Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric oxide (NO) and natriuretic peptide (NP) coupled signaling, stimulating phosphorylation changes by protein kinase G (PKG). Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease1,2. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation3. Furthermore, though PDE5A regulates NO-generated cGMP4,5, NO-signaling is often depressed by heart disease6. PDEs controlling NP-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A7,8 is expressed in mammalian heart including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates NP rather than NO-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neuro-hormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of NO-synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phospho-proteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signaling independent of the NO-pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Background Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples. Methods and Results On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry–based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy. Conclusions Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.

ATP is required for normal cardiac contractile function, and it has long been hypothesized that reduced energy delivery contributes to the contractile dysfunction of heart failure (HF). Despite experimental and clinical HF data showing reduced metabolism through cardiac creatine kinase (CK), the major myocardial energy reserve and temporal ATP buffer, a causal relationship between reduced ATP-CK metabolism and contractile dysfunction in HF has never been demonstrated. Here, we generated mice conditionally overexpressing the myofibrillar isoform of CK (CK-M) to test the hypothesis that augmenting impaired CK-related energy metabolism improves contractile function in HF. CK-M overexpression significantly increased ATP flux through CK ex vivo and in vivo but did not alter contractile function in normal mice. It also led to significantly increased contractile function at baseline and during adrenergic stimulation and increased survival after thoracic aortic constriction (TAC) surgery-induced HF. Withdrawal of CK-M overexpression after TAC resulted in a significant decline in contractile function as compared with animals in which CK-M overexpression was maintained. These observations provide direct evidence that the failing heart is "energy starved" as it relates to CK. In addition, these data identify CK as a promising therapeutic target for preventing and treating HF and possibly diseases involving energy-dependent dysfunction in other organs with temporally varying energy demands.

Based on these findings, we postulate a new mechanism of cardiac toxicity based on the PTM-driven accumulation of desmin amyloid-like oligomers. Phosphorylation and cleavage as well as oligomers formation are reduced by treatment (CRT) indicating a relationship between the three. Finally, the decrease of desmin amyloid-like oligomers with CRT or small molecules points both to a general mechanism of HF based on desmin toxicity that is independent of protein mutations and to novel potential therapies.

Rationale One of the physiological mechanisms by which the heart adapts to a rise in blood pressure is by augmenting myocyte stretch-mediated intracellular calcium, with a subsequent increase in contractility. This slow force response was first described over a century ago and has long been considered compensatory, but its underlying mechanisms and link to chronic adaptations remain uncertain. Because levels of the matricellular protein thrombospondin-4 (TSP4) rapidly rise in hypertension and are elevated in cardiac stress overload and heart failure, we hypothesized that TSP4 is involved in this adaptive mechanism. Objective To determine the mechano-transductive role that TSP4 plays in cardiac regulation to stress. Methods and results In mice lacking TSP4 (tsp4−/−), hearts failed to acutely augment contractility or activate stretch-response pathways (ERK1/2 and Akt) on exposure to acute pressure overload. Sustained pressure overload rapidly led to greater chamber dilation, reduced function, and increased heart mass. Unlike controls, tsp4−/− cardiac trabeculae failed to enhance contractility and cellular calcium after a stretch. However, the contractility response was restored in tsp4−/− muscle incubated with recombinant TSP4. Isolated tsp4−/− myocytes responded normally to stretch, identifying a key role of matrix-myocyte interaction for TSP4 contractile modulation. Conclusion These results identify TSP4 as myocyte-interstitial mechano-signaling molecule central to adaptive cardiac contractile responses to acute stress, which appears to play a crucial role in the transition to chronic cardiac dilatation and failure.