Age‐related wild‐type transthyretin amyloidosis (wtATTR) is characterized by systemic deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in the connective tissue of many organs. In the heart, this leads to age‐related heart failure with preserved ejection fraction (HFpEF). The hypothesis tested is that TTR deposited in vitro disrupts cardiac myocyte cell‐to‐cell and cell‐to‐matrix adhesion complexes, resulting in altered calcium handling, force generation, and sarcomeric disorganization. Human iPSC‐derived cardiomyocytes and neonatal rat ventricular myocytes (NRVMs), when grown on TTR‐coated polymeric substrata mimicking the stiffness of the healthy human myocardium (10 kPa), had decreased contraction and relaxation velocities as well as decreased force production measured using traction force microscopy. Both NRVMs and adult mouse atrial cardiomyocytes had altered calcium kinetics with prolonged transients when cultured on TTR fibril‐coated substrates. Furthermore, NRVMs grown on stiff (~GPa), flat or microgrooved substrates coated with TTR fibrils exhibited significantly decreased intercellular electrical coupling as shown by FRAP dynamics of cells loaded with the gap junction‐permeable dye calcein‐AM, along with decreased gap junction content as determined by quantitative connexin 43 staining. Significant sarcomeric disorganization and loss of sarcomere content, with increased ubiquitin localization to the sarcomere, were seen in NRVMs on various TTR fibril‐coated substrata. TTR presence decreased intercellular mechanical junctions as evidenced by quantitative immunofluorescence staining of N‐cadherin and vinculin. Current therapies for wtATTR are cost‐prohibitive and only slow the disease progression; therefore, better understanding of cardiomyocyte maladaptation induced by TTR amyloid may identify novel therapeutic targets.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano‐signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano‐signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1μM mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α‐actinin by 6 hrs. In myosin inhibition, this was accompanied by redistribution of intracellular poly‐ubiquitin K48 to the cellular periphery relative to the poly‐ubiquitin K48 reservoir at the I‐band. Moreover, loading and unloading of the cellular substrate led to a three‐fold increase in post translational modifications (PTMs) when compared to the myosin‐specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve ERK1/2 and FAK activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α‐actinin to increase its propensity to bind F‐actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α‐actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α‐actinin are discussed with respect to cardiac atrophy and heart failure.

Mammalian sperm motility and fertility potential are regulated by ion homeostasis which is controlled by a suite of ion permeable channels and transporters working in concert. While all mammalian sperm share in their goal to navigate the female reproductive tract in search of an egg, the molecular regulation of this process is extremely diverse and differs dramatically among species. Here we report that electrophysiological recordings from sperm cells of different species illustrate marked differences between rodent, human and ruminant sperm on the basis of their ion channel make-up and regulation. Furthermore, localization patterning of ion channels along the length of sperm flagella show species specific configuration. It is from these observed differences that we can now begin to explain the disparity in motility parameters among species as well as determine the effect proper spatiotemporal regulation of ion concentrations have on proper sperm motility.

Forster resonance energy transfer (FRET) can be used as a spectroscopic ruler to measure nanometer-scale distances. The recovery of inter-dye distance depends on a calibration factor known as the Forster critical distance (R 0 ). This distance is currently estimated based on measurements of the quantum yield of the donor dye, the overlap integral between the donor and acceptor dyes, and assumptions about the index of refraction and the relative orientation of the donor and acceptor dye molecules.Here, we report a method to experimentally measure R 0 , using B-DNA as a structural reference. Fifteen donor (Cy3)-labeled oligonucleotides were generated, by placing donor-labeled Thymidines at positions 11, 14, /, 39. A single complementary strand was synthesized with acceptor (AlexaFluor647) at position 10. The strands were annealed, producing dsDNA consisting of a 30 base pair (bp) ruler with a 10 bp cap on each end. For each freely diffusing construct, the mean transfer efficiency (TE) was measured by singlepair FRET (sp-FRET) and ensemble (en-FRET). The TE's as a function of bp were fit to a reduced representation model of B-DNA that provided the absolute inter-dye distances. The reduced model was formulated based on an atomistic model of dye-labeled B-DNA, R 0 was recovered from the fit. We repeated our approach using three different donor/acceptor pairs, each with a different R 0 .

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