Ronald G. Gregg

Cardiac Troponin I Gene Knockout A Mouse Model of Myocardial Troponin I Deficiency

Troponin I is a subunit of the thin filament-associated troponin-tropomyosin complex involved in calcium regulation of skeletal and cardiac muscle contraction. We deleted the cardiac isoform of troponin I by using gene targeting in murine embryonic stem cells to determine the developmental and physiological effects of the absence of this regulatory protein. Mice lacking cardiac troponin I were born healthy, with normal heart and body weight, because a fetal troponin I isoform (identical to slow skeletal troponin I) compensated for the absence of cardiac troponin I. Compensation was only temporary, however, as 15 days after birth slow skeletal troponin I expression began a steady decline, giving rise to a troponin I deficiency. Mice died of acute heart failure on day 18, demonstrating that some form of troponin I is required for normal cardiac function and survival. Ventricular myocytes isolated from these troponin I-depleted hearts displayed shortened sarcomeres and elevated resting tension measured under relaxing conditions and had a reduced myofilament Ca sensitivity under activating conditions. The results show that (1) developmental downregulation of slow skeletal troponin I occurs even in the absence of cardiac troponin I and (2) the resultant troponin I depletion alters specific mechanical properties of myocardium and can lead to a lethal form of acute heart failure.

Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor beta 1 subunit.

The Ca2+ currents, charge movements, and intracellular Ca2+ transients in mouse skeletal muscle cells homozygous for a null mutation in the cchb1 gene encoding the beta 1 subunit of the dihydropyridine receptor have been characterized. I beta null, the L-type Ca2+ current of mutant cells, had a approximately 13-fold lower density than the L-type current of normal cells (0.41 +/- 0.042 pA/pF at + 20 mV, compared with 5.2 +/- 0.38 pA/pF in normal cells). I beta null was sensitive to dihydropyridines and had faster kinetics of activation and slower kinetics of inactivation than the L-type current of normal cells. Charge movement was reduced approximately 2.8-fold, with Qmax = 6.9 +/- 0.61 and Qmax = 2.5 +/- 0.2 nC/microF in normal and mutant cells, respectively. Approximately 40% of Qmax was nifedipine sensitive in both groups. In contrast to normal cells, Ca2+ transients could not be detected in mutant cells at any test potential; however, caffeine induced a robust Ca2+ transient. In homogenates of mutant muscle, the maximum density of [3H]PN200-110 binding sites (Bmax) was reduced approximately 3.9-fold. The results suggest that the excitation-contraction uncoupling of beta 1-null skeletal muscle involves a failure of the transduction mechanism that is due to either a reduced amount of alpha 1S subunits in the membrane or the specific absence of beta 1 from the voltage-sensor complex.

RECOVERY OF CA2+ CURRENT, CHARGE MOVEMENTS, AND CA2+ TRANSIENTS IN MYOTUBES DEFICIENT IN DIHYDROPYRIDINE RECEPTOR BETA 1 SUBUNIT TRANSFECTED WITH BETA 1 CDNA

The Ca2+ currents, charge movements, and intracellular Ca2+ transients of mouse dihydropyridine receptor (DHPR) beta 1-null myotubes expressing a mouse DHPR beta 1 cDNA have been characterized. In beta 1-null myotubes maintained in culture for 10-15 days, the density of the L-type current was approximately 7-fold lower than in normal cells of the same age (Imax was 0.65 +/- 0.05 pA/pF in mutant versus 4.5 +/- 0.8 pA/pF in normal), activation of the L-type current was significantly faster (tau activation at +40 mV was 28 +/- 7 ms in mutant versus 57 +/- 8 ms in normal), charge movements were approximately 2.5-fold lower (Qmax was 2.5 +/- 0.2 nC/microF in mutant versus 6.3 +/- 0.7 nC/microF in normal), Ca2+ transients were not elicited by depolarization, and spontaneous or evoked contractions were absent. Transfection of beta 1-null cells by lipofection with beta 1 cDNA reestablished spontaneous or evoked contractions in approximately 10% of cells after 6 days and approximately 30% of cells after 13 days. In contracting beta 1-transfected myotubes there was a complete recovery of the L-type current density (Imax was 4 +/- 0.9 pA/pF), the kinetics of activation (tau activation at +40 mV was 64 +/- 5 ms), the magnitude of charge movements (Qmax was 6.7 +/- 0.4 nC/microF), and the amplitude and voltage dependence of Ca2+ transients evoked by depolarizations. Ca2+ transients of transfected cells were unaltered by the removal of external Ca2+ or by the block of the L-type Ca2+ current, demonstrating that a skeletal-type excitation-contraction coupling was restored. The recovery of the normal skeletal muscle phenotype in beta 1-transfected beta-null myotubes shows that the beta 1 subunit is essential for the functional expression of the DHPR complex.

Temporal Associations Among Energy Intake, Plasma Linoleic Acid, and Growth Improvement in Response to Treatment Initiation After Diagnosis of Cystic Fibrosis

OBJECTIVE. It is unclear why some patients with cystic fibrosis (CF) succeed (“responders”) in recovering from malnutrition and growth faltering after treatment initiation whereas others fail to do so (“nonresponders”). We conducted a study to test the hypothesis that sustained high energy intake (↑EN) and normal plasma essential fatty acid status are critical determinants of treatment responsiveness within 2 years after diagnosis of CF. METHODS. A total of 71 CF children who had pancreatic insufficiency but not meconium ileus and were enrolled in the Wisconsin CF Neonatal Screening Project were studied. Responders were defined by having achieved adequate weight gain, as indicated by a recovery of weight z score (Wtz) comparable to Wtz at birth (WtzBR) within 2 years of diagnosis. ↑EN and sustained normal plasma linoleic acid level (↑pLA) were defined by achieving energy intake ≥120% of estimated requirement for ≥75% of the time and maintaining plasma LA ≥26% of total fatty acids for ≥75% of the time, respectively. RESULTS. Thirty-two (68%) screened patients and 13 (54%) patients whose CF was diagnosed conventionally recovered WtzBR within 2 years of diagnosis. Screened patients responded at significantly younger ages (mean/median: 6.3/4.3 months) than patients whose CF was diagnosed conventionally (mean/median: 15.8/11.8 months). Proportionately fewer screened patients (33%) achieved ↑EN compared with patients whose CF was diagnosed conventionally (73%). However, more screened patients responded to ↑EN and recovered WtzBR (91%) than patients whose CF was diagnosed conventionally (56%), although this difference was of borderline significance. Compared with having neither ↑EN nor ↑pLA, the likelihood of being a responder was greatest with combined ↑EN and ↑pLA, followed by ↑EN only. The positive associations between ↑EN and ↑pLA to treatment responsiveness remained significant after adjustment for neonatal screening status, baseline height and weight status, and indices of pulmonary disease severity. CONCLUSION. ↑EN and ↑pLA are critical in promoting adequate weight gain in children with newly diagnosed CF.

A cysteine-for-arginine substitution (R614C) in the human skeletal muscle calcium release channel cosegregates with malignant hyperthermia

A point mutation in the human gene for the skeletal muscle calcium release channel (ryanodine receptor [RYR1]) correlates with inheritance of malignant hyperthermia in a family of Northern European descent. The substitution of thymine for cytosine at position 1840 of the RYR1 transcript results in a cysteine-for-arginine substitution at position 614 (R614C) of the amino acid sequence. The mutation was absent in 59 normal individuals from the general population, in 61 additional unrelated malignant hyperthermia-susceptible patients, and in 18 patients with malignant hyperthermia associated with other inherited or congenital diseases. Together with reports of an equivalent mutation in six susceptible pig strains and an identical mutation in one other human pedigree, these findings suggest that the cysteine-for-arginine mutation represents a shared calcium release channel pathogenesis between porcine malignant hyperthermia and a subset of mutations responsible for the human malignant hyperthermia syndrome.

Waisman syndrome, a human X-linked recessive basal ganglia disorder with mental retardation: Localization to Xq27.3-qter

Linkage of the gene responsible for an X-linked early onset parkinsonism disorder with mental retardation (McKusick 311510) to DNA probes that detect restriction fragment length polymorphisms is described. The disease gene is linked to the F8C gene, and to DNA probes detecting polymorphic loci DXS52, DXS15, DXS134, and DXS374 with maximum lod scores at theta = 0 of 5.08, 5.19, 5.00, 5.03, and 4.46, respectively. Multipoint linkage analysis gives a maximum multipoint lod score of 6.75 at the F8C gene. This places the disease gene in chromosomal region Xq27.3-qter.

Assignment of the human gene for the α1 subunit of the cardiac DHP-sensitive Ca2+ channel (CCHL1A1) to chromosome 12p12−pter

A human clone corresponding to the gene encoding the alpha 1 subunit of the cardiac dihydropyridine-sensitive calcium channel (CCHL1A1) has been isolated and partially sequenced. Oligonucleotides based on the human sequence were constructed and used in the polymerase chain reaction to amplify specifically this human gene in human-rodent somatic cell hybrids. Using somatic cell hybrids that contained defined regions of human chromosome 12, the human alpha 1 subunit of the cardiac dihydropyridine-sensitive calcium channel has been assigned to the short arm of chromosome 12 in the interval 12p12-pter.