Deficiency of PDK1 in cardiac muscle results in heart failure and increased sensitivity to hypoxia

Abstract

We employed Cre/loxP technology to generate mPDK1(-/-) mice, which lack PDK1 in cardiac muscle. Insulin did not activate PKB and S6K, nor did it stimulate 6-phosphofructo-2-kinase and production of fructose 2,6-bisphosphate, in the hearts of mPDK1(-/-) mice, consistent with PDK1 mediating these processes. All mPDK1(-/-) mice died suddenly between 5 and 11 weeks of age. The mPDK1(-/-) animals had thinner ventricular walls, enlarged atria and right ventricles. Moreover, mPDK1(-/-) muscle mass was markedly reduced due to a reduction in cardiomyocyte volume rather than cardiomyocyte cell number, and markers of heart failure were elevated. These results suggested mPDK1(-/-) mice died of heart failure, a conclusion supported by echocardiographic analysis. By employing a single-cell assay we found that cardiomyocytes from mPDK1(-/-) mice are markedly more sensitive to hypoxia. These results establish that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure. They also suggest that a deficiency of the PDK1 pathway might contribute to development of cardiac disease in humans.

Fig. 1. Generation and survival of mice lacking PDK1 in skeletal muscle and heart. (A) Diagram illustrating the positions of exons 2–5 and the loxP Cre excision sites of the floxed PDK1 gene. The positions of the PCR primers used to genotype mice described in the Materials and methods are indicated with arrows. (PDK1^{flΔneo/flΔneo}), allele with the loxP sites flanking exons 3 and 4 without the neomycin resistance cassette; (PDK1^–/–), the allele in which exon 3 and 4 have been removed by Cre recombinase resulting in the ablation of the expression of PDK1 beyond exon 2, which includes the kinase and pleckstrin homology domain (Williams et al., 2000). (B) Breeding strategy used for the generation of mice lacking PDK1 in skeletal muscle and heart, where MckCre denotes transgenic mice expressing the Cre recombinase under the muscular creatine kinase promoter. Note that throughout this study, PDK1^{flΔneo/flΔneo}MckCre^+/– mice are termed mPDK1^–/– and PDK1^{flΔneo/flΔneo}MckCre^–/– are termed mPDK1^+/+. (C) PDK1 was affinity purified from the indicated tissues using PIF–Sepharose (as described in the Materials and methods), electrophoresed on a 10% SDS–polyacrylamide gel and immunoblotted with anti-PDK1 antibody. It should be noted that as observed in previous studies (Lawlor et al., 2002; Collins et al., 2003), PDK1 migrates as a doublet, although the reason for this is not known. Abbreviations used are (Qua), quadriceps; (Gastr), gastrocnemius; (Ext), extensor digitalis; and (Dia), diaphragm. Similar results were obtained in three separate experiments using different mice of 5–6 weeks of age. Direct immunoblotting of cell lysates with PDK1 antibody revealed identical findings except that PDK1 immuno-reactive bands were of lower intensity (data not shown). (D) The indicated number of male and female mice were maintained under standard husbandry conditions and the percentage of surviving mice of each age is indicated. (n) denotes the number of each genotype.

Fig. 2. Histological analysis of hearts from mPDK1^+/+ and mPDK1^+/+ mice. At the indicated times the hearts were fixed in 10% formalin, embedded in wax and stained with haematoxilin and eosin. (A, D, G and J) Comparison of a representative image of heart of littermate mPDK1^–/– and mPDK1^+/+ mice of the same sex, before the fixation and staining. (B, E, H and K) Representative longitudinal sections after fixation and staining. (C, F, I and L) Representative micrographs of transversal sections of the muscular fibres. Scale bar is shown at the bottom of each set of panels.

Fig. 3. Echocardiographic analysis of mPDK1^–/– hearts. (A) Repre sentative transversal section of 6-week-old mPDK1^–/– and mPDK1^+/+ mouse heart fixed in 10% formalin, embedded in wax and stained with haematoxylin and eosin. rv, right ventricle; lv, left ventricle. (B) Echocardiographic analysis of 5- to 6-week-old mPDK1^+/+ and mPDK1^–/– mice. LVEDD, left ventricle end diastolic dimensions; LVESD, left ventricle end systolic dimensions; LVareaED, left ventricle area end diastolic; LvareaSD, left ventricle area end systolic; PWD, posterior wall diastolic thickness; PWS, posterior wall systolic thickness; FS, fractional shortening; EF, ejection fraction; BW, body weight; LVEDD/BW, left ventricular end-diastolic dimension corrected for body weight. Data are presented as the mean ± SEM and were compared by Student’s t-test, n = 11 for mPDK^+/+ and 9 for mPDK^–/–.

Fig. 4. Quantitative analysis of muscle mass, cardiomyocyte volume and number. (A) The muscle mass of mPDK1^+/+ and mPDK1^–/– hearts of the indicated ages or after death. The results shown were determined by the Cavalieri method and presented as the average volume ± SD of the results obtained from analysing three separate hearts of each age and genotype. The cardiomyocyte volume (B) and cardiomyocyte number (C) were determined using the dissector principle. The cardiomyocyte number was determined by calculating the nuclei number, assuming one nuclei per cardiomyocyte. The data are presented as the mean ± SD of three separate hearts of each age and genotype.

Fig. 5. Analysis of markers of heart failure. (A) Histological sections of mPDK1^+/+ and mPDK1^–/– hearts of the indicated ages or after death were stained with Picric-Sirius Red dye, which stains collagen in red. The amount of collagen present in these sections was quantified using the Cavalieri method. The data are presented as the mean ± SD of three separate hearts of each age and genotype. Representative micrographs of Picric-Sirius Red-stained 6-week-old mPDK1^–/– and mPDK1^+/+ heart sections are shown. (B) RNA was isolated from mPDK1^–/– and mPDK1^+/+ hearts of 6 weeks of age, and RT–PCR analysis, described in the materials and methods, was employed to assess the mRNA expression levels of atrial natriuretic factor (ANF), β-myosin heavy chain (β-MHC) and 18S ribosomal RNA (18S) as a control. Each lane on the agarose gel represents a different mouse. (C) Cell extracts were prepared from mPDK1^–/– and mPDK1^+/+ hearts of 6 weeks of age and immunoblotted with the indicated antibodies. Each lane on the immunoblot represents a different mouse. (D) Electron microscope sections of mPDK1^+/+ and mPDK1^–/– hearts of the indicated ages. The Z-line thickness was quantitated by counting 135 randomly derived Z-lines from three hearts of each genotype and age. The data are presented as average thickness ± SD.

Fig. 6. Activation of PKB, S6K and PFK2 in heart by insulin. Mice were fasted overnight and injected with either saline for 10–20 min (for the 0 time point control) or 1 mU/g of insulin for the indicated times. The heart was then rapidly extracted and frozen in liquid nitrogen. PKB (A) or S6K (B) were immunoprecipitated from cardiac extract and the activity determined using a quantitative peptide phosphorylation assay. Each point represents the mean activity ± SD of three different hearts with each assayed in triplicate. (C and D) As above except that cell lysates form the indicated hearts were immunoblotted with the indicated antibodies. Each lane represents a different mouse PFK-2 activity (E) and fructose-2,6-bisphosphate (Fru-2,6-P2) levels (F) were measured as described in the Materials and methods. Each point represents the mean activity ± SD of three to five different hearts with each assayed in duplicate.

Fig. 7. Increased sensitivity of isolated mPDK1^–/– cardiomyocytes to hypoxia. Cardiomyocytes were isolated from hearts of 4-week-old mice and processed as described in the materials and methods (A–C) Average values of membrane potential (A), ([Ca²⁺]_i) (B) and diameter ratio (C) of mPDK1^–/– and mPDK1^+/+ cardiomyocytes. The results shown represent mean ± SEM with measurements performed with three to eight cells of each genotype. (D) Field stimulated beating mPDK1^–/– and mPDK1^+/+ cardiomyocytes were loaded with Fura 2-dye and imaged using a epifluorescent camera. At time zero, to induce hypoxia, the cells were subjected to an environment of pO₂ 20 mmHg. Images of the cardiomyocytes were taken at the indicated time points, and cell death is observed as a rounding up of the cardiomyocyte. (E and F) Depict time-courses of ([Ca²⁺]_i (E) and diameter ratio (F) from cells in (D). Arrows indicate time of cardiomyocyte death. (G) The average survival time ± SEM of five to seven different mPDK1^–/– and mPDK1^+/+ cardiomyocytes subjected to hypoxic conditions was quantitated. *P < 0.05. (H and I) Average values of [Ca²⁺]_i (H) and diameter ratio (I) of mPDK1^+/+ cardiomyocytes in extracellular solution without Ca²⁺ (Ca²⁺ free) or pretreated with thapsigargin plus ryanodine (Th+Ryan) prior (0 min) and after 45 min-long exposure to hypoxia. The results shown represent mean ± SEM with measurements performed with four cells under each condition.