Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.

Comparative effects of contraction and angiotensin II on growth of adult feline cardiocytes in primary culture.

The purposes of this study were 1) to determine whether angiotensin II causes growth of adult feline cardiocytes in long-term culture, 2) to compare the growth effects of angiotensin II with those resulting from electrically stimulated contraction, and 3) to determine whether the anabolic effects of contraction are exerted via the angiotensin type 1 receptor. Adult feline cardiocytes were cultured on laminin-coated trays in a serum-free medium. Cardiocytes were either electrically stimulated to contract (1 Hz, 5-ms pulse duration, alternating polarity) or were nonstimulated and quiescent. Quiescent cells were studied as controls and after treatment with angiotensin II (10(-8) M), losartan (10(-6) M; an angiotensin type 1-receptor antagonist), or angiotensin II plus losartan. Contracting cells were studied in the presence and absence of angiotensin II or losartan. In quiescent cardiocytes, angiotensin II treatment on day 7 significantly increased protein synthesis rates by 22% and protein content per cell by 17%. The effects of angiotensin II were completely blocked by losartan. Electrically stimulated contraction on days 4 and 7 in culture significantly increased protein synthesis rate by 18 and 38% and protein content per cell by 19 and 46%, respectively. Angiotensin II treatment did not further increase protein synthesis rate or protein content in contracting cardiocytes. Furthermore, losartan did not block the anabolic effects of contraction on protein synthesis rates or protein content. In conclusion, angiotensin II can exert a modest anabolic effect on adult feline cardiocytes in culture. In contracting feline cardiocytes, angiotensin II has no effect on growth. Growth caused by electrically stimulated contraction occurs more rapidly and is greater in magnitude than that caused by angiotensin II. Growth of contracting adult feline cardiocytes is not dependent on activation of the angiotensin receptor.

Translational initiation factor eIF-4E. A link between cardiac load and protein synthesis.

To define the coupling mechanism between cardiac load and the rate of protein synthesis, changes in the extent of eIF-4E phosphorylation were measured after imposition of a load. Electrically stimulated contraction of adult feline cardiocytes increased eIF-4E phosphorylation to 34% after 4 h, as compared with 8% phosphorylation in quiescent controls. However, eIF-4E phosphorylation did not increase upon electrical stimulation in the presence of 7.5 mM 2,3-butanedione monoxime, an inhibitor of actin-myosin cross-bridge cycling and active tension development. Treatment of adult cardiocytes with either 0.1 microM insulin or 0.1 microM phorbol 12-myristate 13-acetate increased eIF-4E phosphorylation to 23 and 64%, respectively, but these increases were not blocked by 2,3-butanedione monoxime. In canine models of acute hemodynamic overload in vivo, eIF-4E phosphorylation increased to 23% in response to left ventricular pressure overload as compared with 7% phosphorylation in controls. Acute volume overload had no effect on eIF-4E phosphorylation. These changes in eIF-4E phosphorylation account for differences in anabolic responses to acute pressure versus acute volume overload. These data suggest that eIF-4E phosphorylation is a mechanism by which increased cardiac load is coupled to accelerated rates of protein synthesis.

Contraction accelerates myosin heavy chain synthesis rates in adult cardiocytes by an increase in the rate of translational initiation.

The purpose of this study was to determine the mechanism by which contraction acutely accelerates the synthesis rate of the contractile protein myosin heavy chain (MHC). Laminin-adherent adult feline cardiocytes were maintained in a serum-free medium and induced to contract at 1 Hz via electrical field stimulation. Electrical stimulation of contraction accelerated rates of MHC synthesis 28%, p < 0.05 by 4 h as determined by incorporation of [3H]phenylalanine into MHC. MHC mRNA expression as measured by RNase protection was unchanged after 4 h of electrical stimulation. MHC mRNA levels in messenger ribonucleoprotein complexes and translating polysomes were examined by sucrose gradient fractionation. The relative percentage of polysomebound MHC mRNA was equal at 47% in both electrically stimulated and control cardiocytes. However, electrical stimulation of contraction resulted in a reproducible shift of MHC mRNA from smaller polysomes into larger polysomes, indicating an increased rate of initiation. This shift resulted in significant increases in MHC mRNA levels in the fractions containing the larger polysomes of electrically stimulated cardiocytes as compared with nonstimulated controls. These data indicate that the rate of MHC synthesis is accelerated in contracting cardiocytes via an increase in translational efficiency.

Growth effects of electrically stimulated contraction on adult feline cardiocytes in primary culture.

The purpose of this study was to determine effects of long-term electrical stimulation of cardiocyte contraction on protein synthesis rates and total protein content. Adult feline cardiocytes were plated on laminin-coated culture trays and maintained in a serum-free medium consisting of M199 supplemented with ascorbate, bovine serum albumin, creatine, carnitine, taurine, and 10(-7) M recombinant insulin. Cardiocytes were electrically stimulated to contract with use of continuous electrical pulses of alternating polarity at a frequency of 1 Hz and pulse duration of 5 ms. Nonstimulated cardiocytes are normally quiescent and were used as the control group. In control quiescent cardiocytes, protein synthesis rate decreased by 14% between days 1 and 4 in culture and then remained stable through day 7. In electrically stimulated cardiocytes, protein synthesis rates increased by 19% between days 1 and 7. Protein synthesis rates were 18% higher on day 4 and 43% higher on day 7 in electrically stimulated than in quiescent cardiocytes. Protein content per cell was determined by measuring total fluorescence per cell by use of confocal microscopy of fluorescein isothiocyanate-stained cells. Electrical stimulation significantly increased cellular protein content by 52% after 7 days compared with controls. Quiescent and electrically stimulated cardiocytes remained rod shaped, retained their myofibrillar architecture, and were responsive to electrical stimulation over the 7-day period. These data demonstrated that electrically stimulated contraction of adult cardiocytes resulted in cell growth, as assessed by an increase in protein content per cell over 7 days in culture. This increase was due, at least in part, to an acceleration of steady-state protein synthesis rates.

Electrically stimulated contraction accelerates protein synthesis rates in adult feline cardiocytes.

Cardiocytes were induced to contract via electrical field stimulation with an 8 V/cm electrical square-wave pulse of 5 ms at 0.125-2.0 Hz for up to 6 h. Protein synthesis rates were measured as rate of incorporation of [3H]-phenylalanine into total cell protein. Rates of protein synthesis were accelerated 43 +/- 4%, P < 0.001, by 4 h. The acceleration of total protein synthesis showed a frequency dependence between 0.125 and 0.5 Hz. In addition to accelerating rates of total protein synthesis, electrical stimulation of contraction accelerated fractional rates of synthesis of myosin heavy chain by 42 +/- 8%, P < 0.05. Protein synthesis rates were not accelerated upon electrical stimulation using subthreshold voltages. Addition of 100 ng/ml of actinomycin D had no effect on the ability of electrical stimulation of contraction to accelerate protein synthesis. To uncouple excitation-contraction coupling, 2,3-butanedione monoxime (BDM) was used to block actin-myosin cross-bridge interactions. BDM significantly decreased the ability of electrical stimulation to accelerate protein synthesis rates.