Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations.

Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC50 in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 µM concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 µM. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity.

Effects of impulse noise stimulation on electrocorticogram and heart rate.

It is now recognized that high-level impulse noises penetrate the uterus of pregnant sheep, elevate thresholds of fetal auditory-evoked potential and produce damage to fetal inner ear hair cells. However, little is known about functional effects of airborne impulse noise on the late-term fetus. In the present study, the effects of a series of 20 impulses on the behavioral state of 6 fetal sheep were tested. Noise impulses produced 169.3 peak sound pressure level (pSPL) in air. Peak levels recorded near the fetal head averaged 161.1 dB. Impulses delivered to the fetus during periods of NREM sleep resulted in a decrease in average fetal heart rate (FHR) from 185 +/- 22 beats/min (bpm) before stimulation to 174 +/- 23 bpm 2-5 s after stimulation (p < 0.05). During REM sleep, an FHR acceleration occurred (before stimulation: 177 +/- 24 bpm, after stimulation: 189 +/- 31 bpm; p < 0.05). Impulse exposure during NREM sleep resulted in reductions in delta-, theta- and alpha-band powers. As a consequence, total power decreased from 100 to 72 +/- 16% (p < 0.05). During REM sleep, stimulation provoked a short decrease in total band power from 100 to 73 +/- 20% and a similar decrease in the theta- and beta-band powers. The results indicated that impulse noise evoked short-term alterations in FHR and cortical activity. These changes were mediated by auditory brain stem activation that led to cortical desynchronization during both NREM and REM sleep in late-term fetal sheep.

Intra-abdominal sound pressure levels during impulse noise exposure in sheep.

Ambient sound pressure levels (SPLs) created with intense blasts were compared with SPLs recorded in the abdomen of euthanized sheep. Hydrophones were placed in the abdominal cavity at locations referred to as proximal, medial, and distal with respect to a shock tube that created 169-dB peak SPL (pSPL). No differences in pSPL, duration, or rise time were found between recordings in air and at the intra-abdominal proximal position. Significant differences were noted in these variables when recordings in air were compared with recordings made at the medial and distal locations. Intra-abdominal pSPL varied by 20 dB depending on recording location.

Fetal exposures to sound and vibroacoustic stimulation.

Sounds in the environment of a pregnant woman penetrate the tissues and fluids surrounding the fetal head and stimulate the inner ear through a bone conduction route. The sounds available to the fetus are dominated by low-frequency energy, whereas energy above 0.5 kHz is attenuated by 40 to 50 dB. The fetus easily detects vowels, whereas consonants, which are higher in frequency and less intense than vowels, are largely unavailable. Rhythmic patterns of music are probably detected, but overtones are missing. A newborn human shows preference for his/her mother's voice and to musical pieces to which he/she was previously exposed, indicating a capacity to learn while in utero. Intense, sustained noises or impulses produce changes in the hearing of the fetus and damage inner and outer hair cells within the cochlea. The damage occurs in the region of the inner ear that is stimulated by low-frequency sound energy.

The acoustic environment and physiological responses of the fetus.

The acoustic environment of the fetus is composed of continuous cardiovascular, respiratory, and intestinal sounds that are punctuated by isolated, shorter bursts during maternal body movements and vocalizations. The distribution of sounds is confined to frequencies below 300 Hz. Additionally, vibrations on the external surface of the maternal abdomen can induce sounds inside the uterus. The half-round sound pressure contours in the abdomen during vibroacoustic stimulation differ from the circular distribution of contours resulting from airborne sound pressure exposure. The static and dynamic forces of the vibrator and the vibrator distance from the target are also factors in sound transmission. Responses to sound are best described in animals and include changes in behavioral state, brain bloodflow, auditory brainstem response, and local cerebral glucose utilization along the central auditory pathway.

Effects of intense noise exposure on fetal sheep auditory brain stem response and inner ear histology.

OBJECTIVE: To evaluate, in two separate experiments, the effects of intense noise exposures delivered to fetal sheep in utero during a time of rapid auditory development. DESIGN: In the first experiment, auditory brain stem response (ABR) thresholds to clicks and tone bursts were recorded from chronically instrumented fetal sheep in utero before and after exposure of pregnant ewes to intense broadband noise. A single 16 hr exposure was delivered at 113 days gestational age, a time when the ABR is just emerging. Thresholds were compared with an age-matched, nonexposed control group. In the second experiment, fetal sheep at the same gestational age were exposed four times to broadband noise and their cochleae were harvested 20 days later for histological analysis by the use of scanning electron microscopy. Comparisons were made with an age-matched, nonexposed control group. RESULTS: Experiment One: ABR thresholds recorded between 10 to 20 days after the exposure were not as sensitive as thresholds obtained from control fetuses. There was a tendency for thresholds to 0.5 kHz tone bursts to be more affected than thresholds to clicks. Experiment Two: Scanning electron microscopy of the organ of Corti from fetuses exposed to noise from 111 to 114 days gestational age revealed significant damage to inner and outer hair cells in the middle and apical turns of cochleae. Similar hair cell damage was not present in control fetuses. CONCLUSIONS: Intense exogenous noise penetrated the uterus of pregnant sheep and resulted in elevations in ABR thresholds 2 to 3 wk after exposure. In fetuses repeatedly exposed to noise, the middle and apical turns of the cochlea showed greater hair cell damage than found at the same locations in control cochlea. The basal turn of the cochlea was not damaged.

Vestibular caloric responses and behavioral state in the fetal sheep.

Functional activity of the vestibular system in relation to behavioral state of fetal sheep in utero was studied by cooling and heating of the fetal middle ear and skin (control) with implanted copper-tube heat exchangers. Eye movements and fetal cortical activity were assessed before, during, and after 2 min irrigations with water at 6, 46, or 39.5 degrees C (isothermic). Cold water induced slow-phase eye movements toward the irrigated ear followed by saccades toward the opposite ear after a delay of several seconds. The direction of the response reversed with warm water, and saccades were absent during irrigation with body-temperature water. Cold-water irrigations of the skin over the jaw did not result in nystagmus. Arousal-like responses were elicited with thermal stimulation of the ear or facial skin while the fetus was in either rapid eye movement (REM) or non-REM states. Circulation of 39.5 degrees C water through the ear also produced arousal-like responses, possibly due to turbulence-induced noises in the heat exchanger or slight deviations between the irrigation temperature and the actual fetal inner ear temperature. These results suggest that mechanisms responsible for saccade suppression during depressed levels of consciousness (i.e. sleep) are inactive in utero. Fetal behavioral state responsiveness to vestibular and somatosensory thermal stimulation may be of great significance, especially in the premature neonate.

Vestibular function in the fetal sheep.

Little is known about the functional development of the vestibular system before birth. The purpose of this study was to determine whether vestibular response to caloric stimulation could be elicited in the fetal sheep in utero. Late gestational fetal sheep (n = 6) were instrumented through a midline hysterotomy. Copper caloric probes were inserted into the right bulla and beneath the left facial skin. Electrodes were placed in the skull for monitoring of electro-ocular activity. At least 3 days after surgery the probes were irrigated with water (100 ml/minute) at body temperature, 46 degrees C, and 6 degrees C. Cold water infusion of the bulla consistently produced well-recognized, slow-phase deviations followed by saccades directed contralaterally, findings consistent with vestibular nystagmus. The direction of the response reversed with warm water irrigation. The response was absent with irrigation at fetal body temperature. Only random eye movements were observed in response to caloric stimulation of the facial skin, regardless of water temperature. These results demonstrate that the sheep vestibular system is functioning prenatally. The importance of vestibular function for normal fetal brain maturation may be revealed in future studies using this animal model.

Temporary threshold shifts induced by low-pass and high-pass filtered noises in fetal sheep in utero.

Auditory brainstem responses (ABRs) were obtained from nine late gestational age fetal sheep in utero before and after a 16-h exposure to low-pass (cut-off frequency 1.0 kHz) and high-pass (cut-off frequency 1.0 kHz) noises (approximately 120 dB sound pressure level, recorded in air). Bone-conduction ABRs were elicited by broadband clicks and 0.5, 1.0 and 2.0 kHz tone bursts. Following low-pass noise exposure, ABR thresholds and wave IV latencies increased significantly for 0.5 and 1.0 kHz tone bursts. The high-pass noise exposure produced significant shifts in ABR thresholds and wave IV latencies only for the 1.0 kHz tone bursts. These findings confirm previous reports of low-frequency sound transmission into the fetal inner ear.

Effect of abdominal vibroacoustic stimulation on sound and acceleration levels at the head of the fetal sheep.

OBJECTIVE: To measure the vibratory response of the fetal head and abdominal wall in sheep during vibroacoustic stimulation. METHODS: A piezoresistive accelerometer was attached to the skulls of seven sheep fetuses (128-134 days' gestational age), and a miniature hydrophone was attached to the skin overlying the fetal temporal bone. During fetal preparation and vibroacoustic stimulation procedures, ewes were anesthetized and supine. Vibroacoustic stimulation of the maternal abdomen was produced by each of two clinical devices that differed in spectral content, and an electric toothbrush. RESULTS: The approximate fundamental frequencies (f0) and first overtones (f1), as determined by both recordings of intrauterine sound pressure level and fetal head acceleration, were as follows: fetal acoustic stimulator, 75 and 150 Hz; electronic larynx, 150 and 300 Hz; and electric toothbrush, 25 and 50 Hz, respectively. At fundamental frequencies and first overtones, the ranges of fetal head accelerations (expressed in 1/12-octave bands) were as follows: fetal acoustic stimulator, 10-53 and 25-224 mm/sec2; electronic larynx, 10-53 and 18-114 mm/sec2; and electric toothbrush, 33-792 and 8-116 mm/sec2, respectively. Sound pressure levels exceeded 110 dB in all cases. High sound pressure levels in the uterus were proportional to fetal head vibration levels. CONCLUSION: Vibroacoustic stimulation of the surface of the abdomen of pregnant sheep is accompanied by both acoustic and vibratory exposure of the fetus.

Electrocortical and heart rate response during vibroacoustic stimulation in fetal sheep.

OBJECTIVE: Our purpose was to study effects of vibroacoustic stimuli on electrocortical activity and heart rate changes in fetal sheep in utero. STUDY DESIGN: Seven chronically instrumented near-term fetal sheep were repeatedly stimulated by an electronic artificial larynx for 32 seconds during periods of rapid-eye-movement and non-rapid-eye-movement sleep. Responses to vibroacoustic stimulation were obtained by spectral analysis of the electrocorticogram (fast Fourier transform) and by assessment of changes in fetal heart rate and fetal heart rate variability. RESULTS: During non-rapid-eye-movement sleep vibroacoustic stimulation led to electrocorticogram desynchronization that consisted of a marked reduction of delta and theta band power (p < 0.05). A concomitant fetal heart rate decrease and fetal heart rate variability increase were also noted (p < 0.05). During rapid-eye-movement sleep vibroacoustic stimulation induced a significant increase in alpha and beta band power (p < 0.05) and a slight deviation in basal fetal heart rate and fetal heart rate variability (p < 0.05). CONCLUSION: Vibroacoustic stimulation of fetal sheep provokes reproducible changes in fetal electrocortical activity and heart rate patterns. These changes, which are not easily identifiable in gross polygraphic assessments of the fetal behavioral state, are indicative of fetal arousal.

Rates of local cerebral protein synthesis in fetal and neonatal sheep.

During gestation there is likely to be a constantly changing rate of protein synthesis in the brain that may exhibit regional specificity. With the use of the quantitative autoradiographic L-[1-(14)C]leucine method for the determination of local rates of leucine incorporation into cerebral protein (lCPS(Leu)), we have sought to characterize this important process. lCPS(Leu) was measured in nine fetal sheep (118-139 days gestational age) and five newborn lambs (1-5 days of age). In other experiments, the fraction of leucine in the precursor pool for protein synthesis in the brain derived from the arterial plasma was determined to be 0.57 +/- 0.04 (mean +/- SE) in one fetus and two lambs. This value was used in the calculation of lCPS(Leu) in 35 regions of the central nervous system, pineal body, and whole brain. Regardless of age, lCPS(Leu) was highest in the pineal body, brain stem, and hypothalamic nuclei and lowest in white matter. In sensorimotor cortex, corona radiata, pyramidal tracts, and whole brain, lCPS(Leu) was positively correlated with prenatal age (P < or = 0.05). These increases in lCPS(Leu) probably reflect myelination in the cerebrum, which is known to occur in late gestation.

Fetal sheep in utero hear through bone conduction.

PURPOSE: Although the air-conduction pathway is the principal mode of sound transmission to the inner ear, this may not be true for the fetus in utero. The fetus detects and responds to sounds in the maternal environment. Exogenous sounds can reach the fetal inner ear through the ear canal and middle ear system, bone conduction, or both. This study was designed to compare the effectiveness of these two routes of sound transmission by recording cochlear microphonic potentials from the fetus in utero in response to airborne sounds. MATERIALS AND METHODS: Cochlear microphonics (CMs) recorded from one round window (RW) of fetal sheep in utero were obtained in three conditions: (1) head uncovered; (2) head covered with a neoprene hood; and (3) head covered with a neoprene hood fashioned with a hole that permitted the pinna and ear canal to be exposed. Tone bursts (0.5, 1.0, and 2.0 kHz) were delivered through a loudspeaker at high intensities (100 to 135 dB sound pressure level) to the flank of the ewe. CMs were detected with indwelling electrodes, amplified, and averaged. CM input-output functions were obtained from the fetus in each of the three conditions described above. RESULTS: CMs recorded with the head uncovered were more sensitive than were the CMs recorded with the hood in place. There was no difference in sensitivity between the condition during which the head was completely covered and the condition in which the pinna and ear canal are exposed. CONCLUSION: The principal mode of sound transmission into the fetal inner ear is through bone conduction.

Factors affecting human placenta and birth weights and vitality of newborns in a subtropical environment

Os fatores que afetam o peso de nascimento e da placenta e a vitalidade de recém-nascidos humanos na Flórida foram avaliados em bancos de dados de 1480 (conjunto de dados I) e 788 (conjunto de dados II) casos. Este último conjunto foi uma subdivisäo do primeiro, tendo menos casos devido a procedimentos de seleçäo mais rígidos. Estes procedimentos excluíram natimortos ou fetos com valores muito elevados ou baixos em qualquer variável de resposta, sendo que estes últimos foram entäo considerados como casos anormais. Os fatores avaliados afetaram significativamente as variáveis de resposta na maioria dos casos, mas näo necessariamente em ambos os conjuntos de dados. No conjunto de dados II, efeitos do mês do parto foram detectados em todas as variáveis exceto vitalidade aos 5 min. pós- parto (valor II de Apgar). O peso da placenta foi 112g menor nos meses quentes do que nos meses frios. A vitalidade em 1 min. pós-parto (valor I de Apgar) foi afetada pelo mês mas näo de modo sistemático. Os recém-nascidos do sexo masculino tiveram pesos 45g maiores para placenta, para peso corporal (142g) e para peso total (187g). O grupo étnico afetou todas as respostas, exceto o peso placentário. Efeitos da paridade foram detectados para todas as respostas, exceto peso placentário e valor II de Apgar; a duraçäo da gestaçäo afetou todas as respostas, exceto o peso placentário. Os pesos placentário e do recém nascido correlacionaram-se intensamente com sua soma (correlaçäo de parte com o todo), mas näo um com o outro nem com os valores de Apgar. Estes correlacionaram-se intensamente um com o outro. O conhecimento das características das variáveis de resposta e dos fatores que as influenciam deve contribuir consideravelmente para o conhecimento geral dos fatores que afetam as características dos nascimentos humanos.

Myocardial and cerebral oxygen delivery are not adversely affected by cocaine administration to early-gestation fetal sheep.

OBJECTIVES: Cocaine administration to near-term pregnant sheep causes fetal hypoxemia, but oxygen delivery to the heart and brain are preserved because of increased blood flow. We hypothesized that cocaine administration during earlier fetal gestation impairs oxygen delivery to the heart and brain. STUDY DESIGN: Ten pregnant ewes and fetuses at 0.7 term gestation underwent surgical instrumentation. After 48 hours of recovery fetal blood pressure, heart rate, cerebral and myocardial blood flow, and arterial oxygen content were determined before and during cocaine administration to the ewe. RESULTS: Fetal hypoxemia was not noted in these animals. Fetal myocardial blood flow increased from 220 +/- 100 ml per 100 gm per minute to 349 +/- 183 ml per 100 gm per minute (p=0.03), and oxygen delivery increased from 16 +/- 5 ml of oxygen per 100 gm per minute to 22 +/- ml of oxygen per 100 gm per minute (p=0.02). Fetal cerebral blood flow and oxygen delivery remained unchanged. CONCLUSION: Cerebral and myocardial oxygen delivery are unimpeded by maternal cocaine administration in 0.7 term gestation ovine fetuses.

Acceleration of fetal head induced by vibration of maternal abdominal wall in sheep.

OBJECTIVE: The human body is often exposed to significant vibration stress in the workplace, at home, and during recreational activities. The current study was designed to evaluate whether low- to midfrequency vibrations present at the extraabdominal wall would be attenuated across this wall and what the levels of exposure would be once these vibrations reached the fetal head. STUDY DESIGN: Four pregnant sheep were instrumented with acceleration transducers to obtain acceleration levels at the extraabdominal and intraabdominal walls and at the fetal head. Sine-wave vibration stimulation was applied over a frequency range of 3 to 150 Hz at a constant acceleration level of 2.5 m/sec2 (root-mean-square). RESULTS: Vibration of the extraabdominal wall resulted in a frequency-dependent rise in vibration levels at the intraabdominal wall, from 4% to 140% of the input level. At the fetal head a broad peak in response was noted between 6 and 12 Hz, but the overall levels never exceeded 4% of the input level. CONCLUSION: Fetal exposure to localized vibratory stimulation of the maternal abdomen is maximal in the range of 6 to 12 Hz.

Fetal hearing: characterization of the stimulus and response.

Before sounds originating outside the abdomen of pregnant women can reach the inner ear of the fetus, they must first pass through the tissues and fluids surrounding the fetal head. Low-frequency sound energy easily penetrates to the fetal head, less than 5 dB attenuation for frequencies below 500 Hz, whereas higher frequencies are attenuated by up to 20 to 30 dB. The sound energy in amniotic fluid stimulates fetal hearing through a bone conduction route rather than through the external and middle ear systems. During passage through the bones of the skull, sound energy is slightly diminished for frequencies less than 250 Hz (10 to 20 dB), yet significantly reduced for frequencies from 500 to 2,000 Hz (40 to 50 dB). Thus, the fetus in utero can easily detect low-frequency sound energy (< 500 Hz) produced at levels that are comfortably loud for its mother, but probably cannot detect acoustic energy at frequencies higher than 500 Hz.

Vibration of the abdominal segment in pregnant sheep.

Mechanical vibration of the abdominal wall results in a frequency-related distribution of intra-abdominal sound pressure levels. A greater attenuation of applied signals of equal dynamic force occurs as frequency increases. A broad resonance peak exists between 6 and 18 Hz. Transducers fixed to the fetal head show clear increases in acceleration levels during stimulation of the abdominal surface with the artificial electronic larynx. Sine-wave stimulation results in a frequency-dependent increase in vibration levels of the abdominal wall of 4% to 140% of the input levels. At the fetal head, a broad peak in response was noted between 6 and 12 Hz, but the overall levels never exceeded 4% of the input level.

Cocaine does not compromise cerebral or myocardial oxygen delivery in fetal sheep.

Eight time-dated pregnant ewes at 125 days' gestation (145 days = term) underwent surgery for placement of fetal vascular catheters, electrodes for recording fetal behavioural state, and maternal venous catheters. Three days later, fetal cerebral and myocardial blood flow were determined by the coloured microsphere technique under four conditions: (1) during rapid-eye movement (REM) sleep, before fetal cocaine infusion, (2) 30 min after initiation of a cocaine infusion to the fetus at 0.2 mg/kg per min, (3) during REM sleep, before maternal cocaine infusion, and (4) 30 min after initiation of a cocaine infusion to the ewe at 0.3 mg/kg per min. Cocaine infusion directly to the fetal lamb did not cause hypoxaemia or significantly change cerebral or myocardial blood flow or oxygen delivery. Cocaine administered to the ewe led to a drop in fetal oxygen tension from 3.0 +/- 0.5 to 2.5 +/- 0.3 kPa (P < 0.0001) and in fetal oxygen content from 3.8 +/- 0.7 to 2.8 +/- 0.4 mmol O2/L (P < 0.0001). Prior to maternal cocaine administration, fetal cerebral blood flow was 146 +/- 103 mL/100 g per min and during maternal cocaine infusion it went to 184 +/- 147 mL/100 g per min (P = NS) while myocardial blood flow increased from 156 +/- 92 to 333 +/- 178 mL/100 g per min (P < 0.002). This increase in blood flow negated the effects of hypoxaemia so that cerebral oxygen delivery was unaffected while myocardial oxygen delivery increased an average of 67%. It is concluded that cocaine administration to pregnant sheep does not impede fetal cerebral or myocardial oxygen delivery.

Vibroacoustic stimulation with a complex signal: effect on behavioral state in fetal sheep.

An ideal vibroacoustic stimulus for testing fetal reactivity has yet to be developed. In the present study in fetal sheep we tested the effect on behavioral state of an amplitude and frequency-modulated signal produced at the abdominal surface of the ewe. The stimulus was presented during periods of fetal non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep. Evaluation of behavioral state was accomplished by visual observation of strip-chart recordings. Assessed in this manner, vibroacoustic stimulation during NREM sleep consistently resulted in a change to an indeterminate state. However, stimulation during REM sleep failed to have an effect. Additional evaluation applying spectral analysis to the fetal electrocorticogram during stimulation in NREM sleep revealed a marked decrease in delta band power from 100 to 27 +/- 5% and in theta band power from 100 to 40 +/- 4% resulting in a decrease in total power from 100 to 35 +/- 4% (p < 0.05). Stimulation during REM sleep revealed a significant increase in beta band power from 100 to 123 +/- 14%. Vibroacoustic stimulation in both NREM and REM sleep led to an increase in spectral edge frequency, implying central arousal.