Novel treatment of intracerebral hemorrhage with mechanical tissue resuscitation.

OBJECTIVE: The previous laboratory and clinical experience of the authors had demonstrated that application of controlled subatmospheric pressure directly to injured soft tissue can result in increased survival of compromised tissues. Mechanical tissue resuscitation (MTR) is a new concept evolving from these discoveries. The authors' recent studies have demonstrated that traumatic brain injury tissue can also be salvaged. The aim of this study was to examine the effects of MTR application to injuries from intracerebral hemorrhages (ICHs) in a swine model. METHODS: The ICHs in swine were simulated by infusion of autologous artery blood into the right frontal lobe. A specially designed silicone manifold device was introduced directly into the hematoma. Continuous negative pressure at -50 mm Hg was applied through this device. T2- and T2*-weighted MRI, histological H&E staining, and immunostaining were examined. RESULTS: After 1 week of treatment, MTR significantly decreased gross hematoma volume by more than 60%, from 472.62 ± 230.19 mm3 in the nontreated group to 171.25 ± 75.38 mm3 in the MTR-treated group (p < 0.05). Total hypointense volumes measured on T2*-weighted MR images decreased from 791.99 ± 360.47 mm3 in the nontreated group to 371.16 ± 105.75 mm3 in the MTR-treated group (p < 0.05). The hyperintense area on the T2-weighted MR image decreased significantly from 2656.23 ± 426.26 mm3 in the nontreated group to 1816.66 ± 525.26 mm3 in the MTR-treated group (p < 0.05). When ICHs were treated with MTR for 2 weeks, the gross hematomas were reduced by 94%, from 112.23 ± 66.21 mm3 in the nontreated group to 6.12 ± 10.99 mm3 in the MTR-treated group (p = 0.003). MTR significantly decreased the total necrotic tissue volume in H&E staining from 120.42 ± 48.35 mm3 in the nontreated group to 60.94 ± 38.99 mm3 in the MTR-treated group (p < 0.05). The total hypointense volumes on T2*-weighted MR images were significantly reduced, from 385.54 ± 93.85 mm3 in the nontreated group to 220.54 ± 104.28 mm3 in the MTR-treated group (p < 0.05), while their mean T2 hyperintense volume decreased significantly from 2192.83 ± 728.27 mm3 in the nontreated group to 1366.97 ± 463.36 mm3 in the MTR-treated group (p < 0.05). Histology revealed that the capillary diameter in the reactive tissue rim adjacent to the hematoma increased in both the 1- and 2-week MTR-treated groups. Both von Willebrand factor and CD31 signals were detectable in endothelial cells within the hematoma cavity of both MTR-treated groups. CONCLUSIONS: This study demonstrates that local continuous application of controlled subatmospheric pressure to an ICH can safely remove more than half of a clot in 1 week and more than 90% in 2 weeks.

Attenuated tissue damage with Mechanical Tissue Resuscitation in a pig model of spinal cord injury.

Our previous studies on the treatment of spinal cord injuries with Mechanical Tissue Resuscitation (MTR) in rats have demonstrated that it can significantly improve the locomotor recovery and BBB scores. MTR treatment also reduced fluid accumulations by T2-imaging and improved the mean neural fiber number and fiber length in injured sites by fiber tractography. Myelin volume was also significantly preserved by MTR treatment. For further clinical application, a large animal model is necessary to assess this treatment. This study examined the effects of application of MTR on traumatic spinal cord injury in a swine model. Traumatic spinal cord contusion injuries (SCI) in swine were created by controlled pneumatic impact device. Negative pressure at -75 mm Hg was continuously applied to the injured site through open cell silicone manifold for 7 days. In vivo MR imaging for T2 and GRE analysis employed a 3T machine, while a 7T machine was employed for diffusion tensor imaging (DTI) and fiber tractography. Histological HE and Luxol fast blue staining were examined. MTR significantly reduced the mean injured volumes over 46% by T2-imaging in the injured sites from 477.34±146.31 mm3 in non-treated group to 255.99±70.28 mm3 in MTR treated group (P<0.01). It also reduced fluid accumulations by relative T2 signal density in the epicenter of the SCI from 1.62±0.27 in non-treated group to 1.22±0.10 in the MTR treated group (P<0.05). The mean injured tissue volume measured by H&E staining was 303.71±78.21 mm3 in the non-treated group and decreased significantly to 162.16±33.0 mm3 in the MTR treated group (P<0.01). The myelin fiber bundles stained by Luxol blue were preserved much more in the MTR treated group (90±29.71 mm3) than in the non-treated group (33.68±24.99 mm3, P<0.01). The fractional anisotropy (FA) values processed by DTI analysis are increased from 0.203±0.027 in the untreated group to 0.238±0.029 in MTR treatment group (P<0.05). Fiber tractography showings the mean fiber numbers across the impacted area were increased over 112% from 327.0±99.74 in the non-treated group to 694.83±297.86 in the MTR treated group (P<0.05). These results indicates local application of MTR for seven days to spinal cord injury in a swine model decreased tissue injury, reduced tissue edema and preserved more myelin fibers as well as nerve fibers in the injured spinal cord. Keywords: Mechanical tissue resuscitation, Negative pressure treatment, Spinal cord injury, Diffusion tensor imaging, Nerve fiber tractography.

Ameliorating Spinal Cord Injury in an Animal Model With Mechanical Tissue Resuscitation.

BACKGROUND: Traumatic spinal cord injury (SCI) is a major worldwide cause of mortality and disability with limited treatment options. Previous research applying controlled negative pressure to traumatic brain injury in rat and swine models resulted in smaller injuries and more rapid recovery. OBJECTIVE: To examine the effects of the application of a controlled vacuum (mechanical tissue resuscitation [MTR]) to SCI in a rat model under several magnitudes of vacuum. METHODS: Controlled contusion SCIs were created in rats. Vacuums of -50 and -75 mm Hg were compared. Analysis included open-field locomotor performance, magnetic resonance imaging (in vivo T2, ex vivo diffusion tensor imaging and fiber tractography), and histological assessments. RESULTS: MTR treatment significantly improved the locomotor recovery from a Basso, Beattie, and Bresnahan score of 7.8 ± 1.9 to 11.4 ± 1.2 and 10.7 ± 1.9 at -50- and -75-mm Hg pressures, respectively, 4 weeks after injury. Both pressures also reduced fluid accumulations > 10% by T2-imaging in SCI sites. The mean fiber number and mean fiber length were greater across injured sites after MTR treatment, especially with treatment with -50 mm Hg. Myelin volume was increased significantly by 60% in the group treated with -50 mm Hg. CONCLUSION: MTR of SCI in a rat model is effective in reducing edema in the injured cord, preserving myelin survival, and improving the rate and quantity of functional recovery. ABBREVIATIONS: BBB, Basso, Beattie, and BresnahanDTI, diffusion tensor imagingFA, fractional anisotropyMTR, mechanical tissue resuscitationMTR50, mechanical tissue resuscitation with 50-mm Hg subatmospheric pressureMTR75, mechanical tissue resuscitation with 75-mm Hg subatmospheric pressureROI, region of interestSCI, spinal cord injury.

Mechanical tissue resuscitation at the site of traumatic brain injuries reduces the volume of injury and hemorrhage in a swine model.

BACKGROUND: Traumatic brain injuries (TBIs) continue to be a devastating problem with limited treatment options. Previous research applying controlled vacuum to TBI in a rat model resulted in smaller injuries and more rapid recovery. OBJECTIVE: To examine the effects of the application of a controlled vacuum (mechanical tissue resuscitation) to TBI in a large-animal model. The magnitude of vacuum, length of application, and length of delay between injury and the application of mechanical tissue resuscitation were investigated. METHODS: Localized, controlled cortical injuries were created in swine. Vacuums of -50 and -100 mm Hg were compared. Mechanical tissue resuscitation for 3 or 5 days was compared. Delays of 0, 3, or 6 hours between the creation of the TBI and the initiation of mechanical tissue resuscitation were examined. Analysis included histological assessments, computed tomographic perfusion, and magnetic resonance imaging (T2, proton magnetic spectra). RESULTS: A -100 mm Hg vacuum resulted in significantly smaller mean contused brain and hemorrhage volumes compared with -50 mm Hg and controls. Magnetic resonance spectra of treated animals returned to near baseline values. All 10 animals with 5-day mechanical tissue resuscitation treatment survived. Three of 6 animals treated for 3 days died after the discontinuation of treatment. A 3-hour delay resulted in similar results as immediate treatment. A 6-hour delay produced significant, but lesser responses. CONCLUSION: Application of mechanical tissue resuscitation to TBI was efficacious in the large-animal model. Application of -100 mm Hg for 5 days resulted in significantly improved outcomes. Delays of up to 3 hours between injury and the initiation of treatment did not diminish the efficacy of the mechanical tissue resuscitation treatment.

Cerebral blood flow quantification in swine using pseudo-continuous arterial spin labeling.

PURPOSE: To develop quantitative cerebral blood flow (CBF) imaging using pseudo-continuous arterial spin labeling (PCASL) in swine, accounting for their cerebrovascular anatomy and physiology. MATERIALS AND METHODS: Five domestic pigs (2.5-3 months, 25 kg) were used in these studies. The orientation of the labeled arteries, T1bl , M0bl , and T1gm were measured in swine. Labeling parameters were tuned with respect to blood velocity to optimize labeling efficiency based on the data collected from three subjects. Finally, CBF and arterial transit time (ATT) maps for two subjects were created from PCASL data to determine global averages. RESULTS: The average labeling efficiency over measured velocities of 5-18 cm/s was 0.930. The average T1bl was 1546 ms, the average T1gm was 1224 ms, and the average blood-to-white matter ratio of M0 was 1.25, which was used to find M0bl . The global averages over the subjects were 54.05 mL/100 g tissue/min CBF and 1261 ms ATT. CONCLUSION: This study demonstrates the feasibility of PCASL for CBF quantification in swine. Quantification of CBF using PCASL in swine can be further developed as an accessible and cost-effective model of human cerebral perfusion for investigating injuries that affect blood flow.

Deficiency of liver Comparative Gene Identification-58 causes steatohepatitis and fibrosis in mice.

Triglyceride (TG) accumulation in hepatocytes (hepatic steatosis) preludes the development of advanced nonalcoholic fatty liver diseases (NAFLDs) such as steatohepatitis, fibrosis, and cirrhosis. Mutations in human Comparative Gene Identification-58 (CGI-58) cause cytosolic TG-rich lipid droplets to accumulate in almost all cell types including hepatocytes. However, it is unclear if CGI-58 mutation causes hepatic steatosis locally or via altering lipid metabolism in other tissues. To directly address this question, we created liver-specific CGI-58 knockout (LivKO) mice. LivKO mice on standard chow diet displayed microvesicular and macrovesicular panlobular steatosis, and progressed to advanced NAFLD stages over time, including lobular inflammation and centrilobular fibrosis. Compared with CGI-58 floxed control littermates, LivKO mice showed 8-fold and 52-fold increases in hepatic TG content, which was associated with 40% and 58% decreases in hepatic TG hydrolase activity at 16 and 42 weeks, respectively. Hepatic cholesterol also increased significantly in LivKO mice. At 42 weeks, LivKO mice showed increased hepatic oxidative stress, plasma aminotransferases, and hepatic mRNAs for genes involved in fibrosis and inflammation, such as α-smooth muscle actin, collagen type 1 α1, tumor necrosis factor α, and interleukin-1ß. In conclusion, CGI-58 deficiency in the liver directly causes not only hepatic steatosis but also steatohepatitis and fibrosis.

A new method for modulating traumatic brain injury with mechanical tissue resuscitation.

BACKGROUND: Traumatic brain injuries remain a treatment enigma with devastating late results. As terminally differentiated tissue, the brain retains little capacity to regenerate, making early attempts to preserve brain cells after brain injury essential. OBJECTIVE: To resuscitate damaged tissue by modulating edema, soluble cytokines, and metabolic products in the "halo" of damaged tissue around the area of central injury that progressively becomes compromised. By re-equilibrating the zone of injury milieu, it is postulated neurons in this area will survive and function. METHODS: Mechanical tissue resuscitation used localized, controlled, subatmospheric pressure directly to the area of controlled cortical impact injury and was compared with untreated injured controls and with sham surgery in a rat model. Functional outcome, T2 magnetic resonance imaging hyperintense volume, magnetic resonance imaging spectroscopy metabolite measurement, tissue water content, injury cavity area, and cortical volume were compared. RESULTS: There were significant differences between mechanical tissue resuscitation treated and untreated groups in levels of myoinositol, N-acetylaspartate, and creatine. Treated animals had significantly less tissue swelling and density than the untreated animals. Nonviable brain tissue areas were smaller in treated animals than in untreated animals. Treated animals performed better than untreated animals in functional tests. Histological analysis showed the remaining viable ipsilateral cerebral area was 58% greater for treated animals than for untreated animals, and the cavity for treated animals was 95% smaller than for untreated animals 1 month after injury. CONCLUSION: Mechanical tissue resuscitation with controlled subatmospheric pressure can significantly modulate levels of excitatory amino acids and lactate in traumatic brain injury, decrease the water content and volume of injured brain, improve neuronal survival, and speed functional recovery.

Increased CaVbeta1A expression with aging contributes to skeletal muscle weakness.

Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation-contraction (E-C) coupling. Excitation-contraction uncoupling, a deficit in Ca2+ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. Excitation-contraction uncoupling may be caused by alterations in expression of the voltage-dependent calcium channel alpha1s (CaV1.1) and beta1a (CaVbeta1a) subunits, both of which are necessary for E-C coupling to occur. While previous studies have found CaV1.1 expression declines in old rodents, CaVbeta1a expression has not been previously examined in aging models. Western blot analysis shows a substantial increase of CaVbeta1a expression over the full lifespan of Friend Virus B (FVB) mice. To examine the specific effects of CaVbeta1a overexpression, a CaVbeta1a-YFP plasmid was electroporated in vivo into young animals. The resulting increase in expression of CaVbeta1a corresponded to decline of CaV1.1 over the same time period. YFP fluorescence, used as a measure of CaVbeta1a-YFP expression in individual fibers, also showed an inverse relationship with charge movement, measured using the whole-cell patch-clamp technique. Specific force was significantly reduced in young CaVbeta1a-YFP electroporated muscle fibers compared with sham-electroporated, age-matched controls. siRNA interference of CaVbeta1a in young muscles reduced charge movement, while charge movement in old was restored to young control levels. These studies imply CaVbeta1a serves as both a positive and negative regulator CaV1.1 expression, and that endogenous overexpression of CaVbeta1a during old age may play a role in the loss of specific force.

Muscle fibers from senescent mice retain excitation-contraction coupling properties in culture.

In the present study, we test the hypothesis that mouse skeletal muscle in culture retains the fundamental properties of excitation-sarcoplasmic reticulum Ca(2+) release coupling reported for young-adult (3-4 mo) and senescent (22-23) mice. Dissociated flexor digitorum brevis (FDB) muscles from young-adult and senescent mice were cultured for 7 d in a serum-free medium. During this period, the overall morphology of cultured fibers resembled that exhibited by acutely dissociated cells. In addition, survival analysis revealed that more than 70% of the fibers from both young and old mice remained suitable for electrophysiological studies during this same culture period. Charge movement and intracellular Ca(2+) recordings in FDB fibers, voltage clamped in the whole cell configuration of the patch-clamp technique, reproduced the maximal values, and voltage dependence similarly displayed by acutely dissociated cells for both parameters in young-adult and senescent mice. The analysis of the dihydropyridine receptor by immunoblots confirmed, in the culture system, the age-dependent decrease in the expression of this protein. In conclusion, FDB fibers from young-adult and old mice retain the excitation-contraction coupling phenotype during the course of a week in serum-free medium culture.

Motor neuron targeting of IGF-1 attenuates age-related external Ca2+-dependent skeletal muscle contraction in senescent mice.

A population of fast muscle fibers from aging mice is dependent on external Ca(2+) to maintain tetanic force during repeated contractions. We hypothesized that age-related denervation in muscle fibers plays a role in initiating this contractile deficit, and that prevention of denervation by IGF-1 overexpression would prevent external Ca(2+)-dependent contraction in aging mice. IGF-1 overexpression in skeletal muscle prevents age-related denervation, and prevented external Ca(2+)-dependent contraction in this work. To determine if the effects of IGF-1 overexpression are on muscle or nerve, aging mice were injected with a tetanus toxin fragment-C (TTC) fusion protein that targets IGF-1 to spinal cord motor neurons. This treatment prevented external Ca(2+)-dependent contraction. We also show evidence that injections of the IGF-1-TTC fusion protein prevent age-related alterations to the nerve terminals at the neuromuscular junctions. We conclude that the slow age-related denervation of fast muscle fibers underlies dependence on external Ca(2+) to maintain tetanic force in a population of muscle fibers from senescent mice.

The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca2+ channel Cav1.1.

JP-45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), colocalizes with its Ca2+ -release channel (the ryanodine receptor), and interacts with calsequestrin and the skeletal-muscle dihydropyridine receptor Cav1. We have identified the domains of JP-45 and the Cav1.1 involved in this interaction, and investigated the functional effect of JP-45. The cytoplasmic domain of JP-45, comprising residues 1-80, interacts with Cav1.1. JP-45 interacts with two distinct and functionally relevant domains of Cav1.1, the I-II loop and the C-terminal region. Interaction between JP-45 and the I-II loop occurs through the alpha-interacting domain in the I-II loop. beta1a, a Cav1 subunit, also interacts with the cytosolic domain of JP-45, and its presence drastically reduces the interaction between JP-45 and the I-II loop. The functional effect of JP-45 on Cav1.1 activity was assessed by investigating charge movement in differentiated C2C12 myotubes after overexpression or depletion of JP-45. Overexpression of JP-45 decreased peak charge-movement and shifted VQ1/2 to a more negative potential (-10 mV). JP-45 depletion decreased both the content of Cav1.1 and peak charge-movements. Our data demonstrate that JP-45 is an important protein for functional expression of voltage-dependent Ca2+ channels.

Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle.

IGF-1 is a potent growth factor for both motor neurones and skeletal muscle. Muscle IGF-1 is known to provide target-derived trophic effects on motor neurones. Therefore, IGF-1 overexpression in muscle is effective in delaying or preventing deleterious effects of ageing in both tissues. Since age-related decline in muscle function stems partly from motor neurone loss, a tetanus toxin fragment-C (TTC) fusion protein was created to target IGF-1 to motor neurones. IGF-1-TTC retains IGF-1 activity as indicated by [(3)H]thymidine incorporation into L6 myoblasts. Spinal cord motor neurones effectively bound and internalized the IGF-1-TTC in vitro. Similarly, IGF-1-TTC injected into skeletal muscles was taken up and retrogradely transported to the spinal cord in vivo, a process prevented by denervation of injected muscles. Three monthly IGF-1-TTC injections into muscles of ageing mice did not increase muscle weight or muscle fibre size, but significantly increased single fibre specific force over aged controls injected with saline, IGF-1, or TTC. None of the injections changed muscle fibre type composition, but neuromuscular junction post-terminals were larger and more complex in muscle fibres injected with IGF-1-TTC, compared to the other groups, suggesting preservation of muscle fibre innervation. This work demonstrates that induced overexpression of IGF-1 in spinal cord motor neurones of ageing mice prevents muscle fibre specific force decline, a hallmark of ageing skeletal muscle.

Role of sustained overexpression of central nervous system IGF-I in the age-dependent decline of mouse excitation-contraction coupling.

We investigated the effects of exclusive and sustained transgenic overexpression of insulin-like growth factor (IGF)-I in the central nervous system (CNS) on the age-dependent decline in muscle strength, excitation-contraction coupling, muscle innervation and neuromuscular junction postterminal architecture. We found that (1) transgenic IGF-I overexpression in the CNS does not modify the decline in extensor digitorum longus (EDL) and soleus muscle weight with aging and (2) strength significantly decreases in transgenic (Tg) compared to wild-type mice. The latter finding is consistent with (3) the decreased absolute and specific force measured in the EDL muscle in vitro and (4) the decreased charge movement and peak intracellular Ca(2+) mobilization in individual muscle fibers from old IGF-I Tg mice compared to young wild-type mice, which also is associated with (5) decreased dihydropyridine receptor alpha(1)-subunit expression in old compared to young IGF-I Tg mice. (6) Tg IGF-I prevents a change in muscle fiber type that is associated with (7) improved muscle innervation and postterminal neuromuscular structure. (8) IGF-I is expressed extensively across the spinal cord gray matter and the lateral motor column. Our results raise questions about the timing and cell location of CNS IGF-I overexpression necessary to prevent or to ameliorate age-dependent alterations in the structure and function of skeletal muscle.

Extension and magnitude of denervation in skeletal muscle from ageing mice.

In this work we hypothesized that denervation in flexor digitorum brevis (FDB) muscle from ageing mice is more extensive than predicted by standard functional and structural assays used in the past. In addition, we asked whether denervation is a fully or partially developed process. Despite the reported alteration in skeletal muscle innervation, the quantification of the extension and magnitude of denervation in ageing rodents has remained elusive. To address these two questions we utilized a combination of electrophysiological and immunohistochemical assays directed to detecting the expression of tetrodotoxin (TTX)-resistant sodium channels (Na(v)1.5) in FDB muscles from young-adult and senescent mice. Sodium current density measured with the macropatch cell-attached technique did not show significant differences between FDB fibres from young and old mice. The TTX dose-response curve, using the whole cell voltage-clamp technique, showed three populations of fibres in senescent mice, one similar to fibres from young mice (TTX sensitive), another one similar to fibres from experimentally denervated muscle (TTX resistant), and a third group intermediate between these two. Partially and fully denervated fibres added up to approximately 50% of the total number of fibres tested, a number that concurs with the percentage of fibres positive for the Na(v)1.5 channel by specific immunostaining.

External Ca(2+)-dependent excitation--contraction coupling in a population of ageing mouse skeletal muscle fibres.

In the present work, we investigate whether changes in excitation-contraction (EC) coupling mode occur in skeletal muscles from ageing mammals by examining the dependence of EC coupling on extracellular Ca(2+). Single intact muscle fibres from flexor digitorum brevis muscles from young (2-6 months) and old (23-30 months) mice were subjected to tetanic contractile protocols in the presence and absence of external Ca(2+). Contractile experiments in the absence of external Ca(2+) show that about half of muscle fibres from old mice are dependent upon external Ca(2+) for maintaining maximal tetanic force output, while young fibres are not. Decreased force in the absence of external Ca(2+) was not due to changes in charge movement as revealed by whole-cell patch-clamp experiments. Ca(2+) transients, measured by fluo-4 fluorescence, declined in voltage-clamped fibres from old mice in the absence of external Ca(2+). Similarly, Ca(2+) transients declined in parallel with tetanic contractile force in single intact fibres. Examination of inward Ca(2+) current and of mRNA and protein assays suggest that these changes in EC coupling mode are not due to shifts in dihydropyridine receptor (DHPR) and/or ryanodine receptor (RyR) isoforms. These results indicate that a change in EC coupling mode occurs in a population of fibres in ageing skeletal muscle, and is responsible for the age-related dependence on extracellular Ca(2+).

Preservation of motor neuron Ca2+ channel sensitivity to insulin-like growth factor-1 in brain motor cortex from senescent rat.

Despite the multiple effects on mammals during development, the effectiveness of the insulin-like growth factor-1 (IGF-1) to sustain cell function and structure in the brain of senescent mammals is almost completely unknown. To address this issue, we investigated whether the effects of IGF-1 on specific targets are preserved at later stages of life. Voltage-gated Ca2+ channels (VGCC) are well-characterized targets of IGF-1. VGCC regulate membrane excitability and gene transcription along with other functions that have been found to be impaired in the brain of senescent rodents. As the voluntary control of movement has been reported to be altered in the elderly, we investigated the expression, function and responsiveness of high (HVA)- and low-voltage-activated (LVA) Ca2+ channels to IGF-1, using the whole-cell configuration of the patch-clamp and RT-PCR in the specific region of the rat motor cortex that controls hindlimb muscle movement. We detected the expression of alpha 1A, alpha 1B and alpha 1E genes encoding the HVA Ca2+ channels P/Q, N and R, respectively, but not alpha 1C, alpha 1D, alpha 1S encoding the L-type Ca2+ channel in this region of the brain cortex. IGF-1 enhanced Ca2+ channel currents through P/Q- and N-type channels but not significantly through the R-type or LVA channels. IGF-1 enhanced the amplitude but did not modify the voltage dependence of Ca2+ channel currents in young (2- to 4-week-old), young adult (7-month-old) and senescent (28- to 29-month-old) rats. These results support the concept that despite the reported decrease in circulating (liver) and local (central nervous system) production of IGF-1 with ageing, key neuronal targets such as the VGCC remain responsive to the growth factor throughout life.

Insulin-like growth factor-1 prevents age-related decrease in specific force and intracellular Ca2+ in single intact muscle fibres from transgenic mice.

In the present work we test the hypothesis that sustained transgenic overexpression of insulin-like growth factor-1 (IGF-1) in skeletal muscle prevents age-related decreases in myoplasmic Ca2+ concentration and consequently in specific force in single intact fibres from the flexor digitorum brevis (FDB) muscle from the mouse. Measurements of IGF-1 concentration in FDB muscle showed higher levels in transgenic than in wild-type mice at all ages. The specific tetanic force decreased significantly in single muscle fibres from old (286 +/- 22 kPa) compared to young wild-type (455 +/- 28 kPa), young transgenic (423 +/- 43 kPa), and old transgenic mice (386 +/- 15 kPa) (P < 0.05). These results are consistent with measurements in whole FDB muscles. The peak Ca2+ concentration values in response to prolonged stimulation were: 1.47 +/- 0.15, 1.70 +/- 0.29, 0.97 +/- 0.13 and 1.7 +/- 0.22 microM, in fibres from young wild-type, young transgenic, old wild-type and old transgenic mice, respectively. The effects of caffeine on FDB fibres support the conclusion that the age-related decline in peak myoplasmic Ca2+ and specific force is not explained by sarcoplasmic reticulum Ca2+ depletion. Immunohistochemistry in muscle cross-sections was performed to determine whether age and/or IGF-1 overexpression induce changes in fibre type composition. The relative percentages of type IIa, IIx and I myosin heavy chain (MHC) isoforms did not change significantly with age or genotype. Therefore, IGF-1 prevents age-related decline in peak intracellular Ca2+ and specific force in a muscle that does not exhibit changes in fibre type composition with senescence.

Insulin-like growth factor-1 increases skeletal muscle dihydropyridine receptor alpha 1S transcriptional activity by acting on the cAMP-response element-binding protein element of the promoter region.

Previous work from our laboratory has shown that insulin-like growth factor 1 (IGF-1) increases the expression of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1) subunit by regulating DHPR alpha(1S) nuclear transcription. In this study, we investigated the mechanism by which IGF-1 enhances expression of the DHPR alpha(1S) gene. To this end, the promoter region of the mouse DHPR alpha(1S) gene was recently cloned and sequenced and various promoter deletion-luciferase reporter constructs were used. These constructs were transfected into C2C12 cells and IGF-1 effects were measured by recording luciferase activity. IGF-1 significantly enhanced DHPR alpha(1S) transcription in those constructs carrying cAMP-response element-binding protein (CREB) binding site but not in CREB core binding site mutants. Gel mobility shift assay using a double stranded oligonucleotide for the CREB site in the promoter region, and competition experiments with excess unlabeled or mutated promoter oligonucleotide, and unlabeled consensus CREB oligonucleotide demonstrated that IGF-1 induces CREB binding to the DHPR alpha(1S) promoter. IGF-1-mediated enhancement in charge movement was prevented by incubating the cells with antisense but not with sense oligonucleotides against CREB. These results support the conclusion that IGF-1 regulates DHPR alpha(1S) transcription in muscle cells by acting on the CREB element of the promoter.

Charge movement and transcription regulation of L-type calcium channel alpha(1S) in skeletal muscle cells.

Several factors, such as Ca(2+), trophic factors and ageing, regulate dihydropyridine-sensitive receptor (DHPR) alpha(1) subunit expression. However, basic mechanisms of DHPR alpha(1S) expression are unknown. To better understand the regulatory elements that control transcription, the 1.2 kb 5'-flanking region fragment immediately upstream of the mouse L-type Ca(2+) channel or DHPR alpha(1S) gene was isolated and sequenced. Luciferase reporter constructs driven by different promoter regions of mouse DHPR alpha(1S) gene were used for transient transfection assays in muscle C2C12 cells. In these preparations we found that three regions corresponding to CREB, GATA-2 and SOX-5 consensus sequence within the 5'-flanking region of the DHPR alpha(1S) gene are important for DHPR alpha(1S) gene transcription. Antisense oligonucleotides against CREB, GATA-2 and SOX-5 significantly reduced charge movement in C2C12 cells. Charge movement was recorded in the whole-cell configuration of the patch clamp technique. Results from cells transfected with antisense (AS) and sense (S) oligonucleotides and nontransfected cells were compared. Charge movement experiments were fitted to a Boltzmann equation. Maximum charge movement (Q(max)) (nC microF(-1), mean +/- S.E.M.) for S- and AS-CREB was 70.3 +/- 2.9 and 52.8 +/- 3.3, respectively (P < 0.05). The same parameter for S- and AS-GATA-2 was 71.3 +/- 3.9 and 48.2 +/- 2.3, respectively (P < 0.05) and for S- and AS-SOX-5 was 70.4 +/- 4.2 and 45.1 +/- 3.2, respectively (P < 0.05). Values recorded in cells transfected with sense S-CREB, S-GATA-2 and S-SOX-5 oligonucleotides were not significantly different from those recorded in nontransfected cells. This study demonstrates that the transcription factors CREB, GATA-2 and SOX-5 play a significant role in the expression of the skeletal muscle DHPR or L-type Ca(2+) channel alpha(1S).