Introduction

A delicate balance between protein synthesis and degradation maintains muscle trophic homeostasis, including cardiac size and function. A shift in protein homeostasis in favor of synthesis, resulting in increased cell protein content, is an established mechanism for cardiac hypertrophy.¹ The molecular pathways responsible for enhanced protein synthesis and ensuing cardiac hypertrophy have been extensively characterized.² In contrast, the potential contributions of the protein degradation pathways, including the ubiquitin-proteasome system (UPS), in maintaining cardiac protein homeostasis and the pathogenesis of cardiac hypertrophy are less well understood.^3–7

Hypertrophic cardiomyopathy (HCM) is a relatively common genetic disorder and a prototypic form of cardiac hypertrophy.^8,9 More than a dozen causal genes, coding for thick, thin, and Z disk proteins of sarcomeres, have been identified in probands and families with HCM.^8,9 Cardiac hypertrophy, notwithstanding the genetic-based diagnosis, is the clinical diagnostic hallmark of HCM and a major determinant of mortality and morbidity.^10–12 HCM is the most common cause of sudden cardiac death (SCD) in the young athletes and an important cause of morbidity in the older adults.^9,13

Cardiac hypertrophy in HCM is considered secondary to activation of a diverse array of intracellular signaling pathways that collectively promote protein synthesis.^14,15 The role of protein degradation, the opposite end of the spectrum from protein synthesis, in the pathogenesis of HCM is less well recognized.^16,17 Identification and functional characterization of TRIM63 encoding MuRF1 and FBXO32 encoding F-box protein 32 (aka Atrogin 1 or MAFbx) have raised considerable interest in the role of these molecular regulators of cardiac protein degradation in maintaining cardiac structure and function in cardiomyopathies.^4,5,18–20

TRIM63 (protein ID:Q969Q1), also known as muscle-specific RING finger protein 1 (MuRF1), is an E3 ubiquitin ligase that is expressed selectively in cardiac and skeletal muscles.¹⁸ TRIM63 is capable of polyubiquitination and UPS-mediated degradation of thick filament proteins MYH6 and MYBPC3.^21–23 Overexpression of MuRF1 (TRIM63) antagonizes cardiac myocyte hypertrophic response to agonists and increases susceptibility to heart failure in response to pressure overload.^4,24 In contrast, deficiency of MuRF1 (MuRF1^−/−) exaggerates cardiac hypertrophic response to aortic banding.⁶ In view of the increasing recognition of protein degradation pathways in regulating muscle trophic state, we set to delineate the pathogenic role of TRIM63 in cardiomyopathies. Accordingly, we screened HCM probands for mutations in TRIM63 and identified and functionally characterized 3 mutations that invoke impaired protein degradation as a likely pathogenic mechanism for cardiac hypertrophy in human HCM.

Methods

An expanded Methods section is provided in the Online Data Supplement.

Ethical Approval

The institutional review board approved the studies in humans. The participants signed informed consents. The use of mice conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and was approved by the Institutional Animal Care and Use Committee.

Study Population

The discovery study population comprised 302 (250 white) probands with HCM and 339 (262 white) control individuals, all evaluated by history, physical examination, ECG, and echocardiography. Additionally, 751 control individuals were screened by the TaqMan assays for the specific rare variants identified in the HCM probands.

TRIM63 Variants

The coding and exon-intron boundary regions of TRIM63 were sequenced by Sanger sequencing (Online Table I). The sequence output was analyzed using Variant Reporter software and manually by 2 individuals and compared with the TRIM63 reference sequence (hg19, Chromosome 1, NC 000001.10, 26377798.26394121, complement). Variant genotyping was performed using the Allelic Discrimination Assays on a 7900HT SDS (Online Table I).

Multiple Sequence Alignment

Regions encompassing the mutant amino acids from multiple species were aligned using ClustalW (http://www.ebi.ac.uk/Tools/msa/ clustalw2/).

Exclusion of Known Common Causal Genes for HCM

TRIM63 mutation carriers were screened for mutations in MYH7, MYBPC3, TNNT2, TNNI3, TPM1, and ACTC1, known relatively common genes for HCM, by Sanger sequencing.⁹

Genotyping

Family members were genotyped for 5 short tandem repeat (STR) DNA markers located near the TRIM63 locus on 1p34-p33 by PCR and capillary electrophoresis on an ABI 3730xl system²⁵ (Online Figure I).

Cloning and Site-Directed Mutagenesis

Trim63 cDNA was synthesized from mouse total cardiac RNA by reverse transcription and was tagged with a Flag at the 3′ end. The mutations were introduced by site-directed mutagenesis (Online Table I).

Recombinant Lentivirus and Adenoviruses

Flag-tagged wild-type (WT), p.A48V, p.I130M, and p.Q247* Trim63 cDNAs were cloned into the lentiviral expression vector. The lentiviral plasmid containing the Trim63 cDNA and the packaging plasmids were transfected into 293T cells to generate the viruses.

Flag-tagged Trim63 cDNAs were cloned into adenovirus expression vector by homologous recombination. The plasmid DNA was mixed with Lipofectamine 2000 and transfected to 293A cells. Cells were harvested 7 to 10 days after transfection, when they reached ≈80% cytopathicity. The crude adenovirus lysates were prepared by freeze/thaw cycles. Viruses were amplified to generate higher-titer viral stocks.

HeLa-His/Biotin-Ubiquitin Cells

An MSCV retroviral plasmid expressing a His6-biotin-ubiquitin fusion protein and retrovirus packaging plasmids was cotransfected into 293T cells. Retroviruses were then used to infect the HeLa cells. Cells were then selected under blasticidin selection to generate stable HeLa-His/Bio-Ub cells.

Adult Ventricular Myocytes

Adult cardiac myocytes were isolated from 4- to 6-month-old mice (FVB background) on retrograde perfusion and enzymatic digestion with collagenase type II and were reintroduced to calcium at a final concentration of 200 μmol/L CaCl₂, in the presence of 2 mmol/L ATP. Myocytes were counted in a hemocytometer and placed on laminin-coated plates in a CO₂ incubator at 37°C.

Adenoviral Infection

Adult cardiac myocytes were transduced with the recombinant adenoviruses at a multiplicity of infection of 100 for 4 hours.^26–28

Doxycycline-Inducible WT and Mutant TRIM63 Transgenic Mice

Cardiac-restricted inducible tet-off transgenic mice expressing TRIM63^WT, TRIM63^A48V, TRIM63^I130M, and TRIM63^Q247* were generated by the conventional methods.^29,30 The regulator mice (Myh6-tTA) and the tet-O target vector were kind and generous gifts from Dr J. Robbins.^29,30

Immunoblotting

Immunoblotting was performed using aliquots of 30 μg of protein extracts as published.³¹

Coimmunoprecipitation

To detect auto-ubiquitination, aliquots of 2 μg/mL of biotin were added to culture media of Hela-His/bio-ubiquitin cells transduced with the recombinant lentiviruses. Cells were cultured in the presence of MG132, a proteasome inhibitor. Cell lysates were sonicated to shear DNA and cell membranes. Strepavidin-agarose slurry was added to precipitate biotinylated proteins followed by electrophoresis and immunoblotting.

Likewise, 500-μg aliquots of adult cardiac myocytes proteins, extracted in the presence of MG132, were precleared with IgG and protein A/G agarose beads, incubated with an anti-ubiquitin antibody, and treated with protein A/G agarose beads. The immunoprecipitates were used for immunoblotting.

Immunofluorescence

Immunofluorescence was performed per published methods.³¹ Briefly, colocalization of ubiquitin and TRIM63 in HeLa-His/Bio-Ub cells was detected on treating the cells with biotin to label ubiquitin. The cells were incubated with a rabbit polyclonal anti-Flag antibody followed by incubation with a mixture of a donkey anti-rabbit antibody conjugated with Alexa Fluor 350 and Streptavidin conjugated with Texas Red. Colocalization in myocytes was performed using a monoclonal anti-Flag antibody conjugated to Cy3, and the nuclei were counterstained with DAPI. To detect colocalization of TRIM63 and α-actinin, adenovirally transduced adult cardiac myocytes were fixed, permeabilized, and incubated with mouse monoclonal anti–α-actinin. The secondary antibody was donkey anti-mouse conjugated with Alexa Fluor 488 dye.

Creatine Kinase Enzymatic Assay

Aliquots of protein (10 μg) were mixed with the reconstituted reagent and incubated at room temperature. The optical density of each sample was read using a microplate reader at absorbance wavelength of 340 nm at 10 minutes, 25 minutes, and 40 minutes. Creatine kinase (CK) activities were calculated based on colorimetric readouts.

Echocardiography

Transthoracic (M-mode and 2D) and Doppler echocardiography were performed to assess cardiac function in the age- and sex- matched nontransgenic and transgenic animals using an HP 5500 Sonos echocardiography unit equipped with a 15-MHz linear transducer.^31–33

Cardiac Morphology and Histology

The heart was explanted, fixed by perfusion, and photographed. Ventricular weight was determined after excising the heart at the atrioventricular groove to isolate atria and the great blood vessels. Thin myocardial sections were stained with hematoxylin and eosin and Masson trichrome. Collagen volume fraction was determined by morphometric analysis using ImageTool 3.0 analysis software (http://ddsdx.uthscsa.edu/dig/itdesc.html).^33,34

Myocyte cross-sectional area (CSA) was determined after staining of the cryosections with 2 μg/mL wheat germ agglutinin conjugated with Texas Red. The sections were mounted in Hard Set mounting medium and examined under fluorescence microscopy.³³

Enrichment of Ubiquitinated Cardiac Proteins

Heart protein lysates (250 μg) were incubated with tandem ubiquitin-binding entities agarose beads. Beads were collected by centrifugation, washed with TBS-T, and the ubiquitin-enriched proteins were eluted for immunoblotting.

Turning Off and On Expression of the TRIM63^Q247* Transgene

To shut down the expression of the transgene, 3-month-old nontransgenic and double transgenic mice were fed with doxycycline at a 500 μg/mL concentration in water for 30 days. Doxycycline was then withdrawn in half of the treated animals to reintroduce expression of the transgene. After 72 hours of reexpression of the transgene, mice were euthanized for molecular analysis.

Quantitative PCR

The mRNA levels of selected markers were quantified by quantitative PCR, using specific TaqMan Gene expression assays, as published.³¹ The list of the probes is shown in Online Table I.

Statistics

Differences among the groups (mean±SD) were compared by 1-way analysis of variance for normally distributed continuous variables. Bonferroni correction for multiple comparisons was applied. Variables that did not follow a normal distribution pattern were compared by Kruskal-Wallis test. Differences in the categorical variables were compared by χ² test. All statistical analyses were performed using STATA v 10.1.

The authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.

Results

Characteristics of the Study Population

The discovery study population included 302 probands, including 250 white individuals with HCM and 339 (262 white) control subjects. Characteristics of the study population were largely similar to those published.³⁵ In brief, men composed 54% of the HCM study population. The mean age of the HCM population was 51.4±16.5 years, and the mean interventricular septal thickness was 1.9±0.47 cm (Online Table II).

TRIM63 Variants

Five rare variants (MAF <0.01) and 1 common variant (MAF=0.17) were detected in the study population (Online Table III). All rare variants were detected in the white subjects. The prevalence of the rare variants was enriched in the white probands with HCM, as the rare variants were present in 11 of 250 (4.4%) probands with HCM and 3 of 262 (1.1%) of the control individuals (χ²=5.1, P=0.024). The rare TRIM63 (RefSeq:NP 115977) variants p.A48V (Chr1:26393843C>T, p.Ala48Val), p.I130M (Chr1:26387768C>G, p.Ile130Met), and p.Q247* (Chr1:26384973C>T, p.Glu247Ter) were exclusive to 5 probands with HCM and absent in 339 control individuals who were sequenced and 751 control individuals who were screened by TaqMan assays. The p.A48V and p.Q247* occurred in 2 unrelated families and were present in the affected but not in unaffected members of the families (Figure 1A). The sizes of the families, nevertheless, were inadequate for cosegregation or a formal genetic linkage. Genotyping of the family members for 5 STR markers and 21 single nucleotide polymorphisms (SNPs) at the TRIM63 locus (1p34–1p33) showed unique haplotypes for individuals with the p.A48V mutation. The finding suggests an independent origin of the mutation, assuming no recombination event at the region between the TRIM63 and the marker. However, individuals with the p.Q247* shared the same haplotype for the STR markers and SNPs. Hence, an independent origin of the p.Q247* in these 2 families could not be established (Online Figure I). The p.E269K SNP (rs61749355, mRNA position 941, c.805G>A, NM 032588.2, GAG>ΑΑΓ) was identified in 2 control individuals and 6 probands with HCM. A rare p.R195C variant was identified in a single control individual who had no history of medical problems and had a normal ECG and echocardiogram. A known common SNP p.E237K (rs2275950, mRNA position 845, G>A) was detected at equal frequency in HCM probands and control subjects (MAF: 0.23 and 0.18, respectively, χ²=5.0, P=0.082). The complete list of nonsynonymous and other variants is shown in Online Table III.

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The p.A48V and p.I130M affected highly conserved amino acids and the p.Q247* variant led to deletion of 106 aa from the 353 aa long full-length protein (Figure 1B through 1E). Residues 48 and 130 are located in RING-type (aa 23–79) and B-box-type (117–159) domains of Zinc finger motif in TRIM63 protein, respectively. Residue 130 is also located at interacting domain with titin (TTN), which includes residues 74 to 218. The p.Q247* mutation is located in the coil-coiled domain (aa 207–269), which typically provides mechanical stability to the proteins. PolyPhen2 prediction positioned the missense variants at the probably damaging category (highest).³⁶ Considering the human molecular genetic data, the p.A48V, p.I130M and p.Q247* were referred to as “mutations.”

Exclusion of Known Causal Genes for HCM

None of the 5 HCM probands who carried the TRIM63 mutations had a putative causal variant in MYH7, MYBPC3, TNNT2, TNNI3, TPM1, and ACTC1, known relatively common genes for HCM.⁹

Phenotypic Expression of TRIM63 Mutations

The phenotype in mutation carrier was notable for moderate to severe left ventricular hypertrophy associated with left ventricular outflow tract obstruction in 4 of 6 individuals requiring either surgical septal myectomy or transcatheter septal ablation (Online Table IV).

Mutations Impair Auto-Ubiquitination of TRIM63

E3 ligases are known to undergo auto-ubiquitination, particularly in the absence of their primary substrates. This intrinsic feature of E3 ligases afforded the opportunity to test the effects of the p.A48V, p.I130M, and p.Q247* mutations on TRIM63 ligase activity.³⁷ Transduction of the HeLa-His/biotin-ubiquitin cells with the recombinant lentiviruses expressing either Flag-tagged TRIM63^WT, TRIM63^A48V, TRIM63I^130M, or TRIM63^Q247*, followed by coimmunoprecipitation (Co-IP), showed near complete loss of auto-ubiquitination in cells transduced with the TRIM63^Q247* lentiviral construct (Figure 2A through 2C). Likewise, immunofluorescence staining of the transduced HeLa-His/biotin-ubiquitin cells for ubiquitin and TRIM63 (Flag) showed no discernible auto-ubiquitinated TRIM63 in the cells transduced with the TRIM63^Q247* viruses (Figure 2D). Quantitative analysis also showed 60% to 70% reductions in the levels of auto-ubiquitinated TRIM63^A48V and TRIM63^I130M (Online Figure II).

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Mutations Impair Ubiquitination of TRIM63 Substrates in Adult Cardiac Myocytes

To detect whether mutations affected binding to and ubiquitination of TRIM63 known substrates, cardiac myocytes were transduced with the recombinant adenoviruses and levels of ubiquitinated and total MYH6 and MYBPC3 were determined by Co-IP and immunoblotting. Co-IP of ubiquitinated MYH6 and MYBPC3 with an anti-ubiquitin antibody, performed in the presence of MG132 to prevent degradation of ubiquitinated proteins, showed a near complete absence of ubiquitinated MYH6 and MYBPC3 in cardiac myocytes transduced with Ad5/CMV/TRIM63^Q247* viruses (Figure 3A and 3B). As compared with TRIM63^WT, levels of coprecipitated MYH6 and MYBPC3 were modestly reduced in cardiac myocytes transduced with recombinant viruses expressing TRIM63^A48V protein but were largely unchanged in myocytes transduced with the TRIM63^I130M viruses (Figure 3A and 3B). Because FBXO32 (Atrogin 1 or MAFbx), a muscle E3 ligase, is known to target calcineurin, we determined ubiquitination of PPP3CB (protein phosphatase 3 catalytic subunit β) in cardiac myocytes transduced with the WT or mutant TRIM63 constructs.³⁸ As shown in Figure 3A and 3B, level of ubiquitinated PPP3CB was increased in myocytes transduced with the TRIM63^WT construct as compared with nontransduced myocytes. In contrast, level of ubiquitinated PPP3CB was drastically reduced in cardiac myocytes transduced with TRIM63^Q247* viruses and moderately in the TRIM63^I130M group.

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Mutations Impair UPS-Mediated Degradation of MYH6 and MYBPC3 in Adult Cardiac Myocytes

To determine effects of impaired ubiquitination of mutant TRIM63 on protein levels of known substrates, we performed immunoblotting to detect and quantify MYH6 and MYBPC3 protein levels in cardiac myocytes transduced with the recombinant viruses in the presence or absence of MG132. As shown in Figure 3C, levels of MYH6 and MYBPC3 were reduced significantly in cardiac myocytes transduced with the adenoviruses expressing TRIM63^WT in the absence of MG132, as compared with nontransduced cells. The findings confirm the previous data identifying thick filament proteins as targets of TRIM63.³⁹ In contrast, levels of MYH6 and MYBPC3 protein were increased in cells treated with the adenoviruses expressing TRIM63^A48V, TRIM63^I130M, and TRIM63^Q247* as compared with myocytes expressing TRIM63^WT (Figure 3C and 3E). Treatment with MG132 equalized levels of MYH6 and MYBPC3 in the experimental groups, supporting UPS-mediated degradation of MYH6 and MYBPC3 in TRIM63^WT group (Figure 3D and 3E). Protein level of cardiac troponin I was unchanged in cells expressing the TRIM63^WT protein and was similar in all experimental groups.

Mutations Reduce Localization of TRIM63 to Sarcomere Z-Disk

To determine whether TRIM63 mutations affected its localization in the sarcomere, cardiac myocytes were transduced with the recombinant viruses and costained with anti-Flag (TRIM63) and anti–α-actinin antibodies. As shown in Figure 4, TRIM63^WT was colocalized with α-actinin at the Z-disk (r=0.814). In contrast, TRIM63^I130M and TRIM63^Q247* had a diffuse expression pattern with minimal colocalization with α-actinin (r=0.115 and r=0.199, respectively). TRIM63^A48V showed a moderate reduction in colocalization with α-actinin (r=0.518).

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Mutations Impair UPS-Mediated Degradation of MTOR-S6K Hypertrophic Signaling Pathway in Adult Cardiac Myocytes

To identify the mechanism(s) by which TRIM63 mutations cause HCM, we focused on the mammalian target of rapamycin (MTOR) pathway, not only because it is a major cardiac hypertrophic signaling pathway but also because F-box proteins, such as FBXW7, are known to target phosphorylated MTOR (p-MTOR) for ubiquitination and degradation.⁴⁰ Immunoblot analysis of protein extracts of adult cardiac myocytes transduced with the recombinant adenoviruses in the absence of the UPS inhibitor MG132 showed reduced level of p-MTOR in myocytes expressing the TRIM63^WT protein (Figure 5A). In contrast, p-MTOR protein level in cardiac myocytes transduced with the mutant TRIM63 constructs was significantly higher as compared with the TRIM63^WT group. Inhibition of the UPS by MG132 led to equalization of the p-MTOR level in all experimental groups, including in the TRIM63^WT group. Total MTOR protein level was unchanged in all experimental groups in both MG132-treated and untreated cells.

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To further investigate reduced p-MTOR levels in the TRIM63^WT group, we detected and quantified levels of total and p-S6K1 and p-AKT1, downstream targets of MTORC1 and MTORC2, respectively, by immunoblotting. In accord with the reduced level of p-MTOR, level of p-S6K1 but not total S6K1 was also significantly reduced in cardiac myocytes expressing TRIM63^WT but not in any of the mutant TRIM63 groups (Figure 5B). In contrast, protein level of p-AKT1 and total AKT1 were unchanged in cardiac myocytes transduced with the WT or mutant Trim63 constructs. Treatment with MG132 normalized reduced level of p-S6K1 in the TRIM63^WT group and equalized in all groups.

To determine whether p-MTOR was the direct target of TRIM63, we performed Co-IP using an anti-Flag antibody in immunoprecipitation and an anti p-MTOR antibody for immunoblotting and repeated the Co-IP in the reverse order. Despite 6 independent Co-IP reactions and using different antibodies, no significant Co-IP of p-MTOR with TRIM63 in any of the experimental groups was detected. Likewise, no significant Co-IP of p-S6K1 with TRIM63 was detected in any of the experimental groups. The null results of Co-IP studies might simply reflect the experimental conditions and yet might also indicate an indirect targeting of p-MTOR by TRIM63 through targeting other components of the MTORC1 complex. Therefore, we determined levels of p-Raptor, total Raptor, and GβL by immunoblotting. Levels of p-Raptor and GβL were largely unchanged and there were not significant differences in their levels among the experimental groups (Online Figure III).

Mutations Had No Discernible Effects on CK Activity

TRIM63 is implicated in regulating CK enzymatic activity.⁴ To determine whether mutations affected CK enzymatic activity, CK enzymatic activity was quantified serially in adult mouse cardiac myocytes transduced with the recombinant adenoviruses. TRIM63 mutations had no significant effects on CK protein levels or CK enzymatic activity (Online Figure IV).

Inducible Expression of Mutant TRIM63 Leads to Cardiac Hypertrophy

Because each human genome contains about 100 loss-of-function variants,⁴¹ to further substantiate the causal role of the TRIM63 mutations identified in human patients with HCM, we generated doxycycline-inducible double transgenic mice, transcriptionally regulated by the Myh6 promoter.^29,30 Expression of the transgene was detected by immunoblotting of cardiac protein extracts in 3 tTA:TRIM63^A48V, 1 tTA:TRIM63^I130M, 2 tTA:TRIM63^Q247*, and 1 tTA:TRIM63^WT independent lines (Figure 6A). To determine relative levels of the Flag-tagged transgene TRIM63 and endogenous TRIM63 proteins, cardiac protein extracts were probed by immunoblotting using a pan-TRIM63 antibody. Level of the transgene protein was lower than that of the endogenous TRIM63 protein in tTA:TRIM63^WT, tTA”TRIM63^A48A, and tTA:TRIM63^Q247* but was higher in the tTA:TRIM63^I130M double transgenic mice (Online Figure V). Morphological and histological analysis showed increased ventricular weight/body weight by approximately 15% to 20% (Online Figure VI) and myocyte CSA by about 1.6- to 2-fold in the 3 mutant TRIM63 groups, particularly in tTA:TRIM63^Q247* group, as compared with tTA:TRIM63^WT or nontransgenic mice (Figure 6B). There was also an approximately 3-fold increase in interstitial fibrosis in the tTA:TRIM63^I130M group as compared with nontransgenic mice (Online Figure VII). Interstitial fibrosis was not increased significantly in other mutant TRIM63 groups.

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Echocardiographic evaluation of cardiac size and function showed increased left ventricular wall thickness, mass, and mass index in the mutant TRIM63 groups, except for the TRIM63^I130M, as compared with nontransgenic or tTA:TRIM63^WT mice (Online Table V). Left ventricular fractional shortening was similar to that in the nontransgenic mice, except in the TRIM63^I130M, which was decreased modestly. Expression of the TRIM63^WT was associated with increased left ventricular end diastolic diameter and modestly decreased wall thickness, as compared with nontransgenic mice. However, left ventricular fractional shortening was preserved.

Mutations Reduce Ubiquitination of Total Protein, MYH6, and MYBPC3 and Activate the MTOR-S6K Pathway in the Heart

To assess the effects of the mutations on cardiac protein ubiquitination, we enriched myocardial protein extracts for ubiquitinated proteins by passing the extracts through tandem ubiquitin-binding entities agarose beads and probing the enriched proteins by immunoblotting using an anti-ubiquitin antibody. The findings were notable for a greater than 2-fold increase in the level of ubiquitinated cardiac proteins in the tTA:TRIM63^WT mice (Figure 7A). In contrast, levels of ubiquitinated total protein were reduced in the mutant tTA:TRIM63 groups, as compared with TRIM63^WT mice, and was near absent in the tTA:TRIM63^Q247* mice. These findings are in accord with the in vitro data in the HeLa-His/biotin-ubiquitin cells and Co-IP studies in virally transduced adult cardiac myocytes. Likewise, levels of ubiquitinated TRIM63^A48V and TRIM63^Q247* were reduced significantly but less so in the TRIM63^I130M as compared with TRIM63^WT (Figure 7A, lower panel).

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To analyze levels of the specific sarcomere proteins, we determined protein levels of TRIM63, MYH6, and MYBPC3 in the cardiac protein extracts by immunoblotting. As shown in Figure 7B and 7C, MYH6 level were reduced in the tTA:TRIM63^WT mice, as compared with nontransgenic mice, but were higher in the mutant tTA:TRIM63 groups as compared with the tTA:TRIM63^WT mice but equal to that in the nontransgenic mice, in accord with the in vitro data in cultured cardiac myocytes. MYBPC3 level was only increased in the tTA:TRIM63^Q247* mouse heart.

Immunobloting was performed to detect and quantify levels of the main components of TORC1 complex. There was an approximately 30% reduction in p-MTOR level in the tTA:TRIM63^WT group as compared with nontransgenic mice. Notably, p-S6K1 level in the heart was dramatically increased in the mutant tTA-TRIM63 group, as compared with nontransgenic or TRIM63^WT mice (Figure 7D through 7F).

Because overexpression of TRIM63 has been shown to affect Mybpc3 mRNA level, we also assessed mRNA levels of Myh6, Mybpc3, Mtor, Ppp2cb, and Rps6kb1 (S6k) in the heart of WT and mutant TRIM63 transgenic animals.²⁰ There were no significant differences in the mRNA levels of the selected molecules (Online Figure VIII).

Switching on Expression of Mutant TRIM63 Proteins in the Heart Activates the MTOR-S6K and Calcineurin-NFAT1 Pathways

The switch on and off bigenic mouse approach afforded the opportunity to turn off expression of the mutant TRIM63 protein and then analyze induction of expression of the molecular regulators and markers of cardiac hypertrophy. In this set of experiments, tTA:TRIM63^Q247* was used as this mutation was associated with the most dramatic phenotype in isolated adult cardiac myocytes and in transgenic mice. Accordingly, expression of the TRIM63^Q247* was turned off in the tTA:TRIM63^Q247* mice on daily administration of doxycycline for 30 days and then it was turned on in half of the animals—by withdrawing doxycycline (Online Figure IX). Immunoblotting performed 3 days after turning on expression of the mutant transgene showed concordant increase in the expression levels of p-MTOR, pS6K1, and RCAN1.4 and normalization of these proteins on turning off in the mutant TRIM63 bigenic mice (Figure 8A and 8B). In conjunction with activation of the hypertrophic signaling pathways, mRNA levels of Myh7, Nppa, and Nppb, quantified by quantitative PCR, were increased 3 days after turning on and were normal on turning off expression of the mutant TRIM63^Q247* protein (Figure 8C). Echocardiographic assessment of cardiac size and function showed normal wall thickness and systolic function during the period when the transgene expression was shut down as well as 3 days after turning on expression of TRIM63^Q247* protein.

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Discussion

We provide human molecular genetic and in vitro and in vivo functional evidence to implicate TRIM63, encoding MuRF1, an E3 ubiquitin ligase, as a likely causal gene for human HCM. The p.A48V, p.I130M and p.Q247* variants were exclusive to the HCM study population (whites) and were not detected in over 500 control white individuals. The small size of the families was not permissive to genotype-phenotype cosegregation analysis, which is a limitation of the study. The nonsense (p.Q247*) and the p.A48V variants recurred in 2 families. The nonsense variant led to premature truncation and loss of approximately one-third of the protein. The p.A48V and p.I130M variants affected highly conserved amino acids and were predicted—in silico—to be probably damaging to protein structure and function. Functionally, the variants had loss-of-function effects on E3 ubiquitin ligase activity, as detected in specialized ubiquitin-tagged HeLa cells, virally transduced adult cardiac myocytes, and transgenic hearts. Furthermore, expression of the mutant TRIM63 in the heart through inducible transgenesis led to cardiac and myocyte hypertrophy in mice, activation of the MTOR-S6K and calcineurin-RCAN1.4 pathways and expression of the hypertrophic markers. The truncating TRIM63^Q247* had the most prominent in vitro and in vivo phenotypic effects. Collectively, the human molecular genetic data along with the functional studies in adult cardiac myocytes and transgenic mice support the causal role of TRIM63 in the pathogenesis of human HCM. The findings implicate impaired degradation of cardiac proteins as a pathogenic mechanism for cardiac hypertrophy and activation of the MTOR-S6K and calcineurin-RCAN1.4 pathways as the responsible pathways involved in the pathogenesis of HCM caused by TRIM63 mutations.

The main biological function of UPS in the heart is clearance of the misfolded and damaged proteins. Likewise, TRIM63 (MuRF1) and FBX032 (commonly known as atrogin-1 or MAFbx), the major components of the UPS in the heart, are implicated in the removal of mutant sarcomere proteins in HCM.²⁰ In addition, a mismatch between UPS capacity and accumulation of substrates is commonly observed in various forms of cardiac hypertrophy and dysfunction, which might actively participate in the pathogenesis of cardiomyopathies.^3,5,16,17 The findings of the present study extend the biological functions of UPS in the heart and provide direct genetic and functional evidence to implicate defective protein degradation as the initial impetus for induction of cardiac hypertrophy in HCM. These discoveries advocate the notion that cardiac hypertrophy might also be a disease of impaired protein degradation.

TRIM63 mutations led to loss of E3 ligase activity, as reflected by reduced auto-ubiquitination and ubiquitination of the known substrates. The mutations, however, exhibited variable functional effects and cardiac phenotypes, which are not unanticipated but rather inherent to phenotypic expression of mutations in various genes, as best illustrated for the phenotypic plasticity of LMNA mutations.⁴² The phenotype was more prominent for the truncating TRIM63^Q247* mutation, which apparently, at least in part, had escaped the nonsense mediated decay (NMD) mechanism, an established mechanism for haploinsufficiency resulting from the nonsense mutations in HCM.^17,43 Stable expression of the truncated TRIM63, observed in HeLa cells transduced with a lentiviral construct, in adult cardiac myocytes infected with an adenoviral construct and in the heart of transgenic mice, might reflect the use of cDNA in these experiments as opposed to the genomic fragment. Alternatively, the relatively distal location of the p.Q247* mutation from the translation initiation site might afford the opportunity for the stable expression of a 246 aa protein. The truncated TRIM63 protein exhibited a near total loss of the E3 ligase activity, as demonstrated by near total loss of auto-ubiquitination and ubiquitinylation of TRIM63 substrates MYH6 and MYBPC3. In addition, the level of ubiquitinated calcineurin (PPP3CB) was drastically lower in cardiac myocytes transduced with the TRIM63^Q247* viruses. Thus, the biological and functional data are the strongest for the TRIM63^Q247*.

Expression of TRIM63^WT was associated with reduced levels of MYH6 and MYBPC3, known substrates for TRIM63.³⁹ However, protein level of cardiac troponin I was unchanged, which has been observed in some but not other studies.^23,39 Reduced MYH6 and MYBPC3 levels and increased level of ubiquitinated PPP3CB were associated with mild thinning of the interventricular septum and left ventricular enlargement as compared with nontransgenic mice but no significant change in left ventricular mass, mass index, or fractional shortening at the baseline. Overexpression of TRIM63^WT in the heart is known to render the heart susceptible to failure in response to stress.⁴

The findings also implicate activation of MTOR-S6K (MTORC1 pathway) and calcineurin in the pathogenesis of HCM caused by the TRIM63 mutations and hence link the protein degradation and synthesis pathways. These findings suggest MTORC1 and PPP3CB but not MTORC2 pathways as the potential direct or indirect targets of TRIM63 in the heart. Despite reduced levels of p-MTOR and PPP3CB on overexpression of TRIM63, we could not demonstrate coprecipitation of TRIM63 and p-MTOR or PPP3CB. Thus, the changes in the p-MTOR, p-S6K1, and RCAN1.4 levels might be secondary to activation of the hypertrophic program instigated by the TRIM63 mutations. Nonetheless, activation of p-MTOR/p-S6K and calcineurin/RCAN1.4 pathways in isolated myocytes and the mouse heart support the causal role of TRIM63 variants in the pathogenesis of HCM. The precise mechanism(s) by which a homeostatic shift in favor of reduced protein degradation instigates a cardiac hypertrophic response remain unknown.

Several alternative mechanisms might also account or influence the phenotypic consequences of TRIM63 mutations. The truncating mutation, which eliminates a major part of the coiled coil domain, might interfere with the mechanical stability of the protein and affect its interactions with proteins other than MYH6 and MYBPC3. The p.A48V mutation is located in RING-type domain, which mediates ubiquitin-ligase activity by binding to ubiquitination enzymes as well as the substrates.⁴⁴ The p.I130M and p.Q247* also affected subcellular distribution of TRIM63 and its colocalization with the α-actinin. The p.I130M mutation is located in the B-box type domains of Zinc finger motif, which is also the interacting domain with titin (residues 74–218 on TRIM63 and repeats A168/A169 in titin), adjacent to the titin kinase domain.^45,46 TRIM63 is involved in regulating muscle trophic homeostasis through interactions with a number of proteins including glycogen phosphorylase and FOXO (Forkhead box, subgroup O).⁴⁵ Whether TRIM63 mutations influence titin kinase activity or ubiquitination of other substrates is unknown. The crystal structure of the B-Box domain of TRIM63 has been resolved.⁴⁷ The isoleucine at position 130 resides in a groove near the interacting domain with the muscle-type CK.⁴⁸ Substitution of methionine, which has a side branch, for isoleucine might affect its interactions with CK. However, under the conditions of our experiments, we detected no significant effects of over expression of wild-type or mutant TRIM63 on CK enzymatic activity in virally transduced cardiac myocytes. It is also possible that reduced E3 ligase activity of the mutant TRIM63 proteins impairs clearance of misfolded and damaged sarcomere proteins, which might partially incorporate into the sarcomere and cause sarcomere dysfunction leading to cardiac hypertrophy and dysfunction. Likewise, reduced clearance of damaged and misfolded TRIM63 substrate proteins could lead to their accumulation in the endoplasmic reticulum, inducing an unfolded protein response, which might contribute to the pathogenesis of cardiac phenotype.⁴⁹ Therefore, various alternative or additive mechanisms—beyond those delineated in the present studies—might be responsible for the hypertrophic effects of TRIM63 mutations in humans. Although we did not detect significant differences in the levels of WT and mutant TRIM63 proteins in transduced cardiac myocytes and in the transgenic hearts, differences, beyond the sensitivity of the immunoblotting, could exist in the expression levels of WT and mutant TRIM63 that might confound data interpretation.

In conclusion, we provide molecular genetic evidence in humans and functional data in isolated cardiac myocytes and in inducible transgenic mouse models to implicate TRIM63 as a likely causal gene for human HCM. The discoveries provide evidence for impaired degradation of sarcomere proteins as a mechanism for the pathogenesis of HCM in humans. Consequently, sarcomeres are not only the contractile units of striated muscles but are also signaling hubs for regulation of muscle trophic homeostasis and hence, cardiac size and function.

Sources of Funding

This work is supported in part by grants from the National Heart, Lung, and Blood Institute ( R01-HL088498 and R34HL105563 ), NIA ( R21 AG038597-01 ), a Burroughs Wellcome Award in Translational Research (No. 1005907), TexGen Fund from Greater Houston Community Foundation , and the George and Mary Josephine Hamman Foundation.

Disclosures

None.

Footnotes