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Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogs.

Schenk HC, Krampfl K, Baumgärtner W, Tipold A - BMC Vet. Res. (2012)

Bottom Line: Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation.Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany. henning.schenk@freenet.de

ABSTRACT

Background: The recognition of functional muscular disorders, (e.g. channelopathies like Myotonia) is rising in veterinary neurology. Morphologic (e.g. histology) and even genetic based studies in these diseases are not able to elucidate the functional pathomechanism. As there is a deficit of knowledge and skills considering this special task, the aim of the current pilot study was to develop a canine muscle cell culture system derived from muscle biopsies of healthy client-owned dogs, which allows sampling of the biopsies under working conditions in the daily veterinary practise.

Results: Muscular biopsies from 16 dogs of different age and breed were taken during standard surgical procedures and were stored for one to three days at 4°C in a transport medium in order to simulate shipping conditions. Afterwards biopsies were professionally processed, including harvesting of satellite cells, inducing their proliferation, differentiating them into myotubes and recultivating myotubes after long-term storage in liquid nitrogen. Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation. Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.

Conclusion: We have developed a canine muscle cell culture system, which allows sampling of biopsies from young and old dogs of different breeds under practical conditions. Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

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Whole-cell currents recorded from a canine myotube (mature dog, id 13). Currents were recorded in response to a series of test potentials ranging from −50 mV to +45 mV in −5 mV steps after an initial hyperpolarisation to −120 mV from a holding potential of −100 mV.
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Figure 4: Whole-cell currents recorded from a canine myotube (mature dog, id 13). Currents were recorded in response to a series of test potentials ranging from −50 mV to +45 mV in −5 mV steps after an initial hyperpolarisation to −120 mV from a holding potential of −100 mV.

Mentions: To demonstrate the feasibility of patch-clamp measurements on the cultured canine myotubes, voltage-gated ion channels were evaluated in a pilot study. The applied series of depolarisation pulses initiated whole-cell currents of ion channels in 4 different cells grown out of 3 different muscle biopsies (dog id 5; 8; 13) (Figure 4). By the modalities of the used pulse protocol and the shape of the recorded currents we can state that voltage-gated channels were activated. Due to the small sample size of so far measured cells (n= 4) and the preliminary nature of the electrophysiological study neither evaluation of the kinetics of the recorded data nor the exact characterisation of these channels by different pharmacological tools were performed.


Canine muscle cell culture and consecutive patch-clamp measurements - a new approach to characterize muscular diseases in dogs.

Schenk HC, Krampfl K, Baumgärtner W, Tipold A - BMC Vet. Res. (2012)

Whole-cell currents recorded from a canine myotube (mature dog, id 13). Currents were recorded in response to a series of test potentials ranging from −50 mV to +45 mV in −5 mV steps after an initial hyperpolarisation to −120 mV from a holding potential of −100 mV.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539935&req=5

Figure 4: Whole-cell currents recorded from a canine myotube (mature dog, id 13). Currents were recorded in response to a series of test potentials ranging from −50 mV to +45 mV in −5 mV steps after an initial hyperpolarisation to −120 mV from a holding potential of −100 mV.
Mentions: To demonstrate the feasibility of patch-clamp measurements on the cultured canine myotubes, voltage-gated ion channels were evaluated in a pilot study. The applied series of depolarisation pulses initiated whole-cell currents of ion channels in 4 different cells grown out of 3 different muscle biopsies (dog id 5; 8; 13) (Figure 4). By the modalities of the used pulse protocol and the shape of the recorded currents we can state that voltage-gated channels were activated. Due to the small sample size of so far measured cells (n= 4) and the preliminary nature of the electrophysiological study neither evaluation of the kinetics of the recorded data nor the exact characterisation of these channels by different pharmacological tools were performed.

Bottom Line: Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation.Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover, Germany. henning.schenk@freenet.de

ABSTRACT

Background: The recognition of functional muscular disorders, (e.g. channelopathies like Myotonia) is rising in veterinary neurology. Morphologic (e.g. histology) and even genetic based studies in these diseases are not able to elucidate the functional pathomechanism. As there is a deficit of knowledge and skills considering this special task, the aim of the current pilot study was to develop a canine muscle cell culture system derived from muscle biopsies of healthy client-owned dogs, which allows sampling of the biopsies under working conditions in the daily veterinary practise.

Results: Muscular biopsies from 16 dogs of different age and breed were taken during standard surgical procedures and were stored for one to three days at 4°C in a transport medium in order to simulate shipping conditions. Afterwards biopsies were professionally processed, including harvesting of satellite cells, inducing their proliferation, differentiating them into myotubes and recultivating myotubes after long-term storage in liquid nitrogen. Myogenic origin of cultured cells was determined by immunofluorescence, immunohistology and by their typical morphology after inducing differentiation. Subsequent to the differentiation into myotubes feasibility of patch-clamp recordings of voltage gated ion channels was successfully.

Conclusion: We have developed a canine muscle cell culture system, which allows sampling of biopsies from young and old dogs of different breeds under practical conditions. Patch clamp measurements can be carried out with the cultured myotubes demonstrating potential of these cells as source for functional research.

Show MeSH
Related in: MedlinePlus