J Appl Physiol

90: 1125–1136, 2001.

highlighted topics
Plasticity in Skeletal, Cardiac, and Smooth Muscle
Invited Review: Pathophysiology of cardiac muscle
contraction and relaxation as a result of alterations
in thin filament regulation
OLGA M. HERNANDEZ,1 PHILIPPE R. HOUSMANS,2 AND JAMES D. POTTER1
1
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine,
Miami, Florida 33136; and 2Department of Anesthesiology, Mayo Foundation, Rochester, Minnesota 55905

Hernandez, Olga M., Philippe R. Housmans, and James D.

Potter. Invited Review: Pathophysiology of cardiac muscle contraction
and relaxation as a result of alterations in thin filament regulation. J
Appl Physiol 90: 1125–1136, 2001.—Cardiac muscle contraction depends
on the tightly regulated interactions of thin and thick filament proteins
of the contractile apparatus. Mutations of thin filament proteins (actin,
tropomyosin, and troponin), causing familial hypertrophic cardiomyopa-
thy (FHC), occur predominantly in evolutionarily conserved regions and
induce various functional defects that impair the normal contractile
mechanism. Dysfunctional properties observed with the FHC mutants
include altered Ca2⫹ sensitivity, changes in ATPase activity, changes in
the force and velocity of contraction, and destabilization of the contractile
complex. One apparent tendency observed in these thin filament muta-
tions is an increase in the Ca2⫹ sensitivity of force development. This
trend in Ca2⫹ sensitivity is probably induced by altering the cross-bridge
kinetics and the Ca2⫹ affinity of troponin C. These in vitro defects lead
to a wide variety of in vivo cardiac abnormalities and phenotypes, some
more severe than others and some resulting in sudden cardiac death.
familial hypertrophic cardiomyopathy; troponin; tropomyosin; actin;
myocardium

THIS BRIEF REVIEW WILL SUMMARIZE some recent insights
into thin filament regulation of cardiac muscle contrac-
tion and relaxation. Several mutations in regulatory
proteins of the thin filament have been described that
change the regulation of contraction and relaxation,
and, recently, their impact on in vivo and in vitro
contractility was studied. Within the framework of
current knowledge, we will also offer some suggestions
for directions for further research.
REGULATION OF CONTRACTION BY THIN
FILAMENT PROTEINS
Contraction of vertebrate striated (skeletal and car-
diac) muscle is activated by the binding of Ca2⫹ to the
Address for reprint requests and other correspondence: J. D. Pot-
ter, Dept. of Molecular and Cellular Pharmacology, Univ. of Miami
School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136 (E-mail:
jdpotter@miami.edu).
http://www.jap.org 8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
Ca2⫹-binding subunit troponin C (TnC) of the troponin
complex, which, together with troponin I (TnI), tropo-
nin T (TnT), and tropomyosin (Tm), forms the regula-
tory system of the contractile apparatus (21, 72, 79,
96). Contraction occurs when the myosin head in the
thick filament interacts with actin in the thin filament
causing the two filaments to slide past each other. The
troponin complex in the thin filament regulates the
actin-myosin interaction. Figure 1 summarizes the
changes in the thin filament during activation of con-
traction. In the absence of Ca2⫹, the troponin complex
exists in a closely held conformation. TnI inhibits ac-
tin-myosin binding by interacting with actin-Tm and
fixing the troponin complex on actin through TnI’s
interaction with TnT (14, 15, 26, 68). This inhibitory
interaction of TnI with actin is completely eliminated
when Ca2⫹ binds to the amino-terminal Ca2⫹-specific
sites (only site II in cardiac muscle) of TnC and the
carboxyl terminus of TnI dissociates from the actin-Tm
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1126
Fig. 1. Schematic diagram of the reg-
ulation of muscle contraction by tropo-
nin. TnC, TnI, and TnT, tropomyosin
C, I, and T, respectively. N, amino ter-
minus; C, carboxyl terminus (78a).
complex, which is then followed by the movement of
Tm into a nonblocking position, allowing myosin to
bind to actin (15, 26, 68). The exact function of TnT is
still somewhat controversial, but it is thought to stabi-
lize the troponin complex and to affect the Ca2⫹ sensi-
tivity of actomyosin ATPase activity and the level of
ATPase activation and/or force development (23, 44,
66, 68). The extended amino acid terminus of TnT may
interact with actin and with the overlapping regions of
adjacent Tm molecules (28). TnC, an EF-hand protein,
transfers the Ca2⫹ signal, eliciting contraction to the
fiber, whereas the main function of TnI is to inhibit the
actomyosin ATPase activity (40). TnT directly inter-
acts with Tm (66), TnI (66), and TnC (68). Conforma-
tional changes in TnC affected by Ca2⫹ binding (re-
viewed in Ref. 16) ultimately evoke the interactions
between the thin filament proteins. In addition, TnI
interaction with the other troponin subunits is affected
by TnI phosphorylation (1, 32, 62) and by the oxidation
state of Cys48 and Cys64 at the carboxyl terminus of
TnI when interacting with TnT (9).
MUTATIONS OF THIN FILAMENT PROTEINS IN
FAMILIAL HYPERTROPHIC CARDIOMYOPATHY
Familial hypertrophic cardiomyopathy (FHC) is an
autosomal dominant disease, characterized by left ven-
tricular hypertrophy, myofibril disarray, and sudden
cardiac death (SCD). Numerous studies have shown
that FHC is caused by one of many missense or dele-
tion mutations in various genes that encode for ␤-my-
osin heavy chain (3, 13, 20, 24, 92, 93), ventricular
myosin light chains 1 and 2 (12, 17, 67), myosin binding
protein C (88), titin (75), actin (50, 65), ␣-Tm (10, 33,
57, 59, 71, 84, 87, 90), TnT (2, 11, 36, 43, 51, 84–86, 89,
90), and TnI (34, 37, 55, 77). Whereas individuals with
␤-myosin heavy chain mutations, in general, have a
higher level of cardiac hypertrophy, those with TnT
mutations have less hypertrophy but a higher inci-
dence of SCD in young adults (90). To date, 15 human
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INVITED REVIEW
cardiac TnT mutations have been associated with
FHC: I79N, R92Q/W/L, R94L, A104V, F110I, R130C,
⌬E160, E163K/R, S179F, E244D, R278C, and a muta-
tion that arises from abnormal splicing of intron 16
(G13 A) (2, 18, 27, 36, 51, 58, 84, 86, 90, 91). Other
mutations associated with FHC found in thin filament
proteins are the cardiac Tm mutations A63V, K70T,
V95A, D175N, and E180G/V (10, 33, 57, 59, 71, 84, 87,
90); the TnI mutations R145G/Q, R162W, ⌬K183,
S199N, G203S, K206Q (34, 37), and an exon 8 deletion
mutant (55); and four missense mutations in actin (50,
65). Table 1 summarizes the clinical manifestations of
each of these mutations in ␣-Tm, actin, TnI, and TnT.
The mechanism by which these mutations alter cardiac
contraction and relaxation is still largely unknown,
although some studies suggest that changes in myofi-
brillar Ca2⫹ sensitivity can lead to diastolic dysfunc-
tion and sensitivity to dysrhythmias, which at times
cause sudden death. Tables 2 and 3 summarize the
results from in vitro studies and from studies on trans-
genic animals, respectively, that examine the mecha-
nisms of these mutations and pathologies associated
with FHC. We will now highlight several key observa-
tions that relate to the mechanisms of FHC, in partic-
ular those caused by TnT mutations.
TnT. Several mutations in TnT are responsible for
mechanical abnormalities in the sarcomeric complex.
Replication-defective recombinant adenovirus vectors
for gene transfer of I79N and R92Q mutant cardiac
TnT cDNAs into fully differentiated adult cardiac cells
in primary culture show a regular periodic pattern of
immunolabeled TnT localization (74). In this system,
unlike others mentioned below, direct force measure-
ments demonstrated marked desensitization of sub-
maximal Ca2⫹-activated tension (74). It was suggested
that the decrease in Ca2⫹ sensitivity may be attributed
to a change in the secondary structure caused by the
mutation of the incorporated protein (85) and that this
 INVITED REVIEW 1127

Table 1. Clinical studies of FHC associated with thin filament mutations

Protein Mutation Phenotype Ref.

␣-Tm A63V Hypertrophy of left ventricle and progression to dilated cardiomyopathy; high risk of sudden 59, 94
death; ST segment depression and T-wave inversion
K70T Hypertrophy of left ventricle and progression to dilated cardiomyopathy; high risk of sudden death 59, 94
V95A Spanish-American Family; affected family members only; mild cardiac hypertrophy; high risk of 33
sudden death (11 of 13 deaths were sudden)
D175N Myocyte hypertrophy; myofibril disarray; interstitial fibrosis; varying degree of left ventricular 10
hypertrophy; favorable prognosis
Finnish Study indicating that 25% of HCM were due to ␣-Tm mutation 30
Heart failure and atrial fibrillation 59
FHC patients of diverse origin; only heterozygotes affected 83, 84
Left ventricular hypertrophy in affected individuals only 88
E180G FHC patients of diverse origin; only heterozygotes affected 84
E180V Early onset (14 yr) of hypertrophic nonobstructive cardiomyopathy; left ventricular hypertrophy 71
progressing to severe systolic dysfunction
Actin E99K FHC showing abnormal electrocardiograms and variable pathology 65
P164A De novo, early onset (17 mo) showing abnormal electrocardiograms 65
A295S FHC mostly asymptomatic; heterogeneous phenotype; abnormal electro- and echo-cardiograms; 50
septal hypertrophy and progressive dilation; ventricular tachycardia and diastolic dysfunction
A331P Case study; de novo; early onset (8 yr); abnormal electrocardiograms 65
Tnl R145G Ventricular hypertrophy associated with FHC 34
R145Q Ventricular hypertrophy 34
R162W Apical hypertrophy (apical HCM) 34
⌬K183 Apical FHC and variable ventricular hypertrophy; good prognosis 34
Variable pathology and prognosis with significant penetrance 37
S199N Caucasian family with left ventricular dysfunction, arrhythmia, dyspnoea, angina, and 49a
palpitations; apex hypertrophy, sudden death (2 of 9 deaths)
G203S Apical hypertrophy (apical HCM) 34
K206Q Ventricular hypertrophy associated with FHC 34
⌬Exon8 Associated with mild FHC (left ventricular hypertrophy) 55
TnT I79N Poor prognosis; left ventricular thickening and early sudden death; 75% penetrance; some 90
individuals with mutation unaffected
Associated with FHC 84, 85
R92Q Associated with FHC (affected individuals only); poor prognosis including left ventricular 90
thickening and early sudden death
R92W Minimal hypertrophy and low disease penetrance with high risk of sudden death and poor 51, 52
prognosis
Abnormal Q waves; larger left ventricle; impaired systolic function (asymmetric septal 36
hypertrophy); 11% with mild outflow obstruction
R92L French family with variable hypertrophy; no history of sudden death 8, 18
R94L Myocyte disarray and high risk of sudden death in the absence of myocardial hypertrophy 86
A104V Moderate left ventricular hypertrophy and risk of sudden death 58
F110I Variable hypertrophy; good prognosis; some with mutation unaffected 2
Not affected (small group, n ⫽ 5 from 2 families) 36
Gene dose effect; 4 of 9 heterozygotes with moderate HCM and 2 homozygotes with severe HCM 43
Associated with FHC; present in affected individuals 90
R130C 6 mutations in 3 families; left ventricular hypertrophy 36
⌬E160 Poor prognosis, including left ventricular thickening and early sudden death; associated with FHC; 90
present in affected individuals
Systolic dysfunction 36
E163K Associated with FHC; present in affected individuals 90
E163R Septal hypertrophy 36
S179F Case study; homozygous died at 17 yr with profound left and right ventricular hypertrophy; 26
heterozygous parents and 3 siblings unaffected
E244D Associated with FHC; present in affected individuals 90
R278C Case study of 57-yr-old male with FHC with left ventricular hypertrophy; assymptomatic at 47 10a
Associated with FHC, present in affected individuals 90
Int16(G1 3 A) Found in all affected family members 91
Poor prognosis, including left ventricular thickening with 75% penetrance; high risk of sudden 91
death
Found in affected and clinically unaffected family members with disease haplotype only 84
FHC, familial hypertrophic cardiomyopathy; HCM, hypertrophic cardiomyopathy; Tm, tropomyosin; TnI and TnT, troponin I and T, respectively.

change in structure could subsequently modify the ing and a high risk of sudden death (56, 91). A glycine
actin-Tm interaction of the myofibril (28). to alanine mutation in the intron 16 splice donor site of
The TnT mutation in the intron 16 splice donor site TnT was identified in all affected and three clinically
is associated with pronounced left ventricular thicken- unaffected adults of a family with a history of FHC

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1128 INVITED REVIEW

Table 2. In vitro analysis of thin filament mutations associated with FHC

Protein Mutation Phenotype Condition Ref.

␣-Tm A63V Increases calcium sensitivity of force production Ad a

47
K70T Increases calcium sensitivity of force production Ada 47
D175N Increases velocity of actin translocation RFb 4
Increases calcium sensitivity Ada RFb MF 4, 7, 57
No change in force or velocity RFb 4, 7
Reduces actin affinity; conformational changes when switched from off to on state RFb 21
No effect on calcium sensitivity of force production Ada 47
E180G Weaker affinity for actin; increase velocity of actin translocation RFb 5
No change in force or velocity RFb 4
Increases calcium sensitivity RFb Adc 4, 47
Conformational changes when switched from off/on state RFb 21
Increase in force produced at submaximal calcium activation levels Ada 47
TnI R145G Reduces inhibition of actin-Tm-activated myosin ATPase activity; increases calcium RFb 11
sensitivity of ATPase regulation
Increases calcium sensitivity; suppresses maximum ATPase activity; reduces MFa 80
inhibitory effect of TnI (impairs actin-Tm-TnC interaction); reduces maximum
inhibition
R162W Reduces inhibition of actin-Tm-activated myosin ATPase activity; increases calcium RFb 11
sensitivity of ATPase regulation; stronger affinity for TnC in the presence of
calcium
TnT I79N Reduces isometric force by 25%; increases sliding speed which varied with ionic RFb 28
strength
Increases sliding speed RFe 42
Increases unloaded shortening velocity IM 78
May change actin-Tm interaction RFb 28
Reduces affinity/stability of troponin complex formation, inducing alternation of Ada 74
submaximal force generation
Reduces calcium sensitivity of force production Ada, IM 74, 78
Increases calcium sensitivity of force development SFb 54, 79
No change in calcium sensitivity or binding with Tn complex RFe 42
No change in cooperativity and maximum force SFb 54
Increases calcium sensitivity of ATPase activity without change in maximum level MFb 95
of ATPase activity; impairs inhibitory action of TnI
R92Q Time-dependent impairment of contractility (reduces fractional shortening and Adc 45
peak velocity of shortening)
a
Reduces affinity/stability of troponin complex formation, inducing alternation of Ad 74
submaximal force generation
Increases unloaded shortening velocity IM 78
Reduces calcium sensitivity of force production Ada IM 74, 78
Increases calcium sensitivity of force development SF RFb MFb
b
54, 79, 95
Possible abnormal protein folding due to altered solubility RFd 85
Potentiates maximum level of ATPase activity MFb 95
Reduces ATPase activation RFb 79
Reduces Tn-complex affinity RFb 95
No change in cooperativity and maximum force SFb 54
F110I No increase in calcium sensitivity SFb 61
Increases calcium sensitivity of force development SFb 79
Reduced activation of actin-Tm activated myosin-ATPase RFb
Increases maximum force without affecting cooperativity SFb 61
Reduced Tn affinity for actin-tropomyosin; possible abnormal protein folding due to RFd 85
altered solubility
⌬E160 Increases calcium sensitivity of ATPase activity; reduces calcium-dependent MFb 25
cooperativity
Reduces calcium sensitivity of force production IM 78
Increases TnC calcium affinity RFd 85
E163K Small change in calcium sensitivity; increases ATPase activation RFb 79
E244D Increases maximum force without affecting cooperativity; increases calcium SFb 61
sensitivity of force generation
Reduces Tn-complex affinity for actin-tropomyosin RFd 85
Potentiates maximum level of ATPase activity without calcium sensitization MFb 95
R278C Decreases maximal force and cooperativity SFb 53
Increases in calcium sensitivity of force development SFb 53, 79
Reduces ATPase inhibition RFb 79
Increases calcium sensitivity and potentiates maximum levels of ATPase activity MFb 95
Int16(G1 3 A) Reduced activation of actomyosin ATPase activity (with calcium) for both mutants RFb 56
(delEx16 and delEx15/16); reduces affinity for TnI (delEx16 and delEx15/16); no
change in calcium-independent inhibition or calcium-dependent activation
(delEx16 and delEx15/16)

Continued

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 INVITED REVIEW 1129

Table 2.—Continued
Protein Mutation Phenotype Condition Ref.
b
Increases calcium sensitivity of force generation; decreases cooperativity of SF 60
contraction-independent TnI phosphorylation (delEx16 and delEx15/16); reduces
calcium-activated maximum force in delEx15/16
Prevents switching off at low-calcium concentrations; reduces inhibition; increases RFb 69
velocity (delEx15/16)
b
Reduces activation and inhibition force including that of ATPase activation SF 79
(delEx15/16)
Reduces ATPase inhibition (delEx15/16) RFb
Reduces ATPase activation; reduced Tn affinity for actin-tropomyosin (delEx16 and RFd 85
delEx15/16)
Disruption of sarcomeric structure in 14–21% of cell; reduces calcium-activated IM 91
force of contraction
RF, reconstituted filament; IM, isolated myotubles from transfected primary quail myocytes; Ad, adenovirus infection of human protein;
SF, reconstituted skinned fiber. a Infection of rat cardiomyocytes; b human protein expressed in E. coli; c infection of feline cardiomyocytes;
d
bovine protein expressed in E. coli; e embryonic rat protein (rat I91N ⫽ Hu I79N) expressed in E. coli.

(84). This mutation results in two atypical truncated
transcripts that translate into a short and a long pep-
tide with a loss of 28 or 14 amino acids residues in the
carboxyl end plus 7 new amino acids in the shorter
protein (90, 91). Actomyosin ATPase assays of troponin
complexes reconstituted with the truncated proteins
show that activation of the ATPase activity is greatly
reduced for both mutants in the presence of Ca2⫹ (56).
The carboxyl terminus of TnT interacts with both TnC
and TnI, and, predictably, these truncated proteins
have a lower affinity for TnI (56) and, when reconsti-
tuted in a troponin complex, for actin-Tm (85). This
change in the affinity of the truncated TnTs for partic-
ular proteins or filament complexes may account for
the inadequate regulatory function. Transgenic mice
expressing low levels of truncated TnT (⬃5%) have
myocyte disarray, atrial hypertrophy, reduced ventric-
ular mass, and overall fewer and smaller cardiomyo-
cytes (resulting in a smaller heart) than their wild-type
litter mates, whereas homozygous mice (twice the ex-
pression of truncated TnT) died within 24 h of birth
(81). The surviving transgenic mice showed diastolic
dysfunction and cardiac arrhythmias postexercise con-
sistent with impaired contractility.
Some TnT mutations cause an increase in unloaded
myofilament sliding speed indicative of defective acto-
myosin cross-bridge activity and an increased or de-
creased Ca2⫹ affinity (85). A faster myofilament sliding
speed was observed for I79N (42), R92Q (78), E244D,
and the intron 16 (G13 A) truncated TnT mutant,

Table 3. Transgenic models of FHC associated with thin filament mutations

Protein Mutation Phenotype Transgenic Ref.

␣-Tm D175N Slower contraction and relaxation; normal ventricular function but weaker response Mice 57
to exercise and ␤-adrenergic stimulation
Increased calcium sensitivity; variable myocyte disarray, hypertrophy, and fibrosis (Mouse ␣-Tm)
TnI R145G Myocyte disarray, interstitial fibrosis, premature death; increased calcium sensitivity, Mice 31
hypercontractility, diastolic dysfunction
TnT I79N No cardiac hypertrophy even with exercise, normal survival; large increase in calcium Mice 49
sensitivity of ATPase activity and force relaxation; increase in rate of force
activation and in rate of force relaxation; decreases maximal force/cross-section
area and ATPase activity; loss of sensitivity of pH-induced shifts in calcium-
dependent force and computer simulations, suggesting a decrease in apparent off-
rate of calcium from TnC and increase cross-bridge detachment rate g
R92Q Variable myocyte disarray and interstitial collagen content; reduced left ventricular Mice 41
ejection fraction; systolic and diastolic dysfunction
Normal systolic but dysfunctional diastolic; cardiomyocyte disarray; increases Mice 63
interstitial collagen
Smaller left ventricles; dose-dependent fibrosis and increased mitochondria; increases Mice 82
atrial natriuretic factor and ␤-MHC mRNAs; increased basal sarcomeric activation;
impaired relaxation and shorter sarcomere lengths; hypercontractility and diastolic
dysfunction
Int16(G1 3 A) Diastolic dysfunction and sudden death; cardiac arrhythmias postexercise; Rat (delEx16) 18
myofibrillar disarray, no hypertrophy (delEx15/16 produced no transgenics; high
frequency of resorption and still births)
Cardiomyopathy, smaller hearts, diastolic and milder systolic dysfunction Mice (mouse 81
␣-Tm)
Myofibril disarray, reduced number and size of myocytes
Expression ⬎5% lethal (delEx15/16)
Data are for human TnT unless otherwise stated.

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1130
whereas ⌬E160 appears to decrease unloaded sliding
speed of the filament (85). The shift in sliding speed of
the I79N, R92Q, and ⌬E160 TnT mutants is also ac-
companied by changes in Ca2⫹ sensitivity of force or
ATPase activity (reviewed in Ref. 85).
The TnT-I79N mutation is of special interest because
it poses the highest risk of SCD in young adults (90). At
present, there is no clear understanding of why this
TnT-I79N mutation is associated with increased SCD.
Several investigators have demonstrated an in vitro
effect of the TnT-I79N mutation on the contractile
properties of cardiac and skeletal muscle with conflict-
ing results. Lin et al. (42), using rat cardiac TnT
containing a mutation in an equivalent position to the
TnT-I79N mutation in humans, showed that this mu-
tant TnT had a normal affinity for actin-Tm and con-
ferred normal Ca2⫹ sensitivity to actomyosin ATPase
activity. The regulated thin filaments, however, moved
50% faster over heavy meromyosin (HMM) than con-
trol filaments in an in vitro motility assay. Additional
measurements utilizing the same system revealed that
HMM exerted reduced isometric force on single thin
filaments reconstituted with the TnT-I79N mutant
(28). Sweeney et al. (78) reported that TnT-I79N trans-
fected quail skeletal muscle myotubules had decreased
Ca2⫹ sensitivity of force production, whereas the un-
loaded shortening velocity was increased by about two-
fold. An embryonic isoform of rat TnT-I79N expressed
in adult rat cardiac myocytes induced a decreased Ca2⫹
sensitivity of isometric force (74). Our results on
TnT-I79N reconstituted porcine fibers (79) are in ac-
cord with Morimoto et al. (54), who demonstrated that
TnT-I79N reconstituted skinned rabbit trabeculae in-
creased the Ca2⫹ sensitivity of contraction. A recent
study confirmed increased Ca2⫹ sensitivity of myofi-
brillar ATPase activity of TnT-I79N reconstituted rab-
bit cardiac myofibrils (95). Part of the disparity is likely
due to the different in vitro assays used by these
investigators, illustrating the need to study the effect
of the mutations in an in vivo system.
Until now, a transgenic model for the TnT-I79N has
not been reported, although other mutant TnT trans-
genic mice have been described (Table 3). Miller et al.
(49) examined a wild-type (Tg-WT) and two I79N-
transgenic mouse lines (Tg-I79N) of human cardiac
TnT (hcTnT) driven by a murine ␣-myosin heavy chain
promoter. The levels of expression of either Tg-WT or
Tg-I79N, relative to mouse cTnT, were 71% (WT) or
35% and 52% in the two I79N lines. Extensive charac-
terization of the Tg-I79N lines compared with Tg-WT
and/or non-Tg mice demonstrated the following: nor-
mal survival and no cardiac hypertrophy even with
chronic exercise in all groups, large increases in the
Ca2⫹ sensitivity of ATPase activity and force in
skinned fibers, a substantial increase in the rate of
force activation and a small increase in the rate of force
relaxation in flash photolysis experiments, signifi-
cantly lower maximal force/cross-sectional area and
ATPase activity, and a loss of sensitivity to pH-induced
shifts in the Ca2⫹ dependence of force correlated with
hcTnT-I79N expression levels (49).
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INVITED REVIEW
When native TnT is replaced with mutant I79N TnT,
an increased filament speed is not unexpected. Alter-
ations in the actin interaction surface may allosteri-
cally change actomyosin binding and cross-bridge ki-
netics. If changes in actomyosin interactions increase
the rate of cross-bridge dissociation from actin, peak
isometric force will be reduced and maximal filament
sliding velocity increases. The increased Ca2⫹ sensitiv-
ity of force in reconstituted skinned cardiac fibers in
which native TnT was replaced with TnT-I79N (79)
and in skinned cardiac fibers of transgenic mice (49) is
not readily explained by changes in cross-bridge kinet-
ics alone. The results from skinned cardiac fiber stud-
ies, flash photolysis force transients, and force-pCa
relations (49) were reproduced by computer simula-
tions that integrate Ca2⫹ binding to intracellular Ca2⫹
buffers and predict force generation based on a modi-
fication of the model of Robertson et al. (73) and a
two-state cross-bridge model (48) that utilized an ex-
ponential dependence of the TnC off-rate for Ca2⫹
(Ca2⫹-specific site II) on force (29, 38). Simulations
postulate that an increase in the apparent rate of
actomyosin cross-bridge detachment (g) was not suffi-
cient to account for all experimental observations. The
combination of an increased affinity of TnC for Ca2⫹
and an increase in g reproduced all experimental ob-
servations in skinned fibers that contain the human
TnT-I79N. An increased g was also inferred by others
from their in vitro motility assays (28, 42) or TnT-I79N
transfected myotubes (28), and a consistent picture
regarding this mutation is emerging based on results
from several different approaches. Moreover, the cross-
bridge effects brought about by this mutation are dis-
tinct from the calcium effects, and it is possible that the
former arise from an alteration in the interactions
between TnT, Tm, and actin, whereas the latter arise
from altered TnT and TnC interactions. The predicted
increased affinity of TnC for Ca2⫹ still requires exper-
imental verification.
Compared with wild-type TnT, relaxation of force in
TnT-I79N containing myocardium is slowed by a de-
creased affinity of TnC for Ca2⫹; however, this effect is
somewhat attenuated by an increase in g. The twitch
contraction operates from a pCa range of ⬃7.7 at rest
to a peak systolic value of 5.7. With the use of these
pCa values, simulated peak twitch force in steady-state
conditions in fibers containing Tg-I79N was shown to
be higher than in fibers that contained Tg-WT (Fig. 2).
Simulations for intact living myocardium further pre-
dict a faster rise of force, a slower isometric relaxation,
and an increased residual force or resting tension at
the onset of the next contraction. If twitch amplitudes
in both Tg-WT and Tg-I79N are the same or are nor-
malized, isometric relaxation of Tg-I79N myocardium
is slower than in the WT, as observed in isovolumetri-
cally contracting isolated heart (Fig. 2) (35). The higher
basal contractile state, the increased rate of contrac-
tion, and the slower relaxation (35) in TnT-I79N myo-
cardium would have the following consequences. First,
the contractile reserve is decreased, and an increase in
heart rate and/or of contractility, such as after isopro-
 INVITED REVIEW
Fig. 2. Simulation of Ca2⫹ transients and force during twitches.
Time courses are shown of the intracellular Ca2⫹ concentration
transient (top) and of the corresponding force (middle) for isometric
twitches of intact ventricular myocardium during repetitive stimu-
lation at 400-ms intervals in steady-state control conditions for the
TnT transgenic wild-type (WT; solid lines) and for the TnT trans-
genic I79N mutation (dashed lines). Bottom: normalized force traces
for the same twitches, demonstrating a slower isometric relaxation
in I79N myocardium than in WT. The TnT-I79N mutant data were
obtained by decreasing the apparent dissociation rate of Ca2⫹ from
TnC from 300 to 88 s⫺1 and increasing the cross-bridge apparent
dissociation constant from 10 to 20 s⫺1.
terenol administration, would jeopardize relaxation
and cause diastolic dysfunction. An increased contrac-
tility and heart rate would increase diastolic intra-
cellular Ca2⫹ concentration, cause intracellular Ca2⫹
overload, and dysrhythmias. These predictions seem to
hold true for Tg-I79N mice challenged with isoproter-
enol (35), which demonstrated an impaired inotropic
response, relaxation impairment, and fatal dysrhyth-
mias. It is likely that these mechanisms contribute to
the mortality observed in patients with a TnT-I79N-
based FHC, especially in stimulated states of contrac-
tility such as seen during vigorous exercise or during
inotropic interventions.
The E244D mutation of TnT also appears to increase
Ca2⫹ sensitivity (61). The TnT-E244D mutation has a
shorter side chain due to the absence of a methyl group
in the aspartic acid residue, yet the charge is con-
served. The spatial difference due to the lack of a
methyl group becomes significant if it 1) alters TnT
secondary structure, 2) alters protein-protein interac-
tion within the troponin complex, 3) induces or elimi-
nates structural changes in the troponin complex re-
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1131
quired for its regulatory properties, or 4) produces
other effects. At least one of these conditions appears to
have an impact on thin filament regulation. Skinned
cardiac fiber reconstituted with the E244D TnT in-
creased the maximum force and increased the Ca2⫹
sensitivity of the force generated compared with wild-
type TnT (61). The E244D mutation is located in a
region of the carboxyl terminus of TnT that interacts
with Tm, and changes in TnT-Tm interaction may alter
actomyosin regulation. Indeed, the E224D mutation
was found to have a lower affinity for actin-Tm than
wild-type TnT (85). This mutation has been found in
affected individuals of families with FHC having poor
prognosis (90).
The F110I TnT mutation is associated with variable
cardiac morphologies and prognosis (2, 36, 43, 90). This
mutation, located in a region of TnT that interacts with
actin and Tm (16), also increased the maximum force
in skinned muscle fibers but did not influence the Ca2⫹
sensitivity of force generation (61).
Another TnT transgenic animal model reported by
Oberst et al. (63) was generated for the human cardiac
TnT-R92Q mutation using a murine cTnT promoter.
The level of expression in transgenic lines (wild-type
and R92Q) varied from 1 to 10% of the total cTnT pool.
Diastolic dysfunction and myocyte disarray were ob-
served in the mutant mice but not in wild-type mice
(63). The same TnT-R92Q mutant was expressed in
transgenic mice, in which the level of R92Q expression
varied from 30 to 92% (82). A murine cTnT cDNA and
a rat ␣-myosin heavy chain promoter were used in the
latter study. The R92Q hearts had a significant induc-
tion of atrial natriuretic factor and ␤-myosin heavy
chain transcripts, interstitial fibrosis, and mitochon-
drial pathology. Isolated cardiac myocytes from these
R92Q transgenic mice had increased basal sarcomeric
activation, impaired relaxation, and shorter sarcomere
lengths. Isolated working hearts showed hypercontrac-
tility and diastolic dysfunction, both of which are com-
mon findings in patients with FHC (82). Transgenic
mice expressing 30%, 67%, and 92% R92Q TnT show a
dose-related induction and progression of atrial hyper-
trophy but a constant and age-independent decrease in
ventricular mass. Analogous to the cardiomyocytes and
hearts of the truncated TnT transgenic mice (above),
the R92Q myocytes and hearts are smaller than their
wild-type counterparts. The left ventricle contains both
hypertrophied and dying myocytes with abnormal his-
topathologies, which are particularly prevalent in the
higher expressing mice (82). The mice expressing 67%
R92Q displayed higher heart rates and diastolic dys-
function, prolonged contraction and relaxation, and
possible early activation of ATPase activity. The dia-
stolic dysfunction is attributed to an increase in Ca2⫹
sensitivity, as demonstrated in in vitro studies by Mo-
rimoto et al. (54), Szczesna et al. (79), and Yanaga et al.
(95) or a deregulation of intracellular Ca2⫹. It is rea-
sonable to assume that a change to a more polar amino
acid residue in the Tm-interacting domain of TnT will
affect troponin interactions as shown by a reduction of
the stability of troponin complex formation containing
1132
R92Q (74, 79). In a recent paper (41), a murine ␣-my-
osin heavy chain promoter was used to produce a
transgenic mouse expressing human cardiac TnT-
R92Q. The level of protein expression was relatively
low, and the mutant mice demonstrated myocyte dis-
array and excess interstitial collagen. Interestingly,
none of these transgenic mice demonstrated significant
cardiac hypertrophy.
Exchange studies of TnT-R278C vastly increased
Ca2⫹ sensitivity, more than any other TnT mutation
known hitherto (53, 79). Reconstituted skinned cardiac
fibers containing the R278C mutation in the globular
carboxyl terminal domain of TnT greatly increases the
Ca2⫹ sensitivity of force development (79). TnT resi-
dues 188–288, characterized as the T2 proteolytic frag-
ment having an unusually large number of positively
charged residues, have been reported to interact with
Tm, TnI, and TnC (reviewed in Refs. 16 and 70). The
interaction of the TnT carboxyl terminus with Tm
appears to be Ca2⫹ dependent (76). Mutation of a basic
amino acid to a neutral in the carboxyl end of TnT may
induce steric changes within the troponin complex,
altering the contacts between the regulatory subunits
in response to Ca2⫹. Ohtsuki’s group (53) showed that
this TnT mutation induced a higher level of sub-half-
maximum force as a result of a decrease in maximum
force and cooperativity, although only a slight increase
in Ca2⫹ sensitivity was observed.
TnI. The R145G TnI mutation identified in patients
with FHC constitutes a change in charge in an evolu-
tionarily conserved residue (34). We observed impair-
ment in both ATPase assays and in the relaxation of
force in skinned cardiac muscle preparations incorpo-
rating the R145G mutation (Lang R, Zhao J, Hous-
mans PR, and Potter JD, unpublished observations).
Another group reported that actin-Tm-activated myo-
sin S1 ATPase assays using troponin complexes recon-
stituted with R145G TnI revealed no change in activa-
tion but induced minimal inhibition and increased the
Ca2⫹ sensitivity of regulation compared with wild-type
TnI complexes (11). The switch from a positively
charged to a neutral residue in the R145G TnI muta-
tion occurs in a region that interacts with the amino-
terminal domain of TnC and possibly actin (reviewed
in Ref. 16) and, consequentially, may alter the dynam-
ics or influence the regulatory mechanism of the tropo-
nin complex. We detected a slight increase in the ␣-he-
lical content in circular dichroism studies of the R145G
mutant protein in addition to an increase in the affinity
between TnC and R145G TnI in the presence of Ca2⫹
and Mg2⫹ vs. Mg2⫹ alone (Lang R, Zhao J, Housmans
PR, and Potter JD, unpublished observations). The
large change in the ability of this mutant TnI to inhibit
contraction, combined with the increased Ca2⫹ sensi-
tivity of contraction, would likely lead to severely im-
paired diastolic function.
␣-Tm. Six missense mutations in the cardiac ␣-Tm
have been linked to FHC (Table 1). In vitro analysis of
several missense mutations in the cardiac ␣-Tm gene
have also shown increased Ca2⫹ sensitivity (see Table
2). The A63V mutation, associated with poor prognosis
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INVITED REVIEW
and a risk of sudden death in individuals with FHC
(59, 94), was found to increase the Ca2⫹ sensitivity of
force production in the isometric force recordings of
single adult rat myocytes that expressed this mutant
␣-Tm by means of an adenovirus vector (47). Three
other mutations associated with FHC, K70T, E180G,
and D175N, showed a smaller increase in Ca2⫹ sensi-
tivity of force production, with the smallest increase
reported for the D175N mutation (4, 47). The D175N
Tm mutation is associated with variable prognosis and
pathophysiologies; however, favorable prognosis in
some cases have been documented (10, 59, 87). The
D175N ␣-Tm mutation in transgenic mice causes im-
paired contractility and relaxation and enhances Ca2⫹
sensitivity (57). The increases in Ca2⫹ sensitivity of
force production, as reported by Michele et al. (47)
(A63V ⬎ K70T ⬎ E180G ⬎⬎ D175N ⫽ wild type),
appear to be directly related to the severity of the
disease. Several lines of transgenic mice overexpress-
ing the D175N Tm mutation at ⬍40% and 60% of total
Tm myofibrillar content suggest that the degree of
physiological abnormalities associated with the muta-
tion is dependent on the amount of mutant ␣-Tm in-
corporated in the filaments (57). Mice expressing 60%
of the mutant protein had reduced contraction and
relaxation rates during exercise compared with the
mice expressing ⬍40% of the D175N protein or com-
pared with the wild-type littermates (57). The abnor-
mal cardiac performance of the D175N transgenic mice
was attributed to an increase in Ca2⫹ sensitivity,
which was observed in skinned fiber preparations. Pro-
tein-protein interactions within the troponin-Tm com-
plex may mediate the regulatory dysfunction because
both D175N and E180G are within a region considered
to interact with TnT, whereas A63V and K70T are in
repeat sequences and may alter Tm-actin interaction
(reviewed in Refs. 6, 16, 70).
␣-Actin. In 1999, the first ␣-actin mutation linked to
FHC was reported in a Danish family. The A295S
␣-actin mutation was found in 13 individuals with
different disease phenotypes and a variable age of
disease onset (as early as 4 yr old) (50). Only one
individual was asymptomatic, whereas 3 of the 13 with
the mutation showed more severe phenotypes, which
included pronounced hypertrophy, ventricular tachy-
cardia, and diastolic dysfunction (50). The only other
report identifying cardiac actin mutations linked to
FHC is a recent study describing the phenotypes of
family members of three individuals with different
actin mutations. In this study, two de novo mutations
(P164A and A331P) were associated with early-onset
(8 yr and 17 mo of age, respectively) left ventricular
hypertrophy and repolarization abnormalities (65).
The third actin mutation, E99K, was found to be auto-
somal dominant, with affected individuals having a
later onset of the disease, variable hypertrophy, and
diastolic dysfunction (65). FHC-linked mutations in
other genes were excluded in both studies. The four
missense cardiac actin mutations are located near or
within regions that interact with other actin molecules,
troponin components, or the myosin head and may
 INVITED REVIEW
either destabilize the sarcomeric structure or alter the
force generation during the cross-bridge cycle of con-
traction. Defective myofilament regulation or function
may be due to reduced or enhanced filament protein-
protein interaction caused by replacement of a neutral
or acidic amino acid with a polar or basic amino acid
(A295S and E99K missense mutations); a faulty actin-
actin interaction may affect calcium signal transduc-
tion by altering the Tn-Tm-actin interaction. A change
in the secondary structure of actin, possibly induced by
replacement or addition of the ␣-helical-destabilizing
amino acid proline (as in the case of P164A and
A331P), could also conceivably alter the native envi-
ronment in actin that interacts with itself or other
filament components and ultimately affect force gener-
ation and regulation. These cardiac actin mutations
were only recently identified; therefore, little is known
about their functional consequences.
SUMMARY AND CONCLUSIONS
Thin filament proteins provide for a very tight reg-
ulation of cardiac contraction and relaxation. Each of
several mutations profoundly impacted basal contrac-
tility and the time course of relaxation. In most muta-
tions, Ca2⫹ sensitivity of force development was in-
creased; in some cases, filament sliding velocity was
also increased (Table 2). The myofilament is a highly
interacting system in which even a slight change in
interaction between any of the filament components
may alter the Ca2⫹ sensitivity of force generation. TnT
plays a key role in maintaining the Ca2⫹-dependent
competition of TnC and actin-Tm for the inhibitory
region of TnI (64). The biophysical consequences of one
amino acid substitution in a critical part of TnT will
alter the balance of power between the competing ac-
tin-Tm and TnC sites for the inhibitory region of TnI.
McKay et al. (46) illustrated the precision of filament
protein interactions by describing a skeletal TnC mu-
tant (without Ca2⫹-binding site I, similar to the cardiac
TnC in that cardiac TnC has no functional Ca2⫹ site I),
with a “delicate energetic balance” between Ca2⫹ and
TnI, binding to the TnC regulatory domain in the
“open” state. It is therefore not surprising that several
TnT mutations, and mutations of other thin filament
proteins, manifest themselves by altering both cross-
bridge kinetics (actomyosin dependent), and the Ca2⫹
affinity of TnC, as recently proposed (49). Impaired
contractile function due to thin filament mutations
would most likely contribute to the progression of com-
pensatory hypertrophy (FHC).
Mutations in thin filament proteins give rise to vary-
ing degrees of hypertrophy and incidences of SCD
(Table 1). One of the most puzzling questions regarding
the pathogenesis of TnT-related FHC is the mecha-
nism of SCD. Individuals with TnT-related FHC tend
to exhibit mild or no ventricular hypertrophy, yet ex-
perience a high frequency of sudden death at an early
age. These events could result from diastolic dysfunc-
tion, from the lack of pH sensitivity of Ca2⫹ sensitivity
of force, from alterations in intracellular Ca2⫹ homeo-
Downloaded from www.physiology.org/journal/jappl (046.161.058.175) on February 25, 2019.
1133
stasis, and/or from other processes. It remains to be
determined whether the occurrence of SCD results
from an altered thin filament environment or whether
additional primary affects of the point mutation act at
the level of the cardiac myocyte. Future work in this
area should provide insights into the molecular mech-
anisms responsible for the induction and progression of
FHC.
This work was supported by National Institutes of Health Grants
AR-45391 (to J. D. Potter), HL-42325 (to J. D. Potter), and GM-36365
(to P. R. Housmans).
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