Copyright © 2005, European Society of Cardiology
The ubiquitin–proteasome system may be involved in the pathogenesis of hypertrophic cardiomyopathy
Max-Planck-Institute of Heart and Lung Research, Experimental Cardiology Benekestr. 2, D-61231 Bad Nauheim Germany
* Private address: Richard-Kuhn-Str. 43 D-69123 Heidelberg, Germany. Email address: hanspeter_vosberg{at}web.de
Received 1 February 2005; accepted 1 February 2005
See also article by Sarikas et al. [7] (pages 33–44) in this issue.
Cardiac morbidity results in the majority of cases from conditions that do not arise in the myocardium itself, but rather in response to extra-myocardial challenges associated with systemic, vascular, or other dysfunctions. A few, not particularly frequent cardiac disorders, however, can be traced to an impairment from within the myocardium. Many, if not most, of these conditions have a genetic cause. Mutations in numerous genes have been identified, most notably in genes coding for proteins involved in contraction and force development, electrical conductance, and in structure and maintenance of the cyto- and nucleoskeleton.
Particularly well studied among the genuinely myocardial disorders have been the cardiomyopathies, clinically well known for decades and characterized by distorted force production affecting either diastole (relaxation) or systole (contraction). Impaired diastole and systole are hallmarks of hypertrophic- (HCM) and dilated cardiomyopathy (DCM), respectively. HCM–the topic of this commentary–has been intensively studied with respect to both inherited causes [1] and disease mechanisms [2]. Established clinical features of HCM are unspecific cardiac symptoms in young adults, asymmetric myocardial hypertrophy with or without left-ventricular outflow obstruction, slow progression, but a high risk of sudden cardiac death [3]. Transmission is dominant, with penetrance being variable. Molecular genetic analysis began in the late 1980s and has produced a wealth of information about mutations in a large number of genes since then. In short, more than 200 mutations were found in 12 genes coding for sarcomeric proteins acting as "molecular motors" in cross-bridge cycling (notably myosin, actin) or in the regulation of this process (predominantly the troponin system, tropomyosin, myosin binding protein-C). HCM was therefore tentatively defined as a "disease of the sarcomere" [4]. In addition to the identification of genes, numerous model studies using isolated native or recombinant molecules, transfected cardiomyocytes, transgenic and gene-targeted animals, predominantly mice, and isolated organ preparations from these animals provided evidence, as expected, that sarcomeric functions are altered.
Not all of the experimental data converged to a common scheme. Myosin isolated from human (slow skeletal) muscle tissue, for instance, with an Arg–Gln exchange in position 403 (the first HCM mutation ever identified in HCM patients), showed a decreased sliding velocity in vitro, whereas the corresponding myosin isolated from gene-targeted mice was faster than normal (reviewed in Ref. [2]). Agreement was obtained, however, with respect to defining the overall mechanism as a "poison polypeptide" mode of action resulting from two types of allelic gene products, mutated and wild type, both present and active in the cell. It should be added (without further discussion) that important mechanistic studies have also been focussed on calcium turnover and altered calcium sensitivity imposed by mutated sarcomeric proteins on the force-producing apparatus (see also Ref. [2]), and on the role of the phosphatase calcineurin and the transcription factor NF-AT3. These components are–as far as transgene studies in animals have shown–involved in the formation of myocardial hypertrophy and in the control of potassium channel density in myocyte membranes [5,6].
A distinct picture of functional consequences of HCM mutations evolved from studies of the cardiac myosin binding protein-C gene (MyBP-C, gene designation MBPC3), which is the subject of the paper by Sarikas et al. [7] in this issue of Cardiovascular Research. Mutations in this gene account for more than 40% of all successfully genotyped HCM patients [8]. The 144-kDa protein has a modular structure consisting of 11 globular domains. It is a major component of thick filaments and has been localized in the C-zone of the A-band of the sarcomere. It has a dual function by stabilizing sarcomeric structure and modulating cardiac contractility controlled by phosphorylation, which in turn depends on adrenergic stimulation and cAMP-regulated protein kinase [9,10].
Mutations in the MyBP-C gene are surprisingly often associated with translational frameshifts leading to truncated proteins. Data obtained from transgenic mice as a model expressing truncated myosin binding protein-C genes do not necessarily reflect the situation in the human myocardium in all details [11,12]. In cardiac tissue of patients known to be carriers of MyBP-C frameshift mutations, the expected truncated proteins were not seen in Western blots in a number of independent studies. These observations suggested that in addition to the "poison polypeptide" mode of action, an alternative mechanism exists defined as "haploinsufficiency", a term describing the absence of the gene product of a mutated allele, with the regular non-mutated protein remaining. A plausible, but speculative, claim would be that one allele is not sufficient for maintaining normal myocardial function [13].
These observations made in diseased tissue can be taken together to suggest that truncated protein is initially made, but then rapidly removed from the cell by proteolytic degradation. Two major degradation machineries exist in eukaryotic cells, lysosomes, and the ubiquitin–proteasome system (UPS). Lysosomes are subcellular compartments surrounded by membranes and specialized to degrade molecules of different types and also large composite structures taken up by endocytosis. The ubiquitin–proteasome system is responsible for about 80% of cellular protein degradation. It relies on a complex, enzymatic cascade that operates essentially in two steps. The first is covalent attachment of multiple ubiquitins (small proteins containing 76 amino acids) to unwanted proteins to label them for breakdown. The second step is the introduction of labelled proteins into 26 S proteasomes, complex particles capable of degrading proteins in a successive manner. These scavenger activities are not only targeted to the removal of useless or potentially dangerous proteins, but they are also involved in the regulation of essential cellular functions such as cell cycle, antigen presentation, membrane remodelling, control of apoptosis, and others. They are also involved in disease mechanisms, for instance in inflammatory responses. It has been hypothesized that the ubiquitin–proteasome system plays an important role in the progression of cardiovascular disease such as atherosclerosis [14].
The authors of the current paper [7] have asked whether the ubiquitin–proteasome system is responsible for the disappearance of truncated MyBP-C. Their study was based on isolated embryonic cardiocytes (from rats) infected with recombinant adenovirus harbouring the human MyBP-C gene in three different versions: normal length, one with 3% missing at the C-terminus of the protein, and one truncated by 80%. Quantitative analysis of expression showed roughly equal values for steady-state transcription, regardless of the extent of the truncation. However, a steep gradient was observed in the amounts of the different MyBP-C proteins, with the shortest protein being the least abundant. Imaging of sarcomeric structures in transfected cells exhibited normal intracellular appearance of full-length MyBP-C, near-normal structures with the 3% truncation, but complete loss of order of the residual quantities of the protein truncated by 80%. Evidence in favour of UPS contributing to the removal of truncated MyBP-C was obtained by the use of two selective proteasome inhibitors, lactacystin and MG132. In the presence of these drugs, the amounts of truncated protein rose significantly, with the most dramatic change seen with the 80% truncation. Thus, the answer to the above question is yes, UPS seems to play a major role in the removal of non-functional MyBP-C. Lyosomes are also involved, but only to a minor extent. These results can be taken to explain the absence of mutated MyBP-C in cardiac tissue of patients, supporting the notion of "haploinsufficiency" being a trigger of disease.
But there is more information in the current paper. Unexpected additional data were presented, which may extend the range of existing concepts of HCM pathogenesis. The new twist comes from the observation that two events occur at the same time in a counterproductive manner: degradation of the unwanted protein and, as a consequence of that, inhibition of the degrading system. Evidence in support of this conclusion was derived from co-expression of MyBP-C and a fluorescent, DsRed-based fusion protein reporter system (designated UbiG76V–DsRed) capable of monitoring the activity of UPS through loss of fluorescence upon proteolysis. Retention of fluorescence (due to non-cleavage) is indicative of UPS inhibition. Whereas normal MyBP-C showed no effect on the disappearance of the reporter, the two mutated versions did, with the 80% truncation being a stronger inhibitor than the 3% truncation. The additional demonstration of aggregates in some of the cells infected with truncated MyBP-C may be explained by assuming that UPS is degrading the useless proteins, but not completely. Residual debris is conceivably deposited in the cells by forming insoluble aggregates. However, this view has to be verified.
It is tempting to speculate. But first, follow-up investigations should corroborate these intriguing findings. If UPS is a major component in the pathogenesis of HCM, and if this system is inhibited as a consequence of its encounter with truncated MyBP-C, a route to a wider range of dysfunctions beyond the sarcomeric system may be open. To mention just one possibility: UPS plays an active role in the control of apoptosis. What if that control gets lost or weakened? Another possibility may be mentioned, too. The contribution of UPS may be restricted to the removal of truncated proteins. But couldn't it be that missense mutations, or at least some of them, also lead to proteins subject to breakdown by UPS–with all of the above consequences? A fraction of these proteins could already have an effect if they were recognized and processed as described. Whatever the details of studies to come, these interesting results allow one to conclude that "poison polypeptide" and "haploinsufficiency" concepts are not wrong, but probably much too simple to explain alone the pathogenic mechanisms of HCM.
 |
References |
|---|
 Top References |
|---|
- Marian A.J. Pathogenesis of diverse clinical and pathological phenotypes in hypertrophic cardiomyopathies. Lancet (2000) 355:58–60.[CrossRef][Web of Science][Medline]
- Fatkin D., Graham R.M. Molecular mechanisms of inherited cardiomyopathies. Physiol. Rev. (2002) 82:945–980.
[Abstract/Free Full Text] - Maron B.J. Risk stratification and prevention of sudden death in hypertrophic cardiomyopathy. Cardiol. Rev. (2002) 10:173–181.[CrossRef][Medline]
- Thierfelder L., Watkins H., MacRae C., Lamas R., McKenna W., Vosberg H.P., et al.
-Tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell (1994) 77:701–712.[CrossRef][Web of Science][Medline] - Wilkins B.J., Dai Y.S., Bueno O.F., Parsons S.A., Xu J., Plank D.M., et al. Calcineurin/NFAT coupling participates in pathological, but not physiological cardiac hypertrophy. Circ. Res. (2004) 94:110–118.
[Abstract/Free Full Text] - Dong D., Duan Y., Guo J., Roach D.E., Swirp S.L., Wang L., et al. Overexpression of calcineurin in mouse causes sudden death associated with decreased density of K+ channels. Cardiovasc. Res. (2003) 57:320–332.
[Abstract/Free Full Text] - Sarikas A., Carrier L., Schenke C., Doll D., Flavigny J., Lindenberg K.S., et al. Impairment of the ubiquitin–proteasome system by truncated cardiac myosin binding protein C mutants. Cardiovasc. Res. (2005) 66:33–44.
[Abstract/Free Full Text] - Richard P., Charron P., Carrier L., Ledeuil C., Cheav T., Pichereau C., et al. Hypertrophic cardiomyopathy–distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation (2003) 107:2227–2232.
[Abstract/Free Full Text] - Kunst G., Kress K.R., Gruen M., Uttenweiler D., Gautel M., Fink R.H. Myosin binding protein C, a phosphorylation-dependent force regulator that controls the attachment of myosin heads by its interaction with myosin S2. Circ. Res. (2000) 86:51–58.
[Abstract/Free Full Text] - Harris S.P., Rostkova E., Gautel M., Moss R.L. Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism. Circ. Res. (2004) 95:930–936.
[Abstract/Free Full Text] - Yang Q., Sanbe A., Osinska H., Hewett T.E., Klevitsky R., Robbins J. A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy. J. Clin. Invest. (1998) 102:1292–1300.[Web of Science][Medline]
- Yang Q., Sanbe A., Osinska H., Hewett T.E., Klevitsky R., Robbins J. In vivo modelling of myosin binding protein C familial hypertrophic cardiomyopathy. Circ. Res. (1999) 85:841–847.
[Abstract/Free Full Text] - Moolman J.A., Reith S., Uhl K., Bailey S., Gautel M., Jeschke B., et al. A newly created splice donor site in exon 25 of the MyBP-C gene is responsible for inherited hypertrophic cardiomyopathy with incomplete disease penetrance. Circulation (2000) 101:1396–1400.
[Abstract/Free Full Text] - Herrmann J., Ciechanover A., Lerman L.O., Lerman A. The ubiquitin–proteasome system in cardiovascular diseases–a hypothesis extended. Cardiovasc. Res. (2004) 61:11–21.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||