Proteolytic N-terminal Truncation of Cardiac Troponin I Enhances Ventricular Diastolic Function

Abstract

In the present study, we developed transgenic mice overexpressing the N-terminal truncated cTnI (cTnI-ND) in the heart to examine its biochemical and physiological significance. Ca 2؉ -activated actomyosin ATPase activity showed that cTnI-ND myofibrils had lower affinity for Ca 2؉ than controls, similar to the effect of isoproterenol treatment. In vivo and isolated working heart experiments revealed that cTnI-ND hearts had a significantly faster rate of relaxation and lower left ventricular end diastolic pressure compared with controls. The higher baseline relaxation rate of cTnI-ND hearts was at a level similar to that of wild type mouse hearts under ␤-adrenergic stimulation. The decrease in cardiac output due to lowered preload was significantly smaller for cTnI-ND hearts compared with controls. These findings indicate that removal of the N-terminal extension of cTnI via restricted proteolysis enhances cardiac function by increasing the rate of myocardial relaxation and lowering left ventricular end diastolic pressure to facilitate ventricular filling, thus resulting in better utilization of the Frank-Starling mechanism.

Deletion of a Genomic Segment Containing the Cardiac Troponin I Gene Knocks Down Expression of the Slow Troponin T Gene and Impairs Fatigue Tolerance of Diaphragm Muscle

“…The genotyping of the double transgenic offspring was performed on genome DNA isolated from tail biopsies using PCR, and the compensation for the loss of endogenous cTnI by cTnI-ND in cardiac muscle was confirmed by Western blotting as described (7,9) (Fig. 2).…”

“…cTnI-ND was originally found as a product of restricted proteolysis in cardiac adaptation to simulated microgravity, which selectively removes the cTnI-specific N-terminal extension and leaves the conserved structure intact (10). cTnI-ND transgenic mice exhibit normal base-line life activity with an increased relaxation of the cardiac muscle (9). Using this postnatally up-regulated (11) transgene allele encoding a non-destructive exogenous TnI, we successfully rescued the postnatal lethality of the Tnni3 gene-deleted mice (12) to produce adult skeletal muscle for investigating the functional effects of ssTnT deficiency.…”

“…We previously developed two transgenic mouse lines overexpressing an N-terminal truncated cTnI (cTnI-ND) driven by an ␣-myosin heavy chain (MHC) promoter (9). cTnI-ND was originally found as a product of restricted proteolysis in cardiac adaptation to simulated microgravity, which selectively removes the cTnI-specific N-terminal extension and leaves the conserved structure intact (10).…”

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

“…To investigate whether the N-terminal region of the cardiac TnI polypeptide chain resulted in incompatibility in bacterial cells, we characterized the expression of an N-terminal truncated mouse cardiac TnI in which the N-terminal 28 amino acids are deleted (16). The 5'-truncated mouse cardiac TnI cDNA cloned in pET3d plasmid was sequenced and verified by small-scale protein expression in E. coli [17] and Western blot identification as described below.…”

“…Absent in skeletal muscle TnI, this region does not contain any known binding sites for other myofilament proteins [3]. Deletion of this segment from cardiac TnI resulted in similar effects on cardiac muscle function in transgenic mice [16]. Therefore, the N-terminal segment of cardiac TnI may serve as a conformational modulator for the core structure and this conformational effect may also be a mechanism for the incompatibility of intact cardiac TnI in bacterial cells.…”