Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

Abstract

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirusmediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca 2؉ -activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension-Ca 2؉ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca 2؉ activation of tension in ssTnIexpressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca 2؉ -activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca 2؉ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure͞ function studies in cardiac myocytes.

Phosphorylation of Troponin I by Protein Kinase A Accelerates Relaxation and Crossbridge Cycle Kinetics in Mouse Ventricular Muscle

“…3 Under control conditions, the muscles from TG mice were different in two respects from WT mouse muscles. First, as seen previously, 3,28,29 TG muscles expressing ssTnI had a higher Ca 2ϩ sensitivity than WT muscles ( Figure 2). This illustrates that the isoform of TnI is a major determinant of Ca 2ϩ sensitivity and helps to explain why neonatal myofibrils, which express ssTnI rather than cTnI, have a higher Ca 2ϩ sensitivity than adult myofibrils.…”

Dissecting the role of the myofilament in diaphragm dysfunction during the development of heart failure in mice

“…Manipulation of the Ca 2ϩ sensitivity of the contractile element can affect the amount of force generated as well as the rate at which it is generated (17,19). Such changes in contractile function can be caused by alterations to the proteins that compose the contractile element (17,22,67). The loss of diaphragm function during the development of HF may therefore be due, in part, to changes to the contractile elements of the muscle fibers composing the diaphragm.…”

Designing Heart Performance by Gene Transfer

“…Along with AAV vectors, Ad vectors represent one of the most efficient means of both in vivo and in vitro cardiac transduction. Adenovirus has been an invaluable reagent for cardiac muscle gene transfer (170,245,294,439,813,858,966).…”

“…Westfall et al (966) first determined the direct functional effects of two TnI isoforms expressed in the heart using adenoviral gene transfer of ssTnI into isolated adult cardiac myocytes. With near full replacement of native cTnI by the ssTnI isoform, there was a concomitant gain in myofilament Ca 2ϩ sensitivity of tension and altered cooperativity demonstrating that TnI isoforms directly influence sarcomeric function.…”