Identification and characterization of cvHsp. A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues.

Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp. cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart. The cvHsp gene mapped to 1p36.23-p34.3 between markers D1S434 and D1S507. The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues. In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding). In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart. Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart. cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with alpha-filamin or actin-binding protein 280. Within cvHsp, amino acid residues 56-119 were shown to be important for its specific interaction with the C-terminal tail of alpha-filamin.

Relationship between biochemical and functional effects of protein phosphatase 1 inhibitors in rabbit cardiac skinned fibers.

Tautomycin (TT) and calyculin A (CyA) are inhibitors of protein phosphatases type 1 and 2 (PP1, PP2). Inhibitors 1 and 2 are specific for PP1, which is the major phosphatase functionally relevant in heart and able to dephosphorylate phospholamban (PLB). TT and CyA maintain PLB in its phosphorylated state, thereby increasing calcium uptake. Rabbit saponin skinned fibers (SF) are used to assess calcium load of the sarcoplasmic reticulum (SR). The present investigation aimed to examine the effects of PP1 inhibitors on SR calcium load assessed by caffeine-induced tension transient (CITT), and to correlate this activity with the PLB phosphorylation state. TT and CyA (100 nm) applied during the uptake phase increased the amplitude of CITT by 10 and 20%, respectively,P<0.05 without effect on the release phase. Both CyA and TT were devoid of calcium sensitizing effect when studied on Triton X-100 SF. After skinning procedure, SF were grinded for biochemical studies. SDS-PAGE electrophoresis and immunoblots using a monoclonal PLB antibody showed that cAMP or Ca2+/calmodulin-dependent protein kinases phosphorylated PLB in an additive fashion. Inhibition of PP1 by inhibitor 1, CyA and TT maintained PLB in its phosphorylated state in a dose-dependent manner. The results of this study in which functional and biochemical experiments in cardiac SF were combined demonstrate that strong correlation exists between the phosphorylation-dephosphorylation cycle of PLB and calcium uptake.