Etiology-Dependent Impairment of Diastolic Cardiomyocyte Calcium Homeostasis in Heart Failure With Preserved Ejection Fraction

Figures

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  Figure 1. T-Tubule Density Is Increased in Patients With HFpEF T-tubules were examined in biopsies taken from nonischemic regions of the myocardium in patients undergoing coronary bypass. (A, top) Confocal images of WGA-stained cryosections, with indicated regions enlarged below. (Bottom) Distance from each cytosolic point to the nearest t-tubule or surface membrane. Scale bar = 10 μm. Overall t-tubule density was increased in heart failure with preserved ejection fraction (HFpEF), both when patients were divided by New York Heart Association (NYHA) functional class (B) or severity of diastolic dysfunction (C). T-tubule density was not significantly altered in patients comorbid for diabetes mellitus (DM) (D). Distances to nearest membrane were shorter in HFpEF (E) and negatively correlated with t-tubule density (F). ∗p < 0.05 vs. control. ncells: control = 59 (4 hearts), NYHA functional class I = 70 (4 hearts), NYHA functional class II = 74 (5 hearts), NYHA functional class III = 27 (3 hearts), E/eʹ <14 = 109 (6 hearts), E/eʹ >14 = 61 (6 hearts), nondiabetes mellitus = 86 (6 hearts), diabetes mellitus = 85 (6 hearts). Data are mean ± SD.

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  Figure 2. T-Tubule Density Is Reduced in Patients With HFrEF (A) T-tubule structure was examined in explanted hearts of heart failure with reduced ejection fraction (HFrEF) patients, and compared with healthy hearts deemed unsuitable for transplant. Colored images indicate distance from each pixel to the nearest t-tubule or surface membrane. Scale bars = 10 μm. (B) Overall t-tubule density was decreased in HFrEF. Intracellular distance to nearest membrane (C) was negatively correlated with t-tubule density (D). ∗p < 0.05 vs. control. ncells: control = 102 (4 hearts), New York Heart Association (NYHA) functional class III = 65 (3 hearts), NYHA functional class IV = 122 (3 hearts). Data are mean ± SD.

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  Figure 3. T-Tubules Are Dilated in HFpEF Patients Without Additional Collagen Infiltration dSTORM super-resolution images from HFpEF (A) and HFrEF (B) patient hearts presented alongside respective control subjects, as raw and binarized images (top and bottom panels). (C) T-tubule widths were more variable in HFpEF than control subjects (p = 0.03), and mean measurements were increased. (D) T-tubule widths in HFrEF hearts were also more variable (p = 0.01), due to a subset with broadened geometries. ntubules = 211 (7 hearts), 162 (6 hearts), and 166 (3 hearts) in control, HFpEF, and HFrEF, respectively. (E) Collagen deposition within t-tubules examined by colabelling with WGA and collagen I and III antibodies. An increased fraction of t-tubules exhibited collagen staining in HFrEF. Less collagen signal/t-tubule was observed in HFpEF. ncells: control = 35 (4 hearts), HFpEF = 50 (6 hearts), HFrEF = 27 (3 hearts). ∗p < 0.05 vs. control. Data are mean ± SD. Abbreviations as in Figures 1 and 2.

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  Figure 4. T-Tubule Density and Ca2+ Release Are Predominantly Maintained in Preclinical Models of HFpEF Isolated cardiomyocytes were examined from post–myocardial infarction (MI) Wistar rats with diastolic dysfunction, hypertensive Dahl/Salt Sensitive (Dahl SS) rats, and obese/diabetic ZSF1 rats. (A) Di-8-ANEPPS stains and mean measurements of t-tubule density (ncells: sham = 91 (3 hearts), post-MI = 136 (4 hearts), low-salt Dahl SS=30 (3 hearts), high-salt Dahl SS = 30 (3 hearts), lean ZSF1 = 77 (3 hearts), and obese ZSF1 = 66 (3 hearts). (B) Ca2+-transient recordings (fluo-4 AM), and mean measurements of synchrony of Ca2+ release and removal. ncells: sham = 38 (4 hearts), post-MI = 48 (4 hearts), low-salt Dahl SS = 23 (3 hearts), high-salt Dahl SS = 21 (3 hearts), lean ZSF1 = 49 (3 hearts), obese ZSF1 = 36 (3 hearts). ∗p < 0.05 vs. control or sham. Data are mean ± SD.

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  Figure 5. Impairment of Diastolic Ca2+ Handling in HFpEF Is Etiology-Dependent Representative 1- and 4-Hz Ca2+ transient recordings and measurements of transient magnitude and decay are presented for myocytes isolated from rats with post-MI diastolic dysfunction (A), hypertensive HFpEF (Dahl SS) (B), and diabetic HFpEF (ZSF1) (C). Caffeine-elicited transients were used to calculate rates of Ca2+ reuptake and removal from the cell, estimating SERCA and NCX activity (right panels). Only cells from diabetic ZSF1 HFpEF hearts exhibited slowed Ca2+ transient decline, as activity of both SERCA and NCX were reduced. ncells: sham = 38 (4 hearts), post-MI = 48 (4 hearts), low-salt Dahl SS = 23 (3 hearts), high-salt Dahl SS = 21 (3 hearts), lean ZSF1 = 49 (3 hearts), and obese ZSF1 = 36 (3 hearts). (D) Western blot data showing levels of SERCA, PLB, phosphorylated PLB (Ser16, Thr17), and NCX. nhearts: sham = 5, post-MI = 5, low-salt Dahl SS = 5, high-salt Dahl SS = 6, lean ZSF1 = 6, obese ZSF1 = 6. ∗p < 0.05 vs. control or sham. Data are mean ± SD. Abbreviations as in Figure 4.

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  Figure 6. T-Tubules and Ca2+ Homeostasis Are Disrupted in HFrEF (A) Di-8-ANEPPS staining revealed lower t-tubule density in post-MI rats with HFrEF (ncells: sham = 57 [4 hearts] and HFrEF = 92 [6 hearts]). (B) Correspondingly, Ca2+ transient recordings (fluo-4AM) revealed desynchronized Ca2+ handling in HFrEF (ncells: sham = 19 [3 hearts] and HFrEF = 23 [3 hearts]). (C) Slowed Ca2+ decline in HFrEF cells was linked to decreased SERCA activity (ncells: sham = 19 [3 hearts] and HFrEF = 23 [3 hearts]). (D) Western blotting revealed reduced SERCA expression, with unchanged expression and phosphorylation of PLB. nhearts: sham = 6, HFrEF = 5. ∗p < 0.05 vs. sham. Data are mean ± SD.

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  Central Illustration. Heart Failure With Preserved and Reduced Ejection Fraction Exhibit Distinct Changes in Cardiomyocyte T-Tubule Structure and Etiology-Dependent Impairment of Diastolic Ca2+ Homeostasis Ventricular dilation and elevated wall stress in heart failure with reduced ejection fraction (HFrEF) promote lower cardiomyocyte t-tubule density in human patients. In rats this is accompanied by desynchronization of Ca2+ release. In contrast, heart failure with preserved ejection fraction (HFpEF) hearts exhibit concentric remodeling and maintained wall stress, linked to high t-tubule density and robust Ca2+ release. Impaired diastolic calcium handling occurs in both conditions, but within HF with preserved EF is limited to diabetic individuals, as shown in rat models. Thus, there are critical etiology-dependent differences in mechanisms for diastolic dysfunction in HF with preserved EF.