Sildenafil and Cardiomyocyte-Specific Cgmp Signaling Prevent Cardiomyopathic Changes Associated with Dystrophin Deficiency

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Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency

M. Khairallah*†, R. J. Khairallah*, M. E. Young‡, B. G. Allen*, M. A. Gillis*, G. Danialou†, C. F. Deschepper§, B. J. Petrof†, and C. Des Rosiers*¶

*Montreal Heart Institute and §Institut de Recherche Clinique de Montre´al, Universite´de Montre´al, Montreal, QC, Canada H3C 3J7; ‡U.S. Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030; and †Meakins–Christie Laboratories and Respiratory Division, McGill University, Montreal, QC, Canada H3A 1A1

Edited by Louis J. Ignarro, University of California, School of Medicine, Los Angeles, CA, and approved April 1, 2008 (received for review November 8, 2007) We recently demonstrated early metabolic alterations in the dys- by histology or echocardiography, mdx mouse hearts are abnor- trophin-deficient mdx heart that precede overt cardiomyopathy mally vulnerable to mechanical stress and workload-induced and may represent an early ‘‘subclinical’’ signature of a defective damage (7). More recently, we simultaneously characterized the nitric oxide (NO)/cGMP pathway. In this study, we used genetic and metabolic and functional profile of these dystrophin-deficient pharmacological approaches to test the hypothesis that enhancing hearts using ex vivo perfusion with carbon 13 (13C)-labeled cGMP, downstream of NO formation, improves the contractile substrates. We demonstrated precocious metabolic and func- function, energy metabolism, and sarcolemmal integrity of the tional abnormalities, which preceded the development of overt mdx heart. We first generated mdx mice overexpressing, in a cardiomyopathy (8), and suggested this may represent an early cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC؉/0). ‘‘subclinical’’ signature of defective nitric oxide (NO) and/or When perfused ex vivo in the working mode, 12- and 20-week-old cGMP signaling. hearts maintained their contractile performance, as opposed to the NO mediates a large number of physiological and pathophys- severe deterioration observed in age-matched mdx hearts, which iological effects via its cellular effector cGMP and cGMP- also displayed two to three times more lactate dehydrogenase independent mechanisms (9). In dystrophin-deficient skeletal release than mdx/GC؉/0. At the metabolic level, mdx/GC؉/0 dis- muscle, neuronal NO synthase (nNOS, one of the enzymes played a pattern of substrate selection for energy production that responsible for NO production) is displaced away from its was similar to that of their mdx counterparts, but levels of citric normal subsarcolemmal location to the cytoplasm, where its acid cycle intermediates were significantly higher (36 ؎ 8%), mislocalization and decreased activity level are thought to suggesting improved mitochondrial function. Finally, the ability of contribute to muscle pathology (10–13). In the dystrophic heart, dystrophin-deficient hearts to resist sarcolemmal damage induced a significant down-regulation of nNOS activity has also been in vivo by increasing the cardiac workload acutely with isoproter- reported to occur (14). Conversely, overexpression of an nNOS enol was enhanced by the presence of the transgene and even transgene in the mdx mouse heart was found to mitigate the more so by inhibiting cGMP breakdown using the phosphodies- inflammation, fibrosis, and electrocardiographic abnormalities terase inhibitor sildenafil (44.4 ؎ 1.0% reduction in cardiomyocyte that develop in older mdx mice (15). However, it remains to be damage). Overall, these findings demonstrate that enhancing determined whether these beneficial effects on dystrophic car- cGMP signaling, specifically downstream and independent of NO diomyopathy can be achieved solely through enhancement of formation, in the dystrophin-deficient heart improves contractile cGMP-mediated signaling, without the requirement for in- performance, myocardial metabolic status, and sarcolemmal integ- creased NO production. This is particularly important because rity and thus constitutes a potential clinical avenue for the treat- drugs that inhibit cGMP breakdown, such as the phosphodies- ment of the dystrophin-related cardiomyopathies. terase 5 (PDE5) inhibitor, sildenafil, are already available for clinical use and have been shown to blunt cardiac hypertrophy cardiomyopathy ͉ isotopes ͉ perfusion ͉ energy metabolism ͉ nitric oxide and improve myocardial function in mice subjected to pressure overload (16). ystrophin is a large subsarcolemmal protein, which exists as Accordingly, in this study, we wished to test the hypothesis that Dpart of a multimolecular network (the dystrophin– early activation of the cGMP pathway, specifically downstream glycoprotein complex) that spans the plasma membrane and and independent of NO formation, is able to confer beneficial links the intracellular cytoskeleton to the extracellular matrix (1, effects on the mdx mouse heart. We first generated mdx mice with cardiomyocyte-specific overexpression of a constitutively 2). Abnormalities of dystrophin are the proximate cause of the ϩ skeletal and cardiac disease associated with Duchenne/Becker active form of guanylyl cyclase (mdx/GC /0). In wild-type ani- muscular dystrophies (DMD/BMD) and, of potentially broader mals, the presence of this transgene, which increases cytoplasmic clinical relevance, have recently been linked to several acquired production of cGMP, inhibits isoproterenol- and pressure over- forms of cardiomyopathy and proposed to be a common pathway for contractile dysfunction in the failing myocardium (3–5). Author contributions: M.K., G.D., B.J.P., and C.D.R. designed research; M.K., R.J.K., M.E.Y., Despite enormous progress in identifying the genetic and bio- and M.A.G. performed research; B.G.A., G.D., and C.F.D. contributed new reagents/analytic chemical abnormalities associated with dystrophin deficiency, tools; M.K., M.E.Y., B.G.A., C.F.D., B.J.P., and C.D.R. analyzed data; and M.K., C.F.D., B.J.P., particularly in skeletal muscle, the functional role of dystrophin and C.D.R. wrote the paper. in the heart needs to be better understood for the design of more The authors declare no conflict of interest. effective treatment strategies for DMD and other dystrophin- This article is a PNAS Direct Submission. related cardiomyopathies. ¶To whom correspondence should be addressed. E-mail: christine.des.rosiers@ The most widely used animal model of DMD, the mdx mouse, umontreal.ca. lacks dystrophin due to a spontaneous nonsense mutation within This article contains supporting information online at www.pnas.org/cgi/content/full/ exon 23 of the murine dystrophin gene (6). We have shown that 0710595105/DCSupplemental. even at 8–10 weeks of age, when no cardiac pathology is evident © 2008 by The National Academy of Sciences of the USA

7028–7033 ͉ PNAS ͉ May 13, 2008 ͉ vol. 105 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710595105 Downloaded by guest on September 30, 2021 load-induced hypertrophy (17). Using validated experimental 16 ) 40

ϩ/0 AB* -3 paradigms (7, 8), we tested the ability of the mdx/GC heart to 14 35 * ·10 withstand: (i) ex vivo perfusion in the working mode, with 12 -1 30 * concomitant evaluation of myocardial contractility and meta- 10 25 8 20

bolic profile parameters at different points in the natural evo- RPP lution of the dystrophic cardiomyopathy; and (ii) an acute 6 15 4 10 increase in the cardiac workload induced in vivo by ␤-adrenergic (mmHg)LVEDP 2 5

stimulation. Further to our findings of improved contractility, 0 (mmHg·beats·min 0 energy metabolism, and cellular integrity in mdx/GCϩ/0 hearts, 12 week-old 20 week-old 12 week-old 20 week-old we then treated young (6-week-old) mdx mice with the PDE5 2.5 180 * inhibitor sildenafil and found similar beneficial effects. Taken C D 160 * ** together, our findings point to defective cGMP signaling as being 2.0 140 120 an important component of disease pathogenesis in the dystro- 1.5 phin-deficient heart and suggest the basis for a previously 100 80 undescribed therapeutic approach to prevent or delay the onset 1.0 (mU/mW) 60 of dystrophin-related cardiomyopathies. 0.5 40 Cardiac power (mW) LDH /LDH Cardiac power 20 0.0 0 Results 12 week-old 20 week-old 12 week-old 20 week-old Transgenic Approach: Cardiomyocyte-Specific Overexpression of Con- stitutively Active GC Improves Sarcolemmal Integrity, Myocardial Fig. 1. Functional and physiological parameters of 12- and 20-week-old ϩ/0 Ϯ Contractile Function, and Mitochondrial Metabolism. To test our isolated working mdx and mdx/GC mouse hearts. Data are means SEM of mdx (solid bars) and mdx/GCϩ/0 (open bars) heart perfusion experiments (n ϭ primary hypothesis that early activation of the cGMP pathway 4 mice per group). Values shown represent averages for the 20- to 30-min can confer beneficial effects on the dystrophin-deficient heart, perfusion period. , P Ͻ 0.05; , P Ͻ 0.001 mdx vs. mdx/GCϩ/0 mouse hearts. ϩ/0 * ** we first analyzed mdx/GC mice and their nontransgenic mdx LVEDP, left ventricular end-diastolic pressure; RPP, rate-pressure product. counterparts using our isolated working mouse heart model perfused with a mixture of substrates mimicking the in vivo milieu. We have reported that even at 12 weeks of age, when no showed that the mdx heart displays a metabolic shift toward a significant echocardiographic or histologic abnormalities are preferential use of carbohydrates for energy production and a present, mdx hearts perfused ex vivo in the working mode suffer significant decrease in mitochondrial citric acid cycle (CAC) 13 13 greater cardiomyocyte damage [as indicated by excessive lactate pool size (8). [U- C3]pyruvate and [1- C18]oleate were used to dehydrogenase (LDH) release] and display a reduced ability to evaluate the contribution of carbohydrates to acetyl-CoA pro- sustain normal contractile function, particularly when supplied duction and exogenous fatty acids to ␤-oxidation, respectively. with glucose as the only substrate (7, 8). In this study, compar- As shown in Fig. 2, at 12 weeks of age mdx/GCϩ/0 hearts isons were made between mdx and mdx/GCϩ/0 hearts at two displayed a similar pattern of substrate selection for energy different ages, 12 and 20 weeks, to determine whether differ- production as nontransgenic mdx (Fig. 2A). However, the mdx/ ences related to expression of the transgene become more GCϩ/0 hearts maintained significantly greater levels CAC inter- pronounced as the disease progresses with advancing age. mediates under these conditions (Fig. 2B), indicating partial At 12 weeks of age, the mdx and mdx/GCϩ/0 groups displayed reversal of the previously demonstrated reduction of the CAC similar values for multiple contractile function parameters when pool in mdx hearts (8). It is noteworthy that we were unable to placed in the working heart mode. However, there was a very do the same metabolic analysis at 20 weeks of age, because these clear deterioration of myocardial function observed with ad- older mdx hearts were incapable of sustaining contractile func- vancing age in mdx hearts, whereas the transgenic mdx/GCϩ/0 tion for the time required to assess metabolic fluxes. mice demonstrated a remarkable ability to maintain a stable To ascertain the effects of the transgene on cardiomyocyte level of contractile performance between 12 and 20 weeks. As sarcolemmal integrity in vivo, an acute increase in cardiac indicated in Fig. 1 and supporting information (SI) Table S1, this workload was induced by isoproterenol infusion. We have shown resulted in significant differences between the mdx and mdx/ GCϩ/0 groups for left ventricular end-diastolic pressure, rate- pressure product, cardiac output, and cardiac power at 20 weeks A B 450 400 * of age. Interestingly, the indices of contractility and relaxation 100 (ϩdP/dt and ϪdP/dt) and the rate-pressure product were sig- 350 300 75 nificantly decreased with age in the mdx group but were main- 250 ϩ/0 OLE/CS OLE/CS MEDICAL SCIENCES tained in the mdx/GC group (Table S1 and Fig. 1B). We 50 200 * confirmed that constitutive overexpression of guanylate cyclase (nmol/g) 150 * 100 contribution (%) 25 in the mdx heart was able to significantly up-regulate cGMP substrate Relative [CAC] intermediates [CAC] 50 Ϯ Ϯ PDC/CS PDC/CS * levels (1.9 0.3 vs. 1.1 0.1 fmol/100 mg of heart wet weight, 0 0 * ϩ/0 +/o e e e e mdx mdx/GC te t t t in perfused mdx/GC animals vs. their mdx counterpart, re- ra a tra al i M spectively; P Ͻ 0.05). Furthermore, in both age groups examined, C lutara uma g Succinat F ϩ/0 -Keto the mdx/GC hearts exhibited lower levels of LDH release α (Table S1). In fact, when expressed relative to cardiac power, LDH release tended to decrease with age in mdx/GCϩ/0 hearts Fig. 2. Flux parameters relevant to citrate synthesis and mitochondrial citric (P ϭ 0.09), whereas it increased significantly (P Ͻ 0.05) in acid cycle (CAC) intermediates in isolated working mdx and mdx/GCϩ/0 mouse nontransgenic mdx hearts (Fig. 1D). hearts. Data are means Ϯ SEM of perfusion experiments performed in isolated ϩ/0 ϭ In the aforementioned perfusion studies, we simultaneously working mdx (solid bars) and mdx/GC (open bars) mouse hearts (n 4 per group). Procedures for the determination of (A) the contribution of carbohy- evaluated the metabolic profile of the hearts using substrates 13 drates (lactate, pyruvate, and glucose) and exogenous fatty acids (oleate) to labeled with stable isotopes ( C). This approach allows for acetyl-CoA formation for citrate synthesis (CS) via pyruvate decarboxylation detailed and simultaneous measurements of the dynamics of (PDC) and ␤-oxidation (OLE), respectively referred to as PDC/CS and OLE/CS cardiac energy substrate metabolism, information not accessible and (B) tissue levels of CAC intermediates are described in Materials and ϩ from static measurements of mRNA or protein expression. We Methods. *, P Ͻ 0.05 mdx vs. mdx/GC /0 mouse hearts.

Khairallah et al. PNAS ͉ May 13, 2008 ͉ vol. 105 ͉ no. 19 ͉ 7029 Downloaded by guest on September 30, 2021 700 2.5 AB18 18 AB*** mdx 16 16 mdx/GC+/0 600 *** 2.0 14 14 *** 12 12 500 1.5 * *** 10 10 *** ng of RNA) ng 400 1.0 8 of RNA) ng 8

*** -4 * -1 mRNA levels mRNA mRNA levels mRNA 6 1 6

300 0.5 α 4 4 anf Heart rate (beats/min) sgc (per 10 (per 200 0.0 2 10 (per 2 Cardiomyocyte damage (%) 0 10 20 30 40 50 60 70 80 90 100 0 0 Time (min) Fig. 4. Changes in gene expression levels for atrial natriuretic factor (anf) Fig. 3. Heart rate responses and quantitation of in vivo cardiomyocyte and the ␣ subunit of soluble guanylyl cyclase (sgc␣ ) in placebo vs. sildenafil- sarcolemmal injury induced by an acute increase in cardiac mechanical stress 1 1 treated mdx mice. Data are means Ϯ SEM of eight freeze–clamped placebo after isoproterenol infusion in mdx and mdx/GCϩ/0 mice. Values are means Ϯ (solid bars) and sildenafil-treated (open bars) mdx mouse hearts. mRNA levels SEM of four to six experiments in mdx (solid bars) or mdx/GCϩ/0 (open bars) were normalized to total myocardial RNA. Statistical analyses were performed mouse hearts. Heart rate was continuously monitored by a single-lead elec- on raw data. , P Ͻ 0.05 for placebo vs. sildenafil-treated mdx mouse hearts. trocardiogram (A), and cardiomyocyte sarcolemmal injury was evaluated by * ϩ using the Evans blue vital dye (B). ***, P Ͻ 0.001 for mdx vs. mdx/GC /0 mouse hearts. with this previous pharmacokinetic study, sildenafil administration to mdx mice induced an increase in cGMP levels in the heart Ϯ Ϯ that hearts from 8- to 10-week-old mdx mice exhibit an abnor- at 45 min (3.2 0.6 vs. 1.7 0.4 fmol/100 mg of heart wet weight, mally increased susceptibility to sarcolemmal damage upon in sildenafil- vs. placebo-treated mdx animals, respectively, P ϭ exposure to this form of acute cardiac stress (7). Although 0.09), with a subsequent return to baseline values by 90 min isoproterenol infusion led to a transient and equivalent increase posttreatment (1.9 Ϯ 0.8 vs. 2.1 Ϯ 0.5 fmol/100 mg of heart wet in cardiac frequency (peaking at Ϸ2.5 times the baseline values) weight, not significant). in both nontransgenic mdx and mdx/GCϩ/0 groups, mdx/GCϩ/0 We then assessed whether sildenafil could improve the resis- hearts were able to maintain this elevated heart rate over a tance of mdx hearts to cardiomyocyte damage induced by significantly longer period (Fig. 3A). Moreover, the level of isoproterenol administration in vivo. As was the case in mdx/ ϩ cardiomyocyte sarcolemmal injury (as determined by Evans blue GC /0 hearts, sildenafil-treated hearts were able to sustain their dye uptake) was decreased compared to nontransgenic mdx mice elevated heart rate response for a significantly longer period (see Fig. 3B), although this difference did not reach statistical (Fig. 5A), while showing at the same time a significantly lower significance (P ϭ 0.1). level of cardiomyocyte sarcolemmal injury (44.4 Ϯ 1.0% decrease; Fig. 5B). Finally, it should be noted that the protective Pharmacological Approach: Sildenafil Treatment of Dystrophin-Deficient effects reported in this study cannot be attributed to increased Mice Reduces Early Signs of Cardiomyopathic Remodeling and Protects expression of the dystrophin homologue utrophin, because nei- Against Workload-Induced Cardiomyocyte Injury. To evaluate the ther immunohistochemistry nor Western blot analysis revealed therapeutic potential of a pharmacological approach, we treated any evidence of utrophin up-regulation within mdx/GCϩ/0 or mdx mice with the PDE5 inhibitor, sildenafil, which is known to sildenafil-treated hearts (data not shown). enhance cGMP signaling by inhibiting its breakdown (16). The treatment was given daily from 6 to 12 weeks of age. Although Discussion mdx/GCϩ/0 did not differ significantly from nontransgenic mdx In this study, we have used complementary genetic and phar- in their myocardial contractile performance at 12 weeks of age macological approaches to demonstrate that enhancing cGMP when studied ex vivo, we wished to exclude any possible super- signaling in dystrophin-deficient hearts: (i) protects cardiomyo- imposed effects of sildenafil on cardiac function related to its cytes against mechanical workload-induced sarcolemmal dam- ability to act as a peripheral vasodilator in vivo. Therefore, the age; (ii) preserves mitochondrial metabolic status; and, most mice were anesthetized and examined in vivo by using echocar- importantly, (iii) prevents the deterioration of myocardial con- diography and direct intravascular pressure monitoring. None of tractile performance usually observed with advancing age in mdx the echocardiographic or other hemodynamic parameters, in- hearts. Our findings are consistent with a previous study by cluding blood pressure, were altered by sildenafil treatment Wehling-Henricks et al. (15), which showed beneficial effects of (Table S2). Despite the lack of changes in myocardial contrac- overexpressing an nNOS transgene in the mdx heart. However, tility in vivo at 12 weeks of age, we have reported that the atrial the latter study did not establish whether the salutary effects of natriuretic factor (anf) gene expression, a precocious marker of nNOS overexpression were mediated by NO-induced activation cardiomyopathic remodeling, is significantly increased in the mdx heart even at this early point in the disease process (8). In this study, we found that, compared with the placebo group, mdx 700 3.0 ABPlacebo mice treated with sildenafil had significantly reduced cardiac 600 Sildenafil 2.5 mRNA levels of anf (Fig. 4A), which suggests that the drug # 2.0 prevents the development of cardiomyopathic remodeling at 500 ** ** * 1.5 early stages of the disease. In addition, a significant down- 400 * 1.0 regulation of the ␣1 subunit of soluble guanylyl cyclase (sgc␣1) 300 0.5

gene expression was also observed in sildenafil- as compared (beats/min) rate Heart 200 0.0 Cardiomyocyte damage (%) with placebo-treated mdx hearts (Fig. 4B), a finding consistent 0 10 20 30 40 50 60 70 80 90 100 with enhanced cGMP-mediated signaling in the group receiving Time (min) the active compound (18). The latter notion was further supported by measurements of myocardial cGMP levels. In fact, Fig. 5. Heart rate responses and in vivo cardiomyocyte sarcolemmal injury induced by an acute increase in cardiac mechanical stress in placebo vs. because previous studies have shown that plasma cGMP levels in sildenafil-treated mdx mice. Values are means Ϯ SEM of four to six experiment mice reach their peak within1haftersildenafil administration in placebo (solid bars) or sildenafil-treated (open bars) mdx mouse hearts, as by the oral route (19), we measured cGMP levels in mdx hearts described in Fig. 3. *, P Ͻ 0.05; **, P Ͻ 0.01, #, P Ͻ 0.001 for placebo vs. at 45 and 90 min after i.p. administration of sildenafil. In keeping sildenafil-treated mdx mouse hearts.

7030 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710595105 Khairallah et al. Downloaded by guest on September 30, 2021 of soluble GC or by cGMP-independent mechanisms. Here, Interestingly, a down-regulation of mitochondrial gene expres- we show that enhancing cGMP signaling per se protects the sion has been reported in dystrophic skeletal muscles (22). dystrophin-deficient heart. Moreover, such cardioprotection Likewise, PGC-1␣-null mice, which display defects of mitochon- does not depend on peripheral systemic effects (as could occur drial metabolism (23), have a reduced ability to tolerate with sildenafil) but is also achieved when cGMP is up-regulated ␤-adrenergic stimulation, similar to mdx mice (7). Therefore, it in a cardiomyocyte-specific manner. Therefore, our data suggest appears conceivable that the mitigation of the dystrophic car- that defective cGMP signaling within cardiac muscle cells may diomyopathy observed in mdx/GCϩ/0 mice is mediated at least in play a fundamental role in the pathogenesis of the cardiomyop- part by improved mitochondrial function. Such a mechanism athy associated with dystrophin deficiency. In addition, our study would appear to reconcile many findings. Indeed, it has been raises the possibility that a new therapeutic approach based on reported that augmented cGMP signaling prevents mitochon- cGMP up-regulation with PDE5 inhibitors could be an effective dria-mediated cell death through reduced opening of the mito- treatment for this condition. chondrial permeability transition pore (24). The opening of this In both skeletal and cardiac muscle, it has been proposed that pore was recently shown to be associated with dysregulated dystrophin participates in signaling functions and acts to mechan- ␤-adrenergic receptor signaling and Ca2ϩ handling (25). In this ically reinforce the sarcolemma, thereby helping to protect muscle regard, it has been reported that intracellular Ca2ϩis elevated in cells against workload-induced membrane damage (7, 20). Consis- the dystrophin-deficient heart at all ages (26), whereas activation tent with this notion, it was reported by Danialou et al. (7) that mdx of the cGMP pathway has been shown to lower intracellular Ca2ϩ hearts are more vulnerable to sarcolemmal damage induced by concentration through activation of cGMP-dependent protein aortic banding or ␤-adrenergic stimulation, both of which substan- kinase (PKG) (27). tially augment the mechanical workload placed on the heart. Using Although the genetic and pharmacological approaches used to a similar experimental paradigm, we report in this study that enhance cGMP signaling in the mdx heart resulted in similar cardiomyocyte-specific overexpression of GC and sildenafil treat- protective effects, there are potential differences in their mech- ment in mdx mice both decreased the level of cardiomyocyte injury anisms of action that deserve some comment. PDE5 inhibition and simultaneously improved the ability of mdx mice to sustain an has been shown to attenuate increases in pulmonary arterial increased heart rate during ␤-adrenergic stimulation in vivo.Itis pressures and pulmonary vascular resistance (28–31), which noteworthy that such differences were observed at the young age of could alter the cardiac workload. Other data suggest that 12 weeks, i.e., at a time where there are no echocardiographic signs sildenafil improves endothelial dysfunction, dilates coronary of cardiomyopathy. At 20 weeks of age, differences in susceptibility arteries, and inhibits platelet activation in patients with heart to cardiomyocyte injury between mdx/GCϩ/0 and nontransgenic failure (32, 33). Sildenafil also significantly reduces exercise- mdx hearts became even more evident in the working heart induced ischemia (33), a potentially important pathogenetic perfused ex vivo, a procedure that may itself be considered a mild mechanism in dystrophin-deficient skeletal muscle (10, 34). In form of ‘‘cardiac stress.’’ In fact, mdx/GCϩ/0 hearts released signif- fact, defects in cGMP signaling are also suggested from studies icantly less LDH despite maintaining a substantially greater work- in dystrophin-deficient skeletal muscle (35, 36), and recently, load, as reflected by an Ϸ3-fold higher value for cardiac power. Asai et al. (37) showed that treatment with tadalafil, another Even more striking, enhancing cGMP signaling in cardiomyocytes known PDE5 inhibitor, ameliorates contraction-induced skeletal was able to mitigate cardiac disease progression, as evidenced by the muscle damage. Finally, sildenafil can cross the blood–brain preservation of myocardial contractility between 12 and 20 weeks barrier to inhibit brain PDE5A (38), which could theoretically of age in mdx/GCϩ/0 hearts, in marked contrast to the substantial affect sympathetic and parasympathetic outflow. Therefore, loss of function observed in mdx hearts during the same time frame. although the resting echocardiographic and hemodynamic pa- We have shown that the ex vivo perfusion environment reveals rameters obtained in sildenafil-treated mice did not reveal any cardiomyopathic changes in mdx hearts, which are otherwise significant differences compared with placebo-treated animals, masked by homeostatic compensatory mechanisms present we cannot exclude the possibility that neurohumoral systemic within the intact animal. Using this approach combined with effects of PDE5 inhibition had some influence on mdx hearts. stable isotope methodology, we documented a metabolic shift in With respect to cardiomyocyte-specific effects of cGMP up- substrate utilization from fatty acid to carbohydrate oxidation regulation, there may also be significant differences between the in mdx compared with dystrophin-expressing control hearts (8). transgenic mdx/GCϩ/0 and sildenafil models. Indeed, we have In the present study, mdx/GCϩ/0 mice displayed improved myo- shown that constitutive activation of guanylyl cyclase in our mdx/ cardial function and resistance of cardiomyocytes to workload- GCϩ/0 model chronically increases tissue cGMP levels. This is in induced injury compared with mdx mice. However, both mdx and agreement with our previous characterization of GCϩ/0 animals, in mdx/GCϩ/0 hearts demonstrated a similar substrate selection for which the expression of the transgene was accompanied by in- energy production, which suggests that the metabolic shift creased guanylate cyclase activity and cGMP concentration in

reported in mdx hearts may be an adaptive response rather than isolated cardiomyocytes compared with nontransgenic littermates MEDICAL SCIENCES a deleterious consequence of cardiac dysfunction. Potential (17). However, sildenafil treatments transiently increase cGMP benefits associated with this change in substrate utilization could tissue concentration, which peaks at Ϸ45 min after drug adminis- include: (i) a greater ATP-to-oxygen ratio associated with tration and returns to control levels within 90 min. Kukreja and glucose oxidation; (ii) a more efficient matching between cyto- coworkers have shown that, despite a short half-life in plasma, a solic glycolysis and mitochondrial oxidation, thereby decreasing single administration of sildenafil induces cardioprotective effects the production of protons, a factor that can promote detrimental up to 24 h, suggesting it is not essential to sustain high plasma levels calcium overload; and (iii) improved ion pump function linked of sildenafil to induce the protective effects (31, 39, 40). Similarly, to glycolytic flux (for review, see ref. 21). in this study, the enhanced protection of mdx cardiomyocytes However, beyond the shift in substrate selection, it is impor- against mechanical workload-induced sarcolemmal damage was tant to note that the CAC pool size was significantly increased documented 20 h after the last sildenafil injection. In fact, our gene ϩ/0 in perfused mdx/GC as compared with mdx hearts, suggesting expression data, namely the decreased expression of anf and sgc␣1 an improved mitochondrial metabolic status in the former genes after sildenafil administration, do suggest long-term cellular group. Such a change would be expected to enable the mdx heart changes associated with cGMP signaling. to enhance CAC flux and hence mitochondrial energy produc- Another potential difference between the transgenic mdx/ tion, especially under conditions of high energy demand such as GCϩ/0 and sildenafil models relates to the compartmentation of the increased workload induced by isoproterenol infusion in vivo. cGMP signaling within cardiomyocytes. Sildenafil inhibits

Khairallah et al. PNAS ͉ May 13, 2008 ͉ vol. 105 ͉ no. 19 ͉ 7031 Downloaded by guest on September 30, 2021 PDE5, which is specific for cGMP and exerts a specific spatio- metabolic studies and (ii) optimize perfusion conditions for the more temporal control on the pool of intracellular cGMP synthesized advanced cardiomyopathy found in 20-week-old mdx mice. In each perfu- 13 by soluble GC but not particulate GC (41). In our transgenic sion experiment, [U- C3]pyruvate [initial molar percentage enrichment ϩ/0 (MPE), 99%] was used to assess the contribution of carbohydrates to energy mdx/GC model, the transgene codes for the GC catalytic 13 production and [1- C18]oleate (initial MPE, 100%) for that of fatty acids fragment of the natriuretic peptide receptor A, a soluble cyto- (n ϭ 4 per group). Definitions of the 13C terminology and detailed descrip- plasmic protein that has constitutive GC activity (17). The exact tions for: (i) measurements of the 13C enrichment and concentrations of subcellular localization of this engineered protein remains to be CAC intermediates by gas chromatography coupled with mass spectrome- determined, but differences in the spatiotemporal distribution or try and (ii) calculations of flux ratios relevant to substrate selection for kinetics of cGMP turnover within cardiac muscle cells may be citrate synthesis have been published (42). responsible for the slight differences in the effects of the genetic and pharmacological approaches. In Vivo Manipulation of Cardiac Workload and Quantification of Cardiomyocyte In conclusion, this study demonstrates that enhanced cGMP Sarcolemmal Damage. Our previously described protocol was used to acutely signaling improves contractile performance, sarcolemmal integrity, increase cardiac workload in vivo and induce cardiomyocyte sarcolemmal injury (7) in mdx (n ϭ 7), mdx/GCϩ/0 (n ϭ 5), placebo (n ϭ 5), and sildenafil- (n ϭ and mitochondrial metabolic status in dystrophin-deficient hearts. 7) treated mdx mice. This protocol, which involves isoproterenol infusion (0.1 These findings are compatible with the notion that defects in the mg/kg) and bolus injection of Evans blue (5 ␮g/␮l solution in saline; 5 ␮l/g body cGMP signaling pathway play a crucial role in the pathogenesis of weight over 1 min) into the jugular vein, is sufficiently sensitive for detecting the cardiomyopathy associated with dystrophin deficiency. Further- therapeutic effects (44, 45). more, our data indicate that evolution of the dystrophic cardiomyopathy can be modified independently of the pathology progressing Echocardiography and Cardiac Hemodynamic Measurements. After 6 weeks of in other muscle groups, because cGMP generated in a cardiomyo- daily treatment, the placebo (n ϭ 7) and sildenafil- (n ϭ 10) treated mdx cyte-restricted manner protects the dystrophin-deficient heart mice were anesthetized with the aforementioned ketamine/xylazine mix- against stress-induced injury and from a progressive age-dependent ture. Cardiac hemodynamics were measured by using a 1.4-F Millar micro- manometer-tipped catheter (Millar Instruments) inserted into the left loss of myocardial function. Because there are currently safe and ventricle via the right carotid and through the aortic valve in a retrograde well tolerated pharmacological means to enhance cGMP signaling, fashion. Data were collected and analyzed using IOX software (Emka such drugs could provide the basis for a new therapeutic strategy Technologies). In a different set of experiments, placebo and sildenafil- based on PDE5 inhibition in the dystrophic heart. Further studies treated mdx mice (n ϭ 8 for each group) were subjected to two- are required to determine whether PDE5 inhibition can delay, dimensional guided M-mode echocardiography after anesthesia with 2.5% prevent, or even reverse the onset of dystrophic cardiomyopathies isoflurane (Forane) in 0.5 liter per min of oxygen. Left ventricular internal in DMD/BMD patients. It also appears warranted to evaluate the diameters were measured during diastole and during systole, along with potential benefit of this treatment for patients suffering from the septal and posterior wall thickness. Fractional shortening (percentage) and left ventricular mass (gram) were calculated from published equations (46). acquired forms of cardiomyopathy in which dystrophin abnormal- Mice were randomly selected for testing, and the echocardiographer was ities have been reported. blinded to the mouse treatment status.

Materials and Methods Analyses of mRNA and Protein Expression. Total RNA was extracted and Reagents and Animals. Sources of the chemical reagents and biological reverse-transcribed by using standard methods, followed by quantitative products used in this study have been reported (7, 8, 17, 42). Genetic real-time PCR as described in ref. 8. Gene expression analyses were carried out up-regulation of the cGMP pathway in mdx mice was achieved by cross- in 10- to 12-week-old hearts freeze-clamped in the afternoon (light phase) to breeding female mdx mice (The Jackson Laboratories), with heterozygous avoid any potential circadian variation. The following genes were examined: male C56Bl/6 mice expressing a catalytic fragment of the constitutively (i) anf, a marker of early cardiac remodeling (47) and (ii) sgc␣1, a marker for activated GC domain of the atrial natriuretic factor receptor, under the activation of the cGMP pathway (18). Transcript levels were normalized to control of the cardiac-specific ␣-myosin heavy chain promoter as described total RNA content. Immunohistochemistry and Western blot analysis were (17). Because the dystrophin locus is on chromosome X, all males resulting used to localize and quantify cardiac utrophin levels according to described from this breeding were dystrophin-deficient (mdx), and 50% were het- standard methods (48). erozygous for the GC transgene (mdx/GCϩ/0), as confirmed by Southern blot analysis and/or PCR (17). To avoid the possible artifactual influence of gene cGMP Measurements. Twenty-five milligrams of powdered frozen mouse heart inactivation by insertion, all transgenic animals used for experiments were tissue (n ϭ 4–6 hearts), homogenized by sonication in 350 ␮l of 0.1 M HCl. heterozygous for the transgene. Treatment with the PDE5 inhibitor, silde- cGMP was measured in acetylated supernatants using a RIA kit (Biomedical nafil (pure substance obtained from Pfizer), was initiated at 6 weeks of age Technologies), with modifications to match protocols described by others in male mdx mice and continued over a 6-week period, with daily i.p. using an ELISA (49). injections (0.7 mg/kg per day). Control animals were injected in the iden- tical manner with equivalent volumes of the drug’s saline vehicle. Treat- Statistical Analysis. Data are expressed as means Ϯ SEM (n ϭ 4–8 experiments). ments were begun before the onset of any known cardiomyopathy in mdx Statistical significance was defined as P Ͻ 0.05 using Student’s unpaired t test mice (43). All animal experiments were approved by the local ethics com- or ANOVA (one- or two-way), followed by a Bonferroni selected-comparison mittee and performed according to the guidelines of the Canadian Council test. on Animal Care. ACKNOWLEDGMENTS. We thank Johanne Bourdon for assistance in cardiac Functional and Metabolic Phenotyping of Isolated Working Hearts. The proce- tissue analysis, Dr. Linda Yahiaoui for performance of utrophin Western blot dures for in vivo working mouse heart perfusions in the semirecirculating analyses, Sylvie Picard for cGMP measurements, and Dr. Guy Charron for his mode have been described in detail (42). Briefly, working mouse hearts expertise in animal breeding and genotyping. We thank Dr. Jean-Claude were perfused for 30 min with a semirecirculating modified Krebs– Tardif and Francine Poulin for their assistance in echocardiographic measurements. This work was supported by the Canadian Institutes of Health Research Henseleit buffer containing physiological concentrations of substrates (11 ␮ [Grants 9575 (to C.D.R.), 12743 (to B.J.P.), and 74460 (to C.F.D. and C.D.R.)]; the mM glucose, 0.8 nM insulin, 50 M carnitine, 5 nM epinephrine, 1.5 mM Muscular Dystrophy Association (B.J.P.); the Heart and Stroke Foundation of lactate, 0.2 mM pyruvate, 0.7 mM oleate bound to 3% albumin, and 0.1 mM Canada (studentship to M.K.); and the National Heart, Lung, and Blood EDTA). Preload and afterload were respectively set at 12.5 and 40 mmHg to: Institute (Grant HL-074259-01, to M.E.Y.). B.J.P. is a National Research Scholar (i) obtain similar cardiac power between mdx and mdx/GCϩ/0 for the of the Fonds de la Recherche en Sante´Que´bec.

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