walls of large blood vessels.


Muscular arteries

medium-sized vessels known as distributing arteries. The multiple elastic layers in elastic arteries become restricted to two well-defined elastic layers in muscular arteries, the inner elastic lamina between the intima and the media and an outer elastic lamina that defines the boundary between the media and adventitia. The intima is thin and contains endothelial cells and a relatively thin layer of connective tissue. The media is primarily comprised of smooth muscle cells. The adventitia is com- paratively thick and contains collagen and elastin.


Smaller muscular arteries

These lose the outer elastic lamina, but retain the inner. The intima is comprised of the endothelial cell layer and a thin layer of collagenous material. They have a relatively large layer of smooth muscle in the media which can control vessel diameter. The adventia is about the same width as the media, and usually merges with surrounding con- nective tissue.



Arterioles contain an inner lining of endothelial cells on a thin basement mem- brane. This is immediately surrounded by 1-2 layers of smooth muscle cells, and outer colla- genous tissue which typically blends in with surrounding connective tissue. Arterioles are the gatekeepers to local capillary beds and can greatly restrict, if need be, the flow of blood through them. Metarterioles and arteriole-venule shunts can connect larger arterioles and venules and their vasoconstriction/ vasodilation can direct blood flow through, or permit bypass of capillary beds.



Capillaries are the smallest vessels (about 5-15 μm inner diameter) and have 1-2 endothelial cells surrounding the lumen. There is no muscular layer, but the endothelial cells are often surrounded by cells called pericytes, which are relatively unspecialized cells that can give rise to smooth muscle cells during vessel growth and wound healing, and may be contractile in nature. These are surrounded by collagenous fibrils which connect the capillary to adjacent connective tissue. Most of the molecular traffic between the vascular system and tissues occurs through the capillary endothelium, much of the exchange by diffusion. Two main types of capillaries are recognized, continuous and fenestrated.



contractile cells that wrap around the endothelial cells of capillaries and venules throughout the body.


continuous capillaries

In continuous capillaries the endothelial cells form an uninterrupted lining, although transfer across the lining can occur via pinocytotic vesi- cles.


fenestrated capillaries

In fenestrated capillaries there are pores or fenestrations in the endothelial cells. In some fenestrated endothelia the pores are covered by a thin diaphragm. A good example of fenestrated endothelium is found in the glomerulus of the kidney. Fenestrations permit bulk flow of plasma past the endothelial boundary. In some places, such as in sinusoidal capillaries, the en- dothelial cells are separated by wide pores (discontinuous) that are large enough to permit red blood cells to pass, as in the spleen. Fenestrations (discontinuities) in the liver sinusoids permit blood plasma to directly contact the liver cells.


Post-capillary venules

Capillaries empty into these vessels which initially are similar structurally, but have a larger diameter. They also have surrounding pericytes. Blood flow is slow and leukocytes tend to primarily diapedese through vessel walls at these sites. The endothelium is responsive to vasoregulatory substances such as serotonin and histamine, making these regions more sensitive to controlled permeability. Larger venules begin to get 1-2 layers of smooth muscle in their media (muscular venules), with thin layers of connective adventitia often merging with surrounding connective tissues.



Veins differ from arteries in being relatively thin-walled, and are often seen as collapsed structures in ordinary histological specimens. Although blood pressure is low in veins, the wall thickness increases with their diameter. Small veins have an intimal layer of endothelial cells and no inner elastic lamina. The media is primarily smooth muscle of 2-4 layers. Adventitia is collagenous and again often seen to blend in with surrounding connective tissue. Medium-sized veins have a similar endothelial lining and muscle layer, with an increasing thickening of the tunica adventitia which is the thickest layer. Large veins contain a thin intima of endothelial cells and a media of interlayered smooth muscle and collagen fibers (about 5-7 layers), with very small amounts of elastin fibers. The tunica adventitia is thick and in larger veins contain vasa vasorum. Circulation in veins is by hydrostatic pressure and is aided by contraction of smooth muscle and and the compression of surrounding skeletal muscles. This is particulary important in veins of the legs in helping to overcome gravity. Veins often contain one-way flap valves to prevent backflow. Loss of function of flap valves can lead to expansion of veins, which is particulary evident in veins of the leg (varicose veins).


Pulmonary vasculature

The pulmonary arteries function under lower pressure (about 15-25 mm Hg) and hence their structure is different from systemic arteries in being less thick walled.



Lymphatics flow only one way, from tissues to empty into the blood on the right and left side near the junction of the internal jugular and subclavian veins. They actually begin as nothing more than small spaces in connective tissues which connect to larger spaces lined by a very thin layer of squamous endothelium. At the size of small veins, they are immediately discernable, having only a single, very thin endothelial layer and little discernable outer layers, other than their direct connectivity to interstitial connective tissue. Lymphocytes are often visible, but red cells are absent. The lymph itself often stains a light color, which is not observed with the plasma of blood vessels. Larger lymphatics show a loosely-defined connective tissue structure outside the endothelium. Flap-like valves are abundant, and lymphatics have on-line filters (lymph nodes) where leukocytes are detained and undergo interactions.


arteriovenous shunts

the diversion of blood from an artery directly to a vein.



Anastomoses are defined as connections between arteries and veins that permit collateral circulation to occur within tissues. Should a vessel become occluded or if pressure prevents flow of blood to an area, then that area can be alternatively supplied by flow from an anastomosing artery.


End arteries

End arteries supply a section of a tissue that cannot have an alternate arterial supply hence occlusion of a vessel prevents blood flow in that area. This is found, for example, in the arterial supply to sections of the kidney and segments of the lung.


Portal systems

Portal systems begin in a capillary bed and end in a capillary bed. These are found, for example, in the hypothalamic-anterior pituitary portal system and the hepatic portal system.


Pampiniform plexus

A countercurrent arrangement between an artery and venous net- work. This arrangement is found in the spermatic cord for optimal heat exchange.


How do you determine patient’s prognosis with lung cancer?

Look at genes that do DNA repair (ERCC and RMM). These increase prognosis but decrease chance of response to platinum based chemotherapy. Also look at stage of the cancer, Small cell vs. large cell, and type of tumor and tissue.


To determine criteria predicting patient’s sensitivity to the therapies.

Look at the patient’s cell type, molecular markers, histology. Clinical information is used as well.


To explain what "targeted therapy" means for cancer therapy.

To design and develop targeted therapies, specifically designed to “attack” the molecular targets that are tumor specific. Find agents that work against specific biologic pathways. Ex: Non-small cell lung cancer: can choose bevacizumab or pemetrexed, blocks VEGF pathway or the formation of purine and pyridimine precursors.


demonstrate that exact histological classification matters.

Certain tissue types will make the patient susceptible to toxicity or will confer specificity and effectiveness to a given treatment.


What does biomarkers mean and how can they be used for therapy decision.

Two types of biomarkers: Prognostic: reflect natural history of disease independent of therapy- based on the tumor and the patient themselves. Predictive: reflects the impact of a therapeutic intervention (predicts response to treatment).


lung caner

is diagonesed at a very late stage, kills more ppl than colorectal, breast, pancreas, and prostate combined. 220,000 new cases per year in US, 160,000 deaths per year. Is very heterogeneous disease (probably the most in cancers). It is not only based on morophology but also based on molecular profiling. Only a few of these mutations are drivers, most are just passengers. Screening with low dose CY led to 20% reduction in lung cncer mortalty 6.7% better survival in all cause mortality reduction. It is now recommended by US prevention task force and is now covered by medicare/medicaid. a problem is that there is a 95% false positive rate. we need better imaging and biomarkers in order to reduce the false positive rate



the "treatment of disease by inducing, enhancing, or suppressing an immune response". Cancer immunotherapy attempts to stimulate the immune system to reject and destroy tumors. I


skeletal muscle

are long cylindrical cells also called muscle fibers or myofibers. They are 50-100 μm in diameter and several cm long. They contain hundreds of nuclei, all on the periphery of the cell (a centrally located nucleus in skeletal muscle is indicative of a fiber that has been damaged and repaired). One of the most striking aspects of skeletal and cardiac muscle is the precise and repeating alignment of the myofilaments that creates the striated appearance. Skeletal muscle cells come in several types: slow twitch and two types of fast twitch. Stains can be used to show the random distribution of fiber types in a muscle. This distribution has been referred to as a “checkerboard” pattern, and this pattern can change if a muscle is partially denervated and later reinnervated. nucleus is on periphery. no gap junctions


Cardiac muscle

cells have a single nucleus and are much smaller in diameter and shorter than skeletal muscle fibers. One of the distinguishing features of cardiac muscle is the intercalated disc, which serves two functions. First, it physically ties together adjacent cells so that they don’t pull apart when contracting. Second, it contains gap junctions (within interculated discs, sep the myocytes) for the transmission of electrical current from one cell to the next (for propagation of the action potential) in order for the heart to contract synchronously.


Smooth muscle cells

have a single nucleus (like cardiac cells) but are even thinner in diameter than cardiac cells, being 2-5 μm in diameter. They are spindle shaped with the nucleus near the center. They are not striated (hence the name – smooth), but use the same proteins to generate force as the skeletal and cardiac cells (regulation of contraction is different. are also based on actin and myosin with Ca as regulater, but diff regulation. nucleus is in center and cells are smaller, also have gap junctions. Much smaller diameter (5um compared to 50-100um in skeletal and 50 um in cardiac).



Each striated muscle cell contains a series of repeating functional units called sarcomeres. These are the basic units of contraction. They are defined as extending from one Z line to the next Z line. Force is generated as thick and thin filaments slide past each other.



A muscle fiber (single cell), contains bundles of contractile filaments and each myofibril is covered with its own network of sarcoplasmic reticulum. The myofilaments and their interaction are described here. There are two primary filaments: thin (actin) and thick (myosin). In addition to these structural myofilaments are two regulatory proteins: tropomyosin and troponin.


Thin Filaments

The primary protein that forms the thin filament is actin, which is attached to z disc. Actin exists as either G-actin (globular-single units) or F-actin (filamentous). It is the F-actin that makes up the thin filaments. Each thin filament is one μm long. Filamentous actin is double stranded and helical (an analogy is that two strings of pearls are held side by side and twisted). Bound to the actin filaments are two important regulatory proteins, tropomyosin and troponin. Troponin is bound to one end of the tropomyosin.


Thick Filaments

The thick filament is made of myosin (470,000 Daltons). Myosin is composed of six proteins that are really three pairs: one pair of large, heavy chains and two pairs of small, light chains. Each of the heavy chains forms a long alpha-helical region with a globular head. The alpha-helical regions of each of the heavy chains wraps around the other to form a long rod with the globular heads near each other. The short, light chains are associated with the globular heads (see Figure below). The exact position of the light chains is not known. The rod-like region of a myosin molecule associates with other myosins such that the head of different myosins are staggered both along the length of the thick filament and around the circumference (see the figure below). The thick filaments are 1.6 μm long and contain 300-400 myosins. What fixes their length at 1.6 μm is not known (nor is it known why thin filaments are 1.0 μm). Additional proteins that have been identified that associate with actin and myosin may regulate the lengths of the filaments. It is the myosin heads that are the region of interaction with actin, and the actin-myosin combine to contain the ATPase activity.


Cross Bridge Interactions

As intracellular Ca rises, troponin binds the Ca and exposes binding site of myosin. The myosin head can now bind to actin. Does it need ATP at this point in the cycle to generate force? No, it is as if the spring is already compressed, and the energy is released upon binding. The myosin head rotates, relative to the neck region where the light chains bind. Thus, as soon as myosin binds, it exerts a force (about 5 pN, or the force exerted by gravity on a single bacterium) and causes a shortening of the sarcomere of about 8 nm. After the myosin has “pulled” on the actin, does it just fall off? No, the myosin is stuck to the actin until ATP binds to myosin. Binding of ATP allows myosin to dissociate from actin and hydrolysis of ATP also occurs, putting myosin into a high-energy state (compressing the spring).



a two-stranded alpha-helical coiled coil protein found in cell cytoskeletons. Tropomyosin is rod shaped and binds to 6-7 actin molecules of one strand. In the relaxed state, the binding of myosin to actin is prevented because the binding site on actin is covered by tropomyosin. As intracellular free calcium rises, troponin binds the Ca++ and undergoes a conformational change. Each troponin is bound to the end of one of the rod-like tropomyosin molecules and induces a conformational change in the tropomyosin that exposes the binding sites.



sits at end of tropomyosin, moves in response to increase of Ca (similar structure to calmiacin), causes tropomyosin to move. There are different genes for skeletal and cardiac. If there is heart damage can assay for cardiac version with antibody circulating in blood.


If each myosin-actin cycle results in a displacement of 8 nm, how do we get muscle shortening of centimeters?

There are two answers: (1) Lots of sarcomeres in series will summate linearly. (2) Many myosin-actin cycles occur during a single contraction. The rate of myosin turnover is faster in fast twitch muscle than in slow twitch muscle (different myosin genes). The myosin in fast twitch muscle cycles about 20 times per second, but in slow muscle it only goes about 5 times per second.


Smooth muscle regulation

The regulation of cardiac muscle contraction is like that of skeletal muscle. Smooth muscle regulation is very different. First, smooth muscle contains no troponin, but calcium is still the key regulatory molecule. Increased calcium in the smooth muscle cell binds to calmodulin and the Ca-calmodulin binds to CaM kinase and activates it so that one of the light chains on the myosin head is phosphorylated. The phosphorylated myosin is able to bind to actin and force is generated. This process is much slower than that of skeletal and cardiac muscle. The phosphorylation of myosin is slow and the rate of ATP hydrolysis (cross bridge turnover) is slow. It can take a second to generate full force. Removal of Ca is accomplished by Ca pumps and Na-Ca exchangers in the sarcolemma. This removal of Ca will lead to inactivation of the kinase, and the myosin is then dephosphorylated by a phosphatase. Smooth muscle can also remain in the state where myosin and actin remain bound and locked in a contracted state without consuming ATP



the mutation of this gene is responsible for Duchenne muscular dystrophy. It is a large, filamentous protein that is associated with both actin (not the actin of the thin filaments, but cortical actin beneath the plasma membrane) and the surface membrane. It is part of a complex of proteins, some of which span the plasma membrane and bind to extracellular matrix molecules like laminin. Thus, dystrophin links the cytoskeleton with the extracellular matrix.



an enormous protein that links the myosin thick filaments to the Z-line. Part of the titin molecule is bound to myosin and is not very flexible. The region of titin between myosin and the Z-line is extensible. The idea is that titin keeps the myosin thick filaments centered in a sarcomere.



is also a large protein that is associated with actin thin filaments and is thought to be important for keeping the thin filaments organized. Although these proteins are not a major fraction of the myofilaments, they contribute a large fraction of the passive tension in a muscle.



Although these proteins are not a major fraction of the myofilaments, they contribute a large fraction of the passive tension in a muscle. At the Z-line α-actinin is a molecule that crosslinks actin filaments.


familial hypertrophic cardiomyopathy (FHC).

About half of all cases of sudden death due to cardiac arrest in young athletes are due to familial hypertrophic cardiomyopathy (FHC). These athletes usually have no prior indication of heart problems. The wall of the left ventricle is much thicker than normal, and this is not simply a result of exercise training. The majority of people with FHC have mutations in the cardiac myosin heavy chain (specifically in the head region that interacts with myosin and even more specifically, in two regions of the head: one that binds actin and one that binds ATP). In some cases FHC is due to mutations in troponin. Thus, single amino acid mutations in the regulatory regions of a myofilament protein or a regulatory protein produce muscle disease.


Events occurring during a single contraction

In normal, relaxed muscle [Ca]i is low (<0.1 μM). Membrane events occur which lead to an increase in cytoplasmic free Ca++. Each skeletal muscle cell of a mammal is innervated at one spot. This synaptic contact is usually near the center of the cell. (An exception to this single innervation occurs in some extraocular eye muscles). An action potential in the motor axon causes release of neurotransmitter (acetylcholine (ACh)). ACh diffuses across the synaptic cleft and binds to a receptor (AChR) in the muscle post-synaptic membrane. The AChR is an ion channel that opens and causes depolarization. The depolarization in turn will open Na channels and an action potential is initiated. The action potential propagates in both directions from the endplate, and this electrical signal occurs very fast compared to the contraction of the cell (a couple of msec for the action potential and 50-100 msec for the contraction). [Think about what would happen if some sarcomeres in the center of the fiber started to contract long before the sarcomeres near the ends.]


How does this electrical signal get transformed into a rise of [Ca+2]i?

One way you might guess would be to open Ca+2 channels that are voltage-gated and have Ca+2 flow into the cell as Ca+2 moves down the voltage and concentration gradient. In cardiac and smooth muscle, voltage-activated Ca+2 channels and calcium entry are indeed important. But for skeletal muscle one can remove all extracellular calcium and still get contraction. Moreover, surface channels alone would be insufficient because the diffusion time for Ca+2 to go from the surface to the interior is too long, i.e. contraction of myofilaments near the plasma membrane would occur before Ca+2 could get to the interior. If Ca+2 doesn’t come from the outside, what is the source? A membrane compartment called the sarcoplasmic reticulum (SR) contains the Ca+2. This is a general feature of many cell types: the Ca+2 that is readily released or stored is in smooth endoplasmic reticulum, which is similar to muscle SR. How is Ca+2 released from the SR? Is the depolarization of the surface transmitted to the S.R.? There is a pathway for transmission of the action potential to the interior via the transverse tubule (t-tubule) membrane. We know that the t-tubule membrane is important from the classical experiment of Huxley and Taylor (1958). They pushed the tip of an extracellular microelectrode gently against the surface membrane and depolarized a small patch of the cell. If the electrode tip was over the opening of a t-tubule, then a local contraction would occur, and if it was not over an opening, there was no contraction. We also know that depolarization propagates into the t-system (i.e. there are Na channels in the t-tubules). But the depolarization in the t-tubule membrane does not go directly to the SR. There is no electrical continuity between the SR and the t-system. How do we know this? We can apply a potential to the membrane and ask how much charge it takes to change the membrane potential. Remember that membranes are like a capacitor. And the charge a capacitor stores is proportional to the area. If the SR were electrically connected, it would take a lot more charge to change the potential than was experimentally measured.


Summary: Action potential travels toward tendons and also inward into the t-system

The membrane depolarization in the t-system is translated into Ca+2 release from the SR; this is called E-C coupling (excitation-contraction coupling) and for many years it was a mystery how this worked.


What are the structural components involved in E-C coupling?

The myofilaments are bundled into myofibrils. Remember that each myofibril is wrapped in its own SR. The end of the SR at its contact with the t-tubule is called the terminal cisterna and contains a protein called calsequestrin, each molecule of which binds ∼50 Ca+2. At the apposition of the SR and the t-tubule, there are proteins that are electron-dense and look dark in electron microscope pictures, and this region is called the triad. In the past few years we have learned quite a bit about these proteins that connect the t-tubule and SR. Two protein complexes are involved: one in the t-tubule and the other in the SR; DHPR (dihydropyridine receptor) and RyR (ryanodine receptor). The present theory is that depolarization causes a conformational change in the DHP receptor that in turn causes the calcium release channel to open and Ca flows out of the SR.



a calcium-binding protein of the sarcoplasmic reticulum. The protein helps hold calcium in the cisterna of the sarcoplasmic reticulum after a muscle contraction, even though the concentration of calcium in the sarcoplasmic reticulum is much higher than in the cytosol. It also helps the sarcoplasmic reticulum store an extraordinarily high amount of calcium ions.


DHPR (dihydropyridine receptor)

a complex of several membrane proteins and is in the t-tubule membrane. One of the subunits of the DHP receptor is a voltage-gated Ca channel.


RyR (ryanodine receptor)

in the SR membrane and is a Ca release channel.


malignant hyperthermia (MH)

an abnormal calcium release channel in the SR causes a disease called malignant hyperthermia (MH). Patients with this disease have a catastrophic rise in body temperature when they are given volatile anesthetics (such as halothane) for surgery. The frequency of occurrence is about 1/15,000. When these cases were first reported, mortality was about 70%. In 1976 the mortality was 28%. It is now down around 10% because the treatment is to give intravenous dantrolene. In summary, people with this “disease” function fairly normally (the pigs, however, also have anxiety-related attacks) except when given anesthetics. The anesthetic alters the SR Ca+2 release channel such that Ca+2 release occurs without its normal requirement for a conformational change in the DHP receptor. The steady Ca+2 leak from the SR activates the Ca+2 ATPase to pump Ca+2 back into the SR and a futile, heat-producing cycle occurs and is lethal if not corrected. Mutations in the Ca+2 release channel also cause a disease known as central core disease (CCD). In fact the same mutation can cause MH in some people and CCD in others.



Dantrolene injection is done before the anesthesia if it is known that the patient is susceptible. Dantrolene blocks muscle contraction by blocking Ca+2 release from the SR. This led to the suggestion that the anesthetic elevated Ca+2 in muscle, which caused utilization of ATP and heat generation. What has been recently discovered is that in an animal model (pigs that are inbred for malignant hyperthermia), the Ca+2 release channel in the SR is abnormal.


muscular dysgenesis

The other protein at the triad, the DHP receptor, is the cause of a disease (in mice) called muscular dysgenesis. These mice look normal when born but cannot breathe and die. They lack the DHP receptor in skeletal muscles and thus E-C (excitation-contraction) coupling is interrupted. If muscle cells from the mutant mouse are cultured and injected with DNA coding for the DHP receptor, the cells will now contract when depolarized. This is not only strong evidence that the DHP receptor is responsible for the defect but also shows that gene therapy is possible. The rescue of the muscle cells in the tissue culture dish didn’t help the mouse, and it is not feasible to inject each muscle cell in the animal. One approach used in clinical trials for people with Duchenne muscular dystrophy was to inject normal myoblasts and hope that enough of these will fuse and express the normal dystrophin protein. At the present, this therapy has not been very successful because almost all the injected cells die. The mutant mouse is also instructive about differences in E-C coupling between skeletal and cardiac muscle. Remember that the mouse is fine until it is born and has to use its skeletal muscles to breathe. Contraction in the heart is normal. Cardiac muscle also has DHP receptors and calcium release channels at the triad. Why does the heart E-C coupling function normally? The cardiac DHP receptor is a different gene product (very similar protein but different genes) and that gene is unaffected. If the mutant skeletal muscle cells that lack a DHP receptor are injected with the cardiac gene, they show cardiac E-C coupling; i.e. calcium entry is required for calcium release from the SR. Thus, the type of E-C coupling is dependent on the DHP receptor.


Summary of single contraction

Ca+2 is released from the SR (and primarily from the terminal cisternae), diffuses to the myofilaments, binds to troponin, the conformation of tropomyosin is altered and allows myosin and actin to interact. What terminates the response? Ca+2 ATPase pumps in the SR membrane transport Ca+2 back into the SR and bring cytoplasmic Ca+2 back to a low level (<0.1μM). The muscle then relaxes.


Summary of E-C coupling for skeletal muscle

1. Action potential in motor nerve. 2. Acetylcholine (ACh) release. 3. ACh receptor binds ACh and opens, causing depolarization. 4. An action potential propagates down the fiber. 5. Action potential and depolarization also occurs in t-tubules. 6. Protein links at t-tubules/SR junction (triad) are altered to allow Ca+2 release from SR. 7. Ca+2 binds to troponin, alters conformation of tropomyosin and exposes myosin-actin binding site. 8. As long as Ca+2 and ATP are present the myosin-actin cycle continues. 9. Relaxation: Ca+2 ATPase pumps Ca+2 back into SR, lowering cytoplasmic Ca+2 and tropomyosin again blocks the myosin-actin binding site.


E-C coupling for Cardiac Muscle

Cardiac muscle is almost identical to skeletal muscle in structure and in control of Ca+2 release from the SR. The difference is that Ca+2 entry is required to trigger Ca+2 release by the Ca+2-release channel of the SR since the cardiac Ca+2-release channel binds Ca


E-C coupling for Smooth muscle.

Smooth muscle on the other hand does not need the t-system or SR. Smooth muscle cells are so thin that Ca+2 entering via Ca+2 channels in the surface membrane can easily diffuse to the center of the cell (however, some smooth muscle cells do have a rudimentary SR).



All three muscle types use the sliding filament organization of muscle contraction. The major regulator of contraction is Ca+2. What else affects the tension produced? The length of the muscle fiber is important - the concepts are both fundamental and simple. The relationship between length and tension for a single sarcomere is shown in the figure below. This concept is very important for understanding congestive heart failure. The explanation for the shape of the curve is based on the overlap of the myosin head groups and the actin filament. When the muscle fiber is stretched so much that there is no overlap between actin and myosin, no tension can be generated. Tension increases linearly as the amount of overlap increases. When the actin filaments move into the central region of the thick filaments where there are no myosin head groups, tension plateaus. When the shortening causes the actin filaments to interdigitate in the middle of the sarcomere, tension begins to decrease.


Innervation of muscle

Each muscle is innervated by a group of motor neurons in the spinal cord. Damage to these motor neurons or to the nerve produces paralysis of the muscle. Each motor neuron innervates only one muscle and in that muscle it innervates a subset of the total muscle fibers. The muscle fibers innervated by a motor neuron are termed the motor unit. It is a unit because each time the motor neuron fires an action potential all the muscle fibers innervated by the neuron contract in unison. The size of a motor unit varies not only from muscle to muscle but also within a single muscle. Muscles that perform fine movements (e.g. finger muscles or extraocular eye muscles) tend to have small motor units (tens of muscle fibers for finger muscles and as few as 3 fibers for extraocular muscles) while large muscles that make gross movements have large motor units (hundreds of muscle fibers). For a single muscle there may be dozens of motor neurons that innervate it and the range of motor unit sizes is large. Size recruitment of motor units during voluntary movement: small motor units are recruited first and progressively larger motor units are recruited as the strength of contraction is increased. This allows a fine control of movement. Cardiac and smooth muscle on the other hand are innervated but can function without nervous innervation. They have an innate excitability that is modulated by excitatory and inhibitory innervation. In contrast to skeletal muscle cells which are not electrically coupled, both cardiac and smooth muscle cells are linked by gap junctions.


Muscle fiber types

Mammalian skeletal muscle fibers can be grouped into three classes: slow, fast, and an intermediate. These cells have different myosin isoenzymes, different proportions of mitochondria and oxidative enzymes, different resistance to fatigue, and different speeds of contraction. The slow oxidative fibers are used for postural or relatively maintained contractions. These tend to be reddish in color due to their high myoglobin content. The intermediate fibers are fast with both glycolytic and oxidative enzymes. The fast twitch fibers have high glycolytic content and are used for rapid bursts of activity (sprinting or jumping, for example). Virtually all muscles are a mix of all three types of fibers. Each motor unit, however, is homogeneous.


Graduation of tension

We can grade tension in skeletal muscle in several ways. (1) Increase the frequency of action potentials. This will increase tension until a maximal (tetanic) contraction is achieved. (2) Recruit additional motor units. This increases tension until all motor neurons innervating the muscle are stimulated. (3) Changing the length of the muscle is a minor factor for skeletal muscle because it normally operates near the optimal length. Grading tension in cardiac and smooth muscle is very different from that of skeletal muscle. They both respond to neurotransmitters and hormone-like molecules. They are also strongly influenced by the length of the cell since this length is not fixed by attachments to bone.


satellite cells

Each skeletal muscle cell has very closely associated cells called satellite cells. These are stem cells and are the source of new myoblasts to repair injured muscle. For example, if a muscle fiber is so seriously damaged that the cell degenerates, the satellite cells on the surface of that fiber will divide and fuse, forming a new muscle cell. Satellite cells are responsive to a large number of signaling molecules (fibroblast growth factor (FGF), insulin growth factor (IGF), hepatocyte growth factor (HGF), NF- κ B, nitric oxide (NO) and myostatin (a member of the TGF-β family)). The damaged muscle cell produces factors such as LIF (leukemia inhibitory factor) that triggers proliferation of the satellite cells. There is recent evidence (2011) that connective tissue fibroblasts interact with satellite cells to regulate the proliferation of each cell type. In addition, the fibroblasts prevent the premature differentiation of satellite cells. Thus, there are complex interactions that regulate the number of stem cells for muscle repair and the amount of connective tissue in the regenerated muscle. Although not proven, it is generally thought that in Duchenne muscular dystrophy the cells are weakened and damaged by the absence of the dystrophin protein. Satellite cells are then continually fusing to repair the muscle fibers until the satellite cells are depleted or lose the ability to keep up with the muscle degeneration.


adaptation during exercise

As you exercise, more myofilaments are added and the muscle fibers get larger. A part of this response to exercise is that satellite cells will also divide and fuse to provide the nuclei and protein synthesis machinery to support the extra volume of the muscle cell.


What types of repair take place in smooth muscle?

Smooth muscle can repair itself; the cells can dedifferentiate, enter mitosis and regenerate new muscle cells. This extraordinary ability to proliferate may contribute to the occurrence of smooth muscle tumors, such as leiomyosarcoma. This is a relatively rare disease. Smooth muscle tumors can occur anywhere in the body since blood vessels contain smooth muscle.


What types of repair take place in cardiac muscle?

There are no satellite cells in cardiac muscle. And there is little or no repair of damage after a heart attack. The damaged area contains scar tissue produced by fibroblasts. There have been recent reports of generation of new cardiac muscle cells by circulating stem cells from the bone marrow, but this is not well established yet, and some reports have contradicted this concept. There is also research focused on using control of transcription factors to convert cardiac fibroblasts into myocytes.


Effect of exercise

Hypertrophy (bigger cells) or hyperplasia (more cells)? Lifting weights to increase strength does not add new skeletal muscle fibers. The cross sectional area of each cell is increased; i.e. new myofibrils are formed. Conversely during atrophy (e.g. when a limb is put in a cast) the cross sectional area of individual muscle fibers decreases. Can we change fast fibers into slow and vice versa? If muscle biopsies are taken from world champion athletes as well as normal individuals, and the proportion of fast to slow fibers is plotted, the results are diagrammed below. The champion marathoners and sprinters fall at the extremes of the curve. Are the fiber types you have due to genetics or training? This is controversial. Textbooks state that fast fibers are not converted to slow fibers with exercise. It is however possible to convert fibers by experimentally cross-innervating a nerve from a muscle that is primarily fast to one that is primarily slow. Artificially imposed electrical activity will also convert fiber types. Under normal, physiological situations, the most that is observed is a shift from fast to the fast intermediate and an increase in the oxidative capacity of the cells. Thus, sprinters are born, not made.


Fatigue during exercise.

Muscle fatigue is a complex phenomenon. In a general sense, fatigue is reduced performance during prolonged or intense activity. It is both a decrease in force production and speed of contraction. In principle, fatigue can be due to impairment at any point from the motor neuron to the events at the SR and myofilament interactions. The major causes of fatigue do not appear to involve the motor neuron, the neuromuscular synapse, action potential initiation at the endplate, or action potential propagation along the surface membrane. The steps that are affected are (1) propagation of the action potential into the t tubule, (2) release of Ca+2 from the SR, (3) effect of Ca+2 on the myofilament interaction and (4) force generation by the myofilaments. Let us look at the causes of each of these in more detail below. At high frequency stimulation, potassium builds up and sodium is reduced in the restricted space of the t-tubular network. This can lead to an action potential of reduced amplitude or even action potential failure in the inner part of the t system. In this case the outer part of the fiber contracts but not the center of the fiber. However, this type of fatigue will recover within seconds after the start of recovery because the exchange time for diffusion in the t system is only a few seconds. The more typical type of fatigue is due to metabolic changes, primarily an increase in inorganic phosphate and a decrease in pH (from 7 to 6.5). Skeletal muscle burns ATP to contract, one for each myosin-actin cycle of force generation and one for each Ca+2 ion pumped back into the SR. But ATP levels change very little during strenuous contraction because phosphocreatine regenerates the ATP, and the result is a decrease in phosphocreatine and an increase in creatine and inorganic phosphate. The precise reasons that increased inorganic phosphate and hydrogen ions lead to a decreased Ca+2 release from the SR are not known, but many intracellular molecules affect the Ca+2 release channel of the SR. The effect of Ca+2 on troponin is reduced during fatigue (possibly because hydrogen ions compete with Ca+2 for binding to troponin) causing fewer myosin-binding sites to be exposed. Both elevated phosphate and hydrogen ions reduce the force generated by myosin pulling on actin, but it is not known what part of the conformational change is affected.


Smooth muscle- some further complexities.

Although smooth muscle does not require SR for contraction, some types of smooth muscle do have SR. This illustrates a basic concept: generalizations are hard to make about smooth muscle because there are so many different types with distinct functional properties. Innervation of smooth muscle is complex. Innervation is by sympathetic and parasympathetic neurons. A variety of peptides and CNS neurotransmitters are also utilized. Moreover, some transmitters are inhibitory (i.e. direct inhibition of muscle contraction). The most recently discovered transmitter is NO (nitric oxide). This chemical produces relaxation of smooth muscle. NO is produced by endothelial cells and also by some neurons. It causes relaxation of a variety of smooth muscles including cerebral arteries, coronary arteries, and arterial smooth muscle of the penis. NO binds to a receptor that increases levels of cGMP. Viagra and Cialis work by preventing the breakdown of cGMP and thus, enhance NO action. Some smooth muscles produce action potentials but these are Ca+2, not Na action potentials. Longitudinal muscle of the gut, uterus and bladder are examples of action potential muscles (peristaltic or propagating contractions are important consequences). Arterial smooth muscle, trachea, and the gastric fundus lack action potentials.


HCM: Cellular Consequences

Majority of mutations = missense mutations in structural genes. Disease mechanisms still incompletely understood. Muscle Cell Phenotype: Cardiomyocyte and cardiac hypertrophy -> organ hypertrophy; myocyte disarray -> function compromised; interstitial and replacement fibrosis > propensity to arrhythmia; dysplastic intramyocardial arterioles -> ischemia. sarcomers (increased contratile forces that arent as functional) are inafficient causing myocytes to grow


HCM: Phenotype in Patients

Majority: asymptomatic throughout life but some have dyspnea, angina, and syncope but some experience sudden cardiac death and some of those die suddenly (enriched in athletes?) (mortality is <1% per person/year). Clinical Presentations: Cardiac murmur (if LV outflow obstruction); Cardiac ‘Pump’ Failure (dyspnea, angina); Arrhythmia (syncope/sudden death); Sports/Family screening. Diagnosis: Echocardiogram, EKG, MRI, Family history, genetic testing, chest-X ray.



Myostatin is a secreted growth differentiation factor that is a member of the TGF beta protein family that inhibits muscle differentiation and growth in the process known as myogenesis. Myostatin is produced primarily in skeletal muscle cells, circulates in the blood and acts on muscle tissue, by binding a cell-bound receptor called the activin type II receptor. Mutations in both copies of the human myostatin gene results in individuals that have significantly more muscle mass and hence are considerably stronger than normal. Furthermore, blocking the activity of myostatin may have therapeutic application in treating muscle wasting diseases such as muscular dystrophy.


Myostatin and Skeletal Muscle Growth

Muscle growth: Mature muscle cells ~cannot divide. To increase length: recruit myoblasts (add nuclei). To increase girth: recruit myoblasts and increase size and # of myofibrils (add sarcomeres). Myostatin: Muscle Growth Regulator (Inhibitor). Normally made and secreted by muscles as a negative feedback for muscle growth. May be raised in AIDS patients with muscle wasting


Malignant Hyperthermia

Genetic disorder (dominant): RYR1 mutations ~70%. Environmental disorder: exposure to anesthesia. Typically inhalation agents (halothane) and/or succinylcholine. Prevalence: 1:5,000 – 1:100,000 anesthesia exposures. ~600 cases in US/year (Wisconsin, Michigan, West Virginia). Phenotype: Hypermetabolism, skeletal muscle damage, hyperthermia. Death if untreated (~70% mortality untreated)


Mechanism of malignant hyperthermia

Altered regulation and an abnormally increased release of calcium from the sarcoplasmic reticulum into the sarcoplasm. The associated increased amounts of intracellular calcium result in an increase in oxygen consumption and anaerobic metabolism and cause the muscle rigidity and the clinical scenario described above.4,5 Calcium transport from the sarcoplasmic reticulum into the sarcoplasm is an integral component of the normal excitation-contraction process and is mediated by the ryanodine receptor, isoform 1 (RYR1).6 The calcium-regulating channel of RYR1 is located in the membrane of the sarcoplasmic reticulum and is directly influenced by depolarization via the transverse tubule system, which causes a structural change of the voltage-sensitive L-type (dihydropyridine) calcium channel.7 RYR1 is closely associated with other proteins that may influence calcium regulation, and include FK-506 binding protein and triadin. However, only RYR1 and dihydropyridine have been directly implicated in the pathophysiology of the MH process. Inherited susceptibility to MH is conferred by an abnormal skeletal muscle RYR1 or associated structure that allows abnormal calcium release when exposed to an anesthetic triggering agent.8 The mechanism by which an anesthetic triggering agent causes the abnormal calcium release that initiates the acute MH crisis is unknown, but prolonged RYR1 channel opening has been demonstrated in an experimental model.9 Dantrolene sodium, the treatment for MH, is known to inhibit calcium release via RYR1 antagonism.


Exposure to triggering anesthetic agent with malignent hyperthermia

Exposure of an individual who has a genetic susceptibility (ryanodine receptor [RYR1] or dihydropyridine receptor [DHP] mutation) to an anesthetic triggering agent (ie, volatile inhalational anesthetic agent, succinylcholine, or both) may result in malignant hyperthermia. This reaction is caused by an altered calcium balance between the lumen of the sarcoplasmic reticulum (SR) and the sarcoplasm. Normally, muscle cell depolarization is sensed by the DHP receptor, which is thought to signal RYR1 opening by a direct physical connection. In malignant hyperthermia, accumulation of abnormally high levels of calcium in the sarcoplasm causes uncontrolled anaerobic and aerobic metabolism and sustained muscle cell contraction. This results in the clinical manifestations of respiratory acidosis, metabolic acidosis, muscle rigidity, and hyperthermia. If the process continues unabated, adenosine triphosphate (ATP) depletion eventually causes widespread muscle fiber hypoxia (cell death, rhabdomyolysis), which manifests clinically as hyperkalemia and myoglobinuria and an increase in creatine kinase. Dantrolene sodium binds to RYR1, causing it to favor the closed state, thereby reversing the uninhibited flow of calcium into the sarcoplasm.


Immediate Management of malignant hyperthermia

Stop inhalation agents, succinylcholine. Hyperventilate with 100% O2. Bicarbonate 1-2mg/kg as needed/ Dantrolene 2.5 mg/kg. Cool patient: gastric lavage, surface cooling, wound cooling. Treat arrhythmias: (don’t use Ca channel blockers). Support patient (watch for acidosis, renal failure, electrolyte abnormalities, clotting abnormalities)


Becker muscular dystrophy (BMD)

is characterized by later-onset skeletal muscle weakness; individuals remain ambulatory into their 20s. Despite the milder skeletal muscle involvement, heart failure from DCM is a common cause of morbidity and the most common cause of death. Mean age of death is in the mid-40s. DMD-associated DCM is characterized by left ventricular dilation and congestive heart failure. Female carriers of DMD mutations are at increased risk for DCM. mutation: Smaller, in-frame deletions, Mild muscle complaints (some w/o symptoms), High CK, Later-onset cardiomyopathy



actin and myosin



cardiac muscle cell


fast twitch

two types, one fatigues quickly (lots of mit) and the other fatigues rapidly, contracts rapidly than relaxes, there is a mix of these fibers in all muscle fibers.


slow twitch

one type, slow to contract, does not fatigue quickly (lots of mit)


myosin structure

has actin binding site and nucleotide binding pocket on head region. Wrapped around the shaft of the α-helical neck are two light chains. These chains stiffen the neck so that it can act as a lever arm for the head. Central region without head


Other (Secondary) causes of cardiac hypertrophy

Hypertension, Aortic valve disease, Congenital heart disease, Obesity


Calcium-Induced Calcium Release (CICR)

Cardiac DHPR and RyR are encoded by different genes from those expressed in skeletal muscle. The process in cardiac muscle is called Calcium-Induced Calcium Release (CICR). Ca entry is required for Ca release by the cardiac RyR.


Key Difference between E-C coupling of skeletal and cardiac muscle

Observation: No extracellular calcium is needed for skeletal muscle to contract. Remove extracellular Ca from heart and contraction disappears. Explanation: Ca is needed via DHPR entry to trigger Ca release from the SR in the heart but is not required in skeletal muscle. DHPRs in skeletal muscle make direct physical contact with the RyR to change its conformation and produce Ca release.


Clinical Signs of Malignant Hyperthermia

Specific: Muscle rigidity, Masseter spasm ; Increased CO2 production; Rhabodomyolysis; Hyperthermia. Non-Specific: Tachycardia; Tachypnea; Acidosis (Respiratory); Hyperkalemia


Grading Tension

Skeletal Muscle: 1) action potential frequency and 2) motor unit recruitment Cardiac and smooth muscle: 1) hormones and neurotransmitters and 2) length-tension. Increasing action potential frequency in nerve and muscle until muscle is producing tetanic (maximal) tension.



Myofibrils have become smaller. The muscle atrophies but does not lose muscle fibers; each muscle fiber has become smaller in diameter.


Duchenne Muscular Dystrophy

Caused by big deletions and frameshift. X-linked disease. 1:3500 males. Dystrophin (DMD) mutations. Boys: Onset of skeletal muscle disease ages 3-5 years; Abnormal gait (walking, running, climbing; toe-walking); Gowers’ sign; Calf pseudohypertrophy; High creatinine kinase (1000s); Mild intellectual disability; ~Wheelchair bound by age 11; ~Death in 20s (cardiopulmonary); Cardiomyopathy 100% by 18 yrs. Reproductive fitness ~0. treatment with corticosteroids