BIOCHEMISTRY OF CARDIOVASCULAR SYSTEM

AGNES O. KWENANG

Topic : A. Metabolic regulation in cardiac muscle.
General learning objective Student should be able to understand the general feature of metabolism and its regulation in cardiac muscle.

Specific learning objectives When student have mastered this topic, student should be able to: 1. explain the heart is a largely aerobic organ 2. explain myocardial energy metabolism and its regulation 3. describe cholesterol and lipoprotein turnover correlated with atherosclerosis.

Introduction
The heart is a largely or completely aerobic organ and contain negligible energy reserves in the form of glycogen or lipid. Therefore, the heart also must have a continuous supply of energy.

As prerequisite knowledge, we shall assume that the student has a degree of familiarity with the basic concepts of mechanism of enzyme action, metabolic pathways, electron transport chain and oxidative phosphorylation, and the role of ATP. We shall mainly discuss about cardiac energy metabolism, the regulation of glycolysis and lipolysis.

1. The heart is a largely aerobic organ. CARDIAC MUSCLE
The heart is a muscular organ but one that must maintain continuous rather than intermitten activity. Thus heart muscle, except for short periods of extreme exertion, relies entirely on aerobic metabolism.

It is therefore richly with mitochondria; they comprise up to 40%-50% of its cytoplasmic space, whereas some types of skeletal muscle are nearly devoid of mitochondria.

The heart can metabolize fatty acids, keton bodies, glucose, pyruvate, and lactate. The heart muscle can utilize fatty acids and lactate as energy sources in addition to glucose and ketonbodies.

Fatty acids are the resting heart’s fuel of choice but, upon the imposition of heavy work load or the work load increases dramatically, the heart greatly increases its rate of consumption of glucose or begin to use glucose, which is derived mostly from its own relatively limited glycogen store.

Fig 1. The energy the heart uses to perform pumping the blood is generated through the hydrolysis of ATP to ADP and Pi. ATP constantly generated by the mitochondria that are abundant in heart muscle cells with small amount relatively: 5 umols/g. ATP turn over rate in ATP pool is very fast : 1015 sec.

ATP is used for contraction: 60-70%, and 40% ATP is also used for ion pump by the Ca2+ATPase and Na+/K+-ATPase of the sarcolemma, by the Ca2+-ATPase of SR (Sarcoplasmic Reticulum) to store Ca2+ due to possibly for relaxation in diastole phase , and by biosynthetic processes.

2. Myocardial energy metabolism Mitochondria Fig 2. About 90% of the ATP is synthesized by oxidative phosphorylation from fatty acid oxidation:60-90% and glucose + lactate oxidation: 10-40% in the mitochondria and about 10% by glycolysis, that take place in the cytosol.

Mitochondria are strictly dependent on O2, they mainly oxidize fatty acids (note: of all the food we eat fat has the highest calorie value) and pyruvate arising from the glycolysis of glucose.

Mitochondrial regulation of cell function. Situation in heart muscle. In cardiac muscle, constant contraction utilizes ATP, converting to ADP. There are large number of mitochondria in heart muscle, ensuring that oxidation by the heart is essentially always aerobic.

Campbell1982.138-139
Fig 3

The Regulation of Glucose Oxidation
(Fig.4) A. Activation of glucose transport muscle by insulin or exercise.
into

B. Inhibition of hexokinase by its product, glucose 6-phosphate.

C. Phosphofructokinase is inhibited by ATP (not shown); the inhibition is relieved by AMP and relief is augmented by the product of the reaction, fructose 1,6bisphosphate. The enzyme is inhibited by citrate, which enters the cytosol from mitochondria by an antiport mechanism.

D. The conversion of triose phosphates to pyruvate depends upon availability of ADP, which is also a substrate. Unless ATP is being utilized, the ADP concentration will fall and triose phosphate oxidation will slow.

E. The extent of regulation of pyruvate exchange across the inner mitochondrial membrane is not known. F. The pyruvate decarboxylase component of pyruvate dehydrogenase is inactivated by phosphorylation. Phosphorylation is accelerated by NADH or acetyl coenzyme A, and is inhibited by ADP. G. The citric acid cycle, as well as the electron transport shuttles, hinges upon the availability of ADP to maintain oxidative phosphorylation (not shown).(Fig.5).
Goldstein,1983,3ed,478-479.

Acetyl ko.a
citrate
(-)

Oxaloacetate

*

NADH NAD+

Isocitrate
NADH→ (-) ADP  (+) NAD+ NADH

Malate Fumarate
NAD+

*

α - ketoglutarate

*

QH2 Q

*NADH
succinate
GTP GDP

CoASH

(-)

succinylcoa

CoASH

The Regulation of the Citric Acid Cycle
Since the CAC is in the major routes of fuel combustion in many cells, there must be some control of the rate at which it proceeds (Fig.4). It wouldn’t do to have the oxidative machinery going full tilt like a runaway boiler at times of rest nor would it to do to have it only sluggishly responsive when there is an immediate demand for ATP.

The starred reactions require oxidized coenzymes, and the ratio of oxidized to reduced coenzyme is governed by the availability of ADP and Pi for oxidative phosphorylation (OP). Other specific controls prevent irreversible steps in the cycle from depleting the supply of cofactors and cycle intermediates. The action of negative effectors is indicated by (-); the action of positive effector (+) is indicated by an open arrow next to the reaction arrow.

Stoichiometric Regulation by ADP. Since a major consequence of the action of CAC is the conversion of ADP and Pi to ATP through the mechanism of OP, it follows that the cycle won’t function unless there is supply of ADP and Pi.

ADP concentration as the normal regulating factor, but it may be that the muscular weakness or impaired cardiac performance sometimes seen in patients with low Pi concentration (hypophosphatemia) is due to Pi becoming limiting. (The concentration may be lowered by prolonged use of antacids, which cause passage of insoluble phosphates through the bowel, for example)

Regulation by effectors: a. Citrate synthase reaction. The Km for OAA is in the physiological concentration range; furthermore, citrate is a competitive inhibitor for OAA on the enzyme. The effect is double-barreled.

An accumulation of citrate raises its concentration as an inhibitor, but it also lowers the concentration of OAA as a substrate. Why? Because the complete cycle must function at the same rate to restore the OAA consumed in the first step. Any accumulation of intermediates in the cycle represents a depletion of OAA.

b. Isocitrate dehydrogenase Two important allosteric effects are used here. ADP is a specific activator which lowers the Km for isocitrate. A rise in ADP concentration is a signal of a need for more high-energy phosphate, and the response to this signal includes an accelerated injection of substrate into the CAC.

Isocitrate dehydrogenase is also inhibited by NADH at an allosteric site. Any slowing of OP, which leads to an accumulation of NADH, therefore slows the enzyme in two ways. If the [NADH] is high, the [NAD] must be low, and NAD is required as a substrate for the reaction.

The NADH also combines with the allosteric site to make the enzyme less active. (Any control of Isocitrate dehydrogenase also tends to control citrate synthase because changes in isocitrate concentration are accompanied by changes in citrate concentration. Aconitase rapidly equilibrates the two compound)

c. -ketoglutarate dehydrogenase reaction represents a threat to the supply of Coenzyme A (CoA) for other reaction. Indeed, 70% of the CoA supply in some tissue is present as succinyl CoA under some conditions, even though the enzyme is regulated. The compound tens to accumulate at rest when the phosphate potential is high, owing to the lack of GDP and Pi for its subsequent use.

The extent of accumulation is controlled by constructing the -KG dehydrogenase so that its product, succinyl CoA, is a competitive inhibitor for one of its substrate, CoA. Here again is a doublebarrel effect. A rise in succinyl CoA concentration in itself inhibits, but it also represents a depletion of CoA, causing still more effective inhibition. (Fig 4).

Glodstein,1983,3ed.p437-439.

The Regulation of Mitochondrial Fatty Acid Oxidation
The oxidation of the FAs to acetyl CoA, like the subsequent oxidation of acetyl CoA via the CAC, requires ADP to be available for coupled OP. If there is no demand for high energy phosphate, there is no production of ADP, no electron transport, and no FA oxidation.

However, given a demand for high-energy phosphate, the primary regulation in animal appears to hinge on the amount of substrate available. The 3-oxybutyrates are preferentially used by peripheral tissues when they are available, and high concentration of the FFAs promote the formation of the keton bodies in the liver.
Goldstein 1983,3ed.p.456.

The major energy metabolism pathways. Voet 1995,2nd ed.785-787 Fig 6.

3. Cholesterol and Lipoprotein Turnover correlated with atherosclerosis (CHD)
About two thirds of the cholesterol=ch in the blood is found in LDL. The normal total ch content is 3.1 to 5.7 mM in blood serum, of which roughly one fourth is free ch in the surface of various lipoproteins, and the balance is cholesteryl esters in the lipoprotein cores.

Cholesterol is regarded as a nasty word by many laymen and by my collaborator because of its association with lipoproteins. Anything causing accumulation of the lipoproteins rich in cholesteryl esterchylomicron remnants, IDLs, or LDLs –is almost certain to cause severe atherosclerosis. However, the engulfment of LDLs is a normal device

Ch transporting agent in the blood are LDLs and HDLs
LDL level in blood related to development of atherosclerosis; this result from a chronic inflammatory response that is initiated by the deposition of LDL LDL serves primarily to carry ch molecules from the liver, where they are synthesize and packaged, through the blood to the body’s cells

 HDLs

carries ch in the opposite direction. Excess Ch in transported out of the plasma membrane of the body’s cells directly to circulating HDL particles, which carry ch to the liver for excretion.  High blood levels of LDL are associated with increased of heart disease.  High blood levels of HDL are associated with decreased risk, HDL being called ”good cholesterol”

Cholesteryl ester transfer protein (CETP)
Ch molecules can be transferred from HDL to other lipoprotein particles by an enzyme CETP, an activity that tends to lower HDLch levels. Japanese fam: live more than 100 yr and carry mutations in the CETP A number of small-mw CETP inhibitors in clinical trials, found to greatly increase HDL levels in the blood.

Correlation between the response of inhibitors and CHD is currently under investigation Karp G.2007,5th ed.316-317.

Topic: B. Cardiac marker and Thrombus formation
General learning objective. Student should be able to understand cardiac marker function and thrombus formation

Specific learning objectives
When student have mastered this topic, student should be able to: 1. describe cellular enzyme as biochemical markers of myocardial damage. 2. describe other intracellular protein (non enzyme) as biochemical marker of myocardial damage 3. describe thrombus formation.

Cardiac markers
Introduction Sensitive and specific serologic biomarkers capable of detecting small infarcts (as little as 1.0 g) of dead tissues that may not have been considered an MI in earlier era. Kaplan2003 4th ed.572 Prerequisite knowledge: enzyme kinetics and blood clotting. We’ll mainly discuss: cardiac marker and thrombosis.

The cardiac enzymes historically were - creatine kinase, - aspartate amino transferase - lactate dehydrogenase.

A specific CK isoenzyme (CK-MB) is a more specific indicator of cardiac muscle damage than total CK, and can be detected in serum soon(1-3 hr)after an MI has occurred CK-MB is not completely specific for myocardium. Kaplan 2003 4th ed.573.

Aspartate amino transferase (AST) is a less sensitive index of myocardial damage. It is also hepatic enzyme and levels rise following hepatic congestion due to cardiac failure.

Lactate dehydrogenase (LDH) False positive are also problem, but isoenzyme analysis is available. Its main use is in the retrospective Diagnosis of infarction suspected to have occurred some days previously. Hunt 1982 (1) Nr 19-21 Sept.p.915. Fig.1 Enzyme in serum following an uncomplicated MI

18

Enzyme activity (x-upper limit value)

16

14 12
10 8 6

CK-2 = CK-MB

CK Total

GOT 4 LDH-1 2 LDH-Total 1 2 3 4 5 6

n days after chest pain

Cardiac marker non enzyme
Myoglobin: is a haem-containing protein present in cardiac and skeletal muscle, that also rises early following MI and has also been used in the diagnosis of MI, but is a nonspecific test and appear to offer no significant advantage over CK estimation.

Troponins : are protein that regulate muscle contraction. Isoform of two of these protein T and I isoform, are specific to myocardium, and the introduction of assays for cardiac troponin T (cTNT) and I (cTNI) represents a major development in the biochemical detection of MI. It is possible with troponins to detect infarctions that are orders of magnitude smaller than those detectable with other cardiac marker.

In the early hours following MI myoglobin more sensitive. Also suspected reinfarction cannot reliable be detected by troponins for as much as 2 weeks, since it takes this length of time for troponins to return to normal following an MI Gaw A.3rd ed.50-51.

Homocysteine: accumulation of homocysteine in toxic level   arterial damage.  elasticity deminished and narrowed of vessels caused by over growing connective tissue and deminishing elastic tissue. Abbot Diag.Sep.1998.12th.ed.

Troponins are most useful 12 hours or more after MI, when their diagnostic sensitivity is 100%, ie MI can be excluded with confidence with a negative (normal) troponin result if the sample is taken 12 hours or more after onset of chest pain. However, despite their many potential roles. Troponins should not be seen as’perfect markers.

Plasminogen Activator Inhibitor (PAI-1) Fibrinolytic hypofunction as an atherogenic factor : Lp(a) & LDL ch  increase the synthesis of PAI-1 in endothelium Abbot Diag.April 1998.11th ed.

Biomarker of Congestive Heart Failure
 Atrial

Natriuretic Peptide (ANP) The atrial myocardium secretes ANP when stretched. In response to high cardiac filling response,specialized myocytes in the atria secrete ANP.ANP increases excretion of salt and water by renal tubules.It also has a small vasodilating effect.

Brain Natriuretic Peptide (BNP) Ventricel similar secrete BNP, which increases in heart failure. Level of BNP may be used as a blood test for heart failure. Fagan 2002. 2nd ed.15,62.

Thrombus formation
Pathological clotting A clot is called a thrombus. The occlusion of the vessel is a thrombosis. Three types of thrombi: -red thrombus : consist rbc and fibrin; ,stasis (eg.veins), -white thrombus: consist platelets and fibrin,poor in rbc, blood flow is rapid (eg.arteries) -disseminated fibrin deposit in capillaries. Murray K, 25th ed.752.

-

Atherosclerotic plaque formation is initiated by various types of injury to the endothelial cells that line the vessel, including damage inflicted by oxygen free radicals that chemically alter the LDL-ch particles. The injured endothelium acts as an attractant for white blood cells (leucocytes) and macrophages, which migrate beneath the endothelium and remain there.

The macrophages ingest the ox-LDL, which becomes deposited in the cytoplasm as cholesterol-rich fatty droplets. These cells are referred to as macrophage foam cells. Substances released by the macrophage stimulate the proliferation of smooth muscle cells, which produce a dense, fibrous connective tissue matrix (fibrous cap) that bulges into the arterial lumen. Not only do these bulging lesions restrict blood flow, they are prone to rupture, which can trigger the formation of a blood clot and ensuring heart attack. Karp G, 3rd ed.316.

Acute Myocardial Infarction (AMI)
 Development

of atherosclerosis (usually polygenic) Formation of large thrombus in a coronary artery Deprivation of blood supply (ischemia) to myocardium Shift to anaerobic glycolysis  decreased synthesis of ATP, depletion of adenine nucleotide pool

 Increase

of NADH due to inactive terminal electron transport chain; due to lack of oxygen Accumulation of lactic acid and other metabolits, causing increased intracellular osmolarity and altered membrane permeability Decrease of pH in heart muscle cells Increasingly inefficient contraction of heart muscle

 Cessation of contraction Activation of membrane phospholipases, degradation of proteins by proteases, influx of Ca2+Death of affected area of heart muscle. Murray K, 25th ed.855

Conclusion
We have already discussed: A. Metabolic regulation in cardiac organ. 1. Heart is a largely aerobic organ 2. Cardiac energy metabolism and its regulation ( glycolysis and lipolysis) 3. Cholesterol and Lipoprotein turnover correlated with atherosclerosis

B. Biomarker and Thrombosis. 1. Cardiac markers in Acute Myocardial Infarction : cellular enzyme and other intracellular protein (non enzyme) 2. Biomarker in the diagnosis of heart failure. 3. Thrombus formation in pathological clotting

References
1. _______, Abbot Diagnostic Editorial 11th ed.1

April 1998 2. _______, Abbot Diagnostic Editorial 12th ed. September 1998 3. Barany MK (2002): Biochemistry of Muscular Contraction, Homepage PHYB- BCHE, 19751977. University of Illionis, Chicago 4. Campbell PN, Smith AD (1982): Biochemistry Illustrated, International student ed. Wilture Enterprises (Int.) Ltd/Churchill Livingstone. Hongkong.

5. Fagan T, Sunthareswaran (2002): Cardiovascular System, 2nd ed. Elseviere Science Limited. Mosby. Toronto. 6. Gaw A, et al (2004): Clinical Biochemistry. An Illustrated Colour Text, 3rd ed. Elsevier Limited, Churchill Livingstone. Toronto. 7. Goldstein M (1983): Biochemistry a Functional Approach, 3rd ed. IGAKUSHOIN/Saunders. Tokyo. 8. Hunt D & Kertes P (1982): The management of acute myocardial infarction in Medicine International.(1) Nr 19-21, September.p.915.Medical Education (International) Ltd. Oxford.Great Britain.

9. Kaplan LA et al (2003): Clinical Chemistry. Theory, Analysis, Correlation, 4th ed. Mosby an Affiliate of Elsevier, USA. 10.Karp G (2007): Cell and Molecular Biology. Concepts and experiments, 5th ed. John Wiley & Sons. Inc.Asia. 11.Murray RK et al (2000): Harper’s Biochemistry, 25th ed. Appleton and Lange, USA. 12.Voet D & Voet JG (1975): Biochemistry, 2nd ed. John Wiley&Sons.Inc.Singapore.