Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling.
ABSTRACT A neuronal isoform of nitric oxide synthase (nNOS) has recently been located to the cardiac sarcoplasmic reticulum (SR). Subcellular localization of a constitutive NOS in the proximity of an activating source of Ca2+ suggests that cardiac nNOS-derived NO may regulate contraction by exerting a highly specific and localized action on ion channels/transporters involved in Ca2+ cycling. To test this hypothesis, we have investigated myocardial Ca2+ handling and contractility in nNOS knockout mice (nNOS-/-) and in control mice (C) after acute nNOS inhibition with 100 micromol/L L-VNIO. nNOS gene disruption or L-VNIO increased basal contraction both in left ventricular (LV) myocytes (steady-state cell shortening 10.3+/-0.6% in nNOS-/- versus 8.1+/-0.5% in C; P<0.05) and in vivo (LV ejection fraction 53.5+/-2.7 in nNOS-/- versus 44.9+/-1.5% in C; P<0.05). nNOS disruption increased ICa density (in pA/pF, at 0 mV, -11.4+/-0.5 in nNOS-/- versus -9.1+/-0.5 in C; P<0.05) and prolonged the slow time constant of inactivation of ICa by 38% (P<0.05), leading to an increased Ca2+ influx and a greater SR load in nNOS-/- myocytes (in pC/pF, 0.78+/-0.04 in nNOS-/- versus 0.64+/-0.03 in C; P<0.05). Consistent with these data, [Ca2+]i transient (indo-1) peak amplitude was greater in nNOS-/- myocytes (410/495 ratio 0.34+/-0.01 in nNOS-/- versus 0.31+/-0.01 in C; P<0.05). These findings have uncovered a novel mechanism by which intracellular Ca2+ is regulated in LV myocytes and indicate that nNOS is an important determinant of basal contractility in the mammalian myocardium. The full text of this article is available at http://www.circresaha.org.
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ABSTRACT: Complex paracrine interactions exist between endothelial cells and cardiac myocytes in the heart. Cardiac endothelial cells release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1, angiotensin II, adenylpurines) that exert direct effects on myocyte function, independent of changes in coronary flow. Some of these mediators are also generated by cardiac myocytes, often under pathological conditions. This review focuses on the role of NO in this paracrine/autocrine pathway. NO modulates several aspects of “physiological” myocardial function (e.g., excitation-contraction coupling; myocardial relaxation; diastolic function; the Frank-Starling response; heart rate; β-adrenergic inotropic response; and myocardial energetics and substrate metabolism). The effects of NO are influenced by its cellular and enzymatic source, the amount generated, the presence of reactive oxygen species, interactions with neurohumoral and other stimuli, and the relative activation of cyclic GMP-dependent and -independent signal transduction pathways. The relative physiological importance of endothelium- and myocyte-derived NO remains to be established. In pathological situations (e.g., ischemia-reperfusion, left ventricular hypertrophy, heart failure, transplant vasculopathy and rejection, myocarditis), NO can potentially exert beneficial or deleterious effects. Beneficial effects of NO can result from endothelial-type nitric oxide synthase-derived NO or from spatially and temporally restricted expression of the inducible isoform, inducible-type nitric oxide synthase. Deleterious effects may result from (1) deficiency of NO or (2) excessive production, often inducible-type nitric oxide synthase-derived and usually with concurrent reactive oxygen species production and peroxynitrite formation. The balance between beneficial and deleterious effects of NO is of key importance with respect to its pathophysiological role.Pharmacology [?] Therapeutics 05/2000; · 7.79 Impact Factor
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ABSTRACT: Several effects of nitric oxide (NO) on the control of L-type calcium current (ICa) and of calcium handling in cardiomyocytes have been described. Cardiomyocytes have been shown to express in different conditions all types of nitric oxide synthases (NOS), but the role of NO in the regulation of calcium current remains controversial. Previously, we have shown in guinea pig ventricular cells a stimulatory effect of NOS inhibitors on ICa. Here we investigate the intracellular mechanisms involved in the putative inhibitory role of NO on basal ICa in ventricular cells. The stimulatory effect of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) (1 mM) was present also in calcium transient measurements, but only after a preincubation with L-arginine (L-arg, 0.1 mM). The nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 0.5 mM) increased peak ICa in a similar manner to NOS inhibitors in whole-cell voltage-clamp experiments. Also ODQ (1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one, 0.1 mM), a specific inhibitor of a target of NO, the soluble guanylate cyclase, was able to stimulate ICa. The block of type II phosphodiesterase (cGMP-activated) by EHNA (erythro-9-[2-hydroxy-3-nonylladenine, 30 microM) exerted a similar effect on ICa as PTIO and ODQ. Carbachol (CCh, 1 microM) was able to revert the stimulatory effect on ICa observed with PTIO, ODQ, and EHNA. We propose that the increase of basal ICa in guinea pig cardiomyocytes previously observed with L-NMMA depends on the removal of a tonic NO inhibition. This increase of ICa is mimicked by blocking at different steps the cGMP-cascade activated by NO, suggesting a NO-guanylate cyclase mechanism in the basal control of ventricular calcium current.Pflügers Archiv - European Journal of Physiology 03/2001; 441(5):621-8. · 4.87 Impact Factor
Article: Nitric oxide and cardiac function.Circulation Research 10/1996; 79(3):363-80. · 11.86 Impact Factor
Wallis, Stefan Neubauer, Derek A. Terrar and B. Casadei
Claire E. Sears, Simon M. Bryant, Euan A. Ashley, Craig A. Lygate, Stevan Rakovic, Helen L.
Cardiac Neuronal Nitric Oxide Synthase Isoform Regulates Myocardial Contraction and
Print ISSN: 0009-7330. Online ISSN: 1524-4571
Copyright © 2003 American Heart Association, Inc. All rights reserved.
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Cardiac Neuronal Nitric Oxide Synthase Isoform Regulates
Myocardial Contraction and Calcium Handling
Claire E. Sears, Simon M. Bryant, Euan A. Ashley, Craig A. Lygate, Stevan Rakovic,
Helen L. Wallis, Stefan Neubauer, Derek A. Terrar, B. Casadei
Abstract—A neuronal isoform of nitric oxide synthase (nNOS) has recently been located to the cardiac sarcoplasmic
reticulum (SR). Subcellular localization of a constitutive NOS in the proximity of an activating source of Ca2?suggests
that cardiac nNOS-derived NO may regulate contraction by exerting a highly specific and localized action on ion
channels/transporters involved in Ca2?cycling. To test this hypothesis, we have investigated myocardial Ca2?handling
and contractility in nNOS knockout mice (nNOS?/?) and in control mice (C) after acute nNOS inhibition with 100
?mol/L L-VNIO. nNOS gene disruption or L-VNIO increased basal contraction both in left ventricular (LV) myocytes
(steady-state cell shortening 10.3?0.6% in nNOS?/?versus 8.1?0.5% in C; P?0.05) and in vivo (LV ejection fraction
53.5?2.7 in nNOS?/?versus 44.9?1.5% in C; P?0.05). nNOS disruption increased ICadensity (in pA/pF, at 0 mV,
?11.4?0.5 in nNOS?/?versus ?9.1?0.5 in C; P?0.05) and prolonged the slow time constant of inactivation of ICaby
38% (P?0.05), leading to an increased Ca2?influx and a greater SR load in nNOS?/?myocytes (in pC/pF, 0.78?0.04
in nNOS?/?versus 0.64?0.03 in C; P?0.05). Consistent with these data, [Ca2?]itransient (indo-1) peak amplitude was
greater in nNOS?/?myocytes (410/495 ratio 0.34?0.01 in nNOS?/?versus 0.31?0.01 in C; P?0.05). These findings
have uncovered a novel mechanism by which intracellular Ca2?is regulated in LV myocytes and indicate that nNOS
is an important determinant of basal contractility in the mammalian myocardium. The full text of this article is available
at http://www.circresaha.org. (Circ Res. 2003;92:e52-e59.)
Key Words: neuronal nitric oxide synthase ? ventricular ? contraction ? calcium
thelium but also by the myocytes themselves.1However,
whether constitutive myocardial NO production regulates
basal inotropy and calcium (Ca2?) fluxes remains controver-
sial (see reviews1,2). In particular, some studies have shown
that nonisoform specific inhibition of NO synthase (NOS) or
targeted disruption of the endothelial NOS isoform (eNOS)
has no effect on cell shortening or Ca2?handling.3,4Others,
however, have indicated that endogenous NO production may
tonically inhibit myocardial inotropy5,6and the Ca2?current.7
These studies assumed that eNOS was the only constitutive
isoform involved in the autocrine control of myocardial
function. In 1999, however, Xu and collaborators8localized a
neuronal-type NOS isoform (nNOS) to murine and human
sarcoplasmic reticulum (SR). The subcellular localization of a
constitutive NOS isoform in the proximity of an activating
source of Ca2?suggests that endogenous NO may exert a
specific and localized action on ion channels/transporters
involved in Ca2?cycling. In the present study, we report that
targeted disruption of the nNOS gene (nNOS?/?) as well as
t is now well-established that nitric oxide (NO) is consti-
tutively generated within the heart, not only by the endo-
acute pharmacological nNOS inhibition enhances basal left
ventricular (LV) contraction and intracellular Ca2?([Ca2?]i)
transients by increasing Ca2?influx (via the Ca2?current) and
These findings have uncovered a novel mechanism by
which [Ca2?]iis regulated in LV myocytes. We suggest that
nNOS may provide a negative feedback regulation of Ca2?
influx, because increases in [Ca2?]istimulate nNOS synthesis
of NO, which in turn acts to inhibit Ca2?influx. Such
mechanisms would contribute to the maintenance of a tight
control of [Ca2?]iin physiological conditions and may protect
against Ca2?overload in cardiac disease states.
Materials and Methods
Mice homozygous for targeted disruption of the nNOS gene
(B6,129-NOS1tm1plh, nNOS?/?)9were purchased from Jackson Labo-
ratories (Bar Harbor, Maine) and a colony was established at the
John Radcliffe Hospital by backcrossing the nNOS?/?on a C57BL/6
background. N3 littermate mice homozygous for the nNOS gene
(nNOS?/?) were used as controls in most protocols, in others
Original received October 29, 2002; resubmission received February 6, 2003; revised resubmission received February 20, 2003; accepted February 20,
From the Department of Cardiovascular Medicine (C.E.S., S.M.B., E.A.A., C.A.L., H.L.W., S.N., B.C.), Oxford University, John Radcliffe Hospital;
and the Department of Pharmacology (S.R., D.T.), Oxford University, Oxford, UK.
Presented in part at the 75th Scientific Sessions of the American Heart Association, Chicago, Ill, November 17–20, 2002, and published in abstract
form (Circulation. 2002;106[suppl II]:II-178).
Correspondence to Dr Claire E. Sears or Dr Barbara Casadei, Department of Cardiovascular Medicine, Oxford University, John Radcliffe Hospital,
Oxford, OX3 9DU, UK. E-mail email@example.com
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.orgDOI: 10.1161/01.RES.0000064585.95749.6D
by guest on May 31, 2013 http://circres.ahajournals.org/Downloaded from
age-matched C57BL/6 mice were used as wild-type controls as in
previous studies.10,11The treatment of all animals was in accordance
with the Home Office Guidance on the Operation of Animals
(Scientific Procedures) Act, 1986 (H.M.S.O.).
In Vivo Measurements of LV Function
Hemodynamic indices were measured in anesthetized mice (isoflu-
rane) using a A 1.4F Millar Mikro-tip catheter (SPR-671) inserted
into the LV via the carotid artery. At the end of the experiment, the
catheter was withdrawn from the LV to measure aortic pressures.
Reported values represent an average of 20 consecutive cardiac
cycles. Echocardiography was performed simultaneously using an
Agilent Sonos5500 with a 7- to 15-MHz linear-array transducer.
Parasternal short-axis images, 2-D and M-mode, were obtained at the
level of the papillary muscles and stored digitally.
Myocyte Isolation and Techniques
Single LV myocytes were isolated using a standard enzymatic
dispersion technique as described previously.12For electrophysio-
logical recordings, myocytes were superfused with a modified
Tyrode solution (for composition, see expanded Materials and
Methods, available in the online data supplement at http://www.
circresaha.org). Membrane current was measured using the whole-
cell configuration of the patch-clamp technique (Axopatch 200A,
Axon Instruments). Cell length was concurrently monitored with a
video-edge detection system (IonOptix Corp), with a temporal
resolution of ?4.2 ms. Analog signals (current, voltage, and cell
length) were digitized (Digidata 1200A, Axon Instruments) and
stored on-line to computer for subsequent off-line analysis. Calcium
current (ICa) and unloaded cell shortening were elicited at 35?1°C as
detailed in the expanded Materials and Methods section.
Assessment of SR Ca2?Load
The SR Ca2?content was quantified in voltage-clamped cells by
discharging SR Ca2?with a 10 second application of caffeine
(10 mmol/L, using a rapid solution-switching device) and integrating
the resulting Na?-Ca2?exchange (NCX) current, as described pre-
viously.13In a cohort of myocytes from each group, the caffeine-
induced calcium transient (indo-1) was recorded after exposure to
5 mmol/L nickel for 5 minutes,13and the time constant of decay (?)
was calculated in order to compare the contribution of slow Ca2?
extrusion mechanisms in control and nNOS?/?myocytes.
The contribution of SR Ca2?to excitation-contraction (E-C) coupling
in nNOS?/?myocytes was evaluated by assessing ICaand contraction
after application of thapsigargin (10 ?mol/L). Disabling of the SR by
thapsigargin was verified by the absence of an inward current in
response to a 10 mmol/L pulse of caffeine (data not shown).
Measurement of [Ca2?]iTransients
Indo-1 fluorescence was monitored from cells preincubated with the
acetoxymethyl ester of indo-1 (5 ?mol/L, Sigma) for 20 minutes at
room temperature. Cells were field-stimulated to contract at 1 Hz at
35°C. In a cohort of cells from each group, calibration of the indo-1
signal was performed using the method of Terracciano and
MacLeod.14In some experiments, cells were imaged (Fluo-4, 100
?mol/L loaded via the patch pipette) in line-scan mode (2.61 ms per
line) using a Leica TCS-NT confocal microscopy system.
Western blots were performed on LV membrane subfractions using
specific antibodies to the following: ?-1C subunit of the dihydro-
pyridine calcium channel (Alomone); cardiac ryanodine receptor,
calsequestrin, and SERCA2a (Affinity Bioreagents); phospholamban
(Cyclacel); NCX (Abcam); and the plasmalemma Ca2?-ATPase
(PMCA) (Laboratory Vision).
Acute Inhibition of Myocyte nNOS With
L-VNIO is a potent nNOS selective inhibitor. At the concentration
used, its Kifor nNOS inhibition is 120-fold lower than for eNOS
inhibition.15Cohorts of cells taken from the same isolates were either
incubated with L-VNIO (100 ?mol/L) for 30 minutes (in addition, to
ensure intracellular access for the drug, 100 ?mol/L L-VNIO was
also added to the pipette solution) or were stored under normal
conditions and used as control cells. ICa, cell shortening, and SR load
were assessed as outlined above.
Data are expressed as mean?SEM, and n indicates the number of
cells used. A two-way ANOVA was used to compare interactions
between factors. Point-to-point comparisons were performed with a
Student’s t test. Significance was assessed at the P?0.05 level.
Contractile Parameters in nNOS?/?Mice
The role of nNOS in regulating cardiac function was first
investigated in vivo in anesthetized mice with homozygous
deletion of the nNOS gene (nNOS?/?) and their littermate
controls. LV ejection fraction measured by transthoracic echo-
cardiography was significantly greater in the nNOS?/?mice than
in controls (53.5?2.7% in nNOS?/?versus 44.9?1.5% in
heart rate, LV wall thickness, LV mass to body weight ratio, or
aortic blood pressure (Table). Hemodynamic measurements
showed a trend for a load-independent measure of LV contrac-
tility (maximal rate of rise in LV pressure normalized to
instantaneous developed pressure, LV dP/dTmax/IP)16to be
higher in the nNOS?/?animals (P?0.059), and for the ? of LV
isovolumetric relaxation to be slower (Table).
This finding of enhanced LV contraction in the nNOS?/?
mice was confirmed as a single cell phenomenon in isolated
myocyte studies. Cell shortening elicited by a step depolar-
ization to 0 mV was significantly greater in nNOS?/?than in
control myocytes over the physiological voltage range (Fig-
ures 1B and 1C). Qualitatively similar results were obtained
when contraction parameters were assessed at steady state
(average of five 200-ms depolarizing steps to 0 mV from ?40
mV at 1 Hz; Table). In both protocols, time to 50% relaxation
was significantly greater in nNOS?/?myocytes than in
controls. Myocyte length and capacitance did not differ
between groups (data not shown).
nNOS-Dependent Regulation of [Ca2?]i
To investigate whether an increased SR load might contribute
to the enhanced contraction in nNOS?/?myocytes, we mea-
sured the integral of the caffeine-induced NCX current
(which reflects the amount of Ca2?load in the SR).13Both the
amplitude (?1.59?0.6 versus ?1.68?0.1 pA/pF; Figure 2A)
and integral of the NCX current (0.64?0.03 pC/pF in
controls versus 0.78?0.04 pC/pF in nNOS?/?; P?0.05; n?15
and 17, respectively; Figures 2B and 2C) were larger in
nNOS?/?than in controls. The rate of decay of the caffeine-
induced current was similar in the two groups (359.2?12.2
ms in controls versus 386.3?18.9 ms in nNOS?/?), suggest-
ing similar NCX characteristics in both groups. In addition,
the time constant of decline of the caffeine-induced Ca2?
transient in the presence of Ni2?was similar in control and
nNOS?/?myocytes (?, 6.83?1.2 seconds in control versus
5.13?0.7 seconds in nNOS?/?, n?9 in each group; P?0.25),
indicating that there are no significant differences in the slow
mechanisms of Ca2?extrusion between the two groups.
nNOS-mediated regulation of Ca2?handling was con-
firmed in indo-1–loaded myocytes. [Ca2?]i transients had
significantly greater amplitude in nNOS?/?myocytes com-
March 21, 2003
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pared with controls. There was no difference in diastolic
Ca2?, but peak Ca2?was significantly greater (Figure 3A), as
was the delta amplitude of the transient (0.087?0.01 in
nNOS?/?versus 0.063?0.01 in controls; P?0.05). [Ca2?]i
transients recorded using confocal microscopy in fluo-4–
loaded myocytes also had a greater amplitude in nNOS?/?
compared with controls (n?10 and 15, respectively; data not
shown). The time to peak of the Ca2?transients recorded with
either method did not differ between nNOS?/?and control
myocytes (indo-1 myocytes 29.0?0.9 ms in nNOS?/?versus
29.4?1.3 ms in controls; fluo-4 26.8?1.9 ms in nNOS?/?
versus 31.3?2.7 ms in controls; P?NS for both).
A greater SR load and a larger peak [Ca2?]iin nNOS?/?
could reflect tonic inhibition of SR Ca2?-ATPase (SERCA)
activity by nNOS-derived NO, as suggested by Xu et al.8
However, if this were the primary mechanism of action of
nNOS, we would expect to see a faster decay of the [Ca2?]i
transient and faster relaxation in nNOS?/?myocytes. Instead,
TR50was prolonged (Table), and the time constant of decay of
the [Ca2?]itransient was significantly greater in the nNOS?/?
myocytes (102?11 ms versus 148?11 ms; P?0.05; Figure
3B), suggesting that additional mechanisms may be involved.
Calcium Current in nNOS?/?Myocytes
Thus, we investigated whether the increased SR load and
contraction might result from modulation of another impor-
Figure 1. Contractility is enhanced in vivo and in LV myocytes
from nNOS?/?mice. A, Scatter plot to show data for LV ejection
fraction (%) in nNOS?/?(open circles) and control (filled trian-
gles) mice. Ejection fraction was significantly greater in the
nNOS?/?mice (P?0.01, n?10 for both groups). B, Example rec-
ords of unloaded cell shortening (expressed as percent resting
cell length) elicited by a 200-ms depolarizing step from ?40 to 0
mV in control and nNOS?/?myocytes. C, Contraction-voltage
relationship shows percent cell shortening is greater in the
nNOS?/?myocytes (filled squares) than in control myocytes
(open circles) over the voltage range ?30 to ?60 mV (P?0.05,
n?16 and 21, respectively).
Figure 2. SR Ca2?load is enhanced in nNOS?/?myocytes. A,
Example records of currents (in pA/pF) elicited in nNOS?/?and
control myocytes by a 10-second exposure to 10 mmol/L caf-
feine. These currents are carried predominantly via the NCX in
the Ca2?-extrusion mode. B, Integral of the currents in A in
pC/pF. C, Average results of caffeine-induced current integrals,
showing a larger charge and therefore greater SR load in
nNOS?/?myocytes (*P?0.05, n?15 and 17, respectively).
Contraction Data From Controls and nNOS?/?
Parameter Control nNOS?/?
Tau of isovolumetric LV relaxation, s
Systolic blood pressure, mm Hg
Diastolic blood pressure, mm Hg
Heart rate, bpm
Posterior wall thickness, mm
Anterior wall thickness, mm
LV weight : body wt, 10?3
Myocyte cell shortening, % resting length
Myocyte time to 50% relaxation, ms
Max rate of myocyte shortening, ?m/s
Max rate of myocyte relaxation, ?m/s
Sears et alnNOS and Myocardial Contractility
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tant component of E-C coupling, the Ca2?current. We found
that ICa density was significantly greater in the nNOS?/?
myocytes at voltages from ?30 to ?20 mV (Figures 4A and
4B) At 0 mV, ICadensity was ?9.1?0.5 pA/pF in control
myocytes and ?11.4?0.5 pA/pF in nNOS?/?myocytes
(P?0.01). Steady-state activation curves showed that the
voltage at which ICa was half-maximally activated was
?11.2?0.4 mV in controls and ?12.8?0.6 mV in nNOS?/?
myocytes (P?0.04; Figure 4C). The slope of activation was
unaltered (5.3?0.1 mV in controls versus 5.2?0.1 mV in
nNOS?/?). Similarly, the voltage at which ICawas half-inac-
tivated was similar in both groups (?28.5?0.6 versus
?28.7?0.6 mV in nNOS?/?, slopes 4.7?0.1 and 4.4?0.2
mV, respectively; P?NS for both; Figure 4C).
An additional explanation for the increased SR Ca2?load in
nNOS?/?myocytes can be found by looking at the decay
characteristics of ICa. The decay of steady-state ICais best
fitted by a double exponential function, yielding a fast and a
slow time constant (Figure 4D). The fast time constant was
not significantly different between the two groups (6.08?0.3
ms in controls versus 6.8?0.5 ms in nNOS?/?), but the slow
time constant was approximately 38% greater in the nNOS?/?
myocytes (37.3?21.5 versus 26.9?1.6 ms; Figure 4E). The
inward current during slow decay of ICa is thought to
contribute to Ca2?loading of the SR,17and thus its prolon-
gation in nNOS?/?myocytes may underlie their enhanced SR
Ca2?content. Moreover, we found that the steady-state
current at the end of the pulse was more inward (?0.32?0.07
versus ?0.1?0.04 pA/pF; P?0.05) and the integral of ICa
over the whole pulse (an overall measure of Ca2?influx) was
greater in nNOS?/?
myocytes (?0.2?0.01 versus
?0.12?0.01 pC/pF; P?0.05). Both of these findings would
contribute toward enhancing SR Ca2?load.
We also examined whether nNOS disruption and the
resulting increase in the [Ca2?]itransient were associated with
changes in the expression of other Ca2?cycling proteins in the
LV myocardium. We found no differences in the protein level
of the ?-subunit of the L-type Ca2?channel (Figure 3C),
NCX, SERCA, and PMCA (data not shown). However,
expression of both calsequestrin and the ryanodine receptor
Ca2?release channel (RyR) were increased in the nNOS?/?,
whereas phospholamban levels were decreased (Figure 3C).
Does the Increase in ICain nNOS?/?Myocytes
Underlie the Increase in Cell Shortening?
To test whether the enhanced cell shortening in nNOS?/?
myocytes was predominantly a function of the increased Ca2?
influx via ICa, we assessed steady state contraction and ICa
after disabling the SR with thapsigargin. Cell shortening
Figure 3. Calcium transients from nNOS?/?myocytes are larger and slower to decay. A, Average raw data trace showing the indo-1 fluores-
cence ratio (410/495 nm) in control and nNOS?/?myocytes (n?17 and 19, respectively). Transients recorded from nNOS?/?myocytes had
greater peak fluorescence. Using the calibration values obtained, average diastolic [Ca2?]iapproximated to 97 nmol/L in control and 111
nmol/L in nNOS?/?myocytes, and peak [Ca2?]ito 704 nmol/L in control and to 1.3 ?mol/L in nNOS?/?. B, Time course of decay of the Ca2?
transient was significantly slower in the nNOS?/?than in control myocytes (*P?0.05). C, Western blots using antibodies specific to the
?-subunit of the L-type calcium channel (LTCC), cardiac RyR, phospholamban (PLB), and calsequestrin (Cal) in control and nNOS?/?homog-
enate. Average results are from 4 hearts per group and are normalized to the GAPDH signal, where appropriate (LTCC and RyR).
4 Circulation Research
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