Palmitate diet-induced loss of cardiac caveolin-3: a novel mechanism for lipid-induced contractile dysfunction.

Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate), and MCT diet (21% medium-chain triglycerides, no palmitate). We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release

Caveolin-3 expression is decreased in obese human cardiac LV tissue.

<p>Representative immunohistochemistry showing caveolin-3 in normal, obese, obese with cardiomyopathy, and non-obese cardiomyopathy human left ventricular cardiac tissue. In normal human cardiomyocytes (upper left panel) caveolin-3 is expressed at the plasma membrane and the T-tubule system (white arrows). In obesity (lower left panel), the expression of caveolin-3 is drastically decreased in the T-tubule system (black arrows), which can occur independent of associated heart disease as the samples with cardiomyopathy (upper right panel) show, but seem to be enhanced in obese cases with cardiomyopathy (lower right panel).</p

Dietary intervention is sufficient to reverse palmitate-induced caveolin-3 loss.

<p><b>A</b>) Western blots of cardiac tissue of mice maintained on standard, MCT, or palmitate diet for 12 weeks followed by 12 weeks of standard laboratory chow feeding. Caveolin-1 and -3 expression is normal. (Std standard, MCT medium chain triglyceride high fat control diet, P palmitate). <b>B</b>) Ex vivo contractile performance (RPP, pdP/dt and ndP/dt) and calcium sensitivity normalizes after 12 weeks of standard laboratory chow feeding. N = 6 (3 male and 3 female) per diet, linear regression analysis is not significant.</p

Palmitate-induced loss of T-tubular caveolin-3 and loss of striated appearance of the RyR localization.

<p>Intracellular localization of caveolin-1 (<b>A</b>) or caveolin-3 (<b>B</b>) and RyR in isolated cardiomyocytes from standard, MCT or palmitate diet fed mice. Caveolin-1 and -3 localize to the plasma membrane and the T-tubule system in standard diet fed mice (first row of panels in A and B). In MCT diet fed mice caveolin-1 expression decreases (second row of panels in A) but caveolin-3 expression remains unchanged (second row of panels in B). MCT diet does not change the striated appearance of the RyR (compare both middle panels in A and B). Palmitate diet does not change the expression or localization of caveolin-1 (third row panel A), but expression of caveolin-3 decreases and it is absent from the T-tubule system. In these areas the striated appearance of the RyR is lost (arrows in A and B). All confocal experiments were repeated at least 4 times with similar results.</p

Palmitate increases medium and long chain ceramide and sphingomyelin species in left ventricular tissue.

<p><b>A</b>) Levels of different ceramide species in total lipid extracts of left ventricular tissue determined by mass spectrometry after 12 weeks on standard, control or palmitate diet. Medium chain ceramides with C16, C20 and C20∶1 increase in mice fed palmitate for 12 weeks. Long chain ceramides C24 and C24∶1 are equally increased in control and palmitate diet fed mice, however these data did not reach significance. Mean ± SE, n = 3, two-way ANOVA, Bonferroni post-hoc analysis, * p<0.05. <b>B</b>) Sphingomyelin species levels at the 12 week feeding time point. Palmitate feeding led to an increase of medium chain sphingomyelin species C16 and C20. The long chain sphingomyelins, C24 and C24∶1, are equally increased in mice fed control or palmitate diet compared with standard diet fed mice. Mean ± SE, n = 3 (2 male, 1female each group), two-way ANOVA, Bonferroni post-hoc analysis, * p<0.05.</p

Palmitate diet does not change SERCA2a or DHPR protein levels.

<p>Quantification of SERCA (<b>A</b>) and DHPR (<b>B</b>) protein levels in LV tissue from hearts at the 12-week feeding time point. Protein levels were normalized to GAPDH levels. Both proteins are unchanged by the high fat feeding regimen. Means ± SE, n = 6–9 (3male and 3 female for n = 6, or 5 male and 4 female for n = 9).</p

General characteristics of mice at 12 weeks of the feeding regimen.

<p>BW: body weight, HW: heart weight, Bal Vol.: balloon volume  =  left ventricular volume, LV: left ventricle, m: male, f: female. Means ± SE, n = 5–7.</p

Palmitate-induced loss of caveolin-3 from the T-tubule system in the heart.

<p><b>A</b>) Localization of caveolin-1 and -3 in confocal images of isolated cardiomyocytes from mice fed standard, MCT or palmitate diet for 12 weeks. Both caveolins co-localize in cardiomyocytes from standard diet fed mice, whereas caveolin-1 decreases in MCT diet fed mice and caveolin-3 is lost from the T-tubule system in palmitate diet fed mice. <b>B</b>) Western blot for caveolin-1 and -3 for all diets. <b>C</b>) Quantification of Western blots using GAPDH as loading control (top panel) and percent changes in the lower panel. N = 5 (3 male, 2 female), * p<0.05 one-way ANOVA. <b>D</b>) Representative images of immunohistochemistry for caveolin-3 in left ventricular tissue sections of standard, MCT, and palmitate diet fed mice. Caveolin-3 staining is observed at the plasma membrane and the T-tubule system in standard diet fed mice. The overall signal intensity is modestly decreased in MCT diet fed mice, but caveolin-3 also localizes to the plasma membrane and the T-tubule system. In palmitate diet-fed mice there is significant loss of caveolin-3 staining. Some cardiomyocytes do not show any staining for caveolin-3 (white arrows) at the plasma membrane or the T-tubules. N = 4 per diet. <b>E</b>) Cellular membrane fractionations by sucrose gradient centrifugation. HL-1 cardiomyocytes were exposed to increasing concentrations of BSA-complexed palmitate (0.1, 0.2, and 0.4 mM) and then underwent sucrose gradient centrifugation to separate the different membrane systems of the cells. By convention fraction 4 is considered the high buoyant, caveolae fraction and with increasing fraction number, buoyancy decreases and membranes become denser. Exposure to palmitate causes caveolin-3 to move from the caveolae fraction (fraction 4) to the denser membrane fractions (fraction 8–10), which represent SR, ER, and nuclear membranes. Experiment repeated 4 times with similar results.</p

Palmitate diet causes systolic dysfunction in 12 weeks.

<p><b>A</b>) Rate pressure product (RPP) vs. p[Ca²⁺] relationship for mouse hearts at 2 (x), 4 (○), and 12 (▪) weeks of standard, MCT, and palmitate diet. RPP decreased by 30% in mice after 12 weeks on the palmitate diet. Means ± SE, n = 4–8, linear regression analysis through single data points using GraphPad Prism 4. <b>B</b>) pdP/dt vs. p[Ca²⁺] relationship of mice fed standard, MCT, and palmitate diet for 2, 4, or 12 weeks. pdP/dt is decreased by 40% in mice fed palmitate diet for 12 weeks. Means ± SE, n = 4–8, linear regression analysis through single data points using GraphPad Prism 4. <b>C</b>) ndP/dt vs. p[Ca²⁺] relationship for all diets over 2, 4 and 12 weeks. No differences were observed in diastolic contractile performance. Means ± SE, n = 4–8 (50/50 gender split in all groups), linear regression analysis through single data points using GraphPad Prism 4.</p