Davies, Shireen A.

Dow, Julian T.

Kettlewell, Sarah

Smith, Godfrey L.

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Correlation Of Levels And Time Course Of Intracellular And Mitochondrial Calcium Signals In Isolated Adult Cardiac Cells 

Wednesday, March 4, 2009 625a3220-Pos Board B267
Mitochondria Dynamics in Cardiac Myocytes
Aristide Chikando1, Mariusz Karbowsky1, Jonathan Lederer1,2.
1University of Maryland Biotechnology institute, Baltimore, MD, USA,
2University of Maryland Baltimore, Baltimore, MD, USA.
Mitochondrial dynamics are driven by the inner mitochondrial membrane po-
tential, and have significant implications in cell function. Fission and fusion
are well established characteristics of mitochondrial dynamics in most cell
types. These movements are believed to be geared toward optimizing energy
delivery to metabolic sites within the cell. In cardiac cells, mitochondria dy-
namics and their implications in cell function are poorly studied. The canonical
role of mitochondria in ATP production suggests a more involved role for car-
diac mitochondria dynamics into regulation of cellular function. In this study,
mitochondria dynamics in adult rat, and neonatal, cardiac myocytes, are inves-
tigated. The functionality of proteins involved in the fusion and fission of mi-
tochondria are assayed, using recently developed mitochondria targetted tools.
We show that while adult cardiac myocytes exhibit relatively dormant mito-
chondria dynamics, mitochondria in neonatal hearts exhibit dynamics that
are comparable to that of primary cell types, albeit more organized. Finally,
we investigate the effects of the regulation of expression levels of mitochon-
drial fission and fusion proteins on calcium signaling in both cardiac cell types.
3221-Pos Board B268
PPARa Activation Reduced Remodelling And Improved Left Ventricular
Function In Chronic Pressure/volume Overload Induced Heart Failure
Antonius Baartscheer1, Stephan Schafer2, Barbara Albrecht2,
Jan W.T. Fiolet1.
1Academic Medical Center, Amsterdam, Netherlands, 2Cardiology Research,
Bayer Schering Pharma, Wuppertal, Germany.
Activation of peroxisome proliferator-activated receptor-a (PPARa) in the
heart is associated with a decrease of hypertrophy and fibrosis, but its overall
effect on remodelling in the setting of heart failure has not been evaluated.
We investigated the effect of PPARa activation by a selective and potent
PPARa agonist BAYPP1: in a volume and pressure overload model of heart
failure in rabbits. Volume and pressure overload was induced by induction of
aortic valve insufficiency, followed by abdominal aortic banding 3 weeks later.
Compound treatment started after induction of valve insufficiency. The devel-
opment of HF was examined echocardiographically and electrocardiographi-
cally after 3 months. Action potentials and sodium and calcium handling
were measured in left ventricular myocytes.
Rabbits with volume and pressure overload showed substantial hypertrophy
and impairment of contractility compared to sham operated rabbits (table). Ad-
ditionally increased lung weight, presence of ascites, increase QT duration and
QRS complex and a high rate of premature death were present in HF animals.
Cellular remodelling was demonstrated by: 1) increased myocyte dimensions,
prolongation of the action potential, increased cytoplasmic sodium and dis-
turbed calcium handling: a) increased diastolic calcium b) decreased amplitude
and prolongation of the calcium transient c) decreased SR calcium content e)
increased propensity to evoke calcium after-transient. Chronic treatment with
the PPARa agonist BAYPP1 attenuated development of hypertrophy and abol-
ished development of HF and cellular and ionic remodelling even more pro-
nounced compared to ACE inhibition by Enalapril.
3222-Pos Board B269
Correlation Of Levels And Time Course Of Intracellular And Mitochon-
drial Calcium Signals In Isolated Adult Cardiac Cells
Sarah Kettlewell, Shireen A. Davies, Julian T. Dow, Godfrey L. Smith.
University of Glasgow, Glasgow, United Kingdom.
The contribution of mitochondria to Ca2þ homeostasis in the adult cardiac my-
ocyte is uncertain. We have successfully targeted a genetically encoded Ca2þ
sensitive inverse pericam to the mitochondria of primary mammalian ventric-
ular cardiomyocytes by adenoviral mediated expression (AdMitycam). Cyto-
plasmic Ca2þ ([Ca2þ]cyt) and mitochondrial Ca
2þ ([Ca2þ]mit) were monitored
simultaneously in adult rabbit cardiomyocytes by loading AdMityCam express-
ing cells with Fura2. [Ca2þ]cyt and [Ca
2þ]mit were recorded during voltage
clamp stimulation (0.5Hz). The sarcoplasmic reticulum (SR) was loaded at
a range of [Ca2þ] by increasing [Ca2þ]0 (1.8-7.2mM) and by b-adrenergic stim-
ulation (500nM isoproterenol). Beat-to-beat Ca2þ transients were observed in
both [Ca2þ]cyt and [Ca
2þ]mit. The relationship between mean [Ca
2þ]cyt and
[Ca2þ]mit was unity at [Ca
2þ] below ~133nM, above this value
[Ca2þ]mit>[Ca
2þ]cyt. Under conditions of spontaneity [Ca
2þ]mit>[Ca
2þ]cyt
(204554.0nM vs. 118526.7nM respectively). In electrically stimulatedevents the kinetics of the [Ca2þ]mit transient upstroke (time-to-peak (TTP))
was significantly slower than cytoplasmic. [Ca2þ]mit TTP decreased with in-
creasing SR Ca2þ load whilst [Ca2þ]cyt TTP increased. The time to 50% decay
of the [Ca2þ]mit in these electrically stimulated events however was signifi-
cantly faster than cytoplasmic. b-adrenergic stimulation further decreased the
TT50% decay of the [Ca2þ]cyt by ~25% and [Ca
2þ]mit by ~60%. The mecha-
nism behind the effects of isoproterenol on the kinetics of the mitochondrial
Ca2þ signal is unknown but may be linked to known effects of b-adrenergic
stimulation on Mg2þ extrusion. Whilst the kinetics of the electrically triggered
[Ca2þ]cyt and [Ca
2þ]mit events differed, the changes in [Ca
2þ]cyt and [Ca
2þ]mit
during spontaneous SR Ca2þ release events were similar in time course. These
data suggest that Ca2þ accumulation by cardiac mitochondria depends on the
time course and magnitude of the cytoplasmic Ca2þ signal and the presence
of b-adrenergic stimulation.
3223-Pos Board B270
Tracking Membrane Potential, Calcium and REDOX Activity in Single
Mitochondria During Normal Cardiac Cell Stimulation
Moradeke A. Bamgboye1, Julio Altamirano2, W. Jonathan Lederer1.
1UMB - UMBI, Baltimore, MD, USA, 2UMBI-MBC, Baltimore, MD, USA.
Mitochondria are well recognized as ‘‘the powerhouse’’ of the cell. In cardiac
cells they may also play a role in Ca2þ signaling, redox signaling and cell death.
They are therefore important in maintaining cardiac cellular homeostasis. By
coupling fuel oxidation to ion transport across their inner membrane they create
an electrochemical gradient. This electrochemical gradient is used to drive en-
ergy production to meet the cellular needs; it also drives the transport of ions,
such as Ca2þ, across the inner mitochondrial membrane, in turn, Ca2þ regulates
mitochondrial activity. The mitochondrial membrane potential is therefore an
accurate indicator of the energetic state of the cell. The cardiac action potential
continuously changes the potential of the cell membrane resulting in Ca2þ entry
and further Ca2þ release from the sarcoplasmic reticulum (SR) triggering cell
contraction. In freshly dissociated rat ventricular myocytes, we use confocal
imaging of the fluorescent membrane potential indicator TMRE and the fluo-
rescent Ca2þ indicator Rhod-2 trapped in the mitochondrial matrix to tracked
changes in mitochondrial membrane potential and mitochondrial Ca2þ
([Ca2þ]m) in individual mitochondria in response to electrical stimulation
and caffeine induced SR Ca2þ release. We also tracked changes in mitochon-
drial NADH by measuring NADH auto fluorescence in single mitochondria un-
der the same experimental conditions. We conclude that the intrinsic functional
link between the electrical activity of the cardiac cell (due to SR Ca2þ release)
and mitochondrial dynamics can be faithfully studied in real time by tracking
mitochondrial membrane potential, Ca2þ or NADH in single mitochondria.
3224-Pos Board B271
Comparison of pH-dependence of Carbonic Anhydrase Activity in vitro
and in Living Cells
Francisco C. Villafuerte, Pawel Swietach, Shalani Patiar, Adrian L. Harris,
Richard D. Vaughan-Jones.
University of Oxford, Oxford, United Kingdom.
Carbonic anhydrase (CA) plays an important role in acid-base homeostasis. It
catalyses carbonic buffering, increases intracellular Hþ-mobility and facilitates
the activity of pHi regulatory transporters. In vitro, CA activity is known to be
inhibited by a fall of pH. This observation has been largely overlooked when
considering mechanisms of pHi regulation. There is no information, however,
on the pH-sensitivity of CA in intact cells. We have therefore investigated the
pH-sensitivity of CA both in vitro, and in situ in living cells. In vitro CA activ-
ity was assessed from the time-course of pH change after addition of CO2-sat-
urated water to a buffered solution (pH 8.0-6.5) containing purified bovine
CA2, membrane fragments of HCT116 cells stably transfected with CA9, or
cardiac homogenates from rat ventricle. In situ CA activity was assessed
from the rate of CO2-induced fall of pHi in rat superfused ventricular myocytes,
AM-loaded with the pH-fluorophore, carboxy-SNARF-1. The pH-sensitivity
was estimated by fitting a kinetic algorithm to the pH time-course data. Results
show that both in vitro and in situ CA-activity displays strong pH-dependence,
activity declining with a fall of pH. Apparent pK and Hill coefficient (nH)
values for in vitro pH-dependence were 7.00 and 1.10 respectively for purified
CA2, 6.85 & 1.73 for CA9, and 7.15 and 0.80 for cardiac homogenates. The
intracellular pKa and nH values for generic cardiac CA were 7.20 and 2.50.
These results imply that the enzyme’s contribution to both intracellular and ex-
tracellular pH-regulation may vary dynamically over the physiological pH
range.
Work funded by British Heart Foundation, Cancer Research UK, and Royal
Society.


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Correlation Of Levels And Time Course Of Intracellular And Mitochondrial Calcium Signals In Isolated Adult Cardiac Cells

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