Deborah M. Fine

Cardiac Expression of a Mini-dystrophin That Normalizes Skeletal Muscle Force Only Partially Restores Heart Function in Aged Mdx Mice

Duchenne muscular dystrophy (DMD) affects both skeletal and cardiac muscle. It is currently unclear whether the strategies developed for skeletal muscle can ameliorate cardiomyopathy. Synthetic mini-/micro-dystrophin genes have yielded impressive skeletal muscle protection in animal models. The 6-kb DeltaH2-R19 minigene is particularly promising because it completely restores skeletal muscle force to wild-type levels. Here, we examined whether expressing this minigene in the heart, but not skeletal muscle, could normalize cardiac function in the mdx model of DMD cardiomyopathy. Transgenic mdx mice were generated to express the DeltaH2-R19 minigene under the control of the alpha-myosin heavy-chain promoter. Heart structure and function were examined in adult and very old mice. The DeltaH2-R19 minigene enhanced cardiomyocyte sarcolemmal strength and prevented myocardial fibrosis. It also restored the dobutamine response and enhanced treadmill performance. Surprisingly, heart-restricted DeltaH2-R19 minigene expression did not completely normalize electrocardiogram and hemodynamic abnormalities. Overall, systolic function and ejection fraction were restored to normal levels but stroke volume and cardiac output remained suboptimal. Our results demonstrate that the skeletal muscle-proven DeltaH2-R19 minigene can correct cardiac histopathology but cannot fully normalize heart function. Novel strategies must be developed to completely restore heart function in DMD.

Evaluation of circulating amino terminal-pro-B-type natriuretic peptide concentration in dogs with respiratory distress attributable to congestive heart failure or primary pulmonary disease

OBJECTIVE To evaluate assessment of circulating amino terminal-pro-B-type natriuretic peptide (NT-proBNP) concentration as a means to discriminate between congestive heart failure and primary pulmonary disease in dogs. DESIGN Prospective case series. ANIMALS 46 dogs with signs of respiratory distress or coughing. PROCEDURES All dogs underwent physical and thoracic radiographic examinations. Dogs with evidence of heart disease (eg, murmur, arrhythmia, or large cardiac silhouette detected by radiography) also underwent echocardiography. Dogs with no evidence of heart disease or failure were included if they underwent bronchoalveolar lavage (with cytologic examination and bacterial culture of the lavage fluid). Blood samples for NT-proBNP assay were obtained within 12 hours of the diagnosis of heart failure or prior to bronchoalveolar lavage in dogs with primary pulmonary disease. Circulating concentrations of NT-proBNP were compared between groups and correlated with radiographic and echocardiographic measures of cardiac size. RESULTS Congestive heart failure and primary pulmonary disease were diagnosed in 25 and 21 dogs, respectively. Dogs with congestive heart failure had significantly higher median serum or plasma NT-proBNP concentration (2,554 pmol/L; interquartile [25% to 75%] range, 1,651.5 to 3,475.5 pmol/L) than dogs with primary pulmonary disease (357 pmol/L; interquartile range, 192.5 to 565.5 pmol/L). Radiographic vertebral heart score and echocardiographic left atrial-to-aortic diameter ratio were not correlated with NT-proBNP concentration. Left ventricular end-diastolic diameter (measured echocardiographically) and NT-proBNP concentration were weakly correlated. CONCLUSIONS AND CLINICAL RELEVANCE Serum or plasma NT-proBNP concentration assessment may be useful for discrimination of congestive heart failure from primary pulmonary disease in dogs with respiratory distress or cough.

Evaluation of Pimobendan and N‐Terminal Probrain Natriuretic Peptide in the Treatment of Pulmonary Hypertension Secondary to Degenerative Mitral Valve Disease in Dogs

BACKGROUND Pimobendan is a positive inotrope and vasodilator that may be useful in the treatment of pulmonary hypertension (PHT) secondary to degenerative mitral valve disease. HYPOTHESIS Pimobendan decreases the severity of PHT measured echocardiographically and improves quality-of-life scores. Changes in N-terminal probrain natriuretic peptide (NT-proBNP) concentrations will reflect improvement in severity of PHT. ANIMALS Ten client-owned dogs with peak tricuspid regurgitant flow velocity (TRFV) > or =3.5 m/s. METHODS Prospective short-term, double-blinded, crossover design, with a long-term, open-label component. Short term, dogs were randomly allocated to receive either placebo or pimobendan (0.18-0.3 mg/kg PO q12 h) for 14 days. After a 1-week washout, they received the alternative treatment for 14 days, followed by pimobendan open-label for 8 weeks. RESULTS Short-term comparison: peak TRFV decreased in all dogs on pimobendan compared with placebo from a median of 4.40 (range, 3.2-5.6) to 3.75 (range, 2.4-4.8) m/s (P < .0001). NT-proBNP concentration decreased after treatment with pimobendan from a median of 2,143 (range, 450-3,981) to 1,329 (range, 123-2,411) pmol/L (P= .0009). All dogs improved their quality-of-life score (P= .006). In the long-term comparisons, peak TRFV decreased in all dogs from a median of 4.28 (range, 3.5-5.7) to 3.52 (range, 2.4-5.0) m/s (P < .0001). No significant changes in NT-proBNP or quality-of-life scores were detected. CONCLUSIONS AND CLINICAL IMPORTANCE Pimobendan lowered severity of measurable PHT, improved quality-of-life scores, and decreased NT-proBNP concentrations short-term. Long term, only the reduction in TRFV was maintained.

The PTPN11 loss-of-function mutation Q510E-Shp2 causes hypertrophic cardiomyopathy by dysregulating mTOR signaling

The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to induce the phenotype. Furthermore, the pathomechanism was triggered pre- but not postnatally. However, postnatal rapamycin treatment could still reverse already established HCM, which may have important therapeutic implications.

Age-matched comparison reveals early electrocardiography and echocardiography changes in dystrophin-deficient dogs

The absence of dystrophin in the heart leads to Duchenne cardiomyopathy. Dystrophin-deficient dogs represent a critical model to translate novel therapies developed in mice to humans. Unfortunately, little is known about cardiophysiology changes in these dogs. We performed prospective electrocardiographic and echocardiographic examinations at 3, 6 and 12 months of age in four normal and three affected dogs obtained from the same litter. Affected dogs showed growth retardation and serum creatine kinase elevation. Necropsy confirmed cardiac dystrophin deficiency and histopathology. Q/R ratio elevation and diastolic left ventricular (LV) internal diameter reduction were the most consistent findings in affected dogs at all ages. At 6 and 12 months, dystrophic dogs also showed significant reduction of PR intervals, LV end diastolic/systolic volumes and systolic LV internal diameters. Epicardial and endocardial slope times were significantly reduced in affected dogs at 12 months. These results establish the baseline for evaluating experimental therapies in the future.

TEAD-1 Overexpression in the Mouse Heart Promotes an Age-dependent Heart Dysfunction

TEA domain transcription factor-1 (TEAD-1) is essential for proper heart development and is implicated in cardiac specific gene expression and the hypertrophic response of primary cardiomyocytes to hormonal and mechanical stimuli, and its activity increases in the pressure-overloaded hypertrophied rat heart. To investigate whether TEAD-1 is an in vivo modulator of cardiac specific gene expression and hypertrophy, we developed transgenic mice expressing hemagglutinin-tagged TEAD-1 under the control of the muscle creatine kinase promoter. We show that a sustained increase in TEAD-1 protein leads to an age-dependent dysfunction. Magnetic resonance imaging revealed decreases in cardiac output, stroke volume, ejection fraction, and fractional shortening. Isolated TEAD-1 hearts revealed decreased left ventricular power output that correlated with increased βMyHC protein. Histological analysis showed altered alignment of cardiomyocytes, septal wall thickening, and fibrosis, although electrocardiography displayed a left axis shift of mean electrical axis. Transcripts representing most members of the fetal heart gene program remained elevated from fetal to adult life. Western blot analyses revealed decreases in p-phospholamban, SERCA2a, p-CX43, p-GSK-3α/β, nuclear β-catenin, GATA4, NFATc3/c4, and increased NCX1, nuclear DYKR1A, and Purα/β protein. TEAD-1 mice did not display cardiac hypertrophy. TEAD-1 mice do not tolerate stress as they die over a 4-day period after surgical induction of pressure overload. These data provide the first in vivo evidence that increased TEAD-1 can induce characteristics of cardiac remodeling associated with cardiomyopathy and heart failure.

Supravalvular mitral stenosis in a cat.

A 3-year-old, 4-kg, castrated male domestic shorthair cat presented with signs of progressive respiratory distress. Thoracic radiographs showed pulmonary edema and pleural effusion. Echocardiography revealed a perforate membrane immediately above the mitral valve that divided the left atrium into proximal and distal chambers. The left auricle was proximal to the dividing membrane and connected to the markedly enlarged proximal left atrial chamber, consistent with the diagnosis of supravalvular mitral stenosis (SMS). Position of the obstructing membrane relative to the left auricle distinguishes SMS from cor triatriatum sinister (CTS). In CTS, the left auricle is distal to the dividing membrane and connects to the distal left atrial chamber.

Influence of Beta Blockers on Survival in Dogs with Severe Subaortic Stenosis

Background Subaortic stenosis (SAS) is one of the most common congenital cardiac defects in dogs. Severe SAS frequently is treated with a beta adrenergic receptor blocker (beta blocker), but this approach largely is empirical. Objective To determine the influence of beta blocker treatment on survival time in dogs with severe SAS. Methods Retrospective review of medical records of dogs diagnosed with severe, uncomplicated SAS (pressure gradient [PG] ≥80 mmHg) between 1999 and 2011. Results Fifty dogs met the inclusion criteria. Twenty‐seven dogs were treated with a beta blocker and 23 received no treatment. Median age at diagnosis was significantly greater in the untreated group (1.2 versus 0.6 years, respectively; P = .03). Median PG at diagnosis did not differ between the treated and untreated groups (127 versus 121 mmHg, respectively; P = .2). Cox proportional hazards regression was used to identify the influence of PG at diagnosis, age at diagnosis, and beta blocker treatment on survival. In the all‐cause multivariate mortality analysis, only age at diagnosis (P = .02) and PG at diagnosis (P = .03) affected survival time. In the cardiac mortality analysis, only PG influenced survival time (P = .03). Treatment with a beta blocker did not influence survival time in either the all‐cause (P = .93) or cardiac‐cause (P = .97) mortality analyses. Conclusions Beta blocker treatment did not influence survival in dogs with severe SAS in our study, and a higher PG at diagnosis was associated with increased risk of death.

Echocardiographic assessment of hemodynamic changes produced by two methods of inducing fluid deficit in dogs.

BACKGROUND Hydration status is important to the cardiovascular system because of its effects on preload. Decreased preload can alter echocardiographic measurements of systolic and diastolic function, potentially confounding interpretation of results. HYPOTHESIS/OBJECTIVES Mild fluid deficits are associated with measurable echocardiographic changes that are validated by physical and biochemical markers of decreased intravascular volume. ANIMALS Twenty-five healthy staff/student-owned dogs with no evidence of cardiac or renal disease. METHODS Prospective, interventional laboratory study. Dogs were randomly assigned to water deprivation (WD) alone for 8 hours (n = 13) or to furosemide treatment (FTx, 2.5mg/kg IV) followed by WD for 8 hours (n = 12). Echocardiograms, biochemical sampling, and physical parameters were measured at baseline, and after 4 and 8 hours. RESULTS Both protocols induced fluid deficit as indicated by significant (P < .00001) decreases in weight at 4 hours (WD, 1.1%; FTx, 3.7%) and 8 hours (WD, 2.7%; FTx, 4.5%). Furosemide significantly decreased left ventricular end-diastolic volume (54.3 +/- 19.3-42.1 +/- 17.3 mL, P < .0001), cardiac index (4.2 +/- 1.1-2.9 +/- 0.9 L/min/M2, P < .0001), and mitral valve E wave velocity (0.79 +/- 0.2-0.66 +/- 0.2 m/s, P = .0004). These changes were accompanied by significant increases in blood urea nitrogen concentration (13.8 +/- 2.6-14.8 +/- 2.7 mg/dL, P = .04), vasopressin concentration (1.4 +/- 1.2-3.3 +/- 1.9 pg/mL, P = .045), and PCV (49.8 +/- 4.5-53.2 +/- 6.5%, P = .006). Effects of water deprivation alone were similar, but less pronounced. CONCLUSIONS AND CLINICAL IMPORTANCE Mild fluid deficits have measurable hemodynamic effects in dogs. Hydration status should be considered when evaluating cardiac function by echocardiogram.

Duchenne Cardiomyopathy Gene Therapy

Duchenne cardiomyopathy is a heart disease resulting from the loss of cardiac dystrophin. It significantly reduces the life quality and shortens lifespan in Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy patients and carriers. Gene replacement therapy with adeno-associated viral vector (AAV) and gene repair therapy with exon skipping hold great promise for restoring dystrophin expression and ameliorating cardiomyopathy. The last few years have witnessed tremendous advances towards Duchenne cardiomyopathy gene therapy. The infrastructure (animal models and functional assays) is now available for comprehensive preclinical studies. Essential parameters, such as the therapeutic threshold, have also been defined. Together with the recent developments in novel AAV vectors and modified antisense oligonucleotides, clinical application of Duchenne cardiomyopathy gene therapy may become a reality in the near future.