Abstract P417: Cardiomyopathy-associated Variant In Troponin T Tail Domain Promotes Disruption Of Both Frank-starling Mechanism And Cardiac Myofilament Performance

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) tail region has been linked to familial hypertrophic cardiomyopathy (HCM), arrhythmia, and sudden cardiac death. It has been reported that inotropic stimulation with high extracellular Ca 2+ or isoproterenol led to diastolic dysfunction in both isolated and in vivo HcTnT-I79N mice hearts. Although HcTnT-I79N effects are acknowledged to be dependent on the inotropic state of the cardiac muscle, little is known about how this pathogenic variant affects the Frank-Starling law of the heart. To further investigate the functional and structural consequences of this deadly variant in a stretch-dependent manner, cardiac tissues were harvested from non-transgenic (NTg) control mice and transgenic mice bearing HcTnT-I79N. Left ventricular papillary muscle bundles were permeabilized and mounted for mechanical measurements. Sarcomere length (SL 1.9, 2.1 or 2.3 μm) was set at pCa 8 using HeNe laser diffraction and then Ca 2+ -dependence of isometric force, sinusoidal stiffness (SS, 0.2% PTP length oscillation) and rate of tension redevelopment ( k TR ) were measured. We observed that HcTnT-I79N tissue exhibited increased myofilament Ca 2+ -sensitivity of force, increased SS, slower k TR at all levels of Ca 2+ -activation, and diminished length-dependent activation (LDA). Small-angle X-ray diffraction revealed that HcTnT-I79N permeabilized cardiac muscles exhibit smaller myofilament lattice spacing at longer SLs (2.1 μm and 2.3 μm) compared to NTg. Using 3% Dextran T500 to osmotically compress the myofilament lattice (SL 2.1 μm), HcTnT-I79N showed no change in myofilament lattice spacing and little change in contractile indices associated with LDA. Interestingly, upon osmotic compression, HcTnT-I79N displayed a decrease in disordered relaxed state (DRX, ON state) of myosin and an increase in super-relaxed state (SRX, OFF state) of myosin. We conclude that altered cardiac myofilament performance, lack of responsiveness to osmotic compression, and reduced LDA observed with HcTnT-I79N are partially due to a combination of smaller myofilament lattice and disturbed ON and OFF states of myosin.

Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment

Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Bisphenol S rapidly depresses heart function through estrogen receptor-β and decreases phospholamban phosphorylation in a sex-dependent manner

The health effects of the endocrine disruptor Bisphenol A (BPA) led to its partial replacement with Bisphenol S (BPS) in several products including food containers, toys, and thermal paper receipts. The acute effects of BPS on myocardial contractility are unknown. We perfused mouse hearts from both sexes for 15 min with physiologically relevant doses of BPS or BPA. In females BPS (1 nM) decreased left ventricular systolic pressure by 5 min, whereas BPA (1 nM) effects were delayed to 10 min. BPS effects in male mice were attenuated. In both sexes ER-β antagonism abolished the effects of BPS. Cardiac myofilament function was not impacted by BPS or BPA in either sex, although there were sex-dependent differences in troponin I phosphorylation. BPS increased phospholamban phosphorylation at S16 only in female hearts, whereas BPA reduced phosphorylation in both sexes. BPA decreased phospholamban phosphorylation at T17 in both sexes while BPS caused dephosphorylation only in females. This is the first study to compare sex differences in the acute myocardial response to physiologically relevant levels of BPS and BPA, and demonstrates a rapid ability of both to depress heart function. This study raises concerns about the safety of BPS as a replacement for BPA.