Kish L. Golden

Castration reduces mRNA levels for calcium regulatory proteins in rat heart.

Sex-related differences in the cardiac phenotype have been well established. This study was designed to determine whether androgens regulate myocardial gene expression and play a role in the sex-related differences in the myocardial phenotype. Gonadectomized male rats were treated with testosterone, and myocardial gene expression was examined in whole heart using quantitative real-time PCR. Gonadectomy produced a substantial decrease in mRNA levels for the androgen receptor, Na+/Ca2+ exchanger, L-type calcium channel, and β1-adrenergic receptor (β1AR). Supplementation of testosterone in castrates produced a fivefold increase in androgen receptor mRNA levels. Testosterone treatment of castrates produced almost a sixfold increase in Na+/Ca2+ exchanger mRNA, a tenfold increase in L-type calcium channel mRNA accumulation, and a fourfold increase in β1AR mRNA levels. Increased calcium channel expression, β1AR expression, and Na+/Ca2+ exchanger expression together may alter cytosolic calcium. These results provide the first evidence that testoster-one regulates expression of myocardial calcium regulating genes and thus may play a role in modulating the cardiac phenotype in males.

Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase differentially regulates contractile function in cardiac myocytes from normotensive and spontaneously hypertensive rats: role of Ca2+ regulatory proteins.

Hypertension leads to impaired contractile function. This study examined the impact of inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by thapsigargin or cyclopiazonic acid (CPA) on cardiac contractile function in ventricular myocytes from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Mechanical properties were examined including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (±dL/dt). Intracellular Ca2+ transients were evaluated as fura-2 fluorescent intensity (FFI), excitation-induced change in FFI (ΔFFI=peak-basal), and fluorescence decay rate (τ). Expression of Ca2+ regulatory proteins SERCA2a, Na+−Ca2+ exchanger (NCX), and phospholamban (PLB) were assessed by reverse transcriptase polymerase chain reaction and Western blot. SHR rats exhibited elevated blood pressure. SHR myocytes displayed decreased PS±dL/dt, peak FFI, and ΔFFI; shortened TPS; prolonged τ with normal TR90; and basal FFI compared with WKY myocytes. Inhibition of SERCA with thapsigargin (5μM) or CPA (10 μM) significantly depressed PS±dL/dt, baseline FFI, and ΔFFI, and prolonged TPS, TR90, and τ in WKY myocytes. However, SHR myocytes were relatively insensitive to thapsigargin or CPA with only TPS and TR90 prolonged. Both mRNA and protein expressions of NCX and PLB were significantly enhanced, whereas SERCA2a protein abundance was reduced in SHR rats compared with the WKY group. Our data suggest that inhibition of SERCA function differentially affected cardiac contractile function in ventricular myocytes from normotensive and hypertensive rats possibly through reduced SERCA2a, elevated PLB, and NCX expression under hypertension.

Gonadectomy alters myosin heavy chain composition in isolated cardiac myocytes.

Sex differences in cardiac function have been identified. Studies suggest that the presence of testosterone in males may contribute to the observed differences in cardiac function. Our laboratory has shown previously that testosterone treatment of gonadectomized adult male rats enhances contractility of isolated rat ventricular myocytes. In this study we tested the hypothesis that gonadectomy and hormone replacement influences contractility by altering myosin heavy chain (MHC) composition. To test this hypothesis we analyzed myosin isoform expression in ventricular myocytes isolated from castrated rats displaying a decrease in myocyte contractile velocity and compared them to castrates treated with testosterone that displayed normal myocyte shortening velocity. Sixteen weeks after castration isolated rat ventricular myocytes displayed a 90% (p<0.001) decline in MHC-α mRNA levels and over a twofold (p<0.01) increase in MHC-β transcripts when compared to sham-operated controls. Consistent with these changes we also observed a substantial decline in the ratio of MHC-α to MHC-β protein expression. A reversal in myosin heavy chain composition was achieved following testosterone replacement. These studies provide the first direct evidence that testosterone replacement in gonadectomized animals enhances contractility via transcriptional and translational control of myosin heavy chain composition in isolated rat ventricular myocytes. The influence of testosterone on MHC composition in males may underlie some of the observed sex differences in cardiac function.

Altered Ca2+ homeostasis and impaired mitochondrial function in cardiomyopathy.

Altered Ca2+ homeostasis and myocyte death is a major characteristic of the hereditary cardiomyopathy in the dystrophic hamster. Despite numerous studies, the mechanisms that link calcium dysregulation and cell death in this animal model remain unclear. We have shown previously that the maintenance of mitochondrial Ca2+ homeostasis is essential for cell survival, and that loss of mitochondrial Ca2+ is closely correlated with cell death in cultured cells. Here, we have further investigated the role of mitochondrial Na+-Ca2+ exchange (NCE) in the development of cardiomyopathy in the dystrophic hamster. We found that the myocyte death was associated with elevated NCE activity together with a reduced level of matrix Ca2+, and impaired mitochondrial energetics. The upregulation of NCE activity in myopathic heart was also accompanied by enhanced expression of the sarcolemmal Na+-Ca2+ exchange (NCX) without alterations in the L-type Ca2+ channel expression. Treatment of dystrophic hamsters with diltiazem (a potent inhibitor of NCE and Ca2+ channels) prevented the occurrence of cell death and restored the normal expression of NCX. Our findings implicate the dysregulation of both sarcolemmal and mitochondrial Na+-Ca2+ exchange in cell death in the myopathic heart.

Norepinephrine regulates the in vivo expression of the L-type calcium channel

The α1c subunit (DHP receptor) of the L-type Ca2+ channel is important for calcium homeostasis in cardiac muscle. The DHPr provides the primary mechanism for calcium influx during contraction. Published results demonstrate three in vitro signaling pathways that are important in the regulation of DHPr gene expression in neonatal cardiac myocytes, the protein kinase A (PKA), protein kinase C (PKC) pathways, and intracellular calcium. To determine whether these pathways are important in vivo, we treated adult rats with infusions of isoproterenol, or norepinephrine at 200 μg/kg/h and assessed DHPr mRNA and protein levels. Following a 3-day infusion isoproterenol (ISO) and norepinephrine (NE) produced a small but insignificant reduction in DHPr mRNA levels. When the infusions were continued for 7 days isoproterenol increased DHPr mRNA accumulation to control levels while NE stimulated a 35% increase in DHPr mRNA levels and a 35% increase in protein abundance when compared to controls (p < 0.05). Furthermore, contractility and Ca2+ transient measurements of isolated cardiac myocytes from NE infused animals also display shortened duration of contraction/relaxation and increased intracellular free Ca2+ (DFFI) in response to electrical stimulation (p < 0.01). We conclude norepinephrine treatment alters DHPr mRNA and protein levels, and augments excitation-contraction coupling, and thus may be important for modulating cardiac calcium homeostasis in vivo.

Effect of Insulin on Iodide Uptake in Mouse Mammary Gland Explants

Studies were carried out primarily to assess the role of insulin in regulating iodide uptake in the mammary gland. Using cultured mammary gland explants from virgin and pregnant mice (12–14 days into gestation), insulin (1 μg/ml) was shown to stimulate iodide uptake after a 2-day exposure period. The effect of insulin was manifested by itself, as well as in the presence of cortisol and prolactin. Optimal iodide uptake was observed when tissues were treated with all three lactogenic hormones (insulin, cortisol, and prolactin). In a time-course experiment, the effect of insulin alone was initially observed after a 10-hr treatment; the effect was maintained for 30 hr. In dose-response studies, 1 ng/ml insulin elicited a significant effect after 24 hr in culture; a maximal effect was achieved with 50–100 ng/ml insulin. The optimal cortisol concentration for a maximum stimulation of iodide uptake was 10–7M. In a quantitative Western blot analysis employing an antibody to the sodium-iodide symporter, insulin stimulated an upregulation of the transporter protein after a 4-, 8-, or 20-hr treatment with insulin. Perchlorate and thiocyanate abolished the insulin effect on iodide uptake, further suggesting that the insulin response occurs via a stimulation of the sodium-iodide symporter. Clearly, insulin is an important and essential hormone in the lactogenic hormone complex for regulating iodide uptake in the mammary gland, but maximal expression of iodide uptake is only expressed when all three lactogenic hormones are present.

Effects of Prolactin on Galactosyl Transferase and α-Lactalbumin mRNA Accumulation in Mouse Mammary Gland Explants

Abstract Studies were carried out to determine the possible mechanism(s) by which prolactin (PRL) stimulates α-lactalbumin and galactosyl transferase activity in cultured mouse mammary tissues. In earlier studies it was shown that the onset of the PRL stimulation of galactosyl transferase activity occurs between 4 and 8 hr after adding prolactin to cultured mouse mammary gland explants, and a maximum effect is evoked by 24 hr. In contrast, an effect of prolactin on α-lactalbumin activity occurred 24 hr after adding PRL to mammary gland cultures, but not at earlier times. In the present studies, it is shown that prolactin effects an increased tissue accumulation of mRNAs for both α-lactalbumin and galactosyl transferase after a 4 to 6 hr culture with prolactin. The lowest concentration of prolactin that stimulates the accumulation of both galactosyl transferase and α-lactalbumin mRNA is approximately 10 ng/ml, and a maximum response is achieved with 200 ng/ml. Thus, it is likely that the effect of PRL on the activity of galactosyl transferase is causally associated with the effect of prolactin on the accumulation of its mRNAs. However, the PRL stimulation of α-lactalbumin activity is not temporally associated with the tissue accumulation of the mRNA for α-lactalbumin.

Gonadectomy and androgen replacement alter cardiac performance in conscious adult male rats

Gender disparities in cardiac function have been described. Yet the extent to which gender related differences in cardiac performance are due to the presence of sex-specific biological factors are unclear. We used a longitudinal study aimed at examining whether castration and androgen replacement affects cardiac performance in conscious adult male rats. Adult male rats were implanted with Piezoelectric transit-time gauges and radio telemetry devices to measure regional myocardial segment length and hemodynamic variables before and after castration and after androgen replacement. Androgen withdrawal accelerated average heart rates by 7% (p = 0.010). Heart rate was lowered to intact values when androgens were restored to normal physiological levels (p = 0.004). Mean arterial pressure was not affected by androgen deprivation and androgen replacement. However, androgen withdrawal produced a 40% decrease in the velocity of circumferential shortening and a 46% reduction in the rate of myocardial relaxation. Androgen supplementation completely restored contractile function. These results provide the first evidence that androgen withdrawal and androgen replacement produces dramatic alterations on cardiac performance in conscious animals and demonstrates the significance of androgens as a cardioregulatory hormone in males. Sex steroids are likely contributors to gender-related differences in cardiac function.

An Update of Recent Trials with Vildagliptin, a Dipeptidyl Peptidase-4 Inhibitor for the Treatment of Type 2 Diabetes

Objective: To review the clinical efficacy and tolerability of vildagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, for the treatment of type 2 diabetes, based on recent Phase 3 trials. Data Sources: Primary literature and review articles were obtained via a MEDLINE search (2005–November 2007) using the search terms diabetes, vildagliptin, and LAF-237. Additional data from abstracts presented at clinical meetings were included when appropriate. Study Selection: Nine double-blind, randomized, multicenter, parallel-group trials and vildagliptin studies were identified and reviewed. Data Synthesis: Vildagliptin, a selective DPP-4 inhibitor, has been shown to produce clinically significant reductions in hemoglobin A1c (A1C) levels when used as monotherapy (0.6–1%) or in combination with other glucose-lowering agents (mean decrease 0.7%). Phase 3 trials indicated a 24- to 52-week sustained effect on reduction of blood glucose in patients with type 2 diabetes. The primary endpoint for all trials was change from baseline A1C. In the intent-to-treat population, baseline A1C was compared with end-of-study A1C. More than 2,500 patients with type 2 diabetes enrolled in monotherapy trials; an additional 2,119 participated in combination therapy trials. Males outnumbered females, and all groups included obese patients. The most common adverse effects reported were nasopharyngitis, headache, and dizziness. Vildagliptin was weight-neutral and produced a rate of hypoglycemia similar to that of placebo. Conclusions: Vildagliptin appears to be a promising agent for the management of type 2 diabetes.