Kadon K. Hintz

Phytoestrogenic isoflavones daidzein and genistein reduce glucose-toxicity-induced cardiac contractile dysfunction in ventricular myocytes.

Epidemiological evidence suggests a reduction in the incidence of coronary heart disease, cancer and osteoporosis in populations with a high dietary intake of plant estrogen or phytoestrogen. The clinical benefit of phytoestrogens in cereals, vegetables and medicinal plants is attracting increasing attention for the general public. In the present study, we examined the effect of phytoestrogenic isoflavones daidzein and genistein on glucose toxicity‐induced cardiac mechanical malfunction simulating diabetic cardiomyopathy. Adult rat ventricular myocytes were isolated and maintained for 24 hours in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) medium in the absence or presence of isoflavones daidzein (50 µM) or genistein (20 µM). Cardiac contractile indices were evaluated using an IonOptix® MyoCam system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time‐to‐PS (TPS) and time‐to‐90% relengthening (TR90). Myocytes maintained in HG medium displayed altered mechanical function simulating in vivo diabetes including reduced PS, ± dL/dt and prolonged TR90 associated with normal TPS compared to those from NG myocytes. Interestingly, these HG‐induced mechanical dysfunctions were abolished by co‐incubation of daidzein or genistein. However, daidzein but not genistein itself depressed PS in NG myocytes. Neither daidzein nor genistein affected any other mechanical parameters tested in NG myocytes. Collectively, these data suggest that the phytoestrogenic isoflavones daidzein and genistein may reduce glucose toxicity‐induced cardiac mechanical dysfunction and thus possess therapeutic potential against diabetes‐associated cardiac defects.

Prediabetic insulin resistance is not permissive to the development of cardiac resistance to insulin-like growth factor I in ventricular myocytes

Resistance to insulin-like growth factor I (IGF-1)-induced cardiac contractile response has been reported in diabetes. To evaluate the role of prediabetic insulin resistance to cardiac IGF-1 resistance, whole body insulin resistance was generated with dietary sucrose and contractile function was evaluated in ventricular myocytes. Mechanical properties were evaluated using an IonOptix system and intracellular Ca(2+) transients were measured as changes in fura-2 fluorescence intensity (Delta FFI). After 8 weeks of feeding, sucrose rats displayed euglycemia, hepatomeglay and normal heart size, and glucose intolerance, confirming the presence of insulin resistance. Myocytes from sucrose-fed rats displayed decreased peak shortening (PS), reduced resting FFI, increased intracellular Ca(2+) clearing, associated with normal duration of shortening and relengthening compared to myocytes from starch-fed rats. IGF-1 (10(-10)-10(-6) M) caused a similar concentration-dependent decrease in PS in both groups. Only the highest concentration of IGF-1 elicited an inhibition on Delta FFI in sucrose myocytes. In addition, the IGF-1-induced response was abolished by the IGF-1 receptor antagonist H-1356 in both groups, and by the nitric oxide synthase inhibitor L-NAME in starch but not sucrose myocytes. These results indicated prediabetic insulin resistance alters cardiac contractile function at the myocytes level, but may not be permissive to cardiac contractile resistance to IGF-1.

Acute exposure of ceramide enhances cardiac contractile function in isolated ventricular myocytes

The sphingolipid ceramide, a primary building block for all other sphingolipids, is associated with growth arrest, apoptosis, and lipotoxic dysfunction. Interestingly, ceramide may attenuate high glucose‐induced myocyte dysfunction, produce Ca2+ influx, and augment smooth muscle contraction. To determine the role of ceramide on cardiac excitation–contraction (E–C) coupling, electrically paced adult rat ventricular myocytes were acutely exposed to a cell‐permeable ceramide analog (10 pM–100 μM) and the following indices were determined: peak shortening (PS), time‐to‐P., time‐to‐90% relengthening, and the maximal velocity of shortening and relengthening (±dLdt). Intracellular Ca2+ properties were assessed using fura‐2AM fluorescent microscopy. Our results revealed a concentration‐ and time‐dependent increase of PS in ventricular myocytes in response to ceramide associated with an increase in ±dLdt. The maximal increase in PS was ∼35% from control value and was maintained throughout the first 20 min of ceramide exposure. However, the ceramide‐induced increase in PS was not maintained once the exposure time was beyond 20 min. Acute exposure of ceramide significantly enhanced intracellular Ca2+ release, although at a much lower concentration range. The ceramide‐induced augmentation of PS was not significantly affected by inhibition of phosphatidylinositol (PI)‐3‐kinase, protein kinase C (PKC), ceramide‐activated protein phosphatase (CAPP), and nitric oxide (NO) synthase. Our data suggest that ceramide acutely augments the contractile function of cardiac myocytes through an alternative mechanism(s) rather than PI‐3‐kinase, PKC, CAPP, or NO.

Prenatal ethanol exposure alters ventricular myocyte contractile function in the offspring of rats

Fetal alcohol syndrome (FAS) is often associated with cardiac hypertrophy and impaired ventricular function in a manner similar to postnatal chronic alcohol ingestion. Chronic alcoholism has been shown to lead to hypomagnesemia, and dietary Mg2+ supplementation was shown to ameliorate ethanol-induced cardiovascular dysfunction such as hypertension. However, the role of gestational Mg2+ supplementation on FAS-related cardiac dysfunction is unknown. This study was conducted to examine the influence of gestational dietary Mg2+ supplementation on prenatal ethanol exposure-induced cardiac contractile response at the ventricular myocyte level. Timed-pregnancy female rats were fed from gestation day 2 with liquid diets containing 0.13 g/L Mg2+ supplemented with ethanol (36%) or additional Mg2+ (0.52 g/L), or both. The pups were maintained on standard rat chow through adulthood, and ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical properties were evaluated using an IonOptix™ soft-edge system, and intracellular Ca2+ transients were measured as changes in fura-2 fluorescence intensity (ΔFFI). Offspring from all groups displayed similar growth curves. Myocytes from the ethanol group exhibited reduced cell length, enhanced peak shortening (PS), and shortened time to 90% relengthening (TR90) associated with a normal ΔFFI and time to PS (TPS). Mg2+ reverted the prenatal ethanol-induced alteration in PS and maximal velocity of relengthening. However, it shortened TPS and TR90, and altered the ΔFFI, as well as Ca2+ decay rate by itself. Additionally, myocytes from the ethanol group exhibited impaired responsiveness to increased extracellular Ca2+ or stimulating frequency, which were restored by gestational Mg2+ supplementation. These data suggest that although gestational Mg2+ supplementation may be beneficial to certain cardiac contractile dysfunctions in offspring of alcoholic mothers, caution must be taken, as Mg2+ supplementation affects cell mechanics itself.

Comparison of cardiac excitation-contraction coupling in isolated ventricular myocytes between rat and mouse

Transgenic animals offer many advantages for physiological study. The mouse is the most extensively utilized mammalian model for gene modification. Isolated ventricular myocytes are pivotal for assessment of cardiac function by allowing direct cellular and environmental manipulation without interference from compensatory mechanisms that may exist in vivo. This study was designed to compare the basic excitation-contraction coupling properties of mouse and rat ventricular myocytes. Cardiac myocytes were isolated from age- and gender-matched mice (FVB and C57BL/6) and rats (Sprague-Dawley (SD) and Wistar). Mechanical and intracellular Ca2+ properties were measured with an IonOptix SoftEdge system, including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening and relengthening (+/-dL/dt), and intracellular Ca2+ fura-2 fluorescence intensity and decay rate (tau). Resting cell length was variable among the different species or strains. PS from FVB group was significantly higher than the SD group. TPS and TR(90) were significantly shorter in mice. +dL/dt was similar among all groups whereas -dL/dt was significantly faster in the C57BL/6 group compared to the rat groups. Resting intracellular Ca2+ was lower in mice than in rats, and Ca2+-induced Ca2+ release was variable among the four groups. Intracellular Ca2+ decay was slower in Wistar compared to all other groups. The myocytes from C57BL/6 did not respond to increases in extracellular Ca2+. Myocytes from the FVB group exhibited a lesser reduction in PS in response to elevated stimulus frequency. These data suggest that inherent differences between strains or species should be taken into consideration when comparing results from these different animal models.