Cardiac RNase Z edited via CRISPR-Cas9 drives heart hypertrophy in Drosophila

Abstract

Cardiomyopathy (CM) is a group of diseases distinguished by morphological and functional abnormalities in the myocardium. It is etiologically heterogeneous and may develop via cell autonomous and/or non-autonomous mechanisms. One of the most severe forms of CM has been linked to the deficiency of the ubiquitously expressed RNase Z endoribonuclease. RNase Z cleaves off the 3’-trailer of both nuclear and mitochondrial primary tRNA (pre-tRNA) transcripts. Cells mutant for RNase Z accumulate unprocessed pre-tRNA molecules. Patients carrying RNase Z variants with reduced enzymatic activity display a plethora of symptoms including muscular hypotonia, microcephaly and severe heart hypertrophy; still, they die primarily due to acute heart decompensation. Determining whether the underlying mechanism of heart malfunction is cell autonomous or not will provide an opportunity to develop novel strategies of more efficient treatments for these patients. In this study, we used CRISPR-TRiM technology to create Drosophila models that carry cardiomyopathy-linked alleles of RNase Z only in the cardiomyocytes. We found that this modification is sufficient for flies to develop heart hypertrophy and systolic dysfunction. These observations support the idea that the RNase Z linked CM is driven by cell autonomous mechanisms.

Attention LSTM U-Net model for Drosophila melanogaster heart tube segmentation in optical coherence microscopy images

“…When this dataset was analyzed histologically, the average heart wall thickness for tinC-Cas9 > gZ + larvae was ∼4.3 µm and for tinC-Cas9 > RNZ KO larvae was ∼7.2 µm. Though our measurements are larger, they are still consistent with the findings of the original publication, where histologic treatment of tissue may cause shrinkage, explaining the discrepancy in measurements [ 10 ].…”

“…Using this method, we characterized the heart wall thickness of tinC-Cas9 > gZ + and tinC-Cas9 > RNZ KO larvae from our previous study, where the heart wall thickness measurements were performed by measuring the transverse histological sections [ 10 ]. We averaged the measurements at the end diastolic phase and the end systolic phase separately for the two larvae genotypes, as Fig.…”

“…To demonstrate the heart wall thickness measurements, we utilized the OCM dataset of a Drosophila model of cardiac hypertrophy published by Migunova et al in 2023 [ 10 ]. As reported in that study, cardiomyocyte-specific loss of RNase Z leads to heart wall overgrowth and heart dysfunction.…”

“…We acquired the standard OCM videos of Drosophila lines with CRISPR mediated heart-specific knockout of RNase Z, tinC-Cas9 > RNZ KO , and appropriate control tinC-Cas9 > gZ + . The tinC-Cas9 > RNZ KO larvae were reported to have a profound heart wall hypertrophy compared to the controls [ 10 ]. We included n = 16 tinC-Cas9 > gZ + larvae and n = 34 tinC-Cas9 > RNZ KO larvae for in vivo heart wall thickness analysis.…”

“…Drosophila’s cardiac morphology has been analyzed using histology and micro computerized tomography (microCT) measurements. Histological methods produce 2D ex vivo tissue staining and can be used to characterize the heart wall thickness in disease models of cardiac hypertrophy [ 10 – 12 ]. However, the histological fixation procedure is complicated and time-consuming, and tissue may also become distorted during the procedure [ 13 ].…”