Human ApoE2 Endows Stronger Contractility in Rat Cardiomyocytes Enhancing Heart Function

Figures

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  Figure 1. hApoE2 rats showed enhanced heart function, which influenced the diastolic blood pressure of the carotid artery but not distant tail blood pressure. (A) Heart function of different rats was measured and compared by M-mode echocardiography. (B–E). Left ventricular ejection fraction (LVEF, (B,D)) and left ventricular fractional shortening (LVFS, (C,E)) measured by echocardiography were compared between different male rats at different ages. (F,G). LVEF and LVFS of male and female hApoE2 rats were compared. (H). Blood pressure was detected through arterial cannulation of the left carotid artery in rats under anesthesia. (I). Blood pressure measured with a tail-cuff technique (CODA® Noninvasive Blood Pressure System) was compared between awake rats. (J). Arterial strain measured in the abdominal aorta of different rats was compared. (K). Heart rate of different rats under awake condition. One-way ANOVA followed by a Tukey post hoc test. a vs. b, A vs. B, p < 0.05, NS, no significant difference. Rat number was indicated by symbols in the figures.

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  Figure 2. Multiple tests demonstrated that hApoE2 rats did not exhibit detrimental heart remodeling. (A,B) Heart weight normalized by body weight (A) and tibia bone length (B) were compared between different rats. (C) Left ventricular end-diastolic anterior wall thickness (LVAWd) measured at postnatal age 6 months were compared. (D) Left ventricular heart wall thickness represented by the ratio of the left ventricular radius and wall thickness was compared via analysis of the Hematoxylin and eosin (HE)-stained cross-sections of the hearts. (E) Representative HE-stained cross-sections of the rat hearts were shown. (F) Average cell area of cardiomyocytes from cross-sectioned rat hearts was compared. (G). Cell membranes of cross-sectioned rat cardiomyocytes were stained by CF555 wheat germ agglutinin (WGA) conjugates in red. Nuclei were stained by DAPI in blue. (H) Representative tracings for pulse wave Doppler for the rat mitral annulus velocity were shown. E and A wave ratio, as a marker of the left ventricular diastolic function and stiffness, were compared in different male rats at postnatal 6 months (I) and between male and female hApoE2 rats at postnatal 6 months (J). (K) Images of…

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  Figure 3. Isolated adult hApoE2 cardiomyocytes showed stronger cellular contractility, which is not due to the mechanisms regulating actin and myosin filament assembly. (A) DyLightTM 554 Phalloidin stained the F-actin labeling the thin filaments (red) of the cardiomyocytes isolated from adult rats. Nuclei were stained by DAPI in blue. (B) The relaxed sarcomere length of isolated adult cardiomyocytes was compared. (C) Representative traces of sarcomeric contractions are shown. Isolated cardiomyocytes were stimulated by 5 to 8 volts pacing stress at 1 Hz. (D) Percent change of sarcomere length during the contraction was compared between cell groups. (E) The departure velocity, a value indicating the maximal rate of change during the contraction, was compared. (F) The time to 75% of maximal departure velocity indicating how fast contraction happened was compared. (G) The time to 75% baseline, representing the time that sarcomere restored 75% of length change between the shortest length after stimulation, and initial baseline length were compared (noting that the sarcomere may not back to the baseline length during the relaxation phase). (H) The single exponential tau, the exponential decay…

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  Figure 4. ApoE2 expressing cardiomyocytes exhibited elevated energy production through mitochondrial respiration/fatty acid β-oxidation. Western blotting detected the protein expression levels of rat ApoE (anti-rApoE) and human ApoE (anti-hApoE) in heart lysate of adult rats (A), H9C2 cells transfected with different human ApoE isoforms (B), and rat neonatal cardiomyocytes transfected with different human ApoE isoforms (C). Seahorse XF assays, a method to detect the mitochondrial function, were applied to measure the oxygen consumption rate (OCR) using isolated cardiomyocytes of adult rats at age 7–8 months (D), transfected H9C2 cells (E), and transfected rat neonatal cardiomyocytes (F). Representative Seahorse XF assay traces, basal OCR, ATP production rate, maximal OCR, and proton leak were compared between groups of cells. Seahorse data were normalized with total cellular protein content using BCA assays. Data were summary of 17–45 reads from 4 independent assays for each group. (G–I) The expression levels of fatty acid transporting proteins (anti-FAT/CD36) on cell membrane and carnitine palmitoyltransferase 1 (anti-CPT1) on the outer membrane of mitochondria, detected with Western…

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  Figure 5. Intracellular Ca2+ transients indicated that Ca2+ increased more and more quickly after stimulation in isolated adult hApoE2 cardiomyocytes. (A) Representative traces of intracellular Ca2+ transients are shown. Fura2-AM signal was used as an index of intracellular Ca2+ concentration. (B) The basal concentration of intracellular Ca2+ indicated by the Fura2-AM signal was compared in isolated relaxed adult cardiomyocytes. (C) Percent change of Fura2-AM signal was compared in rat cardiomyocytes after electric stimulation of 5 to 8 volts at 1 Hz. (D) The departure velocity, indicating the maximal rate of Ca2+ change, was compared. (E) The time to 75% of maximal departure velocity, indicating how fast the signal increased, was compared. (F) The time to 75% baseline, representing the time that the Ca2+ signal dropped 75% of total change, a parameter of Ca2+ reuptake speed, was compared during relaxation. (G) The single exponential tau, equaling the time taken for switching on Ca2+ reuptake during the relaxation phase, was compared as well. A greater tau value means a longer Ca2+ reuptake time. (H–K) Western blotting was applied to detect the proteins responsible for intracellular Ca2+…

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  Figure 6. The possible mechanism of how ApoE2 is responsible for enhanced heart function. hApoE2 rat shows enhanced left ventricular function in comparison to hApoE3, E4, WT, and ApoE KO rats via echocardiography. Isolated adult hApoE2 cardiomyocytes showed stronger cellular contractility and higher intracellular Ca2+ concentration after electric stimulation. Two mechanisms might contribute to the potent contractility of hApoE2 cardiomyocytes: sufficient energy supply and potent Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). The former was a result of robust mitochondrial fatty acid β-oxidation and enhanced fatty acid uptake by fatty acid transporting protein CD36. The latter was a consequence of the considerable Ca2+ release from the sarcoplasmic reticulum through increased ryanodine receptors (RyR) accompanied by the slightly decreased expression of Ca2+ reuptake protein sarcoplasmic reticulum Ca2+ ATPase (SERCA). The red-colored objects denote those augmented signals in hApoE2 cardiomyocytes. The green-colored objects denote the suppressed signals. (Created with BioRender.com).