Research ArticleLaminopathies

Temsirolimus Activates Autophagy and Ameliorates Cardiomyopathy Caused by Lamin A/C Gene Mutation

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Science Translational Medicine  25 Jul 2012:
Vol. 4, Issue 144, pp. 144ra102
DOI: 10.1126/scitranslmed.3003875

Abstract

Mutations in the lamin A/C gene (LMNA), which encodes A-type lamins, cause a diverse range of diseases collectively called laminopathies, the most common of which is dilated cardiomyopathy. Emerging evidence suggests that LMNA mutations cause disease by altering cell signaling pathways, but the specific mechanisms are poorly understood. We show that the AKT–mammalian target of rapamycin pathway is hyperactivated in hearts of mice with cardiomyopathy caused by Lmna mutation and that in vivo administration of the rapamycin analog temsirolimus prevents deterioration of cardiac function. We also show defective autophagy in hearts of these mice and demonstrate that improvement in heart function induced by pharmacological interventions is correlated with enhanced autophagy. These findings provide a rationale for treatment of LMNA cardiomyopathy with rapalogs and implicate defective autophagy as a pathogenic mechanism of cardiomyopathy arising from LMNA mutation.

Introduction

Laminopathies are a diverse group of diseases arising from mutations in LMNA, the gene that encodes the A-type lamins A and C (1). A-type lamins are intermediate filament proteins of the nuclear lamina, a proteinaceous meshwork lining the inner nuclear membrane. Although A-type lamins are expressed in most differentiated mammalian somatic cells, specific mutations in LMNA lead to tissue-selective diseases affecting striated muscle, adipose tissue, or peripheral nerve, or more generalized diseases with features of accelerated aging, such as Hutchinson-Gilford progeria syndrome (HGPS). The most prevalent laminopathy is dilated cardiomyopathy (hereinafter referred to as LMNA cardiomyopathy) with variable skeletal muscle involvement, which includes autosomal Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. Patients with LMNA cardiomyopathy have higher rates of deadly arrhythmias and a relatively early onset of heart failure compared to individuals with most other inherited dilated cardiomyopathies (2). The pleiotropic nature of laminopathies, as well as the mechanisms involved in the pathogenesis of specific diseases such as LMNA cardiomyopathy, is poorly understood.

Emerging evidence suggests that LMNA mutations that cause abnormalities in the nuclear lamina lead to dysregulation of signaling pathways that underlie disease pathogenesis (1). For example, impaired canonical Wnt signaling contributes to HGPS by reducing the expression of genes encoding extracellular matrix components (3). Notably, inducing autophagic clearance of the lamin A variant responsible for HGPS with the autophagy-activating compound rapamycin improves cellular phenotypes associated with the disease (4). In LMNA cardiomyopathy, extracellular signal–regulated kinases 1 and 2 (ERK1/2) signaling is hyperactivated in the hearts of human subjects with LMNA cardiomyopathy and in LmnaH222P/H222P mice, an animal model of the disease (5, 6). Reducing ERK1/2 signaling to basal levels in hearts of LmnaH222P/H222P mice ameliorates cardiomyopathy (68), demonstrating that ERK1/2 hyperactivation contributes to pathogenesis.

AKT signaling, which is predominantly triggered by insulin or insulin-like growth factor, is also implicated in the development of cardiomyopathy. Although AKT is generally accepted to promote cell survival in response to acute ischemic stress (9, 10), more recent evidence suggests that AKT activation also contributes to the pathogenesis of cardiomyopathy and that blocking downstream signaling prevents the disease (11, 12). Concurrent activation of both ERK1/2 and AKT signaling has also been documented in failing hearts of human subjects (13, 14) and in hearts from mice with cardiomyopathy induced by overexpression of the Gsα subunit (15). We therefore have investigated the involvement of AKT signaling in the pathogenesis of LMNA cardiomyopathy.

Results

AKT–mammalian target of rapamycin signaling is enhanced in LMNA cardiomyopathy

We examined AKT phosphorylation, which is indicative of its activation, in hearts of wild-type and LmnaH222P/H222P mice that develop cardiomyopathy at 4, 8, 12, and 16 weeks of age (Fig. 1A) (16). To ensure that the observed phenotypes were not secondary to the development of cardiomyopathy, we chose 4 weeks as our earliest time point because male LmnaH222P/H222P mice do not develop detectable cardiac abnormalities until 8 weeks of age (16). Full activation of AKT requires phosphorylation on two separate sites, threonine 308 (T308) and serine 473 (S473). As early as 4 weeks of age, we detected increased AKT phosphorylation on both S473 and T308 (Fig. 1A). As the mice aged, AKT phosphorylation (S473 and T308) in hearts increased further, peaking at 12 weeks of age (Fig. 1A).

Fig. 1

AKT-mTORC1 is activated in LMNA cardiomyopathy. (A) Western blot analysis of phospho-serine 473 AKT [pAKT(S473)], phospho-threonine 308 AKT [pAKT(T308)], total AKT, phosphorylated mTOR (pmTOR), total mTOR, phosphorylated S6 (pS6), and total S6 in hearts of 4- to 16-week-old LmnaH222P/H222P (LmnaH222P) and wild-type (LmnaWT) mice (top). Numbers on top of blots denote samples from individual hearts. Representative blots from two independent experiments. Quantification of pmTOR, pAKT, and pS6 normalized to total mTOR, AKT, and S6, respectively, presented as fold change over LmnaWT (bottom); n = 4. (B) Western blot of pmTOR, mTOR, pAKT(S473), pAKT(T308), and total AKT in hearts of 20-week-old LmnaH222P mice treated with DMSO or selumetinib for 4 weeks (top). Numbers on top of blots denote samples from individual hearts. Quantification of pmTOR and pAKT normalized to total mTOR and AKT, respectively, presented as fold change over DMSO (bottom); n = 6. (C) Western blot of pAKT (S473 and T308), total AKT, and α-tubulin in hearts from human subjects with (LMNAmt) or without (LMNAwt) LMNA cardiomyopathy (top). Numbers on top of blots denote samples from individual hearts. Quantification of pAKT(S473) and pAKT(T308) normalized to total AKT, presented as fold change over LMNAwt (bottom); n = 3.

Activated AKT activates mammalian target of rapamycin complex 1 (mTORC1) (1722), which contains mTOR, a protein kinase that forms two functionally distinct complexes, mTORC1 and mTORC2. mTORC1 is a central regulator of metabolism and cell growth (23) that is implicated in the pathogenesis of cardiomyopathy (11, 12, 2426), and mTORC2 catalyzes phosphorylation of AKT on S473 (23). To determine whether mTOR activation occurred concurrently with AKT activation, we assessed its phosphorylation in the same heart samples. Similar to AKT phosphorylation, we observed enhanced mTOR phosphorylation at 4 weeks, which increased further with age (Fig. 1A). We observed increased levels of phosphorylated ribosomal protein S6, a downstream target of mTORC1 starting at 8 weeks, confirming that mTORC1 was activated. These results demonstrate activation of AKT-mTOR signaling in hearts of LmnaH222P/H222P mice before the onset of clinically detectable cardiomyopathy.

Activated ERK1/2 can catalyze an inhibitory phosphorylation on tuberous sclerosis complex (27), a repressor of mTORC1, hence potentially activating it. Because ERK1/2 is hyperactivated in hearts of LmnaH222P/H222P mice (5), we determined whether enhanced ERK1/2 signaling contributed to the increased phosphorylation of mTOR. We examined mTOR phosphorylation in hearts of LmnaH222P/H222P mice treated systemically with selumetinib (1 mg kg−1 day−1), an inhibitor of the ERK1/2-activating kinase MEK1/2 (mitogen-activated or extracellular signal–regulated protein kinase kinases 1 and 2), from 16 to 20 weeks of age. This dose of selumetinib blocks cardiac ERK1/2 phosphorylation and improves heart function in LmnaH222P/H222P mice (6). Selumetinib reduced mTOR phosphorylation in the heart relative to controls given dimethyl sulfoxide (DMSO) (Fig. 1B). We also observed consistent reduction in AKT phosphorylation after ERK1/2 inhibition (Fig. 1B), which may further contribute to the reduced mTOR phosphorylation. These results show that ERK1/2 hyperactivation contributes to the enhancement of mTOR phosphorylation in hearts of LmnaH222P/H222P mice.

To test whether the changes we observed in hearts of LmnaH222P/H222P mice also occur in human disease, we examined phosphorylated AKT levels in left ventricular tissue from human subjects with confirmed mutations in the LMNA gene (table S1). Compared with controls, enhanced AKT phosphorylation (on T308 and S473) was observed in ventricular tissue from human subjects with LMNA cardiomyopathy (Fig. 1C). This observation established that enhanced AKT-mTORC1 signaling is a feature of LMNA cardiomyopathy in humans.

Given the enhanced AKT-mTORC1 signaling in hearts of LmnaH222P/H222P mice, we hypothesized that reducing mTORC1 activity would prevent progression of cardiomyopathy. We blocked mTORC1 in 14-week-old LmnaH222P/H222P mice with daily intraperitoneal injections of temsirolimus (5 mg kg−1). After 2 weeks of treatment, mice were analyzed by echocardiography and sacrificed for biochemical analyses. Temsirolimus reduced phosphorylated mTOR and S6 in hearts compared to DMSO placebo (Fig. 2A). Previously reported effects of temsirolimus on AKT phosphorylation (increased and decreased phosphorylation on T308 and S473, respectively) (2830) were also observed, further demonstrating a pharmacological effect (fig. S1, A and B). At 16 weeks, LmnaH222P/H222P mice exhibit significantly increased left ventricular end-diastolic and end-systolic diameters and markedly decreased fractional shortening compared to wild-type mice (7, 8, 16). After temsirolimus treatment, overall heart size was reduced compared to placebo controls (Fig. 2B). Motion-mode (M-mode) echocardiography showed that left ventricular diameters were significantly smaller and fractional shortening was significantly greater in temsirolimus-treated mice than in controls (Fig. 2, C and D). Temsirolimus also reduced the mRNA levels of NppA and NppB, genes that encode natriuretic peptide precursors that stimulate vasodilation and vascular fluid egress to compensate for ventricular dilatation, but not those of Col1a1, Col1a2, or Fn1, which encode collagens and fibronectin involved in fibrosis (Fig. 2E). Levels of atrial natriuretic peptide (ANP), encoded by NppA, were also decreased (Fig. 2F). Hence, mTORC1 inhibition improves left ventricular diameters and apparent contractility in LmnaH222P/H222P mice but may not prevent cardiac fibrosis.

Fig. 2

mTORC1 inhibition ameliorates cardiomyopathy in LmnaH222P/H222P mice. (A) Western blot of pmTOR, total mTOR, pS6, and total pS6 in hearts of 16-week-old LmnaH222P/H222P mice treated with DMSO or temsirolimus (Temsir) for 2 weeks (left). Numbers on top of blots denote individual heart samples. Quantification of pmTOR and pS6 normalized to total mTOR and S6, respectively, presented as fold change over DMSO (right); n = 5. (B) Representative hearts of 16-week-old LmnaH222P/H222P mice treated with DMSO or Temsir. Scale bar, 1 cm. (C) Representative M-mode echocardiographic tracings of 16-week-old LmnaH222P/H222P mice treated with DMSO or Temsir. (D) Graphic representations and table with numerical values for left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and fractional shortening (FS) of hearts of 16-week-old LmnaH222P/H222P mice treated with DMSO or Temsir; n = 8. (E) qPCR analysis on NppA, NppB, Col1a1, Col1a2, and Fn1 mRNAs in hearts of 16-week-old LmnaH222P/H222P mice treated with DMSO or Temsir. Tnni3 mRNA was used as internal control; n = 5. (F) Western blot of ANP and GAPDH in hearts of 16-week-old LmnaH222P/H222P mice treated with DMSO or Temsir (top). Numbers on top of blots denote samples from individual hearts. Quantification of ANP normalized to GAPDH, presented as fold change over DMSO (bottom); n = 5.

Autophagy is impaired in LMNA cardiomyopathy

Given that mTORC1 blockade in hearts of LmnaH222P/H222P mice prevented progression of cardiomyopathy, we explored putative pathogenic mechanisms engendered by activated mTORC1. We focused on macroautophagy (autophagy), which involves lysosome-dependent degradation of cytoplasmic cargo enclosed in a double-membrane vesicle termed the autophagosome (31, 32). Because mTORC1 inhibits autophagy and defective autophagy has been linked to cardiomyopathy (3335), we explored autophagic responses in hearts of LmnaH222P/H222P mice.

Autophagic flux can be broadly categorized into two general steps: (i) vesicle nucleation and formation of autophagosomes and (ii) lysosome-mediated autophagosome degradation. We assessed the conversion of microtubule-associated protein 1 light chain 3β from its nonlipidated (LC3B-I) to lipidated form (LC3B-II) during autophagosome formation (31, 36). Hearts from 12- and 16-week-old LmnaH222P/H222P mice exhibited slightly variable but generally reduced steady-state levels of LC3B-II compared to wild-type mice (Fig. 3, A and B). Because autophagic flux is dynamic, reduced LC3B-II levels can indicate either impaired formation or enhanced degradation of autophagosomes. Therefore, we assessed the expression of p62/SQSTM1 (p62), an LC3B-binding protein that is degraded along with LC3B-II during vesicle degradation (37). p62 expression was enhanced at 12 weeks and increased further by 16 weeks (Fig. 3, A and B). Increased p62 levels did not correlate with its mRNA expression (fig. S2). Accumulation of p62 coincident with reduced LC3B-II is suggestive of defective autophagosome formation rather than rapid degradation. This occurred despite increased expression of beclin1, an activator of autophagy necessary for vesicle nucleation, at 16 weeks (Fig. 3A) (32). These data suggest that, despite increased beclin1 expression, autophagosome formation was progressively impaired in hearts of LmnaH222P/H222P mice.

Fig. 3

Autophagy is impaired in hearts of LmnaH222P/H222P mice. (A) Western blot of nonlipidated LC3B (LC3B-I), lipidated LC3B (LC3B-II), p62, beclin1, and GAPDH in hearts of 4- to 16-week-old LmnaH222P/H222P (LmnaH222P) and wild-type (LmnaWT) mice (left). Numbers on top of blots denote individual mouse heart samples. (B) Quantification of LC3B-II and p62 normalized to GAPDH, presented as fold change over LmnaWT (right); n = 6; *P = 0.03; **P = 0.002; #P = 0.04. (C) Western blot of LC3B-I, LC3B-II, p62, and α-tubulin in hearts of 16-week-old LmnaH222P and LmnaWT mice treated with chloroquine (Chloroq) or vehicle (Ctrl) for 10 days (top). Numbers on top of blots denote individual heart samples. Quantification of LC3B-II and p62 normalized to α-tubulin, presented as fold change over Ctrl (bottom); n = 3. (D) Western blot of LC3B-I, LC3B-II, p62, and α-tubulin in hearts of 16-week-old LmnaH222P and LmnaWT mice fasted for 24 hours (Fasted) or fed ad libitum (Fed) (top). Numbers on top of blots denote individual heart samples. Quantification of LC3B-II and p62 normalized to α-tubulin, presented as fold change over Fed (bottom); n = 3.

To further verify that autophagosome formation is impaired in vivo, we treated 15-week-old LmnaH222P/H222P mice with chloroquine (50 mg kg−1 day−1), a lysosomal inhibitor that reduces autophagosome degradation (38). Administration of chloroquine for 10 days resulted in a significant increase in LC3B-II and p62 in hearts of wild-type mice (Fig. 3C), demonstrating autophagosome accumulation. However, chloroquine had insignificant effects on LC3B-II levels in hearts of LmnaH222P/H222P mice (Fig. 3C), confirming that autophagosome formation is impaired. Likewise, no significant change was observed in p62 levels in hearts of LmnaH222P/H222P mice, which were elevated before chloroquine administration. To further characterize the impairment, we examined autophagic responses in hearts of wild-type and LmnaH222P/H222P mice fasted for 24 hours. In hearts of wild-type mice, fasting led to a significant increase in LC3B-II, whereas p62 levels, although decreased relative to fed wild-type mice, were not significantly different (Fig. 3D). No significant differences were observed in LC3B-II and p62 levels between fed and fasted LmnaH222P/H222P mice, suggesting that fasting-induced autophagic responses were also defective in hearts of LmnaH222P/H222P mice.

To eliminate the influence of cardiac fibroblasts, we assessed autophagic responses in isolated ventricular cardiomyocytes from 12-week-old LmnaH222P/H222P mice. No phenotypic differences were observed between cardiomyocytes from wild-type and LmnaH222P/H222P mice when assessed by light microscopy (Fig. 4A). We then deprived cardiomyocytes of glucose for 2 and 4 hours and examined LC3B-II and p62 levels. Glucose-deprived cardiomyocytes from wild-type mice exhibited enhanced LC3B-II, whereas both basal and glucose-deprived LC3B-II levels in cardiomyocytes from LmnaH222P/H222P mice were comparatively reduced (Fig. 4B). Levels of p62 were also higher in LmnaH222P/H222P cardiomyocytes, indicative of impaired autophagic flux, and were unaffected by glucose deprivation. These results establish defective autophagy as a feature of cardiomyocytes from LmnaH222P/H222P mice. To test whether similar phenotypes exist in the human disease, we examined LC3B-II and p62 in left ventricular tissue from human subjects. Virtually no LC3B-II was detected in tissue from human subjects with LMNA cardiomyopathy compared to unaffected controls (Fig. 4C). Moreover, we observed increased p62 levels in hearts with LMNA cardiomyopathy. These results show that autophagy is impaired in human hearts with LMNA cardiomyopathy and support the hypothesis that defective autophagy underlies LMNA cardiomyopathy pathogenesis.

Fig. 4

Reactivation of autophagy is associated with improved cardiac function. (A) Photomicrographs of cardiomyocytes from 12-week-old LmnaH222P/H222P (LmnaH222P) and wild-type (LmnaWT) mice. Scale bar, 120 μm. (B) Representative Western blot of LC3B-I, LC3B-II, p62, and α-tubulin in cardiomyocytes from LmnaH222P and LmnaWT mice deprived of glucose (Gluc−) for 0, 2, and 4 hours (top). Quantification of LC3B-II and p62 normalized to α-tubulin, presented as fold change over LmnaWT (bottom); n = 3; *P = 0.011; #P = 0.046. (C) Western blot of LC3B-I, LC3B-II, p62, and GAPDH in hearts from human subjects with (LMNAmt) or without (LMNAwt) LMNA cardiomyopathy (top). Numbers on top of blots denote samples from individual patients. Quantification of LC3B-II and p62 normalized to α-tubulin, presented as fold change over LMNAwt (bottom); n = 3. (D) Western blot of LC3B-I, LC3B-II, p62, and α-tubulin in hearts of 16-week-old LmnaH222P/H222P mice treated with DMSO or Temsir (left). Numbers on top of blots denote samples from hearts of different individuals. Quantification of LC3B-II and p62 normalized to α-tubulin, presented as fold change over DMSO (right); n = 5. (E) Immunofluorescence micrographs of heart sections from 16-week-old LmnaWT, DMSO-treated LmnaH222P, and Temsir-treated LmnaH222P mice probed with antibodies against α-actin and ubiquitin. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain. Representative figures from two independent hearts per group are shown. Scale bars, 30 μm. (F) Immunofluorescence micrographs of heart sections from 16-week-old LmnaWT, DMSO-treated LmnaH222P, and Temsir-treated LmnaH222P mice probed with antibodies against α-actin and lamin A/C. DAPI was used as a nuclear counterstain. Representative figures from two independent hearts per group are shown. Scale bars, 30 μm. (G) Representative Western blot of LC3B-I, LC3B-II, p62, and GAPDH in hearts of 20-week-old LmnaH222P/H222P mice treated with DMSO or selumetinib (top). Numbers on top of blots denote individual heart samples. Quantification of LC3B-II and p62 normalized to α-tubulin, presented as fold change over DMSO (bottom); n = 6. (H) Schematic of the proposed pathogenesis of LMNA cardiomyopathy. See the text for details.

AKT activation can inhibit autophagy through inhibitory phosphorylation of Forkhead box O3a (FoxO3a) (39, 40). We did not observe significant differences in phosphorylated FoxO3a or expression of mRNAs encoded by genes associated with a FoxO3a-activated autophagy in hearts of wild-type and LmnaH222P/H222P mice (fig. S3, A and B). This ruled out the possibility that AKT inhibited autophagy via FoxO3a in our study.

Temsirolimus reactivates autophagy

To test whether the beneficial effect of temsirolimus in the heart may be mediated by enhancing autophagy, we measured LC3B-II and p62 expression in hearts of temsirolimus-treated LmnaH222P/H222P mice. Compared to placebo controls, hearts from temsirolimus-treated mice exhibited increased LC3B-II and reduced p62 expression (Fig. 4D), an expression profile indicative of enhanced autophagy. Temsirolimus did not alter the expression of beclin1 or phosphorylated ERK1/2 (fig. S4).

Defective autophagy should lead to accumulation of ubiquitinated proteins (32), which should be degraded by temsirolimus-induced autophagy. To verify whether this is the case in the hearts of LmnaH222P/H222P mice, we performed immunofluorescence microscopy on heart sections from 16-week-old wild-type, DMSO-treated LmnaH222P/H222P, and temsirolimus-treated LmnaH222P/H222P mice with anti-ubiquitin antibodies. Antibodies against α-actin, which detect the I band of sarcomeres, were used to identify cardiomyocytes. In hearts from wild-type mice, we mainly observed diffuse fluorescence that colocalized with α-actin labeling (Fig. 4E). In contrast, we observed large aggregates of ubiquitinated proteins in hearts of DMSO-treated LmnaH222P/H222P mice, and temsirolimus treatment noticeably reduced these aggregates (Fig. 4E). Similarly, insoluble aggregates of lamin A/C, in the form of intranuclear foci, have been proposed to contribute to the pathogenesis of laminopathies (4). We therefore labeled cardiac tissue with anti–lamin A/C antibodies to assess whether lamin A/C aggregates were visible in the hearts of LmnaH222P/H222P mice. We did not observe obvious lamin A/C aggregates, and temsirolimus had no observable effects on lamin A/C expression (Fig. 4F). This result was confirmed by Western blot analysis demonstrating comparable lamin A/C expression in hearts of DMSO- and temsirolimus-treated LmnaH222P/H222P mice (fig. S4). However, consistent with previous studies (7, 8), we observed irregular, misshapen nuclei in hearts of LmnaH222P/H222P mice.

Reduction of ERK1/2 signaling also ameliorates cardiomyopathy in LmnaH222P/H222P mice (68). To verify whether this was associated with enhanced autophagy, analogous to that observed with temsirolimus treatment, we measured LC3B-II and p62 expression in hearts of selumetinib-treated LmnaH222P/H222P mice. Selumetinib treatment increased the level of LC3B-II while reducing p62 in hearts of LmnaH222P/H222P mice (Fig. 4G), consistent with enhanced autophagy. Together, our results link reactivation of autophagy to improved cardiac function after ERK1/2 or mTORC1 blockade. They further suggest that temsirolimus may be beneficial as a treatment for LMNA cardiomyopathy.

Discussion

Our findings support a model for the pathogenesis of LMNA cardiomyopathy (Fig. 4H) in which the LmnaH222P mutation results in aberrant activation of both the ERK1/2 and the AKT signaling cascades that converge on mTORC1. Activated mTORC1 inhibits autophagic responses and reduces tolerance to energy deficits. In this scenario, the heart is unable to compensate for increased or fluctuating energy demand and, over time, develops muscle damage and dilated cardiomyopathy. The beneficial effects of MEK1/2 inhibitors such as selumetinib and PD98059 in LMNA cardiomyopathy (68) may be a result in part of reducing mTORC1 activities. Moreover, through undefined mechanisms, MEK1/2 inhibition also reduces AKT phosphorylation, which can further decrease mTORC1 activities. Temsirolimus and other rapamycin analogs similarly block mTORC1 activity and could reactivate autophagy and prevent the progression of cardiomyopathy.

The exact mechanisms by which defects in nuclear lamins cause dysregulated AKT-mTORC1 and MEK-ERK1/2 signaling remain to be elucidated. A-type lamins control ERK1/2 signaling output by acting as a molecular scaffold that allows efficient interaction between ERK1/2 and c-fos (41). Perhaps ERK1/2 feedback inhibitory mechanisms are disrupted by amino acid substitutions in A-type lamins that cause cardiomyopathy. Alternatively, extracellular factors may play a role. For example, Gene Ontology analysis of mRNA expression in hearts from LmnaH222P/H222P mice hinted at the involvement of the insulin-like growth factor signaling pathway (5). Intracellular propagation of insulin-like growth factor signaling can be triggered by both the AKT and the ERK1/2 cascades (42).

As pathways that mediate cell growth and proliferation, both AKT and ERK1/2 signaling have been implicated in hypertrophic cardiomyopathy, in which the myocardium exhibits abnormal thickening. The demonstration that AKT and ERK1/2 are hyperactivated in human LMNA cardiomyopathy, which is characterized by left ventricular dilatation with a thinned left ventricular wall, suggests that hypertrophic and dilated forms of cardiomyopathy share a putative pathogenic mechanism(s) emanating from deregulated cell signaling. Moreover, the convergence of the AKT and ERK1/2 cascades on mTORC1 implies a central role for mTORC1 in cardiomyopathy. Indeed, the involvement of mTOR in cardiac pathophysiology is evolutionarily conserved from fruit flies to mammals (24, 43). Enhanced mTOR signaling is seen in mouse models of hypertrophic cardiomyopathy, and inhibition of mTORC1 with rapamycin ameliorates the disease (11, 2426). Nevertheless, cardiac-specific ablation of mTOR (44) or raptor (45), an essential component of mTORC1, results in dilated cardiomyopathy, suggesting that a threshold level of mTORC1 activity is necessary for proper cardiac homeostasis and response to physiological stress.

We have also shown that pharmacological mTORC1 inhibition reactivates autophagy and prevents cardiac damage in a mouse model of LMNA dilated cardiomyopathy. Furthermore, we have shown that inhibition of ERK1/2 signaling, previously shown to be beneficial in the same model of cardiomyopathy, enhances autophagy in the heart. On the basis of these findings, it could be hypothesized that combining MEK1/2 and mTORC1 inhibitors to treat LMNA cardiomyopathy may be synergistic, given that ERK1/2 and mTORC1 trigger divergent mechanisms that contribute to LMNA cardiomyopathy (for example, induction of genes involved in fibrosis elicited by ERK1/2 hyperactivation). In this regard, we performed a pilot study with a small group of mice (n = 3) that were treated with temsirolimus alone or in combination with selumetinib for 2 weeks from 18 to 20 weeks of age. This preliminary study showed no apparent benefit of adding selumetinib to temsirolimus (factional shortening of 19.1 ± 6.7 SD for temsirolimus alone compared to 14.5 ± 4.6 SD for combination treatment with P = 0.38). Future studies optimizing drug doses, treatment duration, and the age of initial treatment will be required to definitively assess the therapeutic benefit of a combination treatment or to improve treatment outcomes by reduced dosing of drugs to avoid adverse events associated with higher doses.

In a report, also in this issue of Science Translational Medicine, Ramos et al. (46) show that rapamycin improves cardiac function and prolongs survival in Lmna−/− mice, another model of LMNA cardiomyopathy, providing additional evidence that rapalogs may be a useful treatment for humans with this disease.

Materials and Methods

Animals

The Columbia University Medical Center Institutional Animal Care and Use Committee approved all protocols that used vertebrate animals, and the investigators adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. LmnaH222P/H222P mice were generated and genotyped as described (16). Only male mice were used. Selumetinib treatment has been described (6). For temsirolimus treatment, 14-week-old LmnaH222P/H222P mice were given temsirolimus (5 mg kg−1 day−1) or DMSO for 2 weeks by intraperitoneal administration. Chloroquine, dissolved in phosphate-buffered saline, was similarly injected into 15-week-old mice (fed ad libitum) at 50 mg kg−1 day−1 for 10 days. Autophagy was induced in vivo by fasting mice (given only water) for 24 hours.

Human subjects

Heart tissue from human subjects with LMNA cardiomyopathy was obtained from Myobank–AFM of the Institut de Myologie. Control human heart samples without patient identifiers were obtained from the National Disease Research Interchange (table S1). All tissue samples were obtained with appropriate approvals and consent from the Institut de Myologie and National Disease Research Interchange (not specifically for this study) and provided without patient identifiers.

Isolation of mouse cardiomyocytes

Cardiomyocytes were isolated as described (47). Briefly, the heart was removed and the aorta was cannulated. After Ca2+-free buffer was perfused for 2 min, collagenase I/II (0.3 mg ml−1; Liberase TH, Roche) solution was perfused through the coronary arteries for 6 min with Ca2+ at 12.5 μM. Left ventricular tissue was teased apart and pipetted to release individual cells. After enzymatic dispersion, Ca2+ concentration in the buffer containing bovine serum albumin (3.5 mg ml−1) was elevated in three steps up to 400 μM. This method yielded an ~99% pure population of cardiomyocytes that were ~80% viable in culture.

RNA isolation and quantitative polymerase chain reaction

Total RNA was isolated with TRIzol (Invitrogen). Complementary DNAs were generated from 1 μg of RNA primed with a 1:1 ratio of random hexameric primers: oligo(dT) with SuperScript II Reverse Transcriptase (Invitrogen). Quantitative polymerase chain reaction (qPCR) was performed on ABI 7300 Real-Time PCR System (Applied Biosystems) with SYBR Green (USB). qPCR primers for NppA, NppB, Col1a1, Col1a2, and Fn1 have been described (8). All other primers are listed in table S2. Gapdh and Tnni3 mRNAs were assessed to ensure equal fidelity in enzymatic reactions and equal loading between samples. Gapdh mRNA was used as internal control to normalize qPCR unless otherwise indicated. Fold changes in gene expression were determined by the ΔΔCt method and are presented as fold change over untreated or wild-type controls.

Protein extraction and Western blot analysis

Whole-cell extracts were isolated with radioimmunoprecipitation assay buffer (Sigma) containing a protease inhibitor cocktail (Roche), 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium vanadate. Samples (15 to 30 μg) were loaded for SDS–polyacrylamide gel electrophoresis. The following antibodies were purchased from Cell Signaling Technology: beclin1 (catalog no. 3738), phospho-ERK1/2 (9101), LC3B (2775), phospho-mTOR (Ser2448, 2971), mTOR (2972), phospho-AKT (Ser473, 4060; Thr308, 4056), phospho-S6 (Ser240/244, 2215), S6 (2217), phospho-FoxO3a (Thr32, 9464), FoxO3a (2497), and ubiquitin (3936). The following antibodies were purchased from Santa Cruz Biotechnology: ERK1/2 (sc-94), lamin A/C (sc-20681), and α-tubulin (sc-12462-R). Antibodies against p62/SQSTM1 (610832), α-actin (MA1-21597), AKT (05-796), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AM4300) were purchased from BD Biosciences, Thermo Scientific, Millipore, and Ambion, respectively. Quantification of blots was performed with ImageJ (48), normalized to loading controls as indicated, and presented as fold change over untreated or wild-type controls.

Transthoracic echocardiography

LmnaH222P/H222P mice were anesthetized with 1 to 2% isoflurane and placed on a heating pad (37°C) attached to an electrocardiographic monitor. Echocardiography was performed with a Vevo 770 Imaging System (VisualSonics) equipped with a 30-MHz transducer. Parameters were measured for at least three cardiac cycles. A blinded echocardiographer, who was unaware of the treatment received by any animal, performed the examinations and interpreted the results.

Statistical analysis

GraphPad (Prism Software Inc.) was used to perform statistical analyses. Statistical significance between groups of animals analyzed by echocardiography was analyzed with a corrected parametric test (Welch’s t test), with a value of P < 0.05 being considered significant. To validate results, we performed and concordance-checked a nonparametric test (Wilcoxon-Mann-Whitney test) (table S3). For all other experiments, a two-tailed Student’s t test was used with a value of P < 0.05 considered significant. Values with error bars shown in the figures are means ± SEM. Sample sizes are indicated in the figure legends.

Supplementary Materials

www.sciencetranslationalmedicine.org/cgi/content/full/4/144/144ra102/DC1

Fig. S1. Effect of temsirolimus on AKT phosphorylation.

Fig. S2. p62 mRNA expression in hearts of LmnaH222P/H222P mice.

Fig. S3. Analysis of FoxO3a signaling in hearts of LmnaH222P/H222P mice.

Fig. S4. Effect of temsirolimus on lamin A/C, beclin1, and phosphorylated ERK1/2 expression.

Table S1. Heart tissue samples from human subjects.

Table S2. Primers used for qPCR analyses.

Table S3. Echocardiography data with additional statistics.

References and Notes

  1. Acknowledgments: We thank G. Bonne for providing LmnaH222P/H222P mice and F. Sera for technical assistance with echocardiographic analysis. Funding: J.C.C. is supported by a Ruth L. Kirschstein National Research Service Award Individual Fellowship from the NIH (F32-HL094037). J.P.M. is supported by a Mentored Clinical Scientist Development Award from the NIH (1K08HL105801). This work was supported by grants from the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR048997) and Muscular Dystrophy Association (MDA172222) to H.J.W. Author contributions: J.C.C. and H.J.W. generated the hypotheses and designed experiments. J.C.C. performed experiments and generated data in all figures and Supplementary Materials. A.M. and W.W. designed and performed experiments treating and analyzing LmnaH222P/H222P mice in Fig. 2. S.I. and S.H. performed the echocardiographic analysis in Fig. 2. J.P.M. designed the experiments with isolated cardiomyocytes in Fig. 4. H.J.W. checked and reviewed the data analyses. J.C.C. and H.J.W. wrote the manuscript. All authors reviewed and edited the manuscript before submission. Competing interests: H.J.W. and A.M. are inventors on a pending patent application (PCT/US09/42614) on methods for treating and/or preventing cardiomyopathies by ERK and c-Jun N-terminal kinase inhibition filed by the Trustees of Columbia University in the City of New York. H.J.W. and J.C.C. are inventors on a provisional patent application on the use of rapamycin and rapamycin analogs for the treatment of dilated cardiomyopathies currently planned for filing by the Trustees of Columbia University in the City of New York.
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