Am J Physiol Heart Circ Physiol 306: H291–H297, 2014.
First published November 15, 2013; doi:10.1152/ajpheart.00715.2013.
Rapid Report
Lasting alterations of the sodium current by short-term hyperlipidemia as a
mechanism for initiation of cardiac remodeling
M. Biet,1 N. Morin,1 O. Benrezzak,2 F. Naimi,1 S. Bellanger,1 J. P. Baillargeon,2 L. Chouinard,2
N. Gallo-Payet,2 A. C. Carpentier,2 and R. Dumaine1
1
Department of Physiology and Biophysics, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke,
Sherbrooke, Quebec, Canada; and 2Department of Medecine (Endocrinology), Faculté de Médecine et des Sciences de la
Santé, Université de Sherbrooke, Sherbrooke, Quebec, Canada
Submitted 18 September 2013; accepted in final form 9 November 2013
process ultimately results in a decrease of contractility, slowing
of diastole, ventricular hypertrophy, and in later stages by
systolic dysfunctions that progress to decompensate heart failure (1). The mechanisms underlying lipid-induced cardiac
dysfunction is poorly understood (3), but the observations
suggest that early remodeling of cardiac electrical currents and
changes in regulation of intracellular calcium in cardiomyocyte
are involved.
The cardiac sodium current (INa) is an interesting candidate
to explain both the electrical and calcium disturbances caused
by exposure of the heart to high fatty acid concentration. INa
controls heart excitability and modulates ventricular repolarization (8). Moreover, INa “window current ” regulates intracellular sodium concentration and will directly influence the
rate of exchange of the sodium-calcium exchanger and diastolic relaxation. In this study we tested the hypothesis that
changes in INa are part of the early cardiac remodeling occurring during increased fatty acid delivery to lean tissues and
potentially contribute to the initial increase in cellular calcium.
To characterize the earliest changes in INa remodeling, we
elevated plasma concentration of nonesterified fatty acids
(NEFAs) using intravenous lipid infusion in dog (6, 10), a
model very close to human cardiac electrophysiology. Our
results indicate that an acute (8 h) Intralipid and heparin (IH)
infusion was sufficient to induce long-lasting, electrical remodeling of INa, consistent with an increase in intracellular calcium
and alteration of cardiac excitability.
dog; sodium channels; patch clamp; insulin
METHODS
that cardiac dysfunction ultimately leading to CHF is primarily caused by coronary (ischemic) heart disease (CHD) and hypertension (14). However, in
many cases, such as diabetes, patients remain at increased risk
of heart failure even after adjusting for concomitant risk and in
absence of hypertension or CHD. These observations indicate
that ventricular dysfunction may develop in absence of hemodynamic impairments (11, 31, 32) and suggest that other
mechanisms than cardiovascular dysfunction are involved in
the development of CHF. Impaired dietary fatty acid uptake in
adipose tissues leading to increased cardiac fatty acid uptake is
associated with early myocardial contractile dysfunction (5, 19,
25). In the settings of insulin resistance and diabetes, this
THE CONVENTIONAL WISDOM HOLDS
Address for reprint requests and other correspondence: R. Dumaine, Dept.
de Physiologie et Biophysique, Fac. de Médecine et Sciences de la Santé
(CHUS), Univ. de Sherbrooke, Sherbrooke Qc, J1H 5N4 (e-mail: robert.
[email protected]).
http://www.ajpheart.org
All animal procedures conformed to the Canadian Institutes of
Research, Guide for the Care and Use of Laboratory Animals (No.
036-05), the principles of laboratory animal care (National Institutes
of Health Publication No. 85-23, Revised 1985), and were approved
by the Institutional Animal Ethics Review Committee of the University of Sherbrooke.
Animal preparation. Female mongrel dogs weighing between 25
and 35 kg and at least 1 yr old were used in this study. Dogs were
neither pregnant nor lactating. Only dogs considered healthy after
physical examination, cardiopulmonary auscultation, blood analysis,
and standard biochemistry profiling were used. The animals were
housed in individual kennels (4 ⫻ 3 m) under controlled conditions of
temperature (21°C) and photoperiod (12-h:12-h light-dark cycles) and
supervised by a veterinarian.
Dogs were fed the Purina dog chow real chicken Pro-plan at the
same time once a day for a period of 2 to 3 wk before any experiments
to ensure weight stabilization. Purina Pro-plan (Nestlé, Mississauga,
Ontario, Canada) is a standard diet comparable with other previously
reported (17). The Pro-plan was served according the National Research Council recommendation for canine maintenance. Dogs had
free access to water.
0363-6135/14 Copyright © 2014 the American Physiological Society
H291
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Biet M, Morin N, Benrezzak O, Naimi F, Bellanger S, Baillargeon JP, Chouinard L, Gallo-Payet N, Carpentier AC, Dumaine
R. Lasting alterations of the sodium current by short-term hyperlipidemia as a mechanism for initiation of cardiac remodeling. Am J
Physiol Heart Circ Physiol 306: H291–H297, 2014. First published
November 15, 2013; doi:10.1152/ajpheart.00715.2013.—Clinical and
animal studies indicate that increased fatty acid delivery to lean
tissues induces cardiac electrical remodeling and alterations of cellular calcium homeostasis. Since this may represent a mechanism
initiating cardiac dysfunction during establishment of insulin resistance and diabetes or anaerobic cardiac metabolism (ischemia), we
sought to determine if short-term exposure to high plasma concentration of fatty acid in vivo was sufficient to alter the cardiac sodium
current (INa) in dog ventricular myocytes. Our results show that
delivery of triglycerides and nonesterified fatty acids by infusion of
Intralipid ⫹ heparin (IH) for 8 h increased the amplitude of INa by
43% and shifted its activation threshold by ⫺5 mV, closer to the
resting membrane potential. Steady-state inactivation (availability) of
the channels was reduced by IH with no changes in recovery from
inactivation. As a consequence, INa “window” current, a strong
determinant of intracellular Na⫹ and Ca2⫹ concentrations, was significantly increased. The results indicate that increased circulating
fatty acids alter INa gating in manners consistent with an increased
cardiac excitability and augmentation of intracellular calcium. Moreover, these changes could still be measured after the dogs were left to
recover for 12 h after IH perfusion, suggesting lasting changes in INa.
Our results indicate that fatty acids rapidly induce cardiac remodeling
and suggest that this process may be involved in the development of
cardiac dysfunctions associated to insulin resistance and diabetes.
Rapid Report
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ALTERATIONS OF INa BY HYPERLIPIDEMIA
check the position of implanted vascular catheters and to collect
organs and tissues.
Myocytes were obtained by enzymatic dissociation as previously
described (9). Briefly, a left ventricular wedge was cut and perfused at
35°C through a coronary artery for 10 min with Ca-free Tyrode
solution, supplemented with 2 mmol/l EGTA and 0.1% BSA. Perfusion was switched to Tyrode solution containing 0.1 mM Ca and 230
U/ml collagenase (CLS 2, Worthington, Freehold, NJ) and recirculated for 10 –20 min until the tissue became discolored and mushy.
The wedge was then removed and minced, and tissues were gently
stirred in beakers containing the enzymatic solution. The supernatant
containing dissociated cells was kept in 10-ml tubes and stored in
Krebs solution containing (in mmol/l) 100 potassium glutamate, 10
potassium aspartate, 25 KCl, 10 KH2PO4, 2 MgSO4, 20 taurine, 5
creatine, 0.5 EGTA, 20 glucose, 10 HEPES, and 2% BSA, supplemented with 0.2 mM CaCl2.
Electrophysiology. Dissociated myocytes were placed in a chamber
mounted on the stage of an inverted microscope (Nikon Diaphot,
Tokyo, Japan) and superfused with solution containing (in mmol/l)
120 choline-Cl, 10 NaCl, 5 NaOH, 2.8 Na acetate, 4 KOH, 0.5 CaCl2,
1.5 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). Tetraethyl ammonium (5 mmol/l), CoCl2 (1 mmol/l), and BaCl2 (5 mmol/l)
were used to block transient outward (Ito), L-type calcium (ICaL), and
inward rectifier (IK1) currents, respectively. Membrane currents were
measured in the whole cell configuration of the patch-clamp technique
as previously described (13). All recordings were obtained at room
temperature (22°C) using an Axopatch 200B amplifier (Axon Instruments, Union City, CA), equipped with a CV-201A head stage (Axon
Instruments, Foster City, CA). Patch pipette had electrical resistance
between 1 and 3 M⍀ when filled with a solution containing (in
mnol/l) 15 NaCl, 5 KCl, 120 CsF, 1.0 MgCl2, 4 Na2-ATP, 10 EGTA,
and 10 HEPES (pH 7.2 with CsOH). All the solutions were adjusted
at 300 mosmol/l with sucrose. Currents were filtered at 5 kHz and
digitized at 10 kHz. Data acquisition and analysis were performed
using pCLAMP programs V9.2 (Axon Instruments), EXCEL (Microsoft), and ORIGIN 7.0 (Microcal Software, Northampton, MA)
softwares. Whole cell capacitance and series resistance compensation
(85%) were optimized to minimize the capacitive artifact and reduce
voltage errors. Sodium window current was calculated using the
classical Hodgkins and Huxley model based on the overlap of the
steady-state inactivation and activation curves using the equation
INa ⫽ GNa·m3·h·(Vm ⫺ ENa) where GNa, m, h, Vm and ENa represent
the maximum conductance, the fraction of current activated and
inactivated, the membrane potential, and the sodium reversal potential
respectively, as described in Figs. 3–5.
Statistical analysis. Data are expressed as means ⫾ SE. Comparison between saline-infused dogs and IH-treated dogs was performed
using a two-way ANOVA.
RESULTS
Infusion of IH (0.5 U·kg⫺1·min⫺1) for 8 h significantly
increased the levels of triglycerides and NEFAs for the whole
duration of the infusion and resumed to normal levels thereafter (Fig. 1A). Corticotrophin (adrenocorticotropic hormone)
and cortisol were measured during and after IH treatment to
eliminate the possibility of stress-related artefacts in our measurements. Figure 1B shows that plasma concentrations of both
hormones remained stable and comparable between sham- and
IH-treated animals. Moreover, the increase in circulating NEFAs
during infusion did not change the levels of insulin or glucose
neither during IH nor infusion in the following postprandial period
(Fig. 1C), indicating that our treatment did not induce diabetes in
these animals.
Chronic exposure to high serum concentrations of lipids
could lead to onset of type 2 diabetes and eventually triggers
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Surgical procedures. Presurgical setup, anesthesia, and surgical
procedures were conducted under aseptic conditions to prevent infections. Dogs were fasted 16 h before the beginning of surgery. On the
day of the surgery, dogs were premedicated with subcutaneous
acepromazine maleate (0.1 mg/kg), as a sedative to reduce apprehension and atropine sulfate (0.02 mg/kg) as an anticholinergic agent used
to prevent throat secretions. General anesthesia was induced with
thiopental sodium (15 mg/kg iv) and maintained throughout surgery
with 1–2.5% isoflurane and pure oxygen. Arterial O2 saturation from
pulse oximetry, heart rate, and ECG were monitored during anesthesia. Fluid hydration was maintained with Ringer lactate solution (30
drops/min). Once dogs were anesthetized, they were placed on the
heated table (37°C), and the anterior portion of the neck was opened
by median incision (7 cm), and conjunctive, adipose, and muscular
tissues were dissected. Two premounted PRN catheters (Silastic
catheter) were prepared. PRN is an injection port adapter (BD luer-lok
adapter with short 0.75-in. offset flow path) that serves as line of
access to vascular catheters kept internalized under the skin. One
sterile Silastic catheter (0.03-in. inner diameter ⫻ 0.065-in. outer
diameter, Down Corning, Midland, MIID) was inserted into the
isolated jugular vein and advanced up to the right atrium for continuous peripheral infusions (33). In addition, one sterile Silastic catheter
(0.04-in. inner diamter ⫻ 0.085-in. outer diameter) was inserted into
the left carotid artery and advanced into the aortic arch for arterial
sampling. After completion of surgery, dogs were equipped with an
Elizabethan collar and an adapted jacket (Lomir, Canada) to protect
catheters and PRN from any damages. Flushes with saline and heparin
solutions were performed twice a day until the day of use for blood
collection.
Buprenorphine (0.02 mg/kg sc) and Longisil antibiotics (penicillin
10,000 U/lbs im) were given immediately after surgery to prevent pain
and septicemia. Local infection at the sites of catheters implantation
was controlled by Flamazine applied every day topically. All dogs
completely recovered within 7 days as established from full return to
normal values of renal, hematology and metabolic parameters.
After surgery (7–10 days), dogs (control and experimental groups,
respectively) received saline or Intralipid as 20% triglyceride emulsion (0.02 ml·kg⫺1·min⫺1) plus heparin (0.5 U·kg⫺1·min⫺1) for 8 h
(from time ⫺510 min to ⫺30 min). The latter was given to stimulate
lipoprotein lipase activity to hydrolyze the triglycerides that are
infused. At time 0, dogs were fed with an isocaloric meal. During the
three experimental periods [saline or Intralipid infusion (⫺510 to ⫺30
min), basal period (⫺30 to 0 min), and the postprandial period (0 –540
min)], blood was collected and plasma samples were prepared for
biochemistry and metabolic analysis. The total volume of blood
withdrawn did not exceed 20% of total blood volume of the animal.
Plasma cortisol and adrenocorticotropic hormone concentrations
were determined using a human antibody (MP, Biomedicals)-based
radioimmunoassay method and a commercial RIA kit (Immunocorp,
MP, Biomedicals), respectively, adapted for dog. Total NEFAs in
plasma were quantified using a commercially available colorimetric
assay (NEFA C kit; Wako Chemicals). Plasma triglyceride concentration was determined using a colorimetric assay (Trig/GB, Boehringer Mannheim/Roche Diagnostics).
Samples for determination of glucose and insulin were collected
into tubes containing Na2 EDTA and trazylol (7,700 KIU/ml, calbiochem) to inhibit proteolysis. Plasma glucose concentration was determined by the glucose oxidase method (540 nm). Plasma insulin was
measured using a specific canine insulin enzyme-linked immunoassay
kit (Cedarlane, Burlington, CA).
Heart excision and cardiomyocyte dissociation. Immediately after
the last blood sampling (⬃12 h after IH treatment), animals were
sedated with a mix of Atravet (0.25 mg/kg im) and heparin (5,000 U)
for 30 min to avoid blood coagulation and then anesthetized with
pentobarbital sodium (25 mg/kg iv). The beating heart was quickly
removed by an incision at the fifth intercostal space causing the
euthanasia of the animal. Necropsy was systematically performed to
Rapid Report
H293
ALTERATIONS OF INa BY HYPERLIPIDEMIA
Sham (n= 10)
0
60
Sham (Saline; n=11)
IH (n= 7)
*
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
180
300
420
Plasma Triglycerides
(mg/ml)
Plasma NEFA (nmol/l)
A
IH (n=8)
2.5
*
2.0
1.5
1.0
0.5
0.0
0
60
Infusion Time (min)
180
300
420
Infusion Time (min)
4
3
Cortisol (ng/ml)
Sham (n=6)
IH (n=5)
2
1
0
0
60
180
300
80
40
20
0
420
0
Infusion Time (min)
Sham (n=10)
60
180
300
420
Infusion Time (min)
10
IH (n=7)
3.2
Plasma Glucose (mM)
Plasma Insulin (ng/ml)
C
Sham (n=9)
IH (n=5)
60
2.4
1.6
0.8
0.0
Sham (n=10)
IH (n=7)
8
6
4
2
0
-600 -400 -200
0
200 400 600
-600 -400 -200
0
Infusion (min)
Postprandial (min)
Infusion (min)
Postprandial (min)
remodeling of the heart, leading to diabetic cardiomyopathy.
Polyunsaturated fatty acids applied in vitro are known to alter
the kinetic and to reduce expression of the cardiac INa heterologously expressed in HEK cells and in isolated cardiomyocytes (2, 16, 21, 22, 30, 34 –36). We therefore tested whether
early electrophysiological remodeling occurs following IH treatment by measuring the amplitude of the cardiac INa. Figure 2A
shows that IH infusion slightly increased the amplitude of the
maximum peak current from ⫺41.4 ⫾ 1.9 to ⫺58.4 ⫾ 7.4
pA/pF. Current-voltage analysis of the recordings showed a
shift from ⫺30 to ⫺35 mV of the maximum current voltage
(Fig. 2B). The threshold for activation of the current was
similarly shifted by ⫺5 mV, indicating changes in the voltage
dependence of activation of the channels. Interestingly, maximum amplitude remained larger in IH-treated cells for the 3
days, during which we were able to keep dissociated myocytes
alive and reliably measure INa (Fig. 2C). Analysis of INa
activation revealed that IH treatment shifted the voltage dependence of opening of the channels by ⫺10 mV (Fig. 2D).
Maximum conductance obtained as the slope of the linear
portion of the I–V relationship was similar in sham- and
IH-treated animals (Fig. 2E), thus indicating that the lipid
200 400 600
infusion mainly affected the gating of the current rather than
expression of new channels. To determine if a change in the
availability of the sodium channels participates to the voltage
shift in maximum current, we next assessed INa voltage dependence of inactivation. Fig. 3, A and B, shows that IH treatment
induced a small, albeit significant, ⫺5 mV shift in midinactivation potential, indicating that availability of the channels will
be more importantly reduced at depolarized potentials in IHtreated animals. This effect is not consistent with the increased
INa observed on the I–V relationship (Fig. 2B) and indicates
that the increase in maximal INa amplitude is mostly due to a
change in channels activation gating.
The cardiac refractory period is highly dependent on recovery of sodium channels from their inactivated state and is
involved in many types of arrhythmias such as reentry. To test
for changes in recovery from inactivation, we used the doublepulse protocol shown in Fig. 3C. Statistical analysis (F-test) of
the fit to data (Fig. 3D) did not reveal a significant difference
in the kinetic of reactivation of the channels.
The overlap of the activation and inactivation curves creates
semistable conditions where a fraction of sodium channels
remains in transition between the inactivated (closed) and
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Plasma ACTH (ng/ml)
B
Fig. 1. Monitoring of plasma hormones and free
fatty acids concentrations. A: nonesterified fatty
acids (NEFAs) and triglyceride plasma concentrations were significantly increased in Intralipids-heparin (IH)-treated dogs compared
with sham-infused animals during the 8-h IH
treatment. *P ⬍ 0.05; data are means ⫾ SE.
B: plasma concentrations of stress hormones
adrenocorticotropic hormone (ACTH) and
cortisol remained similar in saline- and IHinfused dogs. Corticotropin (ACTH) level
was not altered during the 8 h of IH perfusion
and thereafter. Cortisol levels tend to be
slightly higher (not significant) at the onset on
the treatment (first 2 h) but were similar and
remained stable in both conditions during the
subsequent 8 h of perfusion. C: plasma levels of
insulin (left) and glucose (right) during infusion
(⫺540 to 0 min) of saline (sham) or IH and the
following 8-h postprandial period (0 to 540
min) without infusion. Data are means ⫾ SE.
Rapid Report
H294
ALTERATIONS OF INa BY HYPERLIPIDEMIA
A
30 ms
-30 mV
-120 mV
0
Sham
IH
2 nA
2 ms
C
Membrane potential (mV)
-40
-20
70
*
60
-20
-40
I NA, Max (pA/pF)
-60
50
40
30
20
10
-60
0
IH
Sham
IH
IH
Sham
D
E
1.0
2.5
0.8
2.0
G NA, Max (pS/pF)
GNa/GNa,Max
Sham
0.6
0.4
0.2
1.5
1.0
0.5
0.0
0.0
-60
-40
-20
0
Membrane potential (mV)
activated (open) states and vice versa. As a consequence, a
very small but persistent inward “window” current (IW) exists
in the voltage range of this overlap. IW thus contributes to
establish the resting membrane potential and modulates the
sodium gradient between the intra- and extracellular milieus.
Figure 4 shows that IH infusion shifted the voltage range where
IW is active closer to the normal myocytes resting membrane
potential (-80 mV) and increased its maximum amplitude.
DISCUSSION
We demonstrated that IH infusion increases plasma free
fatty acids and triglycerides concentration without inducing
hyperinsulinemia, hyperglycemia, or changes in circulating
stress hormones levels. These results confirm that the alterations of INa are linked to alterations of the lipid content and/or
delivery to the heart. We found that IH increased the maximal
amplitude of INa by 41% and shifted the voltage dependence of
activation of the channels toward more negative potentials.
Physiologically, a negative shift in INa activation lowers the
voltage threshold for activation of sodium channels and is a
well-known mechanism to increase cardiac excitability that
will be further enhanced by the increase in INa amplitude. Our
results also indicate that IH infusion had smaller effects on
inactivation of the INa that could not account for the observed
changes in the I–V relationship.
Alterations of the lipid content in cardiomyocyte plasma
membrane are known to modulate the gating of INa. In rat
cultured cardiomyocytes and cardiac muscle, eicosapentaenoic
acids, docosahexahenoic acids, and other polyunsaturated fatty
acids (PUFAs) increased the threshold for action potential
firing (67, 68), had inhibitory effect on INa, and enhanced the
kinetic of inactivation (69, 66). Similar effects were also
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B
INa (pA/pF)
Fig. 2. Increased plasma concentration of free fatty
acids shifted the activation of the cardiac sodium
current (INa) toward more negative potentials. A: protocol (inset) and representative current recordings
from left ventricular cardiomyocytes of saline(sham) and IH-infused dogs. B: current-voltage relationship (I–V) of INa and corresponding maximum
current amplitude (C). I–V relationship was obtained from measurements of INa elicited by a 30-ms
pulse to indicated voltage from a holding potential
of ⫺120 mV. Peak INa was normalized to the
capacitance of their respective cells. Current densities (in pA/pF) were plotted against their respective
test potential in saline (n ⫽ 11) and IH (n ⫽ 11)
conditions. D: conductance was calculated as the
ratio INa/(Vm ⫺ ENa), where Vm is membrane potential and ENa is sodium reversal potential, and
normalized to maximum conductance (GNa; slope of
the linear portion of the I–V relationship) to obtain
the fraction of channels activated at each voltage.
ENa represents the zero current membrane potential.
Data were fitted to a standard Boltzmann distribution function with midactivation potentials (V½) of
⫺44.4 ⫾ 0.2 and ⫺33.7 ⫾ 0.2 mV, respectively, for
the IH- and sham-infused dogs. (P ⬍ 0.05, F-test).
E: maximum conductance was not significantly different between the two conditions (2.0 ⫾ 0.2 and
2.2 ⫾ 0.1 pS/pF for sham and IH conditions, respectively). *P ⬍ 0.05 vs. sham.
Rapid Report
H295
ALTERATIONS OF INa BY HYPERLIPIDEMIA
A
-10 mV
500 ms
B
Sham
IH
1.0
-120 mV
INa / INa,Max
0.8
0
0.6
0.4
0.2
0.0
-100
INa,Max
-80
-60
-40
5 nA
4 ms
D
C
S1
S2
-20 mV
IH
Sham
1.0
-120 mV
Δt (ms)
IS2 / IS1
0.8
0
0.6
0.4
0.2
0.0
5 nA
50 ms
0
20
40
60
80 120 140
Δt (ms)
observed in NaV1.5 channels expressed in HEK cells (71, 65,
72, 73). Part of these results may be explained by changes in
the fluidity of the plasma membrane surrounding the sodium
channels (66). Such hypothesis is consistent with specific
alterations of the activation gating of the channel since the
structures involved in activation are transmembrane segments
imbedded in the plasma membrane, whereas the inactivation
gate of the channel is in the hydrophilic intracellular milieu.
However, the voltage shift in INa gating linked to changes in
membrane fluidity are opposite to the effects of in vivo exposure to IH we report here. While species differences might be
involved, it is important to note that in previous in vitro
experiments, myocytes were exposed for short period of time
(10 to 50 min) to lipids. Therefore, only acute effects of
exposure to fatty acids were measured. It is also possible that
other in vivo adaptive mechanisms may contribute to modulate
myocardial INa. The opposite results in our in vivo experiments
suggest that other modulatory mechanisms, possibly related to
by-products of ␤-oxidation are activated over the course of 8 h.
Surprisingly, short-term IH infusion was sufficient to induce
chronic changes in INa gating that could still be recorded 12 h
after the dogs were left to recover from treatment. Such early
electrophysiological remodeling of the cardiac myocytes is a
new finding. Our results therefore indicate that circulating
NEFAs can remodel the heart within a time frame as short as
a few hours and well before the onset of insulin resistance or
detection of type 2 diabetes.
Another interesting finding is the negative voltage shift in
sodium window current caused by IH infusion. The consequences of this are twofold. First, it will increase the inward
(leak) sodium current close to ⫺80 mV, thus causing a depolarization of the normal resting membrane potential. Combined
with our observation of a negative shift in activation and
augmented INa amplitude, this will increase cardiac excitability. Second, the augmentation of the window current near the
resting membrane potential of the cell will increase the sodium
influx and the intracellular sodium concentration, thereby reducing the sodium gradient. The sodium gradient between the
intra- and extracellular compartments plays a key role in
regulating calcium homeostasis by directly regulating the turnover of the sodium-calcium exchanger. A reduction in the
sodium gradient may therefore translate into a diminished
extrusion of intracellular calcium. This mechanism is well
characterized and contributes to the calcium overload observed
during hypertension, cardiac hypertrophy, and heart failure (7,
15, 18, 24, 27).
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Membrane potential (mV)
Fig. 3. Short-term increase in plasma free fatty
acids shifts the availability of sodium channels
toward more negative potentials. A: representative current recordings at a test pulse of ⫺10 mV
following a series of conditioning pulses (⫺140
to ⫹40 mV) in increments of 5 mV from a
holding potential of ⫺120 mV (inset). B: inactivation curves (availability) were obtained by
plotting the ratio of INa to its maximum value
against the conditioning pulse voltage. Data were
fitted against a Boltzmann distribution function
and yielded mid-inactivation potentials (V½) of
⫺66.7 ⫾ 0.4 and ⫺70.8 ⫾ 0.2 mV for sham (n ⫽
8) and IH (n ⫽ 14) conditions, respectively (P ⬍
0.05). C: recovery from inactivation is not
changed by short-term exposure to increased
level of NEFAs. Representative INa recordings
during application of a standard electrophysiological double pulse protocol (S1-S2, 20 ms) to
measure recovery of INa from inactivation (inset).
D: recovery from inactivation expressed as the
fraction of the initial current (IS1) elicited during
the second pulse (IS2) and plotted against the
interpulse interval duration (⌬t) in sham (n ⫽ 8)
and IH (n ⫽ 14) conditions. Data are presented as
means ⫾ SE and were fitted to a two exponential
distribution function (solid line).
Rapid Report
H296
ALTERATIONS OF INa BY HYPERLIPIDEMIA
IH
Sham
0.08
0.08
0.06
0.06
0.04
0.04
0.02
0.02
G / G Max
I / I Max
A
0.00
0.00
-70
-60
-50
-40
-30
-20
Membrane Potential (mV)
B
Membrane Potential (mV)
0.0
-80
-70
-60
-50
-40
-30
-20
I W (pA/pF)
-0.1
-0.2
-0.3
-0.4
-0.5
Fig. 4. Intralipid infusion increased the sodium window current. A: overlapping
area between the activation (G/GMax) and inactivation (I/IMax) curves in shamand IH-infused animals shown in Figs. 2D and 3B. B: corresponding window
current (IW) calculated from data in A using standard Hodgkin and Huxley
formalism, as described in METHODS.
During cardiac ischemia, the metabolism of the myocardium
becomes anaerobic and free fatty acids accumulate. In severe
cases, the release of catecholamine induced by ischemia accentuates the release of fatty acids from adipose tissues. Clinical observations show that elevated plasma concentration of
free fatty acids is associated with an increased incidence of
arrhythmias during myocardial infarction, whereas restoring
glycolysis to the myocardium has a protective effect (28). Our
results showing that fatty acids lower the voltage threshold for
activation of INa and increased its window current suggest that
an increase in heart excitability and a rise in intracellular
sodium are part of the pathophysiological mechanisms, leading
to the increase incidence of arrhythmias.
Support for modulation of cardiac electrical currents by free
fatty acids as a cause of arrhythmias also comes from studies
of other cardiac ion channels. Among them, the ultrarapid
potassium current (IKur), the delayed rectifier [IK (IKr and IKs)],
and the ICaL (26, 37). IKur contributes primarily to repolarization in the atrium, and its role in the ventricle seems minimal.
Therefore, its inhibition by PUFAs is likely to increase action
potential duration and refractoriness in atria. Ventricular repolarization on the other hand is initiated by activation of IKr and
ACKNOWLEDGMENTS
We thank Jean Philippe Gagné for technical contribution to the surgical
procedures.
GRANTS
This work was funded by grants from the Canadian Institute of Health and
the Heart and Stroke Foundation of Canada (to R. Dumaine).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00715.2013 • www.ajpheart.org
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-80
IKs, which will to a large extent determine the duration of
ventricular action potential (APD). Fish oil supplementation,
however, showed no effect of the dietary supplement on IKr or
IKs but did increase the amplitude of ICaL and the Ito (37).
Inhibition of IKr and IKs or a combined increase in ICaL and a
decrease of Ito by PUFAs can prolong APD and the QT interval
on the electrocardiogram ECG, a known index for cardiac
arrhythmias.
Increased plasma free fatty acid concentration is well known
to decrease insulin secretion by pancreatic ␤-cells and considered a risk factor to develop insulin resistance and ultimately
type 2 diabetes. Interestingly, most studies in nongenetically
altered diabetic mice report an increase in intracellular calcium
levels without significant changes in the amplitude of ICaL (4,
20, 23, 29). These findings argue against a role for ICaL in the
increase in intracellular calcium observed in type 2 diabetes.
Our results on the other hand provide evidence that alteration
of INa may significantly contribute to the process. Studies have
demonstrated that diabetic hearts are characterized by an increase in cellular calcium, ultimately leading to heart failure.
Alterations in the activity of the sarcoplasmic reticulum Ca2⫹
ATPase pump were proposed as a potential mechanism to
explain that cardiompathies once type 1 and 2 diabetes is well
established (12, 38, 39). However, these studies did not provide
an initiation mechanism leading to impaired Ca2⫹ handling.
Our results suggest a new paradigm by which changes in INa
gating modulate the sodium gradient to alter calcium homeostasis as plasma free fatty acids increase and insulin resistance
develops. We further demonstrate that cardiac adaptation to
elevated plasma NEFAs occurs within a relatively short period.
In this context, a few repeated exposures to high-fat plasma
concentrations might be sufficient to chronically remodel the
heart. It is therefore tempting to speculate that cardiac remodeling is triggered by free fatty acids well before insulin resistance and type 2 diabetes can be clinically detected.
In summary, we show that 8-h infusion of PUFAs in dog
increased INa amplitude by 43%, lower its voltage threshold for
activation, and increase its window current. These findings are
consistent with an increased excitability and intracellular calcium concentration that potentially contribute to ventricular
arrhythmias associated to an increase in plasma free fatty acid
concentration such as the ones observed during ischemia or
diabetes. However, our animal model, while suited to study the
effects of acute in vivo exposure to high plasma levels of free
fatty acids, does not take into account the chronic hyperlipidemia and hyperglycemia that concomitantly develop during
diabetes. Therefore, further studies are warranted to determine
if chronic exposure of dogs to fatty acids will result in the
development of diabetic cardiomyopathies and if this is accompanied by changes in INa gating.
Rapid Report
ALTERATIONS OF INa BY HYPERLIPIDEMIA
AUTHOR CONTRIBUTIONS
M.B., N.M., O.B., F.N., S.B., and L.C. performed experiments; M.B., O.B.,
F.N., J.-P.B., L.C., N.G.-P., A.C.C., and R.D. analyzed data; M.B., N.M., and
R.D. prepared figures; M.B., N.M., O.B., F.N., S.B., J.-P.B., L.C., N.G.-P.,
A.C.C., and R.D. edited and revised manuscript; M.B., N.M., O.B., F.N., S.B.,
J.-P.B., L.C., N.G.-P., A.C.C., and R.D. approved final version of manuscript;
O.B., J.-P.B., N.G.-P., A.C.C., and R.D. conception and design of research;
J.-P.B., N.G.-P., A.C.C., and R.D. interpreted results of experiments; R.D.
drafted manuscript.
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