Assessing the cardiac safety of β-d-N4-hydroxycytidine (NHC) and Molnupiravir on hiPSC-Derived Cardiomyocyte Platform at Physiologically Relevant Concentrations

Technical Summary: Assessing the Cardiac Safety of β-d-N4-hydroxycytidine (NHC) and Molnupiravir on hiPSC-Derived Cardiomyocytes

Problem Statement
Molnupiravir, a nucleoside analog approved for emergency use in treating SARS-CoV-2, functions by inducing lethal mutagenesis in viral RNA. However, as a prodrug of β-d-N4-hydroxycytidine (NHC), it raises theoretical concerns regarding off-target effects on host nucleic acid metabolism and mitochondrial function, particularly in high-energy-demand tissues like the heart. While direct clinical evidence of Molnupiravir-induced cardiotoxicity is limited, the potential for mitochondrial injury and arrhythmogenicity necessitates rigorous preclinical evaluation. Furthermore, existing preclinical data often lack representation from Southeast Asian populations, whose genetic variability in ion channels and drug metabolism may influence cardiotoxic risk. This study addresses the need to evaluate the cardiac safety profile of Molnupiravir and NHC using a human-relevant model at physiologically relevant concentrations.

Methodology
The researchers established a robust platform using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from a healthy Vietnamese donor. The study employed the following workflow:

• Cell Model: hiPSCs (line VRISGi004-A) were differentiated into cardiomyocytes using the StemMACS™ CardioDiff Kit XF, followed by metabolic purification using glucose-free media supplemented with sodium L-lactate to enrich for cardiomyocytes (>80% purity).
• Drug Exposure: Purified hiPSC-CMs were treated with Molnupiravir or NHC at concentrations ranging from 1 to 40 µM for 5 days. Concentrations were selected to reflect clinical plasma levels (1–20 µM) and supraclinical levels (>30 µM) to assess safety margins.
• Assessments:
  • Viability: Measured via MTT assays and Calcein-AM/Propidium Iodide (PI) double-staining.
  • Structural Integrity: Sarcomere organization was analyzed using immunofluorescence for α-actinin, quantified via deep learning (D-SarcNet) for Z-disk organization and SarcGraph algorithms for sarcomere length and width. Mitochondrial morphology and network complexity were assessed using MitoTracker Green staining and skeleton analysis.
  • Functional/Contractile Analysis: Spontaneous beating properties were analyzed using MUSCLEMOTION software.
  • Transcriptional Profiling: qRT-PCR was performed to evaluate the expression of cardiac transcription factors, structural genes, ion channels (CaV1.2, NaV1.5, hERG), hypertrophic/fibrotic markers, and mitochondrial biogenesis genes.

Key Results

1. Cytotoxicity: Neither Molnupiravir nor NHC significantly affected cell viability or morphology at clinically relevant concentrations (1–20 µM). Even at supraclinical concentrations (30–40 µM), cell survival remained above 90%, with only a modest reduction in viability observed for NHC at the highest doses.
2. Sarcomere Organization:
   • At 10 µM, both compounds maintained Z-disk alignment comparable to controls.
   • At 30 µM, distinct phenotypic differences emerged. NHC treatment caused subtle Z-disk misalignment and a significant reduction in sarcomere width (from 1.41 ± 0.06 µm to 1.36 ± 0.12 µm). Conversely, Molnupiravir at 30 µM enhanced Z-disk definition, resulting in a more regular arrangement and an increase in sarcomere width (1.49 ± 0.09 µm).
   • Sarcomere length and orientational order parameters remained unchanged across all conditions.
3. Mitochondrial Structure: Unlike the fragmentation induced by oxidative stress (H2O2 control), neither drug caused mitochondrial fragmentation. Instead, both compounds at 30 µM increased mitochondrial area and network complexity (branching and junction density), suggesting an adaptive or protective remodeling rather than toxicity.
4. Gene Expression:
   • Stability: Expression of pluripotency markers, core cardiac transcription factors (GATA4, NKX2.5), and structural genes (MYH6, MYH7, TNNT2) remained largely stable.
   • Ion Channels: Both compounds downregulated the CACNA1C gene (encoding CaV1.2), with Molnupiravir showing a stronger effect. No significant changes were observed in KCNH2 (hERG) or SCN5A (NaV1.5).
   • Remodeling Markers: At 30 µM, both drugs significantly downregulated the hypertrophic marker ANP and fibrotic markers (COL1A1, TGFβ1).

Significance and Claims
The authors claim that their study provides the first comparative assessment of Molnupiravir and NHC cardiotoxicity using hiPSC-CMs derived from a Southeast Asian (Vietnamese) donor, addressing a gap in preclinical diversity. The primary significance of the work lies in establishing that:

• Safety at Therapeutic Doses: Neither compound causes direct cardiotoxicity, cell death, or significant mitochondrial damage at clinically relevant concentrations.
• Compound-Specific Structural Effects: While generally safe, supraclinical doses induce distinct, compound-specific structural alterations. NHC appears to mildly disrupt sarcomere integrity, whereas Molnupiravir may promote sarcomeric alignment, potentially reflecting a maturation-like effect.
• Transcriptional Signatures: The downregulation of CACNA1C and hypertrophic/fibrotic genes suggests specific molecular interactions that warrant caution, particularly in patients with preexisting cardiovascular conditions, though these do not currently indicate overt toxicity.
• Platform Utility: The study validates the use of patient-specific hiPSC-CMs as a predictive platform for screening antiviral safety, highlighting the importance of considering genetic background in cardiotoxicity research.

The authors conclude that while Molnupiravir and NHC appear safe for acute use, the observed structural and transcriptomic alterations at supraclinical levels necessitate further investigation into their long-term functional implications, particularly for vulnerable cardiac populations.