Transcripts and Protein Expression of Different HMW Non-Myofibrillar Tpm1 Isoforms in Human iPSC-CMs During...

Area of Science:

• Molecular Biology
• Cell Biology
• Stem Cell Research

Background:

• Mammals possess four tropomyosin (TPM) genes, with TPM1 producing myofibrillar and non-myofibrillar isoforms via alternative splicing.
• Non-myofibrillar TPM1 isoforms, including those with exon 9d or 9a peptides, regulate actin dynamics in non-muscle cells.
• Understanding TPM1 isoform expression during cardiomyocyte differentiation is crucial for cardiac development and regeneration research.

Purpose of the Study:

• To investigate the expression patterns of high-molecular-weight (HMW) non-myofibrillar Tpm1 isoforms during human induced pluripotent stem cell (hiPSC) differentiation into cardiomyocytes (CMs).
• To evaluate the protein expression and localization of specific Tpm1 isoforms during hiPSC-CM development.
• To determine the functional role of the Tpm1.5 isoform in myofibril organization in both hiPSC-CMs and other muscle cell types.

Main Methods:

• Quantitative reverse transcription PCR (qRT-PCR) with isoform-specific primers to analyze Tpm1 transcript expression.
• Western blotting using antibodies against Tpm1-exon 6a and Tpm1-exon 9d, including 2D Western blotting and mass spectrometry for protein analysis.
• Transfection of YFP-Tpm1.5 fusion constructs into embryonic chicken cardiomyocytes and hiPSC-CMs, followed by immunofluorescence microscopy.

Main Results:

• Transcripts for non-myofibrillar Tpm1 isoforms (excluding Tpm1.3 and Tpm1.14) peaked at Day 15 of hiPSC-CM differentiation.
• Protein expression of Tpm1.4, Tpm1.5, Tpm1.6, and Tpm1.7 increased until Day 15 but decreased significantly by Day 20.
• YFP-Tpm1.5 fusion protein successfully integrated into myofibrils of hiPSC-CMs and chicken cardiomyocytes/skeletal muscle, despite antibody recognition issues with native protein.

Conclusions:

• Non-myofibrillar Tpm1 isoforms are transiently expressed during early hiPSC-CM differentiation, suggesting a temporary role in cardiac development.
• The Tpm1.5 isoform, previously considered non-muscle specific, demonstrates the ability to incorporate into cardiac and skeletal myofibrils.
• Conformational differences exist between nuclear and cytoplasmic Tpm1 proteins, impacting antibody binding and cellular localization.