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Cardiac myocyte targeted MerCreMer transgenic mice expressing tamoxifen-inducible Cre driven by the α-myosin heavy chain promoter are increasingly used to control gene expression in the adult heart. Here we show tamoxifen-mediated MerCreMer nuclear translocation can induce severe transient dilated cardiomyopathy in mice with or without loxP transgenes. The cardiomyopathy is accompanied by marked reduction of energy/metabolism and calcium handling gene expression (e.g. PGC1-α, PPAR-α, SERCA2A); all fully normalized with recovery. MCM-negative/flox-positive controls display no dysfunction with tamoxifen. Nuclear Cre translocation and equally effective gene knockdown without cardiomyopathy is achievable with raloxifene, suggesting toxicity is not simply from Cre. Careful attention to controls, reduced tamoxifen dosing and/or use of raloxifene is advised with this model.
The Cre-loxP system is widely used for selective cell-targeted manipulation of gene expression1 and has been further enhanced by generating tamoxifen-responsive fusion proteins for conditional Cre induction2;3. Targeted cells constitutively express Cre flanked by mutated estrogen receptor ligand-binding domains (MerCreMer, MCM) insensitive to endogenous estrogen but sensitive to tamoxifen (TAM). MCM is cytoplasmic via binding to heat shock protein 90 complex, but this complex dissociates upon TAM-Mer binding, whereupon the MCM targeting sequence sends the construct to the nucleus for Cre-mediated excision of loxP flanked sequences1. Sohal et al. linked MCM with an α-myosin heavy chain (Myh6) promoter to create cardiomyocyte-specific gene targeting3. However, Cre recombinase displays dose dependent cytotoxicity impairing growth and causing DNA fragmentation4;5, and a recent review raised a caution that TAM-stimulated MCM in adult hearts may also adversely influence heart function6. Here we report on these cardiac effects and provide methods to avoid them.
Myh6-MerCreMer+/+ transgenic mice (#005650, Jackson Labs, Bar Harbor, ME)) were used. Myh6-MCM+/−/no-flox were generated by mating to C57Bl/6 mice. Two strains with flox’d alleles coding for either R2 (Tgfbr2fl/fl ) or R1 (Alk5fl/fl) transforming growth factor-β receptors (both on C57Bl/6 backgrounds) were crossed with Myh6-MCM+/− mice to study gene knockdown.
Cardiac function was assessed by serial echocardiography and invasive pressure-volume analysis. Gene expression was determined by real-time PCR, gene knockdown by analysis of mRNA and TGFβ-stimulated Smad2 phosphorylation, and nuclear Cre by immunohistochemistry and immunoblot. Details are provided in the on-line supplement.
In both MCM+/−/Tgfbr2fl/fl and MCM+/−/Alk5fl/fl mice, TAM administered at 20mg/kgBW i.p.× 5d (proposed dose3) was insufficient for gene and functional knockdown (latter assessed by suppression of TGFβ-stimulated Smad2 phosphorylation, Fig. 1A,B, Online Fig 1A, B). Increasing the dose to 80mg/kgBW×5d (i.p) resulted in 60% mortality by 6 days after TAM treatment due to severe cardiomyopathy (Online Fig 2). Oral delivery of the same dose for 7d was tolerated (no mortality) and effective at gene and functional TGFβ-receptor knockdown attained (Fig 1A,B, Online Fig 1A,B). However, a marked though reversible dilated cardiomyopathy (Fig 1C, supplemental Fig 1) was also observed in both flox’d/MCM+/− models and MCM+/− mice without a flox’d transgene. MCM-negative controls ±flox’d genes (e.g. MCM−/−/Tgfbr2fl/fl) developed no myopathy at any TAM dose. Cardiac-depression peaked ~3 days after terminating TAM (day 10 of protocol) with fractional shortening declining from 61±1 to 26.5±5% (p<0.01) and end-diastolic dimension increasing (3.2±0.1 to 4.1±1.4 mm,p<0.01) in MCM+/− mice (±flox’d alleles, n=19). In vivo pressure-volume analysis in MCM+/−/no flox mice confirmed marked transient systolic and diastolic depression (Fig. 1D, Online Table), with full recovery observed by day 28 (3-wks after stopping TAM). MCM−/− controls had no TAM-induced dysfunction. Myocardium displayed patchy interstitial mononuclear infiltration at day 10 (mild myocarditis) that resolved by day 28, and no myocyte hypertrophy (Online Fig 3).
TAM-MCM induced cardiomyopathy was accompanied by marked changes in stress response, energy/metabolism, and calcium-handling genes (Fig 2, Online Fig 5). Natriuretic peptide expression (Nppa and Nppb) rose markedly in MCM+/− versus MCM−/− by day-10, then returned to normal, though β-myosin heavy chain (Myh7), which typically rises with cardiac stress, was unchanged. Peroxisome proliferator-activated receptor-alpha (PPARα), PPARγ–coactivator 1 alpha (PGC1α), and transcription factor A-mitochondrial (TFAM), genes centrally involved with coordinating mitochondrial function, energetics and metabolism7 and suspected to play a key role in dilated human cardiomyopathy8, all declined substantially with cardiac depression, and then fully recovered to normal levels. Lastly, both sarcoplasmic reticular ATPase and phospholamban expression declined transiently, correlating with cardiac function. While these changes are observed with various cardiac failure conditions, the insult in this instance started in the nucleus and its striking reversibility unusual. While Cre toxicity might be suspected, reversibility would be less anticipated from DNA fragmentation, particularly in differentiated tissue without a high rate of cell regeneration.
Raloxifene (RAL) is an alternative selective estrogen receptor modulator with similarities but also differences to TAM regulated transcription. This may be due to differential binding to estrogen related receptors (e.g. ERRγ)9 and/or recruitment of different co-activators10. Since RAL interacts with Mer albeit at lower binding affinity11, we tested whether RAL could induce gene knock-down without cardiomyopathy. Because of poor solubility, DMSO was required for i.p. dosing, limiting the dose to ≤40mg/kgBW/day which was suboptimal for gene knockdown in MCM+/−/Alk5fl/fl. However, higher oral doses were feasible, tolerated, and effective. Myh6-MCM+/− mice fed 160mg/kgBW/day RAL p.o. displayed effective gene knockdown but without cardiac dysfunction (Figure3A, B Online Fig 6). Nuclear Cre-targeting was similar with TAM or RAL treatments, shown by histochemistry (Fig 3C) and nuclear-fraction immunoblot (Online Fig 7). Stress, metabolic, and Ca2+-handling gene changes (e.g. Fig 2) were not observed (data not shown). Importantly, gene knockdown efficacy with RAL was similar as with 80 mg/kgBW/day TAM, though required longer (21d) exposure (Fig 3B, Online Fig. 8,9). Recombination (lox-P site excision) was observed earlier with RAL (7d) though at lower levels. Both RAL and TAM resulted in a similar ~10% decline in BW during the first week that subsequently recovered (n=6–7/group, p=0.95, 2-way ANOVA). Lower oral TAM dose (20mg/kgBW/day, 1/3 previously reported12)×21d also induced effective gene knockdown without dysfunction (Online Fig 10).
Our study did not precisely define the mechanism for TAM-MCM cardiac effects, and such analysis falls outside the scope and intent of this report. However, the data raise a novel hypothesis that the cardiotoxicity is not simply due to Cre. First, the striking reversibility is difficult to reconcile with mechanisms of cell damage attributed to Cre, namely targeting pseudo loxP sites to cause DNA fragmentation, cell growth arrest and/or death4;5. Second, the finding that both TAM and RAL induced similar nuclear Cre localization and gene suppression yet with striking differences in cardiac phenotype further questions a Cre-toxicity mechanism. An alternative relates to the specific nature of the ligand-MCM complex. In addition to recruiting different nuclear co-activators that can differentially target transcription10, TAM but not RAL can inhibit ERRγ which along with ERRα plays a central role in bio-energetic regulation13. These differences could alter nuclear interactions that depend or are independent of Cre recombinase. Though TAM exposure at the same dose was not toxic, the MCM construct increases nuclear levels >4 fold which could amplify interactions. While similar energy/metabolic changes often accompany pathologic cardiac stress7;8; here the triggering mechanism involved altered nuclear signaling, so these hibernation-like reversible changes may indeed be primary. Further studies are needed to clarify this hypothesis. Lastly, it remains possible that differences in the time-course and/or nuclear Cre-exposure between TAM and RAL plays some role, and the finding that lower prolonged dosing of TAM was also effective without myopathy might suggest this. However this could also reflect less nuclear exposure to TAM-MCM.
Our results have implications for existing and ongoing research with the MerCreMer model. Studies lacking Myh6-MCM+/−-flox(−) controls should be viewed cautiously, particularly if a significant cardiac phenotype is found within the 1–2 weeks after starting TAM. In such models, gene deletion without cardio-depression (e.g. using raloxifene or longer-term low dose TAM) is required. Both TAM and RAL dosing may need to be individualized depending upon the flox’d gene (perhaps related to gene accessibility and/or expression rate). For TAM, care to include MCM+ controls and provide sufficient recovery time is strongly advised. While RAL avoids the myopathy, the dose required was fairly high in the present flox’d models (lower doses might work for other models). However, mice treated mice with ~10× this dose for up to 3 months had no systemic limiting effects14. As RAL may have anti-hypertrophic effects when given chronically15, MCM+ controls are also advised.
Sources of Funding
Supported by NHLBI: HL-59480, HL-77180, and HL-98297 (DAK), AHA fellowship award (MZ), and AHA Scientist Development Grant (ET).