Jyh-Hung Lin

Hypertrophic cardiomyopathy in pigs: quantitative pathologic features in 55 cases.

naturally occurring hypertrophic cardiomyopathy (HCM) was diagnosed in 55 purebred pigs 6 to 12 months of age. Ten (18%) of the pigs died suddenly during auction or shipment or were found dead by their keepers. The other 45 pigs failed to meet the criteria for brediing stock. Forty-six purebred and 64 hybrid pigs were studied for control. Heart weights were significantly heavier (p < 0.001) in the pigs with HCM (473.5 ± 31.8 g; heart weight [HW]/body weight [BW] ratio 4.6 ± 0.7) than in the purebred (334.4 ± 29.7 g; HW/BW 3.4 ± 0.3) and hybrid (344.3 ± 28.9 g; HW/BW 3.4 ± 0.1) pigs without HCM. The ventricular septum (VS) in the 55 pigs with HCM was significantly thicker (26.0 ± 3.1 mm; p < 0.001) than in the purebred (19.6 ± 2.6 mm) and hybrid (14.1 ± 0.5 mm) pigs without HCM. The left ventricular free wall (LV) was significantly thicker (p < 0.001) in the pigs with HCM (20.0 ± 2.7 mm) than in the purebred (18.1 ± 2.1 mm) and hybrid (15.6 ± 0.3 mm) pigs without HCM. Asymmetric septal hypertrophy was evident because the ratio of VS to LV was significantly greater (p < 0.001) in the pigs with HCM (1.3 ± 0.2) than in the purebred (1.0 ± 0.2) and hybrid (0.9 ± 0.01) pigs without HCM. The anterior portion of the VS appeared to bulge into and impinge upon the left ventricular outflow tract, in which a fibrotic endocardial plaque was often seen. Histologic features included marked muscle cell disorganization in the VS, LV, right ventricular free wall. Abnormal intramural coronary arteries and myocardial fibrosis were seen in most pigs with HCM. Silver impregnation stains showed that there were marked increases in perimysial coils, pericellular weaves, and cell-to-cell struts. Matrix disorientation was evident in the hearts with HCM. Quantitation revealed that the collagen protein in the hearts with HCM (23.8 ± 2.8 μg/mg protein) was significantly higher (p < 0.001) than in the hearts of purebred (15.7 ± 1.8 μg/mg protein) and hybrid (13.9 ± 4.2 μg/m pprotein) pigs without HCM. Total muscle protein in the hearts of the purebred pigs with (51.6 ± 3.3 mg) and without (51.9 ± 3.0 mg) HCM was not different; however, in hearts with HCM (51.6 ± 3.3 mg) it was significantly higher (p < 0.001) than in those of hybrid pigs (47.6 ± 4.4 mg) without HCM. There was 47% to 52% more stainable collagen in the heart with HCM (44.7 ± 5.2 μg collagen/mg protein) than in the purebred (30.3 ± 4.0 μg collagen/mg protein) and hybrid (28.3 ± 8.1 μg collagen/mg protein) hearts without HCM. Gross and histologic features and connective tissue abnormalities in the pigs with HCM were strikingly similar to those in humans, cats, and dogs with HCM. Thus we conclude that spontaneous porcine HCM presents a new and important model for the cardiovascular investigator.

Molecular cloning and characterization of porcine cDNA encoding a 90-kDa heat shock protein and its expression following hyperthermia

We have isolated and sequenced cDNA clones encoding a 90-kDa heat shock protein (HSP90) from a porcine brain cDNA library. The sequence of the 2202-nucleotide coding region showed 88.6% homology with that of the human homologue. Moreover, the deduced amino acid sequence of the porcine hsp90 cDNA was 99.7% identical to that of the human counterpart, with a difference of only three amino acids in a total of 733 residues. Expression of the gene was greatly increased in cultured cells during recovery from heat shock treatment at 45 degrees C for 60 min. Three major transcripts 2.2, 3.0, and 4.1kb in size were detected by Northern blot hybridization. These transcripts were further identified in a whole-pig hyperthermia experiment. These three hsp90 transcripts were constitutively expressed in porcine tissues including kidney, liver, brain, and heart, and their levels were markedly enhanced during recovery from 30-min hyperthermia treatment at 43 degrees C. Furthermore, we found that HSP90 was preferentially expressed in pituitary gland, brain, adrenal gland, and testis, in comparison to the other tissues.

Alteration of endogenous antioxidant enzymes in naturally occurring hypertrophic cardiomyopathy

We have recently developed a porcine model with naturally occurring hypertrophic cardiomyopathy (HCM). Similar to humans, occluded intramural coronary artery and damaged mitochondria are frequently observed in these animals in which the disease is thought to be associated with the local ischemia of myocardium. In view of antioxidant functions involved in the ischemic injury, we measured the expression of endogenous antioxidant enzymes in the tissues with and without HCM. The results showed a significant increase of Cu,Zn‐superoxide dismutase (SOD), but not Mn‐SOD, and decrease of catalase (CAT) activities in the various areas of HCM hearts. It was demonstrated that SOD/CAT ratios in the HCM hearts were significantly higher than those in normals and were found to be dramatically correlated with the severity of cardiac hypertrophy. The altered SOD/CAT ratio was also consistent with increase in lipid damage. We hypothesize that the elevated SOD combined with an inadequate amount of H2O2 scavenging enzyme may lead HCM heart at oxidative stress risk. However, the pathogenic role of imbalanced antioxidant enzyme needs to be further explored.

Purification and characterization of porcine testis 90-kDa heat shock protein (HSP90) as a substrate for various protein kinases.

We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90α. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [γ-32P]ATP/Mg2+ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [γ-32P]ATP·Mg2+ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 α, and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3 α.