Immuno-molecular quantitation of β-isomyosin in Belgrade rats, a model of hypertrophic cardiomyopathy.

Objectives In hypertrophic cardiomyopathies, changes in the αand β isoform of cardiac myosin occur. These changes in a rat model of hypertrophic cardiomyopathy caused by autosomal recessive mutation of the “Divalent Metal Transporter-1” (DMT1) before and after development of cardiomyopathy by dietary iron overload were assessed by two immune-molecular methods.

Methods ELISAs and Immunoperoxidase method with antimyosin 2G42D7 specific for total myosin (tMyo) and R11D10 specific for β-isomyosin were used. Hearts from iron loaded homozygous Belgrade rats with at 11 weeks (heart/body weight =0.48+0.03) were compared to normal age-matched heterozygous Belgrade controls (Heteral, 0.3+0.01, p<0.01) and 4.5 weeks old homozygous Belgrade rats prior to iron overloading. Heart extracts and frozen section were prepared from these 3 groups of rats. After normalization to tMyo concentration in the heart extracts, the tMyo and β-isomyosin compositions were assessed by ELISAs and enhanced immunoperoxidase staining using HRP+ obtained from Akrivis Tech LLC.

Results β-isomyosin concentration in the 4.5 weeks old Belgrade rat hearts (0.3+0.13 μg/2.33 μg of tMyo) was no different from that of 11.5 weeks old Heteral hearts (0.45+0.37, p = NS). However, they were significantly lower than that of the 11.5 weeks old Belgrade hearts (0.92+0.7 μg, p < 0.001). The intensity of immunoperoxidase staining (% absorbance) for tMyo was no different in all 3 groups (20.59+2.33, 20.33+1.76 and 20.95+1.82, n=60 each group, p = NS) respectively. However, those of β-isomyosin were 5.09+1.99 and 4.64+1.69 for the 4.5 weeks Belgrade and 11.5 weeks Heteral hearts respectively (p = NS) and 13.08+2.02 in the 11.5 weeks Belgrade hearts (p <0.001).

Conclusions Monoclonal antimyosin antibodies with 2 different specificities can be used to quantitate the extent of changes in isomyosin expression from heart tissue extracts or by immunoperoxidase method. Quantitative increase in β-isomyosin in this experimental rat model is concordant with the development of hypertrophic cardiomyopathy.

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