Unraveling cardiac mechanical anisotropy: Sarcomeric stiffness in the transverse and longitudinal directions.

Introduction:
Conditions such as heart failure with preserved ejection fraction involve pronounced changes in myocardial passive stiffness – a mechanical property not yet fully understood. Much of the uncertainty arises because ex vivo studies typically assess stiffness by measuring resistance to longitudinal stretch. However, this approach oversimplifies the complex in vivo milieu, where passive forces act in three dimensions. Since transverse stiffness is seldom examined, it is essential to elucidate the contributions of individual sarcomeric proteins to enable more accurate extrapolation from ex vivo findings to physiological conditions.

Methods:
In addition to conventional longitudinal stretch experiments, we employed atomic force microscopy nanoindentation to quantify transverse stiffness. Under paired-testing conditions, we measured stiffness changes following targeted disruption of major myofilament proteins. Quality control measures included immunofluorescence microscopy and SDS-PAGE. Depending on the targeted protein, we performed various treatments: For actin severing, we applied the gelsolin fragment GLN-40 to achieve near-complete F-actin fragmentation; elastic I-band titin was specifically cleaved in a unique transgenic titin cleavage (TC) mouse model by introducing tobacco-etch virus protease (TEVp); and depolymerization of titin-myosin composite filaments was accomplished by treatment with 1 M KCl. 

Results:
Longitudinal stretching of homozygous TC cardiac fiber bundles following TEVp treatment demonstrated a significant reduction of passive peak forces by 71.5%, 56.4%, 53.9%, and 41.3% at 10%, 15%, 20%, and 25% strain, respectively (p<0.001 for all). In isolated cardiomyocytes – absent an extracellular matrix – the decrease in passive stiffness was even larger, with an 83.8% reduction at 20% strain (p<0.001). Actin filaments accounted for 16.5% (p=0.044), 18.7% (p<0.001), 15.2% (p<0.001), and 17.3% (p<0.001) of longitudinal stiffness at 8%, 12%, 16%, and 20% strain in fiber bundles and 33.1% (p<0.001) in cardiomyocytes. Depolymerization of titin-myosin composite filaments revealed contributions of 59.2%, 50.5%, 54.2%, 52.6%, and 51.9% at 5%, 10%, 15%, 20%, and 25% strain, respectively (p<0.001 for all). In contrast, contributions to transverse stiffness differed substantially, highlighting the pronounced anisotropy of cardiac tissue. Atomic force microscopy on sectioned fiber bundles demonstrated that titin contributed only 24.6% (p<0.001) and 20.3% (p<0.001) to transverse passive stiffness at compressive loads of 2 nN and 4 nN, respectively, much less than its longitudinal contributions. Actin filaments contributed 25.7% (p<0.001) and 20.6% (p<0.001) at the same compression levels, slightly higher than their longitudinal roles. High-salt treatment produced reductions in stiffness of 35.6% (p<0.001) and 33.5% (p<0.001), indicating – along with the titin-cleavage results – that myosin exerts a greater influence on transverse than on longitudinal passive stiffness.

Outlook:
To our knowledge, this study is the first to elucidate the precise contributions of sarcomeric proteins to transverse myocardial passive stiffness. By comparing these findings with parallel investigations of longitudinal stiffness, we have quantified cardiac anisotropy. These insights may improve in vivo stiffness prediction, inform in silico modeling, and enhance the relevance of ex vivo studies of cardiac disease.