Transgenic modulation of phospholamban in the rabbit heart. (a) Construct design. The rabbit β-myosin heavy chain (β-MyHC) promoter region was used to drive the rabbit phospholamban coding sequence, flanked by a human growth hormone polyadenylation signal (hGH polyA). The exon-intron structure composing the 5′-untranslated sequence of β-MyHC is denoted by grey rectangles/line respectively. (b) Amino acid sequence of the wild-type phospholamban construct. (c) Representative RNA dot blots of phospholamban and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) expression from two of six different rabbits are shown. Each dot represents an individual rabbit and shows the reproducibility of phospholamban expression within a line. Ventricular RNA isolated from 6-month old rabbits of both genders was probed for phospholamban and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase. Duplicate blots were done and the data normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA expression. (d) Total phospholamban levels normalized to glyceraldehyde 3-phosphate dehydrogenase expression (n = 6/genotype). *P < 0.05 vs. nontransgenic (NTG) based Student’s t-test. (e) Normalized sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase expression was unchanged by phospholamban overexpression. (f) Representative immunoblot of cardiac homogenates isolated from six month old nontransgenic and transgenic rabbit ventricles showing pentameric and monomeric phospholamban separated by SDS-PAGE and probed with specific monoclonal antibodies. Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2a and glyceraldehyde 3-phosphate dehydrogenase levels were also determined. (g) phospholamban expression in the transgenic rabbit hearts was significantly increased relative to the nontransgenic hearts. Glyceraldehyde 3-phosphate dehydrogenase was used as a loading control. *, P < 0.05 vs. nontransgenic. (h) Quantitation of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase levels revealed that the expression in the transgenic rabbit hearts was not changed compared to levels seen in nontransgenic rabbit hearts. (i, j) Skeletal muscle expression of phospholamban in the viable transgenic founder. Shown are multiple dilutions of protein derived from the indicated muscles. Note the robust expression in the slow type soleus and lack of expression in the extensor digitorum longus (EDL) fast twitch muscle.

Skeletal muscle histology of phospholamban rabbits. (a) RNA blots from nontransgenic and an ill, transgenic rabbit at 6 months. An effort was made to obtain samples from overtly healthy tissue in order to avoid titrating RNA levels present at a site in which substantial fatty infiltrates were present. Only modest levels of overexpression in both the soleus and gastrocnemius were detected. (b) Skeletal muscle samples were collected from 6 month old rabbits. No gender differences presented. All sections were stained with Masson’s trichrome and images captured at 10X magnification. An example of an ill F0 animal is also shown. The slow-twitch skeletal muscle in which phospholamban was driven exhibited a dystrophic-like phenotype, as evidenced by the severe muscle wasting in the soleus muscle and, to a lesser extent, in the gastrocnemius, reflecting the mixed fiber-types present in that muscle. This animal had 2.2-fold overexpression of phospholamban at the RNA level in the soleus (data not shown); protein levels were not determined because of the extensive wasting that was present.

Cardiac histology of viable and ill transgenic lines. (a) All right ventricular sections were collected from 6 month old rabbits and stained with Masson’s trichrome. Ventricular myofibers from founder rabbits who were overtly ill exhibited a vacuolated appearance that was not observed in nontransgenic sections. Vacuoles are indicated by arrows. (b) Right ventricular sections taken from 6 month transgenic rabbits (viable line). Shown are areas of myocyte dropout, vacuolization and fibrosis that were not apparent in the nontransgenic hearts.

Phospholamban localization and ultrastructural analyses. (a) Whole heart sections from six month old rabbits were stained with phospholamban antibody (green) and counterstained with desmin antibody (red) to identify cardiac myofibers. No gender-specific differences were observed. Cross-sectional and longitudinal views of phospholamban staining show concentrated perinuclear accumulations in both the nontransgenic and transgenic sections. Magnification; 60X. (b) nontransgenic and transgenic hearts appeared to be identical at the ultrastructural level.

Phospholamban staining of an ill transgenic rabbit. Sections of left ventricle and soleus muscle were taken from an ill, 6 month old transgenic F0 rabbit that was exhibiting overt signs of distress and had to be euthanized, and a 6 month old nontransgenic age-matched control. Sections were stained with phospholamban antibody (green) and counterstained with desmin antibody (red) to identify cardiac myofibers. (a) Both the left ventricle and soleus show signs of pathology. Left ventricle samples show disarray while the soleus exhibits severe muscle wasting. Cross-sectional and longitudinal views of the left ventricle show phospholamban staining to be clumped in the transgenic cardiomyocytes, in contrast to the more evenly distributed pattern in the nontransgenic heart. (b) Few soleus myofibers remained in the ill transgenic rabbit. Phospholamban staining varied widely from fiber to fiber in both the transgenic and nontransgenic soleus. Magnification; 20X.

Cardiac function. Serial echocardiograms were performed on six month old nontransgenic and transgenic rabbits from both genders (n = 8–14/genotype) exposed to increasing doses of dobutamine. (a) Percent fractional shortening (% FS) showed no differences at baseline or in response to increasing levels of dobutamine. (b) Velocity of circumferential fiber shortening (VCFc) was not different between transgenic and nontransgenic hearts. (c, d) Cardiac catheterization also showed no differences between the transgenic and nontransgenic hearts.

Measurements of calcium flux. Sarcoplasmic reticulum calcium (SR Ca²⁺) uptakes were measured from homogenates isolated from six month old nontransgenic and transgenic rabbit left ventricles. Sarcoplasmic reticulum calcium uptake in nontransgenic (squares, n = 3) and transgenic (circles, n = 3) hearts. Maximal velocity of Ca²⁺ uptake did not differ between transgenic and nontransgenic samples (10.23 ± 1.96 and 9.89 ± 0.89 nmol/mg/min, respectively). Inset represents EC₅₀ (μM) of sarcoplasmic reticulum calcium uptake. Data were analyzed by nonlinear regression with Microcal Origin software (version 6.1).