Effect of various diets prescribed in The Indian System of Medicine on the resting potential of cells

Abstract

Our work is primarily to prove that cells of the same tissue would exist with different resting potentials depending on the dietary intake as prescribed in the Indian system of Medicine. We had used the action potentials in the heart muscle as a reference parameter for this purpose. We had used three groups, each group containing 15 in numbers, of Male Wistar rats, which were respectively fed with diets as prescribed for increasing Vata, Pitta and Kapha explained in the Indian system of medicine. We used two control group of rats. All the five groups were fed with balanced diet for a period of three weeks. One group of control rats was sacrificed and we measured action potentials from the intact heart. The action potentials showed an average resting potential of about - 84.5 mV. The second control group of rats was continued with the same diet. The three experimental rats were then fed with vata, pitta and kapha enhancing diets. At the end of another three weeks all the rats in all groups were sacrificed and action potentials were measured from their intact hearts. Intracellular calcium handling plays an important role in cardiac electrophysiology. Using two fluorescent indicators, we developed an optical mapping system that is capable of measuring calcium transients and action potentials at 256 recording sites simultaneously from the intact rat heart. On the basis of in vitro measurements of dye excitation and emission spectra, excitation and emission filters at 515 ± 5 and >695 nm, respectively, were used to measure action potentials with di-4- ANEPPS, and excitation and emission filters at 365 ± 25 and 485 ± 5 nm, respectively, were used to measure calcium transients with indo 1. The percent error due to spectral overlap was small when action potentials were measured (1.7 ± 1.0%, n = 3) and negligible when calcium transients were measured (0%, n = 3). Recordings of calcium transients, action potentials, and isochrone maps of depolarization time and the time of calcium transient onset indicated negligible error due to fluorescence emission overlap. These data demonstrate that the error due to spectral overlap of indo 1 and di-4-ANEPPS is sufficiently small, such that optical mapping techniques can be used to measure calcium transients and action potentials simultaneously in the intact heart. The values of Action potentials in the second control group did not vary much with those in the first control group. The values of Action potentials in the vata food fed rats where very close to the resting potential. Those, fed with Pitta enhancing diet varied between -86.5mV and - 93.5mV. Anil those fed with Kapha enhancing diet had varied between -105.28mV and - 112.28mV. These findings in our view are very important to classify cells based on their resting potentials. The findings prove that the more the resting potential, the lesser the external stimulus needed to excite and generate an Action potential which gives a greater understanding of arrhythmias in cardiac muscles and various nervous and other disorders.

Full text

Russian Journal of Physiology – Heart and Circulatory Physiology 280: H2053-H2060, 2001;
0363-6135/01
Vol. 280, Issue 5, December 2002
Effect of various diets prescribed in The Indian System
of Medicine on the resting potential of cells.
DR. Ravishankar Polisetty and Dr. Nikolskiy Peter Vladislavovich.
The Bakulev Centre for Cardiac Surgery, Russian Medical Academy, Moscow, Russian Federation.
ABSTRACT
Our work is primarily to prove that cells of the same tissue would exist with different
resting potentials depending on the dietary intake as prescribed in the Indian system of
Medicine. We had used the action potentials in the heart muscle as a reference parameter
for this purpose. We had used three groups, each group containing 15 in numbers, of
Male Wistar rats, which were respectively fed with diets as prescribed for increasing
Vata, Pitta and Kapha explained in the Indian system of medicine. We used two control
group of rats. All the five groups were fed with balanced diet for a period of three weeks.
One group of control rats was sacrificed and we measured action potentials from the
intact heart. The action potentials showed an average resting potential of about – 84.5
mV. The second control group of rats was continued with the same diet. The three
experimental rats were then fed with vata, pitta and kapha enhancing diets. At the end of
another three weeks all the rats in all groups were sacrificed and action potentials were
measured from their intact hearts. Intracellular calcium handling plays an important role
in cardiac electrophysiology. Using two fluorescent indicators, we
developed an optical
mapping system that is capable of measuring
calcium transients and action potentials at
256 recording sites
simultaneously from the intact rat heart. On the basis
of in vitro
measurements of dye excitation and emission spectra,
excitation and emission filters at
515 ± 5 and >695 nm, respectively,
were used to measure action potentials with di-4-
ANEPPS, and excitation
and emission filters at 365 ± 25 and 485 ± 5 nm, respectively,
were used to measure calcium transients with indo 1. The percent
error due to spectral
overlap was small when action potentials
were measured (1.7 ± 1.0%, n = 3) and
negligible when calcium
transients were measured (0%, n = 3). Recordings of calcium
transients,
action potentials, and isochrone maps of depolarization time and
the time of
calcium transient onset indicated negligible error
due to fluorescence emission overlap.
These data demonstrate that
the error due to spectral overlap of indo 1 and di-4-ANEPPS
is
sufficiently small, such that optical mapping techniques can be
used to measure calcium
transients and action potentials simultaneously
in the intact
heart. The values of Action
potentials in the second control group did not vary much with those in the first control
group. The values of Action potentials in the vata food fed rats where very close to the
resting potential. Those, fed with Pitta enhancing diet varied between -86.5mV and -
93.5mV. Anil those fed with Kapha enhancing diet had varied between -105.28mV and -
112.28mV. These findings in our view are very important to classify cells based on their
resting potentials. The findings prove that the more the resting potential, the lesser the
external stimulus needed to excite and generate an Action potential which gives a greater
understanding of arrhythmias in cardiac muscles and various nervous and other disorders.

intracellular calcium; electrophysiology; optical mapping; di-4-ANEPPS; indo 1, Vata,
Pitta, Kapha
INTRODUCTION
INTRACELLULAR CALCIUM is an important ion that has many direct effects on the
electrophysiology of the heart. For example,
the effects of intracellular calcium on
membrane calcium channels,
nonselective cation channels, and exchangers can
significantly
influence transmembrane potential (2). Furthermore, there is
considerable
evidence that abnormal intracellular calcium handling
plays an important role in
arrhythmias associated with electrical
alternans (17) and heart failure (29, 37) and during
the
initiation of ventricular fibrillation (26). More recently,
data from isolated myocytes
suggest regional heterogeneities of
intracellular calcium handling associated with heart
failure (15,
24). Therefore, to better understand the mechanistic relationship
between
intracellular calcium handling and arrhythmogenesis, in various diets, a
method for
mapping calcium transients and action potentials simultaneously
from the intact heart is
essential.
Fluorescent indicators of intracellular calcium (12) have been used extensively to
measure calcium transients and absolute
intracellular calcium at the level of the intact
heart (3,
16, 21) and single cell (1). Likewise, voltage-sensitive
fluorescent indicators
have been used to map action potentials
from the intact heart (27) and have been used
extensively to
study cellular mechanisms of arrhythmias (10, 11, 38). Because
the
excitation and emission wavelengths of fluorescent indicators
vary from ultraviolet to
near infrared, it is possible to measure
more than one cellular parameter by using multiple
indicators.
To do so, the spectral overlap must be minimal, such that fluorescence
of one
indicator does not significantly overlap with that of the
other. We developed an optical
mapping system to measure with
high resolution intracellular calcium transients and
action potentials
simultaneously from the intact heart. We demonstrate that there
is
negligible error caused by spectral overlap of indo 1 and di-4-ANEPPS
and that optical
mapping techniques can be used to measure calcium
transients and action potentials
simultaneously with high
resolution.
METHODS
Experiments were carried out in accordance with RF (Russian Federation) Public Health
Service (under the Ministry of Health and Social Dvelopment) guidelines for the care and
use of laboratory animals.
Male wistar rats (n = 15, 120 – 150g) in the first control group
were fed with balanced diet as prescribed by the Ethical committee of the Moscow State
University for about three weeks and then were anesthetized
with pentobarbital sodium
(30 mg/kg ip), and their hearts were
rapidly excised and perfused by an aortic cannula as
Langendorff
preparations with oxygenated (95% O
2
-5% CO
2
) Tyrode solution containing
(mM) 121.7 NaCl, 25.0 NaHCO
3
, 2.74 MgSO
4
, 4.81 KCl, 5.0 dextrose,
and 2.5 CaCl
2
(pH 7.40, 32°C). Perfusion pressure was maintained
at 60-70 mmHg by regulating
coronary perfusion flow with a digital
dual-head roller pump. Hearts were stained by
direct coronary
perfusion for ~10 min with the voltage-sensitive indicator di-4-ANEPPS

(Molecular Probes, Eugene, OR) dissolved in 0.19 ml of ethanol
at a final concentration
of 15 µM and for ~30 min with the calcium-sensitive
indicator indo 1-AM (Molecular
Probes) dissolved in a 0.5-ml solution
of DMSO and Pluronic (20% wt/vol) at a final
concentration of
5 µM. In all experiments, 2,3-butanedione monoxime (10 mM) was
used
to ensure that motion artifact, if present, did not influence
our
results.
Perfused hearts were placed in a custom-built Plexiglas chamber that was attached to a
micromanipulator (18). The mapping
field was positioned over the left anterior
descending coronary
artery, just below its bifurcation with the diagonal coronary
artery.
The anterior surface of the heart was stabilized with
a movable piston against an imaging
window. To avoid epicardial
surface cooling and temperature gradients, the heart was
immersed
in the coronary effluent, which was maintained at a constant temperature
equal
to the perfusion temperature with a heat exchanger located
in the chamber. The
electrocardiogram (ECG) was monitored using
three silver disk electrodes fixed to the
chamber in positions
roughly corresponding to ECG limb leads I, II, and III. ECG signals
were filtered (0.3-300 Hz), amplified (×1,000), and displayed
on an oscilloscope. To
ensure physiological stability of the preparation,
the ECG, coronary pressure, coronary
flow, and perfusion temperature
were monitored continuously throughout each
experiment. Preparations
remained viable for 3-4 h, but the experimental protocols
typically
lasted <1
h.
Spectrofluorometer Measurements
To select optical filters for measuring transmembrane potential and intracellular calcium
simultaneously, in vitro excitation
and emission spectra of indo 1 and di-4-ANEPPS were
obtained with
a spectrofluorometer. In four experiments, hearts were stained
with indo
1 (n = 2) or di-4-ANEPPS (n = 2). Immediately after
they were stained with indo 1, hearts
were perfused with a zero-calcium
Tyrode solution to measure the peak emission of
unbound indo 1.
This peak was chosen, because unbound indo 1 is more efficiently
excited at 365 nm and is closer to the emission spectrum of di-4-ANEPPS
than is the
bound form of indo 1. For all hearts, a portion of
the left ventricle was dissected and
placed in an ultraviolet-grade
cuvette with the epicardial surface at a 45° angle with
respect
to the surface of the cuvette to maximize fluorescence detection.
The cuvette was
then placed in the sample compartment of a spectrofluorometer
(SLM Aminco
8100, Spectronic Instruments, Rochester, NY) maintained
at a constant temperature of
32°C. Excitation and emission scans
were completed within 5
min.
Optical Mapping System
We have developed an optical mapping system that is capable of measuring high-fidelity
fluorescent signals with high spatial
and temporal resolution simultaneously at
256 recording sites
from the intact heart (Fig. 1). Action potentials were measured
using
di-4-ANEPPS with filtered excitation light (515 ± 5 nm;
Omega Optical, Brattleboro,
VT) obtained from a 180-W quartz tungsten
halogen lamp light source (Oriel, Stratford,
CT) directed to the
heart with a liquid light guide. Calcium transients were measured
using indo 1 with filtered excitation light (365 ± 25 nm; Omega
Optical) obtained from a
250-W mercury arc lamp light source (Oriel)
directed to the heart with a second liquid
light guide. Excitation
light from both light guides was directed to the same location
on

the heart. Fluoresced light from the heart was collected by
a tandem lens assembly (22,
30) as shown in Fig. 1. The tandem
lens assembly consisted of four high-numerical
aperature complex
photographic lenses (85 mm F/1.4, 35 mm F/1.4, 105 mm F/2.0, and
105 mm F/2.0; Nikon, Tokyo, Japan) placed facing each other. A
dichroic mirror
(560 nm; Omega Optical) placed between the lenses
passes light of longer wavelengths to
an emission filter (>695
nm; Shott Glass Technologies, Duryea, PA) and a
16 × 16 element
photodiode array (detector 1) and reflects light of shorter wavelengths
to
a second emission filter (485 ± 5 nm; Chroma, Brattleboro,
VT) and a 16 × 16 element
photodiode array (detector 2). Emission
wavelengths were chosen on the basis of the
excitation and emission
spectra obtained using the spectrofluorometer (see RESULTS). All
optical components of the tandem lens system (e.g., lenses, filter
holders, detectors) were
aligned and rigidly mounted to optical
rails. Before each experiment, optical alignment
was verified
with an accuracy of ~35 µm by directing an image of detectors
1 and 2 onto
the charge coupled device videocamera (Pulnix, Sunnyvale,
CA) for display on a video
monitor. Photocurrent from all 256
photodiodes of each detector array was passed
through low-noise
current to voltage converters (Hamamatsu, Hamamatsu City, Japan)
and then underwent postamplification (×1, ×50, ×200, ×1,000) with
AC coupling (10-s
time constant), followed by low-pass antialias
filtering (500 Hz). Signals recorded from
each photodiode and
ECG signals were multiplexed and digitized with 12-bit precision
at
a sampling rate of 1,000 Hz/channel (Microstar Laboratories,
Bellevue, WA). For the
present study, an optical magnification
of ×1.24 resulted in a total mapping field of
1.4 × 1.4 cm with
0.09 cm of spatial resolution. To view, digitize, and store the
position
of the mapping array relative to anatomic features, a
mirror was temporarily inserted
between the lenses of the tandem
lens assembly to direct reflected light to the charge
coupled
device videocamera.

Fig. 1. Optical mapping system for simultaneously recording action potentials
and calcium transients. Filtered [Ex Filter 2 (365 ± 25 nm) and Ex Filter
1 (515 ± 5 nm)] excitation light from a mercury arc lamp (250 W) and QTH lamp
(180 W) is directed by liquid light guides to the same location on the preparation.
Fluorescence is collected by a tandem lens system assembly consisting of
4 complex photographic lenses. A dichroic mirror passes fluorescence of longer
wavelengths to an emission filter (Em Filter 1 >695 nm) and detector array
(Detector 1) and reflects fluorescence of shorter wavelengths to a second emission
filter (Em Filter 2, 485 ± 5 nm) and detector array (Detector 2). A removable
mirror inserted temporarily instead of the dichroic mirror redirects an image of the
preparation to a charge coupled device videocamera. Signals from individual
photodiodes are passed through an array of current-to-voltage (I-V) converters,
amplified, filtered, and multiplexed to a 12-bit analog-to-digital (A/D) converter at
1,000 Hz per recording site. UV, ultraviolet.
The rats in the other four groups (each group containing about 15 rats) were fed with
balanced diet, vata enhancing diet, pitta enhancing diet and kapha enhancing diet
respectively for another three weeks and then using the above mentioned method were
sacrificed and their Action potentials and Calcium transients were measured.

Vata enhancing diet (VED) included fresh uncooked vegetables with less oil content,
cold and raw foods like dry cold cereal etc., raw sprouts, with bitter, pungent and
astringent tastes.
Pitta enhancing diet (PED) included warm, spicy foods with pungent, salty and sour taste.
More night shades were given.
Kapha enhancing diet (KED) included cold and cooked foods with lots of oils. Foods
which were sweet were the chosen foods. Plenty of rie and wheat foods were chosen.
Experimental Protocol
A polytetrafluoroethylene-coated silver bipolar electrode with 1-mm interelectrode
spacing was used to stimulate the anterior
ventricular epicardial surface at twice diastolic
threshold current.
To ensure steady-state conditions, the preparation was paced at
a
constant baseline cycle length of 400 ms. The ECG, perfusion
pressure, flow, and
temperature were checked continuously throughout
each experiment to monitor steady-
state
conditions.
To determine the amount of error caused by spectral overlap of di-4-ANEPPS and indo
1, two protocols were performed in separate
experiments.
Protocol A. Hearts were first perfused with indo 1. The change in fluorescence emission
was measured at >695 nm (i.e., emission filter
normally used for di-4-ANEPPS) using
515 ± 5 and 365 ± 25 nm excitation,
in the presence of indo 1 alone. The change in
fluorescence intensity
measured at >695 nm in the absence of di-4-ANEPPS is a direct
measure of the error due to spectral overlap of indo 1 during
measurement of action
potentials [voltage potential (V
m
) error].
Then the change in fluorescence was measured at
485 ± 5 nm using
365 ± 25 and 515 ± 5 nm excitation to measure intracellular calcium
transients with no error due to spectral overlap of di-4-ANEPPS.
Finally, hearts were
loaded with di-4-ANEPPS, and with both indicators
present, action potentials (V
m
) and
calcium transients (Ca
2+
) were recorded
simultaneously.
Protocol B. Hearts were first perfused with di-4-ANEPPS. The change in fluorescence
emission was measured at 485 ± 5 nm (i.e., emission
filter normally used for indo 1)
using 515 ± 5 and 365 ± 25 nm
excitation, in the presence of di-4-ANEPPS alone. The
change in
fluorescence intensity measured at 485 ± 5 nm in the absence of
indo 1 is a
direct measure of the error due to spectral overlap
of di-4-ANEPPS during measurement
of calcium transients (Ca
2+
error). Then the change in fluorescence was measured at >695
nm using 365 ± 25 and 515 ± 5 nm excitation to measure action
potentials with no error
due to spectral overlap of indo 1. Finally,
hearts were loaded with indo 1, and with both
indicators present,
action potentials (V
m
) and calcium transients (Ca
2+
) were recorded
simultaneously.
Data Analysis
V
m
and Ca
2+
transients recorded simultaneously and error signals (i.e., V
m
error and Ca
2+
error) were recorded from all 256 mapping sites. To quantify
signal magnitude, the

maximum change in fluorescence intensity
corresponding to the maximum change in
fluorescence during the
upstroke of the action potential or the upstroke of the calcium
transient (i.e., peak-to-peak amplitude) was calculated for every
signal including error
signals. Because excitation light and dye
distribution are not uniform across the mapping
field, V
m
and
Ca
2+
error were calculated as a percent error, where peak-to-peak
amplitude
of V
m
error and Ca
2+
error signals were normalized to the peak-to-peak amplitude of
V
m
and Ca
2+
measured in the presence of di-4-ANEPPS and indo 1 at each site
(1)
for protocol A and
(2)
for protocol B.
It is important to note that V
m
and Ca
2+
recordings were made with both indicators
present. For example, V
m
consisted of fluorescence
due to di-4-ANEPPS and an error
signal due to indo 1 (i.e., V
m
error signal). Therefore, V
m
error amplitude was subtracted
from
V
m
amplitude in the denominator (Eq. 1) so that V
m
error was determined
as a
percentage of fluorescence associated with a "pure" action
potential.
The rise times of all optical action potential and calcium transient upstrokes were
calculated as the time required for fluorescence
to change from 10% to 90% of maximum.
Depolarization times were
calculated for all action potential recordings and defined as
the
time from stimulation to maximum positive derivative of the
action potential upstroke
(i.e., dV/dt
max
). The onset of the calcium
transient was calculated at all sites and defined
as the time
from stimulation to when fluorescence increased 25% above minimum
diastolic level. Levels of significance were determined using
a Student's t-test, where
P < 0.05 was considered statistically
significant.
RESULTS
Emission Spectra of Indo 1 and Di-4-ANEPPS
Figure 2 shows the emission spectra of indo 1 (excitation at 365 nm) and di-4-ANEPPS
(excitation at 515 nm) measured in a
representative experiment. Spectra were normalized
to their peak
fluorescence intensities. On the basis of the emission spectra,
filters were
chosen to minimize spectral overlap without significantly
sacrificing signal strength.
Superimposed on the emission spectrum
of indo 1 is the normalized transmittance
characteristics (as
measured by the manufacturer) of the interference filter (shaded
gray)
chosen to measure calcium transients (Ca
2+
emission filter, 485 ± 5 nm). To minimize
contribution from di-4-ANEPPS,
a filter with a narrow bandwidth and sharp cutoff
wavelength was
chosen near the peak emission of indo 1 (480 ± 3 nm, n = 2).
Superimposed
on the emission spectrum of di-4-ANEPPS are the normalized

transmittance
characteristics (as measured by the manufacturer) of the long-pass
filter
chosen to measure action potentials (V
m
emission filter,
>695 nm). To minimize
contribution from indo 1, a filter with
a cutoff wavelength much greater than the emission
spectra of
indo 1 was chosen (V
m
emission filter, >695 nm). As a consequence,
the cutoff
wavelength was significantly greater than the peak
emission wavelength of di-4-ANEPPS
(636 ± 2 nm, n = 2), which
could significantly reduce the magnitude of action potential
recordings.
Fig. 2. Normalized transmittance characteristics of emission filters (gray area)
selected for indo 1 (Ca
2+
emission filter, 485 ± 5 nm) and di-4-ANEPPS
[membrane potential (V
m
) emission filter, >695 nm] superimposed on normalized
emission spectra (solid lines) of indo 1 (excitation 365 nm) and di-4-ANEPPS
(excitation 515 nm). On the basis of the emission spectra, filters were chosen to
minimize spectral overlap and maximize signal strength.
On the basis of the spectra measured, the maximum error due to spectral overlap was
expected to be minimal; however, because
the absolute magnitude of the emission spectra
was not taken into
account, it is possible that the error due to overlap may be greater
than
predicted by the normalized spectra. In addition, with the
use of the chosen filters, the
magnitude of calcium transients
and action potentials may be too small and, thus,
significantly
reduce signal fidelity. It is also possible that fluoresced light
originating off
the central optical axis does not strike the interference
filter (Ca
2+
filter) normal to its
surface. This, theoretically, reduces the
central wavelength of the interference filter by
several nanometers
and, thus, slightly increases the separation between Ca
2+
and V
m
filters. Therefore, a direct measurement of the error
due to spectral overlap and the signal
magnitude of action potentials
and calcium transients over the entire mapping field was
required.
Error Due to Spectral Overlap of Di-4-ANEPPS and Indo 1
To quantify the error due to spectral overlap of di-4-ANEPPS and indo 1, two separate
experimental protocols were followed
(protocols A and B). A representative example of
fluorescence
measurements made at a single recording site during each experimental

protocol is shown in Fig. 3. In protocol A (Fig. 3A), fluorescence
change was first
measured at >695 nm in the presence of indo 1
alone (Fig. 3A, top trace) with both
excitation lights on. The
change in fluorescence intensity represents the error due to
spectral
overlap of indo 1 during measurement of transmembrane potential
(i.e., V
m
error).
Although difficult to see, the morphology of
the V
m
error signal is that of a calcium
transient where fluorescence
decreases on excitation, as expected with indo 1 emission at
>695
nm. Fluorescence change was then measured again at >695 nm, but
in the presence
of indo 1 and di-4-ANEPPS, and is shown plotted
on the same scale (Fig. 3A, bottom
trace). As with fluorescence
due to indo 1, fluorescence due to di-4-ANEPPS at >695 nm
decreased
on excitation (i.e., depolarization). In this case, the total
change in fluorescence
intensity (i.e., V
m
) included fluorescence
changes due to di-4-ANEPPS and indo 1;
however, V
m
error relative
to the change in fluorescence due to di-4-ANEPPS, calculated
using
Eq. 1, was very small (0.84%) and was not visually apparent on
the action potential
recording.
Fig. 3. A: protocol A. Change in fluorescence measured from a single recording
site at >695 nm with both excitation sources on in the presence of indo 1 alone
(top trace, V
m
error) and in the presence of di-4-ANEPPS and indo 1 (bottom
trace, V
m
) drawn on the same scale. The error signal is small compared with the
total change in fluorescence (0.84%). B: protocol B. Change in fluorescence
measured from a single recording site at 485 ± 5 nm with both excitation sources
on in the presence of di-4-ANEPPS alone (top trace, Ca
2+
error) and in the
presence of indo 1 and di-4-ANEPPS (bottom trace, Ca
2+
) drawn on the same
scale. The error signal was smaller than the detectable range of our system.
In protocol B, fluorescence change was first measured at 485 ± 5 nm in the presence of
di-4-ANEPPS alone (Fig. 3B, top trace)
with both excitation lights on. The change in
fluorescence intensity
represents a direct measurement of the error due to spectral overlap
of di-4-ANEPPS emission during measurement of intracellular calcium
(i.e., Ca
2+
error).
The Ca
2+
error signal was undetectable with our system resolution. Fluorescence
change
was measured again using the same Ca
2+
filters with both light sources on and in the

presence of both
di-4-ANEPPS and indo 1 and is shown plotted on the same scale
(Fig.
3B, bottom trace). At this emission wavelength (485 ± 5
nm), fluorescence due to indo
1 decreased on excitation. In this
case, the total change in fluorescence intensity (i.e.,
Ca
2+
) consisted of fluorescence only from indo
1.
We determined that the percent V
m
error and Ca
2+
error were small throughout the entire
mapping field. The percent V
m
error
calculated using Eq. 1 over the entire mapping field
from a representative
experiment is shown in Fig. 4. The average percent V
m
error was
extremely small (0.92 ± 0.94%) and at many sites undetectable.
However, the percent V
m
error was as high as 4%. In contrast,
the percent Ca
2+
error was undetectable with the
resolution of our recording system
across the entire mapping field (not shown). V
m
error
was greater
than Ca
2+
error, indicating greater spectral overlap between di-4-ANEPPS
and
indo 1 at >695 nm than at 485 ± 5 nm. Nevertheless, as shown
in Table 1, the average
percent error due to spectral overlap
was extremely small over all experiments.
Fig. 4. Percent V
m
error over the entire mapping field from a representative
experiment. , Individual recording sites in the mapping field array. Horizontal
bars, mean and SD. Over the entire mapping field, the error due to spectral overlap
is small (0.92 ± 0.94%). In many cases, the error was below the resolution of our
mapping system (i.e., 0%).
Table 1. Summary data for percent V
m
error and percent Ca
2+
error
Table 1. Summary data for percent V
m
error and percent Ca
2+
error

Expt. No.
% V
m
Error
(protocol A)
% Ca
2+
Error
(protocol B)
1
2.9
2
0.9
3
1.4
4
0.0
5
0.0
6
0.0
Mean ± SD 1.7 ± 1.0% 0.0%
Data demonstrate the effect of spectral overlap between indo 1 and di-4-ANEPPS. The
value shown for each individual experiment is the mean value for all 256 recording
sites. For protocol A, percent membrane potential (V
m
) error was very small for all
experiments; for protocol B, percent Ca
2+
error was always less than the detectable
range of our recording system.
Action Potentials and Calcium Transients Measured Simultaneously
After measurement of the error due to spectral overlap of indo 1 and di-4-ANEPPS,
calcium transients and action potentials
were recorded simultaneously during each
experiment (n = 6). A
representative example of an action potential and calcium transient
recorded simultaneously from the same site in the presence of
indo 1 and di-4-ANEPPS is
shown in Fig. 5. In the action potential
recording (Fig. 5, top), a rapid upstroke and all
phases of the
action potential are clearly visible with no apparent artifact
due to spectral
overlap of indo 1 (V
m
error). The calcium transient
recorded from the same site (Fig. 5,
bottom) also shows no noticeable
error due to spectral overlap of di-4-ANEPPS. The
initial rapid
increase in intracellular calcium followed the upstroke of the
action potential
by 7 ms in this example, and the decrease in
intracellular calcium to minimum diastolic
levels occurred well
beyond the repolarization phase of the action potential. The average
rise time of optical action potential upstrokes for all experiments
was 7.9 ± 2.9 and
8.4 ± 3.0 ms in the absence and presence, respectively,
of indo 1 (not significant).
Similarly, the average rise time
of calcium transient upstrokes in all experiments was
14.4 ± 2.2
and 15.9 ± 1.0 ms in the absence and presence, respectively, of
di-4-ANEPPS
(not significant). These data indicate that the extremely
small error due to spectral overlap
of indo 1 and di-4-ANEPPS
does not influence measurements when the change in
fluorescence
and, thus, the error due to overlap are expected to be greatest
(i.e., during the
action potential and calcium transient upstroke).
Fig. 5. A representative example of an action potential and calcium transient
recorded simultaneously from the same site after perfusion of the heart with both
indo 1 and di-4-ANEPPS.

Action potentials and calcium transients were recorded simultaneously from all 256 sites
within the mapping field. Isochrone
maps of depolarization time and the time of calcium
transient
onset relative to the time of stimulation are shown in Fig. 6.
The spread of
depolarization (Fig. 6A) indicates anisotropic propagation
from the site of stimulation
(pacing symbol) with an average conduction
velocity of 54 cm/s along the fast axis of
propagation. Calcium
transients were also recorded from the same 256 recording sites,
and the time of calcium transient onset was calculated for each
site (Fig. 6B). The spatial
pattern of the calcium transient onset
was also anisotropic and, as expected, closely
mirrored that of
electrical activation. The site of earliest depolarization occurred
at 5 ms,
followed by the onset of the calcium transient at 10
ms. The calcium transient onset and
depolarization times were
distributed such that the action potential always preceded the
onset of the calcium transient by, on average, 7.8 ± 2.8 ms over
the entire mapping field.
These data indicate negligible error
due to spectral overlap of indo 1 and di-4-ANEPPS
over the entire
mapping field.
Fig. 6. Contour maps of depolarization time (A) and the time of calcium transient onset (B)
calculated from action potentials and calcium transients recorded simultaneously from
256 sites on the epicardial surface of the intact guinea pig heart. The contour legend represents
both panels, where each contour interval indicates 5 ms and the fiducial point (i.e., 0 ms)
corresponds to the time of stimulation (pacing symbol). As expected, the pattern of
depolarization is mirrored by, after a slight delay, the pattern of calcium transient onset.

Table 2
Experimental
values
Control
group 1
Control
group 2 VED fed rats PED fed rats KED fed rats
Resting
Potential
V, mV - 84.5 ± 3.00 - 85.67 ± 3.00 - 70.45 ± 4.5 - 90 ± 3.5 - 108.78 ± 3.5
As seen from the table the control groups of rats had nearly the same resting potential.
The VHD fed rats had a resting potential which were very near to the threshold potential
which in all rats measured around -65mV ± 2.5. On the contrary the KED fed rats had
lower resting potentials than even the PED fed rats making their cells more stable.
.
DISCUSSION
We have observed the effects of the various diets as described in the Ayurvedic texts
does have impact on the resting potentials of the heart cells. Basing on our results we
state that the cells resting potentials vary between -70 and -110mV approximately (Table
2). The cells in the VED fed rats have a resting potential which is very near to the

threshold potential. It means that the cells would require only a smaller stimulus to excite
an action potential. On the contrary the KED cells appear to be more stable requiring a
stronger stimulus to generate action potential. We have described and validated a new
technique based on optical mapping that is capable of measuring calcium transients and
action potentials simultaneously from the intact heart. Two fluorescent
indicators were
chosen that have previously been used extensively
to measure intracellular calcium (indo
1) and transmembrane potential
(di-4-ANEPPS). We demonstrate that the error due to
spectral overlap
of indo 1 and di-4-ANEPPS is very small. As a result, intracellular
calcium transients and action potentials can be mapped simultaneously
with high signal
fidelity from the same heart with negligible
error. This technique may provide significant
insight into the
cellular mechanisms of arrhythmias associated with abnormal intracellular
calcium
handling.
To simultaneously measure intracellular calcium and transmembrane potential in the
same heart, two fluorescent indicators
were used. The indicators chosen in the present
study were di-4-ANEPPS
(23) for sensing transmembrane potential and indo 1 (12) for
sensing free intracellular calcium concentration. These particular
indicators were chosen
because 1) both have been well characterized
and independently accepted as standard
techniques and 2) the wavelengths
of peak emission are significantly separated,
minimizing spectral
overlap. Figure 2 illustrates this point. Both spectra correspond
to
fluorescence emission at resting membrane potential (di-4-ANEPPS)
and low
intracellular calcium levels (indo 1). The gray areas
in Fig. 2 indicate the wavelengths at
which calcium transients
(Ca
2+
filter) and action potentials (V
m
filter) were measured in
the
present study. As indicated in Fig. 2, when fluorescence is measured
using Ca
2+
or V
m
filters, fluorescence originates from both indicators;
however, the contribution of one is
much larger than that of the
other. For example, the change in fluorescence intensity at
485
± 5 nm (i.e., area under both spectral curves bounded by 485 ±
5 nm) is mostly due to
indo 1; however, a small amount of fluorescence
change may arise from di-4-ANEPPS
and is what we called Ca
2+
error. Likewise, fluorescence changes at >695 nm arise mostly
from di-4-ANEPPS with a small contribution from indo 1 (V
m
error).
If emission spectra
or optical filters were closer in wavelength,
significant overlap would occur, resulting in a
composite signal
consisting of significant fluorescence from di-4-ANEPPS and indo
1.
With a judicious selection of optical filters, we found that the error due to spectral
overlap of indo 1 and di-4-ANEPPS was
sufficiently small, such that calcium transients
and action potentials
could be measured simultaneously from the intact heart with
negligible
error. We found that V
m
error due to fluorescence of indo 1 was,
on average,
extremely small (1.7 ± 1.0%) and at many sites zero.
The variability in error across the
mapping field could be explained
by an unequal distribution of relative intensity of
excitation
light at 365 and 514 nm and/or relative dye concentration. Indeed,
error
variability due to differences in relative fluorescence
intensity of fluo 3/4 and di-4-
ANEPPS was analyzed in a study
by Johnson et al. (14) and was similar in magnitude to
that
measured in the present study. The Ca
2+
error due to fluorescence change of di-4-
ANEPPS was so small
that it was undetectable with the resolution of our mapping
system.
On the basis of the emission spectra and the transmission characteristics
of the
optical filter chosen, this is not a surprise. It is possible,
on the basis of biological and dye
loading variability, that the
contribution of one dye may become much stronger than that
of
the other. In such a case, the error due to overlap might become
significant. It may be

possible to compensate for such differences
in dye loading by adjusting excitation light
intensity. For example,
if action potentials are significantly larger than calcium transients,
then the excitation light used to maximally excite di-4-ANEPPS
can be reduced.
However, reducing excitation intensity would also
lower the signal amplitude of the
action potentials and, thus,
reduce signal fidelity. In the present study, to achieve high
signal fidelity, excitation intensity was maintained at levels
normally used for measuring
action potentials and calcium transients
independently. Finally, our experimental
protocols to measure
error due to spectral overlap were not designed to test the possibility
of indo 1 emission exciting di-4-ANEPPS. Although this is theoretically
possible, it is
unlikely to affect our results, because the change
in fluorescence intensity associated with
indo 1 is much smaller
than the amount of excitation light required to generate significant
fluorescence of di-4-ANEPPS. Experiments specifically designed
to test this possibility
support our conclusion (unpublished
observation).
The error due to spectral overlap of indo 1 and di-4-ANEPPS was negligible, making it
possible to map with confidence calcium
transients and action potentials from the intact
heart with high
resolution. The calcium transient and action potential shown in
Fig. 5
demonstrate a rapid rise in intracellular calcium several
milliseconds after the upstroke of
the action potential. This
result is expected on the basis of the theory of calcium-induced
calcium release (8). The decline of intracellular calcium is
much slower, extending
beyond the repolarization phase of the
action potential when transmembrane potential is
at rest. The
rise time of calcium transients measured in this study is comparable
to that
measured previously (6). Moreover, the rise times of
the calcium transients and optical
action potentials were unaffected
by the presence of both di-4-ANEPPS and indo 1. At all
256 mapping
sites, action potentials and calcium transients exhibited a close
spatial
relationship. The contour maps shown in Fig. 6 demonstrate
the pattern of depolarization
time and time of calcium transient
onset. Throughout the entire mapping field, action
potential propagation
(Fig. 6A), after a delay, is mirrored by the calcium transient
onset
(Fig. 6B). These data provide further evidence that the
error due to spectral overlap of
indo 1 and di-4-ANEPPS is negligible
and that this technique can be used to map with
high resolution
calcium transients and action potential simultaneously in the
intact heart.
In preliminary studies using a similar technique,
it was possible to investigate
intracellular calcium handling
and repolarization alternans (19) and to examine the
relationship
between action potentials and calcium transients during reentrant
excitation
(20).
Intracellular calcium and transmembrane potential have been measured previously in the
same heart (4, 7, 21). However,
in these studies, recordings could be made from only one
site
at a time. This limitation may hinder the investigation of certain
arrhythmia
mechanisms. Recently, using an approach slightly different
from that used in the present
study, Fast and Ideker. (9) developed
a technique for mapping action potentials and
calcium transients
in myocyte cultures. With the use of the voltage-sensitive dye
RH-237
and the calcium-sensitive dye fluo 3, action potentials
and calcium transients were
recorded with negligible error. In
the present study, we measured changes in fluorescence
intensity
of indo 1 and not absolute intracellular free calcium levels.
Nevertheless, indo
1, unlike fluo 3, has a second emission peak
that occurs at ~405 nm, corresponding to the
bound form of indo
1. This peak could be used to further reduce spectral overlap
and,

more importantly, could also be used to measure actual intracellular
calcium levels
throughout the heart using standard ratiometric
imaging techniques (34).
Clinical Implications
The Vata cells thus are very susceptible to extremely low stimuli and are capable of
exciting action potentials even with low stimuli. From this it may be hypothesized that if
say there is an accumulation of more Vata in the Immune cells then it may result in
Hypersensitivity reaction like allergies and asthma etc., If the same is applied to nerve
cells the vata nerve cell may result in sleep disorders and so on. The Pitta cells are stable
and represent the normal metabolic functioning of the body. The Kapha cells are more
stable than the Pitta cells and their excitation requires stronger stimuli. If kapha increases
in say for example, the Immune cells again the immune response occurs only to a
stronger bacterial or allergic stimulus and so hypersensitivity reactions do not occur in
such individuals. There is also a possibility of a symbiotic environment in such
individuals. Here kapha should not be confused with a hefty person. Vata, Pittaand Kapha
are only various Action potential states of cell. In the course of this experiment we also
have concluded the following:
Because intracellular calcium plays a critically important role in the electrophysiology of
the heart, there are several important
clinical implications of abnormal intracellular
calcium handling.
T wave alternans, a known predictor of sudden cardiac death (31),
has
been mechanistically linked to repolarization alternans and
the initiation of ventricular
fibrillation (28) and torsade
de pointes (33). It has been suggested that intracellular
calcium
handling plays a significant role in the cellular mechanisms of
repolarization
alternans (17, 32, 33); however, a causal
relationship has yet to be determined. We have
shown, in a preliminary
study, that in the intact heart, spatial heterogeneity of
repolarization
alternans is closely mirrored by spatial heterogeneity of calcium
transient
alternans (19). Heart failure is another significant
clinical paradigm that is associated with
a high incidence of
sudden cardiac death and the occurrence of ventricular (5)
and atrial
(25) arrhythmias. Intracellular calcium homeostasis
is believed to be significantly altered
in failing hearts due
to, in part, upregulation of Na
+
/Ca
2+
exchanger (35) and impaired
uptake of calcium by the sarcoplasmic
reticulum (13). Such alterations of intracellular
calcium handling
may lead to calcium overload and, in turn, the occurrence of delayed
afterdepolarizations. Delayed afterdepolarizations have been associated
with triggered
arrhythmias in failing hearts (36). Undoubtedly,
the ability to map intracellular calcium
and transmembrane potential
simultaneously in the intact heart will provide new and
important
information concerning the cellular mechanisms of arrhythmias
associated with
abnormal intracellular calcium
handling.
ACKNOWLEDGEMENTS
This work was supported by a grant from Sai Ganga Panacea LLC, a Delaware
corporation, whose Chairmand CEO is DR. Ravishankar POlisetty.

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