Total RNA was extracted from ventricular myocardium using the TRI Reagent (Molecular Research Center Inc., Cincinnati, OH) as described previously (11). The concentration of RNA was determined using Agilent RNA 6000 Nano assay (Agilent technologies Inc., Germany) following the manufacturer’s instruction. For RT reaction, 1 μg of RNA was used as a template to generate complementary DNA using the SuperScript III First-Strand Synthesis Kit (Invitrogen), and the RT was performed by following the manufacturer’s instructions. For the duplex PCR of a gene of interest and GAPDH, 2 μl of solution from the RT reaction and specific primers toward the target gene and GAPDH were used. The mRNA levels of the gene of interest were quantified by PCR at the minimum number of cycles (15 cycles) capable of detecting the PCR products within the linear amplification range. Mouse PDE1A, GFPdgn, and GAPDH were measured using the following primer pairs: PDE1A, 5′-CTAAAGATGAACTGGAGGGATCTTCG GAAC-3′ (forward) and 5′-TGGAGAAAATGGAAGCCCTAATTCAGC-3′ (reverse); GFPdgn, 5′-TCTATATCATGGCCGACAAGCAGA-3′ (forward) and 5′-ACTGGGTGCTCAGGTAGTGGTTGT-3′ (reverse); and GAPDH, 5′-ATGACATCAAGAAGGTGGTG-3′ (forward) and 5′-CATACCAGGAAATGAGCTTG-3′ (reverse).

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