ELECTROPHORETIC SEPARATION AND QUANTITATION OF CARDIAC MYOSIN HEAVY-CHAIN ISOFORMS IN 8 MAMMALIAN-SPECIES

A protocol for sample preparation and gel electrophoresis is described that reliably results in the separation of the alpha- and beta-isofor ms of cardiac myosin heavy chain (MHC-alpha and MHC-beta) in eight mam malian species. The protocol is based on a simple, nongradient denatur ing gel. The magnitude of separation of MHC-alpha and MHC-beta achieve d with this protocol is sufficient for quantitative determination of t he relative amounts of these two isoforms in mouse, rat, guinea pig, r abbit, canine, pig, baboon, and human myocardial samples. The sensitiv ity of the protocol is sufficient for the detection of MHC isoforms in samples at least as small as 1 mu g. The glycerol concentration in th e separating gel is an important factor for successfully separating MH C-alpha and MHC-beta in myocardial samples from different species. The effect of sample load on MHC-alpha and MHC-beta band resolution is il lustrated. The results also indicate that inclusion of a homogenizatio n step during sample preparation increases the amount of MHC detected on the gel for cardiac samples to a much greater extent than for skele tal muscle samples. Although the protocol described in this study is e xcellent for analyzing cardiac samples, it should be noted that the sa me protocol is not optimal for separating MHC isoforms expressed in sk eletal muscle, as is illustrated.