ExcitExcitationation--concontractracttiionon

ccoupling in oupling in

cardiomyocytescardiomyocytes

Dr. Tóth AndrásDr. Tóth András

2+2+ii

„Intracellular free calcium concentration”

TopicsMajor cellular structures involved in E-C coupling

Myofilaments: The end effector of E-C coupling

Sources and sinks of activator Ca

Cardiac action potentials and ion channels*

Ca influx via sarcolemmal Ca channels

Na/Ca exchange and the sarcolemmal Ca-pump

Sarcoplasmic reticulum Ca uptake, content & release

Excitation-contraction coupling

Control of cardiac contraction by SR & SL Ca fluxes

Cardiac inotropy

Ca „mismanagement”

Similarities between cardiac and skeletal muscle EC coupling

� Activated Ca2+ channels trigger the opening of SR

Ca2+ release channels

� APs provide the excitation stimulus used to activate

plasma membrane Ca2+ channels (or DHPRs)

� Resulting elevation in intracellular Ca2+ activates the

contractile machinery

� Both muscle types are striated & contain T-tubules

and highly developed intracellular SR networks

1!

Differences between cardiac and skeletal muscle EC coupling

� The heart contains specialized excitatory tissues (e.g.

SA node) and conductive fibers (Purkinje Fibers)

� The heart is a syncytium of many cells electrically

connected at intercalated discs by gap junctions

� The ventricular AP is 100x longer (250 ms) than that

of skeletal muscle

� Cardiac muscle contains a less developed T-tubule

and SR system

2!

Summary of cardiac EC coupling

� An AP is propagated from an adjacent myocyte via

gap junctions located at the intercalated disc

� AP activates membrane Ca2+ channels causing a

substantial Ca2+ influx during a prolonged AP

� Local increase in myoplasmic Ca2+ triggers a larger

release of Ca2+ from the SR (CICR)

� The global increase in myoplasmic Ca2+ activates the

myofilaments to initiate contraction

� β1-adrenergic stimulation increases contractility by

increasing Ca2+ current, release, and reuptake

3!

Major cellular structures

involved in EC coupling

“Our hero” the cardiac ventricular myocyte

~ 100 x 25 µµµµm

4

Schematic diagram of skeletal and cardiac muscles

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.

Skeletal muscle Cardiac muscle5

!

The „restricted space” located between junctional SR and the sarcolemma 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”

6!

Relative positions of key proteins at the skeletal muscle triad

The SR is filled with

calsequestrin.

The non-junctional SR

surface is covered with

the Ca-pump protein.

RyR foot proteins are

organised in two parallel

rows and protrude from

the SR

A similar array of DHP

proteins exists in the T-

tubular membrane, but

the axis of fourfold

symmetry is rotated and

they lie over alternating

foot structures

7

Structural differences in skeletal & cardiac T-tubule junctions

In contrast to skeletal muscle, where DHPRs are found in very regular

structure, the DHPRs in the heart cells are sparse and less aligned.

8

Myofilaments: the end

effector of ECc

Myofilament proteins

Cardiac Troponin-C

One Ca-specific binding site

(regulation, Kd = 500 nM)

Two Ca-Mg specific binding

sites (stability)

9!

The “sliding filament” mechanism of contraction in cardiac cells

10!

Ca2+

*A + M � ADP � Pi

High actin affinity

A-M � ADP � Pi

ADP+Pi

ADP+Pi

A-M

Pi

ADP

*

A-M ATP

Low actin affinity

*

ATP

ATP

Resting muscle

Rigor Complex

The major steps of the crossbridge cycle in cardiac muscle

11!

The regulation of the contractile force in skeletal & cardiac muscle

The contractile force in skeletal muscle is determined by

A) Contraction summation (tetanus)

B) Activation of further fibers (recruitment)

C) Sarcomer length (myofilament overlap)

The contractile force in cardiac muscle is determined by

A) Intracellular Ca concentration (analog)

(intrinsic regulation)

B) Sarcomer length (myofilament overlap)

(extrinsic regulation)

12!

The length-tension relationship in skeletal & cardiac muscle

13!

Factors which alter cardiac myofilament Ca-sensitivity

Ca-sensitizer agents Hypoxia – ischemia

Positive inotropic agents

14

Force-velocity and force-power curves in cardiac muscle

15!

Sources, sinks and kinetics

of activator Ca

General scheme of Ca cycle in a cardiac ventricular myocyte

(From Bers, 2002)

16!!

Total Ca requirements for myofilament activation

A) Ca sensitivity of the myofilaments (F = Fmax/(1 + (Km/[Ca]i)n)

B) The amount of added total cytosolic (activator ) Ca, required to

activate contractile force

Data shown were measured in „skinned” cardiac fibers (Hill

coefficient n = 2) & intact cardiomyocytes (n = 4)

17

Dynamic Ca changes (Ca transient) during a twitch

Rabbit vetricular

myocyte model

A) Free [Ca]i and change in total cytosolic [Ca]cyt

B) Associated changes in Ca bound to different cytosolic ligands

C) Ca currents and transporter fluxes

!!!

Experimental

determination of the

“exact” [Ca]i value is

practically impossible !!!

18!

Ca content of the cytosol, Ca influx & efflux mechanisms

Extracellular space (ECS ∼ 30% total body volume)

[Ca]: 2 mmol/L ECS x 0.55 L ECS/L cytosol =1000 µmol/L cytosol

Influx: VD Ca channels, Na/Ca exchanger, „leakage” channels

Efflux: Na/Ca exchanger, sarcolemmal Ca-ATPase

Internal sarcolemmal surface

[Ca]: 60 µmol/L cytosol (no role in EC coupling!)

(following very quick removal of extracellular Ca, depolarization

does not produce measurable contraction or [Ca] increase)

Sarcoplasmic reticulum

[Ca]: 50-250 µmol/L cytosol

Influx: SR Ca-release channel (to cytosol!)

Efflux: Sr Ca ATPase (SERCA2) (to SR!)

Mitochondria

[Ca]: 10 000 µmol/L cytosol (in vitro) (PO43- – „matrix loading”)

[Ca]: 100 µmol/L cytosol (in vivo)

Influx: Na/Ca antiport (to cytosol!)

Efflux: Ca uniport (to mitochondrium!)

The role of the individual Ca transporter mechanisms in relaxation

Caffein: release of SR

Ca content, inhibition

of uptake

0Na + 0Ca: inhibition

of Na/Ca exchanger

FCCP: uncoupling

of mitochondria

19

The role of the mitochondrium in intracellular Ca regulation

The Ca cycle across the inner

mitochondrial membrane – changes in

[Ca2+]m are reflected in activities of the

mitochondrial dehydrogenases

Mitochondrial free Ca content

[Ca]m as a function of cytosolic Ca

concentration [Ca]i

Critical [Ca]i

≈ 500 nM

20

Cardiac action potentials

and ion channels*

Cardiac ion channels

21!

Ca influx via sarcolemmal

Ca channels

The role of L-type Ca channels in ventricular myocytes

(From Bers, 2002)

22!

Properties of cardiac L- & T-type Ca channels

23!

Properties of the L-typeCa channel

CaL channel: ∼ 3-5/µm2

DHP receptor: ∼ 20/µm2

24

ICa „positive starcase” - Ca-dependent Ca-current facilitation

The reason for Ca-dependent ICa-facilitation (or inhibition) –” starcase” – is the significant

difference between Ca levels in rest & during permanent activity. In some species (dog,

rabbit, human) the relationship is positive, in other species (rat, mouse) negative

PR: post rest SS: steady state pulse

25

ICa during square pulse and AP-clamp

The amount of

transported Ca is

mainly determined

by the shape -

duration of the AP

Long AP: large

Ca influx

Short AP: less

Ca influx

26

Modulation of ICa by agonists and antagonists

Antagonists: Dihidropiridin (DHP)-family (nifedipine, nitrendipine,

nimodipine, nisoldipine, (+) Bay K 8644,

azidopine, iodipine)

Fenil-alkilamin (ΦAA)-family (Verapamil)

Benzothiazepin (BTX)-family (Diltiazem)

Agonists: (-) Bay K8644

(+) S-202-79, etc.

Agonists: mode 2 („permanently” open state)

(e.g. Bay K 8644 ∼ 0.6ms → ∼ 20 ms)

Antagonists: mode 0 („permanently” closed state)

27!

Dual pathways for activation of ICa by ββββ-adrenergic stimulation

1. Gs → Adenylyl-cyclase → cAMP↑ → PKA

2. Gs → direct effect (AKAP: PKA anchoring protein, PLB: phospholamban)

1.

2.

28

Conclusion

A) L-type Ca channel current (ICa) is the main route of Ca entry into

the cell (vs. leak, Na/Ca exchange, or ICa,T).

B) ICa plays a central role in cardiac EC-coupling and overall Ca

regulation and contraction.

C) The kinetics and amplitude of the ICa during the AP are critical factors

in controlling the amount of Ca released by the SR.

D) Ca which enters as ICa may also contribute directly to the activation

of the myofilaments, and to the replenishment of the SR Ca stores.

E) For a steady state to exist, the amount of Ca influx via ICa must be

extruded from the cell during the same cardiac cycle (e.g. via NCX).

Any uncompensated Ca influx could constitute a progressive Ca load

of the cell.

F) Due to the high conductance of these ion channels, a relative small

number of Ca channels which fail to inactivate (or reactivate), could

lead to substantial Ca gain (especially in depolarized cell). This can

compromize relaxation and contraction, and even be arrhytmogenic.

!

Na/Ca exchange & the

sarcolemmal Ca-pump

Sarcolemmal Ca-transport mechanisms in ventricular myocyte

(From Bers, 2002)

29!

Structural & functional model of the sarcolemmal Ca2+-ATPase

A) Linear representation : 10 TM regions, phospholipid (PL) sensitive site,

calmodulin-binding site (CaM-BD), etc.

B) 2D model: in autoinhibitory state (left), and following

Ca2+-CaM stimulation (right)

30

Kinetic properties of the cardiac sarcolemmal Ca2+-ATPase

Calmodulin binding is an essential condition for physiological activity !!!

31!

A structural model for the Na/Ca exchanger (NCX)

regulation

szabályozásXIP: exchange inhibitory protein

32

Functional model of the “electrogenic” Na/Ca exchanger

A. 2D topological model

B. TM 2, 3, 7, 8, and the α-1

& α-2 loops form the

transport center

Major steps of the transport cycle of the

NCX in cardiac muscle. The exchanger

activity is regulated by intracellular Nai &

Cai concentrations

(E1: inside-open, E2: outside-open)

33

Membrane potential (Em) dependence of NCX current (INa/Ca)

Spike phase of the AP (Em > 0): Ca influx is dominant,

Plateau phase: depending on ion distributions both influx or efflux are feasible,

Repolarization phase: the Ca efflux is dominant (especially if [Ca2+]i is high)

34!

Enhancers and inhibitors of theNa/Ca exchange

35

Changes in ENa/Ca and INa/Ca during an action potential in rabbit ventricular myocyte

„Reverse mode”: Ca2+ influx, Na+ efflux – early phase of AP

„Forward mode”: Ca2+ efflux, Na+ influx – late phase of AP

36!

It is so simple to approximate the exchanger current !!!

37

Competition among Ca transport-mechanisms during relaxation

Heart failure:

INCX ↑↑↑↑↑↑↑↑↑↑↑↑

ISERCA ↓↓↓↓↓↓↓↓↓↓↓↓

38

“Rest decay” of SR Ca content

The rate of the rest decay

is mainly depending on

the ratio of the leakage

currents of the SR and SL

and the activity of the

Na/Ca exchanger

The rate of the rest decay

is quite species

dependent: it is small in

rat, but rather significant

in rabbit

Resting state is not a

physiological state for

the cardiac cell !!!

39

Conclusion

A) Na/Ca exchanger mechanism is essential in myocardial intracellular

Ca regulation

B) Na/Ca exchange is the main means by which Ca (entering the cell

via L-type Ca channels) is extruded from the cell, during both relax-

ation & diastole. By comparison the sarcolemmal Ca-pump (SLCP)

seems relatively unimportant in cardiac muscle.

C) Na/Ca exchange can even compete with the powerful SR Ca-pump

(SERCA) for cytoplasmic Ca (~ 1:2), thus contributing to relaxation

D) Na/Ca exchange can also mediate Ca influx sufficient to activate cell

contraction, but this probably does not occur under normal physio-

logical conditions (where its main role is Ca extrusion).

E) In order for a steady state to be achieved the average amount of Ca

extruded during each cardiac cycle should equal the amount of Ca

influx by L-type Ca channels.

F) Since Na/Ca exchange is the main means by which the cell extrudes

Ca, anything which prevents this Ca extrusion will increase cellular

Ca loading and can lead to Ca overload.

!

Sarcoplasmic reticulum - Ca

uptake, content and release

SR Ca-transport mechanisms in ventricular myocytes

(From Bers, 2002)

40!

SR Ca-pump (SERCA2) structure & steps of Ca transport

Structure:

10 transmembrane spans. 70% of

the protein is on the cytoplasmic

side of the SR membrane (β-strand,

phosphorilation & nucleotid

binding sites, stalk domains and a

hinge)..

A: Ca2+ uptake from the cytosol

B: Ca2+ release to SR lumen

Steps of Ca transport:

E1: 2 high affinity Ca2+ binding sites,

Ca & ATP binding, phosphorilation,

transition to E2 state

E2: lower affinity state, Ca2+ release

to SR, 2 H+ transported to cytosol,

transition to E1 state

A B

41

Phospholamban structure & its effect on SR Ca transport

PLB-SERCA2

interaction:

heterodimer PLB-

SERCA inhibits Ca

transport –

phosphorilation or

Ca binding reduce

inhibition

Ratio: 2(-3) PLB monomer/SERCA2 (non-saturated)

PLB

42

Pharmacological inhibitors of the SR Ca-ATPase (SERCA2)

Thapsigargin (TG) (Kd < 2 pM)

Cyclopiazonic acid (CPA)

2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ)

Major (patho)physiological regulatory factors of the SR Ca-ATPase

Ca: Normally [Ca]i is the limiting substrate for SERCA, thus the amount of

available Cai is the main factor which regulate pump activity

pH: Optimal pH for the SERCA is slightly alkaline (∼ 8). Decrease in pH

(especially pH < 7,4, e.g. acidosis associated with ischemia)

depress the rate of SR Ca-pumping and elongates relaxation

ATP: SERCA has a high affinity ATP site (Kd ∼ 1 µM) which is the substrate

site and a second, lower ATP affinity site (Kd ∼ 200 µM) which

serves a regulatory role. ATP normally is not a limiting factor.

Mg: The actual substrate of SERCA is probably Mg-ATP, thus a decrease in

intracellular Mg concentration may depress its activity

Inhibitors & regulators of the SR Ca-pump

43!

Major factors influencing SR Ca content

SOC: store operated channels

triadin, junctin:

SR structure proteins

44!

Properties of the SR Ca release channel (ryanodine receptor)

MW = 2 260 000 Da

According to some

hypotheses the output

of the RyR Ca channel

is located at the side of

the molecule, thus Ca

ions from the SR may

directly enter the

„restricted space”

45

Ca sparks in isolated ventricular myocytes

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

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.

Fusion of a large

number of sparks

leads to Ca-transient

& contraction !!!

46

Factors which alter

SR Ca release

The RyR macromolecular „signaling

komplex” serves as „final

integrator” site for a number of Ca

regulatory mechanisms !!!

47

Kiriazis 2000

Effects of genetical modulation of Ca-transport mechanisms

48

Conclusion

The SR can accumulate sufficient Ca and release it fast

enough to activate cardiac muscle contraction

Some typical values

In a typical ventricular myocyte there are ~ 2.5*105 DHPR, ~ 1.5-2.5*106

RyR & ~ 0.75-1.25*109 SR Ca-ATPase molecules

Typical Ca spark activity in rest is ~ 50/s for this level of spark activity the

activation of ~ 1000/s RyR is needed (only ~ 0.02% of RyRs)

For peak SR Ca release (~ 3 mM/s) ~ 40 000/s RyR is needed

(only ~ 4% of RyRs)

For total SR Ca release (~ 50 µmol/L citosol) ~ 7500 spark is needed

(only ~ 5% of RyRs)

For the measured Ca influx current via L-type Ca channels (max.1 nA)

~ 2-3% of the channels (DHPRs) are needed (only ~ 5000)

!

Excitation-contraction

coupling (ECc)

In contrast to skeletal muscle in cardiac myocytes external Ca influx is essential for activation of contraction

A) Isolated rat ventricular myocyte B) Frog skeletal muscle fiber

49!

Possible

activators of

cardiac SR

Ca release

50

Ca-induced Ca-release (CICR) mechanism in SR & principles of

local control of Ca release in skinned Purkinje fibers

A. Tension recorded in response

to rapid application & removal

of solutions of indicated [Ca]

B. Relationship between trigger

[Ca] for SR Ca release & the

contraction amplitude resulting

from CICR (pCa = - log[Ca])

Below a given Ca level positive,

above it negative feed-back can be

observed (low [Ca]sm↑↑↑↑, high

[Ca]sm↓↓↓↓ the CICR mechanism) !!!

This is also evident from the fact that

with increasing delay duration tension

is reduced.

Aktivation Inhibition

Delay

duration↑↑↑↑

Ca release depends on both trigger

[Ca] and the rate of Ca change

51!

Diagram of Ca-induced Ca-release (CICR) in cardiac muscle

The two Ca-binding sites of the RyR bind Ca with different kinetics (1: fast, low

affinity binding site, 2: slow, high affinity binding site). Thus, following fast activation

of the RyR receptor its slow inactivation may also be induced by the Ca influx

52!

SR

+

TT

Ca2+

SRTT

Ca2+ Channel Release Channel

+ +Vm

Excitation-contraction coupling in cardiac muscle(Ca2+-Induced-Ca2+-Release)

53

„Local control” theory of EC-coupling in cardiac muscle

Observations: The rate of [Ca] change in the RyR environment either activate

or inactivate SR Ca release (i.e. the RyR).

Apparent junctional colocalization of DHPR & RyR

INa → ([Na]sm↑ → [Ca]sm↑) → SR Ca/release

Observation of localized SR Ca-release events (Ca sparks)

”Common Ca-pool” models could not explain the graded CICR

Hypothesis: RyR activity is modulated by the „fuzzy space” (ie. [Ca]sm)

„Ca-synapse” theory →→→→ 1 DHPR triggers only 1 RyR

„Cluster bomb” theory →→→→ 1 DHPR triggers a cluster of RyRs

Features: Either model could explain graded Ca release and high gain, but the

cluster bomb model does not require an extra large „single RyR” Ca-flux.

Within a cluster of RyRs (couplon) Ca release can be effectively all or

none, the release can be regenerative.

CICR gradation comes largely from recruitment of RyR clusters rather

than varying their Ca flux.

Validity: The local control theory was developed for Ica-induced SR Ca-

release and its validity is unproven for SR Ca-release induced

by different Ca triggers less localized to the junctional region

(NCX, „caged” Ca).

54!!

!

Comparison of EC-coupling in skeletal & cardiac muscle

In skeletal muscle

The physical link between DHPR & RyR is critical for VDCR

Influx of external Ca (ICa) is not required

In cardiac muscle

The physical link between DHPR & RyR is not critical for CICR.

Influx of external Ca (ICa) is crucial

55

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 oversimp-

liplification 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 modulate cardiac Ca release. There is also 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 compelling 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.

!

Control of cardiac contraction

by SR & SL Ca fluxes

56

Species differences in steady state contraction & in post rest recovery of the contractile force

The effect of caffein, &

ryanodin pretreatment

on steady state twitch

contractions in various

cardiac muscle prepa-

rations.

Recovery of twitch force

after 30 s rest in cardiac

muscle preparations in

the absence (top) and

presence (bottom) of

ryanodin

A) [Ca]i-dependence of Ca transport in myocytes

B) Relative Ca-fluxes in ventricular preparations

C) Integrated Ca-fluxes during twitch relaxation

D) Fraction of activator Ca from ICa & SR Ca release

Analysis of cell Ca-fluxes in different species

57

Force-frequency relationship in cardiac muscle

Frequency-dependent changes in contractile force in cardiac myocyte

Force-frequency relationship in rat, rabbit, guinea-pig and human venricular myocyte

58

Conclusion

A) There is a great variation in details of [Ca]i regulation in different

cardiac muscle preparations and conditions. This apparent complexity

can be better understood by considering a small number of common systems

which interact and a few key functional properties that differ among cardiac

preparations.

B) Ca-influx can activate contractions in some hearts, but under normal

conditions in adult mammalian cardiac muscle the SR is the major

source of activator Ca. Ca influx can serve to trigger SR Ca release

and contribute to SR loading for the next contraction.

C) Ca released from the SR can be reaccumulated in the SR, or extruded

by the NCX. In steady state Ca-influx should always be balanced with

Ca-efflux during the carciac cycle.

D) During rest the Ca content of the SR can be gradually depleted by the

NCX & can also be quickly refilled during post rest activity (ICa) in

5-10 contraction). Depending on trans-sarcolemmal [Na]-gradient, rest

can either deplete or fill the SR Ca pool.

E) A dynamic yet delicate balance exists in the control of cardiac [Ca]i

and change in this system can lead to inotrópic & lusitropic effects.

!

Cardiac inotropy

Physiologic regulation of the inotropic state

Major regulatory mechanisms of cardiac muscle inotropy:

1. Sympathetic nerve system 2. Frank-Starling mechanism

3. Force-frequency relationship 4. Adrenergic regulation

5. Vascular function

59!

Hormone receptors and ion transporters in cardiac muscle 1.

Hormone receptors and ion transporters in cardiac muscle 2.

ββββ-adrenergic receptor signaling in ventricular myocytes

Top: activation, desensitization & down-regulation of the β1-receptor

Bottom: differences in G-protein coupling of the three β-receptor types

β-adrenergic mechanisms (through β2,3-receptors) may also mediate inhibitory

(cardioprotective) effects (decreasing contractility by the NO pathway) !!!

60

αααα-adrenergic transduction pathway in ventricular myocytes

αααα-adrenergic regulatory pathways: → G-protein → PLC (& PLD) → IP3+DAG →These products have divergent effects leading to positive inotropy & hypertrophy

61!

Comparison of αααα- and ββββ-adrenergic inotropy

α-adrenergic stimulation

increases Ca-transients to much

less extent than β-adrenergic

stimulation

β-adrenergic stimulation

decreases, α-adrenergic

stimulation increases

myofilament Ca-sensitivity

62!

Diastolic [Ca]i rise is intrinsically inotropic

63!

Effects of acetylstrophanthidin (Na-pump inhibition) on Ca-homeostasis

Acetylstrophanthidin

increases resting Nai

level (A), and

consequently both

[Ca]i & SR Ca

content (B).

Acetylstrophanthidin

increases twitch

force, even with

caffein or ryanodin

64

Consequences of Ca channel inhibition

The Ca channel blocker (nifedipine) nearly abolishes control twitch tension,

even in the presence of caffein, when APD is also significantly reduced (top).

After Na-pump inhibition (by ACS) nifedipine does not abolish tension, despite

even larger reduction in APD (bottom).

65

Conclusion

A) A number of mechanisms are available to increase cardiac inotropy

- hypotermia (experimental)

- β-adrenergic activation (physiological)

- α-adrenergic activation (physiological)

- CaMKII (Ca-CaM-dependent protein kinase) (physiological)

- cardioactive steroids (cardiac glycosids) (therapeutical)

B) From physiological viewpoint the role of β1-adrenergic activation (ANS) is

particularly important (inotropic, chronotropic, lusitropic, etc. effects), but

β2,3-receptors often mediate cardioprotective, inhibitory mechanisms (NO).

C) The significance of α1-adrenergic activation in increasing inotropy in the

human heart is moderate. However, it has an important role in induction of

cardiac hypertrophy (PKC). α1-AR activation enhances Ca sensitivity of the

myofilaments, but does not accelerate relaxation & typically prolong APD.

D) The role of CaMKII is less understood. As activated CaMKII also becomes

autophosphorylated, it may have the ability to integrate [Ca]i signals.

E) Digitalis is the oldest (1785) cardiac inotropic agent and the related cardio-

protective steroids are still among the most efficacious inotropic agents. By

inhibiting Na/K-ATPase, it shifts Na/Ca exchange, increases Ca influx &

decreases Ca efflux. Elevated [Ca]i intrinsically increases inotropy. Over-

dose, however, may cause negative inotropic & arrhythmogenic effects.

!

Ca “mismanagement” &

negative inotropy

Spontaneous Ca release & afterpotentials in cardiac myocytes

EADs may develop as a consequence of ICa reactivation, especially in cases when

APD is significantly elongated (e.g. LQT syndrome)

The main reason for DADs is the activation of Ca-dependent ion channels (e.g.

INA/Ca, ICl(Ca), INS(Ca)) by spontaneous spark activity generated by SR Ca overload

66!

The effect of acidosis on cardiac EC- coupling

C

D

In papillary muscle prepar-

ation pHo was shifted from

7.4 → 6.2. Contractile force

decreased, but Ca-transient

increased (A+B).

Decreased pH substantially

depresses maximal contrac-

tile force (C).

Acid transporters involved in

pHi regulation (D).

67

Some changes which occur during ischemia & reperfusion

68

Hyperthrophic signaling cascades

69

Major changes in EC-coupling during heart failure

Structures with altered

function in heart failure:

NCX (reverse & forward)

Voltage sensor

Phospholamban

SR-Ca-ATPase (SERCA2a)

Relative importance of NCX

increases significantly

Relative importance of

SERCA decreases

significantly.

70!

Positive feedback of maladaptive gene expression

Decreased pump function

is compensated by

(physiological) negative

feedback mechanisms.

Major consequences of

these compensatory

mechanisms are increased

[Ca]i & Ca transient.

[Ca]i increase induces

maladaptive gene-

expression, leading to

further depressed cardiac

pump function (circulus

viciosus).

!!!

!!!

- feedback

+ feedback

71!

Alteration of expression and function in human heart failure

72

Contractile dysfunction and arrhythmogenesis in heart failure

73

Altered calcium handling gene expression in end-stage human HF

74

Conclusion

A) The heart (ventricular myocyte) is a remarkably well tuned system, which can

rapidly vary its contractile output in response to a wide variety of physiologi-

cal stimuli by changing ion currents, Ca handling & myofilament properties.

There are some redundancies in this system, but any major perturbation in

normal [Ca] handling mechanisms still may lead to severe negative inotropy.

B) Ca overload often leads to spontaneous SR Ca-release & Ca-waves, which - if

randomly generated in a large number of cardiac cells - may also substantially

decrease contractile force.

C) Iti (transient inward current: INa/Ca+ ICl(Ca) + INS(Ca)) activated by SR Ca-release

is a major factor in eliciting delayed afterpotentials & aftercontractions.

D) Acidosis is a major consequence of myocardial ischemia & depression at low

pHi of several Ca-transport systems (NCX, SERCA) substantially contribute

to ischemic cardiodepression (decreased contractile force & Ca sensitivity).

E) In progression of the extremely komplex, multifactorial pathomechanisms of

hypoxia, ischemia, reperfusion & heart failure cellular Ca mismanagement

appears to be a common endpoint. Permanently increasing disturbances in Ca-

homeostasis may drastically depress cardiac contractile force & gradually

disables the heart in providing sufficient cardiac output to supply the metabolic

demands of the organism.

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