Jaime Meléndez

Cardiomyocyte Degeneration With Calpain Deficiency Reveals a Critical Role in Protein Homeostasis

Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation.

Effects of antihypertensive treatment on cardiac IGF-1 during prevention of ventricular hypertrophy in the rat

There is some evidence that cardiac rather than circulating insulin-like growth factor-1 (IGF-1) levels contribute to the development of renovascular hypertensive left ventricular hypertrophy (LVH), remaining unknown the effects of antihypertensive drugs on IGF-1 levels. We have assessed here the preventive effects of enalapril, losartan, propanolol and alpha-methyldopa on left ventricle (LV) and circulating IGF-1 levels in a rat model of hypertension and LVH (Goldblatt, GB). Our results show that relative LV mass and the LV content of IGF-1 were significantly lower with all antihypertensive drugs in GB rats (p<0.001). Serum concentrations of IGF-1 were lower in GB rats treated with enalapril, alpha-methyldopa and propanolol (p<0.01), but not in those treated with losartan. These results support the hypothesis that local rather than seric IGF-1 contributes to the development of left ventricular hypertrophy induced by pressure overload in the rat.

SYNTHESIS AND PHARMACOLOGICAL EFFECTS OF 3-METHYL TYRPHOSTIN (2-CYANO-3-[4-HYDROXYPHENYL]-2-BUTENETHIOAMIDE): A NEW PROTEIN TYROSINE KINASE INHIBITOR

A novel tyrphostin (2-cyano-3-[4-hydroxyphenyl]-2-butenethioamide) (CHB) with a methyl group adjacent to the aromatic ring has been synthesized. The new compound inhibits the autophosphorylation of the platelet-derived growth factor receptor kinase and the phosphorylation of phosphatidylinositol 3-kinase by this receptor kinase with an IC of approximately 1 μM. It also decreased the proliferation of a cell line transformed with the keratinocyte growth factor kinase but had no effect on the parent NIH/3T3 cells. Neither cyclic adenosine monophosphate-dependent protein kinase nor protein kinase C activities were affected by the new tyrphostin.