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Forced expression of the cell cycle inhibitor p57Kip2 in cardiomyocytes attenuates ischemia-reperfusion injury in the mouse heart.

Haley SA, Zhao T, Zou L, Klysik JE, Padbury JF, Kochilas LK - BMC Physiol. (2008)

Bottom Line: Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions.The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation.However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 +/- 15 vs 30 +/- 6 mmHg, p = 0.05), rate pressure product (22.8 +/- 4.86 vs 10.4 +/- 2.1 x 103bpm x mmHg, p < 0.05) and coronary flow (3.5 +/- 0.5 vs 2.38 +/- 0.24 ml/min, p <0.05).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Sheila_Haley@brown.edu

ABSTRACT

Background: Myocardial hypoxic-ischemic injury is the cause of significant morbidity and mortality worldwide. The cardiomyocyte response to hypoxic-ischemic injury is known to include changes in cell cycle regulators. The cyclin-dependent kinase inhibitor p57Kip2 is involved in cell cycle control, differentiation, stress signaling and apoptosis. In contrast to other cyclin-dependent kinase inhibitors, p57Kip2 expression diminishes during postnatal life and is reactivated in the adult heart under conditions of cardiac stress. Overexpression of p57Kip2 has been previously shown to prevent apoptotic cell death in vitro by inhibiting stress-activated kinases. Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions. To investigate this hypothesis, we created a transgenic mouse (R26loxpTA-p57k/+) that expresses p57Kip2 specifically in cardiac tissue under the ventricular cardiomyocyte promoter Mlc2v.

Results: Transgenic mice with cardiac specific overexpression of p57Kip2 are viable, fertile and normally active and their hearts are morphologically indistinguishable from the control hearts and have similar heart weight/body weight ratio. The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation. However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 +/- 15 vs 30 +/- 6 mmHg, p = 0.05), rate pressure product (22.8 +/- 4.86 vs 10.4 +/- 2.1 x 103bpm x mmHg, p < 0.05) and coronary flow (3.5 +/- 0.5 vs 2.38 +/- 0.24 ml/min, p <0.05).

Conclusion: These data suggest that forced cardiac expression of p57Kip2 does not affect myocardial growth, differentiation and baseline function but attenuates injury from ischemia-reperfusion in the adult mouse heart.

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Creation of a transgenic mouse with forced expression of p57Kip2 in a cre dependent manner. (A) Recombination scheme displaying the structure of the targeting vector, genomic R26 locus, targeted locus after homologous recombination and recombined locus after cre activation (B) PCR genotype analysis of tail genomic DNA (gDNA) (C) PCR analysis shows that cre-induced recombination is restricted to the heart, and occurs only in the presence of cre recombinase. T= tail gDNA, H = heart gDNA (D) RT-PCR analysis demonstrates robust expression of p57Kip2 message in adult transgenic hearts in the presence of cre recombinase, compared to barely detectable levels in the absence of cre recombinase.
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Figure 1: Creation of a transgenic mouse with forced expression of p57Kip2 in a cre dependent manner. (A) Recombination scheme displaying the structure of the targeting vector, genomic R26 locus, targeted locus after homologous recombination and recombined locus after cre activation (B) PCR genotype analysis of tail genomic DNA (gDNA) (C) PCR analysis shows that cre-induced recombination is restricted to the heart, and occurs only in the presence of cre recombinase. T= tail gDNA, H = heart gDNA (D) RT-PCR analysis demonstrates robust expression of p57Kip2 message in adult transgenic hearts in the presence of cre recombinase, compared to barely detectable levels in the absence of cre recombinase.

Mentions: Since the p57Kip2 cDNA is preceded by a loxP-flanked strong transcriptional termination sequence (tpA), in the absence of cre-recombinase p57Kip2 transcription is terminated prematurely and the generated transgenic mice (R26loxpTA-p57k/+) were phenotypically normal as expected. When these mice were crossed with the Mlc2v- Crek/+ transgenic mice that express cre-recombinase under the transcriptional control of the myosin light chain-2 ventricular (mlc2v) promoter, the cre-mediated excision of the floxed termination sequence led to forced expression of p57Kip2 in ventricular cardiomyocytes. Fifty-three double heterozygous animals (R26loxpTA-p57k/+/Mlc2v-Crek/+) from these crosses have been analyzed. The double transgenic mice developed normally and no defects in embryos or adults were observed. Litter sizes and fertility were similar to those of control mice and offspring were produced in the expected Mendelian ratios (Table 1). None of the R26loxpTA-p57k/+/Mlc2v-Crek/+ mice were prone to early lethality over 2 years of observation suggesting that cardiac specific overexpression of p57Kip2 is well tolerated from the very early stages of myocardial differentiation. Ventricular tissue-specific cre-recombination that allows p57Kip2 over-expression in cardiomyocytes of the compound heterozygous offspring could be detected by PCR as shown in the diagram (Figure 1A, 1C).


Forced expression of the cell cycle inhibitor p57Kip2 in cardiomyocytes attenuates ischemia-reperfusion injury in the mouse heart.

Haley SA, Zhao T, Zou L, Klysik JE, Padbury JF, Kochilas LK - BMC Physiol. (2008)

Creation of a transgenic mouse with forced expression of p57Kip2 in a cre dependent manner. (A) Recombination scheme displaying the structure of the targeting vector, genomic R26 locus, targeted locus after homologous recombination and recombined locus after cre activation (B) PCR genotype analysis of tail genomic DNA (gDNA) (C) PCR analysis shows that cre-induced recombination is restricted to the heart, and occurs only in the presence of cre recombinase. T= tail gDNA, H = heart gDNA (D) RT-PCR analysis demonstrates robust expression of p57Kip2 message in adult transgenic hearts in the presence of cre recombinase, compared to barely detectable levels in the absence of cre recombinase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268709&req=5

Figure 1: Creation of a transgenic mouse with forced expression of p57Kip2 in a cre dependent manner. (A) Recombination scheme displaying the structure of the targeting vector, genomic R26 locus, targeted locus after homologous recombination and recombined locus after cre activation (B) PCR genotype analysis of tail genomic DNA (gDNA) (C) PCR analysis shows that cre-induced recombination is restricted to the heart, and occurs only in the presence of cre recombinase. T= tail gDNA, H = heart gDNA (D) RT-PCR analysis demonstrates robust expression of p57Kip2 message in adult transgenic hearts in the presence of cre recombinase, compared to barely detectable levels in the absence of cre recombinase.
Mentions: Since the p57Kip2 cDNA is preceded by a loxP-flanked strong transcriptional termination sequence (tpA), in the absence of cre-recombinase p57Kip2 transcription is terminated prematurely and the generated transgenic mice (R26loxpTA-p57k/+) were phenotypically normal as expected. When these mice were crossed with the Mlc2v- Crek/+ transgenic mice that express cre-recombinase under the transcriptional control of the myosin light chain-2 ventricular (mlc2v) promoter, the cre-mediated excision of the floxed termination sequence led to forced expression of p57Kip2 in ventricular cardiomyocytes. Fifty-three double heterozygous animals (R26loxpTA-p57k/+/Mlc2v-Crek/+) from these crosses have been analyzed. The double transgenic mice developed normally and no defects in embryos or adults were observed. Litter sizes and fertility were similar to those of control mice and offspring were produced in the expected Mendelian ratios (Table 1). None of the R26loxpTA-p57k/+/Mlc2v-Crek/+ mice were prone to early lethality over 2 years of observation suggesting that cardiac specific overexpression of p57Kip2 is well tolerated from the very early stages of myocardial differentiation. Ventricular tissue-specific cre-recombination that allows p57Kip2 over-expression in cardiomyocytes of the compound heterozygous offspring could be detected by PCR as shown in the diagram (Figure 1A, 1C).

Bottom Line: Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions.The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation.However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 +/- 15 vs 30 +/- 6 mmHg, p = 0.05), rate pressure product (22.8 +/- 4.86 vs 10.4 +/- 2.1 x 103bpm x mmHg, p < 0.05) and coronary flow (3.5 +/- 0.5 vs 2.38 +/- 0.24 ml/min, p <0.05).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Sheila_Haley@brown.edu

ABSTRACT

Background: Myocardial hypoxic-ischemic injury is the cause of significant morbidity and mortality worldwide. The cardiomyocyte response to hypoxic-ischemic injury is known to include changes in cell cycle regulators. The cyclin-dependent kinase inhibitor p57Kip2 is involved in cell cycle control, differentiation, stress signaling and apoptosis. In contrast to other cyclin-dependent kinase inhibitors, p57Kip2 expression diminishes during postnatal life and is reactivated in the adult heart under conditions of cardiac stress. Overexpression of p57Kip2 has been previously shown to prevent apoptotic cell death in vitro by inhibiting stress-activated kinases. Therefore, we hypothesized that p57Kip2 has a protective role in cardiomyocytes under hypoxic conditions. To investigate this hypothesis, we created a transgenic mouse (R26loxpTA-p57k/+) that expresses p57Kip2 specifically in cardiac tissue under the ventricular cardiomyocyte promoter Mlc2v.

Results: Transgenic mice with cardiac specific overexpression of p57Kip2 are viable, fertile and normally active and their hearts are morphologically indistinguishable from the control hearts and have similar heart weight/body weight ratio. The baseline functional parameters, including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), LVdp/dtmax, heart rate (HR) and rate pressure product (RPR) were not significantly different between the different groups as assessed by the Langendorff perfused heart preparation. However, after subjecting the heart ex vivo to 30 minutes of ischemia-reperfusion injury, the p57Kip2 overexpressing hearts demonstrated preserved cardiac function compared to control mice with higher left ventricular developed pressure (63 +/- 15 vs 30 +/- 6 mmHg, p = 0.05), rate pressure product (22.8 +/- 4.86 vs 10.4 +/- 2.1 x 103bpm x mmHg, p < 0.05) and coronary flow (3.5 +/- 0.5 vs 2.38 +/- 0.24 ml/min, p <0.05).

Conclusion: These data suggest that forced cardiac expression of p57Kip2 does not affect myocardial growth, differentiation and baseline function but attenuates injury from ischemia-reperfusion in the adult mouse heart.

Show MeSH
Related in: MedlinePlus