EDUCATION IN HEART VOL 3 - PART 8 potx

The Goldman grading system allowed an estimate of the weighted risk of perioperative cardiac compli-cations based on the presence or absence of clinical factors including the history of

SECTION IX: GENERAL CARDIOLOGY

27 ANAESTHESIA AND THE CARDIAC

PATIENT: THE PATIENT VERSUS THE

PROCEDURE

James B Froehlich, Kim A Eagle

For patients undergoing elective surgery, the most common cause of significant morbidity and

mortality is occurrence of complications related to cardiac disease.1

It is estimated thatapproximately one million patients undergoing surgery each year in the USA suffer aperioperative myocardial infarction.1This is particularly true for those with previous coronarydisease and those facing higher risk surgery Because of this fact, a great deal of research hasfocused on assessing cardiac risk before elective surgery Less attention has been paid to methods

of modifying the risk of cardiac complications attending surgery through medication use or otherstrategies The risk of cardiac complications engenders a sense of conflict in that the patient per-ceives surgery as a threatening foe to be overcome: the patient versus the procedure We would like

to change that paradigm, and encourage an appreciation for the risk inherent to the patient,rather than to the procedure itself That is, preoperative evaluation of the patient’s risk, versus theprocedure’s risk

During the past 10–20 years, the assessment of cardiac risk before surgery evolved a great deal tially, the focus was on appropriate identification of surgical procedures that carried high risk Thefocus then shifted to identifying those patient factors associated with increased risk of cardiac com-plications during surgery Several technical advances were made during this time, including theintroduction of imaging stress tests to assess cardiac ischaemia, such as dobutamine echocardiogram,dobutamine thallium, and adenosine or dipyridamole thallium testing All of these modalities havebeen shown to identify patients at increased risk for cardiac complications of surgery Cardiac cath-eterisation has also been used as a screening modality before elective surgery, though this has notbeen shown to be cost effective, especially given the low overall incidence of severe coronary arterydisease Studies performed during this time period also delineated the clinical factors that identifypatients at increased risk of cardiac complications More recently, efforts have been made to combineboth clinical evaluation and testing in the most efficient and appropriate manner to identify patients

Ini-at risk of cardiac complicIni-ations Finally, recent studies have addressed the effectiveness of medicIni-ations

or interventions to decrease risk in high risk patients

Preoperative cardiac evaluation has several goals:

c evaluate and assess perioperative cardiac risk, and provide this information to both patient andsurgeon for decision making purposes

c optimise appropriateness of preoperative testing and/or intervention

c to the extent possible, adjust care in order to decrease operative risk

c given the prevalence of coronary disease and its complications, assess and intervene to modifylong term risks for cardiovascular disease

We discuss below the current state of preoperative cardiac evaluation and interventions to decreaseperioperative cardiac risk, and offer an approach to the preoperative assessment and perioperativecare of patients with cardiac disease, focusing on risk reduction

Historically, the preoperative assessment of patients before elective surgery was based almostentirely on the clinical evaluation and examination The American Society of Anesthesiology hasused the ASA physical status classification system (1963) to grade perioperative risk This classifi-cation could identify those at extremely high risk of complications from surgery, but did not offermuch sensitivity in assessing patients’ risk The patients in level IV or V were at extremely elevatedrisk, but patients categorised in level III constituted a very wide spectrum of risk and comorbiddisease Furthermore, the ASA classification system does not focus on cardiac risk per se It offers

no consideration for the presence or absence of serious coronary disease in otherwiseasymptomatic or undiagnosed patients

Lee Goldman, then a resident at Massachusetts General Hospital, conducted a study thatidentified clinical factors conferring elevated risk of surgical complications.2

By performing amultivariate logistic regression analysis of a wide range of clinical parameters on 1000 consecu-tive patients undergoing elective surgery at the Massachusetts General Hospital, Goldman and his

colleagues identified clinical markers of increased risk, and

appropriately weighted them based on the epidemiological

risk they conferred The Goldman grading system allowed an

estimate of the weighted risk of perioperative cardiac

compli-cations based on the presence or absence of clinical factors

including the history of recent myocardial infarction,

presence of congestive heart failure, critical aortic stenosis,

significant non-cardiac organ failure or disease, urgency of

surgery, and advanced age The presence of these factors,

par-ticularly when added together, correlated with elevated risk

However, the majority of patients studied did not have

mark-ers of high risk and the index proved to be insensitive for

dis-criminating risk in patients who would be considered

intermediate in risk The Goldman index did not include

evaluation by objective stress testing, nor does it allow one to

infer a plan for appropriate further steps in the evaluation

process

Several other studies have confirmed the utility of clinical

evaluation in identifying patients at increased risk of

significant coronary disease L’Italien and others reviewed the

clinical risk factors of patients undergoing elective vascular

surgery at Massachusetts General Hospital, University of

Massachusetts Medical Center, and the University of Vermont

Medical Center, and analysed these clinical risk assessments

with the results of thallium functional testing, also done

before surgery.3

This group initially identified a small list ofclinical factors that conferred risk based on multivariate

logistic regression analysis These clinical factors are advanced

age, a history of diabetes, myocardial infarction, angina, or

congestive heart failure This group’s findings, corroborated by

other groups, revealed that the absence of any of these clinical

markers of risk conferred a very low risk of complications of

surgery (3% in this study) Likewise, the presence of one or

two of these factors conferred a moderately increased risk

(8%) and the presence of three or more a high risk of death or

myocardial infarction during vascular surgery (18% in this

study)

Paul and colleagues reviewed an extensive database of

car-diac catheterisation results on 878 consecutive patients

undergoing elective vascular surgery at the Cleveland Clinic.4

They reviewed these same five clinical markers of risk, and

observed that the presence of three or more of these clinical

markers was coincident with a high likelihood of three vessel

or left main coronary artery disease Similarly, the absence of

any of these markers of risk was coincident with a very low

likelihood of having severe coronary artery disease on

catheterisation Taken together, these studies of clinical

mark-ers of cardiac risk suggest that patients who are properly

evaluated, and have none of these clinical markers of risk,

have a very low likelihood of suffering cardiac complications

of surgery This finding has recently been corroborated in

clinical trials of the effect of perioperative β blockade on

cardiac complications

NON-INVASIVE TESTING

The introduction of sensitive non-invasive tests for coronary

artery disease, particularly pharmacologic stress tests that

require no treadmill exercise, has greatly influenced the

preoperative assessment of cardiac risk Several early studies

demonstrated a very high sensitivity of these tests for

identi-fying patients at increased risk of perioperative cardiac

complications Most impressively, these results have been

repeatedly duplicated by a large number of investigators In an

important work on the subject, Boucher and others strated that thallium testing before elective vascular surgeryaccurately identified those patients who suffered cardiac com-plications of surgery.5

demon-Furthermore, those patients with a mal thallium study had a very low incidence of cardiaccomplications This was followed by several other studies,which demonstrated essentially similar results Taken to-gether, the clinical studies of thallium testing before vascularsurgery have shown strikingly consistent results These are avery high sensitivity (between 85–100%), but a fairly low spe-cificity for the identification of patients who suffer cardiaccomplications of surgery For this reason, the negative predic-tive value of thallium is quite high, better than 95%, evencombining all current clinical studies The positive predictivevalue, however, is quite low because of the low specificity (aproblem of false positive tests in lower risk patients) Thismakes thallium stress testing a reassuring test when negative,but clinically confusing when positive Such results highlightthe fact that thallium testing is inappropriate as a uniformscreening test, particularly when applied to “low risk”individuals

nor-Fewer studies have examined dobutamine echocardiogram

as a preoperative screening modality; however, the results arequite similar to those found with thallium testing There issimilar sensitivity with the same problem of relatively lowspecificity At institutions that have established proficiency atdobutamine echocardiogram testing, the results are consid-ered interchangeable with thallium testing Dobutamineechocardiography has the advantage of providing informationregarding valvar structure and function

Exercise tolerance testing, without cardiac imaging, alsohas an important role in screening for cardiac risk Exercisetolerance, combined with electrocardiographic interpretation(assuming a normal baseline ECG), has great prognosticpower for the patient with known or suspected coronary dis-ease Similarly, the ability to achieve maximum predictedheart rate without ECG confers a low risk for cardiac compli-cations of elective surgery Because it evaluates exercise toler-ance and gives an idea of the level of stress that may induceinducible ischaemia, exercise testing is generally preferable topharmacologic testing, particularly for long term prognostica-tion

Because of the relatively non-specific nature of functionaltesting, it is best employed as a component of an organisedprogramme for cardiac risk evaluation Proper clinical assess-ment of pre-test probability of significant coronary diseasewill allow more prudent use and interpretation of ischaemiatesting

Invasive testing has been proposed as a screening modalityfor patients undergoing high risk surgery—for example,peripheral vascular reconstructions Hertzer and colleaguesreported from the Cleveland Clinic on the use of routine cath-eterisation on 1000 consecutive patients scheduled for vascu-lar surgery.6

Although they reported a high incidence ofpatients with severe coronary disease, requiring coronarybypass grafting, subsequent review of the data suggests thatmost of those patients with coronary disease sufficientlysevere to warrant revascularisation could be identified onclinical grounds This, and the expense and risk of routinecatheterisation, have led most to consider clinical andfunctional assessment as initial screening for cardiac risk

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METHODS FOR LOWERING PERIOPERATIVE

CARDIAC RISK

Coronary bypass surgery

Recently, attention has turned to evaluating the effectiveness

of methods for intervening to lower risk of cardiac

complica-tions during elective surgery Coronary revascularisation is

one such intervention A retrospective review by Eagle and

colleagues of the CASS (coronary artery surgery study)

registry data supports such a protective effect.7These data

demonstrate that patients undergoing elective vascular

surgery, who had previously undergone coronary artery

bypass grafting, did better than control patients who had

similar amounts of coronary disease, but no surgical coronary

revascularisation This type of analysis does not take into

consideration the cumulative risk of both coronary and

peripheral revascularisation, and so does not necessarily

argue for prophylactic surgical coronary revascularisation

before elective peripheral vascular surgery But it does

suggest a protective effect of prior coronary bypass surgery

Data from the Cleveland Clinic showed similar findings—that

patients with a history of coronary artery bypass grafting,

regardless of clinical risk factors, had lower perioperative

car-diac complication rates surrounding vascular surgery than

patients with coronary disease managed medically These

studies argue that a history of successful coronary artery

bypass surgery confers a lower risk of cardiac complications

surrounding elective surgery

Percutaneous coronary intervention

The discovery of ischaemia on functional testing frequently

leads to consideration of percutaneous revascularisation before

elective vascular surgery This practice has not been subjected to

randomised controlled trials to assess its efficacy Trials are

cur-rently underway for this purpose Previous randomised studies

comparing medical treatment with angioplasty in patients with

stable coronary disease of limited severity have demonstrated

an increased event rate in those patients undergoing

angio-plasty The bulk of this increase came in the form of

periprocedural complications Retrospective studies reporting

the rates of perioperative cardiac complications in patients who

underwent previous preoperative angioplasty and/or coronary

stent placement have shown very mixed results Posner and

colleagues reported a lower rate of cardiac complications among

patients who underwent angioplasty before surgery compared

with a group of patients with coronary artery disease managed

medically.8

This study is uncontrolled for severity of disease or

medical management, however Massie and associates

per-formed a case–control study comparing patients with abnormal

thallium studies who did and did not undergo angiography

before vascular surgery, and found no difference in event rates.9

Hassan and colleagues found similarly low rates of cardiac

complications after non-cardiac surgery among patients in the

BARI (bypass angioplasty revascularization investigation)

study.10This was equally true for patients who had undergone

multivessel percutaneous coronary intervention, as for those

who underwent coronary bypass surgery Finally, Kaluza and

colleagues reported a very high incidence of stent thrombosis,

death, and myocardial infarction in patients undergoing

non-cardiac surgery within two weeks of coronary stent

placement.11

These data raise concern that the strategy of

prophylactic, percutaneous coronary revascularisation before

elective surgery may result in destabilisation of previously stable

coronary disease which offsets the potential advantage of

improving ischaemic thresholds of the heart by reducing severe,

fixed coronary stenoses

Perioperative medical treatmentSeveral recent studies have suggested thatβ blockers decreaserisk of perioperative complications A randomised study byMangano and colleagues evaluated brief courses of peri-operativeβ blockade in patients undergoing a variety of surgi-cal procedures.12The study was small, and demonstrated nodifference in perioperative complication rate However, overthe succeeding two years, the patients who received this briefcourse of perioperativeβ blockade had a lower incidence ofcardiac events This study did not control for medicationsbetween the two groups, but at least raises the question of aprotective effect of perioperative β blockade A more recentstudy by Poldermans and associates randomised only clini-cally high risk patients undergoing elective, major vascularsurgery, to the β blocker bucindolol or placebo.13This studydemonstrated a significant reduction in perioperative cardiacevents, both fatal and non-fatal, with the use of aβ blocker

These patients were givenβ blocker treatment days or weeksbefore surgery Theβ blocker was titrated to a target dose of 10

mg per day, so long as the heart rate remained above 60 beatsper minute These studies, combined with previous investiga-tions that show a protective effect of β blockers for bothambulatory and perioperative ischaemia, support the hypoth-esis that perioperative β blockade decreases cardiac riskamong high risk patients

Finally, several recent studies evaluated the effect of αreceptor agonists in the perioperative period on the incidence

of cardiac events In a large randomised controlled trial ofintravenous α2agonist mivazerol during surgery, Oliver andcolleagues compared outcomes during surgery in patientswho had either a history of coronary artery disease, or thepresence of significant risk factors.14

They found no significanteffect in the patients undergoing non-cardiac surgery in gen-eral, but a significant reduction in both cardiac events anddeath in the subset of patients undergoing vascular surgery

Mangano and colleagues reported the results of a randomisedtrial of the same agent in 300 patients undergoing non-cardiacsurgery, and found no significant effect on cardiac events.15These and other studies at least raise the possibility that intra-operativeα agonists may reduce perioperative cardiac events

These reports certainly raise hope for therapeutic intervention

to lower perioperative risk of cardiac events The initialβ blockerstudy of Poldermans and colleagues demonstrated benefit in ahigh risk cohort of patients undergoing vascular surgery.13

Morerecent data from the same group suggests benefit fromβ block-ade across all risk groups This requires prospective trialvalidation Currently, it seems quite reasonable to use theAmerican College of Cardiology/American Heart Association(ACC/AHA) guidelines to assess risk,16

and considerβ blockade

in any patients at increased risk not already taking them Therole ofα agonists is less clear The above mentioned studies sug-gest some benefit from their use in patients undergoing vascu-lar surgery, but little is known about these patients, and whatthe indications for use of this agent would be

PUTTING IT ALL TOGETHERThe past two decades have answered many questions aboutperioperative cardiac complications, and who is at increasedrisk for them As discussed above, we have a goodunderstanding of what constitutes a high risk patient, andwhat tests are useful in further defining risk The ACC/AHApreoperative evaluation guidelines describe a method of inte-grating these data into an efficient, evidence based approach

to evaluating cardiac risk.16

This approach incorporates three

ANAESTHESIA AND THE CARDIAC PATIENT: THE PATIENT VERSUS THE PROCEDURE

steps: first, a clinical evaluation to determine the patient’s

likelihood of significant coronary disease, and perioperative

cardiac event risk; second, selective use of non-invasive testing

to further refine risk assessment; and third, intervention to

further assess and/or modify cardiac risk This approach

should be taken with the patient’s lifetime risk of cardiac

dis-ease manifestations as the end point, not just the

peri-operative period The following algorithm outlines this

approach (fig 27.1)

The first step in this algorithm is to determine urgency of the

planned surgery Obviously, emergent surgery should proceed

without the delay of cardiac evaluation Any surgical procedures

not felt to be emergent allow for more thorough evaluation of

cardiac risk For patients who have undergone coronary

revascularisation within the previous five years, without any

recurrent symptoms of cardiac disease, further evaluation is

probably unnecessary (step 2) If previous, recent, (within two

years) adequate cardiac evaluation has taken place, without any

change in clinical status, then there is usually no need to repeat

before surgery, if the results indicated low risk (step 3) Finally,

a thorough clinical evaluation should be undertaken todetermine if major markers of risk are present (for which

Figure 27.1 Algorithm for cardiac risk assessment before non-cardiac surgery Hx MI, history of myocardial infarction; CHF, congestive heart failure; DM, diabetes mellitus.

Poor

<4 METs

Moderate to excellent ≥4 METs

Intermediate (>2) clinical predictors

Minor or no clinical predictors

Major clinical predictors (A)

Non-invasive testing

Operating room

Non-invasive testing

Operating room

4 Clinical assessment (Hx MI,CHF, DM, angina, age > 70)

3 Recent coronary evaluation 2

1

Coronary revascularisation within 5 years

If yes, and no recurrent symptoms

Need for non-cardiac surgery Clinical evaluation steps

(1) Emergency surgery

(2) Prior coronary revascularisation

(3) Prior coronary evaluation

(4) Clinical markers of risk?

ischaemic risk by clinical symptoms or non-invasive study

Soci-ety class III or IV)

c Significant arrhythmias

underlying heart disease

c Supraventricular arrhythmias with uncontrolled ventricular rate

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cardiac catheterisation should be considered), or if any of the

five clinical markers of risk are present (history of myocardial

infarction, diabetes mellitus, congestive heart failure, angina,

age > 70 years) A decision about stress testing is based on the

clinical markers of risk present, the patient’s functional capacity

by history, and the expected cardiovascular stress posed by

non-cardiac surgery (fig 27.1)

In this way, a systematic approach, based on the current

lit-erature and validated prediction tools, can guide the

assessment of risk, and the prudent use of further diagnostic

testing of cardiac risk before non-cardiac surgery As stated

above, this systematic approach does not rely on testing, but

incorporates clinical evaluation with objective testing to define

cardiac risk of non-cardiac surgery optimally

REFERENCES

1 Mangano DT, Goldman L Preoperative assessment of patients with known or suspected coronary disease N Engl J Med 1995;333:1750–6.

c Excellent review of the topic.

2 Goldman L, Caldera DL, Nussbaum SR, et al Multifactorial index of cardiac risk in non-cardiac surgical procedures N Engl J Med 1977;297:845–50.

c Seminal work, identifying for the first time the magnitude and nature of the impact on perioperative cardiac risk of several important clinical findings.

3 L’Italien GJ, Paul SD, Hendel RC, et al Development and validation

of a Bayesian model for perioperative cardiac risk assessment in a cohort

of 1,081 vascular surgical candidates J Am Coll Cardiol 1996;27:

779–86.

c This study shows the relative roles of clinical and radiological evaluation of patients before vascular surgery, and the value of combining the two to improve accuracy and decrease testing.

4 Paul SD, Eagle KA, Kuntz KM, et al Concordance of preoperative clinical risk with angiographic severity of coronary artery disease in patients undergoing vascular surgery Circulation 1996;94:1561–6.

c Confirmatory study, documenting the power of clinical risk factors

to predict severity of coronary disease.

5 Boucher CA, Brewster DC, Darling RC, et al Determination of cardiac risk by dipyridamole-thallium imaging before peripheral vascular surgery.

N Engl J Med 1985;312:389–94.

6 Hertzer NR, Beven EG, Young JR, et al Coronary artery disease in peripheral vascular patients: a classification of 1000 coronary angiograms and results of surgical management Ann Surg 1984;199:223–32.

c Early, single centre report of findings on routine cardiac catheterisation in patients undergoing vascular surgery.

7 Eagle KA, Rihal CS, Mickel MC, et al Cardiac risk of noncardiac surgery: influence of coronary disease and type of surgery in 3368 operations CASS Investigators and University of Michigan Heart Care Program Coronary artery surgery study Circulation 1997; 96:1882–7.

8 Posner KL, Van Norman GA, Chan V Adverse cardiac outcomes after noncardiac surgery in patients with prior percutaneous transluminal coronary angioplasty Anesth Analg 1999;89:553–60.

9 Massie MT, Rohrer MJ, Leppo JA, et al Is coronary angiography necessary for vascular surgery patients who have positive results of dipyridamole thallium scans J Vasc Surg 1997;25:975–82; discussion 982–3.

10 Hassan SA, Hlatky MA, Boothroyd D, et al Outcomes of non-cardiac surgery after coronary bypass surgery or coronary angioplasty in the bypass angioplasty revascularization investigation (BARI) Am J Med (in press).

c BARI study data suggesting low rates of cardiac events after either coronary bypass surgery or multi-vessel coronary angioplasty before non-cardiac surgery.

11 Kaluza GL, Joseph J, Lee JR, et al Catastrophic outcomes of non-cardiac surgery soon after coronary stenting J Am Coll Cardiol

2000;35:1288–94.

c One of few observations about the important question of timing of surgery after percutaneous coronary intervention (PCI) This study suggests significant risk associated with stopping antiplatelet agents within 2–4 weeks of PCI.

12 Mangano DT, Layug EL, Wallace A, et al Effect of atenolol on mortality and cardiovascular morbidity after noncardiac surgery Multicenter study

of perioperative ischemia research group N Engl J Med 1996;335:1713–20.

B Estimated functional capacity, based on daily

activities

1 MET

↓

c Can you take care of yourself?

c Eat, dress or use the toilet?

c Walk indoors around the house?

c Walk a block or two on level ground at 2–3 mph or

c Climb a flight of stairs or walk up a hill?

c Walk on level ground at 4 mph or 6.4 km/h?

c Run a short distance?

c Do heavy work around the house like scrubbing floors

or lifting or moving heavy furniture?

c Participate in moderate recreational activities like golf,

bowling, dancing, doubles tennis, or throwing a

baseball or football?

c Participate in strenuous sports like swimming, singles

tennis, football, basketball, or skiing?

>10 METs

METs, metabolic equivalents

C Risk stratification for non-cardiac surgical

procedures

Major (reported cardiac risk often > 5%)

c Emergent major operations, particularly in the elderly

c Aortic and other major vascular

c Peripheral vascular

c Anticipated prolonged surgical procedures associated

with large fluid shifts and/or blood loss

Intermediate (reported cardiac risk 1–5%)

c Carotid endarterectomy

c Head and neck

c Intraperitoneal and intrathoracic

+ Evaluation based on cardiac risk, not pendingsurgery

+ Utilise history, physical, and ECG findings to stratifyclinical risk

+ Further evaluation (for example, stress testing,catheterisation), based on clinical evaluation, andprobability of disease

+ Use stress testing to modify, not co-opt, pre-testinglikelihood of disease

+ Decision regarding stress testing, cardiac tion, or revascularisation, based on algorithm+ β Blockade indicated for higher risk patients undergo-ing vascular surgery

catheterisa-ANAESTHESIA AND THE CARDIAC PATIENT: THE PATIENT VERSUS THE PROCEDURE

13 Poldermans D, Boersma E, Bax JJ, et al The effect of bisoprolol on

perioperative mortality and myocardial infarction in high-risk patients

undergoing vascular surgery Dutch echocardiographic cardiac risk

evaluation applying stress echocardiography study group N Engl J Med

1999;341:1789–94.

14 Oliver MF, Goldman L, Julian DG, et al Effect of mivazerol on

perioperative cardiac complications during non-cardiac surgery in patients

with coronary heart disease The European mivazerol trial (EMIT).

Anesthesiology 1999;91:951–61.

15 Anon Perioperative sympatholysis Beneficial effects of the alpha 2-adrenoceptor agonist mivazerol on hemodynamic stability and myocardial ischemia, MeSPI –Europe research group Anesthesiology 1997;86:346–63.

16 Eagle KA, Brundage BH, Chaitman BR, et al Guidelines for perioperative cardiovascular evaluation for non-cardiac surgery Circulation.

1996;93:1278–317.

c Current ACC/AHA guidelines for cardiac evaluation prior to non-cardiac surgery Contains a very thorough review of the entire English language literature.

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28 MYOCARDIAL MOLECULAR

BIOLOGY: AN INTRODUCTION

Nigel J Brand, Paul J R Barton

The recent publication of draft copies of the human genome sequence from both public and

private sector consortia has fuelled anticipation that eventually, once all genes have been

identified, we will be able to ascertain which of them are involved in human diseases,

includ-ing those affectinclud-ing the cardiovascular system Understandinclud-ing the molecular biology behind both

inherited and acquired disorders is now viewed as essential to provide a full picture of the aetiology

and progression of disease Within the past decade considerable advances have been made in

iden-tifying the genetic basis of myocardial disorders such as familial hypertrophic cardiomyopathy and

dilated cardiomyopathy, as well as the molecular signalling pathways and gene regulatory events

that characterise acquired disease such as pressure overload induced cardiac hypertrophy

Further-more, by defining the molecular processes underlying normal development we may be able to

manipulate immature cell phenotypes such as those of embryonic stem cells or skeletal myoblasts

to replace damaged, terminally differentiated cells such as cardiac myocytes In this review we

out-line the basic principals of gene expression, the different mechanisms by which expression is

regu-lated and how these can be examined experimentally

The blueprint for any organism is contained within its genome in the form of chromosomes and is

written in the universal four “base” language of adenine (A), guanine (G), cytosine (C), and

thym-ine (T) Chromosomes are built of chromatin, double stranded DNA wrapped around a

multi-protein complex core comprised of histone proteins This DNA contains the language (DNA

or nucleotide sequence) that can be read and translated into proteins, and these areas of DNA are

called genes.1 2In higher organisms, ranging from yeast to plants and man, practically all genes are

interrupted, with sequences coding for protein (coding exons) separated by regions of non-coding

DNA called introns The beginning and ends of genes are usually marked by exons that do not

code for parts of the protein, the so called non-coding exons Within coding exons, contiguous

groups of three bases (codons), form the genetic code The 64 (43) individual codons specify for the

20 amino acids from which proteins are made, or signal the start (initiator methionine codon) or

end (stop codon) of translation It is therefore the contiguous order of the codons within a gene

that delineate the linear amino acid sequence of the protein produced

In general, gene expression describes the production of RNA and, subsequently, protein from

a gene This process can be split into three major parts: transcription of the gene in the nucleus

to make primary RNA, splicing of the primary transcript to form the mature messenger RNA

(mRNA) and translation of mRNA in the cytoplasm to produce the protein for which the gene

codes (fig 28.1) Transcription is carried out by an enzyme called RNA polymerase II (RNA pol II)

under the direction of specialised basal transcription factors that form a multi-protein complex

with RNA pol II on the gene promoter.1The promoter contains the start site of transcription,

usu-ally designated +1, which marks the beginning of the first exon of the gene and hence corresponds

to the first nucleotide of the mRNA Binding sites for various transcription factors, which are

DNA binding proteins with highly specific affinities for particular DNA sequences, sequester

tran-scription factors to the gene where they participate in boosting (or, in some cases, repressing) the

level of transcription Transcription factors may bind within the promoter or lie within areas called

enhancers that are located distally—usually upstream, but occasionally downstream, of the

pro-moter Considerable effort has focused on identifying promoter and enhancer sequences

responsi-ble for directing gene transcription and the identification of the factors that act on them.3

Oncebound to their cognate DNA sequences, transcription factors help drive the rate at which RNA pol

II initiates fresh rounds of transcription The polymerase moves along the gene making a primary

RNA copy of one strand of the DNA duplex, copying both exonic and intronic sequences This

pri-mary RNA transcript is subsequently processed to remove the intron derived sequences and the

exons joined together (RNA splicing) Following some 5′ and

3′ modifications, such as the addition of a 3′ poly-adenylic acid

tract (polyA tail), the final mRNA product is exported from the

nucleus to the cytoplasm, where it serves as a template for the

production of protein by the ribosomes Subsequent

post-translational modifications such as cleaving off any propeptide

or leader sequences which direct the protein to its ultimate

destination in the cell or the attachment of phosphate or

acetyl groups to specific amino acid residues may be necessary

to produce the final functional form of the protein

CONTROL POINTS FOR GENE EXPRESSIONAll of the stages of gene expression are points at which regu-lation can be exerted However, the primary point of control is

at the level of transcription Many promoters and enhancers ofmyocardial genes have been cloned and transcription factors

Figure 28.1 The process of gene expression Chromosomes are scaffolds of DNA organised around protein (histones) in units called nucleosomes DNA is unwound from histones before transcription of a gene by RNA polymerase II and transcription factors (coloured) The primary RNA transcript, which is a copy of both exonic (red) and intronic (blue) DNA sequences, is processed subsequently to remove intronic sequences (mRNA splicing) The resulting mRNA is then exported to the cytoplasm for translation and subsequent post-translational modification such as methylation, glycosylation or phosphorylation Detection of mRNA by northern blot is illustrated: the blot shows that mRNA for the slow skeletal isoform of troponin T (TnTs) is expressed only in adult skeletal muscle (Sk) and not in fetal (F) or adult (H) heart or liver (L).

Rehybridisation of the blot with a probe to 18S rRNA (18S) shows presence of RNA in each lane Protein expression is analysed by western blotting: in the example shown, a universal antibody recognising all three troponin I (TnI) isoforms shows distribution of fast skeletal (f), slow skeletal (s) and cardiac (c) isoforms in adult skeletal muscle (Sk) and fetal heart (F) Figure courtesy of KA Dellow.

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active in heart and belonging to a variety of gene families have

been identified in recent years (table 28.1).4–9

The ing observation that can be drawn is that expression of

overwhelm-individual genes is regulated (1) through the coordinate

bind-ing of different types of transcription factors, (2) by

interactions between factors and with ancillary co-factors

such as histone acetylases (HATs) or deacetylases (HDACs),

which do not necessarily bind to DNA themselves, and (3)

through signal transduction pathways which influence their

activity by, for example, phosphorylation.3

Most transcriptionfactors are modular in structure, containing separable protein

domains that carry out a particular function such as DNA

binding, dimerisation with other family members (for

exam-ple, the related bHLH proteins HIF-1α and β, products of

separate genes9

) or serving as transcriptional activation

domains (TADs) to promote high level transcription

Ulti-mately, once bound to DNA transcription factors interact with

other bound proteins and act to increase the rate of RNA

syn-thesis TAD activity, which is often measured by introducing

cloned transcription factors into cells grown in culture (see

below) may be intrinsic or may reflect the binding of a

co-activator or co-factor protein which itself possesses

signifi-cant activation properties For example, myocardin is a

recently identified heart restricted co-factor for the

ubiqui-tously expressed serum response factor (SRF).6

SRF binds tothe promoters of several genes expressed in heart, including

cardiacα actin, and has been shown to interact with many

factors including the homeobox factor Nkx-2.5 and the zinc

finger factor GATA-4 to regulate expression In contrast to

Nkx-2.5 and GATA-4, which are transcriptional activators in

their own right, myocardin does not bind to DNA but

complexes with bound SRF and serves as an extremely potent

co-factor for transcription, promoting up to a thousand-fold

activation in combination with SRF

Transcription factors may be expressed in a highly tissue

restricted manner or at particular developmental stages, in

turn regulating the expression of their target genes For

example, GATA factors are expressed from the earliest

detect-able stages of cardiogenesis and may play a role in gene

regu-lation at this stage.10

Later, Nkx2.5 shows regional variation in

expression and may play a specific role in the developing

myo-cardial conduction system Most transcription factors can be

grouped into gene families on the basis of sequence similarity

in regions of functional importance, such as a DNA binding

domain or a protein dimerisation interface Such a high

degree of sequence homology allows new family members to

be discovered by searching DNA or protein sequence databases

across diverse phyla In this way Nkx-2.5 was identified as the

mammalian homologue of a Drosophila gene called tinman, (named after one of the characters in The Wizard of Oz)

originally identified as a mutation that resulted in lack ofdevelopment of the heart equivalent in the fly, the dorsalvessel.8In humans, mutations in the Nkx-2.5 gene have been

correlated with a variety of cardiac anomalies includingtetralogy of Fallot and idiopathic atrioventricular block.11Currently, there is renewed interest in the role that chroma-tin structure plays in regulating gene expression.12It has longbeen known that when DNA is wrapped around histones inthe form of chromatin, gene activity is silenced, and that thelocalised unwinding of the DNA from chromatin, accompa-nied with histone displacement, is vital to allow gene expres-sion to progress Central to recent studies has been the identi-fication and biochemical characterisation of proteins thatpossess HAT or HDAC activity, adding or removing, respec-tively, acetyl groups from exposed lysine residues on histones

HAT activity correlates with activation of gene expression,while HDAC activity results in repression.13In several cases thefunctions of these proteins have been shown to be intimatelylinked to the state of transcription factors binding to targetgenes For example, the active heterodimeric form of the basichelix-loop-helix transcription factor HIF-1 (table 28.1) senseschanges in partial pressure of oxygen and thus acts as ahypoxia sensor in several systems, including angiogenesis andvascular remodelling.9

Once bound to the DNA of target genesfor regulation, C terminal TADs in the HIF heterodimer inter-act with transcriptional co-activators such as the CREB-binding protein, CBP (In cardiac muscle, the most likelyco-activator is the related protein p300) These large proteinspossess intrinsic chromatin remodelling activity by recruiting

to the DNA still more proteins which allow chromatin tounwind Probably the best understood system is currently thatinvolving retinoid and steroid hormone receptors such as thy-roid hormone receptorα1 (TRα1) that, once it has bound itscognate ligand T3, activates expression of genes such ascardiac α myosin heavy chain.2

The binding of T3 to thehormone binding domain of TRα1 results in a conformationalchange in the proteins’ structure, allowing the receptor tointeract with co-activators The net result is that HAT activitypromotes localised unwinding of chromatin, allowing access

of RNA pol II and basal transcription factors to the DNA Inthe absence of T3, the nuclear receptor still binds to DNA butinteracts instead with co-repressors such as N-CoR and SMRT,which then recruit HDACs to the DNA, leading to chromatincondensation and repression of gene expression.14

The activation of transcription factors by phosphorylation is

a focal point for transducing extracellular stimuli through

Table 28.1 Some examples of key transcription factors expressed in the heart

Factor Gene family DNA binding site Expression pattern Examples of gene regulated

MEF-2 A, B, C, D 4 MADS box family CTA(A/T)4TAG Widely expressed TnIc CK-M

SRF 5 MADS box family CC(A/T) 6 GG (“AcrG box”) Widely expressed α-cardiac actin, SM22, c-fos

Myocardin 6 SAP domain family Does not bind directly to DNA, but

binds to SRF as co-factor

GATA-4, 5, 6 7 GATA family A /TGATA A /g Myocardium, endoderm α-MHC, cardiac TnC, TnIc

Nkx-2.5 8 Homeobox TNNAGTG (high affinity)

C( A /T)TTAATTN (low affinity)

Cardiac mesoderm MLC2v, ANF, cardiac α-actin HIF-1 9 bHLH α/β heterodimers bind CANNTG Ubiquitous; HIF-1β constitutively

expressed, changes in levels of active HIF-1α induced by hypoxia

MLC2v, ANF, cardiac α-actin

α-MHC, cardiac α myosin heavy chain; ANF, atrial natriuretic factor; bHLH, basic helix-loop-helix; CK-M, muscle creatine kinase; HIF-1, hypoxia inducible

factor-1; MEF-2, myocyte enhancer factor-2; MLC2v, ventricular myosin light chain 2; SM22, smooth muscle 22; SRF, serum response factor; TnIc, cardiac

troponin I; TnC, troponin C.

MYOCARDIAL MOLECULAR BIOLOGY: AN INTRODUCTION

MAP kinase signal transduction pathways For example, CBP

associates only with the phosphorylated form of the CREB

protein, a transcription factor that binds the cyclic AMP

response element found in many gene promoters Once bound

to CREB, CBP forms protein–protein interactions with the

basal transcription factor TFIIB, allowing transcription by

RNA pol II to progress Members of the myocyte enhancer

factor-2 (MEF-2) family of transcription factors (table 28.1),

which are widely expressed but appear to be enriched and

have particular roles in skeletal and cardiac muscle, may

regu-late changes in gene expression arising from a hypertrophic

stimulus MEF-2 proteins have been implicated as responders

to MAP kinases activated by hypertrophic stimuli in cardiac

myocytes.15 The hypertrophic agonists endothelin-1 and

phenylepherine activate p38 MAPKs in cultured rat neonatal

cardiac myocytes16, and in vivo p38 activity increases in aortic

banded mice which go on to develop pressure overloaded

hypertrophy (Wang and colleagues 1998, cited in Han and

Molkentin15

) In rat, a similarly induced hypertrophy results in

an increase in DNA binding activity of MEF-2.17

Asdevelopment of hypertrophy is associated with changes in

transcription of various myocardial genes which require

MEF-2, this suggests that MEF-2 may be a direct target for

MAP kinase signalling (fig 28.2)

A second point at which gene expression can be controlled

is at the level of mRNA splicing The primary RNA transcript

of many genes may be alternatively spliced in that certain

exons may be excluded or included from the transcript to

pro-duce the final mRNA In this way, multiple proteins may be

generated from a single gene according to the combination of

exon derived RNA segments that are spliced together The

ver-tebrate tropomyosin (TM) genes are examples of genes

expressed in both cardiac and skeletal muscle that are subject

to complex patterns of alternative mRNA splicing For

exam-ple, two isoforms of theα-TM gene in Xenopus, differ in their

inclusion of alternative 3′untranslated region exons and showrestricted expression in the embryo.18

The XTMα7 isoform isfound in the somites, from which the skeletal muscledevelops, whereas the XTMα2 isoform is expressed in bothsomites and embryonic heart In the adult, XTMα2, but notXTMα7, is selectively expressed in striated muscle and heart.Following translation of the mRNA into polypeptide,production of mature active protein may require several steps,each of which is open to regulation This is well illustrated bythe matrix metallproteinases (MMPs) which are believed toplay a key role in myocardial remodelling.19

MMPs areproduced in an inactive form (as a zymogen) which requirescleavage to produce active enzyme and are further regulated

by specific inhibitory molecules (tissue inhibitors of proteinases or TIMPs) Examining MMP gene expression atthe level of RNA is of importance to understanding gene regu-lation, but may not therefore be a good indicator of MMPenzyme activity Clearly, it is important to understand the bio-logical system in question before deciding which is the mostrelevant level of regulation For the purposes of this review wewill be focusing on the initial stage of gene expression, namelytranscription, and the methods of examining this as well asmonitoring RNA content

metallo-DISSECTING PROMOTER FUNCTION

In order to understand how genes are regulated in the heart,many gene promoters have been isolated and characterisedwith regard to the key regulatory DNA sequences they harbourand the transcription factors that bind them A widely used

method of measuring promoter function is to insert (clone) the

promoter and various lengths of upstream sequence into an

artificial plasmid-based construct in front of a reporter gene

whose expression can be easily monitored Typically the fireflygene luciferase or the bacterial genes chloramphenicol acetyl

Figure 28.2 Linking MEF2 to hypertrophy Hypertrophic stimuli activate intracellular signalling pathways Upstream protein kinases (for example, MEKKs) activate MAP kinase family members (MKKs) which in turn phosphorylate the three MAP kinases p38-MAPK, JNKs, and ERKs MAP kinases have been linked to the phosphorylation of certain transcription factors, for example, p38-MAPK has been shown to activate MEF2 family members (see text for details) This implicates MEF2 proteins as direct transducers of intracellular signalling pathways to bring about some, or all, of the changes in gene expression associated with hypertrophy Figure courtesy of KA Dellow.

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200

transferase (CAT) and β-galactosidase (LacZ) are used for this

purpose Constructs can be introduced into cells in culture by a

variety of transfection techniques, or into whole animals using

transgenic approaches By careful choice of expression

constructs containing progressively smaller deletions, the

posi-tions of DNA sequences responsible for high level transcription,

tissue specificity or specific responses to stimuli (for example,

stretch or agonist induced hypertrophy) can be found The

binding sites for candidate transcription factors can then be

pinpointed accurately through mutagenesis of individual

nucleotides to test the validity of those sequences In this way,

we and others have dissected the promoter of the cardiac

troponin I gene, which is only ever expressed in cardiac muscle

Deletion analysis of the human gene promoter and

upstream sequences has revealed several important

transcrip-tion factor binding sites within 100 nucleotides upstream of

+1 (the conventional notation for promoter sequence is

nega-tive numbering running upstream from the transcription start

site) Among these is an A/T rich element centred around−30

that binds the TATA-box factor, TBP, the octamer protein Oct-1,

and several MEF2 proteins There are two binding sites in the

human TnIc gene for GATA-4 and a C-rich sequence around

−95 that encompasses both binding sites for the zinc finger

factor Sp1 and a CACC-box with the sequence CCCACCCC.20

Mutation of each site in TnIc promoter-CAT reporter

constructs results in a 50–95% reduction in transcriptional

activity when transfected into cultured cardiac myocytes,

sug-gesting that each site serves to bind proteins involved in

maximal transcriptional activity The identity of these proteins

was characterised using the electrophoretic mobility shift

assay (EMSA), also known as band shift assay In this assay, a

radiolabelled double stranded DNA fragment containing a

putative binding site for a transcription factor (usually

referred to as a cassette and generated by annealing short

synthetic oligonucleotides corresponding to the two

comple-mentary strands of DNA) is mixed with an extract of nuclear

proteins prepared from cells or tissue If a protein binds the

cassette, it will retard its migration compared to unbound

cas-sette when subjected to electrophoresis through a

non-denaturing polyacrylamide gel on account of the added mass

of the protein

Mutagenesis of individual nucleotides in the cassette or

titra-tion of unlabelled competitor cassettes in molar excess enable

us to analyse the specificity of interaction between factor and

DNA By incubating the protein–DNA complex with antibody to

putative factors, a “supershift” complex can be obtained (due to

the added mass of the antibody) and the identity of bound

pro-teins thereby confirmed (fig 28.3) Furthermore, by using

nuclear extracts from different cells, an indication of the

distri-bution of the factor can be determined For example, our group

has recently found shown that of four proteins binding the

human TnIc CACC-box, two are members of the widely

expressed Sp family of zinc finger factors while the other two

appear to be expressed only in cardiac myocytes.21

These ments therefore identify the regions involved and the factors

experi-they bind Similar experiments in mouse have been taken

further by showing that only 230bp of the mouse TnIc promoter

are necessary to drive cardiac restricted expression of a LacZ

reporter gene in transgenic mice.22

The role of specific factors in regulating a promoter can be

assessed by simultaneous introduction (co-transfection) of

suitable promoter-reporter constructs with an expression

construct encoding the factor(s) in question For example, the

role of GATA-4 in regulating cardiac specific expression has

been examined in a number of contexts In an elegant

experi-ment a reporter construct containing the promoter andupstream sequences of theα-MHC gene, which contains twobinding sites for GATA-4, was barely active when injected intoskeletal muscle, which lacks endogenous GATA-4.23However,expression could be boosted fourfold by co-injection of anexpression vector for GATA-4 (fig 28.4) In contrast, a mutantreporter construct in which both GATA-4 binding sites hadbeen mutated exhibited only 12% of the activity of thewild-type construct

METHODS FOR MEASURING GENE EXPRESSIONMany techniques have been established for studying genestructure and expression In particular, a variety of methodsfor measuring mRNA have been developed that allowabundance, tissue specificity, and developmental expression to

be followed The relative advantages and disadvantages of themajor techniques are shown in table 28.2

Southerns, northerns, and westernsElectrophoresis of DNA or RNA molecules through an agarose

or polyacrylamide gel matrix form the backbone of some keymolecular biology techniques as the speed and distance thatmolecules migrate is in direct proportion to their size Hence,

Figure 28.3 Visualising DNA–protein interaction by electrophoretic mobility shift assay (EMSA) A double stranded oligonucleotide cassette containing a consensus binding site for GATA factors (A/TGATAA/G) was radiolabelled and incubated with nuclear protein extracts from neonatal cardiac myocytes Lane 1: A specific GATA-4-DNA complex is formed (solid arrowhead) which migrates behind (hence mobility shift) the free probe (FP) Lane 2: The identity of the bound protein is confirmed by addition of a specific antibody which results in a DNA/protein/antibody complex (supershift) (arrow) Specificity of binding can be demonstrated by mutation of the oligonuleotide sequence which results in loss of GATA-4 binding—not shown (see Dellow and colleagues 21 ).

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MYOCARDIAL MOLECULAR BIOLOGY: AN INTRODUCTION