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. 2020 Nov 26;533(1):9-16.
doi: 10.1016/j.bbrc.2020.08.104. Epub 2020 Sep 9.

Optimizing delivery for efficient cardiac reprogramming

Martin H Kang  1 , Jiabiao Hu  1 , Richard E Pratt  1 , Conrad P Hodgkinson  1 , Aravind Asokan  1 , Victor J Dzau  2
Affiliations

Optimizing delivery for efficient cardiac reprogramming

Martin H Kang et al. Biochem Biophys Res Commun. .

Abstract

Following heart injury, cardiomyocytes, are lost and are not regenerated. In their place, fibroblasts invade the dead tissue where they generate a scar, which reduces cardiac function. We and others have demonstrated that combinations of specific miRNAs (miR combo) or transcription factors (GMT), delivered by individual lenti-/retro-viruses in vivo, can convert fibroblasts into cardiomyocytes and improve cardiac function. However, the effects are relatively modest due to the low efficiency of delivery of miR combo or GMT. We hypothesized that efficiency would be improved by optimizing delivery. In the first instance, we developed a multicistronic system to express all four miRNAs of miR combo from a single construct. The order of each miRNA in the multicistronic construct gave rise to different levels of miRNA expression. A combination that resulted in equivalent expression levels of each of the four miRNAs of miR combo showed the highest reprogramming efficiency. Further efficiency can be achieved by directly targeting fibroblasts. Screening of several AAV serotypes indicated that AAV1 displayed tropism towards cardiac fibroblasts. Combining multicistronic expression with AAV1 delivery robustly reprogrammed cardiac fibroblasts into cardiomyocytes in vivo.

Keywords: Cardiac reprogramming; Cardiomyocytes; Stoichiometry; miRNAs.

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Victor Dzau and Conrad Hodgkinson are co-founders of Recardia Therapeutics. Please note that all Biochemical and Biophysical Research Communications authors are required to report the following potential conflicts of interest with each submission. If applicable to your manuscript, please provide the necessary declaration in the box above.

Figures

Figure 1.
Figure 1.. Generation of a multicistronic miR combo.
(A) The endogenous miR-17-92 multicistronic. Pre-miRNAs (grey boxes) and mature miRNAs (black boxes) are shown. (B) Generation of the multicistronic miR combo: The sequences for the mature endogenous miRNAs were replaced with the mature miRNA sequences of the constituent miRNAs of miR combo. As indicated in the figure, lower stems and loops of the endogenous miRNAs as well as the spacing between the endogenous miRNAs were maintained. The region occupied by miR-92a-1 was not used and was removed. (C) Versions of the multicistronic miR combo used in this study.
Figure 2.
Figure 2.. miRNA stoichiometry influences reprogramming efficiency.
(A) Neonatal cardiac fibroblasts were transiently transfected with a plasmid containing one of the four versions of the multicistronic miR combo. After 3 days, miRNA expression was analyzed by qPCR. Expression of each miRNA is shown relative to expression levels in fibroblasts transfected with a construct containing five identical copies of a non-targeting miRNA. (B) Cultured neonatal cardiac fibroblasts were transiently transfected with the four versions of the multicistronic miR combo in vitro. Following transfection, expression levels of the cardiac commitment marker Mef2C (day 3 post-transfection) and the mature cardiomyocyte marker aMHC (day 14 post-transfection) were measured by qPCR. Expression is shown relative to expression levels in fibroblasts transfected with a construct containing five identical copies of a non-targeting miRNA. N=3.
Figure 3.
Figure 3.. AAV1 demonstrates fibroblast tropism.
(A) Cardiac fibroblasts were incubated with an AAV GFP reporter at a wide range of genome copies (GC) per cell. AAV1, 2, 5, 6, 9, and rh10 capsids were used. Imaging and FACS was performed 8 days following infection. Images 160,000 GC/cell. FACS AAV1 10,000 and 160,000 GC/cell shown. (B) Comparison of the number of cardiac fibroblasts and cardiomyocytes expressing the GFP transgene following incubation with 160,000 genome copies per cell using the indicated capsids. GFP+ cells were counted by FACS 8 days following infection. (C) AAV1-GFP was injected into mouse hearts. Three weeks following injection heart sections were immunostained for GFP and the fibroblast marker S100A4. N=3. Representative images are shown.
Figure 4.
Figure 4.. A multicistronic miR combo reprograms fibroblasts into cardiomyocytes in vivo.
Fibroblast-specific protein 1-Cre/tandem dimer Tomato (tdTomato) mice were subjected to either a sham operation or myocardial infarction (MI). Immediately after MI, a control AAV or a single AAV virus containing version 1 of the multicistronic miR combo was injected into the heart. An AAV containing a non-targeting miRNA (negmiR) was used as a control. Eight weeks after injury the entire peri-infarct region was visualized by serial sectioning through the heart tissue. Sections were probed for tdTomato and cardiac troponin-T. For all panels, scale bar, 100 μm, n=3, P values indicated.
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