Fast skeletal muscle troponin T increases the cooperativity of transgenic mouse cardiac muscle contraction

Abstract

1. To investigate the functional significance of different troponin T (TnT) isoforms in the Ca2+ activation of muscle contraction, transgenic mice have been constructed with a chicken fast skeletal muscle TnT transgene driven by a cardiac alpha-myosin heavy chain gene promoter. 2. Cardiac muscle-specific expression of the fast skeletal muscle TnT has been obtained with significant myofibril incorporation. Expression of the endogenous cardiac muscle thin filament regulatory proteins, such as troponin I and tropomyosin, was not altered in the transgenic mouse heart, providing an authentic system for the functional characterization of TnT isoforms. 3. Cardiac muscle contractility was analysed for the force vs. Ca2+ relationship in skinned ventricular trabeculae of transgenic mice in comparison with wild-type litter-mates. The results showed unchanged pCa50 values (5.1 +/- 0.04 and 5.1 +/- 0.1, respectively) but significantly steeper slopes (the Hill coefficient was 2.0 +/- 0.2 vs. 1.0 +/- 0.2, P < 0.05). 4. The results demonstrate that the structural and functional variation of different TnT isoforms may contribute to the difference in responsiveness and overall cooperativity of the thin filament-based Ca2+ regulation between cardiac and skeletal muscles.

Figure 2. Production of transgenic mouse with a heart-specific transgene encoding a fast skeletal muscle TnT isoform

A, transgene construct for the expression of adult chicken breast muscle TnT under the control of the mouse α-MHC gene promoter. Position of the chicken TnT cDNA probe used in Southern blot genotype analysis is outlined on the map. For the Southern blot genotyping, the mouse genomic DNA and the transgene DNA fragment prepared for the pronucleus injection were digested by the restriction enzyme Pvu II. In the transgenic mouse genome which contains tandem repeats of the transgene, two positive Pvu II fragments of 0.95 and 2.0 kb are expected to hybridize with the cDNA probe. The 1.05 kb transgene construct-specific band will not be seen after integration into the chromosome. B, Southern blot genotyping of the transgenic mouse lines. Only the samples from seven positive founders are labelled with their identification numbers to show the various levels of transgene copies integrated into the mouse genome as compared with the 1- and 10-copy standards digested together with normal (wild-type) mouse genomic DNA.

Figure 1. Primary structural comparison between cardiac and skeletal muscle TnTs

The amino acid sequences of the adult chicken breast muscle fast TnT (CfTnT) and adult mouse cardiac TnT (McTnT) were deduced from cDNA sequences. In the primary structure alignment, identical residues are represented by dashes and the gaps inserted to maximize the alignment are represented by asterisks. The exon boundaries are outlined according to rat cardiac (Jin et al. 1992) and fast skeletal muscle (Breitbart & Nadal-Ginard, 1986) TnT genes.

Figure 3. Heart-specific expression of fast skeletal muscle TnT in transgenic mice and incorporation into the cardiac myofibril

A, expression of fast skeletal muscle TnT in the transgenic mouse heart. Total protein extracts from transgenic mouse heart bearing the α-MHC promoter-directed chicken breast muscle TnT transgene (TGM) were analysed together with normal mouse heart and chicken breast muscle (pectoralis) controls by 14 % SDS-PAGE with an acrylamide/bisacrylamide ratio of 180:1. To monitor specific protein expression, Western blots were done using four specific mAbs against chicken breast muscle TnT (6B8), cardiac TnT (CT3), TnI (TnI-1), or tropomyosin (CH1), respectively. The blots revealed a high level expression of the chicken fast skeletal muscle TnT in the transgenic mouse heart but not skeletal muscle (gastrocnemius). The expression of TnI, tropomyosin and the endogenous cardiac TnT was not affected as compared with the normal mouse heart control. B, significant incorporation of the exogenous fast skeletal muscle TnT into the cardiac myofibrils of transgenic mice. The extensively washed transgenic mouse ventricular myofibrils were dissolved in SDS-PAGE sample buffer and analysed by Western blotting using the RATnT polyclonal antiserum. The result shows a significant myofibril incorporation of the fast skeletal muscle TnT in the transgenic mouse cardiac muscle. fTnT, chicken fast skeletal muscle TnT; cTnT, mouse cardiac TnT.

Figure 4. Incorporation of chicken fast skeletal muscle TnT in the transgenic mouse cardiac muscle thin filament

Phase-contrast and immunofluorescence microscopy was carried out on cardiac myofibrils from the transgenic and wild-type mice. The chicken fast skeletal muscle TnT-specific 6B8 mAb fluorescence staining demonstrated an I-band localization of the fast skeletal muscle TnT in the transgenic, but not wild-type, mouse cardiac sarcomeres similar to that in the chicken breast muscle myofibril, indicating a native thin filament association.

Figure 5. Ca²⁺-activated contraction of permeabilized cardiac muscle containing exogenous fast skeletal muscle TnT

A, representative data trace of force during Ca²⁺ activation of skinned trabeculae from control (left) and transgenic (right) hearts. Solution was changed at the breakpoints of the lines below the data traces and the pCa of each solution is displayed. B, the force-pCa curves were normalized to the maximal Ca²⁺- (pCa 4) activated force as 1 (the two superimposed pCa 4 data points are slightly offset for display). The results demonstrate that the transgenic mouse cardiac muscle (○, dotted line) containing the exogenous fast skeletal muscle TnT is activated by Ca²⁺ with identical pCa₅₀ values as compared with the normal mouse cardiac muscles (•, continuous line). However, the slope of the force-pCa curve was significantly steeper for the transgenic vs. normal cardiac muscle (P < 0.05), indicating an increased cooperativity (Hill coefficient = 2.0 ± 0.2 vs. 1.0 ± 0.2).

Figure 6. Distribution of thin filament proteins in the cardiac muscle cells of transgenic and control mice

The dissected transgenic (TG) and wild-type (WT) mouse ventricular muscle tissue strips were extracted with the skinning solution used for the contractility experiments. The distribution of exogenous and endogenous TnT, TnI and tropomyosin was examined by Western blotting using specific antibodies as described in Fig. 3, except that the RATnT antiserum was used at a lower concentration and, therefore, reacted more weakly with the cardiac TnT. The blots demonstrate that there were significant amounts of TnT, TnI and tropomyosin in the soluble fraction. The decrease of cardiac TnT incorporation in the transgenic mouse cardiac myofibril with an increase in the soluble fraction can be visualized. Despite the presence of over-expressed free fast skeletal muscle TnT in the transgenic cardiac myocytes, no significant difference was seen for the distribution of TnI and tropomyosin as compared with the wild-type cardiac muscle.