Sarcomere length-dependent activation (sLDA) of cardiac filaments describes the maximum force performance and the calcium sensitivity, both of which increase with the sarcomere length during the diastolic state. This phenomenon constitutes the cellular basis of the Frenk-Starling law of the heart. Myofilament contractility is generated and regulated cooperatively by protein elements on both thin and thick filaments, such as myosin, actin, myosin binding protein C, tropomyosin, and troponin complex. Due to the significant clinical impact of sLDA, its underlying mechanism has been studied for decades, and the consensus is sarcomere stretches cause the interfilamentous spacing to decrease and the myosin heads to become closer to the actin filament, making it easier to form strong cross-bridge and generate higher maximum force. However, the mechanism for sarcomere length-dependent Ca2+ sensitivity regulation remains unclear and controversial. In general, the mechanism is associated with troponin. The troponin complex on the thin filament responds to intracellular calcium binding and releases the inhibition of the myosin-actin strong cross-bridge interaction from the combined inhibition of cTnI, cTnT, and Tm, thus triggering force contraction. Cardiac troponin T (cTnT) anchors the entire troponin complex on the thin filament by attaching it to both tropomyosin (Tm) and the actin filament. Cardiac troponin C (cTnC) binds the Ca2+ ion and interacts with cardiac troponin I (cTnI) to induce a conformational change on itsN domain, moving tropomyosin (Tm) away from the myosin binding sites on the actin filament. This allows the myosin heads to bind with actin filaments and initiate contraction. The troponin complex plays a key regulatory role in Ca2+-induced force generation, and it is likely to be involved in the regulation of Ca2+ sensitivity changes due to the sarcomere length changes.This study aims to investigate how the Ca2+ sensitivity of troponin responds to sarcomere stretching directly and how myosin and the N-domain of MyBP-C, another essential regulatory protein on the thick filament, affect the Ca2+ sensitivity. To study the sensitivity of the structural opening of cTnC/troponin in response to Ca2+ binding during [Ca2+] influx, a pair of fluorescent donor and acceptor was labeled to the 13C and 51C, respectively, of the N-domain cTnC to monitor structural opening uponCa2+ binding. A recombinant ΔSP-cTnI, in which the switch peptide (SP) region of cTnIis replaced by a non-functional G-linker, was designed to block strong cross-bridge formation during Ca2+ binding, thus enabling the study of SL-dependent Ca2+ sensitivity regulation without XB interference. Results show that when the strong XB of the filaments was blocked with ΔSP-cTnI, the SL-dependent Ca2+ remained the same as the filaments with strong XB formation. Myosin binding protein C (MyBP-C), another essential regulatory protein on the thick filament, affects SL-dependent Ca2+ sensitivity, as studied using a transgenic rat model with C0-C2 region truncation. Compared to the filaments of wild-type rats, the SL-dependent changes in Ca2+ sensitivity of the TG rats with N-terminal of MyBP-C truncated was reduced by 30%. To further study the significance of the troponin proteins in the signal transmission of the sarcomere stretching to protein conformational changes, two TEV digestion sites were inserted into the cTnT and cTnI proteins, each right in front of the IT arm of the troponin complex. The Ca2+ sensitivity became largely independent of the sarcomere length changes with the cleavage on the pre-IT arm region of the troponin complex.Our results suggest (1) SL-dependent Ca2+ sensitivity is regulated independently of strong cross-bridge formation but requires free myosin heads, (2) the N-terminal region of MyBP-C helps regulate the Ca2+ sensitivity during sarcomere stretching, and (3) the troponin complex is essential in the signal conversion of sarcomere stretching to Ca2+ sensitivity regulation. This study provides a unique perspective on the mechanism underlying sarcomere length-dependent activation and has established unique approaches for future studies to understand the LDA mechanism fully
