scholarly journals Limited functional and metabolic improvements in hypertrophic and healthy rat heart overexpressing the skeletal muscle isoform of SERCA1 by adenoviral gene transfer in vivo

2008 ◽  
Vol 295 (6) ◽  
pp. H2483-H2494 ◽  
Author(s):  
J. Michael O'Donnell ◽  
Aaron Fields ◽  
Xianyao Xu ◽  
Shamim A. K. Chowdhury ◽  
David L. Geenen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Rate Pressure Product ◽  
Substrate Selection ◽  
Nmr Analysis ◽  
Sprague Dawley ◽  
Important Distinction ◽  
Adenoviral Gene Transfer

Adenoviral gene transfer of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a to the hypertrophic heart in vivo has been consistently reported to lead to enhanced myocardial contractility. It is unknown if the faster skeletal muscle isoform, SERCA1, expressed in the whole heart in early failure, leads to similar improvements and whether metabolic requirements are maintained during an adrenergic challenge. In this study, Ad.cmv.SERCA1 was delivered in vivo to aortic banded and sham-operated Sprague-Dawley rat hearts. The total SERCA content increased 34%. At 48–72 h posttransfer, echocardiograms were acquired, hearts were excised and retrograded perfused, and hemodynamics were measured parallel to NMR measures of the phosphocreatine (PCr)-to-ATP ratio (PCr/ATP) and energy substrate selection at basal and high workloads (isoproterenol). In the Langendorff mode, the rate-pressure product was enhanced 27% with SERCA1 in hypertrophic hearts and 10% in shams. The adrenergic response to isoproterenol was significantly potentiated in both groups with SERCA1. 31P NMR analysis of PCr/ATP revealed that the ratio remained low in the hypertrophic group with SERCA1 overexpression and was not further compromised with adrenergic challenge. 13C NMR analysis revealed fat and carbohydrate oxidation were unaffected at basal with SERCA1 expression; however, there was a shift from fats to carbohydrates at higher workloads with SERCA1 in both groups. Transport of NADH-reducing equivalents into the mitochondria via the α-ketoglutamate-malate transporter was not affected by either SERCA1 overexpression or adrenergic challenge in both groups. Echocardiograms revealed an important distinction between in vivo versus ex vivo data. In contrast to previous SERCA2a studies, the echocardiogram data revealed that SERCA1 expression compromised function (fractional shortening) in the hypertrophic group. Shams were unaffected. While our ex vivo findings support much of the earlier cardiomyocyte and transgenic data, the in vivo data challenge previous reports of improved cardiac function in heart failure models after SERCA intervention.

scholarly journals Consequences of SUR2[A478V] Mutation in Skeletal Muscle of Murine Model of Cantu Syndrome

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1791
Author(s):  
Rosa Scala ◽  
Fatima Maqoud ◽  
Nicola Zizzo ◽  
Giuseppe Passantino ◽  
Antonietta Mele ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Katp Channel ◽  
Ex Vivo ◽  
Channel Modulation ◽  
Flexor Digitorum Brevis ◽  
Inflammatory Edema ◽  
Flexor Digitorum ◽  
Channel Currents

(1) Background: Cantu syndrome (CS) arises from gain-of-function (GOF) mutations in the ABCC9 and KCNJ8 genes, which encode ATP-sensitive K+ (KATP) channel subunits SUR2 and Kir6.1, respectively. Most CS patients have mutations in SUR2, the major component of skeletal muscle KATP, but the consequences of SUR2 GOF in skeletal muscle are unknown. (2) Methods: We performed in vivo and ex vivo characterization of skeletal muscle in heterozygous SUR2[A478V] (SUR2wt/AV) and homozygous SUR2[A478V] (SUR2AV/AV) CS mice. (3) Results: In SUR2wt/AV and SUR2AV/AV mice, forelimb strength and diaphragm amplitude movement were reduced; muscle echodensity was enhanced. KATP channel currents recorded in Flexor digitorum brevis fibers showed reduced MgATP-sensitivity in SUR2wt/AV, dramatically so in SUR2AV/AV mice; IC50 for MgATP inhibition of KATP currents were 1.9 ± 0.5 × 10−5 M in SUR2wt/AV and 8.6 ± 0.4 × 10−6 M in WT mice and was not measurable in SUR2AV/AV. A slight rightward shift of sensitivity to inhibition by glibenclamide was detected in SUR2AV/AV mice. Histopathological and qPCR analysis revealed atrophy of soleus and tibialis anterior muscles and up-regulation of atrogin-1 and MuRF1 mRNA in CS mice. (4) Conclusions: SUR2[A478V] “knock-in” mutation in mice impairs KATP channel modulation by MgATP, markedly so in SUR2AV/AV, with atrophy and non-inflammatory edema in different skeletal muscle phenotypes.

In Vivo and ex Vivo Approaches to Studying the Biomechanical Properties of Healing Wounds in Rat Skin

2013 ◽  
Vol 135 (10) ◽  
Author(s):  
Clare Y. L. Chao ◽  
Gabriel Y. F. Ng ◽  
Kwok-Kuen Cheung ◽  
Yong-Ping Zheng ◽  
Li-Ke Wang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Ex Vivo ◽  
Normal Skin ◽  
Biomechanical Properties ◽  
Skin Wound ◽  
Sprague Dawley ◽  
Rat Skin ◽  
Mechanical Stiffness ◽  
Air Jet

An evaluation of wound mechanics is crucial in reflecting the wound healing status. The present study examined the biomechanical properties of healing rat skin wounds in vivo and ex vivo. Thirty male Sprague-Dawley rats, each with a 6 mm full-thickness circular punch biopsied wound at both posterior hind limbs were used. The mechanical stiffness at both the central and margins of the wound was measured repeatedly in five rats over the same wound sites to monitor the longitudinal changes over time of before wounding, and on days 0, 3, 7, 10, 14, and 21 after wounding in vivo by using an optical coherence tomography-based air-jet indentation system. Five rats were euthanized at each time point, and the biomechanical properties of the wound tissues were assessed ex vivo using a tensiometer. At the central wound bed region, the stiffness measured by the air-jet system increased significantly from day 0 (17.2%), peaked at day 7 (208.3%), and then decreased progressively until day 21 (40.2%) as compared with baseline prewounding status. The biomechanical parameters of the skin wound samples measured by the tensiometer showed a marked reduction upon wounding, then increased with time (all p < 0.05). On day 21, the ultimate tensile strength of the skin wound tissue approached 50% of the normal skin; while the stiffness of tissue recovered at a faster rate, reaching 97% of its prewounded state. Our results suggested that it took less time for healing wound tissues to recover their stiffness than their maximal strength in rat skin. The stiffness of wound tissues measured by air-jet could be an indicator for monitoring wound healing and contraction.

Ex vivo gene transfer to mature skeletal muscle by using adenovirus helper cells

2004 ◽  
Vol 6 (2) ◽  
pp. 155-165
Author(s):  
Shigemi Kimura ◽  
Makoto Ikezawa ◽  
Baohong Cao ◽  
Yasunari Kanda ◽  
Ryan Pruchnic ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Helper Cells ◽  
Ex Vivo Gene Transfer

scholarly journals Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model

2021 ◽  
Vol 18 ◽  
pp. 347-354
Author(s):  
Masashi Noda ◽  
Kohei Tatsumi ◽  
Hideto Matsui ◽  
Yasunori Matsunari ◽  
Takeshi Sato ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Canine Model ◽  
Gene Transfer Techniques

Abstract 249: Delayed Gene Transfer Increases Transgene Expression in Vein Grafts

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Liang Du ◽  
Jingwan Zhang ◽  
Alexander Clowes ◽  
David Dichek
Keyword(s):  
Gene Expression ◽  
Blood Flow ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Arterial Blood Flow ◽  
Vein Grafts ◽  
Arterial Blood ◽  
En Face

Background Autogenous vein grafts are effective therapies for obstructive arterial disease. However, their long-term utility is limited by stenosis and occlusion. Genetic engineering of veins that prevents intimal hyperplasia and atherosclerosis could significantly improve the clinical utility of vein grafts. We recently reported that a helper-dependent adenoviral vector (HDAd) reduces atherosclerosis 4 wks after gene transfer in fat-fed rabbits and can express a therapeutic transgene (apo AI) in normal rabbit carotids for at least 48 wks. Use of HDAd for vein graft gene therapy will depend on achievement of similarly high and persistent transgene expression in grafted veins. Hypothesis We tested the hypothesis that Ad-mediated transgene expression in grafted veins (at an early time point) can be increased by varying the timing of gene transfer. Methods Rabbit external jugular veins were transduced by exposure to a beta galactosidase (b-gal)-expressing Ad: in situ either without (a) or with (b) immediate arterial grafting; c) ex vivo with grafting after overnight incubation with Ad; d) in vivo immediately after grafting and e) in vivo 4 wks after grafting (n = 6 - 19 veins/group). Transgene expression was measured in veins removed 3 d after Ad exposure by PCR quantitation of b-gal mRNA and by en-face planimetry of blue-stained area. Results B-gal transgene expression was higher in ungrafted veins than in veins grafted immediately after gene transfer (84 ± 17 vs 9.4 ± 2.0 arbitrary units (AU); P < 0.0001). Overnight incubation of veins with Ad increased gene expression ex vivo by 10-fold but neither this nor performing vector infusion immediately after grafting improved gene expression (11 ± 4.7 and 9.1 ± 1.8 AU; P > 0.9 for both vs immediately grafted veins). Delaying gene transfer until 4 wks after grafting significantly increased gene expression, to a level equivalent to transgene expression in ungrafted veins (61 ± 11 AU; P = 0.3 vs ungrafted veins). En face planimetry yielded similar results. Conclusions Exposure of a transduced vein to arterial blood flow is associated with significant loss of transgene expression. Transgene expression in grafted veins is significantly higher when gene transfer is performed 4 wks after exposure of the vein to arterial blood flow.

Skeletal muscle phenotype signaling with ex vivo endurance-type dynamic contractions in rat muscle

2021 ◽  
Author(s):  
Jesper Emil Jakobsgaard ◽  
Jacob Andresen ◽  
Frank V. de Paoli ◽  
Kristian Vissing
Keyword(s):  
Skeletal Muscle ◽  
Translation Initiation ◽  
Ex Vivo ◽  
Contractile Activity ◽  
Specific Protein ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Metabolic Signaling ◽  
Dynamic Endurance

Skeletal muscle phenotype may influence the response sensitivity of myocellular regulatory mechanisms to contractile activity. To examine this, we employed an ex vivo endurance-type dynamic contraction model to evaluate skeletal muscle phenotype-specific protein signaling responses in rat skeletal muscle. Preparations of slow-twitch soleus and fast-twitch extensor digitorum longus skeletal muscle from 4-wk old female Wistar rats were exposed to an identical ex vivo dynamic endurance-type contraction paradigm consisting of 40 minutes of stretch-shortening contractions under simultaneous low-frequency electrostimulation delivered in an intermittent pattern. Phosphorylation of proteins involved in metabolic signaling and signaling for translation initiation was evaluated at 0, 1, and 4 hours after stimulation by immunoblotting. For both muscle phenotypes, signaling related to metabolic events was upregulated immediately after stimulation, with concomitant absence of signaling for translation-initiation. Signaling for translation-initiation was then activated in both muscle phenotypes at 1-4 hours after stimulation, coinciding with attenuated metabolic signaling. The recognizable pattern of signaling responses support how our ex vivo dynamic muscle contraction model can be utilized to infer a stretch-shortening contraction pattern resembling stretch-shortening contraction of in vivo endurance exercise. Moreover, using this model, we observed that some specific signaling proteins adhering to metabolic events or to translation initation exhibited phosphorylation changes in a phenotype-dependent manner, whereas other signaling proteins exhibited phenotype-independent changes. These findings may aid the interpretation of myocellular signaling outcomes adhering to mixed muscle samples collected during human experimental trials.

In vivo leucine oxidation at rest and during two intensities of exercise

1982 ◽  
Vol 53 (4) ◽  
pp. 947-954 ◽  
Author(s):  
P. W. Lemon ◽  
F. J. Nagle ◽  
J. P. Mullin ◽  
N. J. Benevenga
Keyword(s):  
Skeletal Muscle ◽  
Specific Activity ◽  
Muscle Metabolism ◽  
Weighted Average ◽  
Mixed Diet ◽  
Sprague Dawley ◽  
Tracer Dose ◽  
Sprague Dawley Rats ◽  
14Co2 Production

After ingestion of a mixed diet containing a tracer dose (10 muCi) of L-[1–14C]leucine (Leu), 32 male Sprague-Dawley rats (70–90 g) remained at rest (R) or completed 1 h exercise at 80 (E80) or 40% VO2max (E40). 14CO2 production was assessed for 6 h (exercise occurred from h 2 to 3). Four rats were killed at 2, 3, 4, and 6 h (R), at 3 and 6 h (E80), and at 6 h (E40). Determinations were 1) tissue specific activity dpm X mumol-1 from a) mixed skeletal muscle (gastrocnemius, soleus, quadriceps, and hamstrings) and b) liver and 2) radioactivity remaining in the gastrointestinal tract (GIT). Leu oxidized (mumol) was estimated (14 CO2 dpm X tissue sp act dpm-1 X mumol-1) independently from skeletal muscle and liver. Results were 1) 14CO2 production increased in both E80 and E40 compared with R (P less than 0.05), 2) E80 14CO2 increase was greater than E40 (P less than 0.05), 3) GIT absorption was reduced in E80 and E40 compared with R (P less than 0.05), and 4) exercise Leu oxidation (weighted average of tissue estimates) was 26% greater than R (P less than 0.05). The origin and site of the increased Leu oxidation cannot be determined from the present data; however, due to the magnitude of increase in skeletal muscle metabolism relative to other tissues during exercise, it is probable that skeletal muscle plays a significant role.

scholarly journals Image-based modelling of skeletal muscle oxygenation

2017 ◽  
Vol 14 (127) ◽  
pp. 20160992 ◽  
Author(s):  
B. Zeller-Plumhoff ◽  
T. Roose ◽  
G. F. Clough ◽  
P. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Blood Vessel ◽  
Ex Vivo ◽  
Imaging Techniques ◽  
Muscle Oxygenation ◽  
Skeletal Muscle Oxygenation ◽  
Health And Disease ◽  
Vessel Networks

The supply of oxygen in sufficient quantity is vital for the correct functioning of all organs in the human body, in particular for skeletal muscle during exercise. Disease is often associated with both an inhibition of the microvascular supply capability and is thought to relate to changes in the structure of blood vessel networks. Different methods exist to investigate the influence of the microvascular structure on tissue oxygenation, varying over a range of application areas, i.e. biological in vivo and in vitro experiments, imaging and mathematical modelling. Ideally, all of these methods should be combined within the same framework in order to fully understand the processes involved. This review discusses the mathematical models of skeletal muscle oxygenation currently available that are based upon images taken of the muscle microvasculature in vivo and ex vivo . Imaging systems suitable for capturing the blood vessel networks are discussed and respective contrasting methods presented. The review further informs the association between anatomical characteristics in health and disease. With this review we give the reader a tool to understand and establish the workflow of developing an image-based model of skeletal muscle oxygenation. Finally, we give an outlook for improvements needed for measurements and imaging techniques to adequately investigate the microvascular capability for oxygen exchange.

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