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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) decreases μ‐calpain levels and proteolysis of sarcomeric proteins induced by dobutamine (Dob) in mdx hearts. Representative blots (A) and quantification (B) of phospho–cardiac troponin I (p‐cTnI) and total cTnI, phospholamban (PLB), α‐actin, and μ‐calpain from control and Tad‐treated mdx hearts that underwent the Dob stress test. Protein content was normalized to Ponceau Red staining and displayed as arbitrary units (AU) relative to Dob only values. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment.
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jah31697-fig-0002: Tadalafil (Tad) decreases μ‐calpain levels and proteolysis of sarcomeric proteins induced by dobutamine (Dob) in mdx hearts. Representative blots (A) and quantification (B) of phospho–cardiac troponin I (p‐cTnI) and total cTnI, phospholamban (PLB), α‐actin, and μ‐calpain from control and Tad‐treated mdx hearts that underwent the Dob stress test. Protein content was normalized to Ponceau Red staining and displayed as arbitrary units (AU) relative to Dob only values. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment.

Mentions: Because cTnI, another direct target of PKG1α kinase activity, also modifies inotropy,20, 21 we evaluated phosphorylated cTnI levels as a protective mechanism. Indeed, dobutamine infusion depressed phosphorylated cTnI levels in nontreated hearts; however, this was due to loss of cTnI rather than to a change in phosphorylation (Figure 2A and 2B). Both SERCA2 and cTnI are targets for intracellular proteases known as calpains,22, 23 thus we looked for loss of other known calpain targets. The sarcomere‐associated proteins phospholamban and actin were both degraded in dobutamine‐stressed controls (Figure 2A); therefore, preservation of contractility‐related proteins by tadalafil could be due to modulation of protease expression and activation. Accordingly, we found the presence of μ‐calpain (also known as calpain 1), a protease that largely targets sarcomere‐associated proteins,24 to be elevated with dobutamine stress, although it was significantly lower in the tadalafil‐treated hearts (Figure 2B). These data presented a model in which transient cardiac stress in the dystrophic heart, such as that induced by adrenergic stimulation, increased PDE5 content and expression, thereby reducing cGMP content and ultimately leading to cardiac dysfunction by increased proteolysis of sarcomeric proteins. The basis for tadalafil's protective effect likely involves the prevention of damage from transient stressors such as adrenergic responses.


Tadalafil Treatment Delays the Onset of Cardiomyopathy in Dystrophin ‐ Deficient Hearts
Tadalafil (Tad) decreases μ‐calpain levels and proteolysis of sarcomeric proteins induced by dobutamine (Dob) in mdx hearts. Representative blots (A) and quantification (B) of phospho–cardiac troponin I (p‐cTnI) and total cTnI, phospholamban (PLB), α‐actin, and μ‐calpain from control and Tad‐treated mdx hearts that underwent the Dob stress test. Protein content was normalized to Ponceau Red staining and displayed as arbitrary units (AU) relative to Dob only values. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment.
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jah31697-fig-0002: Tadalafil (Tad) decreases μ‐calpain levels and proteolysis of sarcomeric proteins induced by dobutamine (Dob) in mdx hearts. Representative blots (A) and quantification (B) of phospho–cardiac troponin I (p‐cTnI) and total cTnI, phospholamban (PLB), α‐actin, and μ‐calpain from control and Tad‐treated mdx hearts that underwent the Dob stress test. Protein content was normalized to Ponceau Red staining and displayed as arbitrary units (AU) relative to Dob only values. Values are represented as mean±SEM; *P<0.05 vs Dob‐only treatment.
Mentions: Because cTnI, another direct target of PKG1α kinase activity, also modifies inotropy,20, 21 we evaluated phosphorylated cTnI levels as a protective mechanism. Indeed, dobutamine infusion depressed phosphorylated cTnI levels in nontreated hearts; however, this was due to loss of cTnI rather than to a change in phosphorylation (Figure 2A and 2B). Both SERCA2 and cTnI are targets for intracellular proteases known as calpains,22, 23 thus we looked for loss of other known calpain targets. The sarcomere‐associated proteins phospholamban and actin were both degraded in dobutamine‐stressed controls (Figure 2A); therefore, preservation of contractility‐related proteins by tadalafil could be due to modulation of protease expression and activation. Accordingly, we found the presence of μ‐calpain (also known as calpain 1), a protease that largely targets sarcomere‐associated proteins,24 to be elevated with dobutamine stress, although it was significantly lower in the tadalafil‐treated hearts (Figure 2B). These data presented a model in which transient cardiac stress in the dystrophic heart, such as that induced by adrenergic stimulation, increased PDE5 content and expression, thereby reducing cGMP content and ultimately leading to cardiac dysfunction by increased proteolysis of sarcomeric proteins. The basis for tadalafil's protective effect likely involves the prevention of damage from transient stressors such as adrenergic responses.

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.