Skeletal Muscle (and a little cardiac) Excitation-Contraction Coupling

Ed BalogApplied Physiology

555 14th St NW Rm 1303ed.balog@ap.gatech.edu

[Ca2+] 1 mM

Endo/SarcoplasmicReticulum

Ca2+ATP

ADP + Pi

Ca2+

ATP

AD

P +

Pi

Ca2+

Na2+[Ca2+] 100 nM

[Ca2+] 1-2 mM

IP3RRyR

Intracellular Calcium Signaling

Fertilization Stimulus-secretion

coupling Metabolism Muscle Contraction

Learning and Memory Gene regulation Cell death Immune cell activation

Berridge et al., Nature Rev. Mol. Cell Biol. 1:11, 2000.

Muscle Contraction Summary

Buffers

Mitochondria

Troponin& other Ca2+-

binding proteins

Ca2+

Ca2+ Movement in Muscle

SR

Cytoplasm

Flux that increases cytoplasmic Ca2+

Flux that decreases cytoplasmic Ca2+

Determinates of Contractile Force

1. Maximal Calcium Activated Force

2. Calcium Sensitivity

3. Calcium Delivered to Contractile Proteins

Excitation-Contraction (EC) Coupling

The process linking depolarization of the muscle cell surface membrane to the release of Ca2+ from the sarcoplasmic reticulum (SR).

EC coupling controls the [Ca2+] within the muscle cell; [Ca2+] controls force.

Skeletal Muscle Membrane System

Sarcolemma

TriadTransverse Tubule

SarcoplasmicReticulum

Hypotheses:1. Depolarization of the sarcoplasmic reticulum.

2. Chemical messenger from the transverse-tubules to the sarcoplasmic reticulum.

3. Ca2+-induced Ca2+ release (CICR).

4. Physical link between the transverse-tubules and the sarcoplasmic reticulum. This hypothesis also became to be known as depolarization-induced Ca2+ release (DICR)

Calcium-Induced Calcium Release?

Small amount of Ca2+entering the cell from the extracellular fluid triggers much larger SR Ca2+release.

Primary mechanism of EC coupling in the heart.

Skeletal muscle SR Ca2+ release can be triggered via CICR under experimental conditions.

But – Contraction can be elicited in the absence of extracellular Ca2+.

Physical Link?

Primary mechanism in skeletal muscle.

Electronmicrographs show electron-dense structures in triadic junction, called “feet” linking t-tubules and SR.

Dysgenic mouse muscle lacking “feet” also lack EC coupling.

Excitation-contraction coupling: a tale of two Ca2+ channels

aka “Ca2+ release channel” or “junctional foot protein”

Homotetramer; 2 million daltons.

aka “DHPR”, voltage sensor” or “L-type channel”

Heteromultimeric protein w/ 5 subunits.

Origin of charge movement & L-type Ca2+ current.

Ryanodine Receptor

Dihydropyridine Receptor

RyR tetramer

DHPR tetrad

T-tubule membrane

SR membrane

Lamb and Stephenson, Dept of Zoology, LaTrobe University

High K+ High Na+

Posterino, Proc Australian Physiol Pharmacol Soc 32: 28, 2001.

Mechanically Peeled Muscle Fiber

Methods to Study Excitation-Contraction Coupling

Fig. 5. Ca2+ sparks activated by membrane depolarization in skeletal muscle. The figure shows line scan images of sparks activated by small depolarizations (indicated at top) from a holding potential of −90 mV. For the pulses to −70 mV individual, randomly activated sparks are evident during the depolarization in the images and in the fluorescence records from individual, identified triads (below). The lowermost record in each column shows the average elevation of fluorescence from the entire image. The rightmost panel shows a depolarization to −60 mV during which the high frequency of Ca2+ sparks has resulted in a much larger elevation of fluorescence within the fiber, precluding the observation of individual sparks.

Klein and Schneider Prog Biophys Mol Biol 92:308, 2006

[3H]Ryanodine Binding

Ryanodine binds the open channel with high affinity and specificity.

Binding reflects the open state of the channel. Ryanodine

Single Channel Recording in a Planar Lipid Bilayer

LipidBilayer

Channels

Some of the Whole-Cell Measurable Events of Excitation-Contraction Coupling

DHPR (L-type Ca2+ Channel)

Member of voltage-gated ion channel superfamily, which also includes Na+ & K+ channels.

Pentamer; α1 subunit forms pore.

Cav1.1 : skeletal muscle isoform; 1873 aa in humans

Cav1.2: cardiac isoform; 2169 aa in humans

NC

βN

C

+++

+++

+++

+++

α1

N C

γ

α2

N

C

II-III loop and β-subunitvital for skeletal muscle ECC

δC

N

cytoplasm

t-tubulelumen

S

S

10 μA/μF

25 ms

DHPR electrical signals:L-type calcium current Cardiac current is larger

and is fully activated by a ventricular action potential. Ca2+ influx via the cardiac DHPR activates RyR2 via Ca2+ induced Ca2+ release (CICR).

Skeletal muscle current is smaller and is barely activated during a skeletal muscle action potential. The current is not required for contraction.

Cardiac

Skeletal Muscle

Wang et al Biophys J 77:2709, 1999Caputo & de Bolanos J Physiol 289: 175, 1979.

Skeletal Muscle Contraction Does Not Require the Entry of Extracellular Calcium

2.5 mM Ca2+ 0 Ca2+ 2.5 mM Ca2+

1. Voltage dependence of Ica and contraction differ.

2. Ica activation is too slow to contribute Ca entry during an action potential.

3. Skeletal muscle can contract in the absence of extracellular Ca.

Dulhunty and Gage J Physiol 399:63, 1988

Tetanus

Twitch

DHPR electrical signals: Charge movement

Arises from movement of charged amino acids across membrane electric field.

Similar to ion channel gating currents, but larger and slower.

Required for skeletal muscle contraction.

t-tubulemembrane

++

+

++

+ΔV

DHPRtetrad

+

Charge Movement & Ca2+ Release

Top: voltage dependence of skeletal muscle contraction.

A: Intracellular calcium transients recorded from a muscle fiber.

B: T-tubule charge movement records from the same fiber.

Below: Correlation between Charge movement and Ca2+ release rate.

Caputo & de Bolanos J Physiol 289: 175, 1979.

Ryanodine Receptor (RyR)

The open channel binds Ryanodine, an alkaloid derived from the South American plant Ryania speciosa.

Member of intracellular Ca2+ channel family, includes IP3 receptor. Largest known ion channel. Three isoforms: RyR1 (skeletal), RyR2 (cardiac), RyR3 (wide cellular

distribution, low abundance).

Sarcolemma

TriadTransverse Tubule

SarcoplasmicReticulum

Calcium Dependence of RyR1 and RyR2

High affinity calcium binding site – Activates channel when bound. Calcium selective (KCa/KMg ~100) .

Low affinity calcium binding site – Inhibits channel when bound. Relatively unselective for divalent cations (KCa/KMg ~1) .

A IAL

A

I

ALSR lumenal calcium binding site – Activates channel when bound.

Song et al Prog Biophys Mol Biol. 105:145, 2012.

RyR Macromolecular Complex

RyR Channel Modulators (Partial List)

ExogenousCaffeine

RyanodineRuthenium Red

Volatile AnestheticsDepolarizing Muscle

RelaxantsOxidizing/reducing agents

Local Anesthetics4-chloro-m-cresol

Dantrolene

EndogenousAdenine Nucleotides

CalmodulinMg2+

H+

Inorganic PhosphateDihydropyridine Receptor

FKBP12/12.6Reactive Oxygen Species

Nitric Oxide

Arrangement of DHPR and RyR1

4 DHPRs per coupled RyR

RyR1

α1

β

α2

γδ

SR Membrane

T-tubule Membrane

DHPRs

Junctophilin

Serysheva et al PNAS 99:10370, 2002.

RyR1

DHPR tetrads

ToadfishSwim Bladder

(very fast)

MammalianFast-twitch

Muscle

MammalianSlow-twitch

Muscle

DHPR:RyR Arrangement

and Ratio Varies with

Muscle Fiber Type

MammalianCardiac Muscle

RyR2

Individual DHPRs

How are uncoupled skeletal muscle ryanodine receptor

channels opened?

+ + +

+ + +

+ + +

+ + +

+ + +

Direct Coupling Ca2+-Induced Ca2+ Release

Comparison of Cardiac and Skeletal Muscle Excitation-Contraction Coupling

Cardiac Muscle:Calcium-InducedCalcium Release

Skeletal Muscle: Mechanical Coupling

DHPR mediates Ca2+ influx, Ca2+ binds to and activates the underlying RyR.

Charge movement within DHPR and subsequent conformational change activates RyR via direct physical interaction.

Ca2+

++ +++ ++ V

DHPR tetrad(α1s)

RyR1 Ca2+

Ca2+DHPR(α1c)

+

+V t-tubule

RyR2

SR

Two Forms of Ca2+ Entry in Skeletal Muscle

Store-Operated Calcium Entry (SOCE)

Requires depletion of the internal stores & has been best characterized in non-excitable cells.

Requires STIM1 and ORAI Significant SR Ca2+ depletion

required to reach activation threshold for SOCE only achieved during prolonged bouts of ECC.

SOCE is not responsible for refilling the SR during periods of fiber quiescence.

Excitation-Coupled Calcium Entry (ECCE)

Activated following prolonged membrane depolarization

Independent of the calcium stores.

Requires functioning L-type channel and RYR1, but molecular identity of the pore remains undefined although it is likely to involve the L-channel.

Store-Operated Ca2+ Channels

Lewis Nature 446: 284, 2007.

Calcium Transporters in Muscle

SERCA: Sarco/Endoplasmic Reticulum Calcium ATPasePMCA:Plasma Membrane Calcium ATPaseNCX:Na/Ca ExchangeMCU:Mitochondrial Uniporter

SERCA: Sarco/Endoplasmic Reticulum Calcium ATPase

• Encoded by 3 mammalian genes• ~1000 amino acids• 10 TM helices• Located in intracellular organelles: ER

and SR• 3 Cytoplasmic domains (A,N,P)• 2 Ca2+ transported per ATP hydrolyzed• Activity regulated by phospholamban and

sarcolipin

SERCA GenesSERCA1 – Expressed in fast-twitch skeletal muscle. Two splice variants.

SERCA1a in adult fast-twitch skeletal muscle.SERCA1b in embryonic skeletal muscle.

SERCA2 – Expressed in cardiac and slow-twitch skeletal muscle. Three splice variants.

SERCA2a in cardiac and slow-twitch skeletal muscle.SERCA2b low levels in most tissues (“house-keeper”)SERCA2c in embryonic heart cells and mesenchymal stem cells (give rise to muscle and bone)

SERCA3 – Expressed in smooth muscle. Five splice variants.In smooth muscle, blood, and neural cells. Co-expressed with SERCA2b (and others).

Why 3 genes with multiple splice variants?Tune enzyme activity (calcium affinity & maximal velocity) to cell type, ligand sites to modulate activity, binding sites for regulators, other unknown reasons.

PMCA:Plasma Membrane Ca2+-ATPase

• Encoded by 4 mammalian genes• ~1300 amino acids• 10 TM helices• Located in surface and t-tubule

membranes• 3 Cytoplasmic domains (A,N,P)• 1 Ca2+ transported out of cell per ATP

hydrolyzed• Activity regulated by calmodulin

Brini et al. FEBS J 280:5385, 2013

NCX:Sodium-Calcium Exchanger

• Encoded by 3 mammalian genes• ~950 amino acids• 9 TM helices• Located in surface and t-tubule

membranes• 1 Ca2+ transported out of cell per 3 Na+

into cell

• Driven by Na+ potential (Em-ENa)

• Reverses mode during depolarization Red = CalciumGreen = Sodium

MCU:Mitochondrial Calcium Uniporter

• A complex of proteins located in mitochondrial inner membrane.

• Facilitated diffusion of Ca into matrix.

• Driven by large electronegative potential (-180 mV).

Marchi & Pinton, in press J Physiol 2013

Ryanodine Receptor Diseases

RyR1

Malignant Hyperthermia

Central Core Disease

RyR2

Catecholaminergic Polymorphic Ventricular Tachycardia

Arrhythmogenic Right Ventricular Dysplasia

RyR3

None Identified, Yet

Malignant HyperthermiaAn autosomal dominant pharmacogenetic disease characterized

by an unusual metabolic reaction to volatile anesthetics and depolarizing muscle relaxants.

Symptoms include:HypercapniaCyanosisTachycardiaMuscle rigidityRhabdomyolysisHyperthermia

Incidence: 1 in 15,000 anaesthetized children 1 in 50,000 anaesthetized adults

With the introduction of dantrolene the mortality rate has been reduced from ~80% to current ~5%.

About 50% of cases linked to RyR1 mutations.

A1832GG2060CV2117LD2129ER2163CR2163H/PV2168MA2200VT2206M/RV2210FV2212AV2214IV2280II2321VR2336HN2342SE2344DV2346ME2348GA2350T

R367LR401C/G/HI403MQ474HY522S/CR530HR533C/HR533HR552WR614C/LS846LR1043CR1140CS1342GQ1589PP1592LS1728P/FM1729RP1787LM1814K

L13RC35RR 44C/HD60NQ155KR156KE160GR163C/LG165RD166N/GR177CY178CG215EV218IM226KD227VG248RR316LR328WG341R

R2355WF2364VP2366RA2367TG2375AA2428TD2431N/WG2434RR2435H/LA2437VR2452W/QI2453TR2454C/HR2458C/HP2496LR2508G/C/HY2510HE2545DV2550L

R2591WT2596IR2676WD2730H/GG2733DT2787SR2840WE2880KE3104KR3119HR3350HK3367RP3527SE3583QE3584QR3707LQ3756EV3840IR3903Q

I3916MD3986EG3990VS4050YT4081MN4119YΔ4124-4216R4136SI4138TV4234LE4283VT4637A/IG4638DR4645QΔ4647-4648L4650PH4651PP4668SF4684S

K4724QY4733EG4734ER4737W/GL4793PY4796CF4808IL4814FI4817FG4820WL4824PR4825C/PT4826IL4838VV4849IA4856GF4860VR4861C/HΔ4863-4969

Y4864CK4876RM4880TG4891RR4893W/QA4894T/VI4898TG4899R/EA4906VR4914G/TF4921SV4927FΔ4927-4928I4938MD4939EA4940TG4942VF4960YP4973L

0 1000 2000 3000 4000 5000

RyR1 MH/CCD Mutations

Amino Acid Sequence

Increased Sensitivity to RyR Activators Forms Basis for MH Screening

In Vitro Contracture TestCaffeine/Halothane Contracture Test

European Malignant Hyperthermia Group (EMHG)Low threshold = contracture of ≥0.2 g at a concentration of

≤2 mM caffeine or ≤ 2% halothane MH Susceptible(MHS): Low contraction threshold for both

Caffeine & HalothaneMH Equivocal(MHE): Low threshold for oneNormal (MHN): Normal threshold for both

North American Malignant Hyperthermia Group (NAMHG)

Low threshold = contracture of ≥0.3 g at a concentration of ≤2 mM caffeine or ≥0.3 g at ≤ 3% halothane

MH Susceptible(MHS): Low contraction threshold for either Caffeine or Halothane

Normal (MHN): Normal threshold for both

Gallant and Lentz Am J Physiol 262:C422, 1992

R615CHomozygous

R615CHeterozygous

Normal

MacLennan and Phillips Science 256:789, 1992.

How does abnormal Ca2+ regulation cause MH?

Is there a link between MH and exertional heat illness?

Porcine Stress Syndrome

26 soldier with exertional heat illness, all had positive in vitro contracture tests (Bendahan et al Anesth Anag 93: 683, 2001).

3 patients with positive IVC and RyR1 mutations; 2 had history of EHI (Brown et al Br J Anesth 88:508, 2002).

Effectiveness of dantrolene in treatment of heat illness questioned (see Hadad et al Sports Med 34: 501, 2004).

Y522S MH mutation knock-in mouse has an increased sensitivity to heat stress (Chelu et al FASEB J 20:329, 2006).

Cardiac Excitation Contraction Summary

Bers 2002 Nature 425:198.

Catecholaminergic Polymorphic Ventricular Tachycardia

Rare autosomal dominant (RyR2 mutations) or recessive (calsequestrin)

Symptoms:Rare before age 10.None at restVentricular arrhythmias of varying morphology upon exercise or catecholamine administration.SyncopeDeath

Mortality: 30-35% by age 30

CPVT Mutations

In Situ Confocal Imagingof a CPTV Heart

Chen et al Circ Arrhythmia Electrophys 5:841, 2012

CPVT Mechanisms

Calcium Leak from RyR2

Activation of Na/Ca exchange

Inappropriately timed depolarization

Arrhythmia

Sudden Death