Week 2 lecture 4 Comprehensive Study Notes: Skeletal and Smooth Muscle Physiology, Excitation-Contraction Coupling, and Intercellular Junctions

Objectives and Overview of Muscle Physiology and Cellular Junctions

• Class Objectives:

  • Analyze the gross and microscopic anatomy of skeletal muscle.

  • Detail the step-by-step mechanism of skeletal muscle contraction.

  • Examine acetylcholine (ACh) secretion and synaptic transmission at the neuromuscular junction (NMJ) as a foundation for deriving action potentials.

  • Explore smooth muscle structural features, tissue organization, and contraction mechanisms.

  • Investigate gap junction architecture, intercellular communication, associated clinical diseases, and roles in tumor growth suppression.

  • Compare and contrast tight junctions, gap junctions, adherens junctions, and desmosomes/hemidesmosomes in epithelial and cellular organization.

• General Muscle Classification:

  • Skeletal Muscle: Striated, voluntary control, responsible for gross body movements.

  • Cardiac Muscle: Striated, involuntary control, features intercalated discs that electrically and mechanically couple adjacent cardiac muscle cells.

  • Smooth Muscle: Non-striated, involuntary control, primarily localized within the walls of internal organs such as the gastrointestinal tract (gut).

Hierarchical Organization and Anatomy of Skeletal Muscle

• Structural Levels (Gross to Microscopic):

  • Gross Muscle: The intact whole muscle body.

  • Muscle Fasciculus (Fascicle): Bundles of individual muscle fibers grouped within the whole muscle.

  • Muscle Fiber: The individual, multi-nucleated cellular unit of skeletal muscle.

  • Myofibrils: Cylindrical contractile organelles running parallel inside each muscle fiber.

  • Sarcomere: The microscopic contractile unit contained sequentially within myofibrils.

• Connective Tissue Coverings:

  • Epimysium: Dense connective tissue layer surrounding the entire exterior of the whole muscle body.

  • Perimysium: Connective tissue sheath surrounding individual muscle fascicles.

  • Endomysium: Delicate connective tissue layer surrounding each single muscle fiber cell.

• Muscle Fiber Cellular Architecture:

  • Sarcolemma: The specialized plasma membrane enclosing the skeletal muscle fiber cell.

  • Sarcoplasm: The cytoplasm of the muscle fiber cell in which organelles and myofibrils are suspended.

  • Nuclei & Organelles: Muscle fibers are formed by cell fusion, making them single multinucleated cells. Nuclei (one or more per cell) and mitochondria reside in the sarcoplasm floating directly adjacent to and between parallel myofibrils.

  • Sarcoplasmic Reticulum (SR): Membrane-bound intracellular organelle surrounding myofibrils that serves as the primary storage depot for calcium ions ($\text{Ca}^{2+}$). Release of stored calcium into the sarcoplasm triggers muscle contraction.

• Hypertrophy vs. Hyperplasia:

  • Physical exercise and resistance training (e.g., bodybuilding) do not increase the absolute number of muscle fiber cells.

  • Muscle growth (hypertrophy) occurs through the expansion and synthesis of additional myofibrils inside existing muscle fiber cells, increasing cellular mass and bulk.

Myofibril Architecture, Filaments, and Sarcomere Banding

• Sarcomere Structure:

  • Defined as the segment spanning from one $Z$\text{ disc} to the adjacent $Z$\text{ disc}.

  • Forms the repeating functional contractile unit within a myofibril.

• Myofilaments:

  • Thick Filaments: Composed primarily of the protein myosin.

  • Thin Filaments: Composed primarily of the protein actin, along with regulatory proteins troponin and tropomyosin.

• Sarcomere Zones and Bands:

  • $Z$\text{ Disc (Line)}: Dense protein boundary defining the edges of a single sarcomere and serving as the anchor point for thin filaments.

  • $I$\text{ Band}: The light-staining region containing only thin filaments (actin). It spans across the $Z$\text{ disc} between adjacent sarcomeres.

  • $A$\text{ Band}: The dark-staining region spanning the entire length of the thick filaments (myosin). It includes regions where thick and thin filaments overlap.

  • $H$\text{ Zone}: The central region within the $A$\text{ band} that contains only thick filaments (myosin) and no thin filaments when relaxed.

  • $M$\text{ Line}: The central transverse line in the middle of the $H$\text{ zone} that anchors neighboring thick filaments together.

  • Zone of Overlap: The region within the $A$\text{ band} where thick filaments and thin filaments lie parallel and interdigitate.

Mechanism of Skeletal Muscle Contraction and Sarcomere Changes

• Sliding Filament Theory:

  • Contraction occurs as myosin heads bind to actin and walk along thin filaments, pulling the $Z$\text{ discs} closer together towards the central $M$\text{ line}.

  • The total length of the sarcomere shortens during contraction.

  • Filament Length Preservation: The individual physical lengths of thick filaments (myosin) and thin filaments (actin) do not change during contraction.

  • Sarcomere shortening is driven entirely by an increase in the degree of overlap between thick and thin filaments.

• Dynamic Band Behavior During Contraction:

  • $H$\text{ Zone}: Shortens as thin filaments are pulled inward towards the center, reducing the non-overlapped myosin area.

  • $I$\text{ Band}: Shortens as thin filaments slide deeper into the $A$\text{ band}, reducing the non-overlapped actin area.

  • $A$\text{ Band}: Stays the same length. Because the $A$\text{ band} represents the absolute end-to-end length of the myosin thick filaments, its dimensions never change during contraction or relaxation.

  • $M$\text{ Line}: Remains stationary in the center of the sarcomere.

Molecular Structure of Thick and Thin Filaments

• Thick Filament Composition:

  • Composed of aggregated myosin molecules containing flexible tail regions and globular myosin head regions.

  • Myosin Heads: Extend outward toward thin filaments. Contain specific enzymatic sites with $\text{ATPase}$ activity that hydrolyze ATP to drive cross-bridge cycles.

• Thin Filament Composition:

  • $F$\text{-Actin (Fibrous Actin)}: A double-stranded helical polymer composed of individual polymerized globular actin ($G$\text{-actin}) monomers.\n * *ADP Binding*: Adenosine diphosphate (\text{ADP}$) is bound to each$G$\text{-actin}$ monomer at active binding sites.

  • Myosin-Binding Sites: Specific active sites on $G$\text{-actin} monomers where myosin globular heads physically attach.\n * *Tropomyosin*: A long, fibrous protein strand running along the groove of the actin helix. In relaxed muscle, tropomyosin physically covers the myosin-binding sites on actin, blocking cross-bridge formation.\n * *Troponin*: A globular regulatory protein complex bound to both actin and tropomyosin. Contains high-affinity binding sites for calcium ions (\text{Ca}^{2+}).\n\n* **Calcium-Driven Conformational Shift**:\n 1. Free intracellular calcium (\text{Ca}^{2+}) increases in the sarcoplasm.\n 2. \text{Ca}^{2+} binds directly to troponin.\n 3. Troponin undergoes a structural conformational change that pulls tropomyosin away from the active binding sites on actin.\n 4. The uncovered myosin-binding sites are exposed, allowing energized myosin heads to attach and initiate cross-bridge cycling.\n\n# Cross-Bridge Cycle and Mechanics of Rigor Mortis\n\n* **Step-by-Step Cross-Bridge Cycle**:\n 1. *ATP Hydrolysis & Energy Activation*: A myosin head hydrolyzes bound \text{ATP}$into$\text{ADP}$and inorganic phosphate ($\text{P}_i). The released energy reorients, cocks, and energizes the myosin head into a high-energy position.\n 2. *Cross-Bridge Attachment*: The energized myosin head attaches to the exposed myosin-binding site on the actin filament, forming a cross-bridge.\n 3. *Power Stroke*: The myosin head releases \text{ADP}$and$\text{P}_i$, causing the head to pivot/whip towards the center of the sarcomere ($M\text{ line}). This pulling motion slides the thin filament past the thick filament.\n 4. *Cross-Bridge Detachment*: A fresh molecule of \text{ATP}$binds to the myosin head. This$\text{ATP} binding causes the myosin head to detach completely from the active site on actin.\n 5. *Re-cocking*: The new \text{ATP}$is hydrolyzed, returning the myosin head to its cocked position to repeat the cycle as long as$\text{Ca}^{2+} remains available.\n\n* **Molecular Mechanism of Rigor Mortis**:\n * *Definition*: A state of rigid muscular contracture that occurs following death.\n * *Cause*: Cellular respiration stops upon death, halting \text{ATP}$production and depleting intracellular$\text{ATP} pools.\n * *Mechanism*: Without fresh \text{ATP} binding to myosin heads, cross-bridges cannot detach from actin. The myosin heads remain permanently bound to active sites in a rigid, locked state until protein degradation occurs.\n\n# Excitation-Contraction Coupling, Sarcoplasmic Reticulum, and Muscle Tone\n\n* **Excitation-Contraction Coupling Steps**:\n 1. A muscle action potential propagates along the sarcolemma and down into the transverse tubules (T\text{-tubules}}).\n 2. The membrane potential change triggers voltage-sensitive calcium release channels in the sarcoplasmic reticulum membrane to open.\n 3. \text{Ca}^{2+} rapidly diffuses out of the sarcoplasmic reticulum lumen down its high concentration gradient into the sarcoplasm.\n 4. Sarcoplasmic \text{Ca}^{2+}$binds troponin, shifting tropomyosin and permitting actin-myosin cross-bridge power strokes using$\text{ATP}.\n 5. To terminate contraction, active transport pumps (\text{Ca}^{2+}\text{-ATPase}$or$\text{SERCA}$pumps) continuously pump$\text{Ca}^{2+}$out of the sarcoplasm back into the sarcoplasmic reticulum against its concentration gradient using$\text{ATP} energy.\n 6. As sarcoplasmic \text{Ca}^{2+} levels drop, troponin reverts to its original shape, allowing tropomyosin to slide back and block the myosin-binding sites on actin, producing muscle relaxation.\n\n* **Physiology of Muscle Tone**:\n * *Definition*: A continuous, sustained state of partial muscle contraction that keeps muscles firm without producing overt movement.\n * *Neurological Control*: Driven by involuntary nerve impulses originating in the brain that alternately stimulate scattered motor units within a muscle.\n * *Function*: Maintains posture and joint stability; muscle tone increases with regular exercise.\n * *Flaccid Muscle*: Loss of muscle tone occurring when nerve supply to a muscle is cut, damaged, or destroyed, leaving muscle fibers completely unstimulated.\n\n# Neuromuscular Junction Dynamics, Acetylcholine Signaling, and Pharmacology\n\n* **Neuromuscular Junction (NMJ) Structure**:\n * A specialized chemical synapse formed between the axon terminal of a somatic motor neuron and the motor end plate of a skeletal muscle fiber.\n * *Motor End Plate*: The highly folded specialized region of the sarcolemma directly opposite the neuronal axon terminal containing ligand-gated ion channels.\n * *Synaptic Cleft*: The narrow intercellular fluid-filled gap separating the neuronal presynaptic membrane from the postsynaptic motor end plate.\n\n* **Synthesis and Exocytosis of Acetylcholine (ACh)**:\n 1. Synaptic vesicles are formed via budding from the Golgi apparatus in the neuronal soma and transported to the axon terminal via axoplasmic streaming.\n 2. \text{ACh} is synthesized in the presynaptic cytoplasm and actively transported into synaptic vesicles.\n 3. An action potential arriving at the motor neuron axon terminal depolarizes the membrane and opens voltage-gated \text{Ca}^{2+} channels.\n 4. \text{Ca}^{2+}$enters the presynaptic terminal down its electrochemical gradient, causing$\text{ACh}-filled synaptic vesicles to fuse with the presynaptic plasma membrane.\n 5. \text{ACh} is released via exocytosis into the synaptic cleft.\n\n* **Postsynaptic Events and Signal Termination**:\n 1. \text{ACh}$diffuses across the synaptic cleft and binds to nicotinic acetylcholine receptors ($\text{nAChRs}) on the motor end plate.\n 2. Receptor binding opens the ligand-gated cation channels, allowing sodium (\text{Na}^+) to flow rapidly into the muscle cytoplasm down its electrochemical gradient.\n 3. Inflow of \text{Na}^+ depolarizes the motor end plate, generating an end-plate potential that triggers a muscle action potential.\n 4. *Termination*: Acetylcholinesterase (\text{AChE}$) localized in the synaptic cleft rapidly hydrolyzes$\text{ACh} into acetate and choline, closing the channel and preventing endless, unguided muscle stimulation.\n\n* **Pharmacology of the NMJ**:\n * *Acetylcholine-like Agonists (Stimulants)*:\n * *Examples*: Nicotine, Methacholine, Carbachol.\n * *Mechanism*: Bind and activate nicotinic \text{ACh}$receptors directly. Unlike$\text{ACh}, these molecules are **not** destroyed by acetylcholinesterase, leading to prolonged channel opening, sustained depolarization, and persistent end-plate potentials.\n * *Anti-Acetylcholinesterase Agents (AChE Inhibitors)*:\n * *Examples*: Nerve gas compounds, organophosphates, neostigmine.\n * *Mechanism*: Block the enzymatic degradation of \text{ACh}$by inhibiting$\text{AChE}$. Endogenous$\text{ACh} accumulates in the synaptic cleft, causing repetitive channel activation and prolonged stimulation.\n\n# Smooth Muscle Anatomy and Phosphorylation-Dependent Contraction\n\n* **Structural Organization**:\n * *Cell Shape*: Spindle-shaped, single-nucleated cells that lack visible striations.\n * *Lack of Troponin Complex*: Smooth muscle **does not contain** troponin or tropomyosin regulatory complexes.\n * *Morphological Shifts*: In a relaxed state, cells are elongated; upon contraction, cells shorten and bulge outward into a thicker geometry via diagonal overlapping of actin and myosin filaments.\n * *Functional Types*:\n * *Visceral (Single-Unit) Smooth Muscle*: Found in organ walls (gut). Innervating autonomic neurons form synapses with subset cells; gap junctions electrically connect surrounding cells so the tissue contracts together as a single unit.\n * *Multi-Unit Smooth Muscle*: Individual cells are each innervated by distinct autonomic nerve terminals, allowing independent, fine-grained control (e.g., iris of eye, piloerector muscles).\n\n* **Molecular Mechanism of Smooth Muscle Contraction**:\n 1. Intracellular \text{Ca}^{2+} concentration increases in response to neural, hormonal, or mechanical stimulation.\n 2. \text{Ca}^{2+} binds to calmodulin (replacing the function of troponin).\n 3. The \text{Ca}^{2+}-calmodulin complex activates the enzyme **Myosin Light Chain Kinase (MLCK)**.\n 4. Active \text{MLCK}$utilizes$\text{ATP} to phosphorylate regulatory myosin light chains located on the myosin heads.\n 5. Phosphorylation of myosin enables it to bind actin filaments and initiate cross-bridge cycling, shortening the cell.\n\n* **Smooth Muscle Relaxation**:\n 1. Intracellular \text{Ca}^{2+} is pumped out of the cell or back into internal stores.\n 2. Decline in \text{Ca}^{2+}$inactivates$\text{MLCK}.\n 3. The enzyme **Myosin Light Chain Phosphatase** removes the phosphate group from myosin light chains (dephosphorylation), causing cross-bridge detachment and muscle relaxation.\n\n# Structural Architecture and Intercellular Signaling of Gap Junctions\n\n* **Discovery and Basic Properties**:\n * First identified and named in 1966 by Kanno and Loewenstein.\n * Consists of direct intercellular channels spanning a narrow gap of 2\text{--}4\,nm between adjacent plasma membranes.\n * Aggregates of channels cluster into discrete membrane patches termed *gap junction plaques*.\n\n* **Molecular Subunit Architecture**:\n * *Connexin*: The basic transmembrane protein subunit. Each connexin polypeptide spans the lipid bilayer 4\text{ times}$($4\times$transmembrane domains), with both its amino ($N$\text{-terminus}) and carboxyl ($C\text{-terminus}) domains localized in the cytosol. Two extracellular loops contain critical disulfide bridges that mediate inter-cellular docking.\n * *Connexon (Hemichannel)*: Six individual connexin proteins oligomerize within a single membrane to form a hexameric ring with a central pore, called a connexon or hemichannel.\n * *Intact Gap Junction Channel*: Two connexons from opposing adjacent cell membranes align and dock end-to-end across the 2\text{--}4\,nm gap to form a continuous intercellular channel.\n\n* **Permeability and Signaling Pathways**:\n * Allows passive intercellular passage of small molecules, ions, and second messengers (< 1\,kDa$), including cyclic$\text{AMP}$($\text{cAMP}$), calcium ($\text{Ca}^{2+}$), and inositol triphosphate ($\text{IP}_3).\n * *Cell Fate Modulation*:\n * *Pro-Apoptotic (Cell Death)*: Transfer of \text{Ca}^{2+}$,$\text{IP}_3$, or extracellular$\text{ATP} can induce pro-apoptotic signaling via Bax and Bak proteins.\n * *Anti-Apoptotic (Cell Survival)*: Transfer of survival signals can activate pathways involving BCL\text{-}X_L$,$ASK1$,$SRC$,$ERK, and the MAP kinase pathway.\n * *Hemichannel Functions*: Un-docked hemichannels allow movement of molecules between the cytoplasm and extracellular space, including reactive oxygen species (\text{ROS}$) such as hydroxyl radicals, hydrogen radicals, superoxide anions, and uncharged molecules like hydrogen peroxide ($H_2O_2) and hypochlorite ions.\n\n* **Tumor Suppression vs. Metastasis**:\n * In early stage tumor cells and transformed cell lines, connexins act as growth suppressors (tumor suppressors).\n * In advanced stages of cancer progression, gap junction function can shift to promote tumor cell extravasation, invasion, and metastasis.\n\n* **Pathologies Associated with Gap Junction Mutations**:\n * *Sensorineural Deafness*: Disruptions in intercellular potassium (\text{K}^+) recycling pathways mediated by gap junctions in cells supporting the inner ear cochlea.\n * *Charcot-Marie-Tooth Disease*: A inherited neurological/neuromuscular peripheral nerve disorder caused by connexin mutations (named after Jean-Martin Charcot, Pierre Marie, and Howard Henry Tooth; completely unrelated to dental teeth).\n * *Cataracts*: Loss of lens opacity due to impaired metabolite transport across avascular lens fiber cells.\n * *Skin Diseases*: Various dermatological disorders caused by epidermal connexin mutations.\n\n# Molecular Composition, Function, and Pathophysiology of Tight Junctions\n\n* **Functional Mechanics of Tight Junctions (Zonula Occludens)**:\n * Form continuous belt-like seals around the apical boundaries of epithelial and endothelial sheets.\n * Regulate **paracellular transport**: Passive, non-energy dependent movement of ions, solutes, and water through the intercellular space *between* adjacent cells (driven by concentration gradients, electrodiffusion, and osmosis).\n * Contrast with **transcellular transport**: Energy-dependent, directional solute movement directly *through* the cell regulated by cell-specific apical and basolateral membrane transporters.\n\n* **Transepithelial Electrical Resistance (TEER)**:\n * Tight junctions appear as network strands under electron microscopy.\n * The total number of tight junction strands along the apical-to-basal axis is directly proportional to the cell layer's Transepithelial Electrical Resistance (\text{TEER}).\n * Higher strand density elevates electrical resistance, making the paracellular barrier tighter and slower to pass solutes.\n\n* **Molecular Components of Tight Junction Strands**:\n * *Claudins*: Transmembrane proteins that are **necessary and sufficient** to construct tight junction strands and establish paracellular barrier selectivity.\n * *Occludin*: A transmembrane strand protein that helps stabilize junctions but is **not** essential for tight junction strand formation.\n * *JAMs (Junctional Adhesion Molecules, e.g., JAM-1)*: Immunoglobulin superfamily proteins involved in junction assembly and cell adhesion; not strictly required for basic strand formation.\n\n* **Knockout Models and Pathology**:\n * *Occludin Knockout*: Knockout mice display no significant disruption in overall epithelial barrier function, showing occludin is non-essential for barrier integrity.\n * *Claudin Knockouts*:\n * *Claudin-1 Knockout*: Results in postnatal death within 1\text{ day} of birth due to catastrophic epidermal water loss and complete breakdown of the skin barrier.\n * *Brain/Testis Claudin Knockouts*: Causes total loss of central nervous system myelin structures and degeneration of Sertoli cell barriers in testis.\n * *Cochlear Claudin Knockouts*: Causes autosomal recessive deafness via breakdown of the paracellular \text{K}^+ ion gradient required for hair cell depolarization in the inner ear (mechanistically distinct from intercellular gap junction deafness).\n * *Renal Claudin Knockouts*: Causes hypomagnesemia (pathological reduction in magnesium reabsorption) due to selective loss of \text{Mg}^{2+} paracellular flux in the distal convoluted tubules of the kidney.\n\n# Anchoring Junctions: Desmosomes, Hemidesmosomes, Adherens Junctions, and Beta-Catenin Signaling\n\n* **Desmosomes (Macula Adherens)**:\n * Button-like intercellular spots providing extreme mechanical shear resistance across tissue sheets.\n * *Cytoskeletal Anchor*: Tethers intercellular adhesion complexes to intracellular **intermediate filaments** (e.g., keratin in epithelial cells).\n * *Core Proteins*: Transmembrane cadherin proteins desmoglein and desmocollin linked cytoplasmically to plaque proteins plakoglobin and desmoplakin.\n\n* **Hemidesmosomes**:\n * Morphologically resemble "half-desmosomes" located exclusively at the basal cellular membrane.\n * *Function*: Anchors the basal membrane of epithelial cells to the underlying extracellular matrix / basement membrane.\n * *Cytoskeletal Anchor*: Connects extracellular basement membrane attachments internally to intermediate filaments (keratin).\n\n* **Adherens Junctions (Zonula Adherens)**:\n * Cell-to-cell anchoring junctions located just basal to tight junctions.\n * *Cytoskeletal Anchor*: Tethers neighboring cell membranes directly to the intracellular **actin microfilament** cytoskeleton.\n * *Transmembrane Adhesion Molecules*: Calcium-dependent cadherins, specifically **E-cadherin** (Epithelial cadherin) and **N-cadherin** (Neuronal cadherin).\n * *Plaque Complexes*: Cadherins bind directly to \beta$\text{-catenin}$, which binds $\alpha$\text{-catenin}$and$p120$\text{-catenin}$, anchoring the entire complex to actin filaments.

• Epithelial-to-Mesenchymal Transition (EMT) in Cancer:

  • During early carcinoma development, cancer cells undergo an EMT phenotypic switch.

  • Cells downregulate and lose membrane E-cadherin, replacing it with N-cadherin ("cadherin switching"), which weakens cell-cell adhesion and enhances cell motility, invasion, and metastatic potential.

• **Three Subcellular Pools and Roles of Beta-Catenin ($\beta$\text{-catenin})**:\n 1. *Membrane-Bound Pool*: Located at adherens junctions where \beta$\text{-catenin}$ binds the cytoplasmic tail of E-cadherin and links to $\alpha$\text{-catenin} to anchor actin microfilaments.\n 2. *Cytoplasmic Pool*: Unbound cytosolic \beta$\text{-catenin}$ is continually targeted by a multiprotein destruction complex for polyubiquitination and degradation by the proteasome.

  3. Nuclear Pool: When degradation pathways are inhibited or E-cadherin is lost, $\beta$\text{-catenin}$$ accumulates in the cytoplasm and translocates into the nucleus. Inside the nucleus, it acts as a transcriptional co-activator, binding genomic promoter elements to alter gene transcription and drive oncogenic, metastatic programs.