INTRODUCTION
Increased contractility and maladaptive remodeling of the heart resulting in cardiomyocyte hypertrophy and myocardial collagen accumulation occurs in vitamin D receptor knockout (VDRKO) mice and vitamin D-deficient rats.1-3 3 (25[OH]2 D3 ) levels may be a result of inadequate ultraviolet B (UVB) exposure and/or inadequate dietary vitamin D intake, a genetic abnormality of the hepatic 25-hydroxylase activity, or increased 25(OH)2 D3 catabolism.4 2 D3 treatment has been shown to reduce plasma renin activity, angiotensin II levels, blood pressure, and myocardial hypertrophy.5,6 1
Leptin is an adipocyte-derived pleiotropic hormone that is produced primarily by white adipose tissue and is linearly correlated with total body fat mass.7 8-10 2 D3 has been shown to inhibit leptin synthesis in human adipocytes cultured in vitro.11 12 13-15 16 17,18
Although the role of 1,25(OH)2 D3 has been studied in VDRKO mice and in various in vitro and ex vivo experiments, less is known about it's role in relevant animal models of human heart failure.1,19 20,21 cp ), a mutant form of the leptin receptor, which may play a role in the development of heart failure. The SHHF rat develops chronic hypertension and cardiac hypertrophy at an early age and progresses to congestive heart failure by the second year.22 23-25 2 D3 treatment on cardiac hypertrophy and function in SHHF(cp/+) rats fed normal and high-salt diets and determined the influence of 1,25(OH)2 D3 treatment on serum leptin levels in both dietary groups.
MATERIALS AND METHODS
Animals
Male and female SHHF rats (cp /+) (Genetic Models, Indianapolis, Indiana), originating from mating of Koletsky and inbred spontaneously hypertensive rats (SHR), were provided either normal rodent chow (10-week-old rats) or a commercially prepared high-salt diet (8% NaCl) (Harlan Teklad diet TD92012, Madison, Wisconsin; 17-week-old rats). All experiments were conducted in compliance with the Animal Care and Use Committee of the University of Michigan. Animals were not exposed to UV and fluorescent light sources to preclude de novo 1,25(OH)2 D3 synthesis during this time period. All of the rats were provided with access to water and food ad libitum.
1,25(OH)2 D3 Treatment
Animals fed the normal or high-salt diet were given subcutaneous injections of either 15 ng 1,25(OH)2 D3 in 1,2-propanediol (Sigma-Aldrich, St. Louis, Missouri);100 g total body weight) or vehicle (ethanol in 1,2-propanediol) for 13 weeks.
Echocardiogram Measurements
Two-dimensional and M-mode echocardiographic (ECG) images were recorded on rats anesthetized with isoflurane using a GE S10-MHz phased-array transducer, connected to a General Electric, Vivid 7 Ultrasound System and the AnonyMOUSE/rat ECG Screening System (Mouse Specifics, Boston, Massachusetts). Data were analyzed by proprietary software, and all rats' ECG intervals were analyzed by Student's 2-tail t -test.
Tissue Harvest and Measurement of Heart/Tibia and Heart/Brain Ratios
After 13 weeks of treatment, rats were weighed, anesthetized with sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, Illinois), and heparinized (1500 U/kg of body weight) to allow exsanguinations via the inferior vena cava. Hearts were rapidly excised from the chest cavity, washed twice with ice-cold saline, dissected to remove major blood vessels and connective tissue, blotted to remove excess fluid, and weighed. The ventricles were separated and individually weighed. After removing and weighing the tibias and brains, the heart/tibia and heart/brain weight ratios were calculated.
Rat Leptin Determination
Rat leptin was assessed in serum samples according to the manufacturer's instructions using a commercially available enzyme immunoassay kit obtained from Cayman Chemical, Inc. (Ann Arbor, Michigan).
MEASUREMENTS OF SERUM CALCIUM, MAGNESIUM, AND PHOSPHATE
Blood calcium (Arsenazo III), magnesium (Formazan Dye), and phosphate (Phosphomolybdate) were measured by the Special Chemistry Clinical Laboratories, University of Michigan Hospital.
TOTAL COLLAGEN DETERMINATION
Total soluble content was determined using the Sircol Collagen Assay Kit (Biocolor, Northern Ireland) according to the manufacturer's instructions. Briefly, hearts were homogenized in 0.5 M acetic acid containing 1 mg pepsin. Each sample was incubated for 24 hours at 4°C with stirring. After centrifugation, supernatant was assayed. One milliliter of Sircol dye reagent that binds to collagen was added to each sample and the solutions were mixed for 30 minutes. After centrifugation, the pellet was suspended in 1 mL of the alkali reagent included in the kit and read at 540 nm with a spectrophotometer. Collagen standard solution was used to construct a standard curve.
Statistical Analysis
All data are expressed as the mean ± SEM. Statistical comparisons of means were performed using a paired Student's t -test.
RESULTS
Effect of 1,25(OH)2 D3 Treatment on Total Body Weight and Serum Leptin
In rats fed both the normal and high-salt diets, there was a trend for the 1,25(OH)2 D3 treatment to reduce total body weight after 13 weeks, but this effect was statistically significant only in the rats fed the high-salt diet (Table 1 ). As compared with control rats, serum leptin levels were lower in rats fed a normal diet and treated with 1,25(OH)2 D3 , but this effect did not reach statistical significance (P = 0.09; Fig. 1A ). In contrast to this result, 1,25(OH)2 D3 treatment of rats fed the high-salt diet resulted in higher levels of serum leptin (P < 0.05; Fig. 1B ).
TABLE 1: Morphometric Analysis of SHHF Rats Treated with 1,25(OH)2 D3
FIGURE 1: Effect of 1,25(OH)2 D3 treatment on leptin levels in the SHHF (cp /+) rats fed a normal and a high-salt diet. Serum leptin levels in rats fed a normal diet (A) and a high-salt diet (8% NaCl) (B) treated with or without 1,25(OH)2 D3 for 13 weeks. Bars represent the mean ± SEM. n = 5-6 rats/group.
1,25(OH)2 D3 Treatment Reduces Cardiac Hypertrophy
SHHF (cp /+) rats are hypertensive and develop significantly more hypertrophic hearts than normal animals throughout their lifespan. We previously reported that vitamin D3 deficiency induces increases in heart weight, an indication of myocardial hypertrophy.3 2 D3 treatment could directly attenuate increases in heart weight in SHHF (cp/+) rats, we assessed total heart weight, heart to tibia weight, and heart to brain weight ratios following 13 weeks of treatment. As shown in Table 1 , total heart weights were lower for the 1,25(OH)2 D3 -treated fats fed the normal diet. In addition, there were significantly lower (P = 0.07, 1-tail test) heart to tibia weight ratios in 1,25(OH)2 D3 -treated rats fed the normal diet (P = 0.14, 2-tail test) compared with the controls (Table 1 ). Feeding salt-sensitive rats a high-salt diet is known to induce hypertension and left ventricular (LV) cardiac hypertrophy and hasten the development of heart failure.26 cp /+) rats and to determine if 1,25(OH)2 D3 treatment could provide a protective effect. In comparison with the control group, the total heart weight was lower, as was both the heart to brain and heart to tibia weight ratios (Table 1 ), which were significantly lower in the SHHF (cp /+) rats receiving 1,25(OH)2 D3 treatment. The results of these experiments suggest that 1,25(OH)2 D3 attenuates the development of cardiac hypertrophy in SHHF rats.
Effect of 1,25 (OH)2 D3 Treatment on Left Ventricular Myocardial Collagen Content
We assessed total collagen content of the hearts to determine if 1,25(OH)2 D3 influences the extracellular matrix in the myocardial tissue in SHHF (cp /+) rats. As compared with vehicle-treated rats, there was slightly less collagen in the hearts of the 1,25(OH)2 D3 -treated rats fed the control and high-salt diets but, in both dietary groups, these differences were not statistically significant (P = 0.44) and (P = 0.07; Fig. 2 ).
FIGURE 2: Effect of 1,25(OH)2 D3 treatment on myocardial total soluble collagen and left ventricular cardiac myocyte diameter in the SHHF (cp /+) rats. Myocardial total soluble collagen levels were assessed in rats fed a normal diet (A) and a high-salt diet (8% NaCl) (B) treated with or without 1,25(OH)2 D3 for 13 weeks. P values were determined using a paired Student's t -test (mean ± SEM, P < 0.001, n = 40).
1,25(OH)2 D3 Treatment Increases Serum Calcium but Not Magnesium and Phosphate Levels in Rats Fed the High-Salt Diet
Serum calcium, magnesium, and phosphate levels were assessed in the rats fed both the normal and high-salt diet (data not shown). Although calcium levels were higher (P < 0.05) in 1,25(OH)2 D3 -treated rats (normal: 11.6 ± 0.7, high salt: 11.4 ± 0.6 ) relative to the control group (normal: 9.9 ± 0.2, high salt: 9.7 ± 0.1), these were within the normal calcium level for rats. Serum phosphate and magnesium levels were not significantly different in 1,25(OH)2 D3 -treated SHHF rats fed either diet when compared with the control groups.
1,25(OH)2 D3 Reduces Stroke Volume and Left Ventricular Diameter in Rats Fed the High-Salt but Not the Control Diet
Because we observed that 1,25(OH)2 D3 ameliorated cardiac hypertrophy, we assessed the effect of this hormone on cardiac function using echocardiography to estimate stroke volume and LV diameters during diastole and systole. As shown in Figure 3 , 1,25(OH)2 D3 did not alter stroke volume or LV diameters in 1,25(OH)2 D3 -treated rats fed the control diet. However, the hormone treatment resulted in lower stroke volume and LV diameters (both diastole and systole) in rats fed the high-salt diet relative to the control group. Although we did not observe any differences in heart rate, mean arterial blood pressure, or fractional shortening, we did find that relative ventricular wall thickness was greater in the 1,25(OH)2 D3 -treated rats fed the high-salt diet as compared with the controls (Table 2 ).
TABLE 2: Cardiac Contractile Performance in SHHF Rats Treated with 1,25(OH)2 D3
FIGURE 3: Effect of 1,25(OH)2 D3 treatment on stroke volume and left ventricular diameter in the SHHF (cp/+) rats. Stroke volume (A and B) and left ventricular diameters during diastole and systole (C and D) were determined using echocardiography in rats fed a normal diet or a high-salt diet (8% NaCl) and treated with or without 1,25(OH)2 D3 for 13 weeks. Bars represent mean ± SEM with n = 5- 6 rats per group.
DISCUSSION
Using VDRKO mice and other models of vitamin D deficiency, we previously demonstrated that this hormone plays a role in modulating cardiac contractility, hypertrophy, and fibrosis.1-3 20,21 17,18 2 D3 treatment for 13 weeks reduces heart weight, cardiomyocyte hypertrophy, stoke volume, and LV diameter in SHHF rats fed a high-salt diet. These improvements occurred despite increases in serum leptin, an adipokine that is associated with heart failure and is known to contribute to cardiomyocyte hypertrophy.17
SHHF rats develop heart enlargement, myocardial collagen accumulation, LV dilation, and increases in stroke volume in response to overstimulation of the heart via the systemic renin-angiotensin aldosterone systems.27,28 1-3,29 2 D3 treatment for 13 weeks prevents the development of heart enlargement.
In addition, we observed significantly greater relative ventricular wall thicknesses in rats treated with 1,25 (OH)2 D3 . We believe that this observation may be a result of the high salt 1,25(OH)2 D3 -treated rats having a significant reduction in LV diameter. Because relative wall thickness is a ratio of the wall thickness to the diameter, although both decreased in the treated animals, the diameter decreased more, leading to an increase in relative wall thickness. This would be consistent with having diminished pathologic hypertrophy and ventricular dilatation associated with heart failure progression and would not affect compensatory hypertrophy associated with an increased afterload of systemic hypertension.
The mechanism by which 1,25(OH)2 D3 exerts its protection against heart enlargement was most likely a result of a direct effect on cardiomyocytes that express the vitamin D receptor.30 31 2 D3 prevents overstimulation of the heart by decreasing cardiac contractile performance and ultimately cardiomyocyte hypertrophy through a protein kinase C-dependent mechanism.32 2 D3 activates protein kinase C-mediated phosphorylation of Ca2+ cycling and myofilament proteins (phospholamban and troponin I), resulting in a decrease in peak shortening and acceleration of contraction and relaxation of cardiomyocytes.32 2 D3 and other vitamin D compounds alter cardiomyocyte expression of genes known to contribute to ventricular hypertrophy.33 2 D3 treatment, in comparison with the untreated rats, results in improved cardiac function as indicated by lower cardiac output and LV diameters as assessed by echocardiography. Likewise, a report by Bodyak et al has demonstrated that paricalcitol (an activated vitamin D compound) attenuated LV abnormalities associated with dietary sodium in Dahl salt-sensitive rats.33 2 D3 affected peripheral vascular resistance because we did not observe any differences in mean arterial blood pressure between the control and treated animals (Table 2 ).
It is possible that there were indirect effects of 1,25(OH)2 D3 on cardiovascular function in our studies. For example, an 8% NaCl diet is calciuric; the more sodium in renal tubules, the more calcium is excreted. Unless this is replaced, chronic urinary calcium losses will lead to ionized hypocalcemia and secondary hyperparathyroidism (SHPT). The elevation in serum parathyroid hormone promotes intracellular calcium overloading and oxidative stress with the observed responses seen in the heart on a high-salt diet. 1,25(OH)2D3 administration may serve to increase calcium absorption in the gut and calcium reabsorption in the nephron to thereby prevent hypocalcemia and SHPT. In the SHR, there is an increased expression of parathyroid hormone-related peptide gene in blood vessels.34 35 36
Previous reports have suggested that leptin plays a direct role in heart failure by contributing to cardiac hypertrophy in human subjects. Indeed, leptin levels are associated with myocardial wall thickness and LV mass, independent of body mass index and body composition in patients.37,38 39,40 2 D3 . This finding is consistent with the observation that 1,25(OH)2 D3 has been shown to directly reduce leptin production by human adipocytes in vitro.11 2 D3 treatment of SHHF rats fed a high-salt diet resulted in a slight but insignificant decrease in total body weight and a significant increase in serum leptin levels. This result suggests that a high-salt diet has a more potent effect on leptin production in vivo than does 1,25(OH)2 D3 .
Dobrian and colleagues have reported that a high-salt diet increases serum leptin levels in rats in a dose-dependent manner and that dietary salt intake was associated with increases in serum fatty acids and increased adipocyte diameter, a potential trigger for increased leptin synthesis.10,41 cp /+) are lean, have mild hyperleptinemia, and are insulin resistant.25 cp /+ rats responded to increased salt intake in a similar manner as the normal-diet control rats; however, we observed a synergistic effect between a high-salt diet and vitamin D3 that led to an augmentation in serum leptin levels in SHHF rats. In total, whereas others have suggested that leptin may be an important factor in the pathogenesis of heart enlargement associated with heart failure,42 2 D3 to reduce cardiac hypertrophy in the SHHF rat is not dependent on circulating leptin levels.
CONCLUSIONS
In summary, we have observed that 1,25(OH)2 D3 treatment reduced cardiac hypertrophy in SHHF rats fed a normal and a high-salt diet, a previously unrecognized finding that warrants further investigation. We show that this action is independent of effects of the hormone on serum leptin levels in the SHHF rat that is known to be heterozygous for a mutant leptin receptor. The VDR exists in cardiac myocytes and the mechanisms used by 1,25(OH)2 D3 may be to directly activate this receptor.43 2 D3 as an adjunctive therapeutic agent in patients with LV hypertrophy, a strategy that has been used successfully to reduce posterior wall thickness in patients with renal failure.33
ACKNOWLEDGMENTS
Support for this research was provided by NIH Grants HL074894 (R.U.S.) and HL077417(P.M.)
REFERENCES
1. Simpson RU, Hershey SH, Nibbelink KA. Characterization of heart size and blood pressure in the vitamin D receptor knockout mouse.
J Steroid Biochem Mol Biol . 2007;103:521-524.
2. Weishaar RE, Kim SN, Saunders DE, et al. Involvement of vitamin D3 with cardiovascular function. III. Effects on physical and morphological properties.
Am J Physiol . 1990;258:E134-142.
3. Weishaar RE, Simpson RU. Vitamin D3 and cardiovascular function in rats.
J Clin Invest . 1987;79:1706-1712.
4. Zittermann A, Schleithoff SS, Koerfer R. Vitamin D insufficiency in congestive heart failure: Why and what to do about it?
Heart Fail Rev . 2006;11:25-33.
5. Kimura Y, Kawamura M, Owada M, et al. Effectiveness of 1,25-dihydroxyvitamin D supplementation on blood pressure reduction in a pseudohypoparathyroidism patient with high renin activity.
Intern Med . 1999;38:31-35.
6. Park CW, Oh YS, Shin YS, et al. Intravenous calcitriol regresses myocardial hypertrophy in hemodialysis patients with secondary hyperparathyroidism.
Am J Kidney Dis . 1999;33:73-81.
7. Maffei M, Halass J, Ravussin E, et al. Leptin levels in human and rodent: Measurements of plasma leptin and ob RNA in obese and weight-reduced subjects.
Nat Med . 1995;1:1155-1161.
8. Sarraf P, Frederich RC, Turner EM, et al. Multiple cytokines and acute inflammation raise mouse leptin levels: Potential role in inflammatory anorexia.
J Exp Med . 1997;185:171-176.
9. Mancuso P, Gottschalk A, Phare SM, et al. Leptin-deficient mice exhibit impaired host defense in gram-negative pneumonia.
J Immunol . 2002;168:4018-4024.
10. Dobrian AD, Schriver SD, Lynch T, et al. Effect of salt on hypertension and oxidative stress in a rat model of diet-induced obesity.
Am J Physiol Renal Physiol . 2003;285:F619-628.
11. Menendez C, Lage M, Peino R, et al. Retinoic acid and vitamin D(3) powerfully inhibit in vitro leptin secretion by human adipose tissue.
J Endocrinol . 2001;170:425-431.
12. Ahima RS, Flier JS. Leptin.
Annu Rev Physiol . 2000;62:413-437.
13. Haynes WG, Morgan DA, Walsh SA, et al. Receptor-mediated regional sympathetic nerve activation by leptin.
J Clin Invest . 1997;100:270-278.
14. Dong F, Zhang X, Yang X, et al. Impaired cardiac contractile function in ventricular myocytes from leptin-deficient ob/ob obese mice.
J Endocrinol . 2006;188:25-36.
15. Wallace AM, McMahon AD, Packard CJ, et al. Plasma leptin and the risk of cardiovascular disease in the west of Scotland coronary prevention study (WOSCOPS).
Circulation . 2001;104:3052-3056.
16. Barouch LA, Berkowitz DE, Harrison RW, et al. Disruption of leptin signaling contributes to cardiac hypertrophy independently of body weight in mice.
Circulation . 2003;108:754-759.
17. Schulze PC, Kratzsch J, Linke A, et al. Elevated serum levels of leptin and soluble leptin receptor in patients with advanced chronic heart failure.
Eur J Heart Fail . 2003;5:33-40.
18. Doehner W, Pflaum CD, Rauchhaus M, et al. Leptin, insulin sensitivity and growth hormone binding protein in chronic heart failure with and without cardiac cachexia.
Eur J Endocrinol . 2001;145:727-735.
19. Rahman A, Hershey S, Ahmed S, et al. Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice.
J Steroid Biochem Mol Biol . 2007;103:416-419.
20. Gerdes AM, Onodera T, Wang X, et al. Myocyte remodeling during the progression to failure in rats with hypertension.
Hypertension . 1996;28:609-614.
21. Heyen JR, Blasi ER, Nikula K, et al. Structural, functional, and molecular characterization of the SHHF model of heart failure.
Am J Physiol . 2002;283:H1775-1784.
22. Johnsen DD, Kacimi R, Anderson BE, et al. Protein kinase C isozymes in hypertension and hypertrophy: Insight from SHHF rat hearts.
Mol Cell Biochem . 2005;270:63-69.
23. Ahn J, Varagic J, Slama M, et al. Cardiac structural and functional responses to salt loading in SHR.
Am J Physiol . 2004;287:H767-772.
24. Haas GJ, McCune SA, Brown DM, et al. Echocardiographic characterization of left ventricular adaptation in a genetically determined heart failure rat model.
Am Heart J . 1995;130:806-811.
25. Radin MJ, Holycross BJ, Hoepf TM, et al. Increased salt sensitivity secondary to leptin resistance in SHHF rats is mediated by endothelin.
Mol Cell Biochem . 2003;242:57-63.
26. Inoko M, Kihara Y, Morii I, et al. Transition from compensatory hypertrophy to dilated, failing left ventricles in Dahl salt-sensitive rats.
Am J Physiol . 1994;267:H2471-2482.
27. Radin M, Holycross B, Sharkey L, et al. Gender modulates activation of renin-angiotensin and endothelin systems in hypertension and heart failure
J Appl Physiol . 2002;92 935-940.
28. Onodera T, Tamura T, Said S, et al. Maladaptive remodeling of cardiac myocyte shape begins long before failure in hypertension.
Hypertension . 1998;32:753-757.
29. Weishaar R, Simpson R. Vitamin D3 and cardiovascular function in rats.
J Clin Invest . 1987;79:1706-1712.
30. O'Connell TD, Simpson RU. Immunochemical identification of the 1,25-dihydroxyvitamin D3 receptor protein in human heart.
Cell Biol Int . 1996;20:621-624.
31. Emter CA, McCune SA, Sparagna GC, et al. Low-intensity exercise training delays onset of decompensated heart failure in spontaneously hypertensive heart failure rats.
Am J Physiol . 2005;289:H2030-2038.
32. Green JJ, Robinson DA, Wilson GE, et al. Calcitriol modulation of cardiac contractile performance via protein kinase C.
J Mol Cell Cardiol . 2006;41:350-359.
33. Bodyak N, Ayus JC, Achinger S, et al. Activated vitamin D attenuates left ventricular abnormalities induced by dietary sodium in Dahl salt-sensitive animals.
Proc Natl Acad Sci U S A . 2007;104:16810-16815.
34. Noda M, Katoh T, Kurokawa K, et al. Increased expression of parathyroid hormone-related peptide gene in blood vessels of spontaneously hypertensive rats.
Hypertension . 1997;30:1284-1288.
35. Sutherland SK, Benishin CG. Regulation of parathyroid hypertensive factor secretion by Ca2+ in spontaneously hypertensive rat parathyroid cells.
Am J Hypertens . 2004;17:266-272.
36. Delfini E, Petramala L, Caliumi C, et al. Circulating leptin and adiponectin levels in patients with primary hyperparathyroidism.
Metabolism . 2007;56:30-36.
37. Perego L, Pizzocri P, Corradi D, et al. Circulating leptin correlates with left ventricular mass in morbid (grade III) obesity before and after weight loss induced by bariatric surgery: a potential role for leptin in mediating human left ventricular hypertrophy.
J Clin Endocrinol Metab . 2005;90:4087-4093.
38. Paolisso G, Tagliamonte MR, Galderisi M, et al. Plasma leptin level is associated with myocardial wall thickness in hypertensive insulin-resistant men.
Hypertension . 1999;34:1047-1052.
39. Rajapurohitam V, Gan XT, Kirshenbaum LA, et al. The obesity-associated peptide leptin induces hypertrophy in neonatal rat ventricular myocytes.
Circ Res . 2003;93:277-279.
40. Xu FP, Chen MS, Wang YZ, et al. Leptin induces hypertrophy via endothelin-1-reactive oxygen species pathway in cultured neonatal rat cardiomyocytes.
Circulation . 2004;110:1269-1275.
41. Zhang Y, Guo KY, Diaz PA, et al. Determinants of leptin gene expression in fat depots of lean mice.
Am J Physiol Regul Integr Comp Physiol . 2002;282:R226-234.
42. Schulze PC, Kratzsch J. Leptin as a new diagnostic tool in chronic heart failure.
Clin Chim Acta . 2005;362:1-11.
43. Nibbelink KA, Tishkoff DX, Hershey SD, et al. 1,25(OH)2-vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte.
J Steroid Biochem Mol Biol . 2007;103:533-537.
