Vitamin D receptor genetics on extracellular matrix biomarkers and hemodynamics in systolic heart failure.

INTRODUCTION: Vitamin D deficiency has been associated with the development of myocardial hypertrophy and inflammation. These findings suggest that vitamin D status and vitamin D receptor (VDR) genomics may play a role in myocardial fibrosis. The aim of this pilot study was to determine the association between vitamin D levels, VDR polymorphisms, and biomarkers of left ventricular remodeling and hemodynamics. METHODS: In a cross-sectional pilot study, patients with ejection fraction (EF) <40% (and New York Heart Association ≥ II) undergoing right heart catheterization were included in the study. Blood was collected for determination of 25-hydroxyvitamin D level (antibody competitive immunoassay), VDR genotypes (BsmI, ApaI, TaqI, and FokI), and biomarkers (N-terminal propeptide of collagen type III [PIIINP], matrix metalloproteinase 2, and galectin 3). The vitamin D genotypes were determined through the use of pyrosequencing. RESULTS: A total of 30 patients with a mean EF of 17% ± 8% were enrolled. There was a significant association between the BsmI C allele, ApaI G allele, and TaqI A allele, which formed a haplotype block (CGA) for analysis. There were no differences in baseline parameters between patients with the VDR haplotype block (n = 20) and those without (n = 10). Individual genotypes were not associated with any biomarker or hemodynamics. Patients with the CGA haplotype demonstrated significantly higher log PIIINP values (1.74 ± 0.32 mcg/mL vs 1.36 ± 0.31 mcg/mL, P = .0041). When evaluating vitamin D levels below and above the median level (19 ng/mL), there was no significant difference between these 2 groups in regard to biomarker levels for left ventricular remodeling. CONCLUSION: This study has shown that a biomarker for collagen type III synthesis, PIIINP, was associated with the CGA haplotype of BsmI, ApaI, and TaqI single nucleotide polymorphisms on the VDR. These findings suggest that VDR genetics may play a role in myocardial fibrosis in patients with systolic heart failure.

Characterization of vitamin D receptor (VDR) in lung adenocarcinoma.

PURPOSE: The anti-proliferative effects of 1α,25-dihydroxyvitamin D(3) (1,25-D(3), calcitriol, the active form of vitamin D) are mediated by the nuclear vitamin D receptor (VDR). In the present study, we characterized VDR expression in lung adenocarcinoma (AC). EXPERIMENTAL DESIGN: We examined VDR mRNA expression using a quantitative real-time PCR (qRT-PCR) in 100 patients who underwent surgery for lung AC. In a subset of these patients (n=89), we examined VDR protein expression using immunohistochemistry. We also examined the association of VDR protein expression with circulating serum levels of 25-hydroxyvitamin D(3) (25-D(3)) and 1,25-D(3). The antiproliferative effects and cell cycle arrest of 1,25-D(3) were examined using lung cancer cell lines with high (SKLU-1) as well as low (A549) expression of VDR mRNA. RESULTS: Higher VDR expression correlates with longer survival after adjusting for age, sex, disease stage and tumor grade (HR 0.73, 95% CI 0.58-0.91). In addition, there was a positive correlation (r=0.38) between serum 1,25-D(3) and tumor VDR protein expression. A greater anti-proliferative effect of 1,25-D(3) was observed in high compared to low VDR-expressing cell lines; these effects corresponded to G1 cell cycle arrest; this was associated with a decline in cyclin D1, S-phase kinase protein 2 (Skp2), retinoblastoma (Rb) and minichromosome maintenance 2 (MCM2) proteins involved in S-phase entry. CONCLUSIONS: Increased VDR expression in lung AC is associated with improved survival. This may relate to a lower proliferative status and G1 arrest in high VDR-expressing tumors.

CARD-024, a vitamin D analog, attenuates the pro-fibrotic response to substrate stiffness in colonic myofibroblasts.

Intestinal fibrosis is one of the major complications of Crohn's disease (CD) for which there are no effective pharmacological therapies. Vitamin D deficiency is common in CD, though it is not known whether this is a contributing factor to fibrosis, or simply a consequence of the disease itself. In CD, fibrosis is mediated mainly by activated intestinal myofibroblasts during remodeling of extracellular matrix in response to wound healing. We investigated the effects of CARD-024 (1-alpha-hydroxyvitamin D5), a vitamin D analog with minimal hypercalcemic effects, on the pro-fibrotic response of intestinal myofibroblasts to two fibrogenic stimuli: TGFß stimulation and culture on a physiologically stiff matrix. TGFß stimulated a fibrogenic phenotype in Ccd-18co colonic myofibroblasts, characterized by an increase in actin stress fibers and mature focal adhesions, and increased αSMA protein expression, while CARD-024 repressed αSMA protein expression in a dose-dependent manner. Culture of colonic myofibroblasts on physiological high stiffness substrates induced morphological changes with increased actin stress fibers and focal adhesion staining, induction of αSMA protein expression, FAK phosphorylation, induction of fibrogenic genes, and repression of COX-2 and IL-1ß. CARD-024 treatment repressed the stiffness-induced morphological features including stellate cell morphology and the maturation of focal adhesions. CARD-024 repressed the stiffness-mediated induction of αSMA protein expression, FAK phosphorylation, and MLCK and ET-1 gene expression. In addition, CARD-024 partially stimulated members of the COX-2/IL-1ß inflammatory pathway. In summary, CARD-024 attenuated the pro-fibrotic response of colonic myofibroblasts to high matrix stiffness, suggesting that vitamin D analogs such as CARD-024 may ameliorate intestinal fibrosis.

CYP24A1 is an independent prognostic marker of survival in patients with lung adenocarcinoma.

PURPOSE: The active form of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25-D(3)), exerts antiproliferative effects in cancers, including lung adenocarcinoma (AC). CYP24A1 is overexpressed in many cancers and encodes the enzyme that catabolizes 1,25-D(3). The purpose of our study was to assess CYP24A1 as a prognostic marker and to study its relevance to antiproliferative activity of 1,25-D(3) in lung AC cells. EXPERIMENTAL DESIGN: Tumors and corresponding normal specimens from 86 patients with lung AC (stages I-III) were available. Affymetrix array data and subsequent confirmation by quantitative real time-PCR were used to determine CYP24A1 mRNA expression. A subsequent validation set of 101 lung AC was used to confirm CYP24A1 mRNA expression and its associations with clinical variables. The antiproliferative effects of 1,25-D(3) were examined using lung cancer cell lines with high as well as low expression of CYP24A1 mRNA. RESULTS: CYP24A1 mRNA was elevated 8- to 50-fold in lung AC (compared to normal nonneoplastic lung) and significantly higher in poorly differentiated cancers. At 5 years of follow-up, the probability of survival was 42% (high CYP24A1, n = 29) versus 81% (low CYP24A1, n = 57) (P = 0.007). The validation set of 101 tumors showed that CYP24A1 was independently prognostic of survival (multivariate Cox model adjusted for age, gender, and stage, P = 0.001). A549 cells (high CYP24A1) were more resistant to antiproliferative effects of 1,25-D(3) compared with SKLU-1 cells (low CYP24A1). CONCLUSIONS: CYP24A1 overexpression is associated with poorer survival in lung AC. This may relate to abrogation of antiproliferative effects of 1,25-D(3) in high CYP24A1 expressing lung AC.

Interaction between vitamin D receptor with caveolin-3 and regulation by 1,25-dihydroxyvitamin D3 in adult rat cardiomyocytes.

We show that 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and a synthetic non-genotropic vitamin D analog agonist, 1a,25(OH)2-lumisterol (JN), exhibit similar rapid effects on sarcomere shortening (contraction) of isolated adult cardiomyocyte. We also report that the vitamin D receptor (VDR) specifically interacts with caveolin-3 in the t-tubules and sarcolemma of isolated adult rat cardiac myocytes. Confocal immunofluorescence microscopy analysis showed co-localization of VDR and caveolin-3 in the t-tubules and sarcolemma of cardiomyocytes. Co-immunoprecipitation studies using VDR antibodies revealed that caveolin-3 specifically co-precipitates with the VDR and similarly the VDR is co-precipitated with caveolin-3 antibody. VDR is also in association with Serca-2, the sarcoplasmic reticulum Ca2+-ATPase, as demonstrated by co-immunoprecipitation, suggesting a role of VDR in regulating cardiac contractility by direct interaction with Serca-2. Treatment of isolated adult rat cardiomyocytes with 10 nM 1,25(OH)2D3 for 1 h caused decreased association between VDR and caveolin-3. These discoveries of the association between VDR and caveolin-3 and the regulation of this interaction by 1,25(OH)2D3 are fundamentally important in understanding 1,25(OH)2D3 signal transduction in heart cells and suggest a novel mechanism for VDR in the regulation of heart structure and function.

The vitamin D/CYP24A1 story in cancer.

There is increasing evidence linking the incidence of certain cancers to low serum Vitamin D levels. The active metabolite of Vitamin D, calcitriol (1, 25-Dihydroxyvitamin D(3), 1,25(OH)(2)D(3)) apart from a crucial role in maintaining mineral homeostasis and skeletal functions, has antiproliferative, apoptosis and differentiation inducing as well as immunomodulatory effects in cancer. In studying the role of 1,25(OH)(2)D(3) in cancer, it is imperative to examine the potential pathways that control local tissue levels of 1,25(OH)(2)D(3). The enzyme CYP24A1 or 24-hydroxylase converts 1,25(OH)(2)D(3) to inactive calcitroic acid. Extra-renal production of this enzyme is observed and has been increasingly recognized as present in cancer cells. This enzyme is rate limiting for the amount of local 1,25(OH)(2)D(3) in cancer tissues and elevated expression is associated with an adverse prognosis. The gene that encodes CYP24A1 has been reported as an oncogene and may contribute to tumor aggressiveness by abrogating local anti-cancer effects of 1,25(OH)(2)D(3). It is imperative to study the regulation of CYP24A1 in cancer and especially the local metabolism of 1,25(OH)(2)D(3) in cancer cells. CYP24A1 may be a predictive marker of 1,25(OH)(2)D(3) efficacy in patients with cancer as an adjunctive therapy. The following review summarizes the available literature on CYP24A1 as it relates to 1,25(OH)(2)D(3) in cancer and outlines potential ways to inhibit CYP24A1 in an effort to improve the efficacy of exogenous 1,25(OH)(2)D(3).

Relation of serum 25-hydroxyvitamin D to heart rate and cardiac work (from the National Health and Nutrition Examination Surveys).

Vitamin D may protect against cardiovascular disease, but its association with cardiac function is unclear. The aim of this study was to examine the associations of serum 25-hydroxyvitamin D (25[OH]D) with heart rate, systolic blood pressure, and the rate-pressure product (RPP). Data analyses were carried out on 27,153 participants aged > or =20 years, with measurements of serum 25(OH)D, heart rate (from radial pulse), and systolic blood pressure, in the National Health and Nutrition Examination Surveys (NHANES) carried out from 1988 to 1994 and from 2001 to 2006. RPP was calculated as heart rate times systolic blood pressure. Results were adjusted for age, gender, race or ethnicity, body mass index, physical activity, tobacco smoking, co-morbidities, and blood pressure treatment. Compared to participants with 25(OH)D > or =35 ng/ml, the adjusted mean +/- SE heart rate was significantly (p <0.001) higher, by 2.1 +/- 0.6 beats/min, in participants with 25(OH)D <10.0 ng/ml, while mean systolic blood pressure was 1.9 +/- 0.8 mm Hg higher (p <0.05) for participants with 25(OH)D <10.0 ng/ml and 1.7 +/- 0.6 mm Hg higher (p <0.01) for those with 25(OH)D of 10.0 to 14.9 ng/ml. As a consequence, adjusted mean RPP was 408 +/- 110 beats/min . mm Hg higher (p <0.001) for participants with 25(OH)D <10.0 ng/ml and 245 +/- 80 beats/min . mm Hg higher (p <0.01) for participants with 25(OH)D of 10.0 to 14.9 ng/ml, compared to those with 25(OH)D > or =35 ng/ml. In conclusion, these results show that low serum 25(OH)D levels are associated with increased heart rate, systolic blood pressure, and RPP and suggest that low vitamin D status may increase cardiac work. Vitamin D intervention studies are required to confirm these findings.

Membrane localization, Caveolin-3 association and rapid actions of vitamin D receptor in cardiac myocytes.

The active form of vitamin D, 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), mediates both genomic and rapid non-genomic actions in heart cells. We have previously shown that the vitamin D receptor (VDR) is located in the t-tubular structure of cardiomyocytes. Here we show that VDR specifically interacts with Caveolin-3 in the t-tubules and sarcolemma of adult rat cardiac myocytes. Co-immunoprecipitation studies using VDR antibodies revealed that Caveolin-3 specifically co-precipitates with the VDR and similarly the VDR is co-precipitated with Caveolin-3 antibody. Confocal immuno-fluorescence microscopy analysis also showed co-localization of VDR and Caveolin-3 in t-tubules and sarcolemma. The non-genomic effects of the functional VDR were studied in electrically stimulated myocytes isolated from adult rat hearts. Sarcomere shortening and re-lengthening were measured in 1,25(OH)(2)D(3) treated cardiac myocytes. A 1nM treatment decreased peak shortening within minutes, suggesting a rapid effect through the membrane-bound VDR. This novel finding of the interaction between VDR and Caveolin-3 is fundamentally important in understanding 1,25(OH)(2)D(3) signal transduction in heart cells and provides further evidence that VDR plays a role in regulation of heart structure and function.

Genetic variation in CYP27B1 is associated with congestive heart failure in patients with hypertension.

AIMS: We tested the hypothesis that genetic variation in vitamin D-dependent signaling is associated with congestive heart failure in human subjects with hypertension. MATERIALS & METHODS: Functional polymorphisms were selected from five candidate genes: CYP27B1, CYP24A1, VDR, REN and ACE. Using the Marshfield Clinic Personalized Medicine Research Project, we genotyped 205 subjects with hypertension and congestive heart failure, 206 subjects with hypertension alone and 206 controls (frequency matched by age and gender). RESULTS: In the context of hypertension, a SNP in CYP27B1 was associated with congestive heart failure (odds ratio: 2.14 for subjects homozygous for the C allele; 95% CI: 1.05-4.39). CONCLUSION: Genetic variation in vitamin D biosynthesis is associated with increased risk of heart failure.

Vitamin D and cardiovascular disease.

The hormonal derivative of vitamin D, 1,25-dihydroxyvitamin D (1,25[OH](2)D) or calcitriol, has been implicated in many physiologic processes beyond calcium and phosphorus homeostasis, and likely plays a role in several chronic disease states, in particular, cardiovascular disease. Experimental data suggest that 1,25(OH)(2)D affects cardiac muscle directly, controls parathyroid hormone secretion, regulates the renin-angiotensin-aldosterone system, and modulates the immune system. Because of these biologic effects, vitamin D deficiency has been associated with hypertension, several types of vascular diseases, and heart failure. We conducted a MEDLINE search of the English-language literature (1950-2008) to identify studies that examined these relationships; additional citations were obtained from the articles retrieved from the literature search. Treatment with vitamin D lowered blood pressure in patients with hypertension and modified the cytokine profile in patients with heart failure. Measurement of serum 25-hydroxyvitamin D concentration usually provides the best assessment of an individual's vitamin D status. Serum levels below 20 ng/ml represent vitamin D deficiency, and levels above 30 ng/ml are considered optimal. Although the observational data linking vitamin D status to cardiovascular disease appear robust, vitamin D supplementation is not recommended as routine treatment for heart disease until definitive prospective, randomized trials can be carried out to assess its effects. However, such supplementation is often appropriate for other reasons and may be beneficial to cardiovascular health in certain patients.

Macro- and micronutrient dyshomeostasis in the adverse structural remodelling of myocardium.

Hypertension and heart failure are worldwide health problems of ever-increasing proportions. A failure of the heart, during either systolic and/or diastolic phases of the cardiac cycle, has its origins rooted in an adverse structural, biochemical, and molecular remodelling of myocardium that involves its cellular constituents, extracellular matrix, and intramural coronary vasculature. Herein we focus on the pathogenic role of a dyshomeostasis of several macro- (i.e. Ca(2+) and Mg(2+)) and micronutrients (i.e. Zn(2+), Se(2+), and vitamin D) in contributing to adverse remodelling of the myocardium and its failure as a pulsatile muscular pump. An improved understanding of how these macro- and micronutrients account for the causes and consequences of adverse myocardial remodelling carries with it the potential of identifying new biomarkers predictive of risk, onset and progression, and response to intervention(s), which could be monitored non-invasively and serially over time. Moreover, such incremental knowledge will serve as the underpinning to the development of novel strategies aimed at preventing and/or regressing the ongoing adverse remodelling of myocardium. The time is at hand to recognize the importance of macro- and micronutrient dyshomeostasis in the evaluation and management of hypertension and heart failure.

1,25-Dihydroxyvitamin-D3 treatment reduces cardiac hypertrophy and left ventricular diameter in spontaneously hypertensive heart failure-prone (cp/+) rats independent of changes in serum leptin.

A number of investigators have observed insufficient 25-hydroxyvitamin D status in patients with congestive heart failure, suggesting a role for vitamin D insufficiency in the pathogenesis of this disorder. We have observed cardiac hypertrophy and collagen accumulation in rats deficient in vitamin D and in the hearts of vitamin D-receptor knockout mice. Our studies indicate that absence of vitamin D-mediated signal transduction and genomic activation results in cardiomyocytes overstimulation including increased contractility. These events ultimately lead to cardiomyocyte hypertrophy. In this report, we used spontaneously hypertensive heart failure rats cp/+ (hemyzygous for the corpulent gene, a mutant isoform of the leptin receptor) fed a normal and a high-salt diet to assess the potential for activated vitamin D (1,25 dihydroxyvitamin D3) to prevent cardiac hypertrophy and increases in cardiac output. After 13 weeks, as compared with untreated rats, we observed that 1,25 dihydroxyvitamin D3 treatment in rats fed a high-salt diet resulted in lower heart weight, myocardial collagen levels, left ventricular diameter, and cardiac output despite higher serum leptin levels. These studies suggest that 1,25(OH)2D3 treatment may prevent the development of cardiac hypertrophy, an important contributing factor in the progression of congestive heart failure.

Functional vitamin D receptor (VDR) in the t-tubules of cardiac myocytes: VDR knockout cardiomyocyte contractility.

We have previously shown that the active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)(2)D(3)], has both genomic and rapid nongenomic effects in heart cells; however, the subcellular localization of the vitamin D receptor (VDR) in heart has not been studied. Here we show that in adult rat cardiac myocytes the VDR is primarily localized to the t-tubule. Using immunofluorescence and Western blot analysis, we show that the VDR is closely associated with known t-tubule proteins. Radioligand binding assays using (3)H-labeled 1,25(OH)(2)D(3) demonstrate that a t-tubule membrane fraction isolated from homogenized rat ventricles contains a 1,25(OH)(2)D(3)-binding activity similar to the classic VDR. For the first time, we show that cardiac myocytes isolated from VDR knockout mice show accelerated rates of contraction and relaxation as compared with wild type and that 1,25(OH)(2)D(3) directly affects contractility in the wild-type but not the knockout cardiac myocyte. Moreover, we observed that acute (5 min) exposure to 1,25(OH)(2)D(3) altered the rate of relaxation. A receptor localized to t-tubules in the heart is ideally positioned to exert an immediate effect on signal transduction mediators and ion channels. This novel discovery is fundamentally important in understanding 1,25(OH)(2)D(3) signal transduction in heart cells and provides further evidence that the VDR plays a role in heart structure and function.

Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice.

1alpha,25-Dihydroxyvitamin D(3) [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed approximately 41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.

Characterization of heart size and blood pressure in the vitamin D receptor knockout mouse.

Our previous studies showed vitamin D deficiency results in increased cardiac contractility, hypertrophy and fibrosis and has profound effects on heart proteomics, structure and function in rat. In this study we found that the heart in vitamin D receptor knockout (VDR-KO) mice is hypertrophied. Six homozygous VDR knockout (-/-), six wild type (+/+) and six heterozygous (+/-) mice were fed a diet containing 2% Ca, 1.25% P and 20% lactose to maintain normal blood calcium and phosphate levels for 12 months. Tail-cuff blood pressure was performed on all mice. Blood pressure determinations showed no differences in systolic or mean blood pressure in WT (+/+), KO (-/-) or HETERO (+/-) mice at 3 and 6 months. However, decreased systolic BP in the KO mice relative to WT at 9 months of age was observed. ECG analysis showed no significant differences in the intact KO, HETERO or WT mice. The mice were killed at 12 months. Heart weight/body weight ratio was 41% (P<.003) greater in the KO mice versus WT and HETERO was 19% (P<.05) increased versus WT. Other VDR-KO tissues did not display hypertrophy. Cross sectional and longitudinal analysis of the heart myofibrils showed highly significant cellular hypertrophy in VDR-KO mice. Trichrome staining of heart tissue showed marked increase in fibrotic lesions in the KO mice. Analysis of plasma renin activity, angiotensin II (AII) and aldosterone levels showed elevated but not significantly different renin activity in KO versus WT and no significant differences in AII or aldosterone levels. Our data do not support the concept that the renin-angiotensin system or hypertension are the factors that elicit these changes. Data presented here reveal that ablation of the VDR signaling system results in profound changes in heart structure. We propose that calcitriol acts directly on the heart as a tranquilizer by blunting cardiomyocyte hypertrophy.

1,25(OH)2-vitamin D3 actions on cell proliferation, size, gene expression, and receptor localization, in the HL-1 cardiac myocyte.

The steroid hormone 1,25(OH)(2)-vitamin D(3) [1,25D] has been shown to affect the growth and proliferation of primary cultures of ventricular myocytes isolated from neonatal rat hearts. The research presented here shows that the vitamin D receptor [VDR] is present in murine cardiac myocytes (HL-1 cells), and that 1,25D affects the growth, proliferation and morphology of these cells. In addition we show that 1,25D effects expression of ANP, myotrophin, and c-myc. Furthermore, 1,25D effects expression and localization of the VDR within the cell. Murine HL-1 cardiac myocytes were grown and treated with 1,25D in culture, and growth and morphology were assessed with microscopic analysis. Cells were counted and protein levels were evaluated through Western blot analysis. Subcellular localization of the VDR was determined using immunofluorescence and confocal microscopy. 1,25D was found to decrease proliferation and alter cellular morphology of the HL-1 cells. Treatment with 1,25D increased expression of myotrophin while decreasing expression of atrial natriuretic peptide [ANP] and c-myc. 1,25D treatment also increased expression and nuclear localization of the VDR in these cardiac myocytes. Thus 1,25D is an important hormone involved in modulating and maintaining heart cell structure and function.

Calcitriol modulation of cardiac contractile performance via protein kinase C.

Vitamin D(3) deficiency enhances cardiac contraction in experimental studies, yet paradoxically this deficiency is linked to congestive heart failure in humans. Activated vitamin D(3) (1alpha,25-dihydroxyvitamin D(3)) or calcitriol, decreases peak force and activates protein kinase C (PKC) in isolated perfused hearts. However, the direct influence of this hormone on adult cardiac myocyte contractile function is not well understood. Our aim is to investigate whether 1alpha,25-dihydroxyvitamin D(3) acutely modulates contractile function via PKC activation in adult rat cardiac myocytes. Sarcomere shortening and re-lengthening were measured in electrically stimulated myocytes isolated from adult rat hearts, and the vitamin D(3) response (10(-10) to 10(-7) M) was compared to shortening observed under basal conditions. Maximum changes in sarcomere shortening and relaxation were observed with 10(-9) M 1alpha,25-dihydroxyvitamin D(3). This dose decreased peak shortening, and accelerated contraction and relaxation rates within 5 min of administration, and changes in the Ca(2+) transient contributed to the peak shortening and relaxation effects. The PKC inhibitor, bis-indolylmaleimide (500 nM) largely blocked the acute influence of the most potent dose (10(-9) M) on contractile function. While peak shortening and shortening rate returned to baseline within 30 min, there was a sustained acceleration of relaxation that continued over 60 min. Phosphorylation of the Ca(2+) regulatory proteins, phospholamban, and cardiac troponin I correlated with the accelerated relaxation observed in response to acute application of 1alpha,25-dihydroxyvitamin D(3). Accelerated relaxation continued to be observed after chronic addition of 1alpha,25-dihydroxyvitamin D(3) (e.g. 2 days), yet this sustained increase in relaxation was not associated with increased phosphorylation of phospholamban or troponin I. These results provide evidence that 1alpha,25-dihydroxyvitamin D(3) directly modulates adult myocyte contractile function, and protein kinase C plays an important signaling role in the acute response. Phosphorylation of key Ca(2+) regulatory proteins by this kinase contributes to the enhanced relaxation observed in response to acute, but not chronic calcitriol.