Doxycycline Attenuates Protein Aggregation in Cardiomyocytes and Improves Survival of a Mouse Model of Cardiac Proteinopathy

Abstract

Objective

The goal of this preclinical study was to assess the therapeutic efficacy of doxycycline (Doxy) for desmin-related cardiomyopathy (DRC) and to elucidate the potential mechanisms involved.

Background

DRC, exemplifying cardiac proteinopathy, is characterized by intrasarcoplasmic protein aggregation and cardiac insufficiency. No effective treatment for DRC is presently available. Doxy was shown to attenuate aberrant intranuclear aggregation and toxicity of misfolded proteins in non-cardiac cells and animal models of other proteinopathies.

Methods

Mice and cultured neonatal rat cardiomyocytes with transgenic (TG) expression of a human DRC-linked missense mutant αB-crystallin (CryAB^R120G) were used for testing the effect of Doxy. Doxy was administered via drinking water (6 mg/ml) initiated at 8 or 16 weeks of age.

Results

Doxy treatment initiated at 16 weeks of age significantly delayed the premature death of CryAB^R120G TG mice, with a median lifespan of 30.4 weeks (placebo group 25 weeks, p<0.01). In another cohort of CryAB^R120G TG mice, Doxy treatment initiated at 8 weeks of age significantly attenuated cardiac hypertrophy in one month. Further investigation revealed that Doxy significantly reduced the abundance of CryAB-positive microscopic aggregates, detergent-resistant CryAB oligomers, and total ubiquitinated proteins in CryAB^R120G TG hearts. In cell culture, Doxy treatment dose-dependently suppressed the formation of both microscopic protein aggregates and detergent-resistant soluble CryAB^R120G oligomers, and reversed the upregulation of p62 protein induced by adenovirus-mediated CryAB^R120G expression.

Conclusions

Doxy suppresses CryAB^R120G induced aberrant protein aggregation in cardiomyocytes and prolongs CryAB^R120G based DRC mouse survival.

Introduction

Desmin-related myopathy, a well characterized example of proteinopathy, features the presence of desmin-positive protein aggregates in myocytes. Genetic studies linked this disease to mutations in desmin, αB-crystallin (CryAB), and myotilin genes (1). Among these mutations, a missense mutation (R120G) of CryAB is the best studied. Transgenic (TG) overexpression of either mouse or human CryAB^R120G in mouse hearts causes aberrant protein aggregation and cardiomyopathy, recapitulating key features of human desmin-related cardiomyopathy (DRC) (2,3). Recent studies showed that intrasarcoplasmic amyloidosis, a major type of aberrant protein aggregation in DRC and associated cardiomyopathy are reversible upon suppression of CryAB^R120G expression and more remarkably, significantly attenuated by voluntary exercise (4,5). Proteasome proteolytic function is severely impaired in CryAB^R120G TG mouse hearts and aberrant protein aggregation appears to be both responsible for and further exacerbated by proteasome functional insufficiency, forming a vicious cycle (1,6).

It is believed that abnormal protein aggregation and accumulation are deleterious in all proteinopathy, irrespective of the primary etiology. Notably, aberrant protein aggregation in the form of pre-amyloid oligomers has been observed in the majority of failing human hearts resulting from either dilated or hypertrophic cardiomyopathy (4). Moreover, aberrant protein aggregation was recently shown to trigger autophagic activation in pressure overloaded hearts (7). To this end, the well documented CryAB^R120G DRC mice represent a useful animal model for the investigation into the pathogenic role of cardiac aberrant protein aggregation as well as for therapeutic targeting of aberrant protein aggregation in congestive heart failure.

Doxycycline (Doxy) is an FDA-approved second generation antibiotic of the tetracycline family. It is suitable for long term use because of its favorable safety profile. It was demonstrated that Doxy has other important pharmacological actions besides its antibiotic properties. For example, Doxy has been shown to be an inhibitor for matrix metalloproteinases (MMPs) (8). It was also reported that Doxy inhibits the formation of amyloid aggregates both in vitro and in vivo (9). Furthermore, Davis et al reported that Doxy attenuated and delayed toxicity of oculopharyngeal muscular dystrophy possibly by reducing aggregation and inhibiting cell death pathways (9-11). These important recent discoveries prompted us to test whether Doxy has therapeutic value in cardiac proteinopathies. Our study reveals that Doxy significantly attenuates CryAB^R120G-induced aberrant protein aggregation in cardiomyocytes and prolongs the survival of CryAB^R120G DRC mice, providing compelling evidence that Doxy is a promising candidate for a clinical trial to treat cardiac proteinopathies.

Methods

Animals

FVB stable TG mice with cardiomyocyte-restricted overexpression of the mouse CryAB^R120G were used in this study (2). Animal use and care protocols used in this study were approved by the Institutional Committee for the Use and Care of Animals of the University of South Dakota.

Administration of Doxy and echocardiography (Echo)

Doxy (Sigma-Aldrich) was given in drinking water (6mg/ml) containing 5% sucrose, starting at 8 or 16 weeks of age. The control group was given drinking water containing 5% sucrose without Doxy. Echo was performed as described (12).

Neonatal rat cardiomyocyte (NRCM) cultures and adenovirus infection

NRCMs were isolated and cultured as described (6,12). Recombinant adenoviruses expressing hemagglutinin (HA) tagged CryAB^R102G (Ad-HA-CryAB^R120G) or β-galactosidase (Ad-β-Gal) were created as described (6). The viruses were used at a multiplicity of infection of 10 to infect the cultured NRCMs.

Immunofluorescence confocal microscopy and Western blot analyses

Sample preparation, immunofluorescence staining, and Western blot analyses were performed as described (6,12). The antibodies used include the rabbit polyclonal antibodies against CryAB (Stressgen, Victoria, BC), Atg5 (Novus Biologicals), ubiquitin, Atg7 (Sigma), and the mouse antibodies against HA-tag (Santa Cruz, CA) and sarcomeric α-actinin (Sigma), LC3 (Medical & Biological Laboratories Co.), beclin-1 (Santa Cruz), Alexa Fluor 488 anti- rabbit Ig, Alex- Fluor 568 anti-mouse Ig (Invitrogen, Eugene, OR), and horse-raddish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Santa Cruz). Alexa Fluor 568 conjugated phalloidin (Invitrogen) was used to stain F-actin.

Filter-trap assay

This assay was performed as described (4,6).

Statistical methods

The log-rank test was used for the Kaplan-Meier survival analysis. All quantitative data are presented as mean±SD and were analyzed by one-factor or multiple-factor ANOVA using SigmaStat 3.0 software (Systat, Point Richmond, CA), where applicable. The Holm-Sidak test was used for post-hoc pair-wise comparisons. A probability less than 5% was considered statistically significant.

Results

Doxy treatment significantly prolongs survival of mice with CryAB^R120G DRC

CryAB^R120G TG mice (line 134) develop concentric cardiac hypertrophy and diastolic malfunction at 3 months, display overt congestive heart failure between 5-6 months, and die shortly afterwards (2). Due to the expedited course of disease progression in this TG line, we used it to perform a Kaplan-Meier survival analysis on chronic Doxy treatment.

A cohort of 37 CryAB^R120G TG mice was divided randomly into two groups: the Doxy (TG-Doxy, 19 mice) and the placebo (TG-CTL, 18 mice) group. A parallel cohort of NTG littermates was similarly treated to detect any potential adverse effects of Doxy or placebo on normal animals. Echo was performed the day before the initiation of treatment. The treatment was initiated at 16 weeks of age when concentric cardiac hypertrophy and significantly decreased cardiac output were evident (Table 1). Mice of the TG-CTL group showed a median lifespan of 25 weeks similar to what was previously observed in the untreated TG mice (2). However, the premature death was significantly delayed in the TG-Doxy group with 60% of them still alive by the time all TG-CTL mice had died. Their median lifespan was 30.4 weeks, 20.16% longer than that of the TG-CTL group (p<0.01; Figure 1).

Table 1.

Baseline Echocardiography Analyses of Mice Used for Kaplan-Meier Survival Analysis

	NTG	TG-CTL	TG-Doxy
N	23	18	19
Body weight (g)	26±3.7	27±4.2	27±3.6
Heart rate (beats/min)	509±55	414±39*	413±31*
LVPW-d (mm)	0.72±0.09	0.94±0.12*	0.96±0.10*
LVID-d (mm)	3.8±0.34	3.6±0.26†	3.6±0.32†
LVPW-s (mm)	1.12±0.11	1.41±0.22*	1.46±0.18*
LVID-s (mm)	2.23±0.33	1.92±0.26*	1.90±0.31*
FS (%)	41.7±4.5	47.2±4.8*	46.9±5.4*
EF (%)	73.2±5.1	79.1±4.8*	78.8±5.3*
SV (μl)	46±7.6	44±6.6	42±7.4
CO (μl)	23094±4199	18037±3093*	17256±3439*

LVPW, left ventricle posterior wall; LVID, left ventricle internal diameter;-d, end diastole; -s, end systole; FS, fractional shortening; EF, ejection fraction; SV, stroke volume, CO, cardiac output. For all parameters, there is no statistically significant difference between the TG control group (TG-CTL) and the TG Doxy group (TG-Doxy) immediately before Doxy treatment.

Compared to NTG littermates, *: p<0.01, †: p<0.05.

Figure 1. Kaplan-Meier Survival Curves of CryAB^R120G TG Mice Treated with Doxy.

Treatment of line 134 littermate CryAB^R120G TG and NTG mice with Doxy (Doxy) or vehicle control (CTL) was initiated at 16wk of age. No premature death was observed for Doxy or CTL treated NTG mice (Data not shown). The lifespan of CryAB^R120G TG mice was significantly prolonged by Doxy treatment (p<0.01).

The treatment to the NTG cohort was terminated when all mice in the TG cohort died. No difference in animal death was observed between the Doxy- and placebo- treated NTG mice (data not shown).

Doxy treatment attenuates cardiac hypertrophy in DRC mice

To evaluate the effects of Doxy on cardiac hypertrophy and left ventricular function in DRC mice at an earlier time, another cohort of NTG and TG mice was subjected to 4 weeks of Doxy treatment starting at 8wk of age. Previous characterization of this mouse line revealed that cardiac hypertrophy started between 1 and 3 months of age and the down-regulation of α-myosin heavy chain and CryAB^R120G expression was observed at 6 months (2). Hence, choosing the period between 2 and 3 months avoids the potential impact from the down-regulation of TG expression. At 4 weeks after Doxy treatment, Echo assessments revealed significantly increased thickness in the left ventricle posterior wall at the end of both diastole and systole in the TG-CTL mice; but the increases were prevented or significantly attenuated in the TG-Doxy group (Table 2). These indicate that a 4-week Doxy treatment is sufficient to suppress cardiac hypertrophy. The increase in ventricular wall thickness in CryAB^R120G TG-CTL mice was accompanied by decreased internal diameter at end-diastole and end-systole and elevated ejection fraction and fractional shortening. Doxy treatment did not significantly alter ejection fraction, fractional shortening, or the end diastolic left ventricle internal diameter in the TG mice (Table 2). The changes in cardiac mass assessed by Echo were confirmed by gravimetric measurements at terminal experiments. The heart weight to tibial length ratio and the ventricular weight to tibial length ratio were significantly lower in the TG-Doxy group than the TG-CTL group (Figure 2).

Table 2.

Echocardiography Assessments after 4-week of Doxycycline Treatment

Parameters	NTG-CTL	NTG-Doxy	TG-CTL	TG-Doxy
HR (bpm)	479±61	496±57	460±31	440±36
LVPW-d (mm)	0.74±0.07	0.69±0.14	0.93±0.05**	0.82±0.06*††
LVID-d (mm)	3.78±0.15	3.73±0.18	3.59±0.19*	3.52±0.23*
LVPW-s (mm)	1.15±0.11	1.02±0.20	1.62±0.19**	1.36±0.14*†
LVID-s (mm)	2.25±0.11	2.34±0.14	1.75±0.31**	1.79±0.30*
FS (%)	40.37±1.80	37.41±2.59	51.37±6.73**	49.33±5.98**
EF (%)	71.82±2.13	68.18±3.19	82.87±6.22**	81.18±5.70**

Treatment with Doxy or vehicle was initiated at 8wk of age. n=6 for each group. LVPW, left ventricle posterior wall; LVID, left ventricle internal diameter; -d, end diastole; -s, end systole.

Compared with the NTG-CTL, *: p<0.05, **: p<0.01; compared with the TG-CTL, ^†: p<0.05, ^††: p<0.01.

Figure 2. Doxy Reduces Cardiac Hypertrophy in CryAB^R120G TG Mice.

Initiated at 8 weeks of age, a cohort of line 134 TG and NTG mice were treated with Doxy or vehicle (CTL) for 4 weeks and used for gravimetric analyses. Shown are mean ±SD. BW: body weight; HW: heart weight; VW: ventricular weight; T: tibial length. *: p<0.05 vs. the NTG control group (NTG-CTL); #: p<0.05, vs. the TG control group (TG-CTL).

Doxy treatment reduces aberrant protein aggregation in DRC mouse hearts

Since protein aggregation is an important pathogenic process in DRC and Doxy has been shown to suppress intranuclear protein aggregation in non-myocytes, we further tested whether Doxy's protection against DRC was associated with any effect on aberrant protein aggregation in the heart.

Compared with the TG-CTL group, TG-Doxy hearts showed substantially less CryAB-positive protein aggregates by immunofluorescence confocal microscopy (Figure 3), significantly less SDS-resistant CryAB oligomers by filter-trap assays (Figure 4B), and markedly less ubiquitinated proteins (Figure 4C). Doxy treatment did not change total CryAB protein levels (Figure 4A).

Figure 3. Doxy Reduces CryAB-immunopositive Aggregates in CryAB^R120G TG Hearts.

Line 708 CryAB^R120G TG and NTG mice were treated with Doxy or vehicle (CTL) for 2 months, starting at 8wk of age. Paraformaldehyde perfusion-fixed ventricular myocardium was immunostained for CryAB (green) and α-actinin (red). Scale bar = 30μm.

Figure 4. Doxy Inhibits CryAB^R120G Induced Protein Aggregation in Mouse Hearts.

The total and the soluble fractions of myocardial proteins were extracted from mice of the same cohort and treatment as described in Figure 3. A, Total ventricular myocardial proteins were subjected to SDS-PAGE and CryAB levels were analyzed by Western blots. B, SDS-resistant oligomeric CryAB in myocardial proteins was measured by the filter-trap assay. A representative image (upper panel) and the densitometry data (bottom panel) are shown. C, Quantitative Western blot analyses of ubiquitinated proteins in the total myocardial protein extract. A representative image (upper panel) and a summary of densitometry data from 4 repeats (lower panel) are shown. *: p<0.05, vs. NTG; #: p<0.05 vs. TG-CTL. AU: arbitrary unit (the same for other figures).

Doxy dose-dependently inhibits aberrant protein aggregation induced by CryAB^R120G in cultured NRCMs

To determine whether Doxy induced suppression of aberrant protein aggregation in the heart is cardiomyocyte-autonomous, we further tested the effect of Doxy on CryAB^R120G– induced aberrant protein aggregation in cultured NRCMs. One day after the Ad-HA-CryAB^R120G infection, different doses (0.5~10μM) of Doxy were administered daily to the culture media and the cells were collected at 3 or 11 days after treatment. As revealed previously (4,6,13), CryAB-positive protein aggregates were formed in the cytoplasm of cardiomyocytes infected by Ad-HACryAB^R120G but not by Ad-β-Gal. The extent of the aggregates was markedly reduced by Doxy treatment (Figure 5). Western blot analyses showed that Doxy treatments did not discernibly alter the HA-CryAB^R120G protein level in either the soluble or the insoluble fractions (Figure 6A), but the filter-trap assays revealed the SDS-resistant oligomeric forms of HA-CryAB^R120G significantly reduced by Doxy in a dose-dependent manner (Figure 6B, 6C), indicating inhibition of aberrant protein aggregation by Doxy. Moreover, this inhibitory effect appears to be more pronounced at 11 days than 3 days.

Figure 5. Fluorescent Confocal Micrographs of Immunostained NRCMs Overexpressing CryAB^R120G.

NRCMs were infected with Ad-β-Gal or Ad-HA-CryAB^R120G for 24 hours, followed by the addition of various concentrations of Doxy as indicated. After 11 days of Doxy treatment, the cells were fixed and immunostained for HA-CryAB^R120G (green). F-actin was stained by phalloidin (red) and nuclei with DAPI (blue). Scale bar = 50μm.

Figure 6. Doxy Dose-dependently Inhibits CryAB^R120G Induced Protein Aggregation in Cultured NRCMs.

Cultured NRCMs were infected with Ad-HA-CryAB^R120G or Ad-β-Gal for 24 hours before Doxy treatment initiation. A, Three or 11 days after Doxy treatment, the soluble (Sol) and the insoluble (Ins) fractions of proteins were extracted from NRCMs and subjected to Western blot analysis for CryAB. HA-CryAB^R120G (arrows) runs slower than endogenous CryAB. B, Representative images of the filter-trap assays for SDS-resistant HA-CryAB^R120G oligomers in cells at 3 (a) or 11 (b) days of Doxy treatments using anti-HA antibodies. C, Changes in the oligomers detected in 4 repeats. *: p<0.05, **: p<0.01, vs. the 0μM Doxy group. D & E, Western blot analyses of Hsp25 and p62 in cells undergone 11 days of Doxy treatment. **: p<0.01 vs. all HA-CryAB^R120G groups; #: p<0.05, ##: p<0.01, vs. Doxy untreated HA-CryAB^R120G expressing cells.

To determine the potential mechanism of the inhibition of protein aggregation by Doxy, we examined the protein expression of heat shock protein 25 (Hsp25) and p62/SQSTM1. Hsp's play a critical role in preventing protein aggregation (13,14). p62 was shown to promote the formation of ubiquitinated protein inclusion bodies (15,16). As shown in Figure 6D and 6E, p62 in both the soluble and the insoluble fractions and Hsp25 in the insoluble fraction were significantly up-regulated by CryAB^R120G expression, and the upregulation of p62 but not Hsp25 was significantly less in Doxy treated cells vs. vehicle-treated cells.

Activation of autophagy in DRC hearts is not enhanced by Doxy

Autophagy plays an important role in protein quality control (17). Autophagic activation was shown to protect against DRC in mice (18). Hence, we examined the conversion of LC3-I to LC3-II (a commonly used marker of autophagic activation) and several other autophagy-related proteins (19). The protein level of LC3-II, the LC3-II/LC3-I ratio, and a cleaved form of Atg5 in TG-CTL hearts were significantly greater than those in NTG-CTL but no statistically significant difference in these parameters was detected between TG-CTL and TG-Doxy groups (Figure 7A, 7B, and data not shown). In cultured NRCMs overexpressing HA-CryAB^R120G, Doxy at 1μM and 5μM but not 0.5μM significantly reduced LC3-II protein levels but did not alter the LC3-II/LC3-I ratio. Doxy treatment did not change Atg7 and beclin1 protein expression (Figure 7C, 7D). These results indicate that autophagy is activated in the TG heart but Doxy-elicited cardioprotection is independent of autophagy.

Figure 7. Western blot analyses for autophagy-related proteins.

A & B, Total myocardial proteins from mice of the same cohort and treatment as described in Figure 3 were used. Representative immunoblot images are shown in panel A. The open arrow marks a short (or cleaved) form of Atg5. Densitometry results from LC3 immunoblots are summarized in panel B. C & D, Cultured NRCMs were treated with Doxy for 72 hours, starting at 24 hours after Ad-HA-CryAB^R120G infection. Cell lysates were used for the immunoblots. Representative images are shown in panel C. The red arrow marks HA-CryAB^R120G. Densitometry results from the LC3 immunoblots are summarized in panel D. *: p<0.05 vs. the blue and red bar groups; n=4 biological repeats.

Collectively, our in vivo and in vitro experiments demonstrate that Doxy can effectively inhibit aberrant protein aggregation induced by CryAB^R120G, which likely contributes to its protection against DRC.

Discussion

Despite recent advances in understanding genetic basis of DRC (1,3), no effective therapy is available to treat this devastating disease. Using both cell culture and DRC mouse model, the present study reveals for the first time that Doxy can inhibit aberrant protein aggregation in cardiomyocytes, significantly attenuate a DRC-linked misfolded protein induced adverse cardiac remodeling, and effectively prolong the lifespan of a well documented TG mouse model of DRC. These results provide compelling evidence that Doxy is a promising drug candidate to treat DRC.

The dosage and route chosen here for Doxy administration had previously proven effective in treating a mouse model of oculopharyngeal muscular dystrophy (10). It should be noted that Doxy concentration used in the drinking water (6mg/ml) for this study is 6-fold higher than what is commonly used to manipulate transgene expression in the tetracycline-inducible transgenic system. We only tested Doxy here but other tetracycline derivatives, especially those with better tissue permeability (e.g., minocycline), could be as effective or even more effective, as demonstrated in neural proteinopathies (20).

Notably, Doxy treatment in our survival study was initiated at a relatively late stage when DRC pathology and clinical signs are readily detectable (Table 1). The rationale behind this experimental design is to maximize its clinical relevance. It remains to be tested but is very likely that the survival improvement by Doxy would be much greater should the treatment be started earlier. Supporting this prediction, we have observed that a significant attenuation of cardiac hypertrophy without deteriorating cardiac function was detected 1 month after Doxy treatment initiated at 8 weeks of age (Figure 2, Table 2), 8 weeks earlier than the starting point of the survival study.

The mechanisms underlying Doxy's beneficial effects on DRC are potentially quite complex because of Doxy's versatile pharmacological actions. Besides its anti-microbial action, Doxy is also known to inhibit MMPs. By breaking down extracellular matrix, MMPs play important roles in tissue remodeling, cell migration, angiogenesis, and interstitial remodeling (21). Hence, Doxy's MMP inhibition property is believed to contribute to a wide range of its biological effects. Timed administration of Doxy appears to protect cardiac function by modulating post-myocardial infarction remodeling (22-25). We cannot rule out the possibility that Doxy's MMP inhibition property may contribute to its beneficial effects on DRC but two lines of evidence stand against this possibility. First, previous characterization showed no significant interstitial fibrosis in the heart of the DRC mice used here (2). Second, compared with NTG, myocardial activities of MMPs were not increased in TG mice (data not shown). Notably, it was recently reported that Doxy mitigated cardiac remodeling without significantly affecting myocardial MMP activities (26).

Misfolded proteins, when failed to be repaired, are escorted by the chaperones to degradation by the ubiquitin-proteasome system (1). When chaperones and/or the ubiquitinproteasome system are overwhelmed, misfolded proteins undergo aberrant aggregation which produces initially soluble oligomers. If not removed in time, the oligomers will fuse to form large insoluble aggregates. The soluble oligomers are generally believed to be toxic whereas the insoluble aggregates are perhaps not (1). Cardiac toxicity of aberrant protein aggregation was directly demonstrated by the sufficiency of expressing a mutant prion protein or poly-glutamine pre-amyloid oligomers in cardiomyocytes to induce heart failure in mice (27,28). In the present study, we observed that not only insoluble aggregates (Figures 3, 5) but also oligomeric CryAB^R120G (Figures 4, 6) were significantly decreased by Doxy treatment in vivo and in vitro. These data suggest that Doxy may act as a pharmacological chaperone that prevents CryAB^R120G from inducing aberrant oligomerization of endogenous and TG CryAB, allowing the formation of normal CryAB polymers that can exert its normal chaperoning function. Indeed, both tetracycline and Doxy had been shown to interact with prion proteins and reduce in vitro prion protein aggregation and in vivo infectivity (29). Supporting this notion, the same extent of CryAB^R120G protein overexpression, in the presence of Doxy, caused significantly less upregulation of p62 (Figure 6), and less accumulation of ubiquitinated proteins (Figures 4C). Our data suggest that Doxy's inhibition of aberrant protein aggregation of misfolded CryAB^R120G may contribute to its protection against DRC.

Consistent with its presence in the protein aggregates associated desminopathy (30), p62 was significantly up-regulated in both the soluble and insoluble fractions of cultured cardiomyocytes overexpressing CryAB^R120G. Interestingly, the increases in p62 protein levels were significantly attenuated by Doxy (Figure 6D, 6E). p62 is known to interact with both ubiquitinated proteins in the aggregates and autophagosomes and target protein aggregates for selective autophagic degradation (31). p62 also mediates the formation of ubiquitin-positive inclusion bodies in hepatocytes and neurons when autophagy is impaired with varying consequences (16). The role of p62 up-regulation upon aberrant protein aggregation in cardiomyocytes has not been defined but our results implicate that attenuation of p62 upregulation is likely beneficial to the heart and might be an underlying mechanism of Doxy. Consistent with this postulate, the down-regulation of p62 by Doxy treatment did not appear to be caused by autophagic activation because Doxy did not enhance autophagic activation in either the TG heart or NRCMs expressing CryAB^R120G (Figure 7).

It was reported in a TG mouse model of human CryAB^R120G that stress-inducible Hsp's were differentially up-regulated in DRC hearts, with a major increase in Hsp25 expression associated with progression to heart failure and increased mortality (3). However, HSPs such as Hsp22, Hsp70, and HspB8 disrupt oligomer formation induced by CryAB^R120G under certain conditions (13,14). In both CryAB^R120G TG mouse hearts (data not shown) and CryAB^R120G expressing cultured cardiomyocytes, we observed a significant Hsp25 upregulation but Doxy treatment failed to alter it (Figure 6D, 6E). Hence, the suppression of protein aggregation by Doxy is unlikely through an effect on Hsp25.

DRC, by itself, is not a common disease but it exemplifies cardiac proteinopathies featured by intrasarcoplasmic aberrant protein aggregation. Hence, the significance of this study could be far beyond DRC because pre-amyloid oligomers were observed in a large subset of human congestive heart failure resulting from hypertrophic/dilated cardiomyopathies (4). Moreover, aberrant protein aggregates were also observed in pressure overloaded mouse hearts (7).

Interestingly, although chronic Doxy treatment was shown to attenuate isoproterenol and trans-aortic constriction induced cardiac hypertrophy (32), Vinet et al reported that 1 month but not 2 months of low dose Doxy enhanced trans-aortic constriction induced hypertrophy (26). Protective actions of Doxy on rat diabetic cardiomyopathy were also recently reported (25).

In conclusion, the present study provides compelling preclinical evidence that Doxy is a promising drug candidate to treat DRC.

Abbreviations

DRC

desmin-related cardiomyopathy

CryAB

αB-crystallin

TG

transgenic

NTG

non-transgenic

Doxy

doxycycline

MMPs

matrix metalloproteinases

NRCM

neonatal rat cardiomyote

Ad-HA-CryAB^R120G

adenoviruses expressing hemagglutinin tagged CryAB^R102G

β-Gal

β-galactosidase

Hsp

heat shock protein