Rashid Nassar

Force-pCa relation and troponin T isoforms of rabbit myocardium.

We have previously reported the existence of at least four troponin T isoforms in rabbit ventricular muscle and described the changes in their distribution with development. In this report we test whether the proportions of the troponin T isoforms are related to the sensitivity of the myofilaments to calcium. We measured the force-pCa relations in 12 detergent-skinned ventricular strands of cardiac muscle from newborn (2-5-day-old) rabbits. We determined from each strand the amount of each troponin T isoform relative to the total amount of troponin T by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometric scans of Western blots probed with a cardiac-specific troponin T monoclonal antibody, MAb 13-11. To assess the presence of different relative amounts of cardiac and slow skeletal troponin I among the strands, we determined the amount of cardiac troponin I relative to tropomyosin. We determined the Hill coefficient and the pCa for half-maximal force, pCa50, for each strand. pCa50 was related directly to the relative amount of troponin T2 (pslope = 0.037). Our results do not indicate a relation between the Hill coefficient and troponin T2. We also did not find a relation between pCa50 and the cardiac troponin I/tropomyosin ratio, which suggests that the correlation between pCa50 and troponin T2 was not a result of changes in the relative amounts of cardiac and slow skeletal muscle troponin I. Our findings indicate that a relation exists between the force-pCa characteristics of rabbit myocardium and the troponin T isoforms that it expresses, suggesting a role for troponin T in modulating the sensitivity of cardiac myofilaments to calcium.

Developmental changes in the ultrastructure and sarcomere shortening of the isolated rabbit ventricular myocyte.

Sarcomere shortening and ultrastructure of intact isolated myocytes from ventricles of three-week-old and adult rabbits were examined. Cells were fixed and embedded, and after measuring their sarcomere shortening in response to electrical stimulation, they were examined in serial thin sections by electron microscopy. This structure-function analysis showed that adult cells were significantly larger, had longer rest sarcomere lengths, greater amount and velocity of sarcomere shortening, greater velocity of reextension, and shorter contraction duration than immature cells. In immature myocytes, a thin outer shell of myofibrils enveloped a central mass of mitochondria and nuclei, but in adult cells, the cytoskeleton divided the cell into compartments with the mitochondria arranged around and interspersed among the myofibrils. The different arrangement of the organdies and the cytoskeleton at the two ages may account for the shorter rest sarcomere length in the young myocytes and may confer differing internal loads that contribute to their smaller amount and velocity of sarcomere shortening. The corbular and longitudinal sarcoplasmic reticulum were less demarcated in immature than in adult cells. Myocytes from both ages showed postextrasystolic potentiation, suggesting that the sarcoplasmic reticulum modulates calcium at both ages. Restitution of contractility between contractions, obtained by measuring sarcomere shortening of interpolated extrasystoles, was faster in immature than in adult cells and may reflect the structural differences in the sarcoplasmic reticulum. The developmental differentiation in the sarcoplasmic reticulum suggests that changes in compartmentalization of calcium and in the distribution of putative calcium-release sites contribute to the increased contractility of adult myocytes.

Cardiac Troponin T Isoform Expression Correlates With Pathophysiological Descriptors in Patients Who Underwent Corrective Surgery for Congenital Heart Disease

BACKGROUND This study examined cardiac troponin T (cTnT) isoform expression in patients who had undergone surgery at Duke University Medical Center (Durham, NC) between December 1, 1993, and January 31, 1995, to correct congenital heart defects. The human heart expresses four cTnT isoforms (cTnT1 through cTnT4) whose sequence differences result from combinatorial alternative splicing of two exons. We have previously shown that cTnT4 is expressed at higher levels in severely failing hearts from transplant patients. In this study, we tested the hypothesis that congenital heart defects that have a more negative effect on myocardial function increase cTnT4 expression. We used the presence or absence of drug treatment for heart failure or congested circulation before surgery and the duration of inotropic support after corrective surgery as indicators of the pathophysiological state of the heart just before surgery. METHODS AND RESULTS Right atrial appendage tissue was collected from 34 patients, 6 days to 35 years old (median age, 3.4 months). The amounts of the cTnT1 through cTnT4 isoforms, measured as a percentage of total cTnT, were determined from Western blots probed with MAb13-11, a cTnT-specific monoclonal antibody. We found that cTnT4 expression correlated positively with the duration of inotropic support and was higher in patients who received drug treatment before surgery than in those who did not. Furthermore, we found that the percent of cTnT4 was significantly higher in hearts with congenital defects that caused congestive failure than in hearts with tetralogy of Fallot. CONCLUSIONS These findings suggest that in patients with congenital cardiac defects, cTnT4 expression is modulated by heart failure and is increased in hearts that are more hemodynamically stressed.

Soluble Complement Receptor-1 Protects Heart, Lung, and Cardiac Myofilament Function From Cardiopulmonary Bypass Damage

BACKGROUND Host defense system activation occurs with cardiopulmonary bypass (CPB) and is thought to contribute to the pathophysiological consequences of CPB. Complement inhibition effects on the post-CPB syndrome were tested with soluble complement receptor-1 (sCR1). METHODS AND RESULTS Twenty neonatal pigs (weight 1.8 to 2.8 kg) were randomized to control and sCR1-treated groups. LV pressure and volume, left atrial pressure, pulmonary artery pressure and flow, and respiratory system compliance and resistance were measured. Preload recruitable stroke work, isovolumic diastolic relaxation time constant (tau), and pulmonary vascular resistance were determined. Pre-CPB measures were not statistically significantly different between the 2 groups. After CPB, preload recruitable stroke work was significantly higher in the sCR1 group (n=5, 46.8+/-3.2x10(3) vs n=6, 34.3+/-3.7x10(3) erg/cm(3), P=0.042); tau was significantly lower in the sCR1 group (26.4+/-1.5, 42.4+/-6. 6 ms, P=0.003); pulmonary vascular resistance was significantly lower in the sCR1 group (5860+/-1360 vs 12 170+/-1200 dyn. s/cm(5), P=0.009); arterial PO(2) in 100% FIO(2) was significantly higher in the sCR1 group (406+/-63 vs 148+/-33 mm Hg, P=0.01); lung compliance and airway resistance did not differ significantly. The post-CPB Hill coefficient of atrial myocardium was higher in the sCR1 group (2.88+/-0.29 vs 1.88+/-0.16, P=0.023). CONCLUSIONS sCR1 meaningfully moderates the post-CPB syndrome, supporting the hypothesis that complement activation contributes to this syndrome.

A ventilator for magnetic resonance imaging.

Breathing motion severely degrades the quality of magnetic resonance images (MRI) of the thorax and upper abdomen and interferes with the acquisition of quantitative data. To minimize these motion effects, we built an MRI compatible ventilator for use in animal studies. Solid state circuitry is used for controlling ventilation parameters. The ventilator can be triggered internally at frequencies of 0.1 to 30 Hz or it can be triggered externally such as by the MRI pulse sequence. When triggered by the scanner, ventilation is synchronized to occur between image data acquisitions. Thus, image data are obtained when there is no breathing motion and at a minimum lung volume when hydrogen density is maximum. Since the ventilator can be adjusted to operate at virtually any frequency from conventional to high frequency, ventilation can be synchronized to all commonly used repetition times (100 ms to 2000 ms or more; 600 to 30 breaths/min). Scan synchronous ventilation eliminates breathing motion artifacts from most imaging sequences (single and multiple spin echo and inversion recovery). Best image quality is obtained when scan synchronous ventilation is combined with cardiac gating. These methods are also useful for quantitative research studies of thoracic and abdominal organs.

Developmental changes in the expression of rabbit left ventricular troponin T.

We examined cardiac troponin T (TnT) isoform expression in rabbit left ventricular myocardium at three different stages of postnatal development. Using sodium dodecyl sulfate gel electrophoresis (PAGE), we resolved five isoforms: TnT1, TnT2, TnT3, TnT4, and TnT5. TnT1 had the slowest electrophoretic mobility and TnT5 the fastest. The predominant isoforms were TnT2, TnT3, and TnT4. The relative amounts of TnT2, TnT3, TnT4, and TnT5 were examined in myocardium from three age-groups: 3 days (Group 1), 21–22 days (Group 2), and 99–109 days (Group 3). The amount of TnT2 relative to the total amount of TnT (determined by the ratio of the areas under the densitometric curves) decreased significantly (p<0.01) with age from 42 ± 4% in Group 1 to 25 ± 3% in Group 3. In contrast, the relative amount of TnT4 increased with age from 23 ± 2% La Group 1 to 33 ± 4% in Group 3 (p<0.01). The relative amounts of the other two isoforms changed biphasically with development: TnT3 decreased from Group 1 to Group 2 and increased from Group 2 to Group 3. TnT5, a minor isoform, increased from Group 1 to Group 2 and decreased from Group 2 to Group 3. These developmental changes in troponin T expression may account for some of the maturational changes observed in the physiological and biochemical properties of the myocardium.

Sprouting of substance P-expressing primary afferent central terminals and spinal micturition reflex NK1 receptor dependence after spinal cord injury

The primary afferent neurotransmitter triggering the spinal micturition reflex after complete spinal cord injury (SCI) in the rat is unknown. Substance P detected immunohistochemically in the sacral parasympathetic nucleus was significantly higher in 12 SCI rats than in 12 spinally intact rats (P = 0.008), suggesting substance P as a plausible candidate for the primary afferent neurotransmitter. The effects of the tachykinin NK1 receptor antagonist L-733060 on the spinal micturition reflex were then determined by performing conscious cystometry in an additional 14 intact rats and 14 SCI rats with L-733060 (0.1-100 microg) administered intrathecally at L6-S1. L-733060 was without effect in intact rats, but blocked the spinal micturition reflex in 10 of 14 SCI rats and increased the intermicturition interval in 2 of 4 others at doses ranging from 10 to 100 microg. Both phasic and nonphasic voiding contractions, differentiated according to the presence of phasic external urethral sphincter (EUS) activity, were present in most SCI rats. Both types of contractions were blocked by high doses of L-733060. Interestingly, there was a relative decline in phasic voiding contractions at high doses as well as a decline in contraction amplitude in nonphasic voiding contractions. In other respects, cystometric variables were largely unaffected in either spinally intact or SCI rats. L-733060 did not affect tonic EUS activity at any dose except when the spinal micturition reflex was blocked and tonic activity was consequently lost. These experiments show that tachykinin action at spinal NK1 receptors plays a major role in the spinal micturition reflex in SCI rats.

Cardiac muscle following quick-freezing: preservation of in vivo ultrastructure and geometry with special emphasis on intercellular clefts in the intact frog heart.

Intact frog, mouse and finch hearts were quick-frozen on a liquid He-cooled copper block. Adjacent frozen samples from the same heart were processed by freeze-substitution (followed by embedding and thin sectioning), freeze-fracture/etch (followed by platinum/carbon replication) and frozen sectioning (followed by freeze-drying), respectively, and examined with the electron microscope for fidelity of reproducing the in vivo state of heart muscle geometry, especially that of the narrow intercellular clefts between frog cardiac muscle cells. It was concluded that quick-freezing followed by the above procedures accomplishes that and that, therefore, narrow intercellular clefts are an invariant feature of the normal anatomy of frog cardiac muscle, which must be considered in physiological experiments. The methodology showed that quick-freezing through the epicardial surface is capable of producing superb cryopreservation for ultrathin cryosections, as well.

Altered cross-bridge characteristics following haemodynamic overload in rabbit hearts expressing V3 myosin

1 Our goal in this study was to evaluate the effect of haemodynamic overload on cross‐bridge (XBr) kinetics in the rabbit heart independently of myosin heavy chain (MHC) isoforms, which are known to modulate kinetics in small mammals. We applied a myothermal‐mechanical protocol to isometrically contracting papillary muscles from two rabbit heart populations: (1) surgically induced right ventricular pressure overload (PO), and (2) sustained treatment with propylthiouracil (PTU). Both treatments resulted in a 100 % V3 MHC profile. 2 XBr force–time integral (FTI), evaluated during the peak of the twitch from muscle FTI and tension‐dependent heat, was greater in the PO hearts (0.80 ± 0.10 versus 0.45 ± 0.05 pN s, means ±s.e.m.., P= 0.01). 3 Within the framework of a two‐state XBr model, the PO XBr developed more force while attached (5.8 ± 0.9 versus 2.7 ± 0.3 pN), with a lower cycling rate (0.89 ± 0.10 versus 1.50 ± 0.14 s−1) and duty cycle (0.14 ± 0.03 versus 0.24 ± 0.02). 4 Only the ventricular isoforms of myosin light chain 1 and 2 and cardiac troponin I (cTnI) were expressed, with no difference in cTnI phosphorylation between the PO and PTU samples. The troponin T (TnT) isoform compositions in the PO and PTU samples were significantly different (P= 0.001), with TnT2 comprising 2.29 ± 0.03 % in PO hearts versus 0.98 ± 0.01 % in PTU hearts of total TnT. 5 This study demonstrates that MHC does not mediate dramatic alterations in XBr function induced by haemodynamic overload. Our findings support the likelihood that differences among other thick and thin filament proteins underlie these XBr alterations.

Voltage-dependent potassium currents of urethral afferent neurons in diabetes mellitus.

Urethra-to-bladder and urethra-to-urethra reflexes appear to be important for coordination of proper voiding. Diabetes mellitus (DM) is known to result in afferent neuropathy. Neuropathic alterations in electrophysiological properties of urethral afferent neurons may therefore contribute to voiding dysfunction seen in diabetes mellitus. Accordingly, we studied urethral afferent neuronal somata in streptozotocin-induced DM or age-matched vehicle controls by whole-cell patch clamp at 5- or 10-week time points. One week prior to study, Fast Blue was injected into the proximal urethra to label urethral afferent neurons. A previously undescribed diminution of afferent neuronal voltage-dependent potassium currents was a prominent feature of urethral afferent neuropathy in DM, acting to increase neuronal excitability. Thus, unlike bladder afferent neurons, urethral afferent neurons may be hyperexcitable well into DM progression.