A simple adeno-associated virus-based approach for the generation of cardiac genetic models in rats

Abstract

Background: Heart failure is a major health problem and progress in this field relies on better understanding of the mechanisms and development of novel therapeutics using animal models. The rat may be preferable to the mouse as a cardiovascular disease model due to its closer physiology to humans and due to its large size that facilitates surgical and monitoring procedures. However, unlike the mouse, genetic manipulation of the rat genome is challenging. Methods: Here we developed a simple, refined, and robust cardiac-specific rat transgenic model based on an adeno-associated virus (AAV) 9 containing a cardiac troponin T promoter. This model uses a single intraperitoneal injection of AAV and does not require special expertise or equipment. Results: We characterize the AAV dose required to achieve a high cardiac specific level of expression of a transgene in the rat heart using a single intraperitoneal injection to neonates. We show that at this AAV dose GFP expression does not result in hypertrophy, a change in cardiac function or other evidence for toxicity. Conclusions: The model shown here allows easy and fast transgenic based disease modeling of cardiovascular disease in the rat heart, and can also potentially be expanded to deliver Cas9 and gRNAs or to deliver small hairpin (sh)RNAs to also achieve gene knockouts and knockdown in the rat heart.

Imaging of existing and newly translated proteins elucidates mechanisms of sarcomere turnover

“…Protein degradation is mainly thought to be stochastic, although it has been hypothesized that old sarcomeric proteins are identified by quality control pathways and targeted for degradation. 31,32,36 We therefore wanted to determine whether young and old proteins in the sarcomere are turned over at the same rate. We used MYOM1-Halo that has a very strong signal and expressed it in NRVM using an adenoviral vector.…”

“…In addition to the sarcomeric signal we also observed a nuclear accumulation of signal for MYOM1, which likely represents an aggregate, and were therefore careful not to include the nucleus in our analyses. Assuming a first order kinetic 31,32 , we fitted an exponential decay function to the degradation, or a function that gives the difference between 1 and the exponential decay to fit to synthesis and calculated the sarcomeric half-lives of proteins from the fitted curves (Figure 1C). These data show that the sarcomeric half-lives calculated for ACTN2, MYOM1, MYBPC3, and MYL2 were similar.…”

“…The AAVs were produced according to a published protocol 45 , as we previously described 46 . For transduction 50µl of virus containing a total of ~3 X 10^11 viral genomes was delivered intraperitoneally to 4-7-days old mouse pups.…”

Adeno-associated virus vector intraperitoneal injection induces colonic mucosa and submucosa transduction and alters the diversity and composition of the faecal microbiota in rats

“…Either intravenous injection or superior mesenteric artery injection of AAV can successfully induce transduction of the ENS in the stomach, small intestine and large intestine ( Gombash et al., 2014 ; Buckinx and Timmermans, 2016 ; Buckinx et al., 2016 ; Gombash et al., 2017 ). Studies have also shown that IP injection of AAV can lead to successful transduction of adipose ( Queen et al., 2021 ) and heart ( Rotundo et al., 2011 ; Schlesinger-Laufer et al., 2020 ) tissue. AAV IP injection is simple and flexible.…”

Adeno‐associated virus‐based approach for genetic modification of cardiac fibroblasts in adult rat hearts

“…Injection of AAV serotype 9 (AAV9) carrying a CM‐ or CF‐specific promoter with Cre recombinase to mice with a LoxP‐flanked (floxed) gene is a rapid and simple strategy for cardiac gene inactivation in the mouse heart (Francisco et al., 2021 ; Werfel et al., 2014 ). Importantly, this technology has started to be tested for targeting rat CMs in vivo (Schlesinger‐Laufer et al., 2020 ), although to date there are no reports attempting to manipulate genes in rat CFs. Here, we report a simplified CF‐specific rat transgenic model using the AAV9 system, which achieved CF‐specific transgenic expression in adult rat hearts.…”