A Dominant Role of Cardiac Molecular

8/4/2019 A Dominant Role of Cardiac Molecular

1/9

doi:10.1152/physiol.00043.200622:73-80, 2007.PhysiologyAaron C. Hinken and R. John SolaroRelaxationIntrinsic Regulation of Ventricular Ejection andA Dominant Role of Cardiac Molecular Motors in the

You might find this additional info useful...

68 articles, 38 of which can be accessed free at:This article cites

http://physiologyonline.physiology.org/content/22/2/73.full.html#ref-list-1

11 other HighWire hosted articles, the first 5 are:This article has been cited by

[PDF][Full Text]

, October 3, 2008; 283 (40): 26829-26833.J. Biol. Chem.Multiplex Kinase Signaling Modifies Cardiac Function at the Level of Sarcomeric Proteins

[PDF][Full Text], July , 2010; 136 (1): 13-19.J Gen Physiol

R. John Solaro, Katherine A. Sheehan, Ming Lei and Yunbo KeThe curious role of sarcomeric proteins in control of diverse processes in cardiac myocytes

[PDF][Full Text], August , 2010; 109 (2): 610.J Appl Physiol

Rebuttal from Shmuylovich, Chung, and Kovacs

[PDF][Full Text][Abstract], September , 2010; 9 (9): 1804-1818.Mol Cell Proteomics

John Solaro and Peter M. Buttrick

Sarah B. Scruggs, Rick Reisdorph, Mike L. Armstrong, Chad M. Warren, Nichole Reisdorph, R.Identification of Distinct Charged Variants of Regulatory Light ChainA Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and

[PDF][Full Text][Abstract], December 10, 2010; 285 (50): 39150-39159.J. Biol. Chem.

Christine R. Cremo and Josh E. BakerNicholas M. Sich, Timothy J. O'Donnell, Sarah A. Coulter, Olivia A. John, Michael S. Carter,Effects of Actin-Myosin Kinetics on the Calcium Sensitivity of Regulated Thin Filaments

including high resolution figures, can be found at:Updated information and services

http://physiologyonline.physiology.org/content/22/2/73.full.html

can be found at:PhysiologyaboutAdditional material and information

http://www.the-aps.org/publications/physiol

This infomation is current as of May 25, 2011.

ESSN: 1548-9221. Visit our website at http://www.the-aps.org/.Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2007 by the American Physiological Society. ISSN: 1548-9213,developments. It is published bimonthly in February, April, June, August, October, and December by the American Physiological

(formerly published as News in Physiological Science) publishes brief review articles on major physiologicalPhysiology
http://physiologyonline.physiology.org/content/22/2/73.full.html#ref-list-1http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://physiologyonline.physiology.org/content/22/2/73.full.htmlhttp://physiologyonline.physiology.org/content/22/2/73.full.htmlhttp://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://physiologyonline.physiology.org/content/22/2/73.full.html#ref-list-1


2/9

In this review, we consider novel and, in our judg-

ment, under-appreciated aspects of the role of sar-

comeric myosin motors (cross bridges) in regulationof the heart beat. Relatively recent and compelling evi-

dence has substantially expanded the limited textbook

view of the role of cross bridges in the heart beat and

in regulation of contractility. One aspect of this text-

book view is that cross bridges react with actin and

undergo state transitions powered by the free energy

of MgATP hydrolysis that lead to a mechanical transi-

tion impelling thin filaments toward the center of the

sarcomere. A second textbook view is that the actin

cross-bridge reaction is inhibited at diastolic levels of

sarcoplasmic Ca2+ and released from this inhibition

when Ca2+ concentration increases in the sarcomeric

space and Ca2+ binds to troponin C (cTnC). In basal

metabolic states, not all available cross bridges are

engaged in the actin-myosin interaction, and thus a

third textbook view is that major control of contractili-

ty occurs with variations in the number of cross

bridges engaged in the force-generating reaction with

thin filaments. In this perspective, the level of contrac-

tility is governed largely by the amounts and rates of

movements of Ca2+ to and from the sarcomeres.

Our expanded view of the role of the molecular

motors in the heart beat includes processes intrinsic to

the sarcomeres that are significant in sustaining

myocardial stiffness during ejection and determiningthe rate of isovolumic relaxation. We discuss evidence

indicating that these cooperative and mechanical

feedback mechanisms are intrinsic to the sarcomeres

and do not directly depend on membrane-controlled

fluxes of Ca2+. We think this perspective of regulation

of systolic mechanics and relaxation reorients think-

ing with regard to regulation of cardiac function from

a focus on cellular Ca2+ fluxes to a focus on regulation

of processes at the level of the sarcomeres. Central to

these regulatory processes is a role of cardiac molecu-

lar motors beyond simple promotion of force genera-

tion and shortening. It is now evident that, following

the triggering of activation by Ca2+ binding to cTnC,molecular motors are actively engaged in inducing

and sustaining activation of the thin filaments.

Moreover, the strain on active cross bridges, which

occurs during ejection, and the rates of detachment of

cross bridges are also important elements in deter-

mining the duration of systole and the rate of isovolu-

mic relaxation. We discuss evidence that the kinetics

of these sarcomeric processes are rate limiting during

the cardiac cycle and modified by phosphorylation of

regulatory proteins, especially cardiac myosin binding

protein C (MyBP-C) and the thin-filament regulatory

protein cardiac troponin I (cTnI). Finally, discussion is

provided regarding evidence that the kinetics of

molecular motors by drugs targeted to myosin repre-

sents a novel therapeutic approach in treatment of

heart failure.

Structure and Regulation of CardiacMolecular Motor Activity

The molecular motor of heart muscle is a highly asym-

metrical protein consisting of two myosin heavy

chains (MHCs) (FIGURE 1) and two pairs of myosin

light chains (FIGURE 1 illustrates one MHC). The

MHC has a globular head, containing both the actinbinding site and the ATP binding and catalyzing site at

the NH2

terminus of the protein. An -helical COOH-

terminal tail interacts with another MHC in a coiled-

coil motif that combines with rod portions of neigh-

boring myosins to form the thick-filament backbone.

There are two types of MHC expressed in mammalian

myocardium, -MHC and -MHC, existing as dimers

first described by Hoh et al. (16) and termed V1, V

2,

and V3. -MHC generates about three times higher

actin-activated ATPase activities, maximal filament

REVIEW

A Dominant Role of Cardiac MolecularMotors in the Intrinsic Regulationof Ventricular Ejection and Relaxation

Aaron C. Hinkeand R. John Sola

Department of Physiology and Biophyand Center for Cardiovascular Resea

College of MedicUniversity of Illinois at Chicago, Illi

solarorj@uic.

Molecular motors housed in myosins of the thick filament react with thin-filament

actins and promote force and shortening in the sarcomeres. However, other

actions of these motors sustain sarcomeric activation by cooperative feedback

mechanisms in which the actin-myosin interaction promotes thin-filament activa-

tion. Mechanical feedback also affects the actin-myosin interaction. We discuss

current concepts of how these relatively under-appreciated actions of molecular

motors are responsible for modulation of the ejection time and isovolumic relax-

ation in the beating heart.

1548-9213/05 8.00 2006 Int. Union Physiol. Sci./Am. Physiol. Soc.

PHYSIOLOGY 22: 7380, 2007; 10.1152/physiol.00043.2006


3/9

proposed to involve the interactions between the lever

arm of MHC and MyBP-C, thereby constraining move-

ments of the myosin head and affecting the probability

of binding to actin (FIGURE 1).

Thick- and Thin-Filament States inthe Cardiac Cycle

As depicted in FIGURE 1, myosin cross bridges cycli-

cally interact with actin in a process coupled to thehydrolysis of ATP, which drives myocardial force gen-

eration and contraction. Cross bridges are initially in a

weakly bound, non-force-generating rest state (state

1, FIGURE 1) during diastole. FIGURE 1 shows a thin-

filament regulatory unit (RU) consisting of actin-tro-

ponin-tropomyosin in a 7:1:1 ratio. Troponin (Tn) is a

hetero-trimer consisting of cTnC, cTnI, and cTnT. In

the rest state, there is no Ca2+ bound to regulatory sites

on cTnC; cTnI forms a complex with both actin and

cTnT and thus reduces the reactivity of actin for

myosin and holds tropomyosin in a position that

blocks the cross-bridge binding sites on actin (30, 68).

The triggering of force generation and contraction isgoverned by Ca2+ fluxes determined by the dynamics

of electrochemical coupling of Ca2+ release and Ca2+

binding to cTnC. Cross bridges and thin filaments now

enter into a transition state (state 2; FIGURE 1)

74 PHYSIOLOGY Volume 22 April 2007 www.physiologyonline.org

sliding velocity, and greater peak power output than

those preparations containing-MHC (4, 5, 13, 35, 37,

41, 47, 66, 67). -MHC, however, has been reported to

produce twice the cross-bridge force per ATPase cycle

as -MHC, meaning it consumes less energy to main-

tain a given force or a lower tension cost (12, 17, 31, 50,

58). Expression of these isoforms is species dependent

and both developmentally and hormonally regulated

(33) with V3 myosin, a -MHC homodimer predomi-

nately expressed during early development of allmammals and remaining high throughout life in

humans and other large mammals. The V1

cardiac

myosin, a -MHC homodimer, predominates in

mammalian hearts soon after birth and remains high

throughout life in rats and mice (11).

The-helical tail region MHC interacts noncovalent-

ly with the light chains, consisting of one regulatory

light chain (RLC) and one essential light chain (ELC).

The light chains are located toward the neck region of

S1 and help to stabilize the neck and may confer some

regulation of functional activity (65), such as increasing

Ca2+ sensitivity of force (56) and the rate of force devel-

opment (59) (see Ref. 9 for a review). The thick filamentalso contains MyBP-C (FIGURE 1). It has been postulat-

ed that the presence of MyBP-C can alter Ca2+-sensitivi-

ty of force (28), rates of force development, and power

output (27). These functional effects have been

REVIEWS

FIGURE 1. Schematic illustrating the state of cross bridges in the cycle from rest to active statesThin-filament actins are shown with regulatory proteins tropomyosin (Tm) and troponin (Tn) with the Ca2+-binding unit(cTnC) shown in red, the Tm-binding unit (cTnT) in blue, and the inhibitory unit (cTnI) in green. Thick-filament crossbridges (XB) are shown with myosin heavy chain (MHC) and light chains (LC) together with myosin binding protein C(MyBP-C) and titin. See text for further description of the cycle and interpretation of the rate constants.

onMay25,2011

physiologyonline.physiology.o

rg

Download

edfrom
http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/


4/9

determined by the on (kCa

) and off rates (kCa1

) for Ca2+

exchange with cTnC. This transition state involves

altered interactions between TnI and actin, increased

binding of cTnI to cTnC, and a cTnT-dependent shift in

Tm from its blocking position on the thin filament.

These changes in the thin filament permit a shift in

cross bridges toward strongly bound, force-generating

states (state 3; FIGURE 1). The transition state is

homologous to other cross-bridge models whereby a

myosin head has a full compliment of ATP hydrolysisproducts (ADP and Pi) bound and Ca

2+ bound at the

thin filament. The state 2-to-state 3 transition (FIGURE

1; kXB

and kXB-1

) from weak-binding, non-force-gener-

ating cross bridges to strong-binding, force-generating

states is regulated by the kinetics inherent to cross

bridges. The rate-limiting kinetic transition determin-

ing the shift toward the active state appears to be

dependent on the load on the muscle (18) with loads

near peak power output (similar to those encountered

during ejection) determined by the release of Pi

(15)

and loads more near isometric (such as occur during

isovolumic contraction and relaxation) determined by

ADP release (69). An important aspect ofstate 3 is thatstrongly bound cross bridges also induce cooperative

activation of the thin filament by an increase in the

affinity of cTnC for Ca2+ (10, 29, 43). In addition to this

cooperative process within an RU, there is also a coop-

erative activation of near neighbor RU transmitted

through end-to-end interactions between contiguous

Tm strands and possibly through actin-actin interac-

tions (as reviewed in Refs. 9, 40, 63). This mechanism

of propagated activation appears to be particularly

important in cardiac compared with skeletal muscle,

where relatively small changes in strongly bound cross

bridges have significant effects on Ca2+ sensitivity of

force and cross-bridge kinetics (6, 7).

A consequence of these cooperative mechanisms is

illustrated by state 4 in FIGURE 1, which indicates a

population of cross bridges that remain in the active,

force-generating state despite the loss of bound Ca2+.

As discussed below, the lifetime ofstate 4 is an impor-

tant determinant of the duration of ejection. Inasmuch

as the kinetic transition between states 3 and 4 (and

state 2 to 3 to some extent) is dependent on cross-

bridge feedback, mechanisms that alter the number of

active cross bridges will alter this transition. Processes

that may potentially modify the number of active cross

bridges include the sarcomere length, as determinedby the end-diastolic volume, the strength of the inter-

action energies determining cooperativity within and

between RU, and stretch-induced activation (54).

Mechanical feedback associated with sarcomere

shortening also controls the transition from the strong

binding state 4 to the weak binding state 1, thereby

determining the duration ofstate 4. The mechanical

feedback, which is indicated as a unidirectional rate

constant (kvel

) in FIGURE 1, occurs as a result of strain

imposed on active cross bridges with shortening, a

process known as shortening-induced deactivation

(20, 36, 39). Thus the balance of the intensity of the

cooperative mechanisms, which promote the transi-

tion to state 4, and the mechanical feedback, which

promotes dissipation ofstate 4, determine the popula-

tion of cross bridges in state 4. Therefore, these mech-

anisms are likely to be important determinants of the

time to the end of systole.

Another more controversial possibility to increase

cross-bridge availability permitting feedback would beto allow individual cross bridges to interact with the

thin filament multiple times during activation. The

cross-bridge cycle described by FIGURE 1 assumes

cross bridges driving cardiac pump function are tight-

ly coupled to ATP hydrolysis (i.e., one ATP per cross-

bridge cycle). However, questions remain as to the

validity of this assertion. Evidence for a more loosely

coupled system comes from observations by the

Yanagida laboratory (23) and Lombardi et al. (32) that

a single cross bridge can move actin many times the

length of a single cross-bridge swing on an individual

ATP and that cross bridges can reprime following a

power stroke without binding additional ATP, respec-tively. In agreement, based on experimental data con-

cerning length-tension area (analogous to pressure-

volume area) vs. oxygen consumption, Taylor et al.

(62) concluded the cross bridge-to-ATP ratio may be

variable with tightly coupled usage at high loads

becoming more loosely coupled at low loads where

muscle may shorten (reviewed in Ref. 57).

A Dominant Role of ProcessesInvolving Cardiac Molecular Motorsin Left Ventricular Ejection andIsovolumic Relaxation

Ultimately, the reactions of molecular motors in the left

ventricle (FIGURE 1) are responsible for the ejection of

blood into the peripheral circulation. During a beat of

the left ventricle, pressure rises for a time with no

change in ventricular volume and limited internal

shortening of the sarcomeres. During this phase, cross

bridges are recruited into action as Ca2+ rises in the sar-

comeric space. When the aortic valve opens, ejection

begins and the sarcomeres shorten. In this phase, cross

bridges impel the thin filaments toward the center of

the sarcomere. Shortening imposes a strain on reacting

cross bridges and alters the kinetics of the actin-myosin reaction. As systole progresses and blood flows

to the periphery, left ventricle pressure falls, the valve

closes, pressure falls with no change in ventricular vol-

ume, and the strain dependence of actin cross-bridge

kinetics is greatly relieved. Unfortunately, much of our

understanding of the cellular mechanisms governing

these processes (such as the dynamics of excitation

contraction coupling and relaxation) is based on deter-

minations of intracellular Ca2+ and mechanics with

myocytes shortening against zero loads or not

PHYSIOLOGY Volume 22 April 2007 www.physiologyonline.org

REVIEW


5/9

ejection of the blood added to the ventricle during

diastole. FIGURE 2 depicts the time course of Ca2+

bound to cTnC in a region of the A band of sarcomeres

at various time points during pressure changes during

a beat of the left ventricle. The figure illustrates our

perception of the state of the A-band region at various

time points during the beat. Estimation of Ca2+ bind-

ing to the thin filaments is based on a computational

model of Burkoff (2), which is in general agreement

with experimental findings (60) and other models oftransitions in thin-filament RUs during left ventricular

pressure development in a beat of the heart (29).

These models are driven by experimental determina-

tions of the transient change in Ca2+ (29, 44) and by

determinations of kinetics and steady-state binding of

Ca2+ to Tn in myofilaments (43, 49). The estimate is


shortening at all. In the following, we expand gover-

nance of these processes to include prominent, intrin-

sic sarcomeric regulation of these processes and alter-

ations occurring with changes in load.

Differences in kinetics of sarcomeric-bound

Ca2+ and pressure during a heart beat

A major challenge in the investigation of molecular

control mechanisms in regulation of the heart beat is

how to connect the action of the cardiac molecularmotors to global ventricular function. These actions

include state transitions leading to development of

force and shortening, promotion of the activated state

of thin filaments, response to the prevailing pre-load

and afterload, and the relative amounts of Ca2+-bound

to cTnC (related to contractility) that ensures efficient

REVIEWS

FIGURE 2. Relation between Ca2+ bound to cardiac thin filaments (Ca2+-Tn-Tm-Actin) and left ventricular pressure (LVP) in abeat of the heartNote that Ca2+ bound to the thin filament wanes at a time when ejection is still underway. See text for further description of intrinsic sarcomericmechanism sustaining ejection independent of bound Ca2+ and determining rate of isovolumic relaxation. Computation of bound Ca2+ based onBurkhoff (2).

onMay25,2011


rg

Download

edfrom
http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/


6/9

also based on studies by Peterson et al. (44), who

applied a mechanical assay to determine the time

course of the amounts of Ca2+ bound to cTnC, and by

Ishikawa et al. (19), who used a quick-release protocol

and intracellular aequorin to determine cTnC-bound

Ca2+. These studies both demonstrated that the fall in

thin-filament-bound Ca2+ precedes the fall in force in

an isometric twitch of heart muscle.

A significant outcome of these measurements and

models is that they indicate that, during a heart-beat, the time course of Ca2+ bound to the thin-fila-

ment cTnC is nearly over at a time when ejection is

ongoing and isovolumic relaxation has not yet begun.

We interpret this difference in time course of thin-fil-

ament Ca2+ binding and pressure to mean that much

of ejection and relaxation of the ventricle depends on

molecular processes intrinsic to the sarcomere.

These intrinsic molecular processes involve the fol-

lowing significant actions of the cardiac molecular

motors: 1) activation of the thin filaments by cooper-

ative mechanisms sustaining thin-filament activa-

tion despite a fall in thin-filament-bound Ca2+, 2)

sensing the sarcomere length (preload) in a mecha-nism explaining the Frank-Starling law, 3) sensing

the load in a mechanism related to the inverse rela-

tionship between force and velocity, and 4) relax-

ation kinetics related to rates of cross-bridge detach-

ment and intersarcomere dynamics [as described by

Stehle et al. (46)].

Processes intrinsic to the sarcomere control

systolic mechanics and relaxation

Consideration of the change in the state of the A-band

sarcomeric proteins during the transition from diastole

to systole and the return to diastole during a heart beat

serves to describe the interplay between control mech-

anisms extrinsic (such as membrane-dependent regu-

lation of Ca2+ fluxes) and mechanisms intrinsic to the

sarcomere. In the diastolic state, cross bridges are

either blocked from reacting with actin or in a weak,

non-force-generating state (FIGURE 1). The molecular

motors of the heart are not completely passive in dias-

tole in that they are likely to sense the preload. There is

evidence that the state of stretch of the myocardium at

the end of diastole is transmitted to the cross bridges by

titin, which is responsible for the passive force of the

myocyte (8). Pre-load determines the strain on titin,

and it is plausible (although not fully documented) thatthe net effect of the longitudinal and radial strain on

titin alters interfilament spacing or provides a signal

acting through a titin-MyBP-C-cross-bridge interaction

by which the molecular motors sense sarcomere length

(8). In general, it has been thought that the motors

sense sarcomere length via a length-dependent change

in interfilament spacing, which in effect alters the local

concentration of cross bridges at the actin binding sites

and thereby alters the myofilament response to Ca2+

(25, 26, 38). This length-dependent activation of the

sarcomeres provides the cellular mechanism for the

Frank-Starling law. Yet the theory that length depend-

ence of activation is well correlated with changes in

interfilament spacing has not stood the test of direct

determination of the interfilament spacing by X-ray dif-

fraction (25, 26).

With release of Ca2+ into the sarcomeric space, Ca2+

binds to cTnC, and systole begins. The transient

change of intracellular Ca2+ in the sarcomeric space, as

detected by intracellular fluorescent probes, demon-

strates only ~2% of the total cellular Ca2+ that moves

from the extracellular space and sarcoplasmic reticu-

lum (SR) and back during a heart beat (52). Thus thebulk of these Ca2+ ions is bound to buffers including

TnC and other regulatory Ca2+-binding proteins such

as calmodulin (64). With the rise in cTnC-bound Ca2+,

which is shown in FIGURE 2, cross bridges make the

transition from the blocked or weak binding state to a

strong force-generating state. A reasonable estimate is

that ~2025% of available cross bridges are recruited

into the systolic state during resting levels of cardiac

output (24). The reactions of these cross bridges gener-

ate the wall tension responsible for the rising phase of

left ventricular pressure; shortening is limited until suf-

ficient pressure is generated to open the aortic valve.

Removal of Ca2+ from the sarcoplasm by the SR

pump and the Na+/Ca2+ exchanger sets into motion a

process whereby Ca2+ is released from cTnC, and this

is in part responsible for the decay in Ca2+ bound to the

thin filaments. However, as illustrated in FIGURE 2,

this fall in thin-filament Ca2+ binding precedes the fall

in left ventricular pressure, thus indicating that the

actin cross-bridge reactions are sustained even in the

face of a decline in Ca2+-dependent activation.

Moreover, at the time the aortic valve opens, coopera-

tive mechanisms in which thin filament RUs remain

active without Ca2+ bound sustain ejection

(FIGURE 2). Computational models indicate that thisstate occurs by cooperative mechanisms within an RU

in which cross-bridge binding to the thin filament

increases the affinity of cTnC for Ca2+ (step 4 in

FIGURE 1) and by near-neighbor cooperative mecha-

nisms acting longitudinally along the thin fi lament (9,

48). As ejection proceeds against the afterload, cross

bridges enter into a new state in which the strain

imposed by shortening promotes release of ADP from

the cross bridges, an increase in ATP turnover, and an

increase in cross-bridge detachment rate. The effect of


REVIEW

...the time course of Ca2+ bound to the thin-filame

cTnC is nearly over at a time when ejection is ongoin

and isovolumic relaxation has not yet begun.


7/9

a different experimental approach. On the other hand,

when Peterson et al.(44) used ryanodine to slow Ca2+

uptake by SR, the fall in force and cTnC-bound Ca2+

occurred in parallel. Thus, when SR Ca2+ uptake is

slowed down as in heart failure (45), the role of

processes intrinsic to the sarcomeres becomes rela-

tively blunted.

Control of Cardiac Molecular

Motors by Modulation of Thick- andThin-Filament Regulatory Proteins

Phosphorylation of thin- and thick-filament regulatory

proteins that modulate the actin-cross-bridge reaction

also affect duration of ejection and relaxation. Although

multiple sarcomeric proteins are modified by phospho-

rylation (reviewed in Refs. 24, 53), we focus here on

MyBP-C and cTnI inasmuch as the state of these pro-

teins has been reported to affect systolic mechanics.

Both proteins are phosphorylated by protein kinase A at

sites unique to the cardiac variants. In the case of MyBP-

C, compared with controls, mouse hearts lacking

cMyBP-C demonstrate a significantly abbreviated timecourse of systolic elastance (42). There is little change in

the early phase of systole, but hearts lacking MyBP-C

relax prematurely. Sarcomeres lacking MyBP-C also

have faster unloaded shortening velocity than controls

(42). Importantly, PKA-dependent phosphorylation of

MyBP-C, which occurs at a site that interacts with

myosin S2, mimics the effects of its ablation (55). Thus

it is apparent and likely that the state of MyBP-C modi-

fies the transmission of force across the sarcomere as

well as shortening-induced deactivation. This finding

may be of significance regarding the pathology of muta-

tions in MyBP-C, which are a common cause of familial

cardiomyopathy (1).

Among TnI variants, cTnI is unique in having an

NH2-terminal extension of 30 amino acids that contain

PKA-dependent phosphorylation sites.

Phosphorylation of these sites appears pivotal in the

positive inotropic response to -adrenergic stimula-

tion (22) and to changes in frequency (61). However,

the demonstration of both of these effects is most evi-

dent in auxotonically loaded, ejecting hearts and

much less evident in isovolumic hearts, isometric

muscle preparations, or unloaded isolated myocytes.

The increases in ejection indexes, such as stroke work

and the end-systolic pressure-volume relation, are sig-nificantly blunted in hearts expressing slow skeletal

TnI (which lacks the PKA sites) in place of cTnI (22).

However, myocytes from these hearts or isovolumic

hearts demonstrated nearly the same inotropic

responses to -adrenergic stimulation as the controls.

Phosphorylation of cTnI has also been reported to

increase unloaded shortening velocity and power gen-

eration by isolated, skinned cardiac myocytes (14) and

to enhance the effect of sarcomere length on force-

Ca2+ relation (26). Taken together, these results indi-


strain on cross bridges is well accepted to affect

nucleotide binding. For example, in slow skeletal mus-

cle, ADP release is sensitive to the load; the lighter the

load, the greater the shortening velocity, the faster the

release of ADP, and the faster the ATP turnover (51).

This strain dependence of cross-bridge kinetics is the

basis for the Fenn effect. This mechanical feedback

thus promotes the transition of cross bridges from

strong to weak binding states (step 4 in FIGURE 1)

and lowers the affinity of cTnC for Ca2+

. As a result,rate of loss of bound Ca2+ is enhanced. Thus systolic

mechanics reflect a balance between cooperative

activation of thin filaments and shortening-induced

deactivation (29). It is evident that the dissipation of

the cooperative activation is relatively slow. In short-

ening from a long to a short length, Chandra and

Campbell (3) suggest that the sarcomeres remember

the higher force attained at the longer length and

shortening (ejection) is prolonged in comparison to

the situation with no cooperative mechanisms. The

kinetics of the cross-bridge cycle and the relative time

the cross bridges are strongly bound, especially the

rate of detachment of cross bridges, is also an impor-tant determinant of the time in ejection and the iso-

volumic relaxation rate.

Mechanisms at the level of the sarcomere that affect

relaxation include the dissipation of the cooperative

activation of the thin filament and forward detach-

ment of cross bridges as ATP binding to cross bridges

reestablishes the actin-myosin-ADP-Pi weak binding

state (state 1 in FIGURE 1). A third mechanism is a

strain-dependent reversal of the power stroke transi-

tion from the actin-myosin-ADP state to the actin-

myosin-ADP-Pi state (a transition from state 3 to state

2, as illustrated in FIGURE 1). In the case of myofibril

preparations, relaxation is accelerated by small

stretches and by increased Pi

concentration (46).

These common effects have been interpreted by

Poggesi et al.(46) to indicate that, in relaxation, both Pi

binding to cross bridges and altered cross-bridge

strain during relaxation may result in reversal of the

power stroke. If a significant number of the cross

bridges undergo this reversal of the power stroke, then

a significant potential outcome is that weak binding

cross bridges are reestablished without splitting ATP

and ready to engage in the cross-bridge cycle in the

next beat.

Direct tests of the relative significance of processesextrinsic to the sarcomere (Ca2+ uptake by the SR) and

these processes intrinsic to the sarcomere indicate

that the intrinsic processes can be rate limiting. For

example, in the experiments of Peterson et al.(44), the

derivative curve of bound Ca2+ fell to low levels,

whereas force remained high during the onset of relax-

ation in an isometric twitch. This result indicates that

factors other than the velocity of SR Ca2+ uptake are

rate limiting in loaded ejections with shortening. Sys

and Brutsaert (60) reached the same conclusion using

REVIEWS

onMay25,2011


rg

Download

edfrom
http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/


8/9

cate a significant involvement of the cTnI phosphory-

lation in cardiac systolic mechanics and relaxation in

response to -adrenergic stimulation by its effects on

the cooperative activation and mechanical feedback.

Hearts of transgenic mice (TG-cTnIDD) expressing

cTnI, in which the PKA-targeted Ser are mutated to

Asp as a phosphorylation mimetic, also show respons-

es to increases in frequency, indicating an effect of

cTnI phosphorylation on rate-dependent increases in

systolic function (61). Moreover, there was a pro-longed relaxation associated with increased afterload

in nontransgenic control hearts beating in situ when

compared to the TG-cTnI-DD hearts. This difference

was not evident when the hearts were treated with iso-

proterenol. As with the studies on the TG-ssTnI hearts,

frequency-dependent responses in isometric trabecu-

lae from the TG-cTnIDD hearts did not demonstrate

differences from controls (61). These studies reinforce

the role of cTnI phosphorylation on systolic function

and relaxation kinetics and also indicate that the

mechanism is revealed when cross bridges function

against a load as in the ejecting ventricles. It has been

reported that that there is a diminished TnI phospho-rylation in that, which is likely to be an important

mechanism in the blunted force-frequency relation of

failing human hearts.

Novel Pharmaceutical TherapiesTargeting Molecular Motors

Translation of the detailed understanding of sarcom-

eric regulation to therapeutics is an important

research objective, and there is evidence that pharma-

cological modification of molecular motors may be

useful as a therapeutic approach for heart failure. One

aim has been the discovery of small molecules that

directly modify sarcomeric proteins and improve car-

diac function. Early phases of the search for such com-

pounds that enhance myofilament sensitivity to Ca2+

targeted thin-filament proteins. Examples of these

agents are levosimendan and EMD 57033, which bind

to cTnC, and pimobendan, which promotes Ca2+ bind-

ing to cTnC. These agents improves systolic function

in animal models of heart failure (as reviewed in Ref.

21), and levosimendan and pimobendan have pro-

gressed into clinical use. More recently, there has been

a search for agents directly affecting the molecular

motors of the heart. Although further studies arerequired to establish the value of these agents in

human therapeutic application, the initial data are

promising (34). It is apparent that modification of the

myosin by these agents prolongs systolic ejection. This

indicates that these agents alter the balance of cooper-

ative mechanisms and shortening induced deactiva-

tion such that systolic mechanics are enhanced.

Whether modification of troponin and/or MyBP-C by

drugs may also affect this balance appears possible

but has not been investigated explicitly.

References

1. Bonne G, Carrier L, Bercovici J, Cruaud C, Richard P, HainqueB, Gautel M, Labeit S, James M, Beckman J, Weissenbach J, Vosberg HP, Fiszman M, Komajda M, Schwartz K. Cardiacmyosin binding protein-C gene splice acceptor site mutationis associated with familial hypertrophic cardiomyopathy. NatGenet11: 438440, 1995.

2. Burkhoff D. Explaining load dependence of ventricular con-tractile properties with a model of excitation-contraction cou-pling. J Mol Cell Cardiol26: 959978, 1994.

3. Campbell KB, Chandra M. Functions of Stretch Activation inHeart Muscle. J Gen Physiol127: 8994, 2006.

4. De Tombe PP, ter Keurs EDJ. An internal viscous element lim-its unloaded velocity of sarcomere shortening in rat cardiactrabeculae. J Physiol454: 619642, 1992.

5. Fitzsimmons DP, Patel JR, Moss RL. Role of myosin heavychain composition in kinetics of force development and relax-ation in rat myocardium. J Physiol513: 171183, 1998.

6. Fitzsimons DP, Moss RL. Strong binding of myosin modulateslength-dependent Ca2+ activation of rat ventricular myocytes.Circ Res 83: 602607, 1998.

7. Fitzsimons DP, Patel JR, Moss RL. Cross-bridge interactionkinetics in rat myocardium are accelerated by strong bindingof myosin to the thin filament. J Physiol530: 263272, 2001.

8. Fukuda N, Granzier HL. Titin/connectin-based modulation ofthe Frank-Starling mechanism of the heart. J Muscle Res CellMotil26: 319323, 2005.

9. Gordon AM, Homsher E, Regnier M. Regulation of

Contraction in Striated Muscle. Physiol Rev80: 853924, 2000.10. Gordon AM, Ridgway EB. Cross-bridges affect both TnC

structure and calcium affinity in muscle fibers. Adv Exp MedBiol332: 183192; discussion 192184, 1993.

11. Hamilton N, Ianuzzo CD. Contractile and calcium regulatingcapacities of myocardia of different sized mammals scale withresting heart rate. Mol Cell Biochem 106: 133141, 1991.

12. Harris DE, Work SS, Wright RK, Alpert NR, Warshaw DM.Smooth, cardiac, and skeletal muscle myosin force andmotion generation assessed by cross-bridge mechanicalinteractions in vitro. J Muscle Res Cell Motil15: 1119, 1994.

13. Herron TJ, Korte FS, McDonald KS. Loaded shortening andpower output in cardiac myocytes are dependent on myosinheavy chain isoform expression. Am J Physiol Heart CircPhysiol281: H1217H1222, 2001.

14. Herron TJ, Korte FS, McDonald KS. Power output is increasedafter phosphorylation of myofibrillar proteins in rat skinnedcardiac myocytes. Circ Res 89: 11841190, 2001.

15. Hinken AC, McDonald KS. Inorganic phosphate speeds load-ing shortening and increases power output in rat skinned car-diac myocytes. Am J Physiol Cell Physiol287: C500C507,2004.

16. Hoh JFY, McGrath PA, Hale PT. Electrophoretic analysis ofmultiple forms of rat cardiac myosin: effects of hypophysec-tomy and thyroxine replacement. J Mol Cell Cardiol 10:10531076, 1977.

17. Holubarsch C, Goulette RP, Litten RZ, Martin BJ, Mulieri LA,Alpert NR. The economy of isometric force development,myosin isozyme pattern and myofibrillar ATPase activity innormal and hypothyroid rat myocardium. Circ Res 56: 7886,1985.

18. Homsher E, Lacktis J, Regnier M. Strain-dependent modula-tion of phosphate transients in rabbit skeletal muscle fibers.Biophys J 72: 17801791, 1997.

19. Ishikawa T, OUchi J, Mochizuki S, Kurihara S. Evaluation of thecross-bridge-dependent change in the Ca2+ affinity of tro-ponin C in aequorin-injected ferret ventriculr muscles. CellCalcium 37: 153162, 2005.

20. Iwamoto I. Thin filament cooperativity as a major determinantof shortening velocity in skeletal muscle fibers. Biophys J 74:14521464, 1998.

21. Kass DA, Solaro RJ. Mechanisms and use of calcium-sensitiz-ing agents in the failing heart. Circulation 113: 305315,2006.


REVIEW


9/9


REVIEWS

22. Kentish JC, McCloskey DT, Layland J, Palmer S,Leiden JM, Martin AF, Solaro RJ. Phosphorylationof troponin I by protein kinase A accelerates relax-ation and cossbridge cycle kinetics in mouse ven-tricular muscle. Circ Res 88: 10591065, 2001.

23. Kitamura K, Tokunaga M, Iwane AH, Yanagida T. Asingle myosin head moves along an actin filamentwith regular steps of 5.3 nanometres. Nature397:129, 1999.

24. Kobayashi T, Solaro RJ. Calcium, thin filaments,and the intregrative biology of cardiac contractili-ty. Annu Rev Physiol67: 3967, 2005.

25. Konhilas JP, Irving TC, de Tombe PP. Frank-Starling law of the heart and the cellular mecha-nisms of length-dependent activation. PflgersArch 445: 305310, 2002.

26. Konhilas JP, Irving TC, Wolska BM, Jweied EE,Martin AF, Solaro RJ, de Tombe PP. Troponin I inthe murine myocardium: influence on length-dependent activation and interfilament spacing. JPhysiol547: 951961, 2003.

27. Korte FS, McDonald KS, Harris SP, Moss RL.Loaded shortening, power output, and rate offorce redevelopment are increased with knockoutof cardiac myosin binding protein-C. Circ Res 93:752758, 2003.

28. Kunst G, Kress KR, Gruen M, Uttenweiler D,Gautel M, Fink RHA. Myosin binding protein C, aphosphorylation-dependent force regulator inmuscle that controls the attachment of myosin

heads by its interaction with myosin S2. Circ Res86: 5158, 2000.

29. Landesberg A. End-systolic pressure-volume rela-tionship and intracellular control of contraction.Am J Physiol Heart Circ Physiol270: H338H349,1996.

30. Lehman W, Craig R, Vibert P. Ca2+-inducedtropomyosin movement in Limulus thin filamentsrevealed by three-dimensional reconstruction.Nature368: 6567, 1994.

31. Loiselle DS, Wendt IR, Hoh JFY. Energetic conse-quences of thyroid-modulated shifts in ventricularisomyosin distribution in the rat. J Muscle Res CellMotil3: 523, 1982.

32. Lombardi V, Piazzesi G, Linari M. Rapid regenera-tion of the actin-myosin power stroke in contract-ing muscle. Nature355: 638641, 1992.

33. Lompre AM, Nadal-Ginard B, Mahdavi V.Expression of the cardiac ventricular alpha- andbeta-myosin heavy chain genes is developmental-ly and hormonally regulated. J Biol Chem 259:64376446, 1984.

34. Malik F, Teerlink JR, Escandon RD, Clark CP, WolffAA. The selective cardiac myosin activator, CK-1827452, a calcium-independent inotrope,increases left ventricular systolic function byincreasing ejection time rather than the velocity ofcontraction (Abstract). Circulation 114: II441,2006.

35. Martin AF, Pagani ED, Solaro RJ. Thyroxine-induced redistribution of isoenzymes of rabbitventricular myosin. Circ Res 50: 117124, 1982.

36. Martyn DA, Rondinone JF, Huntsman LL.Myocardial segment velocity at a low load: time,

length, and calcium dependence. Am J PhysiolHeart Circ Physiol13: H708H714, 1983.

37. McDonald KS, Herron TJ. It takes heart to win:what makes the heart powerful? News Physiol Sci17: 185190, 2002.

38. McDonald KS, Moss RL. Osmotic compression ofsingle cardiac myocytes reverses the reduction inCa2+ sensitivity of tension observed at short sar-comere length. Circ Res 77: 199205, 1995.

39. McDonald KS, Moss RL. Strongly binding myosincross-bridges regulate loaded shortening andpower output in cardiac myocytes. Circ Res 87:768773, 2000.

40. Moss RL, Razumova M, Fitzsimons DP. Myosin

crossbridge activation of cardiac thin filaments:implications for myocardial function in health anddisease. Circ Res 94: 12901300, 2004.

41. Pagani ED, Julian FJ. Rabbit papillary musclemyosin isozymes and the velocity of muscle short-ening. Circ Res 54: 586594, 1984.

42. Palmer BM, Georgakopoulos D, Janssen PM,Wang Y, Alpert NR, Belardi DF, Harris SP, Moss RL,Burgon PG, Seidman CE, Seidman JG, MaughanDW, Kass DA. Role of cardiac myosin binding pro-tein C in sustaining left ventricular systolic stiffen-ing. Circ Res 94: 12491255, 2004.

43. Pan BS, Solaro RJ. Calcium-binding properties oftroponin C in detergent-skinned heart musclefibers. J Biol Chem 262: 78397849, 1987.

44. Peterson JN, Hunter WC, Berman MR. Estimatedtime course of Ca2+ bound to troponin C during

relaxation in isolated cardiac muscle. Am J PhysiolHeart Circ Physiol260: H1013H1024, 1991.

45. Piacentino V, III, Weber CR, Chen X, Weisser-Thomas J, Margulies KB, Bers DM, Houser SR.Cellular basis of abnormal calcium transients offailing human ventricular myocytes. Circ Res 92:651658, 2003.

46. Poggesi C, Tesi C, Stehle R. Sarcomeric determi-nants of striated muscle relaxation kinetics.Pflgers Arch 449: 505517, 2005.

47. Pope B, Hoh JFY, Weeds A. The ATPase activitiesof rat cardiac myosin isoenzymes. FEBS Lett118:205208, 1980.

48. Rasumova MV, Bukatina AE, Campbell KB.Different myofilament nearest-neighbor interac-tions have distinctive effects on contractile behav-ior. Biophys J 78: 31203137, 2000.

49. Robertson SP, Johnson JD, Potter JD. The time-course of Ca2+exchange with calmodulin, tro-ponin, parvablumin, and myosin in response totransient increases in Ca2+. Biophys J 34: 559569,1981.

50. Rundell VL, Geenen DL, Buttrick PM, de TombePP. Depressed cardiac tension cost in experiment-al diabetes is due to altered myosin heavy chainisoform expression. Am J Physiol Heart CircPhysiol287: H408H413, 2004.

51. Shirakawa I, Chaen S, Bagshaw CR, Sugi H.Measurement of nucleotide exchange rate con-stants in single rabbit soleus myofibrils duringshortening and lengthening using a fluorescentATP analog. Biophys J 78: 918926, 2000.

52. Sipido KR, Wier WG. Flux of Ca2+ across the sar-coplasmic reticulum of guinea-pig cardiac cellsduring excitation-contraction coupling. J Physiol435: 605630, 1991.

53. Solaro RJ. Modulation of Cardiac MyofilamentActivity by Protein Phosphorylation. New York:Oxford Univ. Press, 2001.

54. Stelzer JE, Moss RL. Contributions of stretch acti-vation to length-dependent contraction in murinemyocardium. J Gen Physiol128: 461471, 2006.

55. Stelzer JE, Patel JR, Moss RL. Protein kinase A-mediated acceleration of the stretch activationresponse in murine skinned myocardium is elimi-nated by ablation of cMyBP-C. Circ Res 99:884890, 2006.

56. Stephenson GM, Stephenson DG. Endongenous

MLC2 phosphorylation and Ca2+

-activated forcein mechanically skinned skeletal muscle fibres ofthe rat. Pflgers Arch 424: 3038, 1993.

57. Suga H. Mechanoenergetic estimation of multiplecross-bridge steps per ATP in a beating heart. JpnJ Physiol54: 103108, 2004.

58. Sugiura S, Kobayakawa N, Fujita H, Yamashita H,Momomura S, Chaen S, Omata M, Sugi H.Comparison of unitary displacements and forcesbetween 2 cardiac myosin isoforms by the opticaltrap technique: molecular basis for cardiac adap-tation. Circ Res 82: 10291034, 1998.

59. Sweeney HL, Stull JT. Phosphorylation of myosinin permeabilized mammalian cardiac and skeletalmuscle cells. Am J Physiol Cell Physiol 250:C657C660, 1986.

60. Sys SU, Brutsaert DL. Determinants of force

decline during relaxation in isolated cardiac mus-cle. Am J Physiol Heart Circ Physiol 257:H1490H1497, 1989.

61. Takimoto E, Soergel DG, Janssen PM, Stull LB,Kass DA, Murphy AM. Frequency- and afterload-dependent cardiac modulation in vivo by troponinI with constitutively active protein kinase A phos-phorylation sites. Circ Res 94: 496504, 2004.

62. Taylor TW, Goto Y, Suga H. Variable cross-bridgecycling-ATP coupling accounts for cardiac mecha-noenergetics. Am J Physiol Heart Circ Physiol264:H994H1004, 1993.

63. Tobacman LS. Thin filament-mediated regulationof cardiac contraction. Annu Rev Physiol 58:447481, 1996.

64. Trafford AW, Diaz ME, Eisner DA. A novel, rapidand reversible method to measure Ca buffering

and time-course of total sarcoplasmic reticulumCa content in cardiac ventricular myocytes.Pflgers Arch 437: 501503, 1999.

65. Trybus KM. Role of myosin light chains. J MuscleRes Cell Motil15: 587594, 1994.

66. VanBuren P, Harris DE, Alpert NR, Warshaw DM.Cardiac V1 and V3 myosins differ in their hydrolyt-ic and mechanical activities in vitro. Circ Res 77:439444, 1995.

67. Winegrad S, Weisberg A. Isozyme specific modifi-cation of myosin ATPase by cAMP in rat heart.Circ Res 60: 384392, 1987.

68. Xu C, Craig R, Tobacman LS, Horowitz R, LehmanW. Tropomyosin positions in regulated thin fila-ments revealed by cryoelectron microscophy.Biophys J 77: 985992, 1999.

69. Zhao Y, Kawai M. Kinetic and thermodynamic

studies of the cross-bridge cycle in rabbit psoasmuscle fibers. Biophys J 67: 16551668, 1994.

onMay25,2011


rg

Download

edfrom
http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/http://physiologyonline.physiology.org/

A Dominant Role of Cardiac Molecular

Documents

Transcript of A Dominant Role of Cardiac Molecular