이 작품은 JM, RO1-AR063084to NLW 및 JZ에 RO1-AR057404에 RO1-AG028614에 의해 지원되었다.

1. 삼투 스트레스 관류 시스템 설정 그림 1은 칼슘의 개략적 인 프로토콜이 그대로 골격 근육 섬유에 평가를 불꽃입니다. <ol><li> 두 채널의 최소 함유 관류 시스템의 출구 끝을 위치시킬 세 축 (XYZ) 미세 조작기를 설정. 방법 루어 - -에 및 / 또는 관류 솔루션의 흐름을 끄려면 코리아 크레인이 부착 된 세 개의 일회용 주사기 루어 배럴로 만들어 질 수 있었다. 이러한 관류 채널&gt; 0.2 mm 직경 단일 팁을 통해 관류 액&gt; 1 ㎖ / 분을 전달할 수 있어야한다. </li><li> 관류 시스템, 저장성 타이로드 솔루션을 등장 성 타이로드 해결 한 다른 두 개의 채널을로드합니다. </li></ol> 2. 마우스에서 준비 본래대로 단일 굴근 심지 브레비스 (FDB) 근육 섬유 <ol><li> 마우스 유럽 연합 (EU)에서 발을 제거발목 관절 위의 다리를 절단하여 무거운 해부 가위를 사용하여 NIH와 IACUC 지침에 따라 thanized. </li><li> 발바닥면이 위를 향하고 최소한의 칼슘 2 + 타이로드 솔루션으로 가득 해부 실에 발을 핀. </li><li> 힘줄 절단 조심스럽게 주위 조직에서 근육을 분리하는 힘줄을 당겨 FDB를 해부하다. </li><li> 근육의 깊은 층에 각각의 자리에가 가지 말초 힘줄을 절단하여 절개를 마칩니다. </li><li> 근육 섬유에 추가 손상을 방지 및 콜라게나 소화 솔루션의 해동 나누어지는이있는 튜브에 배치하기 위해 힘줄을 통해 집게와 함께 선택하여 FDB 근육을 이동 37 ℃로 데워진 </li><li> 160 rpm의 속도로 60 ~ 90 분 동안 37 ° C에서 진탕에 똑바로 FDB를 포함하는 튜브를 품어. 이 근육 번들 해리 프로토콜 실험적 동물의 다른 균주에 대해 검사 될 필요특히 막 손상에 취약 그 질병 / 돌연변이 섬유. </li><li> 등장 성 타이로드 솔루션의 700 μL와 튜브에 소화 FDB를 전송하고 부드럽게 팁을 제거하기 위해 점차적으로 깨끗한 면도날로 절단을 통해 직경을 감소 200 ㎖ - 마이크로 피펫 팁의 시리즈를 통해 근육을 여러 번 그려 섬유를 해제 씹다 200 ㎖의 마이크로 피펫의. 질병에서 특히 약한 근육에 손상 섬유를 방지하기 위해 끝이 섬유 다발이 고집하지 않고 통과 할 수 있도록 충분히 그냥 큰 있는지 확인합니다. 너무 작은 구멍을 통해 번들을 강요하지 마십시오. </li><li> 부드럽게 등장 성 1 ㎖를 포함하는 35mm 델타 TPG 요리의 중심에 컷 200 ㎖의 마이크로 피펫으로 도청 및 재현 탁 FDB 섬유 튜브 후 70 ㎖ (총 섬유의 1 / 10)를 인출하여 FDB 섬유를 플레이트 타이로드 솔루션은 요리를 통해 확산을 줄일 수 있습니다. </li><li> D에 그대로 단일 섬유의 수를 결정해부 현미경을 사용하여 ISH. 접시에 3 ~ 4 그대로 섬유를 얻기 위해 FDB 섬유의 추가 분취를 추가합니다. </li><li> 4 ° C에서 추가 격리 된 근육 섬유를 저장하고 6 시간 이내에 사용한다. </li></ol> 3. FLUO - 오전 4시 염료 로딩 및 CA 2 + 영상 (스파크 측정) <ol><li> 관 10 ㎖ 플루오 (Fluo) - 오전 4 주식 (1 ㎜)과에 전송 도금 FDB 섬유와 요리에서 등장 성 타이로드 용액 500 ㎖를 혼합하고 10 mm의 최종 플루오 (Fluo) - 오전 4 농도에 요리에 다시 추가합니다. 또는, 플루로 닉 산 염료 적재 효율을 증가시키기 위해 사용될 수있다. </li><li> 어둠 속에서 실온에서 60 분 동안로드합니다. </li><li> 주의 깊게 자르지 200 ㎖의 플라스틱 마이크로 피펫 팁과 요리에서 플루오 (Fluo) - 오전 4 함유 등장 성 타이로드 용액 500 ㎖를 그려 섬유를 세척하고 신선한 등장 성 타이로드 솔루션의 동일한 볼륨으로 교체. </li><li> 이 과정을 3 번 이상 반복합니다. </li><li> 미토콘드리아의 기능을 시각화하기 위해, 동위 원소 500 ㎖를 전송관 5 ㎖ TMRE 재고 (1 ㎜)로, 혼합, 50 nm의 최종 농도에 다시 요리에 추가로 FDB 섬유와 요리에서 NIC 타이로드 솔루션. </li><li> 10 분 동안 실온에서 배양한다. </li><li> 주의 깊게 500 ㎖를 그려 섬유를 세척 TMRE 함유 포경 200 ㎖의 플라스틱 마이크로 피펫 팁과 요리에서 등장 성 타이로드 솔루션 신선한 등장 성 타이로드 솔루션의 동일한 볼륨으로 교체. </li><li> 이 과정을 3 번 이상 반복합니다. </li><li> 공 초점 현미경에 접시를 전송하고 선명한 줄무늬와 실험을위한 부드러운 sarcolemmal 막과 그대로 섬유를 선택합니다. </li><li> 미세 조작기 컨트롤을 사용하여 멀리 대상 근육 섬유에서와 중앙 오프 관류 시스템을 400 ~ 500 μm의 끝을 배치합니다. </li><li> FDB 섬유는 그 자리에 체재 확인하기 위해 등장 성 타이로드 솔루션의 흐름을 시작합니다. </li><li> 40X 기름 침지 목적으로 플루오 (Fluo) -4에서 오전 형광 신호를 기록하고와 플루오 (Fluo) -4 오전 흥분아르곤 레이저 (여기 파장 488 nm의) 기록 방출 신호 (파장 510-580 nm의). 형광 신호의 세기는 내 Ca 2 + 농도 ([칼슘 2 +] I)의 상대 레벨에 비례한다. </li><li> 섬유상의 삼투압 스트레스를 생산하기 위해 세포 외 용액의 삼투압을 변경하여 칼슘 2 + 과도 상태를 유도한다. 처음에 붓기를 유도하기 위해 100 초 저장성 타이로드 용액 (170 mOsM)와 다음 등장 성 타이로드 용액 (290 mOsM) (60 초 기준 컬렉션)와 섬유를 perfuse합니다. 등장 성 타이로드 용액으로 FDB 섬유를 확대 Perfuse. 일반적으로, 오직 하나의 삼투압 충격 한 델타 TPG 접시 단일 그대로 섬유와 스파크 이벤트 데이터 수집을위한 마지막 10 분에 적용된다. </li><li> 166 LPS (초당 라인, 그리고 EA의 스캔 속도와 XY 이차원 이미지의 시계열을 사용하여 칼슘 2 + 불꽃 (그림 2)의 기록 공간 현지화등장 (1 분 각각의 조건에 대해 3.08 초 / 프레임의 결과로 512 픽셀)로 구성 채널 라인), 저장성 스트레스 (100 초) 및 사후 저장성 스트레스 (5 분). 오프라인 분석을 위해 저장 신호는 실험 종료 후 칼슘 2 + 불꽃의 분포 및 빈도를 알아보고자 하였다. </li><li> 새 접시에 새 섬유를 찾아 칼슘 2 + 과도을 유도하기 위해 동일한 삼투압 충격 프로토콜을 따릅니다. 직접 sarcolemmal 막 아래에 배치 된 공 초점 주사 라인 일분 (즉, 30,000 라인)에 대한 칼슘 2 + 과도를 기록하는 공 초점 라인 스캔 (XT) 모드 (길이 512 픽셀, 2 밀리 초 / 라인 스캔의 샘플 속도를 사용하여 (그림 3 ). 개별 칼슘 2 + 불꽃의 크기와 반응 속도의 분석을위한 linescans의 1 분 간격으로 세 가지를 순차적으로 ()을 기록하기 위해 서로 다른 초점 비행기로 라인 스캔을 변경합니다. </li></ol> 4. 데이터 분석 <ol><li> 다수 상환, 피크 시간 (FDHM에 시간, 상승 시간, XT 라인 스캔, 개별 칼슘 2 + 불꽃 매개 변수의 형태와 반응 속도를 평가 진폭, 즉 (F 0 휴식 칼슘 2 + 형광입니다 F / F 0)에 추적 , 절반 크기의 전체 지속 시간), 기록 된 스파크의 공간 폭 (FWHM, 절반 크기의 전체 폭). 이러한 매개 변수는 칼슘 2 + 플럭스 (진폭)와 같은 RYR 칼슘 2 + 채널의 기본 게이트의 속성을 나타내고, RYRs (기간)의 게이트 속도론. </li><li> 이전에 11,16 바와 같이, 이미지 프로세싱 언어 IDL (대화 형 데이터 언어)을 사용하여 만든 사용자 정의 개발, 반자동 알고리즘으로 디지털 이미지 데이터를 분석합니다. 칼슘 2 +의 고유 반응 속도는 삼투압 스트레스에 의해 FDB 섬유에 의한 칼슘 + 스파크의 경우에 방출하기 때문에 2 + 이벤트를 촉발 CA의 수동 및 자동 기능을 결합하는 것이 좋습니다이러한 사용자 지정 빌드 이미지 분석 프로그램 11 식별 및 처리. 그것은 수동으로 일부 근육 질환 모델에서 ROI (관심 지역)를 식별하는 것이 필요 할 때이 알고리즘은 매우 유용합니다. 투자 수익 (ROI)이 정의되면,이 알고리즘은 자동으로 다음 Excel 파일로 내보낼 수 있습니다 위에서 언급 한 불꽃 매개 변수를 도출 할 수 있습니다. 대안 적으로, 공개 가능 화상 처리 소프트웨어, ImageJ에있는 예 SparkMaster는 칼슘 2 + 불꽃의 운동 특성과 골격근 (17)에서의 공간 분포를 정의하는데 사용될 수있다. </li></ol>

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Biol. 174 (5), 639-645 (2006).</li><li>Weisleder, N., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Ca2++sparks+in+aging+skeletal+and+cardiac+muscle.">Altered Ca2+ sparks in aging skeletal and cardiac muscle.</a> Ageing Res. Rev. 7 (3), 177-188 (2008).</li><li>Yi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+uptake+regulates+rapid+calcium+transients+in+skeletal+muscle+during+excitation-contraction+(E-C)+coupling.">Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling.</a> J. Biol. Chem. 286 (37), 32436-32443 (2011).</li><li>Tjondrokoesoemo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+inositol+(1,4,5)-trisphosphate+receptor+activates+ryanodine+receptor+1+to+mediate+calcium+spark+signaling+in+adult+Mammalian+skeletal+muscle.">Type 1 inositol (1,4,5)-trisphosphate receptor activates ryanodine receptor 1 to mediate calcium spark signaling in adult Mammalian skeletal muscle.</a> J. Biol. Chem. 288, 2103-2109 (2013).</li><li>Martins, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+contribute+to+Ca2++signals+produced+by+osmotic+stress+in+mouse+skeletal+muscle+fibres.">Reactive oxygen species contribute to Ca2+ signals produced by osmotic stress in mouse skeletal muscle fibres.</a> J. Physiol. 586 (1), 197-210 (2008).</li><li>Apostol, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+calcium+signals+induced+by+hyper-osmotic+stress+in+mammalian+skeletal+muscle+cells.">Local calcium signals induced by hyper-osmotic stress in mammalian skeletal muscle cells.</a> J. Muscle Res. Cell Motil. 30 (3-4), 97-109 (2009).</li><li>Jiang, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+acid+adenine+dinucleotide+phosphate+(NAADP)+Activates+Global+and+Heterogeneous+Local+Ca2++Signals+from+NAADP-+and+Ryanodine+Receptor-gated+Ca2++Stores+in+Pulmonary+Arterial+myocytes.">Nicotinic acid adenine dinucleotide phosphate (NAADP) Activates Global and Heterogeneous Local Ca2+ Signals from NAADP- and Ryanodine Receptor-gated Ca2+ Stores in Pulmonary Arterial myocytes.</a> J. Biol. Chem. 288 (15), 10381-10394 (2013).</li></ol>

관심 없음 충돌 선언하지 않습니다.

그대로 골격 근육에 칼슘 2 + 불꽃을 평가하는이 방법은 근육의 생리 및 질병 연구를위한 유용한 도구입니다. 우리는 칼슘 2 + 스파크 응답 (18, 19)뿐만 아니라, 근 위축성 측삭 경화증 (20)을 에이징, 근이영양증 (11) 등, 다른 조건으로 변경 한 것으로 나타났다. 우리의 최근의 연구는 또한 캘리포니아에 골격 근육 섬유 (21) 2 + 불꽃 활성화를 ?...

세포 내 무료 칼슘 2 + ([칼슘 2 +] I) (검토를 위해 Stutzmann과 맛 토손 1 참조)와 같은 신경, 심장, 골격 및 평활근과 같은 흥분 세포에서 여러 세포 기능을 조절하는 다양하고 중요한 보조 메신저입니다. 근질 세망 (SR) 및 T-세관 사이에 칼슘 2 + 동원 및 크로스 토크 (cross-talk)를 규제 (TT) 멤브레인은 근육 생리학의 기본 레귤레이터이다. 또한, 칼슘 2 + 신호의 변화는 다양한 근육 질환의 수축 기능 장애의 기본 메커니즘으로 표시했다. 칼슘 2 + 불꽃이 근질 세망 (SR) 막에 대한 ryanodine 수용체 (RYR) 채널의 오프닝에서 발생하는 초등학교 칼슘 2 + 출시 이벤트를 지역화됩니다. 심장 근육에, 불꽃은 칼슘 2 +에 의한 칼슘 + RELE으로 RyR2 채널의 개방을 통해 자발적으로 발생ASE (CICR) 메커니즘 3-5. 골격근에서 RyR1 엄격히 TT 멤브레인 6,7에서, 전압 센서에 의해 제어된다. 그대로 골격 근육 섬유의 휴식 조건에 따라서 칼슘 2 + 불꽃이 억제되어 거의 검출되지. + 골격 근육 8,9 검출 할 이벤트를 촉발를 최근까지, 필요한 sarcolemmal 막은 RyR1의 전압 센서의 억제를 풀다하기 위해 다양한 화학 물질 또는 기계 &quot;스키닝&quot;방법에 의해 중단 될 수 및 칼슘 허용. 하나의 방법은 이전에 사포닌 세제 (10)에 의해 근육 섬유의 멤브레인의 필수 permeabilization을 설명했다. 2003 년, 우리는 과도 hypoosmotic 스트레스 또는 고 삼투압 스트레스 중 하나는 2 +은 그대로 근육 섬유 (11)의 sarcolemmal 막에 인접한 불꽃 주변 칼슘을 유도 할 수 있다는 것을 발견했다. 이 방법은 이후 칼슘 2 +의 분자 메커니즘 및 변조를 연구하도록 수정되었습니다 </suP&gt; 출시 및 역학 12-16. 여기에서 우리는 그대로 골격 근육에있는 칼슘 2 + 불꽃의 유도, 탐지 및 분석을위한 우리의 실험 방법의 세부 사항을 설명합니다. 우리는 또한 골격 근육, 예를 들어 불꽃 주파수와 진폭 RYR 채널의 개방 가능성을 반영 (Δ F/F0, 개별 칼슘 2 + 불꽃의 원소 속성을 정량화하는 데 사용할 수있는 우리의 맞춤형 스파크 분석 프로그램을 제시하고 SR 내부의 칼슘 2 +로드), 피크 시간 (상승 시간) 및 기간 (FDHM, 스파크의 절반 최대 진폭의 전 기간)뿐만 아니라, 칼슘 2 + 불꽃의 공간적 분포. 또한, 우리는 근육 퇴행 위축과 근 위축성 측삭 경화증과 같은 골격 근육의 다양한 병태 생리 학적 상태에 칼슘 2 + 불꽃을 변경 링크 증거를 제시한다. 이 기술의 장점은 상대적 undam에서 칼슘 2 +를 측정하는 능력을 포함대신, 근육 섬유가 벗겨진 생리적으로 조건에서 기록의 Ca 2 + 불꽃 가까이 허용의 세 세포. 또한, 우리의 맞춤 설계 프로그램은 근육 섬유 관련하여 불꽃의 특성을보다 정확하게 계산을 제공합니다.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td style="width:367px;">Dissect out fibers and check muscle integrity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Dissection tools -scissors, and fine tip forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>DOW CORNING</td>
			<td style="width:119px;">184 SIL ELAST KIT</td>
			<td style="width:367px;">&#160;Make ahead of time for fiber dissection. Mix 5 ml liquid Sylgard components and pour into a 60-mm glass Petri dish and waiting 48 hr to let the Sylgard compound become clear, colorless solid substrate.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60-mm glass petri dish&#160;</td>
			<td style="width:111px;">Pyrex</td>
			<td style="width:119px;">3160</td>
			<td>Fiber dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isotonic Tyrode Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Minimal Ca2+ Tyrode Solution</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hypotonic Tyrode Solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">collagenase type I&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C-5138</td>
			<td>Fiber digestion&#160;</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fluo-4 AM&#160;</td>
			<td style="width:111px;">Invitrogen</td>
			<td>F14201</td>
			<td>Fluorescent Ca2+ imaging dyes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMRE</td>
			<td style="width:111px;">Invitrogen</td>
			<td>T669</td>
			<td>mitochondrial membrane potential fluorescent dyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delta TPG dishes&#160;</td>
			<td>Bioptechs</td>
			<td style="width:119px;">0420041500C</td>
			<td>35 mm glass bottom dish for imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Spark Fit software</td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td>data analysis software</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:195px;">Radiance 2100 laser scanning confocal microscope or equivalent microscope</td>
			<td style="width:111px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">3 axis micromanipulator</td>
			<td style="width:111px;">Narishige International</td>
			<td style="width:119px;">MHW-3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">temperature controllable orbital shaker</td>
			<td style="width:111px;">New Brunswick scientific</td>
			<td style="width:119px;"></td>
			<td>Fiber dissociation</td>
		</tr>
	</tbody>
</table>

assessment of calcium sparks in intact skeletal muscle fibers

항상성 칼슘 2 + 신호를 유지하는 것은 살아있는 세포의 기본적인 생리적 과정이다. 칼슘 2 + 불꽃은 매우 근질 세망 (SR) 막에 채널을 해제 + (RYR) CA ryanodine 수용체에 의해 매개되는 칼슘 2 + 출시 이벤트 2를 지역화 나타나는 가로 무늬 근육 섬유에 신호 + 칼슘의 기본 단위입니다. 적절한 평가 근육의 칼슘 2 + 불꽃 건강하고 병에 걸린 줄이있는 근육의 세포 내 칼슘 2 + 처리 특성에 대한 정보를 제공 할 수 있습니다. 칼슘 2 + 이벤트는 일반적으로 휴면 심근 볼 수있는 불꽃 있지만, 그들은 거의 골격근 섬유 휴식에서 관찰되며, 따라서 골격근 섬유에서 스파크를 생성하고 분석하는 방법에 대한 필요성이있다. 여기에 상세한 F를 사용하여 격리 된 굴근 digitorm 브레비스 (FDB) 근육 섬유에 칼슘 2 + 불꽃을 측정하기위한 실험 프로토콜입니다luorescent 칼슘 2 + 인 지표 및 레이저 스캐닝 공 초점 현미경. 이 방법에서는, 고립 된 FDB 섬유는 등장 성 생리 솔루션에 반환 한 다음 과도 hypoosmotic 스트레스에 노출되어 있습니다. 이러한 조건에서, 강력한 칼슘 2 +는 응답이 젊은 건강한 FDB 근육 섬유 소재 sarcolemmal 막에 인접한 감지 불꽃. 변경된 칼슘 2 +는 응답이 이영 또는 세 골격 근육 섬유에서 검출 불꽃. 이 방법은 최근에 막 분리 된 신호 이노시톨 사이의 크로스 토크 (cross-talk) (1,4,5)-삼인산 수용체 (IP 3 R)와 관련된 것을 증명하고 RYR 캘리포니아 골격근 2 + 불꽃의 활성화에 기여한다. 요약하면, 삼투압 스트레스를 사용하여 우리의 연구는 칼슘을 유도 2 + 불꽃이 세포 내 반응은 근육 생리학 메커니즘을 신호와 마우스 근육 영양 장애의 모델 (MDX 마우스) 또는 근 위축성 측삭 경화증 (ALS 모델 포함 / 질병 상태, 노화를 반영하는 것으로 나타났다).

이전 연구는 과도 hypoosmotic 스트레스 + 주변 칼슘 11. 그림 1은 부드러운 sarcolemmal 막과 특성 분명 줄무늬와 그대로 하나의 근육 섬유의 이미지를 보여줍니다 그대로 근육 섬유의 sarcolemmal 막에 인접한 2 불꽃에 의한 것으로 나타났다. 그림 2는 전형적인 칼슘 2 + 표시 (XY 이미지) 불꽃은 젊고 건강한 쥐에서 FDB 근육 섬유를 팽윤 hypoosmoti...

Watch this Scientific Journal Video about Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers at JoVE.com

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

직접 칼슘 불꽃을 측정하는 방법이 여기에 설명, 칼슘 2 +의 기본 단위는 그대로 골격 근육 섬유의 근질 세망에서 놓습니다. 이 방법은 고립 된 근육 섬유의 ryanodine 수용체에서 칼슘 2 + 릴리스의 삼투 스트레스 매개 트리거를 사용합니다. 역학과 내 Ca 2 + 신호 전달의 항상성 용량은 건강과 질환에서 근육 기능을 평가하기 위해 사용될 수있다.

칼슘의 평가는 그대로 골격 근육 섬유에서 불꽃

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

assessment-of-calcium-sparks-in-intact-skeletal-muscle-fibers

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Biology

15.1K 조회수.  The Ohio State University Wexner Medical Center.  직접 칼슘 불꽃을 측정하는 방법이 여기에 설명, 칼슘 2 +의 기본 단위는 그대로 골격 근육 섬유의 근질 세망에서 놓습니다. 이 방법은 고립 된 근육 섬유의 ryanodine 수용체에서 칼슘 2 + 릴리스의 삼투 스트레스 매개 트리거를 사용합니다. 역학과 내 Ca 2 + 신호 전달의 항상성 용량은 건강과 질환에서 근육 기능을 평가하기 위해 사용될 ?...

비디오: Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

성인 및 배아 골격근의 Microexplant 문화 및 골격 근육 줄기 세포의 분리

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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생물학

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

사포닌 - permeabilized 심장 섬유에 Respirometric 산화 인산화 평가

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

골격근에서 Mitochondrial 절연

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

골격근에서 Progenitors의 정화

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

라이브 세포의 저장소로 작동하는 칼슘 항목의 형광 기반의 측정 : 교양 암 세포에서 골격근 섬유에

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

절연 골격근의 수축성, Fatigability 및 Alternans의 예 VIVO 평가

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Zebrafish 배아 골격근의 레이저 쏜 부상

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

고해상도 호흡율 측정을위한 차동 원심 분리에 의한 골격근에서 본래 미토콘드리아의 분리

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

플라스미드 DNA를 마우스 골격근으로 전기천공

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

말 골격근의 고분해능 불소 호흡 측정

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...