Myofilament Alterations Associated with Human R14del-Phospholamban Cardiomyopathy

A previous study indicated that R14del-PLN mutation resulted in depressed Ca²⁺ kinetics and contractile parameters specific to RV myocytes at 3 months of age [15]. To determine whether R14del-PLN was associated with any alterations in myofilament function, we used skinned myocytes from right and left ventricles of 3-month-old mice and measured the level of maximal force generation (F_max), active force–[Ca] relationships (pCa–force), myofilament calcium sensitivity (EC₅₀) and Hill coefficient (nH), an index of activation of cooperativity at varying calcium concentrations (pCa 10.0 to pCa 4.5) (Figure 1). Strikingly, maximal force from both right and left ventricles was significantly decreased in mutant myocytes compared to WT-PLN controls, but this decrease was more pronounced in the right ventricle (Figure 1A,B,E,F). Further, myofilament calcium sensitivity in both right and left ventricles of mutant skinned myocytes was significantly increased compared to WT-PLN controls (Figure 1C,D,G,H). However, the Hill coefficient (nH: ~2) was not altered. These data suggest that R14del-PLN myocytes need less calcium to reach 50% of the maximal force compared to WT-PLN myocytes, which may be an important compensatory mechanism.

Based on the observed effects of R14del-PLN on contractile parameters, we examined whether the myosin-specific pharmaceutical activator omecamtiv mecarbil (OM, Figure 2) could reverse R14del-PLN-mediated contractile dysfunction and improve the contractile properties of 3-month-old cardiomyocytes in vitro. We also used a myosin-specific pharmaceutical inhibitor, Mavacamten (MYK-461, Figure 3), to determine any changes in contractile force. Contractile parameters, including the amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length, were measured. Parameters of Ca²⁺ transients, including the amplitude at systole and duration of Ca²⁺ decay (tau), were also simultaneously measured. As expected, baseline contractile parameters from right ventricles of R14del-PLN mice were reduced, but no alterations in LV myocytes were observed. Treatment of cardiomyocytes with OM significantly improved the fractional shortening (Figure 2A), contraction velocity (+dL/dt; Figure 2B), and relaxation velocity (−dL/dt; Figure 2C) in RV mutant myocytes, but it had no effects on LV myocytes. However, no parallel stimulatory effects on Ca²⁺ transients of RV mutant myocytes were noted (Figure 2D,E) compared to controls. As a control experiment, we tested the impact of a myosin-specific pharmaceutical inhibitor, mavacamten (MYK-461), on the contractile properties and Ca²⁺ transients in WT-PLN and R14del-PLN myocytes. As expected, MYK-461 decreased the contractility in both WT-PLN and R14del-PLN myocytes similarly, but without affecting Ca²⁺ transients (Figure 3A–E), indicating that inhibition of myosin in the cardiomyocytes of R14del-PLN mice has no benefits. These findings are consistent with our previous data indicating that OM directly enhances myocyte contractility without affecting Ca²⁺ homeostasis [20].

In R14del-PLN patients, symptoms develop most often in the fifth decade with a mean age at presentation ranging from 40 to 48 years [4,5,7]. In addition, the onset of disease appears to indicate a slightly higher frequency in males [7]. Thus, we examined cardiomyocyte contractile parameters and Ca²⁺ kinetics, as well as sex-dependent effects, in the humanized models at 12 months of age, corresponding to the age of patients who typically present with symptoms. In male mice, LV contractile parameters were similar between WT-PLN and mutant hearts (Figure 4A–C). However, fractional shortening (FS) and rates of contraction (+dL/dt) and relaxation (−dL/dt) were all significantly reduced in RV mutant myocytes compared to WT-PLN (Figure 4A–C). These decreases in mutant RV contractile parameters were similar between males and females (Figure 4D–F). Interestingly, mutant females also exhibited depressed rates of contraction and relaxation in LV myocytes (Figure 4D–F). Furthermore, intracellular Ca²⁺-cycling parameters were assessed in isolated cardiomyocytes using the Fura-2 AM fluorescence indicator (2 µM). R14del-PLN elicited decreases in the Ca²⁺ peak of transients and prolongation of the time constant for Ca²⁺ decay (tau) in male RV myocytes (Figure 5A–C). Examination in females revealed similarly depressed parameters specifically in RV myocytes (Figure 5D,E). However, no parallel alterations in LV mutant cells from either males or females were observed (Figure 5).

Echocardiography assessment indicated similar ejection fraction, fractional shortening and cardiac output between these groups (Figure 6B–D), suggesting that in vivo neurohormonal factors may mask the contractile differences in RV myocytes between WTs and mutants. No differences in geometrical parameters or remodeling occurred at 12 months of age, as evidenced by the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) between WT-PLN and R14del-PLN hearts (Figure 6E,F). Furthermore, these parameters were similar between males and females (Figure 6). Therefore, R14del-PLN did not affect in vivo cardiac function or remodeling up to 12 months.

To assess cardiac pathology in 12-month-old WT-PLN and R14del-PLN hearts, we used Picrosirius Red staining of fixed cardiac tissue sections. Pathological analysis revealed significant interstitial fibrosis in both LV and RV tissues from R14del-PLN male and female mice (Figure 7A,C). Quantification of collagen levels indicated twofold increases in mutant males and females compared to WTs (Figure 7B,D). In addition, ultrastructural analysis of longitudinal sections from LV (top) and RV (bottom), using transmission electron microscopy, revealed abnormalities in both males and females from R14del-PLN hearts. Irregular, disarrayed and widened Z-disc patterns (white box) were observed, as well as streaming and smearing of the Z-disc material, in R14del-PLN ventricles (white boxes) (Figure 8A,B). Furthermore, a higher number of lipid droplets were observed in mutant ventricles from both male and female hearts (Figure 9A,B), similar to previous findings in human R14del-PLN patients [21]. These results suggest that structural abnormalities may contribute to the ventricular pro-arrhythmic phenotype in R14del-PLN hearts under stress conditions.

Mice harboring human WT-PLN and the R14del-PLN coding sequence were created by inserting the LoxP-H2B-GFP- 4XpolyA-FRT-Neo-FRT-LoxP-hPLN^WT/R14del cassette into the PLN start codon at exon 2, as previously described [15,32]. The handling and maintenance of animals were approved by the Ethics Committee of the University of Cincinnati. The investigation followed the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health.

Cell contractility and Ca²⁺ transients were measured simultaneously at room temperature (22–23 °C), as previously described [20,33,34,35,36]. Briefly, under basal conditions, cardiomyocytes were incubated with 1.0 μM Ca²⁺-sensitive Fura-2 dye (Invitrogen, Waltham, MA, USA) for 15 min at room temperature and then washed with fresh regular Tyrode’s solution for at least 10 min. Then, cardiomyocytes were placed in a perfusion chamber adapted to the stage of an inverted Nikon eclipse TE2000-U fluorescence microscope. Cells were superfused (Warner Instruments, Boston, MA, USA, six-channel valve controller) with Tyrode’s buffer at room temperature. Sarcomere shortening was assessed using a video-based sarcomere length detection system (Ion-Optix, Milton, MA, USA). To measure intracellular Ca²⁺ transients, Fura-2 fluorescence was excited at 340 and 380 nm and acquired at an emission wavelength of 515 ± 10 nm using a spectrofluorometer (IonOptix, Milton, MA, USA). The emission field was restricted to a single cell with the aid of an adjustable window. Simultaneous measurements of mechanics and Ca²⁺ kinetics were performed at 0.5 Hz in the absence and presence of 100 nmol/L omecamtiv mecarbil (OM) [23], which augments cardiac contractility, and 250 nM mavacamten, a cardiac myosin inhibitor (MYK-461) [24]. Both male and female mice were utilized. Data were analyzed with IonOptix LLC analyzing software (Version 7).

Permeabilized cardiomyocytes from male WT-PLN or R14del-PLN mice were prepared in relaxing solution from frozen tissue and analyzed for pCa–force, as previously described [25]. In brief, 20–30 mg of frozen heart tissue were homogenized twice at 5000 rpm for 7 s and filtered through a 70-µm cell strainer, followed by centrifugation at 100× g for 3 min at 4 °C. To remove the cell membrane, a skinning process was performed by resuspending pelleted cells in relaxing solution [97.92 mM KOH, 6.24 mM ATP, 10 mM EGTA, 10 mM Na2CrP, 47.58 mM potassium propionate, 100 mM BES, 6.54 mM MgCl₂, 1 mM DTT and 100 μM protease cocktail (Sigma-Aldrich, St. Louis, MO, USA)] with 1% Triton-X. The cells were kept on a rotating shaker at room temperature for 10–15 min. Thereafter, the cells were washed twice with two centrifugations at 100× g for 3 min at 4 °C with the relaxing solution to remove Triton. Using the 1600A Permeabilized Myocyte System (Aurora Scientific Inc., Aurora, ON, Canada), skinned myocytes with regular striation patterns were selected and attached to a force transducer (Aurora Scientific Inc., ON, Canada) and a high-speed piezo translator (Thorlabs, Newton, NJ, USA) using optical glue (Norland, Cranbury, NJ, USA). Cells were then exposed to maximum saturating [Ca²⁺] activating solution (pCa 4.5) at the beginning and end of the experimental protocol to test the strength and rundown of cell attachment. Isometric force development was recorded at a sarcomere length of 1.9 µm using the activating solution with varying calcium concentrations (pCa 10.0 to pCa 4.5), while sarcomere length was continuously monitored through Aurora’s High-Speed Video Sarcomere Length (HVSL) measurement system. The recorded total isometric force, after correcting the rundown, was normalized to the cross-sectional area that was calculated by buckling the attached myocyte and measuring the dimensions of the elliptical cell’s two sides. Data were acquired using Aurora’s 600A real data acquisition and analysis system. Force–pCa curves were fit using the modified Hill equation (Force/Force_max = [Ca²⁺]ⁿ/(pCa₅₀ⁿ + [Ca²⁺]ⁿ), where n is the Hill-slope.

Echocardiographic analysis was performed to assess cardiac function using the Vevo 5 2100 Ultrasound system (Visualsonics, Toronto, ON, Canada) as previously described [4]. Briefly, following anesthetization with isoflurane (1.5–2%), M-mode and B-mode images were obtained from a parasternal long-axis view between 2 and 10 mm in depth. Images were analyzed using Vevostrain software (Vevo 2100, v1.1.1 B1455, Visualsonics, Toronto, ON, Canada). All measurements were performed in a blinded manner that included left ventricular (LV) septum, posterior wall and chamber size at end-diastolic (LVEDD) and end-systolic diameters (LVESD). From these values, the FS was obtained with the software following the formula FS = (LVEDD − LVESD/LVEDD) × 100 and EF was calculated via the Teicholz formula by converting the chamber diameter [37,38].

Hearts were excised from anesthetized mice (Euthasol, 200 mg/kg i.p., Virbac AH, Inc., Fort Worth, TX, USA), immediately fixed in 10% neutral buffered formalin (Sigma, Saint Louis, MO, USA) and processed as previously described [15]. Briefly, for histology studies, paraffin-embedded hearts were sectioned (4 μm), deparaffinized, rehydrated, incubated in 0.1% Sirius red in saturated picric acid for 1 h and then washed in acidified water. Paraffin-embedment and slide preparation were carried out by the University of Cincinnati Pathology Department Core Facility. Images were acquired on a Leica DMi8 Widefield microscope with a 20× objective lens using LASX software (Leica Microsystems, 9435 Heerbrugg, Switzerland). The analysis of fibrosis levels in cross sections of LV and RV was performed by calculating the collagen fiber staining with Image J (version 1.48v; National Institutes of Health, Bethesda, Maryland, MD, USA).

For transmission electron microscopy (TEM), RV and LV tissue samples were cut (1 mm²), fixed in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer, post-fixed in 1% osmium tetroxide and processed through a series of ethanol alcohol dehydrations at concentrations of 25%, 50%, 75%, 95% and twice at 100%, incubating at 10-min increments for each step. Then samples were infiltrated and embedded in LX-112 resin. After polymerization at 60 degrees for three days, ultrathin sections (120 nm) were cut using a Leica EM UC7 ultramicrotome and counterstained in 2% aqueous uranyl acetate and Reynold’s lead citrate. Sample preparation and imaging were performed by the Electron Microscopy CORE facility at Cincinnati Children’s Hospital. Images were taken with a transmission electron microscope (Hitachi H-7650, Tokyo, Japan) equipped with a digital camera (Biosprint 16).

Data are expressed as mean ± standard deviation of the mean (SD) for the number of mice, hearts or myocytes, and statistical analyses were performed with two-tailed unpaired t-tests using GraphPad Prism, version 8.4.3. All myocyte data were generated using LV and RV cells from the same mouse on any specific day. Grubbs and ROUT tests were performed to identify statistically significant outliers, and outliers were removed if appropriate. A p-value of <0.05 was considered statistically significant.