In this report, we establish a straightforward method for estimating the equilibrium constant for the creatine kinase reaction (CK K_eq″) over wide but physiologically and experimentally relevant ranges of pH, Mg²⁺ and temperature. Our empirical formula for CK K_eq″ is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we were able to obtain improved estimates of ΔG_ATP, necessitating the reinvestigation of previously reported ADP- and ΔG_ATP-dependent processes. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies and pH is not altered, the maximum Ca²⁺-activated isometric force depends on ΔG_ATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis around the role of submicromolar ADP in force generation.

In this report, we establish a straightforward method for estimating the equilibrium constant for the CK reaction over wide but physiologically and experimentally relevant ranges of pH, Mg2+ and temperature. Our empirical formula for CK Keq” is based on experimental measurements. It can be used to estimate [ADP] when [ADP] is below the resolution of experimental measurements, a typical situation because [ADP] is on the order of micromolar concentrations in living cells, and may be much lower in many in vitro experiments. Accurate prediction of [ADP] is essential for in vivo studies of cellular energetics and metabolism, and for in vitro studies of ATP-dependent enzyme function under near-physiological conditions. With [ADP], we could estimate ΔGATP. Application to actomyosin force generation in muscle provides support for the hypothesis that, when [Pi] varies but not when pH is altered, maximum Ca2+-activated isometric force depends on ΔGATP in both living and permeabilized muscle preparations. Further analysis of the pH studies introduces a novel hypothesis for the role of submicromolar ADP in force generation.

Mutations in genes encoding sarcomeric proteins are a common cause of cardiomyopathy and sudden cardiac death in humans. We evaluated the hypothesis that myofilament dysfunction is coupled to morphological and functional alterations of cardiomyocyte nuclei in a Tnnc1-targeted knock-in (Tnnc1-p.A8V) mouse model of hypertrophic cardiomyopathy (HCM). Tnnc1 is the gene that codes for the isoform of the Ca²⁺-regulatory protein troponin C (cTnC) that is expressed in cardiomyocytes and slow skeletal muscle fibers and resides on thin filaments of sarcomeres in those muscles. This pathogenic mutation in a sarcomere gene alters many aspects of cardiomyocyte function, including sarcomere contractility, cytoplasmic Ca²⁺ buffering, and gene expression. Analysis of myocardial histological sections and isolated cardiomyocytes from adult Tnnc1-p.A8V mouse hearts revealed significantly smaller (cross-sectional area and volume) and rounder nuclei compared to those from age-matched, wild-type control mice. Changes in nuclear morphology could not be explained by differences in cardiomyocyte size or ploidy. Isolated wild-type and mutant cardiomyocyte nuclei, which are embedded centrally within myofibrils, undergo compression during contraction of the cardiomyocyte, indicating that during each heartbeat cardiomyocyte nuclei would be mechanically deformed as well as being exposed to elevated cytoplasmic Ca²⁺. Immunoblotting analysis indicated decreased nuclear localization of cardiac troponin C and decreased histone H4 expression in Tnnc1-p.A8V mouse hearts. Next, we investigated the influence of nucleocytoplasmic transport by immunofluorescence microscopy, and we could not confirm nuclear localization of cardiac troponin C in fixed myocardial tissue from adult mice. However, cardiac troponin C could be detected in healthy human-induced pluripotent stem cell-derived cardiomyocyte nuclei. We conclude that pathological myofilament dysfunction due to a pathogenic, cardiomyopathy-associated mutation can be linked to altered protein composition of cardiomyocyte nuclei and aberrant nuclear morphology.

Fractional laser therapy improves skin texture, range of motion, and quality of life for patients with traumatic scars. Nevertheless, anecdotal evidence suggests declining insurance coverage for laser therapy. We aimed to characterize the landscape of insurance coverage for fractional laser therapy present our six-year reimbursement trends. We cross-sectionally analyzed the 60 largest American health insurers by enrollee size and market share. For each, we identified their laser therapy policy for scar revision and extracted their documentation, prior, and continuing authorization requirements and treatment guidelines. We also collected retrospective institutional claims data from 2017 to 2022 to investigate trends in reimbursement. Of the 60 largest health insurers, we identified 11 (18.3%) policies on scar revision and 40 policies (66.7%) on reconstructive surgery, including scar revision. Nineteen policies considered laser therapy medically necessary with evidence of functional impairment refractory to prior treatment. Three insurers denied laser coverage under any circumstance. Of the 1,531 claims submitted by our institution for burn scar laser therapy, 13.8% were denied. Patients with Medicare (OR_adj, 3.78) or Medicaid (OR_adj, 2.80) had significantly greater odds of coverage than privately insured patients (p<0.01). There was a 14.5% annual reduction in the odds of reimbursement during the study period (OR_adj, 0.86, p < 0.01). Laser therapy is a powerful treatment that is not widely available to patients with traumatic scars. Our institutional data suggest this access may be further eclipsed by decreasing trends in coverage since 2017. Strategies are needed to protect patient access to this life-changing treatment.