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TitleBiochemistry of Hypertrophy and Heart Failure
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Table of Contents
                            Title Page
Copyright Page
Molecular and Cellular Biochemistry:
Preface
Lysophospholipids do not directly modulate Na+-H+ exchange
	Abstract
	Introduction
	Materials and methods
		Materials
		Sarcolemmal membrane preparations
		Measurement of Na+-H+ exchange
		Treatment with lysophospholipids
		Statistics
	Results
	Discussion
	Acknowledgements
	References
Antioxidant enzyme gene expression in congestive heart failure following myocardial infarction
	Abstract
	Introduction
	Materials and methods
		Animal model
		Study groups
		Northern blot analysis
		Western blot analysis
	Results
		General characteristics
		mRNA abundance
		Proteins
	Discussion
	Acknowledgements
	References
Modulation of cardiac remodeling by adenosine: In vitro and in vivo effects
	Abstract
	Introduction
	Adenosine, its metabolism and receptors in the cardiovascular system
	Adenosine and myocardial ischemia
	In vivo effects of adenosine as adjunct to reperfusion on infarct expansion/LV remodeling
	In vitro effects of adenosine on cardiac fibroblast function
	In vitro effects of adenosine on tumor necrosis factor-α. (TNF-α.) release
	In vivo effects of adenosine on post-MI remodeling and scarring
	Conclusions
	Acknowledgements
	References
Right ventricular collagen and fibronectin levels in
patients with pulmonary atresia and ventricular
septal defect
	Abstract
	Introduction
	Materials and methods
		Patients
		Total RNA isolation and RT-PCR
		(Immuno) histochemistry
		Video image analysis
		Statistical analysis
	Results
	Discussion
	Acknowledgement
	References
Amouse model of familial hypertrophic cardiomyopathy caused by a α-tropomyosin mutation
	Abstract
	Introduction
	Materials and methods
		Generation of FHC α-TM 175 and 180 transgenic mice
		RNA and protein analysis
		Histological analysis
		Isolated retrograde perfused heart preparation
		DNA microarray hybridization and analysis
	Results
		Expression of mutant α-TM induces cardiac hypertrophy
		RNA and protein expression in the FHC α-TM 180 hypertrophic hearts
		Assessment of FHC α-TM 180 cardiac function
		DNA microarray analysis of the FHC α-TM 180 hearts
	Discussion
	Acknowledgements
	References
Analysis of postextrasystolic relaxation response in the human heart
	Abstract
	Introduction
	Materials and methods
		Patients
		Hemodynamic assessment
		Statistics
	Results
		Response of - dP/dt
		PRR vs. hemodynamic variables
	Discussion
	Acknowledgement
	References
Differential effects of calpain inhibitors on hypertrophy of cardiomyocytes
	Abstract
	Introduction
	Materials and methods
		Isolation and culture of cardiomyocytes
		Protein content of cardiomyocytes
		Peptidase acti vity of cardiomyocytes
		Statistical analyses
		Animal care
	Results
		Effect of protease inhibitors on hypertrophy
		Effect of phenylephrine and PD150606 on proteaseactivity
	Discussion
	Acknowledgements
	References
Cardiac adaptation to endurance exercise in rats
	Abstract
	Introduction
	Materials and methods
		Endurance exercise training
		Assessment of systolic blood pressure and heart rate
		Echocardiography
		Isolated heart preparations
		Diastolic stiffness
		Purine efflux
		Cardiac energetics
		Collagen distribution by picrosirius red staining and laser confocal microscopy
		Statistical analysis
	Results
	Discussion
	Acknowledgement
	References
Matrix metalloproteinase inhibitors attenuate endotoxemia induced cardiac dysfunction: A potential role for MMP-9
	Abstract
	Introduction
	Materials and methods
		Rat model of endotoxemia and isolated heart perfusion
		Preparation of ventricular homogenates
		Measurement of MMP activity by zymography
		Statistical analysis
	Results
		MMP inhibitors ameliorate endotoxemia induced cardiac dysfunction
		Ventricular MMP-2 activity is decreased during endotoxemia
		Perfusate MMP-2 activities are decreased during endotoxemia
		MMP inhibitors attenuate the LPS induced increase in perfusate MMP-9 activity
	Discussion
		MMP-2 and MMP-9 in cardiomyopathies
		LPS induced activation of MMP-9
		Inhibition of MMPs
		Targets of MMP activity
	Conclusion
	Acknowledgements
	References
Functional and structural characterization of anti-β1-adrenoceptor autoantibodies of spontaneously hypertensive rats
	Abstract
	Introduction
	Materials and methods
		Materials
		Isolated heart preparation
		Heart function
		Isolation and purification of immunoglobulins
		Cell culture and functional characterization
		Protein isolation, electrophoresis, and Western blotting
		Detection of cyclic AMP
		Statistical analysis
	Results
	Discussion
	Acknowledgements
	References
Quinapril inhibits progression of heart failure and fibrosis in rats with dilated cardiomyopathy after myocarditis
	Abstract
	Introduction
	Materials and methods
		Animals and medication
		Hemodynamic study
		Heart weight and histopathology
		Ribonuclease (RNase) protection assay
		Statistical analysis
	Results
		Clinical course
		Body and heart weights
		Hemodynamic parameters
		Quantitative analysis of myocardial fibrosis
		Myocardial mRNA expression of TGF-β1, collagen-III and fibronectin
	Discussion
	Acknowledgements
	References
Calpain-1-dependent degradation of troponin I mutants found in familial hypertrophic cardiomyopathy
	Abstract
	Introduction
	Materials and methods
		Expression and purification of human cardiac troponin molecules
		Reconstitution of human cardiac troponin complexes
		Phosphorylation of cTnI
		In vitro proteolysis with Calpain-I
		Western immunoblot analysis and evaluation of cTnI proteolysis
	Results
	Discussion
	Conclusions
	Acknowledgements
	References
Gender mediated cardiac protection from adverse ventricular remodeling is abolished by ovariectomy
	Abstract
	Introduction
	Materials and methods
		Surgical procedures
		Experimental protocol
		Assessment of ventricular size and function
		Data and statistical analysis
	Results
	Discussion
	Acknowledgements
	References
Protein kinase C isoforrn-selective signals that lead to cardiac hypertrophy and the progression of heart failure
	Abstract
	Introduction
	Protein kinase C isoforms
	Protein kinase C isoform function in cardiomyocytes - lessons from genetic mouse models
	Protein kinase C isoforms as nodal points in cardiomyocyte signaling networks
	Summary and future directions
	Acknowledgement
	References
Contractile effects of adenovirally-mediated increases in SERCA2a activity: A comparison between adult rat and rabbit ventricular myocytes
	Abstract
	Introduction
	Materials and methods
		Adenoviral vectors
		Isolation, culture and infection of adult rat and rabbitventricular myocytes
		Functional characterisation
		Western blotting
		Statistical analysis
	Results
		Contraction amplitude with increasing stimulation frequency
		Beat duration with increasing stimulation frequency
	Discussion
	Acknowledgements
	References
Losartan inhibits myosin isoform shift after myocardial infarction in rats
	Abstract
	Introduction
	Materials and methods
		Animals and experimental myocardial infarction
		Experimental protocol
		Tissue preparation
		Myosin isoform analysis
		Modeling of the LV parameters
		Statistical analysis
	Results
		Effects of Losartan treatment on heart failure incidence and cardiac parameters after myocardial infarction
		Effects of Losartan treatment on myosin isoform distribution after myocardial infarction
		Correlation of myosin isoforms with scar area and calculated myocyte volume
	Discussion
	Acknowledgements
	References
Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion
	Abstract
	Introduction
	Materials and methods
		Cell culture
		Determination of cell number
		Trypan blue-staining
		Gel electrophoresis and immunostaining
		Determination of DNA-ladder formation
		Determination of caspase activity
		High-throughput transcript analysis by quantitative realtime fluorescence PCR
		Statistical analysis
	Results
		Measurement of cell number
		Activity of MAP kinase s and Akt
		Reduction of Akt phosphorylation
		Effect of JNK and p38 blockade on cell survival
		Caspase 7 cleavage and caspase 3 activity
		DNA fragmentation
		Trypan blue staining
		Expression of Bax- and Bcl-2 mRNA
	Discussion
	Acknowledgement
	References
Effect of propranolol on cardiac cytokine expression after myocardial infarction in rats
	Abstract
	Introduction
	Materials and methods
		Animal model
		RNase protection assay (RPA)
		Zymography: Detection of cardiac matrix metalloproteinase activity
		Statistical analysis
	Results
		Hemodynamic measurements
		mRNA Expression of pro-inflammatory cytokines
		MMP IIlRNA expression
		Zymography: Gelatinolytic activity of MMP-2 and MMP-9
		Colligin and collagen mRNA expression
		Expression pattern of the TGF-β isoforms
	Discussion
		Functional alterations
		Cardiac cytokine expression and matrix remodeling
	Acknowledgements
	References
Revisiting the surgical creation of volume load by aorto-caval shunt in rats
	Abstract
	Introduction
	Materials and methods
		Shunt creation
		Shunt patency
		Gene expression
		Data analysis
	Results
		Shunt patency
		Organ to body weight ratios
		Gene expression
	Discussion
	Acknowledgements
	References
Proteomic analysis of Racl transgenic mice displaying dilated cardiomyopathy reveals an increase in creatine kinase M-chain protein abundance
	Abstract
	Introduction
	Materials and methods
		Animal model
		Sample preparation
		Protein separation
		Protein visualization
		Image analysis
		Protein identifi cation
	Results
		Quality control assessment
		Alteration in protein level
		Identification of protein of interest
	Discussion
	Acknowledgements
	References
Adenosine and cardioprotection during reperfusion - an overview
	Abstract
	Introduction
	Adenosine
	Myocardial protection by adenosine
		Reduction of myocardial infarct size
		Attenuation of postischemic dysfunction ('stunned myocardium ')
	References
Index to Volume 251
	Developments in Molecular and Cellular Biochemistry
	Developments in Molecular and Cellular Biochemistry
                        
Document Text Contents
Page 1

BIOCHEMISTRY OF HYPERTROPHY ANDHEART FAILURE

Page 2

Biochemistry of Hypertrophy and
He art Failure

Edited by

LORRIEA. KIRSHENBAUM

Institute of Cardiovascular Sciences
St. Boniface General Hospital
Research Center
351 Tache Avenue
Winnipeg, Manitoba R2H 2A6
Canada

PAWAN K. SINGAL

Institute of Cardiovascular Sciences
St. Boniface General Hospital
Research Center
Faculty ofMedicine
University of Manitoba
Winnipeg, Manitoba R2H 2A6
Canada

Reprinted from Molecular and Cellular Biochemistry, Volume 25 I (2003)

Springer Science+Business Media, LLC

IAN M.C. DIXON

Institute of Cardiovascular Sciences
St. Boniface General Hospital
Research Center
Room 3038 SBGH Research Center
351 Tache Avenue
R2H 2A6, Winnipeg, Manitoba
Canada

Page 78

79

Results Myocardial mRNA expression of TGF-f31, collagen-Ill and
fi bronectin

Clinical course

Four of 15 (27%) rats in V and two of 11 (18%) in QO.2 died
between day 30 and 56. None of the animals in Q2, Q20 or
N died. Although pericardial effusion was observed in most
of the rats in V, little effusion was observed in QO.2, Q2 and
Q20, and no effusion was detected in N.

As shown in Fig. 3, the left ventricular mRNA expression of
TGF-p1,collagen-III andfibronectin weremarkedly up-regu-
lated in V (17.1 ± 6.2, 41.1 ± 5.5 and 4.50 ± 0.07) as com-
pared to those in N (2.78 ± 0.01,7 .87 ± 0.64and not detected;
all p < 0.01). Quinapril treatment (Q20) significantly sup-
pressed the increase in expression of TGF-p 1 mRNA (9.00
± 2.40, p < 0.05), while it also reduced collagen-Ill and
fibronectin mRNA expression albeit to a lesser extent (34.6
± 5.0, NS; and 3.17 ± 0.69, NS).

Body and heart weights

150

~oo
E
5
c,
ell
c
~

~ 50

0

8 -8

a a
86 -2 00
~

~x x
U U
Ql Ql
~ ~

:F4 -4 C>:c
E E
5 5
15 15
C::2 -6 c::
"0 "9+

0 -8

500 5

400
4

'2
-a 3! 300 :c

"' EOJ 5 2Ql
e 200 o,>c:: o:c

100
0

0 -1

150 20

.. 15
100 '11 " II' 0>

-a J:
:c E
E E
5 Q:loCl
o, LJJ
> >
...J 50 ..J

5

0 0

Hemodynamic parameters

Hemodynamic parameters were measuredrandomly selected
rats (8 rats in N and V, and 7 rats in QO.2, Q2 and Q20). As
shown in Fig. 1, the CVP was lower in N and Q20 than in V,
QO.2 and Q2. Mean BP and LVPwere lower in V, QO.2, Q2
and Q20 than in N. LVEDPwas higher and ± dP/dt was lower
inV (14.1 ± 2.0mmHg and +2409± 150/-2318 ±235 mmHg/
sec) than in N (5.0 ± 0.6 mmHg and +6173 ± 191/-7120 ± 74
mmHg/sec; all p < 0.01 ). After quinapril treatment, LVEDP
was decreased and ±dP/dt was increased in a dose-depend-
ent manner (l 0.8 ± 1.8 mmHg and +3211± 307/-2928 ± 390
mmHg/sec in QO.2, 9.4 ± 1.5 mmHg and +2871 ± 270/-2966
± 366 mmHg/sec in Q2, and 6.6 ± 1.5 mmHg and +3569 ±
169/-3960 ± 203 mmHg/sec in Q20).

Although the body weight in Q20 was small, it did not differ
among other three groups with heart failure. The HW and H/
B weresignificantly largerin V (1.28 ± 0.05g and 4.38 ± 0.22
g/kg) than N (0.84 ± 0.03 g and 2.49 ± 0.04 g/kg; both p <
0.01). After quinapril treatment, the HW and HIB were de-
creased in a dose-dependent manner in QO.2 (1.21 ± 0.07 g
and 4.04 ± 0.24 g/kg, NS), Q2 (1.09 ± 0.07 g and 3.81± 0.22
g/kg, NS) and Q20 (0.87± 0.02 g and 2.95 ± 0.08 g/kg; both
p < 0.01 vs. V).

Quantitative analysis of myocardial f ibrosis
c:::::J Group-N
_ Group-V

~ Group-QO.2

~ Group-Q2 ' P<0.05 V5 . Group-V
~ Group-Q20 ' : P<0.01 V5 . Group-V

• P<0.01 V5 . Group-N

Figure 2 shows representative photographs of thin sections
stained with hematoxylin-eosin and Azan-Mallory, The nor-
mal heart showed no fibrosi s (3 ± 1%). Among the four
groups with heart failure, the area of myocardial fibrosis was
the lowest in Q20 (32 ± 4% in V, 22 ± 4% in QO.2, 13 ± 3%
in Q2, and 6 ± I% in Q20; p < 0.01, Q2 and Q20 vs. V).

Fig. I . Effects of quinapril on hemodynamic parameters. Although heart
rate (HR), central venous pressure (CVP), mean blood pressure (mean BP)
andpeak left ventricularpressure (LVP)did notdifferamongthe four groups
with heart failure. left ventricular end-diastolic pressure (LVEDP) and ±dPI
dt improved after quinapril treatment in a dose-dependent manner. LVEDP
and ±dP/dt in Q20 were significantly improved relative to those in V (p <
0.05 and 0.01).

Page 79

80

a e

Fig. 2. The effects of quinapril on myocardia l fibrosis. Figures show representative data for each group. Upper panel : Azan-M allory staining. Lower panel :
hematoxylin-eosin staining. a, N; b, V; c, QO.2; d, Q2; and e, Q20 . Scale bar is 5 mm.

Discussion

In the present study, using a rat model of dilated cardiomy-
opathy induced by autoimmune myocarditis, we examined
the effects of the ACE inhibitor quinapril on survival rate,
progression of heart failure and myocardial fibrosis . We
found that quinapril treatment decreased mortality, heart
weight, myocardial fibrosi s and mRNA expre ssion of TGF-
~ I, and improved hemodynamic functions in rats with dilated
cardiomyopathy after myocarditi s.

A disproportionate accumulation of fibrillar collagen and

other components of the extracellular matri x are seen in the
interstitial space in cases with hypertrophy or dilation of the
left ventricle [20]. This interstitial remodeling probably plays
an important role in the changes that occur in coronary blood
flow, the altered biochem istry of the myocardium, and the
compromised myocardial function in heart failure . Several
lines of evidence suggest that angiotensin-II may be involved
in remodeling of the non-myocyte compartment of the heart :
collagen synthesis of rat cardiac fibroblasts was found to be
increased by angiotensin-II in a concentration-dependent
manner, angiotensin-II also reduced collagenase activity in

1. TGF-~l 2. Collagen- ill 3. Fibronectin

A~

A~
A~ ~a

~ a
~ a

B~

B~ B~

~ b ~b ~ b

N v 020 N V 020 N V 020

Fig. 3. Myocardial mRNA expression of transforming growth factor (TGF)-13 I , collagen-III and fibronectin. Although leve ls of expressio n of TGF-13 I, col-
lagen-III and fibronectin mRNA were increased in V, they were suppressed after quinapril treatment. (A) Probe for target mRNA ; (B) Probe for glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH) mRNA; a, Protected band of target mRNA; b, Protected band of GAPDH mRNA .

Page 155

Developments in Molecular and Cellular Biochemistry

Series Editor: Naranjan S. Dhalla, Ph.D., M.D. (Hon.), FACC

1. VA Najjar (ed.): Biolog ical Effects of Glutamic Acid and Its Derivatives. 1981
2. VA. Najjar (ed.) : Immunologically Active Peptides. 1981
3. VA. Najjar (ed.): Enzyme Induction and Modulation . 1983
4. V.A. Najjar and L. Lorand (eds.): Transglutaminase . 1984
5. GJ. van der Vusse (ed.) : Lipid Metabolism in Normoxic and Ischemic Heart. 1989
6. J.Ee. Glatz and GJ . van der Vusse (eds.): Cellular Fatty Acid-Binding Proteins. 1990
7. H.E. Morgan (ed.): Molecular Mechanisms of Cellula r Growth. 1991
8. GJ. van der Vusse and H. Starn (eds.): Lipid Metabolism in the Health and Diseased Heart .

1992
9. Y. Yazaki and S. Mochizuki (eds.): Cellular Function and Metabolism. 1993
10. J.Ee. Glatz and GJ. van der Vusse (eds.): Cellular Fatty-Acid-Binding Proteins, II . 1993
11. R.L. Khandelwal and J.H. Wang (eds.): Reversible Protein Phospho rylation in Cell

Regulation . 1993
12. J. Moss and P. Zahradka (eds.): ADP-Ribosylation: Metabolic Effects and Regulatory

Functions . 1994
13. V.A. Saks and R. Ventura-C1apier(eds.): Cellular Bioenergetics: Role ofCoupled Creatine

Kinases. 1994
14. J. Slezak and A. Ziegelhoffe r (eds.): Cellular Intera ctions in Cardiac Pathophysiology.

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15. J.A. Barnes , H.G Coore, A.H. Mohammed and R.K. Sharma (eds.): Signal Transduction

Mechanisms. 1995
16. A.K. Srivastava and J.-L. Chiasson (eds.): Vanadium Compounds: Bioch emical and

Therap eutic Applications. 1995
17. J.M.J . Lamers and P.D. Verdouw (eds.): Biochemistry ofSignal Transduction in

Myocardium. 1996
18. E.-G Krause and R. Vetter (eds.): Biochemical Mechan isms in Heart Function. 1996
19. R. Vetter and E.-G Krause (eds.): Biochemical Regulation ofMyocardium. 1996
20. GN. Pierce and W.C. Claycomb (eds.): Novel Methods in Molecular and Cellula r

Biochemistry of Muscle . 1997
21. EN. Gellerich and S. Zierz (eds.): Detection ofMitochondrial Diseases. 1997
22. P.K. Singal, V Panagia and G.N. Pierce (eds.): The Cellular Basis of Cardiovascular

Function in Health and Disease. 1997
23. S. Abdel-a1eem and J.E. Lowe (eds.): Cardiac Metabolism in Health and Disea se. 1998
24. A.K. Srivastava and B. Posner (eds.): Insulin Action . 1998
25. VA. Saks, R. Ventura-Clapier, X. Leverve, A. Rossi and M. Rigoulet (eds.): Bioenergetic s

of the Cell: Quantitative Aspects. 1998
26. GN. Pierce, H. Rupp, T. Izumi and A. Grynberg (eds.): Molecular and Cellular Effects of

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28. M.V. Cohen , J.M. Downey, RJ. Gelpi and J. Slezak (eds.): Myocardial Ischemia and
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29. D.A. Bernlohr and L. Banaszak (eds.) : Lipid Binding Proteins within Molecular and
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37. V. Vallyathan, V. Castranova and S. Shi (eds.) : Oxygen/Nitrogen Radicals: Cell Injury and

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38. J.F.C. Glatz (ed.): Cellula r Lipid Binding Proteins. 2002
39. E. Kardami, L. Hryshko and N. Mesaeli (eds.): Cardiac Cell Biology. 2003
40. J.F. Clark (ed.): Guanidino Compounds in Biology and Medicine . 2003
41. P. Zahradka, J. Wigle and G.N. Pierce (eds.): Vascular Biochemistry. 2003
42. J.S.C. Gilchrist, P.S. Tappia and T. Netticadan (eds.): Biochemistry ofDiabetes and

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