Decreased expression level of apoptosis-related genes and/or proteins in skeletal muscles,but not in hearts,of growth hormone receptor knockout mice

Abstract

The long-lived growth hormone (GH) receptor knockout (GHRKO; KO) mice are GH-resistant due to targeted disruption of the GH receptor (Ghr) gene. Apoptosis is a physiological process in which cells play an active role in their own death and is a normal component of the development and health of multicellular organisms. Aging is associated with the progressive loss of strength of skeletal and heart muscles. Calorie restriction (CR) is a well-known experimental model to delay aging and increase lifespan. The aim of the study was to examine the expression of the following apoptosis-related genes: caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, p53 and cytochrome c1 (cyc1) in the skeletal muscles and hearts of female normal and GHRKO mice, fed ad libitum or subjected to 40% CR for six months, starting at two months of age. Moreover, skeletal muscle caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, Apaf-1, bad, phospho-bad (pbad), phospho-p53 and cytochrome c (cyc) protein expression levels were assessed. Expression of caspase-3, caspase-9, bax and Smac/DIABLO genes and proteins was decreased in GHRKO's skeletal muscles. The Apaf-1 protein expression also was diminished in this tissue. In contrast, bcl-2 and pbad protein levels were increased in skeletal muscles in knockouts. No changes were demonstrated for the examined genes' expression in GHRKO's hearts except for the increased level of cyc1 mRNA. CR did not alter the expression of the examined genes and proteins in skeletal muscles of knockouts versus normal (N) mice. In heart homogenates, CR increased caspase-3 mRNA level as compared with ad libitum mice. Decreased expression of certain proapoptotic genes and/or proteins may constitute the potential mechanism of prolonged longevity in GHRKO mice, protecting these animals from aging; this potential beneficial mechanism is not affected by CR.

Keywords

apoptosis GHRKO mice skeletal muscles heart caspase calorie restriction

Introduction

Altered somatotrophic and insulin signaling pathways can extend longevity. One of the genetic interventions that leads to prolonged longevity in mice is targeted disruption of the fourth exon of the growth hormone (GH) receptor/GH binding protein (Ghr/bp) gene.¹ Mice homozygous for this mutation are known as GH receptor/binding protein knockout mice (GHRKO; Ghr/bp –/–) or ‘Laron dwarf’.¹ These GH-resistant mice live approximately 40% longer than their normal siblings,^2–5 with respect to their average as well as maximal lifespan. GHRKO mice are characterized by a reduced weight and body size, with undetectable levels of GH receptor, high concentrations of serum GH, greatly reduced plasma levels of IGF-I and insulin, and low or normal glucose.^1–3,5–7 The mice in question also have enhanced insulin sensitivity.⁸ Additionally, GHRKO mice show improved oxidative stress resistance, reduced generation of reactive oxygen species and reduced oxidative damage.^4,9,10 These animals also have lower incidence and delayed onset of fatal neoplastic diseases.¹¹

Among many processes that may affect longevity, apoptosis seems to be one of the most important. Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms, playing an essential role in many physiological processes, including nervous system development^12,13 and the embryonic growth of tissues and organs.¹⁴ It is a process in which cells play an active role in their own death (‘cell suicide’) and it is the most common form of eukaryotic cell death. Inappropriate regulation of apoptosis can lead to many pathological conditions and diseases, such as neurodegenerative disorders, cancer and autoimmune diseases.¹⁵ Numerous substances and factors (e.g. free radicals, viruses, bacteria, drugs, ionizing radiation, hormones) can induce apoptosis.

There are two central apoptotic signaling pathways: intrinsic (mitochondrial) and extrinsic. The intrinsic pathway (involving p53, bax, cytochrome c and caspase-9, among others) is initiated by different factors within the cell, such as free radicals, DNA damage, ionizing radiation, chemotherapeutic drugs or hypoxia.^16–18 In turn, the extrinsic (death receptor) pathway (involving, among others, caspase-8) is responsible for the elimination of unnecessary cells during development and immune system education.¹⁷ Although the beneficial role of apoptosis leading to removal of abnormal cells is well known, the entire role of apoptosis in (patho)physiology and potentially in regulation of longevity is still unclear.

It is known that aging is associated with the progressive loss of strength of skeletal and cardiac muscles. Interestingly, analysis of mRNA levels of antiapoptotic IGF-I has indicated that there are differences between skeletal muscles (the decrease of this expression) and hearts (unchanged expression) in GHRKO mice as compared with normal animals.^19,20

In this context, we decided to examine the process of apoptosis (analysing the three caspases [being a subclass of cysteine proteases]: caspase-3, caspase-9 and caspase-8 as well as bax, bcl-2, Smac/DIABLO, p53 and cytochrome c1 [cyc1] mRNA expression) in the skeletal muscles and hearts of GHRKO mice. Moreover, we assessed the protein expression level of caspase-3, caspase-9, caspase-8, bax, bcl-2, Smac/DIABLO, Apaf-1, bad, phospho-bad (pbad), phospho-p53 (pp53) and cytochrome c (cyc) in the skeletal muscle of these long-lived animals.

It is worth recalling that caspase-3 is a main effector (executioner) caspase, responsible for destroying cells and inducing apoptosis; caspase-9 activates the effector caspase-3 (and also caspase-6 and -7) whereas bax (bcl-2-associated X protein) is one of the main proapoptotic bcl-2 family proteins.²¹ Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) is a mitochondrial protein that, after being released from mitochondria into the cytosol, potentiates apoptosis, possibly by neutralizing the IAP (inhibitors of apoptosis proteins) family members and disrupting their ability to inactivate the caspase enzymes.^22,23 Caspase-8 is an initiator caspase, indispensable for induction of the extrinsic apoptotic signaling pathway mediated through death receptors.²⁴ Bcl-2 is one of main antiapoptotic bcl-2 family proteins,²¹ which acts to prevent permeabilization of the outer mitochondrial membrane by inhibiting the action of the proapoptotic proteins Bax and/or Bak.²⁵ Bad is another proapoptotic protein. The disruption of the ratio between pro- and antiapoptotic proteins leads to cytochrome c release through pores in the mitochondrial membrane into the cytosol. The cytochrome c forms a multiprotein complex with procaspase-9 and Apaf-1 (apoptotic protease activating factor-1), called the apoptosome.^26,27 Formation of the apoptosome leads to activation of caspase-9 and caspase cascade and finally to activation of effector caspase-3. p53 protein is a well-known tumor suppressor as well as a very important factor which activates the intrinsic apoptotic pathway.

Calorie restriction (CR) is a well-known experimental model to delay aging and increase lifespan.²⁸ The characteristics of genetically normal mice subjected to CR resemble many phenotypic characteristics of GHRKO mice (reduced body size, reduced plasma IGF-1 and insulin levels, enhanced sensitivity to insulin). Similarly, longevity of normal mice subjected to CR resembles that of GHRKO mice fed ad libitum.²⁹ As yet, it is still not clear which mechanisms involved in CR and also related to Ghr/bp gene disruption are responsible for extended longevity. However, 30% CR initiated at two months of age extends longevity in normal but not in GHRKO mice, suggesting that mechanisms linking CR and GH resistance to aging must overlap.²⁹ Furthermore, it is worth emphasizing that effects of CR on skeletal muscle expression of certain genes involved in insulin action are opposite to those analyzed in the heart.^19,20 This is why we decided to analyze the effect of CR on the process of apoptosis in both of these tissues.

Materials and methods

Animals and CR

The normal and GHRKO mice used in the present study were produced in our breeding colony, developed using animals kindly provided by Dr J J Kopchick (Ohio University). Animals were produced by mating knockout (−/−) males with heterozygous (+/−) females. Normal (+/−) and GHRKO animals (−/−) were separated by phenotypic characteristics. All animal procedures were approved by the Laboratory Animal Care and Use Committee (LACUC) at the Southern Illinois University School of Medicine (Springfield, IL, USA). The mice were housed under temperature- and light-controlled conditions (22 ± 2°C, 12 h light/12 h dark cycle) and fed Lab Diet 5001 chow (PMI Nutrition International, Richmond, IN, USA) containing, among others, 4.5% fat and 23.4% protein. Starting at two months of age, 46 normal and GHRKO female mice were grouped according to average body weight within the phenotype, and divided into four experimental groups: normal-fed ad libitum (N-AL; 10 animals), normal-CR (N-CR; 14 animals), GHRKO ad libitum (KO-AL; 10 animals) and GHRKO-CR (KO-CR; 12 animals). Food-restricted animals were subjected to gradually introduced 40% CR (which corresponded to 60% of the food consumed by their AL counterparts). Water was available at all times to all animals. At eight months of age, the animals were fasted overnight, anesthetized using isoflurane, bled by cardiac puncture and euthanized by decapitation. Hind-limb skeletal muscles and hearts were rapidly collected, starting at 08:00, quickly frozen on dry ice and stored at −80°C until processed.

RNA extraction and complementary DNA transcription

The skeletal muscles and hearts were pulverized in liquid nitrogen. The RNA was extracted from the homogenates of the examined tissues using guanidinium thiocyanate–phenol–chloroform method based on Chomczynski–Sacchi procedure.³⁰ RNA quantity and quality were analyzed on 1.5% agarose gel using electrophoresis. Potentially contaminating residual genomic DNA was eliminated using deoxyribonuclease I (Promega, Madison, WI, USA). Reverse transcription was performed and complementary DNA (cDNA) was synthesized using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) in accordance with manufacturer's instruction.

Real-time polymerase chain reaction

Real-time polymerase chain reaction (RT–PCR) was carried out using the Smart Cycler instrument (Cepheid, Sunnyvale, CA, USA) with iQ SYBR Green Supermix (Bio-Rad Laboratories). The three steps of the PCR included: denaturation at 94°C for two minutes, annealing at 62°C for 30 s with fluorescence reading and extension at 72°C for 30 s. In addition, a melting curve was done for each reaction to evaluate the potential of non-specific products. β ₂-microglobulin (B2M) was used as a housekeeping gene, which was previously validated in our laboratory as the most appropriate gene for normalizing the data.²⁰ Gene expression was assessed by measurement of steady-state levels of mRNA. Relative expression from RT–PCR was calculated from the equation 2^A−B/2^C−D (where A = cycle threshold [Ct] number for the gene of interest in the first control sample; B = Ct number for the gene of interest in the analyzed sample; C = Ct number for the housekeeping gene in the first control sample; D = Ct number for the housekeeping gene in the analyzed sample). The first control was expressed as 1.00 by this equation, and all other samples were calculated in relation to this value. Then, the results in the control group (N-AL) were averaged, and all other outputs were divided by the mean value of the relative expression in the control group to yield the fold change of the expression of genes of interest compared with the control group.

For RT–PCR the following primers were used [gene bank sequence number by each forward primer]: B2M: forward [NM_009735]: 5′-aagtatactcacgccaccca, backward: 5′-aagaccagtccttgctgaag; caspase-3: forward [NM_009810]: 5′-tgcagcatgctgaagctgta, backward: 5′-gagcatggacacaatacacg; caspase-9: forward [NM_015733]: 5′-agcagagagtagtgaagctg, backward: 5′-acacagacatcatgagctcc; caspase-8: forward [AJ007749]: 5′-accgagatcctgtgaatgga, backward: 5′-tgctttcccttgttcctcct; bax: forward [NM_007527]: 5′-ccaccagctctgaacagatc, backward: 5′-cagcttcttggtggacgcat; bcl-2: forward [NM_009741]: 5′-tgggatgcctttgtggaact, backward: 5′-gagacagccaggagaaatca; Smac/DIABLO: forward: [NM_023232] 5′-aagagctgcaccagaaagca, backward: 5′-tctgactgtcaatggcagga; p53: forward [AF151353]: 5′-tcacagtcggatatcagcct, backward: 5′-acactcggagggcttcactt; cyc1: forward [NM_025567]: 5′-tacaagcaggtgtgctcttc, backward: 5′-atcattagggccatcctgga.

Protein extraction and Western blotting

Total proteins were obtained from tissue homogenates. Approximately 100 mg skeletal muscles samples were homogenized in 1 mL ice-cold T-PER Tissue Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL,USA), with Protease Inhibitor Cocktail Kit (Pierce Biotechnology), Phosphatase Inhibitor Cocktail 1 (Sigma-Aldrich, St Louis, MO, USA) and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich). After mixing, homogenates were centrifuged at 16,000 rpm for 30 min. Protein concentrations were assessed using Pierce BCA (bicinchoninic acid) Protein Assay Kit (Pierce Biotechnology) in accordance with the manufacturer's protocol.

Western blot procedure was performed using the following primary antibodies: caspase-3, caspase-9 (mouse specific), bax, caspase-8 (mouse specific), bcl-2, bad, phospho-bad (Ser112), phospho-p53 (Ser15), Apaf-1, cyc (all from Cell Signaling Technology, Beverly, MA, USA), Smac/DIABLO (BioVision, Inc, Mountain View, CA, USA), and secondary goat anti-rabbit or goat anti-mouse antibodies (Calbiochem, La Jolla, CA, USA). Monoclonal anti-β-actin antibody (Sigma-Aldrich) was used, after stripping the membrane, as a control for protein loading.

For Western blotting, protein extracts were mixed with XT Sample Buffer (Bio-Rad Laboratories) and heated in a thermocycler at 99°C for five minutes and then cooled to 4°C. Forty micrograms of the protein was separated electrophoretically using Criterion XT Precast Gel (26 wells; Bio-Rad Laboratories) for 90 min at 150 V. Subsequently, proteins were wet-transferred for 60 min at 100 V onto nitrocellulose membranes (Bio-Rad Laboratories) at 4°C. After the transfer, membranes were rinsed briefly in Tris-buffered saline (TBS; pH 7.6) and blocked with 5% non-fat dry milk or 1% bovine serum albumin in TBS containing 0.05% Tween 20 (TBST) for one hour at room temperature. After blocking, membranes were washed with TBST three times for 15 min each time. Then, the membranes were incubated with the primary antibody specific for the protein of interest, and diluted in the appropriate blocking solution at 4°C overnight with shaking. After incubation, the blots were washed three times (15 min each) with TBST and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody for one hour at room temperature. Horseradish peroxidase activity from secondary antibody was detected using the Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare UK Limited, Little Chalfont, Buckinghamshire, UK). A minimum of six animals per group was analyzed. Photos of blots were taken with Image Reader LAS-4000 (FujiFilm, Tokyo, Japan) and quantified for statistical analysis using Multi Gauge version 3.0 software (FujiFilm Life Science, Tokyo, Japan). The protein level was expressed as the arbitrary unit (AU)/mm². The AU is a unit to measure the emission amount of chemiluminescence material read using LAS. It represents the relative density value accumulated as linear data by a charge-coupled device camera in the image surface.

Statistical analysis

The data are expressed as mean ± standard error of the mean (SEM). To evaluate the effects of the genotype and diet, two-way analysis of variance (ANOVA) was used. A t-test was used to evaluate the effects of diet within genotypes and genotypes within diets. A value of P < 0.05 was considered significant. All statistical calculations were conducted using SPSS version 17.0 (SPSS, Chicago, IL, USA) with α = 0.05. All graphs were made using Prism 4.02 (GraphPad Software, San Diego, CA, USA).

Results

Expression of the four apoptotic genes (caspase-3 [Figure 1a], caspase-9 [Figure 2a], bax [Figure 3a], Smac/DIABLO [Figure 4a]) was decreased in skeletal muscles of GHRKO (KO) mice as compared with normal (N) mice (P = 0.02, P = 0.006, P = 0.035, P = 0.036, respectively). No significant differences were observed for caspase-8 (Figure 5a), bcl-2 (Figure 6a), p53 (Figure 7a) and cyc1 (Figure 8a) mRNA expression in homogenates of the above-mentioned tissue in KO mice in comparison to N mice. In contrast, expression of the seven examined genes (caspase-3 [Figure 1b], caspase-9 [Figure 2b], bax [Figure 3b], Smac/DIABLO [Figure 4b], caspase-8 [Figure 5b], bcl-2 [Figure 6b] and p53 [Figure 7b]) in hearts of KO mice did not differ from values measured in N animals. Only the level of cyc1 mRNA was different, being increased in KO's hearts when compared with N mice (P = 0.001) (Figure 8b). CR did not change the expression of the examined genes in skeletal muscles of knockouts and N mice (Figures 1a–8a). In heart homogenates, CR increased caspase-3 mRNA level as compared with ad libitum (AL) mice (P = 0.008) (Figure 1b). Moreover, CR caused a statistically significant increase of caspase-3 expression in hearts of KO mice as compared with KO-AL animals (P = 0.011) (Figure 1b). The cyc1 mRNA level in hearts was elevated in KO-CR mice as compared with KO-AL (P = 0.043) or to N-CR animals (P < 0.001) (Figure 8b). The expression of other examined genes in hearts was not affected by CR (Figures 2b–7b). The protein expression level of caspase-3 (Figure 9a), caspase-9 (Figure 9b), bax (Figure 10a), Smac/DIABLO (Figure 10b) and Apaf-1 (Figure 13a) was decreased in skeletal muscles of GHRKO mice as compared with N mice (P < 0.001, P = 0.025, P < 0.001, P = 0.022, P = 0.026, respectively). In contrast, bcl-2 (Figure 11b) and pbad (Figure 12b) protein levels were increased in the KOs' skeletal muscles in comparison to N animals (P = 0.006, P = 0.005, respectively). No significant changes in caspase-8 (Figure 11a), bad (Figure 12a), pp53 (Figure 13b) and cyc (Figure 14) protein expression levels between knockouts and N animals were observed. Similarly to gene expression, CR did not alter the level of the examined proteins in skeletal muscles of KOs (pooled KO-AL and KO-CR) versus N mice (pooled N-AL and N-CR) (Figures 9–14). Caspase-3 protein level was decreased in KO-CR as compared with KO-AL (P < 0.001) or N-CR animals (Figure 9a) (P < 0.001). The protein levels of bax (Figure 10a) and Apaf-1 (Figure 13a) were decreased in KO-CR versus N-CR animals (P < 0.001, P = 0.038, respectively). Moreover, CR increased the caspase-9 (Figure 9b) and cyc (Figure 14) protein expression in N mice in comparison with N-AL (P = 0.036, P = 0.002, respectively) or KO-CR mice (P = 0.013, P = 0.036, respectively). Bcl-2 protein level was increased in KO-CR as compared with KO-AL or N-CR animals (Figure 11b) (P = 0.003, P = 0.002, respectively).

Figure 1

Caspase-3 mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a, b – values that do not share the same letter in the superscript are statistically significant (P < 0.05). *P = 0.02 versus N mice (the significance for genotype), **P = 0.008 versus AL mice (the significance for diet intervention)

Figure 2

Caspase-9 mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a, b – values that do not share the same letter in the superscript are statistically significant (P < 0.05). *P = 0.006 versus N mice (the significance for genotype)

Figure 3

bax mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a – values that share the same letter in the superscript are not statistically significant. *P = 0.035 versus N mice (the significance for genotype)

Figure 4

Smac/DIABLO mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a – values that share the same letter in the superscript are not statistically significant. *P = 0.036 versus N mice (the significance for genotype)

Figure 5

Caspase-8 mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a – values that share the same letter in the superscript are not statistically significant

Figure 6

bcl-2 mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a – values that share the same letter in the superscript are not statistically significant

Figure 7

p53 mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a – values that share the same letter in the superscript are not statistically significant

Figure 8

Cyc1 mRNA expression in skeletal muscles (a) and in hearts (b) of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR) (N-AL, n = 10; N-CR, n = 14; KO-AL, n = 10; KO-CR, n = 12). The data from real-time polymerase chain reaction were normalized by the housekeeping gene β 2-microglobulin and expressed as the relative expression. Values are means ± SEM. a, b – values that do not share the same letter in the superscript are statistically significant (P < 0.05). *P = 0.001 versus N mice (the significance for genotype)

Figure 9

Caspase-3 (a) and caspase-9 (b) protein level in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR). The protein level was expressed as the arbitrary unit (AU)/mm². Values are means ± SEM. a, b – values that do not share the same letter in the superscript are statistically significant (P < 0.05). *–P < 0.001 versus N mice (the significance for genotype), **–P = 0.025 versus N mice (the significance for genotype)

Figure 10

bax (a) and Smac/DIABLO (b) protein level in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR). The protein level was expressed as the arbitrary unit (AU)/mm². Values are means ± SEM. a–c – values that do not share the same letter in the superscript are statistically significant (P < 0.05). *P < 0.001 versus N mice (the significance for genotype), **P = 0.022 versus N mice (the significance for genotype)

Figure 11

Caspase-8 (a) and bcl-2 (b) protein level in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR). The protein level was expressed as the arbitrary unit (AU)/mm². Values are means ± SEM. a, b – values that do not share the same letter in the superscript are statistically significant (P < 0.05). *–P = 0.006 versus N mice (the significance for genotype)

Figure 12

bad (a) and pbad (b) protein level in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR). The protein level was expressed as the arbitrary unit (AU)/mm². Values are means ± SEM. a – values that share the same letter in the superscript are not statistically significant. *P = 0.005 versus N mice (the significance for genotype)

Figure 13

Apaf-1 (a) and pp53 (b) protein level in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR). The protein level was expressed as the arbitrary unit (AU)/mm². Values are means ± SEM. a, b – values that do not share the same letter in the superscript are statistically significant (P < 0.05). *P = 0.026 versus N mice (the significance for genotype)

Figure 14

Cyc protein level in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR). The protein level was expressed as the arbitrary unit (AU)/mm². Values are means ± SEM. a, b – values that do not share the same letter in the superscript are statistically significant (P < 0.05)

Discussion

Altered somatotrophic and insulin signaling are among the most important potential mechanisms of extended longevity. Long-lived GHRKO mice with targeted disruption of the Ghr/bp gene¹ are an excellent model system for studies of the control of aging and lifespan. Numerous previous studies sought explanations of the remarkable extended longevity of these animals and identified various potential biochemical and physiological mechanisms, including altered insulin signaling, enhanced insulin sensitivity, resistance to oxidative stress and cancer.^{2–5,8,11,31} However, although it is one of the most important processes involved in the regulation of development and health of living organisms, apoptosis has not been, as far, analyzed in GHRKO mice.

In the present study, the expression levels of four important apoptotic genes (caspase-3, caspase-9, bax and Smac/DIABLO) and five apoptotic proteins (caspase-3, caspase-9, bax, Smac/DIABLO and Apaf-1) were decreased in skeletal muscles of GHRKO mice as compared with N mice. These results, obtained in skeletal muscles, indicate a diminished level of apoptosis, which may be considered as the potentially protective effect for extended longevity of these animals. The decrease of effector (executioner) caspase-3 mRNA and protein expression seems to be the most beneficial result. It was previously reported that the absence of caspase-3 protected against denervation-induced skeletal muscle atrophy in mice.³² Furthermore, the decreased caspase-3 gene and protein expression is consistent with the low blood glucose concentration (observed in GHRKO mice), because high glucose caused an increased activity of caspase-3 (and caspase-8) in mice, resulting in muscle atrophy.³³ In another study, caspase-9 was down-regulated during the postnatal period in murine skeletal muscle and heart with consequent higher heart caspase-9 expression level at adult age.³⁴ The latter result differs from our observations in GHRKO mice showing no change in heart caspase-9 gene expression at adult age (see below) (see Tables 1–3).

Table 1

Relative gene expression (as percent deviation from control group − N-AL) in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR)

Gene	N-AL	N-CR	KO-AL	KO-CR	P value	P value	P value	P value	P value	P value
Gene	(A)	(B)	(C)	(D)	A versus B	C versus D	A versus C	B versus D	AB versus CD (N versus KO)	AC versus BD (AL versus CR)
Caspase-3	100 ± 36%	118 ± 21%	28 ± 5%	74 ± 22%	NS	NS	NS	NS	0.02	NS
Caspase-9	100 ± 31%	114 ± 21%	27 ± 5%	62 ± 19%	NS	NS	NS	NS	0.006	NS
bax	100 ± 26%	195 ± 63%	42 ± 14%	68 ± 18%	NS	NS	NS	NS	0.035	NS
Smac/DIABLO	100 ± 34%	110 ± 22%	35 ± 5%	76 ± 20%	NS	NS	NS	NS	0.036	NS
Caspase-8	100 ± 31%	83 ± 11%	76 ± 9%	73 ± 7%	NS	NS	NS	NS	NS	NS
bcl-2	100 ± 12%	82 ± 10%	104 ± 20%	131 ± 18%	NS	NS	NS	NS	NS	NS
p53	100 ± 32%	88 ± 12%	42 ± 8%	86 ± 21%	NS	NS	NS	NS	NS	NS
cyc1	100 ± 25%	60 ± 18%	79 ± 25%	144 ± 43%	NS	NS	NS	NS	NS	NS

Values are means ± SEM. NS, Not significant

Table 2

Relative gene expression (as percent deviation from control group − N-AL) in hearts of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR)

Gene	N-AL	N-CR	KO-AL	KO-CR	P value	P value	P value	P value	P value	P value
Gene	(A)	(B)	(C)	(D)	A versus B	C versus D	A versus C	B versus D	AB versus CD (N versus KO)	AC versus BD (AL versus CR)
Caspase-3	100 ± 25%	119 ± 20%	84 ± 16%	189 ± 25%	NS	0.011	NS	NS	NS	0.008
Caspase-9	100 ± 31%	165 ± 30%	112 ± 25%	171 ± 47%	NS	NS	NS	NS	NS	NS
bax	100 ± 24%	208 ± 63%	255 ± 96%	420 ± 121%	NS	NS	NS	NS	NS	NS
Smac/DIABLO	100 ± 28%	149 ± 56%	110 ± 26%	193 ± 31%	NS	NS	NS	NS	NS	NS
Caspase-8	100 ± 43%	72 ± 40%	61 ± 28%	54 ± 22%	NS	NS	NS	NS	NS	NS
bcl-2	100 ± 17%	139 ± 28%	155 ± 33%	134 ± 14%	NS	NS	NS	NS	NS	NS
p53	100 ± 44%	91 ± 36%	76 ± 28%	188 ± 59%	NS	NS	NS	NS	NS	NS
cyc1	100 ± 27%	85 ± 17%	140 ± 34%	251 ± 33%	NS	0.043	NS	<0.001	0.001	NS

Values are means ± SEM. NS, Not significant

Table 3

P values for protein level in skeletal muscles of normal (N) and growth hormone receptor/binding protein knockout (GHRKO; KO) mice fed ad libitum (AL) or subjected to 40% calorie restriction (CR)

	A versus B	C versus D	A versus C	B versus D	AB versus CD (N versus KO)	AC versus BD (AL versus CR)
Caspase-3	NS	<0.001	NS	<0.001	<0.001	NS
Caspase-9	0.036	NS	NS	0.013	0.025	NS
bax	NS	NS	NS	<0.001	<0.001	NS
Smac/DIABLO	NS	NS	NS	NS	0.022	NS
Caspase-8	NS	NS	NS	NS	NS	NS
bcl-2	NS	0.003	NS	0.002	0.006	NS
bad	NS	NS	NS	NS	NS	NS
pbad	NS	NS	NS	NS	0.005	NS
Apaf-1	NS	NS	NS	0.038	0.026	NS
pp53	NS	NS	NS	NS	NS	NS
cyc	0.002	NS	NS	0.036	NS	NS

A represents N-AL mice, B represents N-CR mice, C represents KO-AL mice, D represents KO-CR mice. NS, Not significant

In our study, expression of the caspase-9 gene, as noticed above, as well as of caspase-3, bax, Smac/DIABLO, caspase-8, bcl-2 and p53 gene in hearts of GHRKO mice were not changed and did not differ from values measured in N controls. Although the levels of caspase-3, caspase-9, bax and Smac/DIABLO mRNA did not decrease, as seen in the skeletal muscles, but remained unchanged, this also may be considered beneficial, protecting heart function in GHRKO mice. It is known that cardiac myocyte apoptosis is associated with increased DNA damage.³⁵ In such a context, our results showing unchanged expression of these four genes may presumably reflect no significant DNA damage in hearts of GHRKO mice and subsequently no necessity to initiate apoptosis. These results seem to be in accordance with previous observations in hearts of GHRKO mice, revealing mechanisms potentially counteracting the process of apoptosis and, thus, presumably protecting the hearts from aging. Masternak et al. ²⁰ showed no changes in antiapoptotic IGF-I mRNA and increase of IGF-I receptor (IGF-IR) mRNA expression in the hearts of GHRKO when compared with N mice, even though hepatic IGF-I expression and plasma IGF-I levels were profoundly suppressed.¹⁹

The decreased (in skeletal muscles) or unchanged (in hearts) bax mRNA level in GHRKO mice may represent a beneficial effect of GH resistance in these mutants and presumably contribute to their longevity. It has been previously shown that aging up-regulates the expression of bax.³⁶ Hearts of mice lacking the Bax gene (bax −/−) demonstrated reduced infarct size and improved myocardial function, following surgical ligation of the left anterior descending coronary artery.³⁷ Likewise, a cardiac tolerance of Bax knockout mice to ischemia/reperfusion injury was improved when compared with heterozygous (bax +/−) and wild-type (bax +/+) mice.³⁸

In an earlier study, Wencker et al. ³⁹ showed that caspase-8 inhibitors prevented cardiac myocyte death, cardiac dilation and contractile dysfunction in murine model. On the other hand, caspase-8-deficient mice died in utero and showed heart defects associated with abdominal hemorrhage.⁴⁰ In addition, Varfolomeev et al. ⁴¹ demonstrated that caspase-8 deficiency is embryonic-lethal in mice. Thus, unaltered expression of caspase-8 gene and protein level in GHRKO mice also seems to be beneficial for these mutants.

Similarly to GHRKOs' hearts, the expression of caspase-8, bcl-2 and p53 gene was unchanged in skeletal muscles of these long-lived animals.

As in the case of caspase-8, the unchanged p53 gene expression in skeletal muscles and hearts of GHRKO mice may be beneficial for these long-lived mice. p53, apart from its well-known role as a ‘guardian of the genome’, is said to be a mediator of senescence and contributes to aging.^42,43 Studies indicate that mitochondrial p53 levels correlate with mitochondrial DNA (mtDNA) oxidative damage.⁴⁴ Thus, absence of an increase of p53 gene expression may reflect a low level of oxidative damage to mtDNA and also may contribute to GHRKO longevity regulation. Shizukuda et al. ⁴⁵ showed that targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice. p53 inhibitors may be a potentially therapeutic treatment for patients after myocardial infarction (MI) because p53 has been shown to be involved in cardiac rupture after MI, probably via the induction of a proapoptotic pathway.⁴⁶ Interestingly, donor cardiac allografts from p53 knockout mice revealed apoptosis-independent prolongation of survival.⁴⁷ In contrast, loss of p53 in p53 knockout mice heart resulted in a significant increase in mtDNA vulnerability to damage following adriamycin treatment.⁴⁴ Similarly to the gene expression, the level of phosphorylated p53 protein was also unaltered. In conclusion, the role of p53 is multidirectional and is still not fully explained.

No significant differences for bad protein level between N and KO mice were observed. Otherwise, the level of bad protein phosphorylated at Ser112 (pbad) was increased in KOs' skeletal muscles as compared with N mice. It is worth emphasizing that phosphorylation at Ser112 results in the binding of bad to 14-3-3 proteins and in the inhibition of bad binding to bcl-2⁴⁸ what may prevent caspases cascade activation. In this context, the elevated pbad protein level (as well as increase of antiapoptotic bcl-2 protein expression observed in our study) may constitute another beneficial effect for extended longevity in long-lived GHRKO mice.

The cyc1 gene and cyc protein expression did not differ between skeletal muscles of GHRKO and N mice. Instead, in hearts of GHRKO mice, the level of cyc1 was unexpectedly increased, as compared with N mice. Moreover, heart cyc1 gene expression in KO-CR mice was increased as compared with KO-AL as well as to N-CR mice. Nevertheless, it was previously reported that cyc release can occur without loss of membrane potential, which normally precipitates opening of the permeability transition pore.⁴⁹ In addition, cyc is also involved in the electron transport chain. Therefore, the entire analysis of cyc gene and protein expression should take into account its multiple roles in physiological processes.

It must be emphasized that the present study was performed in females. Boland et al. ⁵⁰ demonstrated that 17β-estradiol exerted an antiapoptotic action in skeletal muscle. In another study, 17β-estradiol abrogated apoptosis in murine skeletal muscle cells acting through estrogen receptors.⁵¹ Moreover, it has been shown in a murine model that estrogens displayed myocardial protection in females.⁵² Thus, the gender of animals used in the present study may have contributed to the decrease of certain apoptotic genes and/or proteins (caspase-3, caspase-9, bax, Smac/DIABLO, Apaf-1) in skeletal muscles of the long-living mutants.

It is worth emphasizing that the process of apoptosis was previously analyzed in another kind of long-lived mice – the Ames dwarf. However, the results obtained in these mice were not completely clear. There was a decrease of the proapoptotic caspase-3 expression and a coinciding increase of antiapoptotic bcl-2 gene expression in peripheral blood leukocytes when compared with normal controls.⁵³ On the other hand, procaspase-3 protein levels were increased in Ames dwarf kidney and liver.⁵⁴

CR is a well-known experimental intervention to delay aging and increase lifespan.²⁸ Among its benefits, CR leads to protection of genome integrity and preservation of chromatin structure.⁵⁵ Furthermore, CR increases DNA repair activity and reverses the decrease of this process in aged mice.⁵⁵ Furthermore, CR has been demonstrated to modulate the apoptotic pathways, leading to attenuation of apoptotic signaling.⁵⁶ In our study, CR did not change the expression levels of the examined apoptosis-related genes and proteins in skeletal muscles of knockouts versus normal mice. These results differ from findings obtained by other authors. It has been shown that elevated DNA fragmentation and procaspase-3 level in the aging male Fischer 344 rat gastrocnemius muscle are significantly reduced by CR.^57,58 In other studies, CR counteracted the process of apoptosis in muscles, induced by tumor necrosis factor-α ⁵⁸ and decreased the procaspase-12 protein expression in the gastrocnemius in old rats.⁵⁷ Furthermore, CR reversed the increased level of bax and decreased bcl-2 protein in the kidneys of 24-month-old male Fischer 344 rats.⁵⁹ In contrast, Patel et al. ⁶⁰ demonstrated increased bax protein content in the quadriceps of calorie-restricted (40%) male and female G93A mice (an animal model of amyotrophic lateral sclerosis) as compared with animals fed ad libitum.

In heart homogenates, CR increased caspase-3 mRNA level as compared with AL mice. Moreover, CR caused a statistically significant increase of caspase-3 expression in hearts of KO mice as compared with KO-AL animals. The cyc1 mRNA level in hearts was elevated in KO-CR mice as compared with KO-AL or to N-CR mice. The expression of other examined genes in hearts was not affected by CR. The lack of changes in the expression of most of the examined genes and/or proteins following CR are not completely unexpected. In contrast to extended lifespan and improved insulin sensitivity in normal mice subjected to CR, an identical CR regimen failed to enhance insulin sensitivity or increase average or mean lifespan of GHRKO mice and caused only a small (but statistically significant) increase in the maximal lifespan in females.²⁹ Besides, CR failed to further modify the alterations in insulin signaling in GHRKO mice as compared with normal mice.⁶¹ The insulin signaling cascade in the heart of GHRKO mice was also unaffected by CR.⁶² Similarly, CR did not modify plasma insulin and glucose levels in Ames dwarf mice as compared with normal animals.⁶³ The observations showing diminished effects of CR on the process of apoptosis in skeletal muscles and other tissues and the different results of our study performed in GHRKO mice, may additionally support the hypothesis about the potentially common, and still not fully clear mechanisms of CR and GH resistance, participating in the regulation of physiological processes in these mutants.

In summary, decreased expression of certain proapoptotic genes and/or proteins may constitute the potential mechanism of prolonged longevity in GHRKO mice, protecting these animals from aging; this potential beneficial mechanism is not affected by CR. However, the role of apoptosis in physiology, pathology and aging is still controversial and further studies are needed to determine how the levels of expression of apoptosis-related genes and/or proteins in different organs can contribute to the regulation of longevity.

Author contributions: AG designed and performed the experiment, normal and calorie-restricted mice feeding, RNA and protein extraction, gene expression assessment (using real-time PCR), Western blotting, data analyses and interpretation, and wrote the manuscript. MMM and AB contributed to the experimental design. FW contributed to RNA extraction. MMM, AL, MK-L and AB were involved in the interpretation of the results and review of the manuscript.

Footnotes

Acknowledgements

The present study was supported by NIA, AG 19899, U19 AG023122 and AG31736, NIH/NIA 1R01AG032290, The Ellison Medical Foundation, Southern Illinois University School of Medicine and Polish Ministry of Science and Higher Education (N N401 042638). The authors would like to thank Steve Sandstrom for helping with the editing of the manuscript.

References

Zhou

, Xu

, Maheshwari

, He

, Reed

, Lozykowski

, Okada

, Cataldo

, Coschigano

, Wagner

, Baumann

, Kopchick

. A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 1997;94:13215–20

Coschigano

, Clemmons

, Bellush

, Kopchick

. Assessment of growth parameters and life span of GHR/BP gene disrupted mice. Endocrinology 2000;141:2608–13

Coschigano

, Holland

, Riders

, List

, Flyvbjerg

, Kopchick

. Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and increased lifespan. Endocrinology 2003;144:3799–810

Bartke

, Brown-Borg

. Life extension in the dwarf mouse. Curr Top Dev Biol 2004;63:189–225

Coschigano

. Aging-related characteristics of growth hormone receptor/binding protein gene-disrupted mice. AGE 2006;28:191–200

Kopchick

, Laron

. Is the Laron mouse an accurate model of Laron syndrome? Mol Genet Metab 1999;68:232–6

Bartke

, Chandrashekar

, Bailey

, Zaczek

, Turyn

. Consequences of growth hormone (GH) overexpression and GH resistance. Neuropeptides 2002;36:201–8

Liu

, Coschigano

, Robertson

, Lipsett

, Guo

, Kopchick

, Kumar

, Liu

. Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice. Am J Physiol Endocrinol Metab 2004;287:E405–13

Salmon

, Murakami

, Bartke

, Kopchick

, Yasumura

, Miller

. Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress. Am J Physiol Endocrinol Metab 2005;289:E23–9

10.

Sun

, Steinbaugh

, Masternak

, Bartke

, Miller

. Fibroblasts from long-lived mutant mice show diminished ERK1/2 phosphorylation but exaggerated induction of immediate early genes. Free Radic Biol Med 2009;47:1753–61

11.

Ikeno

, Hubbard

, Lee

, Cortez

, Lew

, Webb

, Berryman

, List

, Kopchick

, Bartke

. Reduced incidence and delayed occurrence of fatal neoplastic diseases in growth hormone receptor/binding protein knockout mice. J Gerontol A Biol Sci Med Sci 2009;64:522–9

12.

Batistatou

, Greene

. Internucleosomal DNA cleavage and neuronal cell survival/death. J Cell Biol 1993;122:523–32

13.

Johnson

Jr , Deckwerth

. Molecular mechanisms of developmental neuronal death. Annu Rev Neurosci 1993;16:31–46

14.

Clarke

. Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl) 1990;181:195–213

15.

Carson

, Ribeiro

. Apoptosis and disease. Lancet 1993;341:1251–4

16.

Budihardjo

, Oliver

, Lutter

, Luo

, Wang

. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 1999;15:269–90

17.

Boatright

, Salvesen

. Mechanisms of caspase activation. Curr Opin Cell Biol 2003;15:725–31

18.

Jin

, El-Deiry

. Overview of cell death signaling pathways. Cancer Biol Ther 2005;4:139–63

19.

Masternak

, Al-Regaiey

, Del Rosario Lim

, Jimenez-Ortega

, Panici

, Bonkowski

, Bartke

. Effects of caloric restriction on insulin pathway gene expression in the skeletal muscle and liver of normal and long-lived GHR-KO mice. Exp Gerontol 2005;40:679–84

20.

Masternak

, Al-Regaiey

, Del Rosario Lim

, Jimenez-Ortega

, Panici

, Bonkowski

, Kopchick

, Wang

, Bartke

. Caloric restriction and growth hormone receptor knockout: effects on expression of genes involved in insulin action in the heart. Exp Gerontol 2006;41:417–29

21.

Coultas

, Strasser

. The role of the Bcl-2 protein family in cancer. Semin Cancer Biol 2003;13:115–23

22.

Srinivasula

, Datta

, Fan

, Fernandes-Alnemri

, Huang

, Alnemri

. Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway. J Biol Chem 2000;275:36152–7

23.

Henry-Mowatt

, Dive

, Martinou

, James

. Role of mitochondrial membrane permeabilization in apoptosis and cancer. Oncogene 2004;23:2850–60

24.

Kruidering

, Evan

. Caspase-8 in apoptosis: the beginning of ‘‘the end’'? IUBMB Life 2000;50:85–90

25.

Ashkenazi

. Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Discov 2008;7:1001–12

26.

Zou

, Li

, Liu

, Wang

. An APAF-1·cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999;274:11549–56

27.

Adrain

, Martin

. The mitochondrial apoptosome: a killer unleashed by the cytochrome seas. Trends Biochem Sci 2001;26:390–7

28.

Weindruch

, Sohal

. Seminars in medicine of the Beth Israel Deaconess medical center. Caloric intake and aging. N Engl J Med 1997;337:986–94

29.

Bonkowski

, Rocha

, Masternak

, Al Regaiey

, Bartke

. Targeted disruption of growth hormone receptor interferes with the beneficial actions of calorie restriction. Proc Natl Acad Sci USA 2006;103:7901–5

30.

Chomczynski

, Sacchi

. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9

31.

Harper

, Salmon

, Chang

, Bonkowski

, Bartke

, Miller

. Stress resistance and aging: influence of genes and nutrition. Mech Ageing Dev 2006;127:687–94

32.

Plant

, Bain

, Correa

, Woo

, Batt

. Absence of caspase-3 protects against denervation-induced skeletal muscle atrophy. J Appl Physiol 2009;107:224–34

33.

Russell

, Rajani

, Dhadda

, Tisdale

. Mechanism of induction of muscle protein loss by hyperglycaemia. Exp Cell Res 2009;315:16–25

34.

Madden

, Donovan

, Cotter

. Key apoptosis regulating proteins are down-regulated during postnatal tissue development. Int J Dev Biol 2007;51:415–23

35.

Barouch

, Gao

, Chen

, Miller

, Xu

, Phan

, Kittleson

, Minhas

, Berkowitz

, Wei

, Hare

. Cardiac myocyte apoptosis is associated with increased DNA damage and decreased survival in murine models of obesity. Circ Res 2006;98:119–24

36.

, Ren

. Influence of cardiac-specific overexpression of insulin-like growth factor 1 on lifespan and aging-associated changes in cardiac intracellular Ca²⁺ homeostasis, protein damage and apoptotic protein expression. Aging Cell 2007;6:799–806

37.

Hochhauser

, Cheporko

, Yasovich

, Pinchas

, Offen

, Barhum

, Pannet

, Tobar

, Vidne

, Birk

. Bax deficiency reduces infarct size and improves long-term function after myocardial infarction. Cell Biochem Biophys 2007;47:11–20

38.

Hochhauser

, Kivity

, Offen

, Maulik

, Otani

, Barhum

, Pannet

, Shneyvays

, Shainberg

, Goldshtaub

, Tobar

, Vidne

. Bax ablation protects against myocardial ischemia-reperfusion injury in transgenic mice. Am J Physiol Heart Circ Physiol 2003;284:H2351–9

39.

Wencker

, Chandra

, Nguyen

, Miao

, Garantziotis

, Factor

, Shirani

, Armstrong

, Kitsis

. A mechanistic role for cardiac myocyte apoptosis in heart failure. J Clin Invest 2003;111:1497–504

40.

Sakamaki

, Inoue

, Asano

, Sudo

, Kazama

, Sakagami

, Sakata

, Ozaki

, Nakamura

, Toyokuni

, Osumi

, Iwakura

, Yonehara

. Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart. Cell Death Differ 2002;9:1196–206

41.

Varfolomeev

, Schuchmann

, Luria

, Chiannilkulchai

, Beckmann

, Mett

, Rebrikov

, Brodianski

, Kemper

, Kollet

, Lapidot

, Soffer

, Sobe

, Avraham

, Goncharov

, Holtman

, Lonai

, Wallach

. Targeted disruption of the mouse Caspase-8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally. Immunity 1998;9:267–76

42.

Tyner

, Venkatachalam

, Choi

, Jones

, Ghebranious

, Igelmann

, Lu

, Soron

, Cooper

, Brayton

, Hee

, Thompson

, Karsenty

, Bradley

, Donehower

. p53 mutant mice that display early ageing-associated phenotypes. Nature 2002;415:45–53

43.

Campisi

. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 2005;120:513–22

44.

Nithipongvanitch

, Ittarat

, Velez

, Zhao

, Clair

DKS

, Oberley

. Evidence for p53 as guardian of the cardiomyocyte mitochondrial genome following acute adriamycin treatment. J Histochem Cytochem 2007;55:629–39

45.

Shizukuda

, Matoba

, Mian

, Nguyen

, Hwang

. Targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice. Mol Cell Biochem 2005;273:25–32

46.

Matsusaka

, Ide

, Matsushima

, Ikeuchi

, Kubota

, Sunagawa

, Kinugawa

, Tsutsui

. Targeted deletion of p53 prevents cardiac rupture after myocardial infarction in mice. Cardiovasc Res 2006;70:457–65

47.

Kown

, Murata

, Jahncke

, Mari

, Berry

, Lijkwan

, Blankenberg

, Strauss

, Robbins

. Donor cardiac allografts from p53 knockout mice exhibit apoptosis-independent prolongation of survival. Transplant Proc 2002;34:3274–6

48.

Zha

, Harada

, Yang

, Jockel

, Korsmeyer

. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14–3–3 not BCL-X_L . Cell 1996;87:619–28

49.

Gross

, McDonnell

, Korsmeyer

. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999;13:1899–911

50.

Boland

, Vasconsuelo

, Milanesi

, Ronda

, de Boland

. 17β-estradiol signaling in skeletal muscle cells and its relationship to apoptosis. Steroids 2008;73:859–63

51.

Vasconsuelo

, Milanesi

, Boland

. 17β-estradiol abrogates apoptosis in murine skeletal muscle cells through estrogen receptors: role of the phosphatidylinositol 3-kinase/Akt pathway. J Endocrinol 2008;196:385–97

52.

Wang

, Crisostomo

, Wairiuko

, Meldrum

. Estrogen receptor-α mediates acute myocardial protection in females. Am J Physiol Heart Circ Physiol 2006;290:H2204–9

53.

Dhahbi

, Li

, Tran

, Masternak

, Bartke

. Circulating blood leukocyte gene expression profiles: effects of the Ames dwarf mutation on pathways related to immunity and inflammation. Exp Gerontol 2007;42:772–88

54.

Kennedy

, Rakoczy

, Brown-Borg

. Long-living Ames dwarf mouse hepatocytes readily undergo apoptosis. Exp Gerontol 2003;38:997–1008

55.

Heydari

, Unnikrishnan

, Lucente

, Richardson

. Caloric restriction and genomic stability. Nucleic Acids Res 2007;35:7485–96

56.

Marzetti

, Lawler

, Hiona

, Manini

, Seo

, Leeuwenburgh

. Modulation of age-induced apoptotic signaling and cellular remodeling by exercise and calorie restriction in skeletal muscle. Free Radic Biol Med 2008;44:160–8

57.

Dirks

, Leeuwenburgh

. Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor, apoptosis-inducing factor, X-linked inhibitor of apoptosis, caspase-3, and caspase-12. Free Radic Biol Med 2004;36:27–39

58.

Phillips

, Leeuwenburgh

. Muscle fiber-specific apoptosis and TNF-α signaling in sarcopenia are attenuated by life-long calorie restriction. FASEB J 2005;19:668–70

59.

Lee

, Jung

, Kim

, Yu

, Chung

. Suppression of apoptosis by calorie restriction in aged kidney. Exp Gerontol 2004;39:1361–8

60.

Patel

, Safdar

, Raha

, Tarnopolsky

, Hamadeh

. Caloric restriction shortens lifespan through an increase in lipid peroxidation, inflammation and apoptosis in the G93A mouse, an animal model of ALS. PLoS One 2010;5:e9386

61.

Bonkowski

, Dominici

, Arum

, Rocha

, Al Regaiey

, Westbrook

, Spong

, Panici

, Masternak

, Kopchick

, Bartke

. Disruption of growth hormone receptor prevents calorie restriction from improving insulin action and longevity. PLoS ONE 2009;4:e4567

62.

Giani

, Bonkowski

, Muñoz

, Masternak

, Turyn

, Bartke

, Dominici

. Insulin signaling cascade in the hearts of long-lived growth hormone receptor knockout mice: effects of calorie restriction. J Gerontol A Biol Sci Med Sci 2008;63:788–97

63.

Argentino

, Dominici

, Al Regaiey

, Bonkowski

, Bartke

, Turyn

. Effects of long-term caloric restriction on early steps of the insulin-signaling system in mouse skeletal muscle. J Gerontol A Biol Sci Med Sci 2005;60:28–34