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Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin ‐ Deficient Hearts

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ABSTRACT

Background: Cardiomyopathy is a leading cause of mortality among Duchenne muscular dystrophy patients and lacks effective therapies. Phosphodiesterase type 5 is implicated in dystrophic pathology, and the phosphodiesterase type 5 inhibitor tadalafil has recently been studied in a clinical trial for Duchenne muscular dystrophy.

Methods and results: Tadalafil was evaluated for the prevention of cardiomyopathy in the mdx mouse and golden retriever muscular dystrophy dog models of Duchenne muscular dystrophy. Tadalafil blunted the adrenergic response in mdx hearts during a 30‐minute dobutamine challenge, which coincided with cardioprotective signaling, reduced induction of μ‐calpain levels, and decreased sarcomeric protein proteolysis. Dogs with golden retriever muscular dystrophy began daily tadalafil treatment prior to detectable cardiomyopathy and demonstrated preserved cardiac function, as assessed by echocardiography and magnetic resonance imaging at ages 18, 21, and 25 months. Tadalafil treatment improved golden retriever muscular dystrophy histopathological features, decreased levels of the cation channel TRPC6, increased total threonine phosphorylation status of TRPC6, decreased m‐calpain levels and indicators of calpain target proteolysis, and elevated levels of utrophin. In addition, we showed that Duchenne muscular dystrophy patient myocardium exhibited increased TRPC6, m‐calpain, and calpain cleavage products compared with control human myocardium.

Conclusions: Prophylactic use of tadalafil delays the onset of dystrophic cardiomyopathy, which is likely attributed to modulation of TRPC6 levels and permeability and inhibition of protease content and activity. Consequently, phosphodiesterase type 5 inhibition is a candidate therapy for slowing the development of cardiomyopathy in Duchenne muscular dystrophy patients.

No MeSH data available.


Tadalafil (Tad) protects mdx hearts from stress induced by dobutamine (Dob). Male mdx mice were control or treated with Tad (n=6) for 8 months, underwent a Dob stress test (A), and were evaluated by echocardiography. B, Baseline echocardiograph measures of ejection fraction and fractional shortening in control and Tad‐treated mdx mice. C, Dob induced changes in ejection fraction and fractional shortening (indicated as percentage change from baseline measurement). Representative blots (D) and quantification (E) of phosphodiesterase type 5 (PDE5), cyclic guanosine monophosphate–dependent kinase 1α (PKG1α), phospho–glycogen synthase kinase 3β (p‐GSK3β) and total GSK3β, phospho–extracellular signal–related kinase 1/2 (p‐ERK1/2) and total ERK1/2, and sarco/endoplasmic reticulum Ca2+‐ATPase 2 (SERCA2) protein content, as normalized to Ponceau Red staining and displayed as arbitrary units (AU) in relation to Dob only values.. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment. F, Pde5 gene expression in control and Tad‐treated mdx hearts in response to Dob stress, as determined by reverse transcriptase polymerase chain reaction using Gapdh as a normalization control. Values are indicated as mean±SEM; *P<0.05 vs both groups (Tukey honest significant difference post hoc test). G, Preservation of SERCA2 levels shows significant inverse correlation to the Dob‐induced change in fractional shortening during the stress test.
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jah31697-fig-0001: Tadalafil (Tad) protects mdx hearts from stress induced by dobutamine (Dob). Male mdx mice were control or treated with Tad (n=6) for 8 months, underwent a Dob stress test (A), and were evaluated by echocardiography. B, Baseline echocardiograph measures of ejection fraction and fractional shortening in control and Tad‐treated mdx mice. C, Dob induced changes in ejection fraction and fractional shortening (indicated as percentage change from baseline measurement). Representative blots (D) and quantification (E) of phosphodiesterase type 5 (PDE5), cyclic guanosine monophosphate–dependent kinase 1α (PKG1α), phospho–glycogen synthase kinase 3β (p‐GSK3β) and total GSK3β, phospho–extracellular signal–related kinase 1/2 (p‐ERK1/2) and total ERK1/2, and sarco/endoplasmic reticulum Ca2+‐ATPase 2 (SERCA2) protein content, as normalized to Ponceau Red staining and displayed as arbitrary units (AU) in relation to Dob only values.. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment. F, Pde5 gene expression in control and Tad‐treated mdx hearts in response to Dob stress, as determined by reverse transcriptase polymerase chain reaction using Gapdh as a normalization control. Values are indicated as mean±SEM; *P<0.05 vs both groups (Tukey honest significant difference post hoc test). G, Preservation of SERCA2 levels shows significant inverse correlation to the Dob‐induced change in fractional shortening during the stress test.

Mentions: Male mdx mice began tadalafil treatment (0.1 mg*mL−1 supplemented in drinking water) at age 4 weeks. After no adverse effects were observed during 8 months of treatment, we investigated the ability of tadalafil to protect the mdx heart from a challenge with the β1‐adrenergic agonist dobutamine, which causes exacerbated cardiac dysfunction in the absence of dystrophin.18, 19 This experiment, outlined in Figure 1A, revealed that although no difference in ejection fraction or fractional shortening existed at baseline (Figure 1B), tadalafil significantly blunted elevation of both ejection fraction and fractional shortening induced by the dobutamine infusion (Figure 1C). Interestingly, the dobutamine challenge increased tadalafil's target protein, PDE5, which is undetectable by immunoblotting in nonstimulated mdx hearts (Figure 1D). Although PDE5 protein levels were not significantly different between control and tadalafil treatment (Figure 1D and 1E), Pde5 gene expression was elevated by dobutamine administration only in the absence of tadalafil (Figure 1F). This suggests there may be a pool of posttranscriptionally repressed Pde5 that is responsive to acute stress.


Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin ‐ Deficient Hearts
Tadalafil (Tad) protects mdx hearts from stress induced by dobutamine (Dob). Male mdx mice were control or treated with Tad (n=6) for 8 months, underwent a Dob stress test (A), and were evaluated by echocardiography. B, Baseline echocardiograph measures of ejection fraction and fractional shortening in control and Tad‐treated mdx mice. C, Dob induced changes in ejection fraction and fractional shortening (indicated as percentage change from baseline measurement). Representative blots (D) and quantification (E) of phosphodiesterase type 5 (PDE5), cyclic guanosine monophosphate–dependent kinase 1α (PKG1α), phospho–glycogen synthase kinase 3β (p‐GSK3β) and total GSK3β, phospho–extracellular signal–related kinase 1/2 (p‐ERK1/2) and total ERK1/2, and sarco/endoplasmic reticulum Ca2+‐ATPase 2 (SERCA2) protein content, as normalized to Ponceau Red staining and displayed as arbitrary units (AU) in relation to Dob only values.. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment. F, Pde5 gene expression in control and Tad‐treated mdx hearts in response to Dob stress, as determined by reverse transcriptase polymerase chain reaction using Gapdh as a normalization control. Values are indicated as mean±SEM; *P<0.05 vs both groups (Tukey honest significant difference post hoc test). G, Preservation of SERCA2 levels shows significant inverse correlation to the Dob‐induced change in fractional shortening during the stress test.
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jah31697-fig-0001: Tadalafil (Tad) protects mdx hearts from stress induced by dobutamine (Dob). Male mdx mice were control or treated with Tad (n=6) for 8 months, underwent a Dob stress test (A), and were evaluated by echocardiography. B, Baseline echocardiograph measures of ejection fraction and fractional shortening in control and Tad‐treated mdx mice. C, Dob induced changes in ejection fraction and fractional shortening (indicated as percentage change from baseline measurement). Representative blots (D) and quantification (E) of phosphodiesterase type 5 (PDE5), cyclic guanosine monophosphate–dependent kinase 1α (PKG1α), phospho–glycogen synthase kinase 3β (p‐GSK3β) and total GSK3β, phospho–extracellular signal–related kinase 1/2 (p‐ERK1/2) and total ERK1/2, and sarco/endoplasmic reticulum Ca2+‐ATPase 2 (SERCA2) protein content, as normalized to Ponceau Red staining and displayed as arbitrary units (AU) in relation to Dob only values.. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment. F, Pde5 gene expression in control and Tad‐treated mdx hearts in response to Dob stress, as determined by reverse transcriptase polymerase chain reaction using Gapdh as a normalization control. Values are indicated as mean±SEM; *P<0.05 vs both groups (Tukey honest significant difference post hoc test). G, Preservation of SERCA2 levels shows significant inverse correlation to the Dob‐induced change in fractional shortening during the stress test.
Mentions: Male mdx mice began tadalafil treatment (0.1 mg*mL−1 supplemented in drinking water) at age 4 weeks. After no adverse effects were observed during 8 months of treatment, we investigated the ability of tadalafil to protect the mdx heart from a challenge with the β1‐adrenergic agonist dobutamine, which causes exacerbated cardiac dysfunction in the absence of dystrophin.18, 19 This experiment, outlined in Figure 1A, revealed that although no difference in ejection fraction or fractional shortening existed at baseline (Figure 1B), tadalafil significantly blunted elevation of both ejection fraction and fractional shortening induced by the dobutamine infusion (Figure 1C). Interestingly, the dobutamine challenge increased tadalafil's target protein, PDE5, which is undetectable by immunoblotting in nonstimulated mdx hearts (Figure 1D). Although PDE5 protein levels were not significantly different between control and tadalafil treatment (Figure 1D and 1E), Pde5 gene expression was elevated by dobutamine administration only in the absence of tadalafil (Figure 1F). This suggests there may be a pool of posttranscriptionally repressed Pde5 that is responsive to acute stress.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cardiomyopathy is a leading cause of mortality among Duchenne muscular dystrophy patients and lacks effective therapies. Phosphodiesterase type 5 is implicated in dystrophic pathology, and the phosphodiesterase type 5 inhibitor tadalafil has recently been studied in a clinical trial for Duchenne muscular dystrophy.

Methods and results: Tadalafil was evaluated for the prevention of cardiomyopathy in the mdx mouse and golden retriever muscular dystrophy dog models of Duchenne muscular dystrophy. Tadalafil blunted the adrenergic response in mdx hearts during a 30&#8208;minute dobutamine challenge, which coincided with cardioprotective signaling, reduced induction of &mu;&#8208;calpain levels, and decreased sarcomeric protein proteolysis. Dogs with golden retriever muscular dystrophy began daily tadalafil treatment prior to detectable cardiomyopathy and demonstrated preserved cardiac function, as assessed by echocardiography and magnetic resonance imaging at ages 18, 21, and 25&nbsp;months. Tadalafil treatment improved golden retriever muscular dystrophy histopathological features, decreased levels of the cation channel TRPC6, increased total threonine phosphorylation status of TRPC6, decreased m&#8208;calpain levels and indicators of calpain target proteolysis, and elevated levels of utrophin. In addition, we showed that Duchenne muscular dystrophy patient myocardium exhibited increased TRPC6, m&#8208;calpain, and calpain cleavage products compared with control human myocardium.

Conclusions: Prophylactic use of tadalafil delays the onset of dystrophic cardiomyopathy, which is likely attributed to modulation of TRPC6 levels and permeability and inhibition of protease content and activity. Consequently, phosphodiesterase type 5 inhibition is a candidate therapy for slowing the development of cardiomyopathy in Duchenne muscular dystrophy patients.

No MeSH data available.