Dong-ik Lee

Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy

Significance Cardiac hypertrophy and dysfunction in response to sustained hormonal and mechanical stress are sentinel features of most forms of heart disease. Activation of non–voltage-gated transient receptor potential canonical channels TRPC3 and TRPC6 may contribute to this pathophysiology and provide a therapeutic target. Effects from combined selective inhibition have not been tested previously. Here we report the capability of highly selective TRPC3/6 inhibitors to block pathological hypertrophic signaling in several cell types, including adult cardiac myocytes. We show in vivo redundancy of each channel; individual gene deletion was not protective against sustained pressure overload, whereas combined deletion ameliorated the response. These data strongly support a role for both channels in cardiac disease and the utility of selective combined inhibition. Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC50, 3–21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management.

Thrombospondin-4 Is Required for Stretch-Mediated Contractility Augmentation in Cardiac Muscle

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.

Cardiomyocyte-Specific Transforming Growth Factor β Suppression Blocks Neutrophil Infiltration, Augments Multiple Cytoprotective Cascades, and Reduces Early Mortality After Myocardial Infarction

Rationale: Wound healing after myocardial infarction involves a highly regulated inflammatory response that is initiated by the appearance of neutrophils to clear out dead cells and matrix debris. Neutrophil infiltration is controlled by multiple secreted factors, including the master regulator transforming growth factor &bgr; (TGF&bgr;). Broad inhibition of TGF&bgr; early postinfarction has worsened post–myocardial infarction remodeling; however, this signaling displays potent cell specificity, and targeted suppression particularly in the myocyte could be beneficial. Objective: Our aims were to test the hypothesis that targeted suppression of myocyte TGF&bgr; signaling ameliorates postinfarct remodeling and inflammatory modulation and to identify mechanisms by which this may be achieved. Methods and Results: Mice with TGF&bgr; receptor–coupled signaling genetically suppressed only in cardiac myocytes (conditional TGF&bgr; receptor 1 or 2 knockout) displayed marked declines in neutrophil recruitment and accompanying metalloproteinase 9 activation after infarction and were protected against early-onset mortality due to wall rupture. This is a cell-specific effect, because broader inhibition of TGF&bgr; signaling led to 100% early mortality due to rupture. Rather than by altering fibrosis or reducing the generation of proinflammatory cytokines/chemokines, myocyte-selective TGF&bgr; inhibition augmented the synthesis of a constellation of highly protective cardiokines. These included thrombospondin 4 with associated endoplasmic reticulum stress responses, interleukin-33, follistatin-like 1, and growth and differentiation factor 15, which is an inhibitor of neutrophil integrin activation and tissue migration. Conclusions: These data reveal a novel role of myocyte TGF&bgr; signaling as a potent regulator of protective cardiokine and neutrophil-mediated infarct remodeling.

Hyperactive adverse mechanical stress responses in dystrophic heart are coupled to transient receptor potential canonical 6 and blocked by cGMP-protein kinase G modulation.

Rationale: The heart is exquisitely sensitive to mechanical stimuli to adapt rapidly to physiological demands. In muscle lacking dystrophin, such as Duchenne muscular dystrophy, increased load during contraction triggers pathological responses thought to worsen the disease. The relevant mechanotransducers and therapies to target them remain unclear. Objectives: We tested the role of transient receptor potential canonical (TRPC) channels TRPC3 and TRPC6 and their modulation by protein kinase G (PKG) in controlling cardiac systolic mechanosensing and determined their pathophysiological relevance in an experimental model of Duchenne muscular dystrophy. Methods and Results: Contracting isolated papillary muscles and cardiomyocytes from controls and mice genetically lacking either TRPC3 or TRPC6 were subjected to auxotonic load to induce stress-stimulated contractility (SSC, gradual rise in force and intracellular Ca2+). Incubation with cGMP (PKG activator) markedly blunted SSC in controls and Trpc3−/−; whereas in Trpc6−/−, the resting SSC response was diminished and cGMP had no effect. In Duchenne muscular dystrophy myocytes (mdx/utrophin deficient), the SSC was excessive and arrhythmogenic. Gene deletion or selective drug blockade of TRPC6 or cGMP/PKG activation reversed this phenotype. Chronic phosphodiesterase 5A inhibition also normalized abnormal mechanosensing while blunting progressive chamber hypertrophy in Duchenne muscular dystrophy mice. Conclusions: PKG is a potent negative modulator of cardiac systolic mechanosignaling that requires TRPC6 as the target effector. In dystrophic hearts, excess SSC and arrhythmia are coupled to TRPC6 and are ameliorated by its targeted suppression or PKG activation. These results highlight novel therapeutic targets for this disease.

Pathological Cardiac Hypertrophy Alters Intracellular Targeting of Phosphodiesterase Type 5 From Nitric Oxide Synthase-3 to Natriuretic Peptide Signaling

Background In the normal heart, phosphodiesterase type 5 (PDE5) hydrolyzes cGMP coupled to nitric oxide– (specifically from nitric oxide synthase 3) but not natriuretic peptide (NP)–stimulated guanylyl cyclase. PDE5 is upregulated in hypertrophied and failing hearts and is thought to contribute to their pathophysiology. Because nitric oxide signaling declines whereas NP-derived cGMP rises in such diseases, we hypothesized that PDE5 substrate selectivity is retargeted to blunt NP-derived signaling. Methods and Results Mice with cardiac myocyte inducible PDE5 overexpression (P5+) were crossed to those lacking nitric oxide synthase 3 (N3−), and each model, the double cross, and controls were subjected to transaortic constriction. P5+ mice developed worse dysfunction and hypertrophy and enhanced NP stimulation, whereas N3− mice were protected. However, P5+/N3− mice behaved similarly to P5+ mice despite the lack of nitric oxide synthase 3–coupled cGMP generation, with protein kinase G activity suppressed in both models. PDE5 inhibition did not alter atrial natriuretic peptide–stimulated cGMP in the resting heart but augmented it in the transaortic constriction heart. This functional retargeting was associated with PDE5 translocation from sarcomeres to a dispersed distribution. P5+ hearts exhibited higher oxidative stress, whereas P5+/N3− hearts had low levels (likely owing to the absence of nitric oxide synthase 3 uncoupling). This highlights the importance of myocyte protein kinase G activity as a protection for pathological remodeling. Conclusions These data provide the first evidence for functional retargeting of PDE5 from one compartment to another, revealing a role for natriuretic peptide–derived cGMP hydrolysis by this esterase in diseased heart myocardium. Retargeting likely affects the pathophysiological consequence and the therapeutic impact of PDE5 modulation in heart disease.

Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These findings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and that the PA2 serves as a microenvironment for their expansion. DOI: http://dx.doi.org/10.7554/eLife.02164.001

Pathological Cardiac Hypertrophy Alters Intracellular Targeting of PDE5 from Nitric Oxide Synthase-3 to Natriuretic Peptide Signaling

Background In the normal heart, phosphodiesterase type 5 (PDE5) hydrolyzes cGMP coupled to nitric oxide– (specifically from nitric oxide synthase 3) but not natriuretic peptide (NP)–stimulated guanylyl cyclase. PDE5 is upregulated in hypertrophied and failing hearts and is thought to contribute to their pathophysiology. Because nitric oxide signaling declines whereas NP-derived cGMP rises in such diseases, we hypothesized that PDE5 substrate selectivity is retargeted to blunt NP-derived signaling. Methods and Results Mice with cardiac myocyte inducible PDE5 overexpression (P5+) were crossed to those lacking nitric oxide synthase 3 (N3−), and each model, the double cross, and controls were subjected to transaortic constriction. P5+ mice developed worse dysfunction and hypertrophy and enhanced NP stimulation, whereas N3− mice were protected. However, P5+/N3− mice behaved similarly to P5+ mice despite the lack of nitric oxide synthase 3–coupled cGMP generation, with protein kinase G activity suppressed in both models. PDE5 inhibition did not alter atrial natriuretic peptide–stimulated cGMP in the resting heart but augmented it in the transaortic constriction heart. This functional retargeting was associated with PDE5 translocation from sarcomeres to a dispersed distribution. P5+ hearts exhibited higher oxidative stress, whereas P5+/N3− hearts had low levels (likely owing to the absence of nitric oxide synthase 3 uncoupling). This highlights the importance of myocyte protein kinase G activity as a protection for pathological remodeling. Conclusions These data provide the first evidence for functional retargeting of PDE5 from one compartment to another, revealing a role for natriuretic peptide–derived cGMP hydrolysis by this esterase in diseased heart myocardium. Retargeting likely affects the pathophysiological consequence and the therapeutic impact of PDE5 modulation in heart disease.

Marked disparity of microRNA modulation by cGMP-selective PDE5 versus PDE9 inhibitors in heart disease

MicroRNAs (miRs) posttranscriptionally regulate mRNA and its translation into protein, and are considered master controllers of genes modulating normal physiology and disease. There is growing interest in how miRs change with drug treatment, and leveraging this for precision guided therapy. Here we contrast 2 closely related therapies, inhibitors of phosphodiesterase type 5 or type 9 (PDE5-I, PDE9-I), given to mice subjected to sustained cardiac pressure overload (PO). Both inhibitors augment cyclic guanosine monophosphate (cGMP) to activate protein kinase G, with PDE5-I regulating nitric oxide (NO) and PDE9-I natriuretic peptide-dependent signaling. While both produced strong phenotypic improvement of PO pathobiology, they surprisingly showed binary differences in miR profiles; PDE5-I broadly reduces more than 120 miRs, including nearly half those increased by PO, whereas PDE9-I has minimal impact on any miR (P < 0.0001). The disparity evolves after pre-miR processing and is organ specific. Lastly, even enhancing NO-coupled cGMP by different methods leads to altered miR regulation. Thus, seemingly similar therapeutic interventions can be barcoded by profound differences in miR signatures, and reversing disease-associated miR changes is not required for therapy success.

NUCLEAR GLYCELALDEHYDE-3-PHOSPHATE DEHYDROGENASE SIGNALING MEDIATES PATHOLOGICAL CARDIAC HYPERTROPHY VIA P300 AND MYOCYTE ENHANCER FACTOR 2

Pathologic stressors disrupt cellular homeostasis and cause various diseases, mediated by stress-responsive epigenetic gene regulatory mechanism. Glycelaldehyde-3-phosphate dehydrogenase (GAPDH) is a classic cytosolic glycolytic enzyme, but has been shown to translocate to the nucleus under stress

New insights into leveraging PKG signalling to treat diseased hearts

Activation of a protein kinase G signalling pathway has been demonstrated to ameliorate a broad range of cardiac disease conditions, including ischemia and infarction, doxorubicin toxicity, protein misfolding disorders, pressure-overload, dilated heart failure, and dystrophin deficiency. The pathways activation depends upon cGMP generated by either NO-sGC or NP-rGC signalling. Growing evidence shows that the former is a target of oxidative stress, depressing the functionality of NO-synthase, NO (by interaction with oxidant species), and sGC. We recently examined the impact of PKG1 oxidation, a disulphide modification at C42 residues in the homodimer subunits, on cardiac stress response. While this oxidation has been previously shown to provide a gain of function in resistance arterioles, in the myocardium, prevention of this oxidation in mice or myocytes expressing a C42S mutant knock-in was protective against pressure overload. The mechanism was not explained on the basis of a change in kinase activity, but rather by differential intracellular targeting likely related to protein interactions. A key target, transient receptor potential canonical-6 ion channel, which transduces hypertrophic and fibrotic signalling via the calcineurin/NFAT pathway, was more suppressed when PKG1 was not oxidized. PKG1 oxidation also appears to alter the capacity of various ways of stimulating the kinase, including PDE inhibition, and sGC or NP stimulation. This introduces PKG1 redox as a major regulator of its signalling effects, that also pathway and ways to activate it. In a second set of studies, we have recently revealed that the highly cGMP selective - PDE9A – is expressed in human and mouse hearts, upregulated in diseased hearts, and plays a role in maladaptive hypertrophy, fibrosis, and dysfunction in hearts exposed to sustained pressure stress. Furthermore, its genetic or selective pharmacological suppression ameliorates this cardiac pathophysiology. Unlike PDE5A, PDE9A targets the NP signalling pathway, effectively bypassing suppression of the NO-sGC cascades as often occurs in diseased hearts due to oxidative stress. PDE9A inhibition remains effective in vivo and in vitro even if NOS is blocked. These studies reveal two novel approaches to better leverage PKG1 activation in the diseased heart, suppression of oxidative modifications of the kinase at C42, and blocking PDE9A to circumvent declines in NO-sGC signalling.