- Email: [email protected]
Signaling pathways that influence extracellular remodeling
Journal of Cardiac Failure Vol. 8 No.6 Suppl. 2002
Signaling Pathways That Influence Extracellular Remodeling RICHARD T. LEE, MD, JAN LAMMERDING, ME Cambridge, Massachusetts
ABSTRACT Background: Remodeling of myocardial tissue requires a rearrangement of cells and extracellular matrix to form the new geometry through processes that are incompletely defined. Exploring new pathways beyond neurohormonal inhibition is essential for developing new therapies for the growing epidemic of heart failure. Methods: One strategy relies on the discovery that progressive ventricular dilation requires matrix metalloproteinases, a family of enzymes that degrade components of matrix but may also participate in activation or release of signaling molecules. Here we will briefly review evidence that matrix metalloproteinase inhibition represents a potential strategy for preventing heart failure. Conclusion: Future applications for understanding cell-matrix interactions, including discovering new pathways with proteomics, will also be discussed. Finally, we propose that defining matrix-remodeling events at the level of the membrane and matrix receptors will be essential, and that new bioengineering tools will provide us with the necessary methods. Key Words: Remodeling, extracellular matrix, genomics, proteomics, stress, strain.
Many therapies in development for heart failure are directed toward interruption of the neurohumoral system. This narrow focus is justified because clinical successes of neurohumoral blockade such as angiotensinconverting enzyme inhibition and aldosterone antagonism contrast with other approaches that have failed clinically, such as stimulating inotropy through phosphodiesterase inhibition.1 However, despite continued successes in refining neurohumoral blockade in heart failure, it is likely that heart failure will remain a progressive disease of epidemic proportions. Thus new approaches to complement neurohumoral strategies are needed. Here we will briefly describe selected aspects of heart failure and extracellular matrix that illustrate future
directions. First, we will discuss the potential and the roadblocks in bringing a specific, well-developed strategy—matrix metalloproteinase (MMP) inhibition—to the bedside in heart failure. One of the compelling new concepts emerging from matrix biology is that the matrix serves as a reservoir of signaling molecules and, in some cases, the matrix proteins themselves transmit critical signals to the cells. Second, we will discuss the importance of using emerging technology to explore new directions in tissue remodeling, both at the broad genomic and proteomic levels and at the microscopic level of the cell membrane/pericellular space.
From the Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Reprint requests: Richard T. Lee, MD, Cardiovascular Research, Partners Research Facility, 65 Landsdowne St., Rm. 279, Cambridge, MA 02139. Copyright 2002, Elsevier Science (USA). All rights reserved. 1071-9164/02/0806-0028$35.00/0 doi:10.1054/jcaf.2002.129262
The premise that MMPs play a role in remodeling ventricular myocardium is no longer in question.2 MMPs are overexpressed in both the acutely remodeling ventricular tissue after infarct and in chronic, nonischemic ventricular myocardium undergoing gradual dilation.3 Furthermore, different MMP inhibitors in different laboratory models of ventricular dilation (and different spe-
Metalloproteinase Inhibition
S339
S340
Journal of Cardiac Failure Vol. 8 No. 6 Suppl. 2002
cies) prevent ventricular dilation.4,5 Finally, transgenic overexpression of MMP-1, a fibrillar collagenase, can directly cause heart failure.6 We initially explored broad-spectrum MMP inhibition in the early remodeling period after myocardial infarction in mice.5 Mice, as with humans, develop progressive left ventricular (LV) enlargement and late systolic dysfunction after occlusion of a coronary artery when the infarction is large; with small infarctions, the left ventricle does not dilate and systolic function remains normal. The first week after infarction is characterized by intense inflammation and matrix degradation, as well as the initiation of the healing response, and MMPs are prominently overexpressed in this period. When we randomized mice to broad-spectrum MMP inhibition or placebo, MMP inhibition attenuated LV dilation, particularly in those mice with greater initial dilation. This experiment suggested that experimental myocardial infarction in mice with targeted deletions of MMPs could be useful for defining the roles of specific MMPs. We then evaluated the role of MMP-3, an enzyme of the “stromelysin” category of MMPs that can degrade many different types of matrix proteins. We did not observe an effect of genetic deletion of MMP-3 on postinfarct remodeling in mice (unpublished observations). However, when we studied mice with deletion of MMP-9, also known as gelatinase B, we observed a reduction in LV remodeling and a decrease in dense fibrillar collagen deposition.7 This study reinforced that the role of specific MMPs in remodeling can be explored in mice, but also raised unexpected questions. For example, we observed compensatory upregulation of MMP-7 in the hearts of mice without MMP-9, suggesting that deletion of MMP-9 leads to accumulation of a substrate that can induce other MMPs.
Pathways of MMP Expression One of the fascinating features of MMP biology is the myriad factors that control activity of these enzymes.8 MMP activity is regulated at the transcriptional level by a variety of growth factors and cytokines, including many factors present in diseased myocardium. Hence autocrine and paracrine pathways and circulating stimuli can induce MMP expression. Physical forces themselves can regulate MMP expression, suggesting that mechanical overload of the myocardium may play a role, although cell deformation experiments have shown both induction9 and suppression10 effects. Physical forces can directly activate transcription factors, such as the AP-1 complex, which tightly controls gene expression of many MMPs. The dominant signaling pathways that activate MMPs in remodeling myocardium are incompletely described.
Tumor necrosis factor-␣ may play a critical role in cardiomyopathies, although we do not yet know the full complement of inflammatory cytokines that are activated in heart failure. After an infarct, infiltrating leukocytes express MMPs and are also sources for inflammatory cytokines, so that it is not surprising that MMP overexpression is particularly prominent during the early phase of LV remodeling and the neutrophil-rich period after ischemia reperfusion.11 MMPs are secreted as inactive zymogens that require processing to active enzymes. Many enzymes and chemicals can activate MMPs, and the dominant controls of activation in vivo remain incompletely defined. Once activated, MMPs can be inhibited by circulating protease inhibitors such as ␣-2-macroglobulin or a series of endogenous tissue inhibitors of metalloproteinases (TIMPS). TIMPs are also under tight transcriptional control and are frequently—but not always—overexpressed in the same regions as MMPs, indicating that cells regulate MMP activity closely after MMPs are secreted.
Bringing MMP Inhibition to the Bedside Despite the availability of compounds that inhibit MMPs and compelling evidence that MMPs mediate pathologic ventricular dilation, several major barriers stand in the way of MMP inhibition reaching the bedside. First, there are at least 20 different MMPs, and the hundreds of orally active MMP inhibitors have varying spectra of inhibition. Thus a clinical MMP inhibition strategy should optimally have a strong scientific basis for choosing a particular spectrum of MMPs; we lack this knowledge currently. Furthermore, thus far pharmacologic MMP inhibition has led to troubling tendonitis that may limit chronic use of these compounds.12 This suggests that MMP inhibition may be most useful for temporally targeted therapy, such as treatment only in the period early after infarct, or that more specific inhibitors that do not cause tendonitis must be developed. In addition, large-scale survival studies in animals must be performed to document the benefit of this strategy. These unanswered questions point out the difficulties in the transition of a strategy from the laboratory to clinical development. During lengthy and expensive clinical trials, it is possible that new information (for example, that inhibition of only one MMP is required or that inhibition during only a few days after infarct is effective) could render a strategy obsolete. Thus more basic research is needed into how MMPs inhibit ventricular dilation, when and which MMPs should be inhibited, and whether these effects are beneficial in the long term.
Signaling pathways that influence extracellular remodeling O Lee and Lammerding
Future Directions in Matrix Remodeling: Seeing the Forest and the Leaves Despite our growing knowledge of the role of extracellular matrix metabolism in ventricular remodeling and heart failure, we are still at the very beginning of understanding the complexities of remodeling tissues. New technologies will play a major role in unraveling these complexities in the next decade. In particular, advances in proteomics will allow us to “see the forest” (ie, the entire protein expression response) of heart failure and ventricular remodeling. This macro view of the remodeling myocardium will undoubtedly yield new directions in heart failure prevention and allow us to consider the complex network of protein-protein interactions. There is also an important gap in our knowledge at the subcellular level: understanding the biomechanics of the myocardial-matrix interface at the molecular level. Many crucial aspects of tissue remodeling probably occur in highly localized regions of the membrane and pericellular space, and this is why using modern bioengineering to “see the leaves” of heart failure is also crucial.
Seeing the Forest: The Role of Proteomics Genomic biology, particularly with DNA microarrays, is already revealing new pathways in cardiac remodeling.13-15 However, gene expression profiles reflect only a fraction of changes that are occurring at the protein level, and proteins are usually the true effectors of cell-signaling pathways. Examining all of the proteins expressed in a given cell or tissue is one of the goals of “proteomics,” which can be loosely considered as the characterization of all proteins.16 With the human genome sequence revealing fewer genes than expected, there is a growing consensus that biologic complexity lies largely in protein functions and interactions, and gene expression information alone cannot unravel that complexity. Until recently, experimental biology has been limited to asking a specific hypothesis regarding the role of one gene or protein in a pathophysiologic process at a time. This strategy has proven useful in drug development because the effects of a compound that inhibits or activates a given protein may be revealed by biologic studies of that specific protein (such as transgenic overexpression in cell culture or in vivo). However, the process of asking one question regarding a single gene or protein ignores most of the interactions that occur between pathways and may overlook critical therapeutic targets for therapy. This is particularly relevant to the
S341
multicellular, dynamic environment of the healing or remodeling myocardium in which the effects of a given protein are best considered in the context of the other proteins. The techniques for proteomic analysis of tissues are still evolving, and currently less than 10% of the human proteome can be analyzed by two-dimensional electrophoresis, the most common approach. Thus proteomic analyses of the myocardium cannot yet approach the range of gene expression by DNA microarrays, which can analyze almost all human genes on a single array. However, rapid advances in mass spectrometry are making proteomic approaches less expensive and increasingly reliable each year. We believe that proteomic approaches will yield essential new information about the matrix and remodeling myocardium. Our unpublished observations suggest that at least hundreds and possibly thousands of proteins are overexpressed in remodeling myocardium, and therefore we have probably only studied a small minority of crucial molecules to date. By looking broadly at the “forest” of proteins in remodeling myocardium, we will be able to see new pathways and potential targets for therapy. For example, differences in profiles of expressed proteins between control and drug-treated myocardium will provide clues to the mechanisms of action of drugs and suggest other proteins that may be drug targets.17
Seeing the Leaves: Understanding Cell-Matrix Interactions Although genomics and proteomics will allow us to take a broader view of remodeling myocardium, we are missing a microscopic understanding of tissue remodeling: the fundamentals of the myocardial cell-matrix mechanical balance at the subcellular level. It is clear that this balance is a key component of heart failure, because intrinsic genetic abnormalities in the sarcomere or cytoskeleton disrupt this balance and lead to cardiomyopathy. Furthermore, extrinsic disruption of cellular biomechanical balance (such as through volume overload) is a common cause of heart failure. Mechanotransduction is defined as the biochemical response of cells to mechanical stimuli. Although mechanotransduction is clearly a major factor in cardiac pathophysiology, the mechanisms by which cardiomyocytes respond to biomechanical stress or strain induced by hemodynamic loading are incompletely defined. One of the reasons that mechanotransduction has been difficult to study is that precisely controlled methods of mechanical stimulation have only recently been developed. These new approaches to mechanotransduction will be required
S342 Journal of Cardiac Failure Vol. 8 No. 6 Suppl. 2002 stimuli. With faster and higher resolution threedimensional microscopy, myocyte mechanics from the matrix through membrane receptors such as integrins to the cytoskeleton and the nucleus will be feasible.
Conclusions
Fig. 1. Magnetic micromanipulator for cellular mechanotransduction experiments. This device is designed to provide a precisely defined stimulus to a magnetic bead coupled to the cell surface through extracellular matrix proteins.
Changes in cell-matrix interactions are insufficiently understood, particularly given the magnitude of the clinical problems that are related to myocardial remodeling. New technologies will allow us to take a broader look at changes in gene and protein expression in remodeling myocardium. Furthermore, we are developing tools that will allow us to probe the relationships between specific matrix receptors and cellular mechanics. These insights may reveal new directions to prevent adverse effects of remodeling.
References to understand the role of the mechanical balance of the cell, membrane, and extracellular matrix and how this balance affects cardiac remodeling. Biophysical methods for delivering precise localized mechanical stimulation to individual cells include the magnetic micromanipulator (Fig. 1). Using controlled magnetic fields, stimulation of particular regions of the cell can be achieved using magnetic beads coated with specific extracellular matrix components or antibodies (Fig. 2). This approach and others hopefully will allow us to understand how cell-matrix interactions affect both myocardial contractility and responses to mechanical
1. Packer M: The development of positive inotropic agents for chronic heart failure: how have we gone astray? J Am Coll Cardiol 1993;22:119A–126A 2. Lee RT, Libby P: Matrix metalloproteinases: not-soinnocent bystanders in heart failure. J Clin Invest 2000; 106:827–828 3. Spinale FG, Coker ML, Bond BR, Zellner JL: Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target. Cardiovasc Res 2000;46:225–238 4. Spinale FG, Coker ML, Krombach SR, Mukherjee R, Hallak H, Houck WV, Clair MJ, Kribbs SB, Johnson LL,
Fig. 2. Measuring mechanical properties of cardiac myocytes with a magnetic micromanipulator. (Left) An adult rat cardiac myocyte is seen, with a microbead on its surface. The microbead is coated with antibodies to an extracellular matrix receptor. (Right) Recordings of cell stiffness can made by measuring displacement of the bead (dark blue) whereas a dynamic force (pink) is imposed. Two spontaneous contractions lead to marked increases in displacement. These methods can be used to study cell stiffness and contractile function with varying mechanical loads through specific matrix receptors.
Signaling pathways that influence extracellular remodeling O Lee and Lammerding
5.
6.
7.
8. 9.
10.
11.
Peterson JT, Zile MR: Matrix metalloproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res 1999;85:364–376 Rohde LE, Ducharme A, Arroyo LH, Aikawa M, Sukhova GH, Lopez-Anaya A, McClure KF, Mitchell PG, Libby P, Lee RT: Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation 1999;99:3063– 3070 Kim HE, Dalal SS, Young E, Legato MJ, Weisfeldt ML, D’Armiento J: Disruption of the myocardial extracellular matrix leads to cardiac dysfunction. J Clin Invest 2000; 106:857–866 Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT: Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 2000;106:55–562 Werb Z, Chin JR: Extracellular matrix remodeling during morphogenesis. Ann N Y Acad Sci 1998;857:110–118 Yang JH, Sakamoto H, Xu EC, Lee RT: Biomechanical regulation of human monocyte/macrophage molecular function. Am J Pathol 2000;156:1797–1804 Yang JH, Briggs WH, Libby P, Lee RT: Small mechanical strains selectively suppress matrix metalloproteinase-1 expression by human vascular smooth muscle cells. J Biol Chem 1998;273:6550–6225 Lindsey M, Wedin K, Brown MD, Keller C, Evans AJ, Smolen J, Burns AR, Rossen RD, Michael L, Entman M:
12.
13.
14.
15.
16. 17.
S343
Matrix-dependent mechanism of neutrophil-mediated release and activation of matrix metalloproteinase 9 in myocardial ischemia/reperfusion. Circulation 2001;103: 2181–2187 Drummond AH, Beckett P, Brown PD, Bone EA, Davidson AH, Galloway WA, Gearing AJ, Huxley P, Laber D, McCourt M, Whittaker M, Wood LM, Wright A: Preclinical and clinical studies of MMP inhibitors in cancer. Ann N Y Acad Sci 1999;878:228–235 Yang J, Moravec CS, Sussman MA, Di Paola NR, Fu D, Hawthorn L, Mitchell CA, Young JB, Francis GS, McCarthy PM, Bond M: Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays. Circulation 2000;102:3046–3052 Friddle CJ, Koga T, Rubin EM, Bristow J: Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy. Proc Natl Acad Sci U S A 2000;97:6745–6750 Stanton LW, Garrard LJ, Damm D, Garrick BL, Lam A, Kapoun AM, Zheng Q, Protter AA, Schreiner GF, White RT: Altered patterns of gene expression in response to myocardial infarction. Circ Res 2000:86:939–945 Pandey A, Mann M: Proteomics to study genes and genomes. Nature 2000;405:837–846 Jin H, Yang R, Awad TA, Wang F, Li W, Williams SP, Ogasawara A, Shimada B, Williams PM, de Feo G, Paoni NF: Effects of early angiotensin-converting enzyme inhibition on cardiac gene expression after acute myocardial infarction. Circulation 2001;103:736–742
Signaling Pathways That Influence Extracellular Remodeling RICHARD T. LEE, MD, JAN LAMMERDING, ME Cambridge, Massachusetts
ABSTRACT Background: Remodeling of myocardial tissue requires a rearrangement of cells and extracellular matrix to form the new geometry through processes that are incompletely defined. Exploring new pathways beyond neurohormonal inhibition is essential for developing new therapies for the growing epidemic of heart failure. Methods: One strategy relies on the discovery that progressive ventricular dilation requires matrix metalloproteinases, a family of enzymes that degrade components of matrix but may also participate in activation or release of signaling molecules. Here we will briefly review evidence that matrix metalloproteinase inhibition represents a potential strategy for preventing heart failure. Conclusion: Future applications for understanding cell-matrix interactions, including discovering new pathways with proteomics, will also be discussed. Finally, we propose that defining matrix-remodeling events at the level of the membrane and matrix receptors will be essential, and that new bioengineering tools will provide us with the necessary methods. Key Words: Remodeling, extracellular matrix, genomics, proteomics, stress, strain.
Many therapies in development for heart failure are directed toward interruption of the neurohumoral system. This narrow focus is justified because clinical successes of neurohumoral blockade such as angiotensinconverting enzyme inhibition and aldosterone antagonism contrast with other approaches that have failed clinically, such as stimulating inotropy through phosphodiesterase inhibition.1 However, despite continued successes in refining neurohumoral blockade in heart failure, it is likely that heart failure will remain a progressive disease of epidemic proportions. Thus new approaches to complement neurohumoral strategies are needed. Here we will briefly describe selected aspects of heart failure and extracellular matrix that illustrate future
directions. First, we will discuss the potential and the roadblocks in bringing a specific, well-developed strategy—matrix metalloproteinase (MMP) inhibition—to the bedside in heart failure. One of the compelling new concepts emerging from matrix biology is that the matrix serves as a reservoir of signaling molecules and, in some cases, the matrix proteins themselves transmit critical signals to the cells. Second, we will discuss the importance of using emerging technology to explore new directions in tissue remodeling, both at the broad genomic and proteomic levels and at the microscopic level of the cell membrane/pericellular space.
From the Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. Reprint requests: Richard T. Lee, MD, Cardiovascular Research, Partners Research Facility, 65 Landsdowne St., Rm. 279, Cambridge, MA 02139. Copyright 2002, Elsevier Science (USA). All rights reserved. 1071-9164/02/0806-0028$35.00/0 doi:10.1054/jcaf.2002.129262
The premise that MMPs play a role in remodeling ventricular myocardium is no longer in question.2 MMPs are overexpressed in both the acutely remodeling ventricular tissue after infarct and in chronic, nonischemic ventricular myocardium undergoing gradual dilation.3 Furthermore, different MMP inhibitors in different laboratory models of ventricular dilation (and different spe-
Metalloproteinase Inhibition
S339
S340
Journal of Cardiac Failure Vol. 8 No. 6 Suppl. 2002
cies) prevent ventricular dilation.4,5 Finally, transgenic overexpression of MMP-1, a fibrillar collagenase, can directly cause heart failure.6 We initially explored broad-spectrum MMP inhibition in the early remodeling period after myocardial infarction in mice.5 Mice, as with humans, develop progressive left ventricular (LV) enlargement and late systolic dysfunction after occlusion of a coronary artery when the infarction is large; with small infarctions, the left ventricle does not dilate and systolic function remains normal. The first week after infarction is characterized by intense inflammation and matrix degradation, as well as the initiation of the healing response, and MMPs are prominently overexpressed in this period. When we randomized mice to broad-spectrum MMP inhibition or placebo, MMP inhibition attenuated LV dilation, particularly in those mice with greater initial dilation. This experiment suggested that experimental myocardial infarction in mice with targeted deletions of MMPs could be useful for defining the roles of specific MMPs. We then evaluated the role of MMP-3, an enzyme of the “stromelysin” category of MMPs that can degrade many different types of matrix proteins. We did not observe an effect of genetic deletion of MMP-3 on postinfarct remodeling in mice (unpublished observations). However, when we studied mice with deletion of MMP-9, also known as gelatinase B, we observed a reduction in LV remodeling and a decrease in dense fibrillar collagen deposition.7 This study reinforced that the role of specific MMPs in remodeling can be explored in mice, but also raised unexpected questions. For example, we observed compensatory upregulation of MMP-7 in the hearts of mice without MMP-9, suggesting that deletion of MMP-9 leads to accumulation of a substrate that can induce other MMPs.
Pathways of MMP Expression One of the fascinating features of MMP biology is the myriad factors that control activity of these enzymes.8 MMP activity is regulated at the transcriptional level by a variety of growth factors and cytokines, including many factors present in diseased myocardium. Hence autocrine and paracrine pathways and circulating stimuli can induce MMP expression. Physical forces themselves can regulate MMP expression, suggesting that mechanical overload of the myocardium may play a role, although cell deformation experiments have shown both induction9 and suppression10 effects. Physical forces can directly activate transcription factors, such as the AP-1 complex, which tightly controls gene expression of many MMPs. The dominant signaling pathways that activate MMPs in remodeling myocardium are incompletely described.
Tumor necrosis factor-␣ may play a critical role in cardiomyopathies, although we do not yet know the full complement of inflammatory cytokines that are activated in heart failure. After an infarct, infiltrating leukocytes express MMPs and are also sources for inflammatory cytokines, so that it is not surprising that MMP overexpression is particularly prominent during the early phase of LV remodeling and the neutrophil-rich period after ischemia reperfusion.11 MMPs are secreted as inactive zymogens that require processing to active enzymes. Many enzymes and chemicals can activate MMPs, and the dominant controls of activation in vivo remain incompletely defined. Once activated, MMPs can be inhibited by circulating protease inhibitors such as ␣-2-macroglobulin or a series of endogenous tissue inhibitors of metalloproteinases (TIMPS). TIMPs are also under tight transcriptional control and are frequently—but not always—overexpressed in the same regions as MMPs, indicating that cells regulate MMP activity closely after MMPs are secreted.
Bringing MMP Inhibition to the Bedside Despite the availability of compounds that inhibit MMPs and compelling evidence that MMPs mediate pathologic ventricular dilation, several major barriers stand in the way of MMP inhibition reaching the bedside. First, there are at least 20 different MMPs, and the hundreds of orally active MMP inhibitors have varying spectra of inhibition. Thus a clinical MMP inhibition strategy should optimally have a strong scientific basis for choosing a particular spectrum of MMPs; we lack this knowledge currently. Furthermore, thus far pharmacologic MMP inhibition has led to troubling tendonitis that may limit chronic use of these compounds.12 This suggests that MMP inhibition may be most useful for temporally targeted therapy, such as treatment only in the period early after infarct, or that more specific inhibitors that do not cause tendonitis must be developed. In addition, large-scale survival studies in animals must be performed to document the benefit of this strategy. These unanswered questions point out the difficulties in the transition of a strategy from the laboratory to clinical development. During lengthy and expensive clinical trials, it is possible that new information (for example, that inhibition of only one MMP is required or that inhibition during only a few days after infarct is effective) could render a strategy obsolete. Thus more basic research is needed into how MMPs inhibit ventricular dilation, when and which MMPs should be inhibited, and whether these effects are beneficial in the long term.
Signaling pathways that influence extracellular remodeling O Lee and Lammerding
Future Directions in Matrix Remodeling: Seeing the Forest and the Leaves Despite our growing knowledge of the role of extracellular matrix metabolism in ventricular remodeling and heart failure, we are still at the very beginning of understanding the complexities of remodeling tissues. New technologies will play a major role in unraveling these complexities in the next decade. In particular, advances in proteomics will allow us to “see the forest” (ie, the entire protein expression response) of heart failure and ventricular remodeling. This macro view of the remodeling myocardium will undoubtedly yield new directions in heart failure prevention and allow us to consider the complex network of protein-protein interactions. There is also an important gap in our knowledge at the subcellular level: understanding the biomechanics of the myocardial-matrix interface at the molecular level. Many crucial aspects of tissue remodeling probably occur in highly localized regions of the membrane and pericellular space, and this is why using modern bioengineering to “see the leaves” of heart failure is also crucial.
Seeing the Forest: The Role of Proteomics Genomic biology, particularly with DNA microarrays, is already revealing new pathways in cardiac remodeling.13-15 However, gene expression profiles reflect only a fraction of changes that are occurring at the protein level, and proteins are usually the true effectors of cell-signaling pathways. Examining all of the proteins expressed in a given cell or tissue is one of the goals of “proteomics,” which can be loosely considered as the characterization of all proteins.16 With the human genome sequence revealing fewer genes than expected, there is a growing consensus that biologic complexity lies largely in protein functions and interactions, and gene expression information alone cannot unravel that complexity. Until recently, experimental biology has been limited to asking a specific hypothesis regarding the role of one gene or protein in a pathophysiologic process at a time. This strategy has proven useful in drug development because the effects of a compound that inhibits or activates a given protein may be revealed by biologic studies of that specific protein (such as transgenic overexpression in cell culture or in vivo). However, the process of asking one question regarding a single gene or protein ignores most of the interactions that occur between pathways and may overlook critical therapeutic targets for therapy. This is particularly relevant to the
S341
multicellular, dynamic environment of the healing or remodeling myocardium in which the effects of a given protein are best considered in the context of the other proteins. The techniques for proteomic analysis of tissues are still evolving, and currently less than 10% of the human proteome can be analyzed by two-dimensional electrophoresis, the most common approach. Thus proteomic analyses of the myocardium cannot yet approach the range of gene expression by DNA microarrays, which can analyze almost all human genes on a single array. However, rapid advances in mass spectrometry are making proteomic approaches less expensive and increasingly reliable each year. We believe that proteomic approaches will yield essential new information about the matrix and remodeling myocardium. Our unpublished observations suggest that at least hundreds and possibly thousands of proteins are overexpressed in remodeling myocardium, and therefore we have probably only studied a small minority of crucial molecules to date. By looking broadly at the “forest” of proteins in remodeling myocardium, we will be able to see new pathways and potential targets for therapy. For example, differences in profiles of expressed proteins between control and drug-treated myocardium will provide clues to the mechanisms of action of drugs and suggest other proteins that may be drug targets.17
Seeing the Leaves: Understanding Cell-Matrix Interactions Although genomics and proteomics will allow us to take a broader view of remodeling myocardium, we are missing a microscopic understanding of tissue remodeling: the fundamentals of the myocardial cell-matrix mechanical balance at the subcellular level. It is clear that this balance is a key component of heart failure, because intrinsic genetic abnormalities in the sarcomere or cytoskeleton disrupt this balance and lead to cardiomyopathy. Furthermore, extrinsic disruption of cellular biomechanical balance (such as through volume overload) is a common cause of heart failure. Mechanotransduction is defined as the biochemical response of cells to mechanical stimuli. Although mechanotransduction is clearly a major factor in cardiac pathophysiology, the mechanisms by which cardiomyocytes respond to biomechanical stress or strain induced by hemodynamic loading are incompletely defined. One of the reasons that mechanotransduction has been difficult to study is that precisely controlled methods of mechanical stimulation have only recently been developed. These new approaches to mechanotransduction will be required
S342 Journal of Cardiac Failure Vol. 8 No. 6 Suppl. 2002 stimuli. With faster and higher resolution threedimensional microscopy, myocyte mechanics from the matrix through membrane receptors such as integrins to the cytoskeleton and the nucleus will be feasible.
Conclusions
Fig. 1. Magnetic micromanipulator for cellular mechanotransduction experiments. This device is designed to provide a precisely defined stimulus to a magnetic bead coupled to the cell surface through extracellular matrix proteins.
Changes in cell-matrix interactions are insufficiently understood, particularly given the magnitude of the clinical problems that are related to myocardial remodeling. New technologies will allow us to take a broader look at changes in gene and protein expression in remodeling myocardium. Furthermore, we are developing tools that will allow us to probe the relationships between specific matrix receptors and cellular mechanics. These insights may reveal new directions to prevent adverse effects of remodeling.
References to understand the role of the mechanical balance of the cell, membrane, and extracellular matrix and how this balance affects cardiac remodeling. Biophysical methods for delivering precise localized mechanical stimulation to individual cells include the magnetic micromanipulator (Fig. 1). Using controlled magnetic fields, stimulation of particular regions of the cell can be achieved using magnetic beads coated with specific extracellular matrix components or antibodies (Fig. 2). This approach and others hopefully will allow us to understand how cell-matrix interactions affect both myocardial contractility and responses to mechanical
1. Packer M: The development of positive inotropic agents for chronic heart failure: how have we gone astray? J Am Coll Cardiol 1993;22:119A–126A 2. Lee RT, Libby P: Matrix metalloproteinases: not-soinnocent bystanders in heart failure. J Clin Invest 2000; 106:827–828 3. Spinale FG, Coker ML, Bond BR, Zellner JL: Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target. Cardiovasc Res 2000;46:225–238 4. Spinale FG, Coker ML, Krombach SR, Mukherjee R, Hallak H, Houck WV, Clair MJ, Kribbs SB, Johnson LL,
Fig. 2. Measuring mechanical properties of cardiac myocytes with a magnetic micromanipulator. (Left) An adult rat cardiac myocyte is seen, with a microbead on its surface. The microbead is coated with antibodies to an extracellular matrix receptor. (Right) Recordings of cell stiffness can made by measuring displacement of the bead (dark blue) whereas a dynamic force (pink) is imposed. Two spontaneous contractions lead to marked increases in displacement. These methods can be used to study cell stiffness and contractile function with varying mechanical loads through specific matrix receptors.
Signaling pathways that influence extracellular remodeling O Lee and Lammerding
5.
6.
7.
8. 9.
10.
11.
Peterson JT, Zile MR: Matrix metalloproteinase inhibition during the development of congestive heart failure: effects on left ventricular dimensions and function. Circ Res 1999;85:364–376 Rohde LE, Ducharme A, Arroyo LH, Aikawa M, Sukhova GH, Lopez-Anaya A, McClure KF, Mitchell PG, Libby P, Lee RT: Matrix metalloproteinase inhibition attenuates early left ventricular enlargement after experimental myocardial infarction in mice. Circulation 1999;99:3063– 3070 Kim HE, Dalal SS, Young E, Legato MJ, Weisfeldt ML, D’Armiento J: Disruption of the myocardial extracellular matrix leads to cardiac dysfunction. J Clin Invest 2000; 106:857–866 Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT: Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 2000;106:55–562 Werb Z, Chin JR: Extracellular matrix remodeling during morphogenesis. Ann N Y Acad Sci 1998;857:110–118 Yang JH, Sakamoto H, Xu EC, Lee RT: Biomechanical regulation of human monocyte/macrophage molecular function. Am J Pathol 2000;156:1797–1804 Yang JH, Briggs WH, Libby P, Lee RT: Small mechanical strains selectively suppress matrix metalloproteinase-1 expression by human vascular smooth muscle cells. J Biol Chem 1998;273:6550–6225 Lindsey M, Wedin K, Brown MD, Keller C, Evans AJ, Smolen J, Burns AR, Rossen RD, Michael L, Entman M:
12.
13.
14.
15.
16. 17.
S343
Matrix-dependent mechanism of neutrophil-mediated release and activation of matrix metalloproteinase 9 in myocardial ischemia/reperfusion. Circulation 2001;103: 2181–2187 Drummond AH, Beckett P, Brown PD, Bone EA, Davidson AH, Galloway WA, Gearing AJ, Huxley P, Laber D, McCourt M, Whittaker M, Wood LM, Wright A: Preclinical and clinical studies of MMP inhibitors in cancer. Ann N Y Acad Sci 1999;878:228–235 Yang J, Moravec CS, Sussman MA, Di Paola NR, Fu D, Hawthorn L, Mitchell CA, Young JB, Francis GS, McCarthy PM, Bond M: Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays. Circulation 2000;102:3046–3052 Friddle CJ, Koga T, Rubin EM, Bristow J: Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy. Proc Natl Acad Sci U S A 2000;97:6745–6750 Stanton LW, Garrard LJ, Damm D, Garrick BL, Lam A, Kapoun AM, Zheng Q, Protter AA, Schreiner GF, White RT: Altered patterns of gene expression in response to myocardial infarction. Circ Res 2000:86:939–945 Pandey A, Mann M: Proteomics to study genes and genomes. Nature 2000;405:837–846 Jin H, Yang R, Awad TA, Wang F, Li W, Williams SP, Ogasawara A, Shimada B, Williams PM, de Feo G, Paoni NF: Effects of early angiotensin-converting enzyme inhibition on cardiac gene expression after acute myocardial infarction. Circulation 2001;103:736–742