Alfred A Walker

MDL-28170, a membrane-permeant calpain inhibitor, attenuates stunning and PKCε proteolysis in reperfused ferret hearts

Objectives : This paper tests the hypothesis that calpains are activated in the ischemic (I)/reperfused (R) heart and contribute to myocardial stunning. Methods : Isolated ferret hearts were Langendorff perfused isovolumically, and subjected to 20 min of global I followed by 30 min of R in the presence or absence of 0.2 μ M MDL-28170, a membrane-permeant calpain inhibitor. Right trabeculae then were isolated from these hearts, skinned chemically, and pCa2+-force curves obtained. Samples of left ventricle were extracted, subjected to SDS-PAGE, and Western analyzed for PKCe and PKMe. Results : Perfused ferret hearts exhibit a 43% decline in left ventricular developed pressure during R. Pre-treatment of hearts with MDL-28170 prior to I significantly improves function during R. Trabecular myofilaments from normal hearts have a KD for Ca2+ of 6.27±0.06; I/R decreased the KD to 6.09±0.04; trabeculae from I/R hearts pre-treated with MDL-28170 have a KD of 6.28±0.04. Western analysis shows ferret hearts to contain a single ≈96 kDa species of PKCe. I/R hearts contain the native PKCe and a ≈25 kDa smaller species of PKCe, which corresponds to PKMe, the calpain proteolyzed form of PKCe. Pre-treatment of I/R hearts with MDL-28170 markedly diminishes PKMe in reperfused hearts. Conclusions : Mechanical stunning during R is sensitive to MDL-28170. Depressed mechanical function is reflected in a hyposensitization of trabecular myofilaments to Ca2+. Western analysis shows that PKMe is present in R hearts.

Comparison of contractile performance of canine atrial and ventricular muscles.

This study compared the contractile performance of a canine right atrial trabecula with that of a macroscopically indistinguishable trabecula isolated from the right ventricular apex. The heart was removed from nine mongrel puppies weighing 6–8 kg and placed in Krebs-Ringers bicarbonate solution. The bathing solution contained only 1.25 mmoles of Ca2+ and was bubbled with a 95% O2–5% CO2 gas mixture. Each atrial trabecula was specially selected from the right atrial appendage. Histologically, these trabeculae showed a remarkable longitudinal orientation of the fibers. At Lmax (the length of the muscle at which developed tension was maximum) under identical conditions of temperature, rate of stimulation, ionic milieu, pH, and O2 and CO2 supply, right atrial trabeculae achieved the same developed and total tensions but in a much shorter time than did ventricular trabeculae. In both muscle groups the maximum developed tension averaged about 2.5 g/mm2. Since Lo (expressed as a fraction of Lmax) was less in atrial muscle than it was in ventricular muscle, we concluded that atrial muscle can be stretched considerably more than can ventricular muscle before optimum length is reached. At any given initial muscle length, the maximum of tension rise for atrial trabeculae amounted to at least twice that for ventricular trabeculae. At any given load up to 1.5 g/mm, the maximum velocity of shortening of an atrial trabecula was about three to four times that of a ventricular trabecula. These results collectively indicate that the contractile performance of the right atrial muscle is in many respects superior to that of the right ventricle, at least under the conditions of these experiments.

Canine atrial and ventricular muscle mechanics studied as a function of age.

Agedependent differences in mechanical performance and morphometric and electron microscopic characteristics of atrial and ventricular trabeculae are described. At 3 months, atrial and ventricular trabeculae develop the same amount of force. At 9 months, the ventricular muscle develops twice as much force as its atrial counterpart, although shortening is almost identical in both muscles. At any age, velocity of shortening of atrial trabeculae is at least twice that of the ventricular muscles. StereologicaJ data indicate that atrial and ventricular working myocytes maintain fixed volume fractions of myofibrils (70%) and mitochondria (25%) between 3 and 9 months of age. A broad frequency distribution of sarcomere lengths was measured at Lmax in muscles of the younger age groups. More than 80% of sarcomeres of adult atrial and ventricular myocytes clustered around 2.05 to 2.25 μm; only 30% of sarcomeres of younger atrial myocytes and 45% of sarcomeres of younger ventricular myocytes were within that length bracket. About 45% of sarcomeres in younger atrial muscles had lengths in excess of 2.35 pm; less than 3% of sarcomeres were longer than 2.35 fun in adult atrial muscles. Sarcomere lengths cluster more and more around the mean with increasing age, suggesting that with maturation there is a more homogeneous recruitment of sarcomeres. At both ages, there is a marked difference between atrial and ventricular myocytes whether examined in terms of morphological development or functional performance. We conclude that any work correlating myocardial structure and function must account for two things: the site from which the muscle was excised and the age of the donor heart.

Maximal twitch tension in intact length-clamped ferret papillary muscles evoked by modified postextrasystolic potentiation.

A modified test of postextrasystolic potentiation achieved with a brief episode of rapid pacing followed by a 6-second pause (RPP maneuver) was used to evoke maximal force in isolated intact ferret right ventricular papillary muscles. Maximal RPP tensions were examined under length-clamped conditions and compared with the steady-state forces obtained when further increases in [Ca2+]0, did not further increase force and to the tensions recorded at the point of saturation of force when similarly length-clamped muscles were subjected to caffeine-induced tetanization. The results show that the calculated maximal twitch tension achieved with RPP is comparable to the 25-35 g/mm2 observed in intact single skeletal muscle fibers. The study also shows that the beat-to-beat decay of the potentiated contraction is exponential. While the amount of the constant fractional beat-to-beat decay is a function of [Ca2+]0, it is not influenced by length. During the decay of potentiation, the ratio of the potentiation of any beat divided by that of the previous beat is a constant, called (x). With certain assumptions, it is shown that (x) is a measure of the fraction of activator calcium taken up by the sarcoplasmic reticulum in each beat and, in the steady state, the fraction of activator calcium that comes from the sarcoplasmic reticulum. The (x) amounted to 33%, 50%, and 65% when [Ca2+]0 was 1.25, 2.50, and 5.0 mM, respectively. Thus, at 1.25 mM [Ca2+]0, some two thirds of the total calcium required to activate the myofilaments comes from the extracellular compartment during excitation and only one third is contributed via release from the sarcoplasmic reticulum. In the region of optimal myofilament overlap, RPP force-length curves are remarkably shallow and almost indistinguishable from the sarcomere length-tension relation observed in skinned single cardiac cells. Tetanus plateau tensions are significantly smaller than RPP forces at any length, and the slope of the tetanus force-length curves is greater than that obtained with RPP. Thus, and by exclusion, we also suggest that caffeine may exert significant downstream inhibitory effects.

Effects of ryanodine on contractile performance of intact length-clamped papillary muscle.

Extent, time course, and underlying mechanisms of the negative inotropic effect of ryanodine were examined in 22 length-clamped ferret right ventricular papillary muscles paced 12/min at 25° C. After 60 minutes of exposure to 5 μM ryanodine a new steady state was attained with developed forces averaging 10-15% of maximum twitch force. Ryanodine does not pharmacologically excise the sarcoplasmlc reticulum (SR) in this preparation. Ryanodine does not appreciably inhibit the ability of the SR to take up Ca2+ as evidenced by the potentiated beats obtained after a short pause that are nearly as large after ryanodine as before. On comparing equipotent beats before and after ryanodine, we found that ryanodine actually increases the rate at which Ca2+ is released during the twitch if the SR Ca2+ stores are equal or similar. The evidence for this conclusion is a larger maximum rate of tension rise and briefer time to peak tension after ryanodine. Since ryanodine increases the time that SR Ca2+ release channels are open and decreases their conductivity, it must follow that the former effect predominates over the latter in our experiments. Ryanodine increases the leakiness of the SR during diastole probably by inhibiting closure of SR Ca2+ release channels. The evidence for this conclusion is as follows: the early peak of the restitution curves after ryanodine, the brevity of the time required for a rested state contraction after ryanodine, and the small amplitude of the steady-state contraction at a rate of 12/min. The SR leaks even in the absence of ryanodine, but if external Ca2+ is so high that Ca1+ loss from the cell is slowed or a Ca2+ leak into the cell through the sarcolcmma cancels the SR leak, then the effects of the SR leak are minimized. The evidence for this conclusion is the time required for rested-state contraction to occur or the slope of the descending limb of restitution curve; however, in presence of ryanodine even high external Ca2+ cannot prevent rapid depletion of SR Ca2+ stores. Even though we have presented evidence for a mechanism whereby ryanodine increases the number of open SR Ca release channels in both systole and diastole, we do not mean to imply that most of them stay open in diastole; the SR would leak too fast to accumulate any Ca for the potentiated beat. Thus, probably most channels close after being open a certain length of time, even in the presence of ryanodine.

Differential inotropic actions of ethmozine and ethacizin (diethylamine analog of ethmozine)

The direct inotropic actions of ethmozine and of its diethylamine analog, ethacizin, were studied in the presence of muscarinic and beta-adrenoreceptor blockade in 12 ferret right ventricular papillary muscles. In each muscle ethmozine caused a small but consistent and significant (p less than 0.05) increase in contractile performance, whereas ethacizin significantly (p less than 0.05) diminished contractility. Although both phenothiazines are fast channel blockers, it appears that the net positive inotropic action of ethmozine is due to its stimulatory effect on the slow inward current and that the negative inotropic action of ethacizin is largely due to its recently demonstrated decreases of the slow inward current.