All experimental animal protocols adhered to the Guidelines for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH; publication no. 85–23, revised 1996) and were approved by the institutional Animal Care and Use Committee (ACUC) at the University of Wisconsin-Madison (Protocol ID: M005490) with steps taken to minimize pain and suffering. Healthy human hearts that went unused for organ transplant were obtained from the University of Wisconsin Organ Procurement Organization, Madison, WI, USA, as approved by the University of Wisconsin Institutional Review Board (IRB). Blood collection and testing for analysis of ceramide levels in systemic circulation were approved by the Health Sciences IRB at the University of Wisconsin-Madison, as well as by the IRBs at the University of California-Los Angeles and Georgetown University. We confirm that data collection and analysis involving human subjects conformed with the Declaration of Helsinki, except for registration in a database. All authors understand the ethical principles of The Journal of Physiology and our study complies with the ethics checklist for research with animal and human subjects.

DF19-9-11T.H iPSCs, obtained from the WiCell Research Institute (RRID:CVCL_K054), were cultured in StemFlex media (A3349401, Gibco) according to the manufacturer’s protocol. Briefly, cryopreserved iPSCs were thawed, added to StemFlex media supplemented with 5 μM Y-27632 (562822, BD Biosciences) and plated onto Growth Factor Reduced Matrigel (354230, Corning) coated six-well dishes (1.5×10⁵-3.3×10⁵ cells per well (9.6cm²)). hiPSCs were subsequently incubated at 37°C; 5% CO₂ until they were 70%–90% confluent with media changes every 24–48 h with StemFlex prior to passaging. hiPSCs were passaged every 4–6 days using Versene (15040066, Gibco) to dissociate cells according to the manufacturer’s protocol, which were then resuspended in StemFlex media, and plated onto Matrigel-coated plates at a 1:6 to 1:12 split ratio.

HiPSCs derived from the DF19-9-11T.H line were differentiated into cardiomyocytes (CMs) using a small molecule-directed (GiWi) protocol as previously described (Zhang et al., 2012; Lian et al., 2013). Briefly, hiPSCs maintained on the StemFlex/Matrigel system were dissociated into single cells and seeded onto Matrigel-coated six-well plates at 2.0 × 10^6 cells/well in StemFlex media. Cells were cultured for 5 days in StemFlex media with daily media changes. On day 0, StemFlex media was replaced with 2.5 mL/well RPMI (11875093) supplemented with B27 without insulin (A1895601, Gibco) supplemented with 10 μM CHIR99021 (2520691, GSK-3 inhibitor, Biogems). Precisely 24 h later (Day 1), cell culture media was changed to 3 mL/well RPMI + B27 without insulin. On day 3, 48 h later, the media was changed to 3 mL/well RPMI + B27 without insulin supplemented with 5 μM IWP-2 (6866167, Biogems). Precisely 120 h later (Day 5), the media was replaced with 3 mL RPMI + B27 without insulin. The media was changed to RPMI + B27 complete supplement (with insulin) (17504044, Gibco) on Day 7 and cells were maintained in this media until day 15 with media changes every 24–48 h. On Day 15, cells from wells containing ≥50% beating cells by visual inspection were dissociated with 10x TrypLE (A1217701, Thermo Fisher Scientific) according to the manufacturer’s protocol. Following resuspension in StemFlex media, cells were replated on Synthemax (3535, Corning) coated six-well plates at 2.0 × 10^6 cells/well. Roughly 48 h after replating, hiPSC-CMs were purified using Lactate media, made with RPMI 1640 with no glucose (11879020, Life Technologies), B27 supplement, and 0.02% (2.66 mM) lactate (L1375, Sigma-Aldrich) for 7 days with media changes every 24–48 h. After selection, CMs were maintained in RPMI with B27 supplement until Day 30 at which point hiPSC-CMs were dissociated for hiPSC-ECT generation (Fig. 1A).

DF19-9-11T.H hiPSC-cardiac fibroblasts (CFs) were differentiated as previously described and cultured in FibroGRO-LS media (SCMF001, Millipore Sigma) in uncoated six-well culture plates (Corning) with passaging every 4–5 days (Zhang et al., 2019). Media was replaced every 24–48 h. Low passage numbers (<12) were used for hiPSC-ECT generation.

Day 30 DF19-9-11T.H hiPSC-CMs were visually inspected and only wells containing ≥95% beating cells were dissociated with 10x TrypLE according to the manufacturer’s protocol and counted using a hemocytometer. hiPSC-CMs were subsequently resuspended in fibrin ECT media (60.3% high-glucose DMEM; 20% F12 nutrient supplement; 1 mg/mL gentamicin; 8.75% fetal bovine serum; 6.25% horse serum; 1% HEPES; 1× nonessential amino acid cocktail; 3 mM sodium pyruvate; 0.004% (wt/vol) NaHCO₃1 μg/mL insulin; 400 μM tranexamic acid; and 17.5 μg/mL aprotinin) and incubated for at least 1 h on a rotating platform at 37°C to form small and uniform clusters of viable CMs. DF19-9-11T.H CFs were dissociated using 1× TrypLE (12604013, Thermo Fisher Scientific) according to the manufacturer’s protocol and counted using a hemocytometer. Following rotational culture, 2 × 10^6 hiPSC-CMs were mixed with 2 × 10^5 hiPSC-CFs in 200 μL fibrin ECT media per hiPSC-ECT, a ratio similar to that previously used in the generation of 3D cardiac constructs (de Lange et al., 2021; De Lange et al., 2023). To this cell mixture, 1.25 mg/mL fibrinogen and 0.5 unit of thrombin were added. This cell-matrix mixture was rapidly mixed and loaded onto a 20×3 mm cylindrical mold of FlexCell^™ Tissue Train silicone membrane culture plate (TT4001U) and incubated under pre-programmed vacuum condition for 60 min at 37°C supplied with 5% CO₂ to allow for attachment of the ECT constructs to the nylon tabs at each end of the Tissue Train well (Fig. 1B). Following polymerization of the fibrin matrix, ECTs were fed with ECT media, carefully separated from the plate surface with a sterile pipette and cultured for 30 days with fibrin ECT media changes every 2–3 days. ECT were stretched days 53–60 (Days 23–30 post-ECT generation) using a FlexCell^™ Tissue Train silicone membrane vacuum system (Fig. 1C). The 7-day cyclic stretch protocol was modified from previously published protocols (Prosser et al., 2013; Lu et al., 2021) and set for 0.25 Hz and incrementally increased every 24 h by 3–4% uniaxial elongation, up to 15%. This cyclical and incremental stretch protocol was implemented as nSMase activation is transient under constant mechanical stress (Czarny et al., 2003). During the stretch protocol, all wells were fed every 48 h with ECT medium supplemented with or without GW4869. All unstretched and stretched ECT were fed with 5 mL/well ECT media to maintain a proper volume of cell culture media with concurrent sampling for downstream LC-MS/MS and H₂O₂ analyses.

GW4869 (Thermo Fisher Scientific, 501873728), a specific nSMase inhibitor was first diluted to 1.5 mM in dimethyl sulfoxide (DMSO), aliquoted, and stored at −80 °C. Prior to use, aliquots were thawed at 37°C and further solubilized with 5% methano-sulfonic acid at a 1:20 ratio (methanesulfonic acid:DMSO) to make a 1.43 mM GW4869 solution.

Cryo-sectioning of OCT-embedded ECT at 10 μM and Masson’s trichrome staining (Cat. No. KTMTR2) was performed by the UW-Madison Translational Research Initiatives in Pathology (TRIP) core facility. Masson’s Trichrome staining images were collected using an EVOS cell imaging system at 20x magnification. ECT fibrotic area was evaluated in ImageJ using a custom program calculated as a percentage fibrotic area of total tissue as discussed previously in detail (Glukhov et al., 2012).

Contraction traces were measured in day 60 hiPSC-ECT using protocols like those previously described (de Lange et al., 2021). Briefly, each hiPSC-ECT construct was transferred from the culture dish to a model 801B small intact fiber test apparatus (Aurora Scientific) in Krebs–Henseleit buffer [119 mmol/L NaCl, 12 mmol/L glucose, 4.6 mmol/L KCl, 25 mmol/L NaHCO₃, 1.2 mmol/L KH₂PO₄, 1.2 mmol/L MgCl₂, 1.8 mmol/L CaCl₂, gassed with 95% O₂-5% CO₂ (pH 7.4)]. Day 60 hiPSC-ECT constructs were attached with sutures between a model 403A force transducer (Aurora Scientific) and a stationary arm and perfused with 37°C Krebs–Henseleit buffer at a rate of 1 mL/min, and field stimulation initiated at 1 Hz (2.5 ms, 12.5 V). The longitudinal length of each construct was increased stepwise until maximal twitch force was achieved to establish the Frank–Starling relationship. ECT were allowed to equilibrate for 20 min with constant perfusion. Then, twitch force production was measured with at a 1.5 Hz pacing frequency both at baseline and following 5 min preincubation with 1 μM isoproterenol. Data from force measurements was analyzed using IonWizard 6.0 software (IonOptix). Under each condition, contraction transients of 40–60 successive contractions were collected and averaged. These data were exported to Microsoft Excel and the kinetics of force generation and relaxation were calculated.

Performed by the SMPH Electron Microscopy Facility (UW-Madison), ECT were fixed and prepared for TEM analysis while unstretched to evaluate the maximum membrane mechanical buffering capacity, similarly to previous work (Egorov et al., 2019; de Lange et al., 2021). Briefly, samples were fixed in 2.5% glutaraldehyde, 2.0% paraformaldehyde buffered in 0.1M sodium phosphate buffer for 2 h at room temperature (RT). After osmium post-fixation, dehydration, and embedding, samples were sectioned on a Reichert-Jung Ultracut E ultramicrotome at 80 nm, and post-stained with uranyl acetate and lead citrate. The sectioned samples were viewed at 80 kV on a Philips CM120 transmission electron microscope at 40,000x magnification. Caveolae-like structures were considered 50–100 nm flask-shaped membrane invaginations located within 50 nm from the cell surface. Caveolae-like structures were identified as sub-sarcolemmal (no visible connection to the sarcolemma in the image plane) or visibly integrated into the sarcolemma (flask-like connection to the sarcolemma observed). To quantify the degree of membrane convolution, we defined a membrane convolution index = (L/L_o − 1), where L is the length of membrane contour, and L_o is the length of a straight path connecting the end points of the membrane segment (Fig. 1D).

ECT media lipids were isolated using a modified bligh-dyer method (Ulmer et al., 2018) from cell culture media (200 μL/sample). The bligh-dyer method was performed as previously described but using a 1:10 sample:solvent ratio during sample incubation with ice-cold methanol and chloroform (1:1) to maximize ceramide extraction. Organic samples were completely dried and reconstituted in 9:1 methanol:toluene. As previously described (Kauhanen et al., 2016), LC-MS/MS experiments were performed using a Bruker Impact II quadrupole time-of-flight (QTOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to a Waters nanoACQUITY UPLC system (Waters Corporation, Milford, MA, USA) in positive ion mode. A Waters nanoEase M/Z HSS T3 column (100 Å, 1.8 μm, 300 μm × 100 mm) was used for reversed-phase separation. Mobile phase A was 10 mM ammonium acetate in water with 0.1% formic acid, and mobile phase B was 10 mM ammonium acetate in acetonitrile:2-propanol (4:3, v/v) with 0.1% formic acid (Kauhanen et al., 2016). The lipids eluted from the column were infused into the mass spectrometer using an electrospray ion (ESI) source. Lipids were identified by searching the LC–MS/MS data against the databases downloaded from MassBank of North America (MoNA) and internal databases and using Lipid Species Annotation function in MetaboScape 2022b. Detected ceramide values were adjusted with respect to initial ECT media protein concentration evaluated via Nanodrop.

H₂O₂ production was measured using the supplied Ampled Red Peroxidase Assay kit protocol (Thermo Fisher Scientific, A22188). Briefly, a standard curve generated from the assay kit’s supplied 3% H₂O₂ solution was used to validate that H₂O₂ detection in ECT media samples were within the linear range. Unique ECT media samples were run in triplicate with 50 μL of conditioned ECT media being used for each replicate. Absorbance (560 nm) detected by a Tecan Infinite M200 plate reader was used to determine H₂O₂ production after a 30 min assay incubation at 37°C.

Amplex red sphingomyelinase assay kit was used to quantify nSMase activity in ECT according to the manufacturer’s protocol (Thermo Fisher Scientific, A12220). Briefly, ECT were lysed and sonicated in CHAPs lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5% CHAPS, 1× protease inhibitor (Bimake). 10 μg of ECT protein (ran in triplicate) with and without nSMase inhibition via GW4869 (20 μM) was used during the assay. Fluorometric measurements were collected after 30 min incubation at 37°C using a Tecan Infinite M200 plate reader with excitation/emission set for 545/590 nm.

Ceramide measures were determined from randomly selected adult men and women who were participants in a survey of health and aging among American adults, Midlife in the US (MIDUS). Initially, MIDUS began in 1995–1996 as a survey of adults recruited through random digit dialing and included individuals between 25–74 years of age across the 48 continental states. A second phase was initiated in 2004 when biological samples were obtained from a subset of the original participants who consented to an overnight hospital stay at one of 3 Clinical and Translational Research Centers, either in Madison, WI, Los Angeles, CA, and Washington DC. The lipid data for this analysis were based on 520 adults between 35–86 years of age. A larger panel of biological and clinical measures was determined from the fasted blood samples that were obtained around 0700. As stated previously, sample collection and testing were approved by 3 IRBs (Health Sciences IRB, UW-Madison, as well as by the IRBs at UCLA and Georgetown University. Lipidomic values were generated by mass spectrometry from frozen sera in one batch using (Complex Lipid Panel, Metabolon, Durham, NC). The current analysis focus just on ceramides, but additional information on other lipids can be found in a previous publication (Berkowitz et al., 2022).

Whole ECTs and human left ventricles were snap frozen in liquid nitrogen post-physiological testing and stored at −80°C. RNA was isolated as previously described (Turner et al., 2022). hiPSC-ECTs were homogenized using TRIzol reagent (Ambion, 15596018) and molecular-grade chloroform was added according to the manufacturer’s instructions. After mixing, incubation, and centrifugation, the aqueous phase containing RNA was collected. RNA was further purified using the Qiagen Miniprep kit according to the manufacturer’s instructions. RNA was quantified and quality was assessed using a NanoDrop spectrophotometer (Fisher Scientific). 50–100 ng total RNA was reverse transcribed into first-strand cDNA with the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, 170–8890) following manufacturer’s protocols. 1 ng of resulting cDNA was used for qPCR analysis. Taqman probes for assayed genes and appropriate controls were arrayed in MicroAmp Optical 96-well Reaction Plates (Applied Biosystems, 4483485), and the PCR performed using the TaqMan Gene Expression Master Mix (Applied Biosystems, 4369016). Real-time monitoring of TaqMan fluorescence was performed on the Bio-Rad RT-qPCR system CFX96. An initial activation step of 2 min at 50°C and 95°C for 10 min was followed by 40 cycles of 15 s of denaturation at 95°C and 60 s of annealing/extension at 60 °C. Data were analyzed in Excel, using a ΔΔCT method as previously described (Turner et al., 2022), with GAPDH used as a housekeeping control gene. TaqMan assay IDs used were GAPDH (Hs02786624_g1), ACTA2 (Hs05005341_m1), COL3A1 (Hs00943809_m1), COL1A2 (Hs01028956_m1), TIMP3 (Hs00165949_m1), MMP2 (Hs01548727_m1), TGF-β1 (Hs00998133_m1), TNF-α (Hs00174128_m1), IL-6 (Hs00174131_m1), SMPD4 (Hs04187047_g1), and SMPD3 (Hs00920354_m1). All TaqMan assays were obtained from Applied Biosystems unless indicated otherwise.

Adult (5 to 7-month-old) male and female C57BL/6 mice (RRID:MGI:2159769) (n=8) were used in the study. Hearts were isolated and Langendorff perfused as described previously (Glukhov et al., 2010). Mice were heparinized and anesthetized, and the heart was removed and placed in oxygenated (95% O₂, 5% CO₂) constant-temperature (37°C), modified Tyrode solution of the following composition (in mmol/L): 128.2 mM NaCl, 4.7 mM KCl, 1.19 NaH₂PO₄, 1.05 mM MgCl₂, 1.3 mM CaCl₂, 20.0 mM NaHCO₃, and 11.1 mM glucose (pH=7.35±0.05). After cannulation, the heart was superfused and retrogradely perfused with warmed (37°C) Tyrode solution under constant aortic pressure of 60 mmHg. The heart was paced at the lateral left ventricular midwall with a silver bipolar electrode coated with Teflon except at the tip. As previously described (Pfeiffer et al., 2014) once isolated and connected to Langendorff perfusion apparatus, a fluid-filled length of polyethylene tubing fitted onto a 20-gauge luer adapter was inserted into the left ventricular cavity via the pulmonary vein and mitral valve. A tie was secured around the opening of the pulmonary vein. The left ventricular cavity tube was connected to a reservoir of warmed and oxygenated perfusate, and left ventricular pressure was controlled by the height of the reservoir relative to the heart.

As previously described (Glukhov et al., 2010), coronary perfused hearts were stained by perfusion with voltage sensitive dye (RH-237, S1109, Thermo Fisher Scientific; 5 μL of 1 mg/ml DMSO, in Tyrode solution) for 10 min. Excitation light (530/540 nm) was generated by a 150-W halogen lamp with an excitation filter (530–40 nm) from a constant-current, low-noise, power supply (MHAB-150W, Moritex USA Inc., CA, USA). The emitted light >660 nm was filtered by a long-pass filter (>660 nm; Thorlabs, NJ) for the action potential signal. The fluorescent light emitted from the preparation was long-pass (>660 nm) filtered using an edge pass filter (Thorlabs, NJ) before reaching the camera. Emitted light was directed towards a MiCAM Ultima-L CMOS camera (SciMedia, CA) with high spatial (100×100 pixels, 60±10 μm/pixel) and temporal (2,000 frames/s) resolution. The acquired fluorescent signal was digitized, amplified, and visualized using custom software (SciMedia, CA). A customized Matlab-based computer program was used to analyze optical signals. Maximum upstroke derivative (dV/dt_max) was calculated for each action potential using the normalized optical signal and its derivatives. Activation maps were constructed during constant 8Hz electrical pacing from activation times, which were determined from the dV/dt_max.

For assessing the effect of cyclic stretch on ECT plasma membrane morphology, fibrosis, β-adrenergic stimulation, and stretch-conditioned media, p values were calculated with two-way ANOVA with post-hoc Bonferroni’s multiple comparisons test to test for interactions when comparing multiple groups in cases where the data displayed a normal distribution. If the Shapiro-Wilk test revealed that the data did not have a normal distribution, Kruskal–Wallis ANOVA was used. Statistical significances between normotensive and hypertensive MIDUS participant ceramide data were determined via Welch’s student t-tests. Correlations were evaluated using linear regression with significance calculated using an F-test. Statistical analyses were performed in Origin or GraphPad Prism software and p-values equal or less than 0.05 were considered statistically significant. All confidence intervals (CI) are 95%.

To determine stretch-induced changes in cardiac myocytes, ECTs were subjected to cyclic stretch for 7 days using a previously published protocol (Lu et al., 2021) for cyclical and incremental stretch modified for elevated mechanical stress. Adjusting for human myocardium, we stretched our ECT at 0.25 Hz and incrementally increased every 24 h by 3–4% elongation, up to 15% length increase over unstretched length, based on Prosser et al. (Prosser et al., 2013). TEM analysis revealed that cyclic stretch reduced abundance of caveolae-like structures by 41%, from 1.87 caveolae/μM (CI 1.53–2.20) in unstretched ECTs to 1.11 caveolae/μM (CI 0.86–1.35) in stretched ECT (p=0.002, two-way ANOVA with post-hoc Bonferroni; Fig. 1F). Furthermore, nSMase inhibition via GW4869 (20 μM) applied during the 7-day stretch protocol prevented the effects of cyclic stretch on caveolae-like structure reduction with a 51% increase in stretched ECT treated with GW4869 (1.68 caveolae/μM, CI 1.36–1.98) compared to untreated stretched ECT (1.11 caveolae/μM, CI 0.86–1.35; p=0.045, two-way ANOVA with post-hoc Bonferroni). In stretched ECTs, the reduction in caveolae-like structures was accompanied by a 43% decrease in membrane convolution (0.037, CI 0.028–0.046) compared with unstretched ECTs (0.065, CI 0.049–0.081), indicating increased membrane tension (p<0.001, Kruskal-Wallis Test; Fig. 1G) (Wei et al., 2017; Egorov et al., 2019). As anticipated, incubation of GW4869 did not prevent a significant reduction in membrane convolution (p<0.001, Kruskal-Wallis Test) in stretched ECT (0.033, CI 0.027–0.040) vs. unstretched ECT (0.086, CI 0.057–0.115), indicating that despite increased membrane tension, nSMase inhibition still prevents stretch-induced reductions in caveolae-like structures.

Studies have shown that inflammatory cardiovascular conditions increase ceramide levels in the heart (He et al., 2012) and blood (Jensen et al., 2020); however, the contribution of nSMase to these increased ceramides is unknown. To explore the potential mechano-activation of nSMase and its role in mediating cardiovascular pathology we utilized LC-MS/MS on ECT cell culture media to determine nSMase activity. LC-MS/MS analysis found that cell culture media collected from cyclically stretched ECT media had a 38% increase of short-chain ceramides (1.00, CI 0.86–1.14) compared to unstretched ECT media (1.38, CI 1.16–1.60; Fig. 2A; p=0.004, two-way ANOVA with post-hoc Bonferroni), with no change in long-chain ceramides (Fig. 2B; p=0.560, two-way ANOVA with post-hoc Bonferroni). GW4869 treatment prevented short-chain ceramide production between unstretched (1.16, CI 1.06–1.27) and stretched ECT (1.21, CI 1.04–1.37; p=1.00, two-way ANOVA with post-hoc Bonferroni), indicating nSMase as a key mediator of stretch-induced short-chain ceramide production in cardiac tissue. Importantly, we demonstrate that GW4869-treated ECT lysate has nSMase activity (2553, CI 1886–3320) reduced by 32.5% compared to untreated ECT lysate (3644, CI 2680–4609; p=0.005, paired student’s t test), suggesting that GW4869 is effective in cardiac tissue (Fig. 2E). Furthermore, our data indicate that GW4869 effectively inhibits nSMase3, as RT-qPCR detected SMPD4 (encoding nSMase3) but not SMPD3 (encoding nSMase2) mRNA in ECT or adult human left ventricle and that SMPD4 is the predominant membrane-associated nSMase in mouse left ventricle (Fig. 2F). RT-qPCR analysis also revealed no changes in SMPD4 mRNA, when comparing unstretched (0.068, CI 0.042–0.093) and stretched ECT (0.040, CI 0.024–0.11; p=0.846, two-way ANOVA with post-hoc Bonferroni correction; Fig. 2D). Protein expression of nSMase3 was unchanged between unstretched and stretched ECT (data not shown). This suggests that nSMase3, not nSMase2, in our ECT is the main nSMase mediator of stretch–induced changes in caveolae-like structures and ceramide production. Overall, these findings support our hypothesis of stretch-induced nSMase activation. Another possible stretch-induced pathogenic factor in myocardium is reactive oxygen species (ROS) (Gao et al., 2021) which have also been shown to play a role in the activation of nSMase via NADPH oxidase (NOX) (Hernandez et al., 2000). While we found that cyclic stretch increases ROS production in ECT media, from 0.33 (CI 0.32–0.34) in unstretched media to 0.36 (CI 0.35–0.37; p<0.001, two-way ANOVA with post-hoc Bonferroni correction) in stretched media, it is unaffected by nSMase inhibition with no significant difference between stretched (0.36, CI 0.35–0.37) and stretched+GW4869 (0.37, CI 0.35–0.39; p=1.00, two-way ANOVA with post-hoc Bonferroni correction; Fig. 2C). This suggests that nSMase is downstream of NOX2 during mechano-transduction, but additional studies are required to determine if nSMase mechano-activation is NOX2-dependent.

It has been shown that caveolae play a critical role in the regulation of cyclic adenosine monophosphate (cAMP) signaling and cardiomyocyte response to sympathetic stimulation (Calaghan & White, 2006; Wright et al., 2014). Caveolae loss has been linked to abnormal response to β-adrenergic receptor stimulation (Rybin et al., 2003) and reported for heart failure patients (Böhm et al., 1988). To determine whether the observed stretch-induced reductions in caveolae-like structures modulates ECT response to sympathetic stimulation, we performed contraction testing on unstretched and stretched ECTs. Analysis of ECT twitch amplitude and kinetics before and after treatment with β-adrenergic receptor agonist isoproterenol (1 μM) revealed significant changes in ECT response as demonstrated in Fig. 3. First, we found that stretched ECT exhibited a significantly blunted response to isoproterenol (p=0.024, Kruskal-Wallis test) as indicated by a 11% decrease of the total contraction time to 100% peak force (CT100) in unstretched ECT (−1.13, CI −2.01 to −0.23) compared to stretched ECT (−0.12, CI −0.53 to 0.29; Fig. 3B). This blunted response was prevented by GW4869 treatment, suggesting an nSMase and caveolae-mediated role. In addition, no significant changes in isoproterenol response were observed in time to 50% and 90% relaxation (RT50 and RT90) between unstretched (−1.34, CI −2.65 to −0.028 and −1.55, CI −2.91 to −0.18, respectively) and stretched ECT (−0.60, CI −1.11 to −0.1 and −0.58, CI −1.06 to −0.1; p=0.735 and 0.436, respectively, two-way ANOVA with post-hoc Bonferroni correction; Fig. 3C–D), suggesting that the stretch protocol has a larger effect on excitation-contraction coupling rather than Ca²⁺ reuptake. Looking further, we found that stretched ECT have a 2.0% and 5.5% blunted kinetic response to isoproterenol with respect to contraction times 25–50% and 50–100% (CT25–50 and CT50–100, respectively; p=0.032 and 0.019, respectively, Kruskal-Wallis Test; Fig. 3F–G) but not 0–25% (CT0–25; p=0.663, Kruskal Wallis Test; Fig. 3E). For CT0–25, CT25–50, and CT50–100, unstretched mean ECT relative responses to isoproterenol were −0.48 (CI −0.72 to −0.24), −0.36 (CI −0.51 to −0.20), and −0.28 (CI −0.94 to 0.37), respectively, and stretched ECT responses were −0.23 (CI −0.46 to 0.00), −0.16 (CI −0.23 to −0.08), and 0.27 (CI 0.049 to 0.49). Stretch and nSMase inhibition had no effect on absolute twitch forces, ECT baseline contraction kinetics, or arrhythmogenesis (data not shown) in response to isoproterenol (p=1.00, two-way ANOVA with post-hoc Bonferroni correction; Fig. 4). In addition, stretch and GW4869 had no significant effect on sarcomere length (p=1.00, two-way ANOVA with post-hoc Bonferroni correction; Fig. 1H) further suggesting the effects of caveolae disassembly on ECT contraction, rather than changes to sarcomere ultrastructure. These findings indicate that our cyclic stretch protocol not only alters ECT plasma membrane morphology but has a significant caveolae- and nSMase-mediated effect on ECT β-adrenergic response.

As both chronically elevated stretch (Díez, 2007) and ceramide (Ji et al., 2017) have been linked to the activation of fibrogenesis and inflammatory-associated pathways, we utilized RT-qPCR to evaluate the effect of stretch and nSMase inhibition on our ECTs. We found that stretch and GW4869 have a limited effect on fibrosis. We observed a significant decrease in fibrotic area only between stretched (9.11% fibrosis, CI 7.72–10.50) and stretched+GW4869 ECT (6.52% fibrosis, CI 5.52–7.52; p=0.017, two-way ANOVA with post-hoc Bonferroni correction) but no statistically significant change in non-stretched and stretched ECT (p=0.609, two-way ANOVA with post-hoc Bonferroni correction), determined by Masson’s trichrome staining (Fig. 5A–B). With regard to mRNA expressions, we observed only a significant increase in ACTA2 between unstretched (1.00, CI 0.66–1.23) and stretched ECT treated with GW4869 (2.16, CI 1.49–2.83; p=0.005, two-way ANOVA with post-hoc Bonferroni correction) and unstretched (1.02, CI 0.75–1.29) and stretched ECT both treated with GW4869 (p=0.006, two-way ANOVA with post-hoc Bonferroni correction; Fig. 5C). In contrast, qPCR analysis revealed that stretch did not have a significant effect between unstretched and stretched ECT for other fibrosis-related mRNAs such as COL3A1 (p=0.100, Kruskal-Wallis Test), COL1A2 (p=0.438, Kruskal-Wallis Test), TIMP3 (p=0.474, Kruskal-Wallis Test), MMP2 (p=1.00, two-way ANOVA with post-hoc Bonferroni correction), and TGF-β1 (p=0.603, Kruskal-Wallis Test; Fig. 5D–H). Lastly, mRNAs for the inflammatory cytokines TNF-α and IL-6 were not detected (data not shown).

Publicly available blood lipidomic values from the MIDUS survey of health in the US were evaluated to determine if hypertension was similarly correlated to ceramide levels among middle-aged and older American adults (https://www.midus.wisc.edu/). Serum lipidomic data were acquired from ~120 hypertensive and ~420 normotensive adult participants. To identify significant differences in distinct subgroups, individuals were categorized by sex and age (above or below the age of 50). We found that ceramide 18:0 was increased by 22% and 17% in hypertensive males (0.111 μM, CI 0.096–0.126 vs. 0.090 μM, CI 0.083–0.098) and females (0.113 μM, CI 0.104–0.121 vs. 0.0904 μM, CI 0.088–0.100) over the age of 50 (p=0.013 and p<0.001, respectively, Welch’s t test), respectively, and ceramide 24:1 was increased by 19% in men over 50 (0.76 μM, CI 0.65–0.87 vs. 0.64 μM, CI 0.60–0.68; p=0.039, Welch’s t test; Fig. 6). In men under 50, no ceramides were elevated in hypertensive individuals while ceramides 16:0 and 20:0 were higher by 8% (0.287 μM, CI 0.270–0.304 vs. 0.266 μM, CI 0.256–0.276) and 16% (0.085 μM, CI 0.076–0.094 vs. 0.073 μM, CI 0.067–0.079), respectively, in hypertensive women under 50 years of age (p=0.040 and 0.028, respectively, Welch’s t test; Fig. 7A). Interestingly, ceramides 16:0, 24:0, and 24:1 were elevated by 6% (0.282 μM, CI 0.269–0.295 vs. 0.266 μM, CI 0.256–0.276), 14% (2.17 μM, CI 2.03–2.30 vs. 1.91 μM, CI 1.81–2.01), and 10% (0.65, CI 0.60–0.70 vs. 0.59, CI 0.55–0.62), respectively, (p=0.050, 0.003, and 0.050, respectively, Welch’s t test) in normotensive men under 50 years old, compared to normotensive women under 50 years old (Fig. 7B). These findings suggest that there is an increase in nSMase activity in hypertensive individuals; however, studies with inhibitors of other ceramide producers, such as ceramide synthases (Rodriguez-Cuenca et al., 2015) and acid sphingomyelinase (Pavoine & Pecker, 2009), are needed to confirm this interpretation.

To provide further support for our ECT findings and demonstrate that nSMase is activated during stretch, we applied high-resolution fluorescent optical mapping of electrical activity during acute ventricular pressure loading on wildtype mice (Fig. 8). Using this model, Pfeiffer et al. previously demonstrated that acute myocardial stretch induces conduction velocity slowing (Pfeiffer et al., 2014). We hypothesized that nSMase mediates this stretch-induced conduction slowing, which is supported by Shi et al.’s findings that nSMase inhibition prevents shear stress induced membrane depolarization in vascular tissue (Shi et al., 2020) which could affect sodium channel activation and subsequent conduction velocity slowing. Acute stretch (10 min) via pressure loading of mouse left ventricle resulted in a 29% and 31% decrease in longitudinal (0.71, CI 0.67–0.76 vs. 1.00, CI 0.81–1.18) and transversal (0.69, CI 0.51–0.88 vs. 1.00, CI 0.63–1.38) conduction velocity, respectively (p<0.001, two-way ANOVA with post-hoc Bonferroni correction; Fig. 9A–B), normalized to baseline, with no change in conduction anisotropy in control (2.04, CI 0.91–3.17 at baseline vs. 2.1, CI 0.99–3.21 during loading, p=1.00, two-way ANOVA with post-hoc Bonferroni correction) and GW4869-treated ventricles (2.39, CI 1.92–2.86 at baseline vs. 2.39, CI 2.09–2.69 during loading, p=1.00, two-way ANOVA with post-hoc Bonferroni correction). Upon unloading (20 min), both conduction velocities returned to baseline levels. More importantly, pre-treatment of mouse hearts with the nSMase inhibitor GW4869 (5 μM) completely prevented the effect of stretch-induced conduction velocity slowing (p<0.001, two-way ANOVA with post-hoc Bonferroni correction). Acute stretch also resulted in reversable decrease in maximum upstroke velocity ((dV/dt)_max; Fig. 9C) which was prevented by pre-treatment with GW4869 (9.63, CI 8.12–11.12 vs. 12.23, CI 10.90–13.55; p=0.020, two-way ANOVA with post-hoc Bonferroni correction). Furthermore, we observed a small but significant correlation between (dV/dt)_max and conduction velocity (r²=0.38, p=0.033, F test; Fig. 9D), which may suggest that the stretch-induced diminished activity of Na⁺ channels is a contributor to the observed conduction slowing. These findings identify nSMase as a key mediator of stretch-induced cardiac conduction slowing that can be observed during acute cardiac pressure-overload.