PRKAR1A deficiency impedes hypertrophy and reduces heart size

Figure 1. Generation of cardiac‐specific PRKAR1A heterozygous knockout mice. (a) Breeding strategy for cardiac‐specific PRKAR1A heterozygous knockout (cPRKAR1A +/‐) mice: PRKAR1A Flox/+/ Mlc2v‐Cre +/‐. (b) Neonatal cPRKAR1A +/‐ mice and littermates were born with the expected Mendelian ratio from a total of 159 mice. (c) Body weight of 3‐month‐old male cPRKAR1A +/‐ and PRKAR1A Flox/+ control mice (Control, n = 13; cPRKAR1A +/‐, n = 12). (d) DNA isolated from control or cPRKAR1A +/‐ heart (ventricle) was used for genotyping PCR. PRKAR1A deletion band was only detected in the cPRKAR1A +/‐ heart. (e) DNA isolated from heart (ventricle), skeletal muscle, brain, and stomach of the cPRKAR1A +/‐ mouse was used for genotyping PCR. PRKAR1A deletion band was only detected in the heart, but not in other tissues of the cPRKAR1A +/‐ mouse. (f,g) Heart protein lysates from 3‐month‐old male mice were subjected to immunoblotting (n = 4 per group). Values are mean ± SEM and analyzed using two‐tailed Student's t test. ** p < .01

Figure 2. Disruption of PRKAR1A induced PKA activation without altering cardiac function. (a) Western blot analysis of control and cPRKAR1A +/‐ heart protein lysates extracted from 3‐month‐old male mice. PKA activity was assessed using phospho‐PKA Substrate (RRXS*/T*) antibody, and GAPDH served as a loading control. Arrowheads indicate elevated levels of phosphorylated PKA substrates (bands 1–3) in cPRKAR1A +/‐ heart. (b) NRCMs were transfected with control (siGFP) or PRKAR1A siRNA (siPRKAR1A) for 48h. PKA activity was assessed using phospho‐PKA Substrate (RRXS*/T*) antibody, with β‐actin as a loading control. NRCMs incubated with the adenylyl cyclase agonist forskolin (FSK, 10 µM) for 24 h served as a positive control for PKA activation. (c‐l) Cardiac function was assessed using echocardiography (Control, n = 13; cPRKAR1A +/‐, n = 12). (c,d) Left ventricular EF (ejection fraction) and FS (fractional shortening). (e,f) IVS;s (interventricular septal thickness at end systole), IVS;d (interventricular septal thickness at end diastole). (g,h) LVPW;s (left ventricular posterior wall thickness at end systole), LVPW;d (left ventricular posterior wall thickness at end diastole). (i,j) LVID;s…

Figure 3. PRKAR1A deficiency impeded hypertrophic growth during heart development. (a) Left ventricular (LV) mass was assessed using echocardiography (Control, n = 13; cPRKAR1A +/‐, n = 12). (b) Heart weight to body weight ratio (Control, n = 6; cPRKAR1A +/‐, n = 7). (c) Heart weight to tibia length ratio (Control, n = 6; cPRKAR1A +/‐, n = 7). (d) Cardiomyocyte cross‐sectional area was analyzed by staining with wheat germ agglutinin (WGA, red), cardiomyocyte marker cardiac troponin T (cTnT, green), and nuclei (DAPI, blue). At least 250 cardiomyocytes were measured per group (Control, n = 6; cPRKAR1A +/‐, n = 7). Scale bar = 20 µm. Values are mean ± SEM and analyzed using two‐tailed Student's t test. * p < .05, ** p < .01

Figure 4. PKA activation inhibited α1‐adrenergic receptor‐mediated hypertrophy. (a,b) NRCMs were transfected with control (siGFP) or PRKAR1A siRNA (siPRKAR1A) prior to incubation with the α1‐adrenergic receptor agonist phenylephrine (PE, 50 µM) for 48 h. (a) Immunofluorescence staining for cardiac troponin T (cTnT, green) and nuclei (DAPI, blue) in NRCMs. Scale bar = 20µm; (b) Cardiomyocyte cell surface area was analyzed using ImageJ. (c,d) NRCMs were incubated with PE (50 µM) for 48 h in the presence of the adenylyl cyclase agonist forskolin (10 µM). (c) Immunofluorescence staining for cTnT (green) and nuclei (blue). Scale bar = 20 µm; (d) Cardiomyocyte cell surface area was analyzed using ImageJ. At least 250 cardiomyocytes were measured per group (n = 4). Two‐way ANOVA with Sidak test. ***p < .001; ****p < .0001

Figure 5. Silencing of PRKAR1A resulted in mitochondrial elongation. (a) H9c2 myoblasts were transfected with control (siGFP) or PRKAR1A siRNA (siPRKAR1A), followed by staining with MitoTracker red to label mitochondria (Red) and DAPI to label nuclei (blue). Boxed areas are enlarged on the left. Scale bar = 20µm; (b) Percentage of H9c2 cells with fragmented or elongated mitochondria. At least 500 cells were counted (n = 3). Two‐way ANOVA with Sidak test. (c) Mitochondrial length. At least 15 mitochondrial particles of 40 cells were measured (n = 3). Two‐tailed Student's t test. ** p < .01, *** p < .001

Figure 6. Knockdown of PRKAR1A provoked PKA‐dependent phosphorylation and inactivation of the mitochondrial fission protein Drp1. (a,b) NRCMs were transfected with control (siGFP) or PRKAR1A siRNA (siPRKAR1A) for 48h, then treated with PKA inhibitor (H89, 5µM) or vehicle for 4h. Cell lysates were immunoblotted using indicated antibodies with β‐actin as a loading control. (a) Representative images; (b) Quantitation of phospho‐Drp1 levels. Two‐way ANOVA with Sidak test. * p < .05; (c) NRCMs transfected with siGFP or siPRKAR1A were subjected to immunofluorescent staining for Drp1 (green) and the mitochondrial marker COX IV (red). Mitochondrial and cytosolic localized Drp1 appears yellow and green, respectively, in the merged image. Scale bar = 10µm. (d) Manders’ overlap coefficients for COX IV and Drp1. Light gray bars indicate COX IV‐associated Drp1, and dark gray bars indicate Drp1‐associated COX IV. At least 45 cells were evaluated (n = 4). Two‐tailed Student's t test. *** p < .001

Figure 7. Inhibition of Drp1 attenuated α1‐adrenergic receptor‐mediated hypertrophy. (a,b) NRCMs were pretreated with the Drp1 inhibitor Mdivi 1 (50 µM) for 1h followed by incubation with PE (50 µM) for 48h. (a) Immunofluorescence staining for cTnT (green) and nuclei (DAPI, blue). Scale bar = 20 µm. (b) Cardiomyocyte cell surface area. Two‐way ANOVA with Sidak test. *** p < .001. (c) Schematic summary. Developmental cues initiate physiologic cardiac hypertrophy through α1‐adrenergic receptor (α1‐AR)‐mediated, Drp1‐dependent mitochondrial fission. PRKAR1A deficiency impedes hypertrophic heart growth, likely through PKA‐dependent Drp1 inactivation. Arrow, activation; bar‐headed line, inhibition