Systems Physiology Final Exam

Biological Signals

Ligand/receptor (hormones, growth factors, cytokines, morphogens)

Chemical Signals

Signal gradients (ions)

Physical Signals

• Mechanical properties of cells and their environment. e.g stiffness of surrounding environment

• Forces: fluids, loading, compression…

Mechnobiology

Cells can sense their environment

Mechanotransduction

Mechanical force influencing cellular signalling and function

Cell Environment

= cells and and extracellular matrix

Cells + Extracellular Matrix

Cells produce extracellular matrix components. Functions of the ECM include the important mechanical and signalling roles, critical for cell, tissue and organ function.

2 Main Types of ECM

Basement Membrane and Interstitial Matrix

Basement Membrane

Surrounds most tissues including cell sheets (epithelial, endothelial) and cells/bundles. Specific components include type IV collagen, laminin, nidogen, perlecan. Found in adipose tissue, epithelial sheets, blood capillaries, and muscle tissue for example.

Interstitial Matrix

A 3D porous, amorphous gel surrounding connective tissue cells (fibroblasts) composed of collagen and elastin.

2 Main Types of Extracellular Matrix Components

Structural, fibrous proteins (biopolymers) and non-structural, non-fibrous proteins.

Structural, Fibrous Proteins of ECM

Collagens, elastins, and laminins

Non-Structural, Non-Fibrous Proteins of ECM

• Glycoproteins/Proteoglycans

• Hyalectans

• Matrillins

• Non-fibrillar collagens

Collagens

• Largest protein component in mammals

• 28 Types

• Monomer, cleaved by proteinases and assembled into fibrils.

• Cross-linked by lysyl oxidase

4 types of Collagens

• Type I - Bone, Skin, Tendon, Heart, Vessels (strength)

• Type II - Cartilage

• Type III - Reticulum, Heart, Blood Vessels (bit elastic)

• Type IV - Basement Membrane

Elastins

• Provides elasticity to tissues: major component of skin, arteries, lungs

• Length can extend 2X

• Tropoelastin (monomer)

• Elastic fibres consist of an elastin core surrounded by microfibrils (fibrillin-1, fibulin-5)

• Cross-linked by lysyl oxidise

Laminins

• Important in basement membrane, mediates cell adhesion

• 60 possible heterotrimers

Glycoproteins/Proteoglycans

• Proteins with carbohydrates/glycosaminoglycans (GAGs)

  • Chondroitin sulphate (CS)

  • Keratan sulphate (KS)

  • Dermatan sulphate (DS)

  • Heparan sulphate (HS)

• Hyaluronic acid: (HA) (cartilage, synovial fluid)

• Aggrecan: (CS, KS, cartilage)

• Fibronectin: (basement membrane)

• Perlecan: (HS, basement membrane)

• Decorin: (CS, DS, interstitial matrix, associates with type I collagen)

ECM Roles

Anchorage, migration barrier, migration track, signal reservoir, low affinity co-receptor, signal presenter, functional fragments, and biomechanical force.

Matrix Remodelling/Turnover

Extracellular matrix remodelling and turnover is common in most tissues to repair/replace = maintain homeostasis Cells produce matrix biopolymers/proteoglycans, but not all and not to the same integrity as during development. Ex. Elastin has a half-life of ~70 years

Matrix Degrading Enzymes

• Matrix Metalloproteinases (MMPs)

• Inhibited by

  • Tissue inhibitors of metalloproteinases (TIMPs)

  • a disintegrin and metalloproteinases (ADAMs)

  • ADAMs with thrombospondin motifs (ADAMTSs)

Release of Protein Fragments from Matrix Degradation

• Degradation can release protein fragments with specific function = Matrikines

• Cleavage of the C terminus of perlecan releases endorepellin = promotes angiogenesis (formation of new blood vessels)

• Ex. for repair processes but can be bad in disease such as diabetes

What adhere cells together?

Integrins that connect to the actin cytoskeleton through focal adhesion.

Integrin Subunits

• 24 integrins, heterodimeric: 17 ⍺ subunits, 8 β subunits

• Form combinations which are largely cell type specific

  • β1 can bind 12 different ⍺

  • β4, 5, 6 and 8 can only bind 1 ⍺

  • Most ⍺ only bind 1 type of β

  • ⍺4 and ⍺v can bind multiple different β

Integrin Ligands

Can be matrix proteins

• Proteins with RGD or integrin interacting domains

  • RGD = amino acids: arginine, glycine, aspartic acid

• E.g. collagen, elastin, GAGs

Can also be growth factors, small molecules, other receptors etc.

• Eg VEGF, thrombospondin, TGF-

3 Types of Cell Junctions

Gap junctions, adherens junctions, and tight junctions

Types of Cell Receptors

GPCR, ion channels, transporters, enzyme linked receptors and nuclear receptors. All can signal to produce a cellular response and co-ordinated cell responses affect tissue level functions.

Functions of the Heart

Generate contraction force, maintain CO, HR, BP within range, and withstand fluidic forces.

2 Types of Forces in the Heart

• Active = contraction of chambers

• Passive = blood flow e.g., passive force of blood flowing through valves (pressure and shear)

Layers of the Heart Wall (inside → out)

Myocardium, endocardium, epicardium, pericardial cavity, parietal layer of serous pericardium, and fibrous pericardium.

Myocardium Cellular Composition

Consists of myocardial cells as well as fibroblasts, capillaries, and other types of cells.

Cells in the Heart

• 11 major cells types

• Non-cardiomyocytes (CMs) dominate heart cellular composition

• Ventricular and atria cardiomyocytes differ - istinct CM populations

• High heterogeneity

• Each region of the heart contains multiple different cell types

• Each cell type within a region has distinct populations that differ in their gene expression and function

• All these cell types and sub-types coordinate to maintain homeostasis and perform organ function

Sub-Populations of Heart Cells

• Fibroblasts

• Endothelial cells (line blood vessels)

• Smooth muscle cells and pericytes 

• Myeloid cells

• Lymphoid (tissue resident)

• Neuronal

• Adipose

Fibroblasts in the Heart

• Responsible for maintenance of ECM

• Distinct populations in each heart region

Smooth Muscle Cells and Pericytes of the Heart

SMCs surround larger arteries and pericytes surround capillaries.

Myeloid Cells of the Heart

• Macrophages/monocytes

  • Monocyte-derived (from blood)

  • Tissue-resident

• Dendritic

Collagen in the Heart

Most abundant matrix component in the heart, produced by cardiac fibroblasts. Scaffold for CMs and non-CMs (ECs and FBs), signals for proliferation, differentiation etc.

Endomysium and Perimysium

• Endomysium = connective tissue around muscle fibres

• Perimysium = connective tissue around bundles of muscle fibres

Purpose of Endomysium and Perimysium

• Functions of these are for synchronised contraction

• Passive expansion (when the ventricles are filling) 

• Overstretch prevention 

• Conduction

Collagen Type I (fibrillar)

• Main collagen = confers tensile strength

• Spatially organised by proteoglycans decorin, biglycan and Col XV

• More found in perimysium

• Contributes to overstretch prevention

Type III Collagen (fibrillar)

• Thin fibres, contributes to elasticity

• More in endomysial

• Contributes to passive expansion

Type I and Type III Collagen Ratio and Type I Collagen Changes

• Type I – Type III ratio influences mechanical properties (contraction and relaxation)

• Type I Collagen increases with age

• Decreases the ability for passive expansion which decreases cardiac output

Heart Valves

• Both semilunar and tricuspid valves have 3 leaflets, bicuspid/mitral valve has 2 leaflets

• Include aortic semilunar valve, pulmonary semilunar valve, tricuspid valve, bicuspid valve

3 Layers of the Valves

• Ventricularis (inflow side) (Atrialis)

• Spongiosa

• Fibrosa (outflow side)

Ventricularis (Atrialis)

• Elastic fibres, fibrillin-1, fibrillin-2 and fibulins

• Stretch and recoil

• Inflow side

Spongiosa

• HA (hyaluronic acid) and proteoglycans; versican, decorin and biglycan.

• Cushion blood pressure forces, assist in re-alignment of collagen and elastin fibers, and resist delamination

Fibrosa

• fibrillar collagens I, III and V

• stiffness and ensure leaflet integrity

• outflow side

Secretion of ECM

Valvular ECs and interstitial cells secrete the most matrix of all heart cells.

Fibrillin 1 Gene (FBN1) Mutations

• Marfan’s syndrome = connective tissue disorder

• Thickening of the AV valves and mitral and/or tricuspid valve prolapse, and aortic aneurysm.

• Get backflow because the valves don’t close properly

Layers of Vasculature (outside → inside)

1. Adventitia - connective tissue and external elastic lamellae

2. Media - smooth muscle cells and internal elastic laminae

3. Intima - endothelium and basement membrane

Arteries vs Veins

• Same layers, but vein have a thinner media layer

• Arteries pressure reservoir

• Veins volume reservoirs

Arterial Wall

• Single cell layer of endothelium

• SMCs in layers separated by concentric elastic laminae

• Stiffness and strength mediated by collagen and elastin (50-70%)

• Elastin is essential for vessel architecture and function

Forces Against Arteries

• Arterial stiffness (force to deform vessel during systole)

• Wall strength (max force applied before vessel failure/rupture)

Main Fibrillar Collagens in Elastic Arteries

Type I, III, and V. III in media (want in the middle to stretch), I in adventitia (prevent overstretch), collagen IV (basement membrane) and VI.

How are the mechanical properties of fibrillar proteins measured?

By stretching the tissue (applying tensile force) which can be plotted on a stess-strain curve.

Stress-Strain Curve

Extension (strain) is measured with applied force (stress). Young’s Modulus (gradient – linear region, aka elastic modulus) as a measure of ‘stretchiness’ of a tissue.

Collagen and Elastin Plotted on a Stress-Strain Curve

5 Steps of the Cardiac Cycle

The hearts cardiac cycle can be broken down into five steps that can then be grouped into: systole (state of contraction) and diastole (state of relaxation - ⅔ time spent in diastole)

Late Diastole

Both sets of chambers are relaxed and ventricles fill passively (note this stage starts with already about 65mL of blood in the left ventricle - it is never empty).

Atrial Systole

Atrial contraction forces a small amount of additional blood into ventricles (the arrow on the graph points to the end diastolic volume (EDV).

Isovolumic Ventricular Contraction

First phase of ventricular contraction which pushes AV valves closed but does not create enough pressure to open the semilunar valves.

Ventricular Ejection

As ventricular pressure rises (to about 80 mm Hg) and exceeds pressure in the arteries, the semilunar valves open and blood is ejected (get to end systolic volume (ESV).

Isovolumic Ventricular Relaxation

As ventricles relax, pressure in ventricles fails. Blood flows back into the cusps of semilunar valves and snaps them closed.

Sino Atrial (SA) Node

“Pacemaker” myocytes with unstable membrane potential capable of spontaneous depolarization. Action potential conduction velocity ~1 m/s. Never reaches a flat line on graph = unstable.

Atrioventricular (AV) Node

Electrically connects atria to the ventricles. AV node slows the action potential conduction velocity to 0.05 m/s to allow time for atria to contract.

Bundle of His and Purkinje Fibres

Conduct the electrical potential through the entire ventricle. Action potential conduction velocity 3-5 m/s.

i_f and i_b^a Ion Channels in Cardiomyocytes

Supply inward depolarizing current (Na⁺) during initial part of pacemaker potential.

i_Na-Ca^b Ion Channels in Cardiomyocytes

Important contribution to later part of pacemaker depolarization by providing inward flow of Na⁺.

i_Ca Ion Channels in Cardiomyocytes

Contribute to last third of pacemaker potential and generates slow-rising action potential by inward movement of Ca²⁺.

i_Kv Ion Channels in Cardiomyocytes

Delayed rectifier, voltage-operated channel that supplies decaying current during pacemaker potential and also supplies repolarization current via outward movement of K⁺.

Myocardial Autorhythmic Cells

Also known as pacemaker cells and the heart beats rhythmically from action potentials they generate. Speed of depolarisation sets the heart beat and can be altered. Resting membrane potential of a myocyte is -80 to -90 mV.

2 Main Types of Heart Cells

• Myocardial Contractile Cells (approx. 99%): They are responsible for contracting and relaxing to pump blood throughout the body.

• Myocardial Autorhythmic Cells (approx. 1%): They initiate and propagate electrical signals, setting the heart's rhythm and coordinating contractions

How to measure the activity of ion channels?

Using electrophysiology - patch clamp. Can do single channel or whole cell recordings.

Organisation of Sarcomeres

Myocyte is packed with contractile bundles called myofibrils (1 μm wide). Myofibril is composed of contractile units called sarcomeres.

Sarcomeres

Are 1.8-2.0 μm in length and consist of two filamentous proteins between two thin partitions (Z Lines). Thick myosin bands occupy the centre of the sarcomere arranged in parallel (A band). Thin actin bands are rooted in the Z line (I band) and then cross over the myosin filaments into the A band. On the sarcolemma, at each Z line are transverse tubules (T-tubules).

Contraction of Sarcomeres

Contraction occurs when actin slides into spaces between myosin. I band shortens but A band does not. Crossbridge cycling mediates constriction and occurs in three stages.

Crossbridge Cycling

1. Rest: Actin-binding sites (yellow star) are blocked

2. Binding site exposed: Ca2+ displaces troponin-tropomyosin complex

3. Flexion: the myosin head flexes towards the centre of the sarcomere pulling the Z line with it

Contractile Force and Energy Source

• Contractile force is determined by the number of cross-bridges formed

• ATP provides the energy for the flexion

• Mitochondria occupy 30-35% of the cell volume of myocytes to provide the ATP

Pre-Load

The stretch of myocardium or end-diastolic volume of the ventricles.

Effect of Preload

Frank-Starling and Length-Tension Relation

Increasing preload increases the contractile force upon stimulation. i.e. The more stretched the sarcomere in cardiac muscle, the more sensitive it is to Ca2+ and the more force it produces.

Effect of Adrenaline on Contraction

Increases calcium in intracellular calcium and increase in the calcium stores as well. This increases heart rate, contraction force, and speed of contraction.

Effect of Endothelin-1 on Contraction

IP₃ is produced which binds to IP₃ receptors on the SR, stimulating calcium release. DAG activates PKC which increases the opening probability of L-type calcium channels.

Effect of Caffeine on Contraction

• At high doses, caffeine can activate Ca2+ release from SR via ryanodine receptors

• At lower concentrations, caffeine can inhibit phosphodiesterase’s (PDE’s) and thus increase free cAMP

• Caffeine can also inhibit IP3 and adenosine receptors at mM concentrations

Effect of Ouabain on Contraction

• Inhibits the Na+/K+ pump, this keeps Na+ inside the cell. Na+ gradient regulates Ca2+ exchangers and voltage regulated Ca2+ channels

Features of Vascular Smooth Muscle

• Actin and myosin structures are very different to cardiac myocytes

• Troponin not present, instead constriction dependent on phosphorylation

• SR Ca2+ store accounts for 1-4% of cell

• SR have inositol 1,4,5 triphosphate receptors and ryanodine receptors

Myogenic Tone

Resistance arteries sense pressure and respond with constriction. Vascular smooth muscles depolarize and voltage gated Ca2+ channels open. Artery contracts against pressure reducing radius and blood flow with increased wall tension.

Smooth Muscle Membrane Potential and Ion Channels

• Membrane potential -60 mV

• K+ channels and Na+/K+ exchanger generate the negative potential

• Stimulation of the K+ channels mainly contributes to vasodilation

• Depolarization opens membrane bound L-Type calcium channels increasing constriction

• Constriction can occur independent of depolarization

Calmodulin Role in Smooth Muscle Contraction

• Increased calcium leads to calcium binding to calmodulin

• Calmodulin activates myosin light chain kinase

• Phosphorylated myosin light chain kinase leads to constriction

• As an additional pathway myosin light chain is sensitized to calcium stimulation via inhibition of myosin light chain phosphatase

Transcytosis

Transport within the cell

Endothelium

The inner cellular lining of all blood vessels and the lymphatic system and is one cell layer thick. Flat 1-2 μm thick, 10-20 μm in diameter, ‘cobblestone’ appearance.

Tight Intercellular Junctions in Endothelium

Tight intercellular junctions to maintain integrity of the vessel wall. Toxic substances (e.g. nicotine) open up these junctions and allow large molecules to pass through the wall, this can lead to disease.

Pinocytosis/Macropinocytosis

• Concentrations of small vesicles adjacent to cell membranes

• Transport of solutes and fluid into/across the cell from the blood

• Facilitates bulk exchange (gases, metabolites, nutrients and proteins)

Caveolae

• A special type of endocytotic/transcytotic vesicle

• Contain caveolin, a transmembrane protein associated with receptors, enzymes and ion channels

• Caveolin-mediated endocytosis of specific factors (ex. LDL)

Weibel-Palade Bodies

• Elongated storage granules specific to endothelial cells

• Main cargo = von Willebrand Factor (vWF) monomers

• Also contain interleukin (IL)-8, endothelin 1, endothelin-converting enzyme, tissue plasminogen activator (tPA), angiopoietin

EC Arrangement

• ECs respond to changes in the blood (biochemical and physical)

• ECs are shear sensitive (they need to respond to changes in flow)

• More aligned in the direction of flow in arteries compared to veins

SMCs and ECs

• SMCs are arranged circumferentially in layers 

• SMCs need to provide pulsatile force

• ECs act as a semipermeable barrier between blood and SMCs

Forces Experienced by Endothelial Cells

• Contact-derived stresses: topography (thickness, fibres and pores), curvature, stiffness

• Flow-derived stresses: shear stress, pressure, tensile strain

Major Roles of Ecs

Metabolism, barrier integrity, vascular tone, mechanotransduction, injury repair, hemostasis, and angiogenesis.

Vascular Tone

The degree of constriction or contraction of vascular smooth muscle within blood vessel walls relative to their maximum diameter. ECs control vascular tone by responding to various hormones, neurotransmitters and vasoactive factors.