Thrombospondin-4 is Necessary for the Increased Calcium Cycling Associated with the Slow Force Response

Abstract

Thrombospondin-4 (TSP4) is a matricellular protein found in the heart and upregulated in heart failure; however, its role in cardiac regulation is unknown. Our previous work showed that mice lacking the TSP4 gene (tsp4-/-) respond normally to acute (seconds) pressure overload, but rapidly deteriorate minutes after. Thus, we hypothesize that TSP4 is involved in the slow force response (SFR), the second phase of stretch-mediated adaptation to loading, discovered a century ago, yet not fully understood. One proposed mechanism for the SFR is an increase in ERK phosphorylation that leads to increased Na+/H+ exchanger-1 activity. The latter increases intracellular Na+, causing the Na+/Ca2+ exchanger (reverse mode) to rise intracellular Ca2+, therefore increasing force. To investigate this, cardiac papillary muscles were isolated from tsp4-/- and wild-type (WT) littermates. Muscles were stretched from 92% of the length that generated maximum force (Lmax ) to 98% Lmax. This length was maintained for 15 min while force and Ca2+ transients (fura-2AM) were simultaneously recorded. The immediate rise in force without change in Ca2+ (Frank-Starling) was similar in both groups. However, whereas a positive SFR occurred in WT (33 ± 7%, n=5), tsp4-/- muscle displayed a negative SFR (−14 ± 2%, n=5). The difference in force was accompanied by a rise in Ca2+ in WT but not in tsp4-/- (7 ± 2% vs. 1± 2%, P<0.05). Next, hearts were excised from mice 15 minutes following transaortic constriction (pressure overload) and phosphorylation of ERK1/2 and Akt was found to be decreased in the tsp4-/- mice (P<0.05). We conclude that TSP4 is necessary for cardiac adaptation to stretch. Its absence blunts the SFR (force and Ca2+ rise). Moreover, TSP4 seems to be involved in this mechanism upstream of ERK1/2 and Akt.