Sarcomere activation in striated muscle requires both thin filament–based and thick filament–based activation mechanisms. Recent studies have shown that myosin heads on the thick filaments undergo OFF to ON structural transitions in response to calcium (Ca2+) in permeabilized porcine myocardium in the presence of a small molecule inhibitor that eliminated active force. The changes in X-ray diffraction signatures of OFF to ON transitions were interpreted as Ca2+ acting to activate the thick filaments. Alternatively, Ca2+ binding to troponin could initiate a Ca2+-dependent crosstalk from the thin filament to the thick filament via interfilament connections such as the myosin binding protein-C. Here, we exchanged native troponin in permeabilized porcine myocardium for troponin containing the cTnC D65A mutation, which disallows the activation of troponin through Ca2+ binding to determine if Ca2+-dependent thick filament activation persists in the absence of thin filament activation. After the exchange protocol, over 95% of the Ca2+-activated force was eliminated. Equatorial intensity ratio increased significantly in both WT and D65A exchanged myocardium with increasing Ca2+ concentration. The degree of helical ordering of the myosin heads decreased by the same amount in WT and D65A myocardium when Ca2+ concentration increased. These results are consistent with a direct effect of Ca2+ in activating the thick filament rather than an indirect effect due to Ca2+-mediated crosstalk between the thick and thin filaments.
Calcium has a direct effect on thick filament activation in porcine myocardium
Disclosures: T.C. Irving reported personal fees from Edgewise Therapeutics and grants from Bristol Meyers Squibb outside the submitted work. W. Ma reported personal fees from Edgewise Therapeutics outside the submitted work. No other disclosures were reported.
This work is part of a special issue on Myofilament Structure and Function.
- Award Id(s): 1R01HL171657,5R01HL157169
- Award Id(s): P30 GM138395
- Award Id(s): P30 AR074990
- Award Id(s): DE-AC02-06CH11357
Saffie Mohran, Timothy S. McMillen, Christian Mandrycky, An-Yue Tu, Kristina B. Kooiker, Wenjing Qian, Stephanie Neys, Brayan Osegueda, Farid Moussavi-Harami, Thomas C. Irving, Michael Regnier, Weikang Ma; Calcium has a direct effect on thick filament activation in porcine myocardium. J Gen Physiol 4 November 2024; 156 (11): e202413545. doi: https://doi.org/10.1085/jgp.202413545
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