Discovery of Nelutroctiv (CK-136), a Selective Cardiac Troponin Activator for the Treatment of Cardiovascular Diseases Associated with Reduced Cardiac Contractility.

Cardiac myosin activation has been shown to be a viable approach for the treatment of heart failure with reduced ejection fraction. Here, we report the discovery of nelutroctiv (CK-136), a selective cardiac troponin activator intended for patients with cardiovascular conditions where cardiac contractility is reduced. Discovery of nelutroctiv began with a high-throughput screen that identified compound 1R, a muscle selective cardiac sarcomere activator devoid of phosphodiesterase-3 activity. Optimization of druglike properties for 1R led to the replacement of the sulfonamide and aniline substituents which resulted in improved pharmacokinetic (PK) profiles and a reduced potential for human drug-drug interactions. In vivo echocardiography assessment of the optimized leads showed concentration dependent increases in fractional shortening and an improved pharmacodynamic window compared to myosin activator CK-138. Overall, nelutroctiv was found to possess the desired selectivity, a favorable pharmacodynamic window relative to myosin activators, and a preclinical PK profile to support clinical development.

Sodium-glucose co-transporter 2 Inhibitors Act Independently of SGLT2 to Confer Benefit for Heart Failure with Reduced Ejection Fraction in Mice.

Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are novel, potent heart failure medications with an unknown mechanism of action. We sought to determine if the beneficial actions of SGLT2i in heart failure were on- or off-target, and related to metabolic reprogramming, including increased lipolysis and ketogenesis. The phenotype of mice treated with empagliflozin and genetically engineered mice constitutively lacking SGLT2 mirrored metabolic changes seen in human clinical trials (including reduced blood glucose, increased ketogenesis, and profound glucosuria). In a mouse heart failure model, SGLT2i treatment, but not generalized SGLT2 knockout, resulted in improved systolic function and reduced pathologic cardiac remodeling. SGLT2i treatment of the SGLT2 knockout mice sustained the cardiac benefits, demonstrating an off-target role for these drugs. This benefit is independent of metabolic changes, including ketosis. The mechanism of action and target of SGLT2i in HF remain elusive.

Titin-truncating variants in hiPSC cardiomyocytes induce pathogenic proteinopathy and sarcomere defects with preserved core contractile machinery.

Titin-truncating variants (TTNtv) are the single largest genetic cause of dilated cardiomyopathy (DCM). In this study we modeled disease phenotypes of A-band TTNtv-induced DCM in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using genome editing and tissue engineering technologies. Transcriptomic, cellular, and micro-tissue studies revealed that A-band TTNtv hiPSC-CMs exhibit pathogenic proteinopathy, sarcomere defects, aberrant Na+ channel activities, and contractile dysfunction. These phenotypes establish a dual mechanism of poison peptide effect and haploinsufficiency that collectively contribute to DCM pathogenesis. However, TTNtv cellular defects did not interfere with the function of the core contractile machinery, the actin-myosin-troponin-Ca2+ complex, and preserved the therapeutic mechanism of sarcomere modulators. Treatment of TTNtv cardiac micro-tissues with investigational sarcomere modulators augmented contractility and resulted in sustained transcriptomic changes that promote reversal of DCM disease signatures. Together, our findings elucidate the underlying pathogenic mechanisms of A-band TTNtv-induced DCM and demonstrate the validity of sarcomere modulators as potential therapeutics.

Structural and Thermodynamic Model for the Activation of Cardiac Troponin.

Cardiac troponin is a regulatory protein complex located on the sarcomere that regulates the engagement of myosin on actin filaments. Low-molecular weight modulators of troponin that bind allosterically with the calcium ion have the potential to improve cardiac contractility in patients with reduced cardiac function. Here we propose an approach to the rational design of troponin modulators through the combined use of solution nuclear magnetic resonance and isothermal titration calorimetry methods. In contrast to traditional approaches limited to calcium and activator-bound troponin structures, here we analyzed the structural and thermodynamic impact of an activator in the context of the troponin functional cycle. This led us to propose a rationale for developing an efficacious troponin activator.

MPFL reconstruction: technique and results.

Patellar instability is a common problem, and medial patellofemoral ligament (MPFL) injury is inherent with traumatic patellar dislocations. Initial nonoperative management is focused on reconditioning and strengthening the dynamic stabilizers of the patella. For those patients who progress to recurrent instability, further investigation into the predisposing factors is required. MPFL reconstruction is indicated in patients with recurrent instability and insufficient medial restraint due to MPFL injury. A technique of MPFL reconstruction is outlined. This procedure may also be performed in combination with other realignment procedures.

Suture placement near the musculotendinous junction in the supraspinatus: implications for rotator cuff repair.

BACKGROUND: Transosseous-equivalent rotator cuff repair has an increased incidence of medial rotator cuff failure compared with single-row repair. No studies have evaluated the influence of the proximity of the suture row to the musculotendinous junction (MTJ) on cyclic gapping and failure properties. HYPOTHESIS: A single row of horizontal mattress sutures placed within the supraspinatus tendon lateral to the MTJ will experience less gap formation and higher failure loads than a similar suture row placed at the MTJ. STUDY DESIGN: Controlled laboratory study. METHODS: Paired supraspinatus tendons were isolated from human cadaveric specimens and resected at the tendon insertion to the humerus. Randomized within a pair, a single row of 4 horizontal mattress sutures was placed either in the tendon 5 mm lateral to the MTJ or at the MTJ. The tied sutures secured the tendon to a fixture that ensured consistent placement of the suture row in the tendon and static fixation of the row. The muscle belly was gripped in a cryoclamp, and a servohydraulic materials testing machine was used to provide uniaxial tensile deformation for 500 cycles at 1 Hz, followed by load to failure at 1 mm/s. Fiducial markers with video tracking were used to quantify gap formation at the suture line, while the materials testing machine recorded loading for the cyclic and failure tests. RESULTS: During cyclic loading, both constructs experienced gross initial gap formation, followed by progressive gap formation that plateaued after cycle 200. The MTJ specimens had significantly higher mean cumulative gapping than the tendon specimens: 3.6±1.0 mm versus 2.4±0.6 mm, respectively (P=.012). The tendon specimens had significantly higher mean loads to failure than did the MTJ specimens: 567.1±121.8 N versus 434.2±148.1 N, respectively (P=.013). The mean failure displacement did not differ between groups for the tendon and MTJ: 5.7±2.5 mm versus 4.5±2.0 mm, respectively (P=.144). CONCLUSION: A horizontal suture row placed at the MTJ has inferior mechanical properties (increased gapping, decreased load support) as compared with a suture row placed 5 mm laterally within the tendon. CLINICAL RELEVANCE: The integrity of rotator cuff repair may be compromised if sutures are placed too close to the MTJ.

MPFL reconstruction: technique and results.

MPFL reconstruction is a viable option for treating patients with recurrent patellar instability, in whom nonoperative methods have failed to provide relief. It is important to evaluate patients for predisposing factors for patellar instability. This technique of MPFL reconstruction uses a reliable method to obtain anatomic tunnel position. Rigid fixation with interference fit in bone tunnels allows early range of motion and rehabilitation and minimizes concern for graft failure.

External fixation of femoral defects in athymic rats: Applications for human stem cell implantation and bone regeneration.

An appropriate animal model is critical for the research of stem/progenitor cell therapy and tissue engineering for bone regeneration in vivo. This study reports the design of an external fixator and its application to critical-sized femoral defects in athymic rats. The external fixator consists of clamps and screws that are readily available from hardware stores as well as Kirschner wires. A total of 35 rats underwent application of the external fixator with creation of a 6-mm bone defect in one femur of each animal. This model had been used in several separate studies, including implantation of collagen gel, umbilical cord blood mesenchymal stem cells, endothelial progenitor cells, or bone morphogenetic protein-2. One rat developed fracture at the proximal pin site and two rats developed deep tissue infection. Pin loosening was found in nine rats, but it only led to the failure of external fixation in two animals. In 8 to 10 weeks, various degrees of bone growth in the femoral defects were observed in different study groups, from full repair of the bone defect with bone morphogenetic protein-2 implantation to fibrous nonunion with collagen gel implantation. The external fixator used in these studies provided sufficient mechanical stability to the bone defects and had a comparable complication rate in athymic rats as in immunocompetent rats. The external fixator does not interfere with the natural environment of a bone defect. This model is particularly valuable for investigation of osteogenesis of human stem/progenitor cells in vivo.

Distinct phenotypes and regenerative potentials of early endothelial progenitor cells and outgrowth endothelial progenitor cells derived from umbilical cord blood.

The capability of postnatal neovascularization makes circulating endothelial progenitor cells (EPCs) promising for regenerative medicine and tissue engineering. Using EPCs isolated from umbilical cord blood, this study aimed to clarify the transition of functional properties from early EPCs (e-EPCs) to outgrowth EPCs (og-EPCs) for potential applications in regenerative medicine. Mononuclear cells were collected from umbilical cord blood via density gradient centrifugation and further negatively selected by CD45. EPCs were sorted from mononuclear cells by the expression of CD34. e-EPCs (7 days of culture) and og-EPCs (3 weeks of culture) were characterized by morphology, intake of acetylated low-density lipoprotein, vessel-cord formation, cell surface phenotype and the expression of angiogenic genes. e-EPCs and og-EPCs were also compared for osteogenic differentiation under the stimulation of BMP-2. Chemotaxis by SDF-1 was compared among og-EPCs and the first- and second-day attached e-EPCs. Based on the expression of angiogenic genes, e-EPCs possessed few angiogenic properties in vitro and og-EPCs were angiogenic. e-EPCs, however, expressed significant CXCR4 and migrated toward the SDF-1 gradient. og-ECPs did not express CXCR4 and showed no response to SDF-1. During culture, gaining an angiogenic phenotype by og-EPCs is associated with the loss of homing potential. These contrast properties determine different potentials of e-EPCs and og-EPCs in regenerative medicine.

Can computed tomography predict hip stability in posterior wall acetabular fractures?

BACKGROUND: In a pilot study, two-dimensional (2-D) CT assessment of posterior wall fracture fragments predicted hip stability with small fracture fragments and instability for large fracture fragments. QUESTIONS/PURPOSES: To confirm the previous findings, we determined whether there is sufficient observer consistency and accuracy to predict hip stability in posterior wall acetabular fractures for this CT assessment method and assessed its ease of clinical use. METHODS: We selected 10 fractures having variable characteristics with known clinical outcome and created three study participant groups, based on level of training, for evaluation. Each observer reviewed the CT scans from the 10 fractures and applied the method in two separate sessions, the second after at least a 1-month washout period. RESULTS: Participants reported subjective ease in using the method, averaging 5 minutes (range, 3-11 minutes) for each assessment. Intraobserver and interobserver reliability were both greater than 0.80 regardless of the level of experience. Although sensitivity was 90%, specificity was only 61% after comparison with examination under anesthesia (EUA). Inappropriate nonoperative treatment would have occurred in 6% of cases and inappropriate operative treatment in 16%. CONCLUSIONS: This method for assessing hip instability is reliable, reproducible, and easy to learn and use. However, as a diagnostic tool in the clinical setting, it is useful only for fractures involving greater than 50% of the posterior wall owing to limited accuracy. For fractures less than 50%, EUA should be performed to determine hip stability.

Comparison between rigid and flexible systems for drilling the femoral tunnel through an anteromedial portal in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to compare the differences in femoral tunnel length and distance to the lateral anatomic structures when using standard and flexible guide pins for anterior cruciate ligament (ACL) femoral tunnel drilling through a medial portal. METHODS: Using a medial arthroscopic portal in 10 cadaveric knees, we sequentially drilled straight and flexible guide pins into the center of the ACL femoral footprint using the same starting point. We recorded the interosseous length and distances to the peroneal nerve and the femoral origin of the lateral collateral ligament (LCL) for each pin. RESULTS: The mean interosseous length was 43.5 mm for the flexible pin and 37.1 mm for the straight pin (P = .01). The mean distance to the peroneal nerve was 42.3 mm for the flexible pin and 37.8 mm for the straight pin (P = .33). The mean distance to the femoral origin of the LCL was 26.1 mm for the flexible pin and 13.4 mm for the straight pin (P = .003). CONCLUSIONS: The use of commercially available flexible reamers and 42 degrees femoral guides results in longer femoral interosseous tunnel length than can be achieved with a straight guide pin. Femoral interosseous length consistently of 40 mm can be achieved with this technique and cannot be replicated with a rigid straight pin. This is advantageous for femoral tunnel drilling in an anatomic ACL reconstruction that uses suspensory fixation devices. There is minimal risk to the peroneal nerve and the femoral origin of the LCL unless lateral femoral wall blowout occurs. CLINICAL RELEVANCE: Flexible pins allow longer femoral tunnels and safer distances from the LCL by use of a medial portal technique.