Insights into cardiac VCP Deficiency Dysfunctions using Zebrafish and Human iPSC-Cardiomyocytes

Background: Valosin Containing Protein (VCP) is an ATPase playing a critical role in various cellular processes, particularly in regulating protein turnover and maintaining the integrity of cellular structures. Mutations in VCP are implicated in various disorders like cardiomyopathy and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD). Although the importance of VCP is well-recognized, creating animal models with complete VCP loss is challenging due to the protein’s critical roles in development, often resulting in embryonic lethality. This limitation complicates our understanding of VCP’s molecular functions in mature tissues, particularly in the heart. To address this gap, we investigated the effects of VCP deficiency in cardiac muscle cells using zebrafish and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).

Methods and Results: We generated a stable VCP knockout zebrafish line using CRISPR/Cas9 technology. The vcp^-/- zebrafish embryos showed significant impairment in cardiac function and fail to survive beyond 72 hours post fertilization (hpf). Here, we observed severe heart abnormalities, including disrupted myofibrils, accumulation of lysosomes, presence of inclusion bodies and mitochondrial degeneration. Furthermore, proteasomal dysfunction was evident through a notable increase in ubiquitinated proteins, highlighting the role of VCP in protein homeostasis and organelle quality control within cells.

To further explore VCP’s roles in the human heart, we used shRNA (AAV transduction) to knock down VCP in iPSC-derived cardiomyocytes. Since VCP interact with pathways involved in Ca²⁺ homeostasis, we investigated calcium dynamics. We observed a significant increase in decay time of Ca²⁺ transients, indicating altered Ca²⁺ reuptake in the sarcoplasmic reticulum, which might lead to slower relaxation during muscle contraction. This suggests that VCP is critical for proper Ca²⁺ handling in cardiomyocytes, likely through its interactions with calcium-regulating proteins.

Conclusions: Our VCP knockout and knockdown models provide insights into the molecular mechanisms underlying VCP deficiency and are valuable tools for understanding its contribution to cardiomyopathies.