Download Basics of skeletal muscle electrophysiology electrophysiolo

Basics of skeletal muscle
electrophysiology
Tóth András, PhD
Topics
• Structure
• Contraction and relaxation
• Activation
• Excitation-contraction coupling
• Action potential
• Ion channels*
• Calcium homeostasis
Structure
Skeletal muscle
Cardiac muscle
In skeletal muscle the SR is greatly enlarged at the terminal cisternae, the
diameter of the T-tubules is relatively narrow.
In cardiac muscle T-tubules are much larger in diameter and the SR is more
sparse, but includes junctional couplings with the external sarcolemma, as well, as
the T-tubules. Myofibrils are also more irregular. Mitochondria are plentiful.
Schematic diagram of skeletal and cardiac muscles
Skeletal muscle fibre types
•
Voluntary muscles contain a variety of fibre types which are specialised for particular tasks. Most
muscles contain a mixture of fibre types although one type may predominate. The pattern of gene
expression within each voluntary muscle cell is governed by the firing pattern of its single motor
neurone. Motor neurones branch within their target muscle and thereby control several muscle
fibres, called a motor unit. The high precision eye muscles have only a few fibres in each motor
unit, but the muscles in your back have thousands. All the cells in a motor unit contract in unison
and they all belong to the same fibre type:
•
Type 1 or slow oxidative fibres have a slow contraction speed and a low myosin ATPase activity.
These cells are specialised for steady, continuous activity and are highly resistant to fatigue. Their
motor neurones are often active, with a low firing frequency. These cells are thin (high surface to
volume ratio) with a good capillary supply for efficient gas exchange. They are rich in mitochondria
and myoglobin which gives them a red colour. They are built for aerobic metabolism and prefer to
use fat as a source of energy. These are the marathon runner's muscle fibres.
•
Type 2A or fast oxidative-glycolytic fibres have a fast contraction speed and a high myosin
ATPase activity. They are progressively recruited when additional effort is required, but are still
very resistant to fatigue. Their motor neurones show bursts of intermittent activity. These cells are
thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. They
are rich in mitochondria and myoglobin which gives them a red colour. They are built for aerobic
metabolism and can use either glucose or fats as a source of energy. These are general purpose
muscle fibres which give the edge in athletic performance, but they are more expensive to operate
than type 1.
•
Type 2B or fast glycolytic fibres have a fast contraction speed and a high myosin ATPase activity.
They are only recruited for brief maximal efforts and are easily fatigued. Their motor neurones
transmit occasional bursts of very high frequency impulses. These are large cells with a poor
surface to volume ratio and their limited capillary supply slows the delivery of oxygen and removal
of waste products. They have few mitochondria and little myoglobin, resulting in a white colour
(e.g. chicken breast). They generate ATP by the anaerobic fermentation of glucose to lactic acid.
These are sprinter's muscle fibres, no use for sustained performance.
Contraction - relaxation
The length-tension relationship in skeletal & cardiac muscle
The contractile force in skeletal muscle is determined by
A) Contraction summation
(tetanus)
B) Sarcomer length (myofilament overlap)
C) Activation of further fibers (recruitment)
The contractile force in cardiac muscle is determined by
A) Intracellular Ca concentration (analog)
(intrinsic regulation)
B) Sarcomer length (myofilament overlap)
(extrinsic regulation)
The regulation of the contractile force in skeletal & cardiac muscle
Activation
Comparison of motor nerve terminal, the neuromuscular junction and a skeletal muscle fibre, showing ion channels (numbered 1–8) whose mutation
leads to disease. Mutations in several AChR subunits (1) cause myasthenia (muscle weakness). Autoimmune channelopathies are indicated by binding
of antibodies (stars), which leads to internalization and downregulation of channel number. Loss of presynaptic K+-channel function (KV1.1, KCNA1)
(2) leads to increased transmitter release and enhanced muscle contraction, whereas downregulation of presynaptic Ca2+ channels (3) or AChR (1)
function causes myasthenia, by preventing neurotransmitter release, or binding, respectively. Gain-of-function mutations in the muscle Na+ channel (4,
SCN4A), and loss-of-function mutations wild-type and mutant (red trace) Na+ current showing the persistent inward current produced by mutations
associated with myotonia and/or periodic paralysis. c, Unlike normal muscle (above), action-potential firing in myotonic muscle (below) continues after
stimulation has ceased, producing a sustained contraction.
The „restricted space” located between junctional sarcosplasmic reticulum (JSR) and the
junctional sarcolemma (JSL) forms a local intracellular compartment which, has a very
special role in both EC coupling and calcium homeostasis.
In this space changes in Na+, K+ & Ca2+ concentrations are significantly greater than in
all other compartments of the cytosol.
L-type Ca channel & NCX protein densities in the junctional sarcolemma are also much
higher than in any other regions of the sarcolemma.
The „restricted space”
Excitation-contraction coupling
(ECC)
A) Isolated rat ventricular myocyte
B) Frog skeletal muscle fiber
In contrast to skeletal muscle in cardiac myocytes external Ca influx is
essential for activation of contraction
In contrast to skeletal muscle, where DHPRs are found in very regular
structure, the DHPRs in the heart cells are sparse and less aligned.
Structural differences in skeletal & cardiac T-tubule junctions
In skeletal muscle
The physical link between DHPR & RyR1 is critical for VDCR
Influx of external Ca (ICa) is not required
In cardiac muscle
The physical link between DHPR & RyR2 is not critical for CICR
Influx of external Ca (ICa) is crucial
Comparison of EC-coupling in skeletal & cardiac muscle
A) Two Ca sparks
(2D confocal
fluorescence)
B) Single Ca spark
(line-scan image)
C) [Ca]i computed
from the image
D) Surface plot of
[Ca]i during a Ca
spark
Fusion of a large
number of sparks
leads to Ca-transient
& contraction !!!
The elementary event of Ca-release from the SR is the local „spark”, which often
occurs during rest in a stochastic manner. 6-20 RyRs contribute to a single spark, which
starts at the T-tubule and increases [Ca2+]i in ∼ 10 ms to a peak value of 200-300 nmol.
The reason for its time dependent decrease is Ca diffusion and Ca reuptake.
Calcium sparks
Similarities between skeletal muscle and
cardiac EC coupling
Both muscle types are striated & contain T-tubules and
highly developed intracellular SR networks
APs provide the excitation stimulus used to activate
plasma membrane DHPRs (or Ca2+ channels )
Activated DHPRs (or Ca2+ channels) trigger the opening
of SR Ca2+ release channels
Resulting elevation in intracellular Ca2+ activates the
contractile machinery
Differences between skeletal muscle and
cardiac EC coupling
The skeletal muscle contains highly developed, cardiac
muscle contains a less developed T-tubule and SR system
The skeletal muscle does not contain, while the heart contains
specialized excitatory tissues (SA node) and conductive fibers
(Purkinje fibers)
Each skeletal muscle fiber is independent, while the heart is a
syncytium of many cells electrically connected at intercalated
discs by gap junctions
The skeletal muscle AP is 100x shorter (2.5 ms) than that of
the ventricular myocardium
Ca-sensitizer agents
Positive inotropic agents
Hypoxia – ischemia
Factors which may alter Ca-sensitivity
Conclusion
A) In a simplified manner the 3 muscle types can serve as models for the 3 major
mechanisms of SR Ca-release (VDCR: skeletal muscle; CICR: cardiac
muscle; IP3ICR: smooth muscle) This is an oversimpliplification since all 3
mechanism may be present and functional in all 3 muscle types.
B) In skeletal muscle VDCR seems to be the crucial initiating process, however,
CICR may be very important in recruiting RyRs (∼ 50%) which are not
physically coupled to T-tubule tetrads. IP3 can also induce Ca release
(IP3ICR), but its significance is not yet clear.
C) In cardiac muscle CICR is the essential EC-coupling mechanism. IP3 may also
modulate cardiac Ca release. There is some evidence for a functional direct
link between the SL and the SR (and possibly VDCR). The significance of
this link is not yet clear.
D) In smooth muscle there is evidence for both IP3ICR & CICR. There is also
evidence for that the IP3ICR interacts with a different plasma membrane Ca
channel (TRP), involved in CCE where the signal is retrograde from IP3R to
TRP.
Action potential
Calcium homeostasis in skeletal
muscle
INFLUX
=
EFFLUX
ICa
+
+
INCX ↓↓
+
TRIGGER
-
Ca-transient
CDI
1
EFFLUX
≠
ICa↑
+
TRIGGER
INFLUX
=
EFFLUX
ICa
+
TRIGGER
CDI
“Empty” cell
INCX ↓
+
Ca-transient
CDI
2
Steady-state
INFLUX
3
EFFLUX
≠
ICa ↑↑
INCX
+
TRIGGER
INFLUX
INCX
+
Ca-transient
During “refill”
CDI
4
Ca-transient
Steady-state
Principles of “autoregulation” of SR Ca2+ content in the heart
This mechanism does not work in skeletal muscle
Modes of regulated Ca2+ entry across the plasma membrane. Ca2+ can enter cells by any of several general
classes of channels, including voltage-operated channels (VOC), second messenger-operated channels
(SMOC), store-operated channels (SOC), and receptor-operated channels (ROC). VOCs are activated by
membrane depolarization, and SMOCs are activated by any of a number of small messenger molecules, the
most common being inositol phosphates, cyclic nucleotides, and lipid-derived messengers (diacylglycerol
and arachidonic acid and its metabolites). SOCs are activated by depletion of intracellular Ca2+ stores, and
ROCs are activated by direct binding of a neurotransmitter or hormone agonist (Ag). In addition, under some
conditions, Ca2+ can enter cells via the Na-Ca2+ exchanger (NCX) operating in reverse mode.
Model of SOCE in skeletal muscle. SOCE in skeletal muscle displays rapid kinetics compared to nonexcitable cells. STIM1 localization may account for these kinetic differences. Electron micrographs of
skeletal muscle from STIM1 gene trapped mice revealed STIM1 protein aggregates located in membranes
of the terminal cisternae and the para-junctional SR. The junctional STIM1 pool is located near or
complexed with Orai1 and can respond rapidly to store depletion.
Although recent studies have shown that STIM1 activation by store depletion suppresses L-type voltageoperated calcium (Cav1.2) channels, whether STIM1 plays a similar role in the regulation of L-type
channels in skeletal muscle which expresses the Cav1.1 isoform is currently unknown.
STIM & ORAI: two major players in the SOC(cer) field
Structure and distribution of STIM proteins. (A) Comparison of the sequence domains of STIM1 and
STIM2 proteins including the EF-hand Ca2+ binding regions, sterile-alpha motifs (SAM), glycosylation
sites (hexagons), transmembrane domains (TM), coiled-coil regions, ezrin–radixin–moesin-like domains
(ERM), and proline-rich domains (P). (B) Domain topologies for STIM1 and STIM2—a number of
potential serine/threonine phosphorylation sites existing on the N-terminal domains of the proteins are
shown (red P). Lower right panel: distribution of STIM1 and STIM2 in the PM and ER. While STIM1 can
be observed in both the ER and PM, the STIM2 protein appears to be exclusively in the ER.
Models for the coupling between STIM1 and Orai1 in the activation of store-operated Ca2+ entry. (A)
Simplest model in which aggregated STIM1 in the ER interacts directly with Orai1 in the PM. (B) STIM1
in the PM is the primary site of a interaction of aggregated STIM1 in the ER; the plasma membrane
STIM1 protein then mediates interaction with and activation of Orai1in the PM. (C) STIM1 aggregated in
the ER activates PM Orai1 channels by direct activation as in (A) but STIM1 in the PM exerts a
modulatory role on this channel activation by interacting with aggregated STIM1 in the ER.