The muscle-specific chaperone protein Melusin is a potent cardioprotective agent Guido Tarone and Mara Brancaccio
Department of Molecular Biotechnology and Health Sciences, University of Torino, Via Nizza 52, 10126 Torino, Italy. guido.tarone@unito.it; phone: 0039-011-6706433; Fax: 0039-011-6706432
Abstract
Melusin is a protein selectively expressed in skeletal muscles and heart and highly conserved in vertebrates. Melusin is part of the heat shock protein 90 machinery and act as molecular chaperone in controlling cardiomyocyte survival and adaptive hypertrophy signalling pathways in the heart in response to different stress conditions. The role of melusin has been extensively investigated in genetically modified mice over the past years disclosing an important cardioprotective function of this unique muscle-specific chaperone protein in different pathological conditions. This review highlights the findings in animal models and the molecular mechanisms underlying melusin cardioprotective function.
Melusin protection in animal model of cardiomyopathies
Melusin (also known as ITBP1BP2) was isolated in a two-hybrid screening as protein binding to the cytosolic domain of β1 integrin, the plasma membrane receptors that mediate cell attachment to the extracellular matrix and connect contractile cytoskeleton to the plasma membrane [3].
Melusin is selectively present in skeletal muscles and heart and its expression is turned on during myogenesis in developing embryos reaching a plateau level in newborn and adult mice. Melusin function in heart has been investigated mainly by generation of loss and gain of function genetic mouse models (see summary in Table 1). Null mice are healthy and fertile and do not show defects in striated skeletal and cardiac muscle development and structure. In physiological conditions, myocardial function is fully retained in the absence of melusin in terms of left ventricle (LV) diameters, wall thickness and contractility [2] indicating that neither cardiac development nor basal function are affected by the absence of melusin in normal physiological conditions. Melusin function, however, is becoming apparent when the left ventricle is subjected to stress conditions such as aortic stenosis caused by surgical aortic banding. In such conditions cardiomyocytes lacking melusin fail to activate the hypertrophy program needed to tackle the increased workload. Indeed, while wild-type mice subjected to 4 weeks of aortic stenosis develop a typical compensatory hypertrophy with retained contractile function, melusin null mice show an impaired cardiomyocyte
hypertrophy and develop accelerated left ventricle dilation, chamber wall thinning and depressed contractility [2].
The key role of melusin in heart response to stress is further demonstrated by a gain of function transgenic mouse model in which melusin is selectively overexpressed in the heart [8]. Melusin overexpression induces mild cardiomyocytes hypertrophy in basal condition and slightly increased left ventricle wall thickness without altering contractile function nor leading to cardiomyopathy in aged mice [8]. Importantly, moreover, when heart is subjected to prolonged aortic stenosis conditions (12 weeks surgical aortic banding) melusin overexpression prevents the transition toward dilated cardiomyopathy and heart failure allowing to maintain concentric compensatory hypertrophy and full contractile function [8]. This cardioprotective function is characterized by reduced infiltration of inflammatory cells, absence of fibrosis, increased capillary density and reduced cardiomyocyte apoptotic death [8], a pattern of cardiac remodelling closely resembling that induced by physical exercise.
The left ventricle pressure overload model described above mimic human pathological conditions such as chronic aortic stenosis, long-standing systemic arterial hypertension or left ventricle outflow tract obstruction, which impose a biomechanical load that trigger an initial adaptive left ventricle hypertrophy useful to match the requirement for increased contractile power. With persistence of the biomechanical noxia imposed by aortic stenosis, adaptive hypertrophy becomes exhausted and left ventricle chamber undergo wall thinning, and contractility decreases to a point that can no longer match the body requirement. This process is accompanied by loss of cardiomyocytes, deposition of stromal tissue and reduced density of capillary vessel a process known as maladaptive cardiac remodeling characterizing heart failure conditions.
Such maladaptive remodeling can also arise in consequence of myocardial infarct [20]. In this frequent pathological condition, ischemia due to coronary vessel occlusion causes irreversible damage to the cardiac tissue which is progressively replaced by a scar of non contractile connective tissue. . When such damage involves significant portion of the left ventricle, cardiac rupture can occur in the early phase after the ischemic insult. The portion of the myocardium not interested by the ischemia undergoes deep remodelling in response to the haemodynamic stress imposed by the loss of infarcted portion. A poor response in this “recovery” phase can lead to maladaptive remodelling with consequent heart failure. Melusin overexpression in the heart, exerts a dual protective action following myocardial infarct induced by left descending coronary permanent ligation. In the early phase, it reduces inflammation and protects against cardiac rupture [40]. Interestingly, melusin protection is gender dependent being effective in male mice, but barely
appreciated in female. This can be explained by the much higher susceptibility of male mice to myocardial infarction-induced cardiac rupture.
Melusin overexpression in the heart also results in an improved response of the surviving myocardial tissue allowing better recovery of contractility and reduced left ventricle dilation and wall thinning during a 2 weeks recovery phase after coronary ligation [40].
The permanent coronary ligation infarct model used in the experiments reported above represents a very severe pathological condition which only partially mimics the human pathology where prompt coronary re-opening is the current standard of cure. Such important maneuver, however, causes further tissue damage due to sudden re-oxygenation. In fact, re-establishing coronary flow rapidly restores the trans-sarcolemmal proton gradient, exacerbating intracellular Na+
and Ca2+ overload induced during the ischemic phase. High cytosolic Ca2+ levels can have profound
negative effects on the cardiomyocyte in reperfusion [25], inducing contractile dysfunction as well as opening of the mitochondrial permeability transition pore with consequent increased reactive oxygen species (ROS) generation and the initiation of pro-death pathways [18]. This event, known as ischemia reperfusion injury, causes increased cardiomyocyte death and tissue injury paradoxically amplifying the initial ischemic damage. Melusin overexpression reduces the infarct size in an ex-vivo whole heart setting (Langendorff) of transient ischemia, clearly protecting from reperfusion injury [28].
As discussed above, the pathophysiological conditions leading to heart failure in consequence of pressure overload, myocardial infarction and ischemia/reperfusion injury are quite distinct. In spite of these differences, all these pathological events result in excess cardiomyocyte workload and activation of common salvage pathways aimed to sustain cardiomyocyte function and survival. The ability of the muscle specific chaperone melusin to sustain these pathways (see below) can explain the broad cardioprotective action of melusin in all these pathophysiological conditions. Altered melusin expression in experimental and human cardiomyopathies
Melusin expression is upregulated during skeletal muscle regeneration [3] and in the heart left ventricle during the first week of pressure overload imposed by aortic banding [8, 35]. This timing coincides with the initial compensatory hypertrophy response of the left ventricle to the pressure overload conditions. However, after 12 weeks of pressure overload, when dilation of the left ventricle and loss of contractility occurs, melusin level are decreased [8]. Initial increase followed by late decrease of melusin level was also reported in a volume overload dog model [9]. Melusin levels in the left ventricle also directly correlate with the rate of left ventricle pressure rise in systole (LVdp/dt) and fractional shortening in a rat myocardial infarct model analyzed at
also present in a cohort of 17 human aortic stenosis patients with severe heart failure and undergoing heart transplantation [4] (Table 1). Other authors have reported no alteration of melusin expression level in the heart of five idiopathic dilated cardiomyopathy and eight ischemic dilated cardiomyopathy patients undergoing heart transplantation [29]. Whether this apparent discrepancy with the results in aortic stenosis patients can be ascribed to different pharmacological treatment or different etiology of the dilated cardiomyopathy remain to be established. Melusin is also expressed in the myocardial atria where it is upregulated in response to neurohumoral stimuli such as
arginine(8)-vasopressin, angiotensin-II and endothelin [1]. Melusin down-regulation in
different cardiac pathological conditions and the functional analysis of transgenic mice, suggest that increasing melusin expression in the left ventricle can represent an important therapeutic approach (see last paragraph).
Mutation of melusin gene in human cardiomyopathies
The functional properties described above point to a possible role of melusin as “modifier gene” whose alteration in sequence or expression could be responsible for increased susceptibility to develop cardiomyopathy in the presence of different pathological stress stimuli. This consideration prompted the search for melusin gene mutations in cardiopathic patients. Surprisingly, this analysis, carried out by three independent laboratories, indicated that mutation of melusin gene is a very rare event. Two missense mutations (His13Tyr, Ala313Gly) were described in probands with a history of familiar cardiomyopathy [27, 32]. The His13Tyr mutation causes the substitution of a conserved amino acid residue in the CHORD I domain (Cysteine and Histidine-rich domain) and was present in the family of a patient with hypertrophic cardiomyopathy [27] (Figure 1). The Ala313Gly mutation is present in the carboxy terminal region linking the CS (domain shared by CHORD containing protein and Sgt1) to the acidic domain (Figure 1) and was identified in the family of patients with dilated cardiomyopathy [32]. The causative significance of these mutations in the context of the cardiomyopathy, however, is unclear since their segregation in the family members did not always correlate with the cardiomyopathy. One additional silent mutation and a nucleotides duplication, as well as a number of single nucleotide polymorphisms were described in a screening involving 1550 patients and healthy control subjects [27]. Overall these data indicate a very high degree of conservation of the melusin gene within the populations analyzed and tend to exclude that genetic variations of melusin gene can have a significant impact on the epidemiology of cardiomyopathy.
The cardioprotective function of melusin [8] [40] is associated with an adaptive cardiac remodelling characterized by increased cardiomyocyte hypertrophy and survival as well as with decreased inflammatory cell infiltrate and deposition of fibrosis. Cardiomyocytes from melusin overexpressing heart, in fact, show increased diameter and length both in basal conditions and after chronic stress stimuli [8]. Notably, the hypertrophy marker alpha skeletal actin, an actin isoform upregulated in human failing heart as well as in experimental cardiomyopathy models, is downregulated in melusin overexpressing hearts both in basal conditions and after pressure overload induced by aortic stenosis [8]. At the same time, melusin overexpression induces increased levels of atrial natriuretic peptide [2, 8], a well known protective agent in heart failure [26].
Melusin overexpression also prevents cardiomyocyte apoptotic death in response to pressure overload [8] myocardial infarct [40] or ischemia reperfusion injury [28]. Cultured cardiomyocytes overexpressing melusin, moreover, are protected from apoptosis induced by oxidative stress [33], thus indicating that melusin overexpression can sustain cardiomyocyte survival in response to different myocardial pathological conditions.
Melusin overexpression also reduces inflammatory cell infiltrate and fibrosis [8, 40]. The mechanisms involved in these functions have not been investigated in details, but reduced inflammatory reaction is paralleled by decreased expression of CXCL10 and increased expression of Pgf, two inflammatory cytokines involved in recruiting leukocytes to inflamed tissues [24] and in promoting reparative inflammation [6] respectively. Moreover, decreased level of TGF-beta have been detected in melusin overexpressing heart subjected to chronic pressure overload consistently with the decreased collagen synthesis and deposition [8].
The pro-hypertrophy and pro-survival function of melusin are dependent on its ability to potentiate the extracellular signal-regulated kinases ERK1/2 and the AKT (also known as protein kinase B) signalling. Elevation of melusin expression in the heart is sufficient to induce increased level of phosphorylation of both AKT and ERK1/2 in basal conditions [8]. Moreover, phosphorylation of these signalling proteins in response to acute surgical aortic stenosis is potentiated by melusin overexpression [8]. Activation of AKT and ERK1/2 signaling is required for the melusin-induced cardiomyocyte hypertrophy as demonstrated by the use of specific pharmacological inhibitors in cultured cardiomyocytes [8, 33]. Melusin-dependent activation of these pathways, moreover, is required for cardiomyocyte protection from apoptosis in response to oxidative stress [33] as well as to reduced tissue damage and infarct size in an ischemia reperfusion injury ex vivo model [28]. Overall, these data demonstrate that the cardioprotective function of melusin is dependent on its ability to potentiate these signalling pathways, likely through melusin chaperone function. In fact, as discussed in the following paragraph, melusin belongs to the class of
small chaperone proteins [14] and it binds to, and cooperates with, the large heat shock protein 90 (Hsp90) [35].
Melusin chaperone function in ERK1/2 and AKT signaling
Chaperones are a large family of molecules assisting protein folding, assembly of supra-molecular structures and performing protein quality control during intense protein translation activity [19, 37]. The best known chaperones are Hsp70 and Hsp90 which primarily recognize hydrophobic amino acid side-chains exposed by non-native proteins and promote their folding through ATP-regulated cycles of binding and release. Different class of chaperones are usually acting in concert to assist protein folding and their action is tightly controlled by so called co−chaperones, a class of molecules that mediate the specificity of a chaperone reaction by choosing the target protein, presenting it to Hsp70 or Hsp90, and coordinating the cycle of binding and release [5, 37].
The fact that accumulation of misfolded proteins in the heart plays a key role in the onset and progression of cardiac hypertrophy and of different forms of cardiomyopathies, is an emerging concept. In fact, mechanical stretching, hypertrophic growth and oxidative stress can induce misfolded protein accumulation, causing cardiomyocyte death or dysfunction [38]; [44]. The ability of chaperone protein to assist protein folding, however, is not only crucial during protein synthesis, but is instrumental to signal transduction. In fact, activation and deactivation of signal transduction proteins involves conformational switches which require chaperone assistance for maximal efficiency. In particular Hsp90 regulates stability and activation state of a wide range of signal transduction proteins including several tyrosine and serine/threonine kinases such as pp60/v−src, Wee−1, Cdk4, Raf, AKT, PI3K and PDK1 [12, 41, 45] in addition to the extensively characterized steroid hormone receptor[31]. The crucial importance of chaperone function in signal transduction is highlighted by the fact that Hsp90 inhibitors induce tumor cell growth arrest and apoptosis by blunting a wide range of signalling pathways [15, 22].
Indeed melusin, in addition to Hsp90, binds a number of signalling molecules, including the mitogen activated protein kinases c-Raf, MEK1/2 and ERK1/2, the Focal Adhesion Kinase (FAK) and the scaffold protein IQGAP1 [33] (Figure 2 and Table 2). Interestingly, in vivo analysis on transgenic mice subjected to surgical banding of the aorta demonstrated that melusin-bound ERK1/2 are activated following biomechanical stress in a FAK and MEK1/2 dependent manner [33].
The melusin interactor, IQGAP1, is a scaffold protein for the MAPK cascade, capable to bind to all the components of the cascade (Raf, MEK1/2 and ERK1/2) allowing their sequential activation [10, 34]. Indeed absence of IQGAP1 in the melusin complex leads to defective ERK1/2
activation in response to biomechanical stress. Moreover, in vitro analysis on neonatal cardiomyocytes demonstrated that both FAK and IQGAP1 are required for melusin-dependent cardiomyocyte hypertrophy and protection from apoptosis through the regulation of ERK1/2 activity [33]. The crucial importance of IQGAP1 as MAPK scaffold downstream of melusin is also confirmed in vivo. In fact, in absence of IQGAP1 mouse hearts display defective ERK1/2 activation and undergo unfavorable cardiac remodeling with accelerated dilation, contractile dysfunction and increased cardiomyocyte apoptosis upon prolonged biomechanical overload [34].
The ERK1/2 pathway is activated by multiple stimuli in addition to mechanical stretching including adrenergic, neuro-endocrine and growth factors. These stimuli can result in either adaptive or maladaptive cardiac response [39] depending on the specific molecules targeted by the activated ERK1/2. This property is crucially affected by the scaffold proteins involved in coordinating the assembly and subcellular localization of the Raf–MEK1/2–ERK1/2 cascade. A number of different proteins are known to act as scaffold for the Raf–MEK1/2–ERK1/2 cascade among which β-arrestin, Gβγ subunits, the four-and-a-half LIM domain- protein 1(FHL1), kinase suppressor of Ras 1 (KSR1) and Ras GTPase-activating-like protein 1 (IQGAP-1). Interestingly melusin binds to IQGAP-1, but does not interact with KSR1 or other known ERK scaffolds indicating a unique selectivity [33] of the melusin dependent ERK1/2 pathway. Based on these data it can be hypothesized that melusin, together with the scaffold protein IQGAP1, is responsible for the organization of a specific signalosome complex allowing activation of target proteins responsible for adaptive remodeling (Figure 2).
Melusin can also bind to p85 (Table 2), the regulatory subunit of class IA PI3Ks, promoting its lipid kinase activity [43] and, thus, potentially controlling downstream signalling including the AKT/GSK3beta pathway. Interestingly, the major melusin interacting protein IQGAP1, in addition to Raf, MEK and ERK, can also bind AKT [34] suggesting a possible co-operation of melusin-IQGAP1 complex in the coordinated activation of AKT.
Melusin is also known to interact with the serine/threonine phosphatase PP5 [21], with the S100 proteins A6, A4 and A1 [11] as well as with the cytoplasmic domain of the beta1 integrin subunit [3] (Table 2). While the functional consequences of these interactions have not been investigated, it can be hypothesized that binding to the PP5 phosphatase can have an impact on the melusin dependent ERK1/2 signaling since a major PP5 substrate is the Raf kinase [42].
The S100 proteins are ubiquitous and multifunctional EF-handed Ca2+ binding proteins. Melusin
co-immunoprecipitates with S100A1 and S100A4 [11] and the short sequence between the CS domain and the C-terminal acidic region of melusin (see Figure 1 and Table 2) binds to the S100 protein in a Ca2+ dependent manner [11]. The melusin CS domain is also known to bind to the cytoplasmic
domain of the integrin beta1 subunit raising the possibility that Ca2+-dependent S100A binding can regulate interaction of melusin with the integrin cytoplasmic region.
Structural basis of melusin chaperone function
Melusin consists of two zinc binding domains called CHORDs (Cysteine and Histidine-rich domains), a CS domain (shared by CHORD containing protein and Sgt1) [36] structurally similar to -crystallin and p23 chaperone proteins [13] and an C-terminal Ca2+ binding domain highly
enriched in aspartic and glutamic acid residues [3] (Figure 1).
The -crystallin-like CS domain is a structural feature common to a class of heat shock proteins belonging to the small chaperone family [14]. Indeed melusin is endowed with intrinsic chaperone activity as demonstrated by its ability to protect protein substrates from heat induced aggregation [35]. A third feature characterizing melusin chaperone function is its ability to bind the well known heat shock protein 90 (Hsp90) [35]. Hsp90, in association with a number of different co-chaperones, assists folding of several target proteins. Melusin, however, does not behave as an Hsp90 substrate [35], but it is rather acting in cooperation with Hsp90 to regulate its chaperone function and/or directing it toward specific target proteins.
The structural basis of melusin binding to Hsp90 has been investigated in some detail. The terminal region of melusin containing CHORD I–II domains directly binds to the amino-terminal ATPase domain of Hsp90 [35]. Binding is increased with adenine nucleotides (ADP and ATP) and Ca2+ [21, 35]. CHORD domains are 60-amino acid modules that bind two zinc ions. They
are usually arranged in tandem and are highly conserved during evolution from plant to mammals. Six cysteine and two histidine residues are invariant within the CHORD domain (Figure 1). Three other residues are also invariant and some positions are confined to positive, negative, or aromatic amino acids. The crystal structure of CHORD domain of Rar1, a protein involved in innate immunity in plants, has been resolved and the Hsp90 binding residues have been identified [46]. CHORD domains also bind to the CS domain [46], a structure present in melusin itself as well as in the Sgt1 and in small chaperone family proteins [14]. Indeed, melusin CHORD domains bind to the CS domain present in the carboxy terminal region of the protein in a two hybrid assay with isolated melusin fragments (unpublished results). This property implies that melusin can form dimers and/or concatamers, a property common to several members of the “small chaperone” protein family and known to be critical in the regulation of chaperone function [14]. Indeed, melusin has been described to form homocomplexes in vitro [21]. The protein surface and the amino acid residues involved in the CHORD/CS binding have been defined by resolving the crystal structure of the CHORD of Rar1 bound to the CS domain of the Sgt1 protein [46].
Interestingly, nucleotides can modify both melusin-melusin interaction as well as the melusin binding to Hsp90. In fact, Rar1-CHORD domain directly interacts with the ADP bound to the nucleotide pocket of Hsp90 [46]. Moreover, ADP and ATP, likely binding to melusin itself, increase both melusin/melusin binding as well as its binding to Sgt1 and to the protein phosphatase 5 [21]. In addition, Ca2+, which is known to bind to melusin, enhances the interactions of melusin
with Hsp90 and Sgt1. The ability of nucleotides and Ca2+ to modulate melusin interactions with
several molecular partners [3, 11, 21], clearly add complexity to the dynamics of the melusin/Hsp90 chaperone system and highlights the need to define the functional consequences on chaperone function.
Concluding remarks
The muscle specific chaperone protein melusin is required to promote cardiomyocyte compensatory hypertrophy program and survival in response to a number of different stress conditions. Such function requires activation of both ERK1/2 and AKT signalling pathways via the assembly of a signalosome complex which includes the heat shock protein 90 and the scaffold protein IQGAP1. Melusin levels are depressed in chronic pathological heart conditions such as in failing heart of aortic stenosis patients [4] as well as in experimental models of pressure overload [8], volume overload [9] and myocardial infarct [17, 40]. At the same time forced expression of the protein in transgenic mice demonstrated strong cardioprotective effect in response to pressure overload, myocardial infarction and ischemia reperfusion injury [8, 28, 40] in the absence of detrimental action on basal heart function. Thus, increasing melusin expression in cardiomyocytes could represent an innovative and powerful therapeutic approach for a number of different cardiac pathological conditions. The innovative aspects, in particular, consist in the muscle restricted expression of melusin as well as in its chaperone function. The cardiac and striated muscle-restricted expression of the protein indicates a selective and dedicated function of melusin in the molecular environment of contractile muscle cells. The chaperone function implies the ability to orchestrate a network of biochemical events specifically directed to sustain cellular function and survival in response to stress stimuli. A delivery protocol capable to selectively target melusin gene to cardiomyocyte in vivo will be a powerful approach. Adeno associated viruses (AAVs), and the cardio tropic AAV9 serotype in particular, are the most attractive viral vectors for in vivo gene delivery at present allowing efficient and stable transgene expression in the absence of risk of insertional mutagenesis and of significant inflammation or immune response [7, 30]. The AAVs are the current mode for percutaneous intracoronary delivery of SERCA2 gene therapy in heart failure clinical trials [16]. Taking in consideration the highly encouraging results of these initial gene
therapy clinical trials [23], the possible extension of this approach to other therapeutic targets can be considered after appropriate safety tests in preclinical animal models.
Acknowledgments
This work was supported by funding from Telethon grant GGP12047 to GT, MIUR Prin 2010RNXM9C_002 to GT; MIUR PRIN 2010J8RYS7_007 to MB.
Conflict of interest: Mara Brancaccio and Guido Tarone are cofounders and scientific consultants for Target Heart Biotec, a company that develops melusin recombinant protein as a drug to counteract cardiomyopathies.
References
1. Aro J, Tokola H, Ronkainen VP, Koivisto E, Tenhunen O, Ilves M, Szokodi I, Ruskoaho H, Rysa J (2013) Regulation of cardiac melusin gene expression by hypertrophic stimuli in the rat. Acta Physiol (Oxf) 207:470-484 doi:10.1111/apha.12044
2. Brancaccio M, Fratta L, Notte A, Hirsch E, Poulet R, Guazzone S, De Acetis M, Vecchione C, Marino G, Altruda F, Silengo L, Tarone G, Lembo G (2003) Melusin, a muscle-specific integrin beta1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload. Nat Med 9:68-75 doi:10.1038/nm805
3. Brancaccio M, Guazzone S, Menini N, Sibona E, Hirsch E, De Andrea M, Rocchi M, Altruda F, Tarone G, Silengo L (1999) Melusin is a new muscle-specific interactor for beta(1) integrin cytoplasmic domain. J Biol Chem 274:29282-29288
doi:10.1074/jbc.274.41.29282
4. Brokat S, Thomas J, Herda LR, Knosalla C, Pregla R, Brancaccio M, Accornero F, Tarone G, Hetzer R, Regitz-Zagrosek V (2007) Altered melusin expression in the hearts of aortic stenosis patients. Eur J Heart Fail 9:568-573 doi:10.1016/j.ejheart.2007.02.009
5. Caplan AJ (2003) What is a co-chaperone? Cell Stress Chaperones 8:105-107 doi:http://dx.doi.org/10.1379/1466-1268(2003)008<0105:WIAC>2.0.CO;2
6. Carnevale D, Cifelli G, Mascio G, Madonna M, Sbroggio M, Perrino C, Persico MG, Frati G, Lembo G (2011) Placental growth factor regulates cardiac inflammation through the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-alpha-converting enzyme axis: crucial role for adaptive cardiac remodeling during cardiac pressure overload. Circulation 124:1337-1350 doi:10.1161/CIRCULATIONAHA.111.050500
7. Chirmule N, Propert K, Magosin S, Qian Y, Qian R, Wilson J (1999) Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther 6:1574-1583
doi:10.1038/sj.gt.3300994
8. De Acetis M, Notte A, Accornero F, Selvetella G, Brancaccio M, Vecchione C, Sbroggio M, Collino F, Pacchioni B, Lanfranchi G, Aretini A, Ferretti R, Maffei A, Altruda F, Silengo L, Tarone G, Lembo G (2005) Cardiac overexpression of melusin protects from dilated
cardiomyopathy due to long-standing pressure overload. Circ Res 96:1087-1094 doi: 10.1161/01.RES.0000168028.36081.e0
9. Donker DW, Maessen JG, Verheyen F, Ramaekers FC, Spatjens RL, Kuijpers H, Ramakers C, Schiffers PM, Vos MA, Crijns HJ, Volders PG (2007) Impact of acute and enduring volume overload on mechanotransduction and cytoskeletal integrity of canine left ventricular myocardium. Am J Physiol Heart Circ Physiol 292:H2324-2332 doi: 10.1152/ajpheart.00392.2006
10. Ferretti R, Sbroggio M, Di Savino A, Fusella F, Bertero A, Michowski W, Tarone G, Brancaccio M (2011) Morgana and melusin: two fairies chaperoning signal transduction. Cell Cycle 10:3678-3683 doi:10.4161/cc.10.21.18202
11. Filipek A, Michowski W, Kuznicki J (2008) Involvement of S100A6 (calcyclin) and its binding partners in intracellular signaling pathways. Adv Enzyme Regul 48:225-239 doi: 10.1016/j.advenzreg.2007.11.001
12. Fujita N, Sato S, Ishida A, Tsuruo T (2002) Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1. J Biol Chem 277:10346-10353
doi:10.1074/jbc.M106736200
13. Garcia-Ranea JA, Mirey G, Camonis J, Valencia A (2002) p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families. FEBS Lett 529:162-167 doi:S0014579302033215
14. Garrido C, Paul C, Seigneuric R, Kampinga HH (2012) The small heat shock proteins family: the long forgotten chaperones. Int J Biochem Cell Biol 44:1588-1592
doi:10.1016/j.biocel.2012.02.022
15. Gaspar N, Sharp SY, Eccles SA, Gowan S, Popov S, Jones C, Pearson A, Vassal G,
Workman P (2010) Mechanistic evaluation of the novel HSP90 inhibitor NVP-AUY922 in adult and pediatric glioblastoma. Mol Cancer Ther 9:1219-1233
doi:10.1158/1535-7163.MCT-09-0683
16. Greenberg B, Yaroshinsky A, Zsebo KM, Butler J, Felker GM, Voors AA, Rudy JJ, Wagner K, Hajjar RJ (2014) Design of a phase 2b trial of intracoronary administration of
AAV1/SERCA2a in patients with advanced heart failure: the CUPID 2 trial (calcium up-regulation by percutaneous administration of gene therapy in cardiac disease phase 2b). JACC Heart Fail 2:84-92 doi:10.1016/j.jchf.2013.09.008
17. Gu R, Zheng D, Bai J, Xie J, Dai Q, Xu B (2012) Altered melusin pathways involved in cardiac remodeling following acute myocardial infarction. Cardiovasc Pathol 21:105-111 doi:10.1016/j.carpath.2011.03.002
18. Halestrap AP, Pasdois P (2009) The role of the mitochondrial permeability transition pore in heart disease. Biochim Biophys Acta 1787:1402-1415 doi:10.1016/j.bbabio.2008.12.017 19. Hartl FU, Hayer-Hartl M (2009) Converging concepts of protein folding in vitro and in
vivo. Nat Struct Mol Biol 16:574-581 doi:10.1038/nsmb.1591
20. Heusch G, Libby P, Gersh B, Yellon D, Bohm M, Lopaschuk G, Opie L (2014)
Cardiovascular remodelling in coronary artery disease and heart failure. Lancet 383:1933-1943 doi:10.1016/S0140-6736(14)60107-0
21. Hong TJ, Kim S, Wi AR, Lee P, Kang M, Jeong JH, Hahn JS (2013) Dynamic nucleotide-dependent interactions of cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones Chp-1 and melusin with cochaperones PP5 and Sgt1. J Biol Chem 288:215-222 doi:10.1074/jbc.M112.398636
22. Hostein I, Robertson D, DiStefano F, Workman P, Clarke PA (2001) Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis. Cancer Res 61:4003-4009
23. Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B, Yaroshinsky A, Zsebo KM, Dittrich H, Hajjar RJ (2011) Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation 124:304-313 doi:10.1161/CIRCULATIONAHA.111.022889
24. Lee EY, Lee ZH, Song YW (2009) CXCL10 and autoimmune diseases. Autoimmun Rev 8:379-383 doi:10.1016/j.autrev.2008.12.002
25. Murphy E, Steenbergen C (2008) Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol Rev 88:581-609 doi:10.1152/physrev.00024.2007
26. Nishikimi T, Maeda N, Matsuoka H (2006) The role of natriuretic peptides in cardioprotection. Cardiovasc Res 69:318-328 doi:10.1016/j.cardiores.2005.10.001 27. Palumbo V, Segat L, Padovan L, Amoroso A, Trimarco B, Izzo R, Lembo G,
Regitz-Zagrosek V, Knoll R, Brancaccio M, Tarone G, Crovella S (2009) Melusin gene
(ITGB1BP2) nucleotide variations study in hypertensive and cardiopathic patients. BMC Med Genet 10:140 doi:10.1186/1471-2350-10-140
28. Penna C, Brancaccio M, Tullio F, Rubinetto C, Perrelli MG, Angotti C, Pagliaro P, Tarone G (2014) Overexpression of the muscle-specific protein, melusin, protects from cardiac ischemia/reperfusion injury. Basic Res Cardiol 109:418 doi:10.1007/s00395-014-0418-9 29. Pfister R, Acksteiner C, Baumgarth J, Burst V, Geissler HJ, Margulies KB, Houser S, Bloch
W, Flesch M (2007) Loss of beta1D-integrin function in human ischemic cardiomyopathy. Basic Res Cardiol 102:257-264 doi:10.1007/s00395-006-0640-1
30. Ponnazhagan S, Erikson D, Kearns WG, Zhou SZ, Nahreini P, Wang XS, Srivastava A (1997) Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells. Hum Gene Ther 8:275-284 doi:10.1089/hum.1997.8.3-275 31. Pratt WB, Toft DO (1997) Steroid receptor interactions with heat shock protein and
immunophilin chaperones. Endocr Rev 18:306-360 doi:10.1210/edrv.18.3.0303
32. Ruppert V, Meyer T, Richter A, Maisch B, Pankuweit S (2013) Identification of a missense mutation in the melusin-encoding ITGB1BP2 gene in a patient with dilated cardiomyopathy. Gene 512:206-210 doi:10.1016/j.gene.2012.10.055
33. Sbroggio M, Bertero A, Velasco S, Fusella F, De Blasio E, Bahou WF, Silengo L, Turco E, Brancaccio M, Tarone G (2011) ERK1/2 activation in heart is controlled by melusin, focal adhesion kinase and the scaffold protein IQGAP1. J Cell Sci 124:3515-3524
doi:10.1242/jcs.091140
34. Sbroggio M, Carnevale D, Bertero A, Cifelli G, De Blasio E, Mascio G, Hirsch E, Bahou WF, Turco E, Silengo L, Brancaccio M, Lembo G, Tarone G (2011) IQGAP1 regulates ERK1/2 and AKT signalling in the heart and sustains functional remodelling upon pressure overload. Cardiovasc Res 91:456-464 doi:10.1093/cvr/cvr103
35. Sbroggiò M, Ferretti R, Percivalle E, Gutkowska M, Zylicz A, Michowski W, Kuznicki J, Accornero F, Pacchioni B, Lanfranchi G, Hamm J, Turco E, Silengo L, Tarone G,
Brancaccio M (2008) The mammalian CHORD-containing protein melusin is a stress response protein interacting with Hsp90 and Sgt1. FEBS Lett 582:1788-1794 doi: 10.1016/j.febslet.2008.04.058
36. Shirasu K, Lahaye T, Tan MW, Zhou F, Azevedo C, Schulze-Lefert P (1999) A novel class of eukaryotic zinc-binding proteins is required for disease resistance signaling in barley and development in C. elegans. Cell 99:355-366
doi:http://dx.doi.org/10.1016/S0092-8674(00)81522-6
37. Taipale M, Jarosz DF, Lindquist S (2010) HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat Rev Mol Cell Biol 11:515-528 doi:10.1038/nrm2918 38. Tarone G, Brancaccio M (2014) Keep your heart in shape: molecular chaperone networks
for treating heart disease. Cardiovasc Res 102:346-361 doi:10.1093/cvr/cvu049
39. Tarone G, Sbroggio M, Brancaccio M (2013) Key role of ERK1/2 molecular scaffolds in heart pathology. Cell Mol Life Sci 70:4047-4054 doi:10.1007/s00018-013-1321-5
40. Unsold B, Kaul A, Sbroggio M, Schubert C, Regitz-Zagrosek V, Brancaccio M, Damilano F, Hirsch E, Van Bilsen M, Munts C, Sipido K, Bito V, Detre E, Wagner NM, Schafer K, Seidler T, Vogt J, Neef S, Bleckmann A, Maier LS, Balligand JL, Bouzin C, Ventura-Clapier R, Garnier A, Eschenhagen T, El-Armouche A, Knoll R, Tarone G, Hasenfuss G (2014) Melusin protects from cardiac rupture and improves functional remodelling after myocardial infarction. Cardiovasc Res 101:97-107 doi:10.1093/cvr/cvt235
41. van der Straten A, Rommel C, Dickson B, Hafen E (1997) The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila. EMBO J 16:1961-1969 doi:10.1093/emboj/16.8.1961
42. von Kriegsheim A, Pitt A, Grindlay GJ, Kolch W, Dhillon AS (2006) Regulation of the Raf-MEK-ERK pathway by protein phosphatase 5. Nat Cell Biol 8:1011-1016
doi:10.1038/ncb1465
43. Waardenberg AJ, Bernardo BC, Ng DC, Shepherd PR, Cemerlang N, Sbroggio M, Wells CA, Dalrymple BP, Brancaccio M, Lin RC, McMullen JR (2011) Phosphoinositide 3-kinase (PI3K(p110alpha)) directly regulates key components of the Z-disc and cardiac structure. J Biol Chem 286:30837-30846 doi:10.1074/jbc.M111.271684
44. Willis MS, Patterson C (2013) Proteotoxicity and cardiac dysfunction--Alzheimer's disease of the heart? N Engl J Med 368:455-464 doi:10.1056/NEJMra1106180
45. Xu Y, Lindquist S (1993) Heat-shock protein hsp90 governs the activity of pp60v-src kinase. Proc Natl Acad Sci U S A 90:7074-7078 doi:10.1073/pnas.90.15.7074
46. Zhang M, Kadota Y, Prodromou C, Shirasu K, Pearl LH (2010) Structural basis for
assembly of Hsp90-Sgt1-CHORD protein complexes: implications for chaperoning of NLR innate immunity receptors. Mol Cell 39:269-281 doi:10.1016/j.molcel.2010.05.010
Figure legends
Figure 1. A) Schematic representation of melusin structural domains (CHORDI, CHORDII and CS domains). B) Sequence alignment among human (Homo sapiens), bovine (Bos Taurus), swine (Sus scrofa), rat (Rattus novergicus) and mouse (Mus musculus) melusin. CHORD domains are underlined and CS domain is double underlined. Asterisks under the sequences indicate invariant residues, colons indicate strongly similar residues and dots indicate weakly similar residues. In bold are highlighted amino acids conserved from plant to mammals CHORD domains.
Figure 2. Schematic representation protein involved in melusin-dependent ERK1/2 signaling in cardiomyocytes.
Table 1. Melusin expression and function in different experimental and pathological settings. Cardiac pathology Experimental model Results References Dilated cardiomyopathy Aortic banding (melusin null mice) Absence of melusin worsens left ventricle contractility and dilation [2] Dilated cardiomyopathy Aortic banding (transgenic mice) Forced melusin expression protects from left ventricle dilation
[8]
Myocardial
infarction Permanent coronary ligation (rat)
Endogenous melusin expression level parallels cardiac
function during cardiac remodeling post- myocardial infarct [17] Myocardial infarction Permanent coronary ligation (transgenic mice) Forced melusin expression protects from acute cardiac rupture and sustains functional recovery [40] Ischemia reperfusion injury Ex-vivo Langendorff technique (transgenic mice) Forced melusin expression reduces infarct size and tissue damage [28] Adaptive remodelling processes Infusion of arginine(8) -vasopressin (AVP) or angiotensin II (Ang II) in rats
Upregulation of left atrial melusin [1] Volume overload induced myocardial remodelling Atrioventricular
block (dog) Upregulation of LV melusin peaks in correspondence to hypertrophic growth [9] Dilated cardiomyopathy in aortic stenosis patients Human heart biopsies Endogenous melusin RNA and protein levels parallels the functional cardiac impairment
Table 2 Melusin interacting proteins.
Interactor Melusin
domain involved
Potential regulator References
Beta 1 integrin CS Ca2+ [3]
Hsp90 CHORD ATP/ADP, Ca2+ [21, 35, 46]
IQGAP-1 CS [33]
ERK1/2 N.D. [33]
FAK N.D. [33]
S100A1, A4, A6, CS Ca2+ [11]
P85-PI3K N.D. [43]
PP5 N.D. ATP/ADP [21]
