Systolic dysfunction in cardiac-specific ligand-inducible MerCreMer transgenic mice - PubMed (original) (raw)

Systolic dysfunction in cardiac-specific ligand-inducible MerCreMer transgenic mice

Michael E Hall et al. Am J Physiol Heart Circ Physiol. 2011 Jul.

Abstract

The Cre-loxP system is a useful tool to study the physiological effects of gene knockout in the heart. One limitation with using this system in the heart is the toxic effect of chronic expression of the Cre recombinase. To circumvent this limitation, a widely used inducible cardiac-specific model, Myh6-MerCreMer (Cre), using tamoxifen (TAM) to activate Cre has been developed. The current study examined cardiac function in Cre-positive C57B/J6 mice exposed to one, three, or five daily doses of a 40 mg/kg TAM to induce Cre activity specifically in the heart. Echocardiography demonstrated no statistically significant differences in systolic function (SF) at baseline as assessed by fractional shortening. In mice exposed to five injections, a significant fall in all determinants of SF was observed 6 days after TAM was initiated. However, SF returned to baseline levels 10 days after TAM initiation although the hearts exhibited significant hypertrophy. Heart weight-to-tibia length ratios were 73 ± 3, 78.5 ± 6, and 87.6 ± 9 mg/cm for one, three, and five TAM injections, respectively. TAM had no effect on cardiac function or hypertrophy in Cre-negative mice. Cre-positive mice receiving five TAM injections had significant reductions in cardiac mitochondrial ATP and significant reductions in the expression of proteins important for the regulation of cardiac oxidative phosphorylation including peroxisome proliferator-activated receptor-γ coactivator-1α and pyruvate dehydrogenase kinase-4. Thus inducible cardiac-specific activation of Cre recombinase caused a transient decline in SF that was dependent on the number of TAM doses and associated with significant hypertrophy and alterations in mitochondrial ATP and important proteins involved in the regulation of cardiac oxidative phosphorylation.

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Figures

Fig. 1.

Fig. 1.

Systolic function as represented by fractional shortening following in 1-, 3-, or 5-day tamoxifen (TAM) treatment in animals expressing _Myh6_-MerCreMer (Cre+) or non-Cre (Cre−) mice. Day 0 is the average of the 2 baseline measurements and days 2, 4, 6, 8, and 10 represent days following the start of TAM administration. *P < 0.05 vs. Cre−/5-day mice.

Fig. 2.

Fig. 2.

Systolic and diastolic left ventricular dimensions for wall thickness and diameter obtained from M-mode measurements of echocardiography following in 1-, 3-, or 5-day tamoxifen treatment in animals expressing myosin heavy chain-MerCreMer or non-Cre controls. A: left ventricular anterior wall systole (LVAWs). B: left ventricular posterior wall systole (LVPWs). C: left ventricular anterior wall diastole (LVAWd). D: left ventricular posterior wall diastole (LVPWd). E: left ventricular end systolic diameter (LVESD). F: left ventricular end diastolic diameter (LVEDD). *P < 0.05 Cre+/5-day vs. Cre−/5-day mice. †P < 0.05 Cre+/3-day vs. Cre−/5-day mice.

Fig. 3.

Fig. 3.

Cardiac mitochondrial ATP levels determined in Cre−/5-day (n = 6) and Cre+/5-day (n = 8) mice at systolic dysfunction nadir (day 6) and at systolic function recovery (day 14). *P < 0.05, compared with Cre−/5-day mice.

Fig. 4.

Fig. 4.

Cardiac phosphorylated (p)-AMPK/total (t)AMPK protein levels in Cre−/5-day (n = 4) and Cre+/5-day (n = 4) mice measured by Western blot analysis. *P < 0.05, compared with Cre−/5-day, day 6. †P < 0.05, compared with Cre+/5-day, day 6. Bars represent breaks in original image separating individual groups that were run in quadruplicate.

Fig. 5.

Fig. 5.

Cardiac peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1-α) protein levels in Cre−/5-day (n = 4) and Cre+/5-day (n = 4) mice measured by Western blot analysis. *P < 0.05, compared with Cre−/5-day mice. Bars represent breaks in original image separating individual groups that were run in quadruplicate.

Fig. 6.

Fig. 6.

Cardiac mitochondrial pyruvate dehydrogenase kinase-4 (PDK-4) protein levels in Cre−/5-day (n = 4) and Cre+/5-day (n = 4) mice measured by Western blot analysis. *P < 0.05, compared with Cre−/5-day, day 6. Bars represent breaks in original image separating individual groups that were run in quadruplicate.

Fig. 7.

Fig. 7.

Cardiac β-tubulin protein levels in Cre−/5-day (n = 4) and Cre+/5-day (n = 4) mice measured by Western blot analysis. P < 0.05, compared with Cre−/5-day, day 6. Bars represent breaks in original image separating individual groups that were run in quadruplicate.

Fig. 8.

Fig. 8.

Cardiac histology from Cre+/5-day and Cre−/5-day mice at day 6 and day 10 post-TAM treatment. A, D, and G: hemotoxylin and eosin (H&E) staining at ×200 in untreated Cre− (A); Cre+/5-day, day 6 (D); and Cre+/5-day, day 10 (G). B, E, and H: periodic acid-Schiff (PAS) staining at ×200 in untreated Cre− (B); Cre+/5-day, day 6 (E); and Cre+/5-day, day 10 (H). C, F, and I: picrosirius red (PSR) staining at ×200 in untreated Cre− (C); Cre+/5-day, day 6 (F); and Cre+/5-day, day 10 (I). H&E staining reveals inflammatory cells seen on day 6 (‡) and condensed nuclear chromatin noted at Systolic function recovery (*). At day 10, intracellular staining on PAS (#) and appearance of increase collagen on PSR (†).

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