Molecular Biology and Its Impact for the Future Robert Roberts, MD

0 ver the past few decades, physicians have watched cardiovascular disease therapy move from largely empiric to more sophis-

ticated treatments based on physiologic principles and, more recently, molecular biology. Cardiology has been slower than other disciplines to move into the arena. Aspects of molecular genetics are nearly 4 decades old, dating from the 1940s including the classic experiments of Watson and Crick on the structure and function of deoxyribonucleic acid (DNA). In much the same manner as cardiology demystified hemodynamics in the 1960s and 1970s and incorporated its principles into everyday prac- tice, the near future will see the use of the molecular biologic techniques that are fundamen- tal to all disciplines of medicine and industry.

MOLECULAR BIOLOGYi THE VERY BASICS The terminology of molecular cardiology will

probably become familiar to both academicians and practitioners within a few years. Research has increased our understanding of cardiac muscle disease; regulation of intracellular ions; myocar- dial injury; cardiac growth and regulation of gene expression; endocrine functions of the heart; blood vessel-endothelium interaction; and regulation of vascular tone, atherosclerosis, and thrombolysis.’ The study begins with an understanding of DNA, a huge molecule composed of monotonously repeat- ing combinations of 4 base pairs. Although the problem can be simply stated, the task is formida- ble, since the amount of DNA in a single cell carries enough genetic information to fill a book of 1 million pages. Put another way, if all the DNA molecules in one person were placed end-to-end, the resulting chain would reach from Earth to the moon 8,000 times. One cell contains 3 billion base pairs, enough to code for 10 million genes. DNA is “selfish,” since less than 1% appears to participate

From the Division of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, Texas.

Address for reprints: Robert Roberts, MD, Chief of Cardiol- ogy, The Methodist Hospital, 6535 Fannin, MS F905, Houston, Texas 77030.

in protein generation; the human genome has between 20,000 and 50,000 genes.

The ability to unravel the enormous maze of DNA came in the 1970s with several Nobel Prize- winning contributions. Early in the decade, it was shown that site-specific recognition and cleavage of DNA were possible by using specific restriction endonucleases that typically recognize 4-6 bases This permitted investigators to know precisely where a DNA molecule had been cut. Since then, more than 300 restriction enzymes have been identified. (‘“Restriction enzyme” refers to the discovery of enzymes in bacteria that restrict for- eign DNA from entering a bacterium’s genome by using the bacterial nuclease.)

The next major advance was the discovery of reverse transcriptase. This enzyme allows genera- tion of complementary DNA from a messenger ribonucleic acid (mRNA) isolated from cell cyto- plasm. This is the reverse of the usual RNA generation sequence, in which DNA is transcribed into mRNA that is then transported to the cyto- plasm. In the presence of a ribosome, the mRNA is translated into protein.

Next, DNA cloning techniques were developed to permit splicing a piece of DNA into a foreign genome with subsequent expression of its product. Thus, the cutting of DNA by means of a site- specific restriction endonuclease and splicing the selected portion into a foreign genome to produce a specific protein were the birth of cloning a recombinant molecule.

Two principal methods are commonly employed for DNA analysis. In restriction fragment length polymorphism (RFLP) analysis, genomic DNA is digested by the bacterial restriction endonuclease, producing fragments that typically range from < 1 to >20 kilobases in size. These fragments are separated by agarose electrophoresis and trans- ferred to a support membrane. The fragments of DNA containing the genes to be studied are detected by hybridization with isolated and labeled segments of the cloned gene.’ A series of l-6 bands can be used to characterize and fingerprint a haplotype.3,4 In a modification of this method, a

A SYMPOSIUM: MECHANISMS OF UNSTABLE ANGINA 3c

synthetic oligonucleotide of about 20 bases is used as the gene probe. For example, this oligonucle- otide can be synthesized to match a mutated fragment of a specific coding region.5

Recently, investigators have used the technol- ogy of the polymerase chain reaction (PCR), which allows direct sequencing of the gene of interest. Theoretically, a “library” of complementary DNAs could be constructed and screened with gene probes to isolate the clone containing the gene of interest, which could then be sequenced. The PCR involves double priming of DNA (or mRNA) and synthesizing multiple copies of the region of the gene between primers.6 Work is conducted at temperatures higher than body temperature in order to accelerate the reactions. With this method, one can define protein molecules in quantities far smaller than is possible with alternative methods, such as 2-dimensional polyacrylamide gel electro- phoresis. Proteins present in small amounts (e.g., tissue plasminogen activators [t-PA]) are readily detected by this method.

MONOCLONAL ANTIBODIES AND CARDIOLOGY The normal immune system responds to

“foreign” substances in a variety of ways, including production of antibodies against multiple antigenic sites, or epitopes, on the offending molecule. The broad range of antibodies produced reflects dif- ferent antigenic determinants on the foreign sub- stance. Classically, monoclonal antibodies are pro- duced by immunizing a mouse with an antigen. The spleen is harvested and plasma cells are isolated and “immortalized” by fusing them with a my- eloma cell line. This process yields a perpetually reproducing hybridoma, which clonally produces an antibody to one of the immunizing antigens7

One problem with this procedure is that it may take up to 18 months to produce products suitable for human use. Another problem is the production of human anti-mouse antibodies (HAMAs). Diag- nostic monoclonal antibodies (e.g., indium-lll- labeled antimyosin to image necrotic myocardium) rarely present a problem, since the half-life is short and, typically, they are administered only once. However, therapeutic monoclonal antibodies (e.g., those used for oncology) may require multiple administrations, with longer in vivo half-lives. To solve this problem, the antigen-binding portion, or F(ab) section, of the HAMA is spliced onto a human antibody. The resulting chimeric mono- clonal antibody appears to induce less HAMA production than the nonhumanized antibody.’

MOLECULAR BIOLOGY AND THE CUNlClAN What can be accomplished with the techniques

just described? There are 4 broad areas of poten- tial importance: (1) it is now possible to perform in vivo structure-function analysis of a single mole- cule (or portion thereof) in the intact, living cell or organism; (2) the gene from a particular molecule can be put into a cell culture or into an intact organism to observe its behavior, and genetic engineering can produce mutations of the mole- cule; (3) diagnostic in situ hybridization can be used to localize DNA or RNA in a biopsy sample (to the cardiologist, this might mean rapid diagno- sis of a viral genome in an endomyocardial biopsy specimen); and (4) so-called “designer” biologicals can be generated, for example, mutated forms of recombinant t-PA (rt-PA) that have specific bio- logic advantages over the naturally occurring prod- uct.

Potential applications in thrombolytic ther- apy: The thrombolytic agents in common use are plasminogen activators that convert the proenzyme plasminogen to the active but nonspecific protease, plasmin. This protease degrades fibrin in blood clots, but also degrades both fibrinogen and other clotting factors (most notably factors V and VIII). During the activation of the fibrinolytic system by plasminogen activators, the circulating inactivator of plasmin, a,-antiplasmin, is rapidly consumed and bleeding may ensue.

The t-PA molecule is composed of 527 amino acids and includes 16 disulfide bonds. There are 3 glycosylation sites and 5 discrete domains in this molecule. These are, in turn, encoded by 14 exons. More than 40 mutant t-PA forms are currently under investigation to improve the pharmacoki- netic profile of this agent. For example, one form being developed contains only the (subunit) krin- gle 2 and the catalytic site. It has a half-life of 60 minutes, may be given as a single injection, and appears to be equivalent or superior in both potency and fibrin specificity.

In general, several approaches to t-PA modifica- tion have been suggested.&“’ In the example just mentioned, deletion mutation of functional do- mains modifies drug clearance and fibrin speci- ficity. Higher fibrin specificity may be achieved by mutation of the plasmin cleavage site, which pre- vents conversion of l-chain t-PA to 2-chain t-PA. Either glycosylation or active site acylation slows drug clearance. In the latter, this occurs in a manner analogous to that used for anistreplase

4c THE AMERICAN JOURNAL OF CARDIOLOGY VOLUME 68 NOVEMBER 4, 1991

(anisoylated plasminogen-streptokinase activator complex [APSAC]).

Newer attempts to “target” fibrinolysis have used a monoclonal antifibrin antibody. This agent binds fibrin, but not fibrinogen, with high affinity and may be linked chemically, immunologically, or by recombinant DNA methods to urokinase, rt- PA, or single-chain urokinase plasminogen activa- tor (scu-PA).~-~ Alternatively, urokinase may be linked to a monoclonal antibody specific for the platelet-fibrinogen receptor complex (glycoprotein IIb/IIIa), yielding enhanced potency with platelet antiaggregant activity.

Other approaches use monoclonal antibodies with specificity for both fibrin and one of the plasminogen activators in order to concentrate the fibrin at the site of the plasminogen activator. The development of chimeric molecules has expanded the field. For example, one such molecule was developed to combine the fibrin affinity of the t-PA with an antibody to macroglobin. However, agents obtained by such exon shuffling have generally been inferior to the parent molecules, possibly because of differences in tertiary structure (protein folding) in comparison to the native molecule.

THE FUTURE: GENETIC ENGINEERING AND HEART FAILURE

A structure-function analysis of cardiac muscle is paramount in properly understanding heart fail- ure. For example, cardiac physiologists have known for decades that cardiac work load is altered by end-diastolic fiber length (Starling’s law). On the cellular level, work may be regulated by biochemi- cal changes, the concept of excitation-contraction coupling. Now, this issue can be approached on the molecular level because of confirming evidence that altered gene expression plays a role in the regulation of cardiac function and hypertrophy.7 In the setting of heart failure, major shifts occur in expressed forms of contractile proteins, sodium- potassium adenosine triphosphatase (Na+,K+- ATPase) and other proteins.11’12 Proto-oncogenes appear to have a role in myocyte proliferation and terminal differentiation and in the regulation of the response to cellular death (as occurs in myocar- dial infarction).

An example is the isolation of the gene for atria1 natriuretic factor. A transgenic mouse may be transfected with atria1 natriuretic factor-promoter region DNA that has been linked to an oncogene. The result is massive right atria1 hypertrophy and supraventricular arrhythmias.13

At present, cardiac therapeutic interventions based on the principles described above are still in the distant future. Study has been hampered by the myocyte resistance to mutation, lack of cell lines in vitro, insufficient neoplasms to study, and difficulty in obtaining adequate tissue serially. Nonetheless, the possibility of specific cardiac gene therapy or the induction of cardiac myocyte replication is now within reach.

Indeed, today more than 80 drugs are produced by genetic engineering, with a major increase predicted over the next decade. The future holds great promise for molecular genetics and molecu- lar cardiology, with major diagnostic and therapeu- tic advances just around the corner.

REFERENCES LHathaway DR, March KL. Molecular cardiology: new avenues for the diagnosis and treatment of cardiovascular diseaseJAm CON Cardiol1989;13:265- 282. 2. Kan YW, Dozy AM. Polymorphism of DNA sequence adjacent to human beta-globin structural gene relationship to sickle mutation. Pmc Natl Acad Sci USA 19?8;75:5631-5635. 3. Dunham I, Sargent CA, Trowsdale J, Campbell RD. Molecular mapping of the human major histocompatability complex by pulsed-field gel electrophoresis. Aoc NatlAcad Sci USA 1987;84723-7241. 4. Gusella JF. DNA polymorphism and human disease. Amu Rev Biochem 1986;55:831-854. 5. Orkin SH, Markham AF, Kazazian HH Jr. Direct detection of the common Mediterranean beta-thalassemia gene with synthetic DNA probes. J Clin Invest 1983;71:775-779. 6. Saiki RY Bugawan % Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified beta-globm and HLA-DQ alpha DNA&h allele-specific oligonucleotide probes. Nature 1986;324:163-166. 7. Haber E. In viva diagnostic and therapeutic uses of monoclonal antldodies in cardiology.Annu RevMed 1986;37:249-261. 8. Bode C, Runge MS, Haber E. Future directions in plasminogen activator therapy. C!in Cardiol1990;13:375-381. 9. Bang NLJ. Tissue-type plasminogen activator mutants. Theoretical and clinical considerations. Circulation 1989;79:1391-1392. 10. Pannekoek H, de Vries H, van Zonneveld AJ. Mutants of human tissue-type plaminogen activator (t-PA): structural and functional properties. F&tio&sis 1988;2312?-127. ll, Mod& E. Chronic adaptations in contractile proteins: genetic regulation, Annu Rev Physiol1987;&.545-554. 11. Orlowski J, Lingrel JB. Tissue-specific and developmental regulation of rat Na, K-ATPase catalytic alpha &form and alpha-l subunit mRNAs. JBiol Chem 1988;263:10436-10462. 13. Field LT. Atrial natriuretic factor-SV40 T antigen transgenes produce tumors and cardiac arrhythmias in mice. Science 1988;239:1029-1033.

A SYMPOSIUM: MECHANISMS OF UNSTABLE ANGINA 5c