Distinct troponin C isoform requirements in cardiac and skeletal muscle

Abstract

The zebrafish mutant silent partner is characterized by a dysmorphic, non-contractile ventricle resulting in an inability to generate normal blood flow. We have identified the genetic lesion in the zebrafish homolog of the slow twitch skeletal/cardiac troponin C gene. Although human troponin C1 (TNNC1) is expressed in both cardiac and skeletal muscle, duplication of this gene in zebrafish has resulted in tissue-specific partitioning of troponin C expression and function. Mutation of the zebrafish paralog tnnc1a, which is expressed predominantly in the heart, results in a loss of contractility and myofibrillar organization within ventricular cardiomyocytes, while skeletal muscle remains functional and intact. We further show that defective contractility in the developing heart results in abnormal atrial and ventricular chamber morphology. Together, our results suggest that tnnc1a is required both for the function and structural integrity of the contractile machinery in cardiomyocytes, helping to clarify potential mechanisms of troponin C-mediated cardiomyopathy.

Figure 1. Characterization of the sil phenotype

Lateral view of wild-type (WT) (A) and sil^m656 mutant (−/−) (B, C) embryos at 2 dpf. The ventricle (v) of the sil mutant embryo (B, C) is visibly compacted and misshapen compared to wild-type (A), while the atrium (a) of the sil mutant (C) has become enlarged. The sil^m656 mutant embryo (B, C) also exhibits pericardial edema and has reduced circulation as compared to wild-type (A). Mutant embryos homozygous for the sil^sk25 allele exhibit the same phenotype.

Figure 2. The sil mutant exhibits a loss of expression of ventricular contractile protein genes

Double staining with an atrial-specific antibody (S46) and an in situ RNA probe for ventricular myosin heavy chain (vmhc) (Yelon et al., 1999) was used to distinguish the atrial and ventricular chambers in wild-type (WT) (A, B) and sil^m656 mutant (−/−) (C-E) embryos. The hearts of wild-type (A) and sil^m656 mutant (C, D) embryos at 34 hpf express markers specific for the two cardiac chambers. However, by 52 hpf, the hearts of sil^m656 mutant (E) embryos no longer express the ventricle specific vmhc marker while both chamber markers are still visible in wild-type embryos (B).

Figure 3. Myofibrillar structure is disorganized in the sil mutant

Transmission electron microscopy was performed to study the structure of ventricular cardiomyocytes from wild-type (WT) (A, B) and sil^m656 mutant (−/−) (C, D) embryos at 50 hpf. (A, B) Myofibrils are easily detectable in the cardiomyocytes from ventricles of wild-type embryos. (C, D) sil^m656 mutant embryos exhibit very sparse and poorly organized sarcomeres. Arrows are used to indicate myofibrils. Bars, 500 nm.

Figure 4. Expression of tnnc1a, tnnc1b, and tnnc2 mRNA in zebrafish

(A, B) The zebrafish tnnc1a gene is expressed throughout the heart of wild-type zebrafish embryos at 30 hpf, indicated by arrows. (C) By 50 hpf, tnnc1a expression is predominantly restricted to the ventricle (v) of wild-type hearts and excluded from the atrium (a). The tnnc1a gene is expressed in cardiac tissue (indicated by the arrow) at both 30 hpf (D) and 50 hpf (D'). The second cTnC gene in zebrafish (tnnc1b) is detected predominantly in skeletal muscle (indicated by arrows) at 30 hpf (E) with decreased expression by 50 hpf (E'). The sTnC gene in zebrafish (tnnc2) is expressed in skeletal muscle (indicated by arrows) at both 30 hpf (F) and 50 hpf (F').

Figure 5. In situ analysis of tnnc1a mRNA expression in sil mutant embryos

In sil^m656 mutant embryos at 1 dpf (D), tnnc1a expression is found throughout the heart as in wild-type (WT) embryos (A). However, by 2 dpf, the expression of tnnc1a in sil^m656 embryos (E, F) has become diffuse compared to wild-type (B, C). No tnnc1a expression can be detected in sil^sk25 mutant embryos either at 1 or 2 dpf (G-I).

Figure 6. Myocardial specific tnnc1a expression rescues the sil mutant phenotype

(A) Depiction of the tnnc1a rescue construct used for injection of zebrafish embryos at the 1-2 cell stage. (B) 2 dpf wild-type embryos coinjected with tnnc1a morpholino (MO), the rescue construct depicted in (A), and transposase RNA. The top row shows an embryo at 4x magnification and the bottom row shows the same embryo at 20x magnification. GFP indicates expression of the construct in the cardiomyocytes of this embryo. (C) Table summarizing the results of rescue experiments in the sil^m656 line, the sil^sk25 line, and wild-type embryos injected with tnnc1a MO. Uninjected controls are compared with injected embryos that either have at least one GFP positive cell (GFP positive) or no GFP positive cells (GFP negative).