Transcript Biochemistry of Cardiac Muscle and Lung

Biochemistry of Cardiac
Muscle and Lung
Jana Novotná
Department of Biochemistry
2nd Faculty of Medicine
Charles University
Specifity of Cardiac
Metabolism

Myocardial function depends on a fine equilibrium
between the work the heart has to perform to meet
the requirements of the body & energy that it is able
to synthesize and transfer in the form of energy-rich
phosphate bonds to sustain excitation-contraction
coupling.

To support high rates of cardiac power, metabolism
is design to generate large amount of ATP.
Specifity of Cardiac
Metabolism
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Heart muscle is highly oxidative tissue.
Mitochondrial respiration produces more than 90% of
energy.
Mitochondria occupy ~30% of cardiomyocyte space.
>95% of ATP formation comes from oxidative
phosphorylation in mitochondria.
~ 60-70% of ATP hydrolysis is used for muscle
contraction, ~30 - 40% for the sarcoplasmic reticulum
(SR) Ca2+-ATPase and other ion pumps.
Regulation of Metabolic
Pathways in the Heart
Glycolysis + b-oxidation  acetyl-CoA  citric acid cycle  NADH, FADH2 
electron transport chain  ATP
W.C. Stanley et all. Physiol. Rev. 85, 2005
Carbohydrate Metabolism
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Glycolytic substrate is derived from exogenous glucose and
glycogen stores.
Glycogen pool in the heart is relatively small (~30 mmol/g wet wt
compared with ~150 mmol/g wet wt in skeletal muscles).
Glucose transport into cardiomyocyte is regulated by
transmembrane glucose gradient and the content of glucose
transporter in the sarcolema – GLUT-4 (lesser extent GLUT-1).
Carbohydrate Metabolism

Insulin stimulation, increased work demand, or ischemia increase
glucose transport and rate of glucose uptake.

Glycolytic pathway converts glucose 6-phosphate and NAD+ to
pyruvate and NADH+H+, generate 2 ATP for each glucose
molecule.
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Pyruvate and NADH+H+ are shuttled to the mitochondrial matrix
to generate CO2 and NAD+ - complete aerobic oxidative
glycolysis.
Carbohydrate Metabolism
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Fosfofructokinase-1 (PFK-1) – key
regulatory enzyme in glycolytic
pathway – catalyzes the first
irreversible step.
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PFK-1 utilized ATP fructose 1,6bisphosphate, is activated by ADP,
AMP and Pi and inhibited by ATP
and fall in pH.

PFK-1 can be also stimulated by
fructose -2,6-bisphosphate (formed
from fructose 6-phosphate by PFK2).
W.C. Stanley et all. Physiol. Rev. 85, 2005
Carbohydrate Metabolism

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) converts
glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate → production of
NADH.
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GAPDH is major regulatory step → accumulation of NADH within
cytoplasm inhibits, and NAD+ activates the GAPDH activity
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Severe ischemia in heart → lactate and NADPH accumulation →
cessation of oxidative metabolism and lactate production.
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Under anaerobic condition pyruvate is converted to lactic acid – nonoxidative glycolysis.
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Lactate is released in the blood stream through specific
monocarboxylate transporter (MCT).
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Critical role of transporter in maintaining the intracellular pH (removes
also the protons produced by glycolysis).
Carbohydrate Metabolism
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In the mitochondria pytuvate is:
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dexarboxylated and oxidized into acetyl CoA by pyruvate
dehydrogenase (PDH)
or carboxylated into oxalacetate by pyruvate carboxylase.
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The control of PDH activity is an essential part of overall
control of glucose metabolism.
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PDH – mitochondrial multicomplex, activity is controlled by
work, substrate and hormones.
Fatty Acid Metabolism
FFA enter the cardiomyocyte by:
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passive diffusion
protein-mediated transport across sarcolema – fatty acid translocase (FAT) or plasma
membrane fatty acid binding protein (FABPpm).
Acyl-CoA synthase (FACS) activates nonesterified FA by esterification to fatty acylCoA.
W.C. Stanley et all. Physiol. Rev. 85, 2005
Fatty Acid Metabolism
Long chain fatty acyl-CoA can be:
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esterified to triglyceride (glycerolphosphate acyltransferase)
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converted to long chain fatty acylcarnitine by carnitine
palmitoyltransferase-I (CPT-I) between inner and outer mitochondria
membranes.
or
W.C. Stanley et all. Physiol. Rev. 85, 2005
Fatty Acid Metabolism
Carnitine acyltranslocase (CAT) transports long-chain acylcarnitine
across the inner membrane in exchange for free carnitine.
Carnitine palmitoyltransferase II (CPT-II) regenerates long chain acylCoA .
W.C. Stanley et all. Physiol. Rev. 85, 2005
Fatty Acid Metabolism
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CPT-1 can be strongly inhibited by malonyl CoA (on the cytosolic
side of the enzyme).
Two isoforms of CPT-1:
 liver CPT-1a and heart CPT-1b
 CPT-1b is 30-fold more sensitive to malonyl-CoA inhibition.
W.C. Stanley et all. Physiol. Rev. 85, 2005
Fatty Acid Metabolism
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Malonyl-CoA - key physiological regulator of FA oxidation in heart (in
malonyl-CoA  FA uptake and oxidation).
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formed from the carboxylation of acetyl-CoA (acetyl-CoA carboxylase – ACC)
from extramitochondrial acetyl-CoA (derived from citrate via ATP-citratelyase
reaction)
rapid rate of turnover in the heart.
ACC activity is inhibited by fosforylation of AMPKacceleration of FA
oxidation.
W.C. Stanley et all. Physiol. Rev. 85, 2005
Fatty Acid Metabolism
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FA undergo b-oxidation generating NADH+H+ and FADH2.
Acetyl-CoA formed in b-oxidation generate more NADH+H+ in
citric acid cycle (CAC).
W.C. Stanley et all. Physiol. Rev. 85, 2005
Interregulation of fatty acid
and carbohydrate oxidation
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The primary physiological
regulator of flux through PDH
and the rate of glucose
oxidation in the heart is fatty
acid oxidation.
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PDH activity is inhibited by
high rate of FA oxidation via
an increase in mitochondrial
acetyl-CoA/free CoA and
NADH/NAD+ which activates
PDH kinase.
Interregulation of fatty acid
and carbohydrate oxidation
Inhibition of FA oxidation
increases glucose and
lactate uptake and
oxidation by:
1.
decreasing citrate levels
and inhibition of PFK
2.
lowering acetyl CoA
and/or NADH levels in
the mitochondria matrix.
Keton Body Metabolism
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During starvation or poorly controlled diabetes the
heart extracts and oxidized ketone bodies (bhydroxybutyrate and acetoacetat).
Low insuline and high fatty acids  ketone bodies.
Ketone bodies become a major substrate for
myocardium.
Ketone bodies inhibit PDH (inhibition of glucose
oxidation) and fatty acid b-oxidation.
Some Aspects of Myocardial
Biochemistry of Heart Failure
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Heart failure reduces the capacity to transduce the
energy from foodstuff into ATP.
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In the advanced stage of HF 
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down regulation in FA oxidation;
increased glycolysis and glucose oxidation;
reduced respiratory chain activity.
Cardiac Muscle and Ischemia
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Coronary artery occlusion → ischemia → significant change in
cell structure, chemistry and function
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loss of contractile function
arrhythmias
cell death
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The decrease of the ATP / ADP, the accumulation of AMP,
inorganic phosphate, metabolic products are removed
(lactate).
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The rapid decline in creatine phosphate - creatine kinase +
ADP → ​ phosphorylation of ADP → ATP (only short-term
mechanism to compensate for reduced ATP production in
mitochondria)
Cardiac Muscle and Ischemia
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Even mild ischemia reduces the concentration of ATP and
creatinephosphate, increases the level of inorganic phosphate
→ activation of glycolysis (glucose needed from the
bloodstream into the heart cells) → increase in the
concentration of pyruvate → conversion by LDH to lactate.
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Prolonged ischemia - the accumulation of substrates (lactate,
NADH+ and H+) → inhibition of glycolysis at the level of
phosphofructokinase and glyceraldehyde-3-dehydrogenase.
Cardiac Markers
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Troponin (T or I) - the
most sensitive and
specific test for
myocardial damage
released during MI from
the cytosolic pool of the
myocytes.
Approximate peak
release in 12 hours in
MI
Cardiac Markers
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Creatin kinase (CK) is relatively specific when
skeletal muscle damage is not present.
CK has two subunits – CK-M (muscle), CK-B (brain)
and mitochondrial CKmi
CK-MM (CK-1) - skeletal muscle 95%, heart 42%,
smooth muscle 2 – 3%
CK-MB (CK2) – skeletal muscle 3%, heart 28%,
smooth muscle 1 – 5%
CK-BB (CK-3) – skeletal muscle 1%, heart 1%,
smooth muscle 87%
Approximate peak release in 10 to 24 hours.
Cardiac Markers
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Lactate dehydrogenase (10 – 24 hours) is not as specific as
troponin.
 LDH is tetramer with 2 subunits – H – heart, M - muscle
 Isoenzymes
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LDH1 (4 H) – heart and red blood cells,
LDH2 (3HM) – heart and reticuloendothelial system,
LDH3 (2H2M) - lungs,
LDH4 (H3M) – kidneys, placenta, pancreas,
LDH5 (4M) – liver and striated muscles
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In normoxia – LDH2 is higher than LDH1
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A high LDH1 level to LDH2 suggest MI
Myoglobin (2 hours) has low specificity for MI – it is high when
muscle tissue is damaged but it lacks specificity.
Cardiac Markers
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Aspartate transaminase (AST)
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was the first used
it is also one of the liver function test
Glycogen phosphorylase isoenzyme BB (GPBB)
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One of 3 isoforms of glycogen phosphorylase exists in heart and
brain tissue
Because of the blood-brain barrier GP-BB can be seen as heart
muscle specific.
One of the "new cardiac markers" which are discussed to
improve early diagnosis in acute coronary syndrome.
Elevated 1–3 hours after process of ischemia.
References
Reviews:
 W.C. Stanley, F.A. Recchia, G.D. Lopaschuk:
Myocardial substrate metabolism in the normal and
failing heart. Physiol. Rev. 85:1093-1129, 2005
 CH. Depré, M.H. Rider, L. Hue: Mechanism of
control of heart glycolysis. Eur. J. Biochem. 258:277290, 1998
 R. Ventura-Clapier, A. Garnier, V. Veksler: Energy
metabolism in heart failure. J. Physiol. 555:1-13,
2003
Biochemistry of
Lungs
Biochemistry of Lungs
• Produce: surfactant
collagen + elastin
mucus (mucopolysacharides + IgA)
•
Activate: angiotensin (ACE in luminal surface of the
•
Inactivate: ROS
pulmonary endothelium)
kinins (hydrolysis of peptide bonds in bradykinin)
serotonin (from mast cells, from blood - MAO)
acetylcholin
detoxication of foreign components
(cytochrom P450 in microsomes)lbiochemistry of
lungs
Pulmonary Surfactant
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surface-active lipoprotein complex formed by type II alveolar cells
complex of proteins and lipids with a hydrophilic and a
hydrophobic region
the hydrophilic head groups facing towards the water and the
hydrophobic tails facing towards the air
reduces surface tension
surface tension is an effect within the surface layer of a liquid that
causes that layer to behave as an elastic sheet
increases pulmonary complience (the ability of the lungs to stretch in a
change in volume relative to an applied change in pressure).
Properties of Surfactant
•
Once secreted to alveolar space, surfactant absorbs
rapidly to the air-liquid interface (a newborn baby´s
first breath).
•
Once in the interface, surfactant films reduces
surface tension when compressed during expiration
(lungs don´t collapse).
•
Surfactant proteins recognize and opsonize
bacterial, fungal, viral surface oligosaccharides.
Lyra, P.P.R.; de Albuquerque Diniz, E.M. Clinics 62: 181, 2007
Structure of Alveolus
http://herkules.oulu.fi/isbn9514270584/html/c273.html
Phospholipides
Nonpolar tail
Polar head
Proteins
Synthesis - epitelial cells
SP-A and SP-D
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large glykosylated proteins ( SP-D
has 355 AA)
 water-soluble
 members of calcium-dependent
carbohydrate-binding collectin family
SP-B and SP-C
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small peptides (35 AA), highly
hydrophobic
 confer surface tension-lowering
properties
 important for spreading of the
surfactant
Proteins
SP-A is responsible for:
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formation of tubular myelin
regulation of phospholipid insertion into the monolayer
modulation of uptake and secretion of phospholipids by type II cells
activation of alveolar macrophages
binding and clearance of bacteria and viruses
chemotactic stimulation of alveolar macrophages
SP-D plays important role in pathogen defence
SP-B and SP-C:
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enhance the biophysical properties of surfactant
assist in rapid insertion of phospholipids into the monolayer and
molecular ordering of the monolayer
Surfactant Metabolism
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DPPC is synthesized
in rER.
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Transferred to the
lamellar bodies together
with SP-B and SP-C (the
lamellar bodies are the
storage and secreting
granules surrounded by
a limiting membrane that
fuses with the plasma
membrane).
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Surfactant secretion
can be stimulated by the
stretching of the type II
epithelial cells, by the
action of beta-agonists,
and purinergic agonists,
such as ATP
Lyra,P.P. R.; de Albuquerque Diniz, E.M. Clinics 62: 181, 2007
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Lamellar bodies and tubular myelin
Lamellar bodies have an acidic internal environment and have high
calcium content.
Control of Surfactant Release
Distortion of cells
Hyperventilation - deep breath, yawn
acetylcholine (large doses)
beta-agonists
purinoreceptors
corticoids (maturity after pre-term birth)
thyroxin
Synthesis
glycerol
glucose from circulation (glykogen)
polar heads
cholin, inositol from circulation
fatty acids
endogenous from lactate
exogenous
Synthesis of DPPC de novo
Glucose
NAD+
glycerol-3-phosphate
Glycogen
NADH
DHAP
palmitoyl-CoA
CoASH
Choline
palmitoyl-G3P
palmitoyl-CoA
ATP
CoASH
ADP
dipalmitoylphosphatidic acid
phosphocholine
H2O
CTP
Pi
PPi
dipalmitoylglycerol
CDP-choline
CMP
DPPC
Function of Surfactant
•
surfactant reduces tension of the air/liquid interface to
near zero
surfactant aids the oxygen to perfuse from the
atmosphere into the pulmonary capillaries
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During the breathing cycle :
DPPC is insert into the monolayer, lowers the
surface tension of air/liquid interface
PG is effective in spreading surfactant
Proteins accelerate the process to lower tension
Reactive Oxygen Species ROS
.
O - + e + H+
2
.
HO2
.
O2- + 2H+ + e
H2O2 + e
.OH + e + H+
.
HO
hydroperoxid radical
2
H+
.
+O 2
hydrogen peroxide
H2 O2
.
OH- + OH
H2 O
superoxid radical
hydroxyl radical
Reactive Oxygen Species ROS
O2 + 2H2
2H2O
Cu,Zn-SOD
O2
e
.
O2
-
e 2H
H2O2
Fe3+
+
Cu
.
e H
.
OH
e H
MPO
OH
H2O
Px
H 2O
HClO
peroxidation of lipids (phospholipids) aldehydes (malonaldehyde)
O3, NO, NOx, SiO2, smoking, infection, radiation, hypoxia/reoxygenation,
ischemia/reperfusion
Reactive Nitrogen Species - RNS
L-arginine
NOS
L-citrulline + NO
HbO2
O2
O2-
nitrate
NO3- + metHb
nitrite
NO2-
peroxinitrite
HOCl + MPO
ONOOH
oxidation
nitration
nitrosation
thiyl radical
S-nitrosothiol
nitrotyrosine
Antioxidant Defence
Free radical scavenging enzymes
Components of antioxidant protection
Enzymatic Antioxidative Defence
tetravalent reduction (ROS generated during oxidative phosphorylation)
acceleration of monovalent reduction:
SOD cytosolic (Cu-Zn)
5
mitochondrial (Mn) 1
extracellular (Cu-Zn)
catalase (heme-containing)
glutathione system GPx (Se)
2 cytosolic, membrane-ass., extracellular
2GSH
NADPH
Rib-6-P
2H2O
GPx
H2O2
GR
reductase
GSSG
NADP+
Glucose
Nonenzymatic Scavengers
vitamin E - lipid peroxyl radicals
vitamin C - O2-, .OH , Fe3+
Fe2+
β-carotene (O2-), uric acid (O2-), glucose (OH),
bilirubin (LOO.)
Fe sekvestration – lactoferrin and transferrin – ferric ions
ceruloplasmin utilize H2O2 for reoxidation of copper
Lung: intracellular enzymes
epithelial lining fluid (GSH 100x higher than in
plasma, catalase, SOD, GPx)
Collagen
90% I and III, type II, V, VIII
Synthesis
Degradation
Deposition
Fibrosis
Emphysema
Degradation – specific MMP´s
TIMP´s – tissue inhibitors of MMP´s