Principal Investigator Yale University, Connecticut, United States
Introduction: Systolic heart failure, caused by insufficient contractility of the left ventricle, affects approximately 6.7 million Americans, and 1 in 4 adults will develop it in their lifetime (Bozkurt et al., 2023). Current therapies have low efficacy and there is no known cure. Cardiac myosin activators such as omecamtiv mecarbil (OM) and danicamtiv have been explored as potential therapies due to their high specificity in recruiting more myosin-actin cross-bridges, increasing the force of contraction. However, in a phase 3 clinical trial, OM was found to reduce hospitalizations by only five percent with no reduction in patient mortality.
Previous work in our laboratory utilizing engineered heart tissues (EHTs) with cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) has shown that OM increases contractile force but also slows muscle relaxation time (Shen et al., 2021). Slower diastole would hinder the refilling of the left ventricle, limiting potential improvements in the pumping of blood to the peripheral tissues.
Prior experiments have shown that intermittent, ‘pulsed’ administration of OM increases contractile force with less diastolic consequences, as compared to tissues treated with a constant dose. For the first time, we attempt to reproduce this phenomenon utilizing a different cardiac myosin activator, danicamtiv, in a female cell line.
Materials and
Methods: Porcine left ventricular tissue blocks were cryosectioned into 150 µm thick slices, laser-cut into 6 mm ribbon-shaped strips, and decellularized with 0.5% SDS in DPBS. The resulting scaffolds, composed of the remnant extracellular matrix, were clipped into polytetrafluoroethylene frames and seeded with hiPSC-CMs differentiated and expanded from a healthy female cell line, SH-5 (Figure 1). EHTs were seeded with 1 million cells: 90% hiPSC-CM, 10% human cardiac fibroblasts.
Following seeding, tissues followed traditional protocol and were cultured in DMEM+B27 for 14 days with three weekly media changed (M/W/F) before separation into four treatment groups for another 7 days of culture. ‘Vehicle’ tissues [n=8] were cultured in DMEM + 0.01% DMSO, ‘Constant – 1 µM’ (C-1µM) [n=7] and ‘Constant – 0.5 µM’ (C-0.5µM) [n=7] tissues were cultured in DMEM with their respective concentrations of danicamtiv, while ‘Pulse – 1 µM’ (P-1µM) [n=8] tissues were cultured with alternating 24-hour blocks of ‘treatment’ with DMEM + 1 µM danicamtiv and ‘washout’ with DMEM + 0.01% DMSO. Media was removed and replenished every 24 hours in all groups. After 7 days of group-dependent treatment, all tissues were subjected to a 24-hour ‘washout’ with DMEM + 0.01% DMSO prior to mechanical testing (Figure 2).
Mechanical testing was performed with a custom setup containing a force transducer and length controller, allowing for electrical pacing and manipulation of tissue stretch. Tissues from all groups were tested in DMEM + 0.01% DMSO, while C-1µM and P-1µM tissues were subjected to additional testing in DMEM + 1 µM danicamtiv.
Results, Conclusions, and Discussions: Measurements of peak force did not provide significant differences between groups but are to be repeated with greater sample size. When normalized by setting the force produced at 0% stretch to 1, the force-length response was consistent in all four groups from -5% to 5% stretch. From 5% stretch until 10% stretch, the force-length relationship in P-1µM tissues is much more consistent with that of the vehicle group compared to that of both constant-dosage groups.
As expected, tissues acutely treated with 1 µM danicamtiv showed marginally decreased time to peak and increased time from peak force to 50% relaxation [RT50] compared to those treated with 0.01% DMSO (0.1334±0.006 vs. 1.565±0.006 s in C-1µM; p=0.0058 [n=6], and 0.1231±0.005 vs. 0.1347±0.005 s in P-1µM; p=0.0346 [n=8]).
Most notably, P-1µM tissues displayed statistically significant smaller RT50 than C-1µM tissues, both with acute administration of 1 µM danicamtiv (0.1347±0.007 [n=8] vs. 0.1565±0.007 s [n=4]; p=0.0093) and in 0.01% DMSO (0.1231±0.005 [n=8] vs. 0.1334±0.005 s [n=6]; p=0.0447) (Figure 3). This indicates that cardiac muscle that has been intermittently treated with danicamtiv provides a faster, more physiological relaxation time during both ‘treatment’ and ‘washout’ periods, compared to that treated with a constant dose. In a human heart, faster diastolic kinetics would allow for increased filling of the ventricle, permitting more pumping of blood to peripheral tissues.
Forthcoming data include western blotting to compare the ratio of myosin to a housekeeping protein and measurements of cross-sectional area with OCT to investigate remodeling of the extracellular matrix and calculate stress. We also intend to perform RNA sequencing to compare the transcriptome during the end of a ‘pulse treatment’ with that at the end of a ‘washout’.
Acknowledgements (Optional): Special thanks to the Amgen Foundation and Yale BioMed SURF for funding my summer research experience, to Dr. Stuart G. Campbell for his constant mentorship and unwavering support, and to our wonderful stem cell technician Xia Li, for preparing and differentiating the various stem cell lines used in our laboratory.
