Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure

Abstract

Heart failure (HF) is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for HF are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced HF, we show that tadalafil treatment improves contractile function, reverses transverse tubule loss, restores calcium transient amplitude and the heart's response to catecholamines. Accompanying these effects, tadalafil treatment normalized BNP mRNA and prevented development of subjective signs of HF. These effects were independent of changes in myocardial cGMP content and were associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterases 2 and 3. We propose that the molecular switch for the loss of transverse tubules in HF and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2 A abundance following phosphodiesterase 5 inhibition.

Figure 1

Tadalafil treatment restores indices of cardiac function in heart failure. (A) Changes in left ventricular dimensions with tachypacing and response to tadalafil treatment (paired data, N = 4 each time point; **p < 0.01 by RM-ANOVA). ‘pre’ denotes before commencement of tachypacing, ‘4wk’ denotes 4-weeks of tachypacing and T1, T2 and T3 denote 1, 2 & 3 weeks of tadalafil treatment. (B) Changes in left ventricular free wall thickness in response to tachypacing and tadalafil treatment (paired data, N = 4 each time point; *, **, *** respectively p < 0.05, 0.01 & 0.001 by RM-ANOVA). (C) Echocardiographic assessment showing short-axis fractional area change in the absence of exogenous catecholamine stimulation: ^*p < 0.05 vs. 4-week time point (tadalafil arm); ^**p < 0.01 vs. HF group at 7 weeks; ^***p < 0.001 vs. pre-pace function (both groups). Statistics by mixed models analysis. N = HF, 5 at all time points; tadalafil, 20 at pre-pace, 19 at 4-weeks and 18 at 7 weeks. (D) Representative systolic calcium transients for control (black), 4-week paced (green), HF (red) and tadalafil (blue) groups. (E) Summary data for systolic calcium transients; *p < 0.05 vs. control; ^#p < 0.05 vs. tadalafil. (n/N = 34/16 control, 6/2 following 4 weeks tachypacing, 7/3 HF, 24/6 Tadalafil). (F) Summary data of echocardiographic assessments following 5 µg/kg/min dobutamine infusion: ^**p < 0.01 vs. HF by one-way ANOVA. N = pre-pacing, 4; 4-weeks, 3; HF, 4; tadalafil, 3. (G) Summary data showing dobutamine induced change in fractional area change derived from paired (repeated measures) data in panel F. Data is expressed as a percentage of the maximum possible response in each group. (H) Summary data showing RR intervals normalized to pre-dobutamine (5 µg.kg/min) RR interval: ^**p < 0.01 vs. pre-dobutamine RR interval by t-test. N = pre-pacing, 5; 4-weeks, 5; HF, 6; tadalafil, 5. (I) Paired data from 5 animals showing RR interval – dobutamine dose response relationship. ^*p < 0.05 by mixed models analysis. (J) Kaplan-Meier plot showing fraction of animals free from subjectively assessed signs of HF (N = HF, 42; tadalafil, 27). Tadalafil treatment commenced 28 days after tachypacing (dashed line): ^***, denotes p < 0.001 (by Cox regression-based test).

Figure 2

Altered abundance of regulators of cyclic GMP in HF and following tadalafil treatment. (A) Summary quantitative PCR assessment of myocardial brain natriuretic peptide (NPPB) mRNA abundance normalized to the housekeeper RPLPO. (B) Summary quantitative PCR assessment of myocardial phosphodiesterase 5 A (PDE5A) mRNA abundance normalized to the housekeepers TBP and YWHAZ. (C) Summary data showing no change in myocardial cGMP content. (D) Changes in protein kinase G abundance assessed by non-denaturing PAGE showing (top) a representative blot, (middle) quantification of total and 75 kDa PKG and, (bottom) quantification of 150 kDa PKG and % total PKG as dimerized form. (E) Altered PDE2A protein abundance showing (top) representative blot and, (bottom) summary data. (F) Altered PDE2A protein abundance showing (top) representative blot and, (bottom) summary data. For all panels *p < 0.05 vs control; ^#p < 0.05 vs tadalafil. N = 8 each group.

Figure 3

Tadalafil normalizes the abundance of key regulators of β-adrenergic signalling. (A) Representative blot (upper) and summary data (lower) showing no change in β₁ adrenergic receptor abundance with tachypacing or tadalafil treatment. (B) Representative blot (upper) and summary data (lower) showing no change in β₂ adrenergic receptor abundance with tachypacing or tadalafil treatment. (C) Representative blot (left) and summary data (right) showing tachypacing induced increased and tadalafil mediated normalisation of GRK2 protein abundance. (D) Representative blot (upper) and summary data (lower) showing tachypacing induced increased and tadalafil mediated normalisation of PP1 protein abundance. (E) Representative blot (upper) and summary data (lower) showing tachypacing induced increased and tadalafil mediated normalisation of PP2A protein abundance. For all panels ^*P < 0.05 vs. control; ^#p < 0.05 vs. tadalafil. N = 8 per group.

Figure 4

Recovery of transverse tubule density with altered orientation following tadalafil treatment. (A) Representative images from cell types indicated showing membrane staining (a), distance maps (b) and skeletonized images (c) from regions of interest shown in panel a. Note the reduction in TT density (a, b) and lateralization of TTs in 4-week paced, HF and tadalafil treated groups (c). Scale bars, 10 µm. (B) Summary data for TT half-distance measurements. (C) Summary data for TT fractional area measurements. For (B,C): n cells/N hearts; control, 30/6; 4-weeks, 25/4; HF, 29/6; tadalafil, 71/6. (D) Representative orientation analysis for TTs in regions highlighted relative to long-axis of cells. 0° represents longitudinally orientated and 90^o transversely oriented tubules. (E) Summary data showing TT transverse to longitudinal orientation ratio. In (B,C,E): ^*p < 0.05 vs control; ^**p < 0.01 vs. control; ^***p < 0.001 vs. control; ^#p < 0.05 vs tadalafil; by linear mixed models analysis. Panels B, C & E show mean ± SEM.

Figure 5

Altered protein abundance of putative regulators of cardiac transverse tubule formation. Representative immunoblots (upper) and summary histograms (lower) for: (A) JPH2; (B) TCAP and (C) MTM1. (D) Changes in AmpII protein abundance showing (upper) representative AmpII immunoblot and (lower) summary data (a); correlation between AmpII protein abundance and TT half distance (b) and TT fractional area (c). Solid lines through data in b. and c. are linear regressions with slopes significantly different from zero (^*p < 0.05). For immunoblots (A–D) N = control, 7; 4-weeks, 6; HF, 7; tadalafil, 8 and mean of 3 technical replicates. ^*p < 0.05 vs. control; ^#p < 0.05 vs. tadalafil; by linear mixed models analysis. For regression analysis: n cells/N hearts; control, 30/ 6; 4-weeks, 25/4; HF, 29/6; tadalafil, 71/6. All data presented as mean ± SEM.

Figure 6

Amphiphysin II drives de novo tubule formation in neonatal rat ventricular myocytes and iPSC derived cardiac myocytes. (A) Assessment of sheep myocardial AmpII isoform expression showing (left to right) representative Qiaxcel run and summary data on sheep cardiac isoform abundance. N = 4 hearts. (B) Representative immunoblot (upper) and summary data (lower) showing up-regulation of AmpII protein abundance following transient transfection of NRVMs. N = 2 isolations. (C) Representative images of NRVMs transfected with mKate2 control (a) vector or AmpII expression vector (b–d). Extracellular Oregon Green 488 imaging showing a transfected and non-transfected (NT) cell (c) and overlay of the mKate2 channel and OG488N channel (from c) showing co-localisation of markers and patency of AmpII driven tubules only in the transfected cell (d). (D) Mean data summarizing percentage of non-transfected (NT), mKate2 control vector and AmpII expression vector transfected cells with tubule structures. N = 6 isolations (non-transfected, 67 cells; mKate2, 23 cells; AmpII, 37 cells) cells in each group. (E) Mean data summarising fractional area of cells occupied by tubules in mKate2 control vector and AmpII expression vector transfected cells. N = 5 isolations (mKate2, 20 cells; AmpII, 29 cells). (F) Representative images showing AmpII driven tubule formation in iCell iPSC derived cardiac myocytes. (G) Mean data summarizing percentage of non-transfected (NT), mKate2 control vector and AmpII expression vector transfected iCell iPSC cardiac myocytes with tubule structures (N = 2 cell batches; non-transfected, 45 cells; mKate2, 17 cells; AmpII, 40 cells). (H) Mean data summarizing fractional area occupied by tubules in iCell cardiac myocytes transfected with mKate2 or AmpII vectors. N = 2 cell batches (makte2, 17 cells; AmpII, 29 cells). ^**p < 0.01 vs mKate2; ^***p < 0.001 vs mKate2; ^#p < 0.05 vs NT; by linear mixed models analysis. Scale bars, 10 µm. Panels A, C, D, F & G show mean ± SEM.