Michael J. Rovetto

The effects of hyaluronidase on interstitial hydration, plasma protein exclusion, and microvascular permeability in the isolated perfused rat heart

Hyaluronic acid, a principal glycosaminoglycan of the cardiac interstitium, may have a role in interstitial hydration, interstitial plasma protein exclusion and microvascular transport process (Wiederhielm, 1976b). We have investigated whether hyaluronidase reduces myocardial hyaluronate concentrations and thereby alters these several physical aspects in the isolated rat heart. Studies were conducted in ischemic, as well as aerobic hearts because of the reported therapeutic efficacy of the enzyme in myocardial ischemia. Two hours of perfusion with hyaluronidase significantly reduced myocardial hyaluronate content. Additionally, hyaluronidase decreased interstitial volume of both aerobic and otherwise edematous ischemic hearts, and prevented ischemic induced increased coronary vascular resistance in ischemic hearts. However, hyaluronidase did not effect the albumin interstitial exclusion volume or microvascular albumin and sorbitol exchange in aerobic hearts. In ischemic hearts, the enzyme did not prevent nor enhance the increase in microvascular permeability which occurred. We conclude that hyaluronate is neither a determinant of interstitial protein exclusion nor microvascular permeability, but plays an important role in interstitial hydration.

Evidence for high molecular weight Na−Ca exchange in cardiac sarcolemmal vesicles

SummaryCardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na−Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na−Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (Mr) of approximately 226,000±20,000sd (n=6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 1∶1 with mobile phase buffer containing 50mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na−Ca exchange activity. Na−Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na−Ca exchange activity were 91±7 and 68±11%, respectively. In the peak fraction, Na−Ca exchange specific activity increased two-to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimumMr of 224,000 Da. Specific45Ca2+-binding SL proteins withMr of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose.These data suggest that native cardiac Na−Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na−Ca exchange protein(s) remains incomplete.

Guanosine metabolism in adult rat cardiac myocytes: ribose-enhanced GTP synthesis from extracellular guanosine

The metabolic fate of transported guanosine was examined in adult rat cardiac myocytes. Freshly isolated cells were incubated with 10 μM or 100 μM [3H]guanosine and the nucleotide products extracted and examined for radiolabel distribution. The data presented show significant incorporation of guanosine into the 5′-nucleotide pool, and a marked stimulation of that incorporation by ribose. An average of 233 pmol/mg cell protein extracellular guanosine was incorporated into the cellular 5′-nucleotides over 90 min at both 10 μM and 100 μM external nucleoside. This appeared primarily as GTP (approx. 204 pmol/mg cell protein in 90 min). Only guanine nucleotides contained radiolabel; adenine nucleotides and IMP remained unlabelled even after 90 min incubation of the cells with [3H]guanosine. Addition of 5 mM ribose to the medium stimulated guanosine incorporation into 5′-nucleotides 1.6-fold (380 pmol/mg protein vs 234 pmol/mg over 90 min at 10 μM guanosine), but did not enhance the amount of guanosine transported into the cells. Intracellular guanosine concentrations exceeded those of the incubation medium at both external guanosine concentrations studied. More [3H]guanosine was salvaged at 100 μM than at 10 μM external guanosine (562 vs 380 pmol/mg protein in 90 min), but only if ribose was present in the medium. We conclude from these studies that guanosine is salvaged by heart muscle, and that at high guanosine levels the rate of guanosine salvage appears dependent on the availability of phosphoribosylpyrophosphate within the cells. At lower guanosine levels in the presence of ribose, cell guanine concentrations limit the rate of guanosine incorporation into 5′-nucleotides.

Forskolin inhibition of hexose transport in cardiomyocytes

The effects of insulin, forskolin, isoproterenol, and epinephrine on 3-O-methylglucose (hexose) transport and cell cyclic AMP levels were determined in adult rat cardiomyocytes. Insulin stimulated hexose transport in these cells an average of 2.5-fold. Initial hexose transport rates at 1 mM hexose were 3.75×10−2 nmol/mg cell protein/second in the absence of insulin, and 8.25×10−2 nmol/mg cell protein/second in the presence of 12.3 μM insulin. Forskolin at 5 μM nearly abolished hexose transport within 3 s of exposure, but did not increase cell cyclic AMP concentrations within 9 s. The apparentKi for hexose transport inhibition was about 0.3 μM forskolin. Epinephrine and isoproterenol at 50 μM increased cell cyclic AMP 4-fold during 9 s exposure, but did not affect hexose transport. Treatment of cells with these catecholamines of forskolin for up to 99 s increased cell cyclic AMP, but only forskolin inhibited hexose transport. We coclude from these results that forskolin acts on hexose transport independent of its action on adenyl cyclase, and that cyclic AMP does not inhibit or stimulate hexose transport.

Molecule specific effects of PKA-mediated phosphorylation on rat isolated heart and cardiac myofibrillar function

Increased cardiac myocyte contractility by the β-adrenergic system is an important mechanism to elevate cardiac output to meet hemodynamic demands and this process is depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility was found to be tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation regulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with unphosphorylated cTnI decreased isometric force production and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. Further, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and reduced recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress both myocyte force and power. Regarding MyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not slow skeletal TnI (ssTnI)) and yielded faster loaded shortening velocity and ∼30% increase in power output. These results add novel insight into the molecular specificity by which the β-adrenergic system regulates myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.