Length-dependent changes in contractile dynamics are blunted due to cardiac myosin binding protein-C ablation

Abstract

Enhanced cardiac contractile function with increased sarcomere length (SL) is, in part, mediated by a decrease in the radial distance between myosin heads and actin. The radial disposition of myosin heads relative to actin is modulated by cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C contributes to the length-dependent activation (LDA) in the myocardium. However, the precise roles of cMyBP-C in modulating cardiac LDA are unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca2+-sensitivity (pCa50) and length-dependent changes in kinetic parameters of cross-bridge (XB) relaxation (krel), and recruitment (kdf) due to rapid stretch, as well as the rate of force redevelopment (ktr) in response to a large slack-restretch maneuver in skinned ventricular multicellular preparations isolated from the hearts of wild-type (WT) and cMyBP-C knockout (KO) mice, at SL's 1.9 μm or 2.1 μm. Our results show that maximal force was not significantly different between KO and WT preparations but length-dependent increase in pCa50 was attenuated in the KO preparations. pCa50 was not significantly different between WT and KO preparations at long SL (5.82 ± 0.02 in WT vs. 5.87 ± 0.02 in KO), whereas pCa50 was significantly different between WT and KO preparations at short SL (5.71 ± 0.02 in WT vs. 5.80 ± 0.01 in KO; p < 0.05). The ktr, measured at half-maximal Ca2+-activation, was significantly accelerated at short SL in WT preparations (8.74 ± 0.56 s−1 at 1.9 μm vs. 5.71 ± 0.40 s−1 at 2.1 μm, p < 0.05). Furthermore, krel and kdf were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, ktr was not altered by changes in SL in KO preparations (8.03 ± 0.54 s−1 at 1.9 μm vs. 8.90 ± 0.37 s−1 at 2.1 μm). Similarly, KO preparations did not exhibit length-dependent changes in krel and kdf. Collectively, our data implicate cMyBP-C as an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL.

Impact of the Myosin Modulator Mavacamten on Force Generation and Cross‐Bridge Behavior in a Murine Model of Hypercontractility

“…Furthermore, Myk461 incubation did not alter cMyBPC phosphorylation status in WT samples (Figure 1B). Phosphorylation levels of cardiac troponin T and cardiac troponin I were not significantly different between WT and KO myocardial samples as previously reported29, 37 (Figure 1B). As expected, cMyBPC was absent in KO samples (Figure 1A).…”

“…Before Myk461 incubation, KO myocardium displayed accelerated k rel (≈107%) compared with WT myocardium (Tables 3 and 4). 29, 37 At pCa 6.1, Myk461 incubation did not alter k rel in WT myocardium, but slowed k rel by ≈36% and ≈51% in KO myocardium at 0.5 and 1.0 μmol/L Myk461, respectively, such that k rel was no longer different between WT and KO myocardium following Myk461 incubations (Table 3). Likewise, k rel was unaffected in WT myocardium at higher Ca 2+ activation (pCa 6.0), but was slowed by ≈27% and ≈35% in KO myocardium at 0.5 and 1.0 μmol/L Myk461, respectively (Table 4).…”

“…Pro‐Q Diamond phosphoprotein stain (Life Technologies) was used to assess myofilament protein phosphorylation in the cardiac samples as described previously29, 33 (Figure 1A). Cardiac myofibrils were prepared by homogenizing frozen ventricular tissue chunks for ≈15 s using a homogenizer (PowerGen 500; Thermo Fischer Scientific) in a relaxing solution containing protease and phosphatase inhibitors (PhosSTOP and cOmplete ULTRA tablets; Roche Applied Science).…”

“…Force‐pCa relationships were determined by measuring the forces generated by the skinned myocardial preparations in a range of pCa solutions that yield submaximal to maximal forces. The apparent cooperativity of force generation was estimated from the steepness of Hill plot transformation of the force‐pCa relationships 29. The force‐pCa data were fit using the equation P/P o =[Ca 2+ ] nH /(k nH +[Ca 2+ ] nH ), where n H is the Hill coefficient and k is the pCa needed to elicit half‐maximal force (ie, pCa 50 ) 29…”

“…Functionally, decreased incorporation of cMyBPC in human HCM samples expressing cMyBPC truncation mutations led to accelerated XB kinetics at submaximal Ca 2+ activations, in part, contributing to cardiac hypercontractility and hypertrophy 27. Mouse models of reduced cMyBPC expression or cMyBPC ablation also exhibit accelerated rates of XB recruitment and XB detachment rates at submaximal Ca 2+ activations,28, 29, 30 in part because reduced expression of cMyBPC radially displaces the myosin heads closer to actin, thereby effectively reducing the distance between actin and myosin and increasing the probability of acto‐myosin XB interactions 31. However, the impact of Myk461 on accelerated XB behavior induced by reduced cMyBPC expression has yet to be investigated.…”