노인과 성인의 단일 근섬유 아형에 따른 수축 특성과 형태의 비교
Age-related changes in contractile properties and morphology on chemically skinned single fibers from young and old human skeletal muscles.
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일반적으로 노화로 인한 근기능의 저하는 근육량 감소와 더불어 근신경계의 기능부전 등으로 인식되고 있지만, 단일 근섬유의 본질적 수축력에 대한 노화의 효과는 여전히 논란이 되고 있다. 노화로 인한 단일 근섬유의 질적 변화를 규명하기 위해, 본 연구에서는 건강한 노인의 골격근을 단일 근섬유로 분리한 뒤, 수축 특성과 횡단면적을 MHC의 발현 유형에 따라 일반 성인의 골격근과 비교하였다. 근생검법을 이용하여 성인 10명(25±2세)과 노인 8명(78±2세)의 외측광근으로부터 근육표본을 추출한 뒤, 이완 용액에서 손상되지 않게 종단면으로 분리한 후, 분리용액에서 24시간 배양(4 °C)하였다. 배양 후 근막구조가 제거된 근다발로 부터 단일 근섬유를 분리하여 실험 장치(등척성 근력 측정기와 고속 모터)에 고정하고 일정한 근절길이(2.6 µm)를 유지하여, 근섬유 길이와 횡단면적을 측정하였다. 활성용액(pCa 4.5)을 사용하여 근섬유를 최대 수축시킨 후 slack검사를 통하여 최대 수축력과 최대 수축 속도를 측정하였다. 측정이 완료된 단일 근섬유 절편은 SDS-PAGE를 이용하여 MHC의 아형을 확인하였다. 실험결과에 따르면, 노인의 type I 근섬유는 일반 성인의 동일아형 근섬유와 비교하였을 때 횡단면적과 최대 수축력(mN)에서 유의한 증가가 나타났다(p< .05). 하지만 횡단면적으로 표준화된 수축력(kN/m2)에서 이러한 차이는 나타나지 않았다. Type IIa 그리고 type IIa/IIx 근섬유에서는 연령에 따른 횡단면적과 최대수축력(mN), 그리고 표준화된 수축력(kN/m2)에서 연령 간 통계적 차이가 없는 것으로 나타났다. 근섬유의 횡단면적과 근력생성능력간의 관계를 분석하기 위한 선형회귀분석 결과에서도, 두 연령 그룹 모두 유의한 선형관계를 나타내었고(p< .001), 그룹 간 유의한 차이가 없었다. 이러한 결과들은 흥분-수축 결합을 배제한 단일 세포 수준에서의 수축력은, type I 근섬유의 형태학적 보상효과와 더불어 노화와 관계없이 유지됨을 증명한다. 따라서 노화에 따른 근기능 저하는 근육 세포의 질적 퇴화보다는, 주로 근육 세포수 감소와 같은 양적 요인에 의한 것으로 사료된다.
Abstract
Studies using human model reported inconsistent results on the intrinsic contractile properties of single muscle fiber. To extensively test aging effect on the innate myofilament characteristics and morphology, functional assay of chemically skinned vastus lateralis muscle fiber(15 °C) were investigated from young(25±2 yrs) and old(78±2 yrs) healthy human subjects(N=10 and 8 for young, and old respectively). Fiber cross sectional area(CSA) was measured by 3 dimensional microscopy, peak Ca2+-activated force and maximal shortening velocity(Vo) were measured by slack test procedure. Fiber myosin heavy chain(MHC) isoform was determined by gel electrophoresis and silver staining. Single muscle fiber assays showed fiber CSA and maximal Ca2+-activated force(mN) in old adults type I fiber were 15 % bigger and 18 % greater compared to the corresponding MHC isoform fibers from young adults(p< .05). However, specific force(maximal Ca2+-activated force normalized by fiber CSA, kN/m2) of type I fiber was identical between age group, which suggested that the greater Ca2+-activated force(mN) of type I fiber is resulted from the bigger fiber CSA. Otherwise, type IIa and IIa/IIx fibers showed no difference on fiber CSA and Po between age groups. There is no difference on shortening velocity in type I and IIa fibers between young and old, type IIa/IIx fiber in old adults showed substantially slower Vo than young adults type IIa/IIx fibers(33 %, p< .05). Linear regression modeling to identify relationship between fiber CSA and force generating ability showed strong direct relationship(p< .001) with no aging effect. Therefore, our result indicated maintained contractile properties of myofilament lattice regardless of age. Collectively, the declines of the muscular strength with aging appeared to be due to the quantitative reduction rather than qualitative impairment of single muscle fiber.
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