Developing cardiac and skeletal muscle share fast-skeletal myosin heavy chain and cardiac troponin-I expression

Abstract

Skeletal muscle derived stem cells (MDSCs) transplanted into injured myocardium can differentiate into fast skeletal muscle specific myosin heavy chain (sk-fMHC) and cardiac specific troponin-I (cTn-I) positive cells sustaining recipient myocardial function. We have recently found that MDSCs differentiate into a cardiomyocyte phenotype within a three-dimensional gel bioreactor. It is generally accepted that terminally differentiated myocardium or skeletal muscle only express cTn-I or sk-fMHC, respectively. Studies have shown the presence of non-cardiac muscle proteins in the developing myocardium or cardiac proteins in pathological skeletal muscle. In the current study, we tested the hypothesis that normal developing myocardium and skeletal muscle transiently share both sk-fMHC and cTn-I proteins. Immunohistochemistry, western blot, and RT-PCR analyses were carried out in embryonic day 13 (ED13) and 20 (ED20), neonatal day 0 (ND0) and 4 (ND4), postnatal day 10 (PND10), and 8 week-old adult female Lewis rat ventricular myocardium and gastrocnemius muscle. Confocal laser microscopy revealed that sk-fMHC was expressed as a typical striated muscle pattern within ED13 ventricular myocardium, and the striated sk-fMHC expression was lost by ND4 and became negative in adult myocardium. cTn-I was not expressed as a typical striated muscle pattern throughout the myocardium until PND10. Western blot and RT-PCR analyses revealed that gene and protein expression patterns of cardiac and skeletal muscle transcription factors and sk-fMHC within ventricular myocardium and skeletal muscle were similar at ED20, and the expression patterns became cardiac or skeletal muscle specific during postnatal development. These findings provide new insight into cardiac muscle development and highlight previously unknown common developmental features of cardiac and skeletal muscle.

Figure 1. sk-fMHC (green color) and cardiac troponin-I (red color) expression (Panel A) and gene expression (Panel B) of skeletal muscle derived stem cell 3D collagen gel bioreactor (MDSC-3DGB).

MDSC-3DGB showed co-localized expression of sk-fMHC and cardiac troponin I. Transcription factor and structural gene expression was increased compared to 2D undifferentiated MDSCs. Scale in panel A indicates 20 µm.

Figure 2. sk-fMHC (green color) and cardiac troponin-I (red color) expression within developing ventricular myocardium and skeletal muscle.

The skeletal muscle fast myosin heavy chain (sk-fMHC) and cardiac troponin-I (cTn-I) expression within the embryonic day (ED13) heart (ED13 Ventricle insert, scale indicates 500 µm). The sk-fMHC was expressed as a typical striated muscle pattern in the developing ventricle and cTn-I expression was near background level at ED13. Somites also express sk-fMHC as a fiber structure and cTn-I was also expressed very weakly similar to the heart (ED13 somite panel). Scales in ED13 ventricle and somite panels indicate 20 µm. At ED20, both heart muscle (ED20 Left Vent Pap M) and skeletal muscle (not shown) express sk-fMHC and cTn-I as a striated muscle pattern. Scales in ED20 and ND4 Vent Pap M panels indicate 20 µm. In the heart, sk-fMHC expression is significantly decreased, and cTn-I expression was increased with striation pattern after neonate day 4, and skeletal muscle significantly decreases its cTn-I expression after neonate day 4 (data not shown). At postnatal day 10 (PND10), left ventricular myocardium does not express sk-fMHC and cTn-I displayed a typical striation pattern (PND10 Left Ventricle panel). Conversely, gastrocnemius muscle expressed sk-fMHC as a typical striation pattern and cTn-I was negative (PND10 skeletal muscle panel). Scales in PND10 panels indicate 20 µm.

Figure 3. Western blot analysis of developing myocardium and skeletal muscle.

Lane 1: ED13; Lane 2: ED20; Lane 3: ND0; Lane 4: ND4; Lane 5: PND10; Lane 6: Adult (8 week-old). Top panel: Left ventricular myocardium, Bottom panel: hind limbs at ED13, ED20, ND0, and ND4, and gastrocnemius muscle in PND10 and adult. At ED13 and ED20, left ventricular myocardium expressed sk-fMHC and cTn-I expression was very weak while skeletal muscle expressed sk-fMHC. During development, sk-fMHC expression in the left ventricular myocardium decreased and increased in skeletal muscle, whereas cTn-I is expressed in the left ventricular myocardium and was negative in skeletal muscle. α-sarcomeric actinin (α-sarcActn) was used to adjust the total protein loading for the electrophoresis.

Figure 4. RT-PCR analysis of cardiac and skeletal muscle transcription factors, sk-fMHC, cTn-I mRNA expression.

Lane 1: ED13 ventricle, Lane 2: ED20 ventricle; Lane 3: ND0 ventricle; Lane 4: ND4 ventricle; Lane 5: PND10 ventricle; Lane 6: Adult ventricle; Lane 7: ED13 hind limbs; Lane 8: ED20 hind limbs, Lane 9: ND0 hind limbs; Lane 10: ND4 hind-limbs; Lane 11: PND10 gastrocnemius muscle; Lane 12: Adult gastrocnemius muscle. We note that ED20 skeletal muscle (hind limbs) mRNA expression patterns are similar to ED20 ventricular myocardium.

Figure 5. Changes in mRNA levels of cardiac and skeletal muscle specific transcription factors within ventricular myocardium and skeletal muscle.

Data are expressed as average ± SD. Cardiac transcription factors, Nkx2.5 and GATA4, and skeletal muscle transcription factor MyoD expression did not change in ventricular myocardium in adulthood, whereas myogenin expression was significantly decreased in ventricular myocardium (P<0.05, ANOVA). Nkx2.5 and GATA4 were significantly decreased during development, and MyoD and myogenin were significantly increased in skeletal muscle during the development (P<0.05). Log₁₀RQ: Relative Quantification of mRNA level compared to the mRNA level in ED13. The ratio is expressed as logarithm with base value of 10 (Log₁₀).

Figure 6. Changes in mRNA levels of sk-fMHC, cTn-I, and cardiac α-MHC within ventricular myocardium and skeletal muscle.

Data are expressed as average ± SD. The sk-fMHC mRNA level was significantly decreased and cTn-I and α-MHC levels were significantly increased in ventricular myocardium (P<0.05, ANOVA). Conversely, sk-fMHC mRNA level was significantly increased and cTn-I and α-MHC were significantly decreased in skeletal muscle. Log₁₀RQ: Relative Quantification of mRNA level compared to the value in ED13. The ratio is expressed as logarithm with base value of 10 (Log₁₀).