Diastolic contributors in cardiomyocytes of a cardiometabolic HFpEF-like mouse model

Figures

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  Figure S1.. Representative tracings from the four-stretch protocol.

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  Figure 1.. Experimental approach to measure diastolic XBs in intact cardiomyocytes. All intact cell experiments were performed at 37°C, and cells were stimulated at 4 Hz. (A) An intact cardiomyocyte was attached to the glass rods that connected to the piezo length controller and the force transducer. (B) A trapezoid stretch-release protocol was applied during a prolonged diastolic interval (pacing paused) to obtain the force–SL relation. The cell was then perfused with 20 mM BDM, a XB inhibitor. The stretch-release was repeated after contraction had completely stopped. (C and D) Representative tracings of SL (C) and force (D) show that, following XB inhibition, the cell gradually ceased beating, accompanied by a progressive increase in baseline SL and a decrease in baseline force. (E and F) Show zoomed-in force tracings before and after XB inhibition. The difference in up-ramp force (highlighted in blue) measured before and after XB inhibition was used to calculate the extent of diastolic XBs.

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  Figure 2.. Diastolic XBs contribute to diastolic stress in intact cardiomyocytes. (A–D) Show diastolic stress over SLs in male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like, respectively. The difference in diastolic stress measured before and after treatment with the XB inhibitor BDM (gray area) represents the extent of XB contribution. (E) The contributions of XB and non-XB stress at SL of 2.15 µm are summarized in E, and the significance of main effects (sex and heart condition) is shown. The data are presented as means ± SE. The sample sizes are 11, 13, 10, and 12 mice for male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like, with 2–4 cells analyzed per mouse. Analysis was performed using a linear mixed model with least significant difference post-hoc comparisons. ns indicates not significant. For simplicity, post-hoc comparisons between male-ctrl vs. female HFpEF-like and male HFpEF-like vs. female-ctrl are not shown.

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  Figure 3.. The extent of diastolic XBs is increased in male HFpEF-like but not in female HFpEF-like cardiomyocytes. (A and B) XB stress across diastolic SLs in cardiomyocytes from control vs. HFpEF-like males (A) and females (B). (C and D) The ratio of diastolic XBs to total diastolic stress across diastolic SLs of control vs. HFpEF-like cardiomyocytes of male (C) and female (D) mice. The data are presented as means ± SE. The sample sizes are 11, 13, 10, and 12 mice for male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like, with 2–4 cells analyzed per mouse. Analysis was performed using a linear mixed model at individual SLs. Simple linear regression was used to compare the slopes of the XB–SL relations (A and B). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

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  Figure 4.. Contribution of microtubules in intact cardiomyocytes with and without XB inhibition. (A–D) To evaluate the contribution of the microtubular network, diastolic stress–SL relation was measured in colchicine-treated and DMSO-treated cardiomyocytes, both before and after treatment with BDM, a XB inhibitor in (A) male-ctrl, (B) male HFpEF-like, (C) female-ctrl, and (D) female HFpEF-like mice. No statistically significant effect of colchicine on diastolic stress is observed in any group, although the male HFpEF-like group shows a trend toward reduced diastolic stress before BDM treatment in colchicine compared with DMSO-treated cells. The data are presented as means ± SE. The sample sizes are 9, 10, 8, and 11 mice for male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like, with 2–4 colchicine-treated cells and 2–4 DMSO-treated cells analyzed per mouse. Analysis was performed using a linear mixed model at individual SLs. The statistical significance of stress before and after BDM is not shown for simplicity.

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  Figure 5.. Microtubules influence the magnitude of diastolic XBs. (A–D) XB stress across SL is shown for colchicine-treated vs. DMSO-treated cells in four experimental groups: male-ctrl (A), male HFpEF-like (B), female-ctrl (C), and female HFpEF-like (D). Colchicine-treated cells exhibit lower diastolic XB levels than DMSO-treated cells in male HFpEF-like (B) and male-ctrl at shorter SLs (A). The data are presented as means ± SE. The sample sizes are 9, 10, 8, and 11 mice for male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like, with 2–4 colchicine-treated cells and 2–4 DMSO-treated cells analyzed per mouse. Analysis was performed using a linear mixed model at individual SLs. *P ≤ 0.05 and ns indicates not significant.

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  Figure 6.. Passive sarcomere stress is increased in cardiomyocytes from both male HFpEF-like and female HFpEF-like mice. (A and B) Diastolic stress measured in cardiomyocytes following combined treatment with BDM and colchicine, which inhibit XB activity and depolymerize microtubules, reflects passive sarcomere stress. (A and B) Passive sarcomere stress is elevated in cardiomyocytes from male HFpEF-like (A) and female HFpEF-like mice (B) compared with their respective controls. (C) The correlation between diastolic XB stress and passive sarcomere stress at a SL of 2.1 µm is plotted for four experimental groups: male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like (C). There is no correlation between sarcomere passive stress and the extent of diastolic XB stress. For A and B, the data are presented as means ± SE. The sample sizes are 9, 10, 8, and 11 mice for male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like, with 2–4 cells analyzed per mouse. Analysis was performed using a linear mixed model at individual SLs, *P ≤ 0.05. For C, the sample sizes include n = 19, 32, 23, and 29 cells from 9, 10, 8, and 11 mice for male-ctrl, male HFpEF-like, female-ctrl, and female…

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  Figure 7.. Ca 2+ handling is altered in cardiomyocytes from male HFpEF-like mice. (A–D) Ca2+ transients in isolated intact cardiomyocytes were measured using Fura-2 under 4 Hz stimulation. The Fura ratio (340/380) at diastole (A), transient amplitude (B), time to peak (C), and time to 50% transient decay (RT50; D) are shown. The data are presented as means ± SE. The sample sizes include n = 334, 337, 226, and 483 cells from 7, 8, 7, and 10 mice for male-ctrl, male HFpEF-like, female-ctrl, and female HFpEF-like. Analysis was performed using a linear mixed model with least significant difference post hoc comparisons. Main effects are shown only when they are statistically significant. Each data point represents the average value of one mouse. For simplicity, post hoc comparisons between male-ctrl vs. female HFpEF-like and male HFpEF-like vs. female-ctrl are not shown.

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  Figure 8.. XB kinetics at the myofibril level are unchanged in HFpEF-like mice. (A–J) The upper panel (A–E) show cardiac myofibril mechanic parameters at pCa 4, while the lower panels (F–J) show the same parameters at pCa 5.6. Displayed are steady-state stress (A and F), rate constant of force development (B and G), duration of the slow phase of relaxation (C and H), rate constant of the slow phase of relaxation (D and I), and rate constant of the fast phase of relaxation (E and J). The data are presented as means ± SE. The sample sizes include n = 5, 6, 5, and 7 mice, with 3–7 myofibrils analyzed per mouse. Analysis was performed using a linear mixed model with least significant difference post hoc comparisons. Each data point represents the average value of one mouse. kACT, rate constant of force development; TREL,slow, duration of the slow phase of relaxation; kREL,slow, rate constant of the slow phase of relaxation; kREL,fast, rate constant of the fast phase of relaxation.