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. 2014 Feb;306(3):H373-81.
doi: 10.1152/ajpheart.00411.2013. Epub 2013 Nov 22.

Normal cardiac function in mice with supraphysiological cardiac creatine levels

Lucia Santacruz  1 , Alejandro Hernandez, Jeffrey Nienaber, Rajashree Mishra, Miguel Pinilla, James Burchette, Lan Mao, Howard A Rockman, Danny O Jacobs
Affiliations

Normal cardiac function in mice with supraphysiological cardiac creatine levels

Lucia Santacruz et al. Am J Physiol Heart Circ Physiol. 2014 Feb.

Abstract

Creatine and phosphocreatine levels are decreased in heart failure, and reductions in myocellular phosphocreatine levels predict the severity of the disease and portend adverse outcomes. Previous studies of transgenic mouse models with increased creatine content higher than two times baseline showed the development of heart failure and shortened lifespan. Given phosphocreatine's role in buffering ATP content, we tested the hypothesis whether elevated cardiac creatine content would alter cardiac function under normal physiological conditions. Here, we report the creation of transgenic mice that overexpress the human creatine transporter (CrT) in cardiac muscle under the control of the α-myosin heavy chain promoter. Cardiac transgene expression was quantified by qRT-PCR, and human CrT protein expression was documented on Western blots and immunohistochemistry using a specific anti-CrT antibody. High-energy phosphate metabolites and cardiac function were measured in transgenic animals and compared with age-matched, wild-type controls. Adult transgenic animals showed increases of 5.7- and 4.7-fold in the content of creatine and free ADP, respectively. Phosphocreatine and ATP levels were two times as high in young transgenic animals but declined to control levels by the time the animals reached 8 wk of age. Transgenic mice appeared to be healthy and had normal life spans. Cardiac morphometry, conscious echocardiography, and pressure-volume loop studies demonstrated mild hypertrophy but normal function. Based on our characterization of the human CrT protein expression, creatine and phosphocreatine content, and cardiac morphometry and function, these transgenic mice provide an in vivo model for examining the therapeutic value of elevated creatine content for cardiac pathologies.

Keywords: cardiac failure; creatine transporter; energy metabolism; phosphocreatins.

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Figures

Fig. 1.
Fig. 1.
Human creatine transporter (hCrT) is expressed in creatine transporter transgenic (CrT-Tg) cardiac muscle. A: Western blot of cardiac tissue homogenates from adult transgenic or wild-type (WT) mice. In CrT-Tg homogenates, a band of ∼55 kDa corresponding to the hCrT protein was detected using a specific rat monoclonal antibody against the human CrT protein isoform. B: cardiac ventricle tissue sections from 8-wk-old CrT-Tg animals or WT age-matched controls were prepared for histological analysis as described in methods. Staining was observed only in CrT-Tg animals (bottom), and localized to the cell surface in a striated pattern.
Fig. 2.
Fig. 2.
High-energy metabolite analysis. Hearts from control or CrT-Tg mice were harvested, and the content of creatine (Cr), phosphocreatine (PCr), ATP, and free ADP was determined by HPLC as described in methods. The number within each bar indicates the number of hearts analyzed. A: CrT-Tg mice hearts had significantly higher Cr content compared with WT mice. B: PCr levels were significantly increased in CrT-Tg levels in 2- and 4-wk-old animals (2.3- to 2.5-fold vs. WT controls). By 8 wk of age, the content of PCr in CrT-Tg mice was the same as WT controls. C: ATP levels were significantly elevated in CrT-Tg at 2 and 4 wk of age but fell below those measured in WT animals in 8-wk-old CrT-Tg hearts. D: total ADP content was significantly elevated in 8-wk-old WT animals. Two- and 4-wk-old animals had similar ADP content, irrespective of transgene expression. E: free ADP content (calculated as described in methods) was elevated in CrT-Tg animals at all time points. F: the PCr-to-ATP ratio was significantly elevated in CrT-Tg animals at 4 and 8 wk of age. G: CrT-Tg animals had significantly greater PCr-to-Cr ratios than age-matched WT controls. H: the ATP-to-ADP ratio (calculated using the measured total ADP content) was significantly higher in 2- and 4-wk-old CrT-Tg animals compared with WT animals. This difference was not observed in 8-wk-old animals. *P < 0.05 vs. WT, age-matched control, 2-way ANOVA, Fisher's least significant difference (LSD). #P < 0.05 vs. same line at 2 wk, 2-way ANOVA, Fisher's LSD.
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