Farid Moussavi-Harami

Contractile properties of developing human fetal cardiac muscle

The contractile properties of human fetal cardiac muscle have not been previously studied. Small‐scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well‐controlled conditions. We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59–134 days. Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression. Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell‐derived cardiomyocytes.

2-Deoxy adenosine triphosphate improves contraction in human end-stage heart failure

We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca(2+) concentrations with dATP compared to ATP. This resulted in an increase in the Ca(2+) sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (ktr) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest that dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development were increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models, show the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure.

Transcription factor CHF1/Hey2 regulates EC coupling and heart failure in mice through regulation of FKBP12.6.

Heart failure is a leading cause of morbidity and mortality in Western society. The cardiovascular transcription factor CHF1/Hey2 has been linked to experimental heart failure in mice, but the mechanisms by which it regulates myocardial function remain incompletely understood. The objective of this study was to determine how CHF1/Hey2 affects development of heart failure through examination of contractility in a myocardial knockout mouse model. We generated myocardial-specific knockout mice. At baseline, cardiac function was normal, but, after aortic banding, the conditional knockout mice demonstrated a greater increase in ventricular weight-to-body weight ratio compared with control mice (5.526 vs. 4.664 mg/g) and a significantly decreased ejection fraction (47.8 vs. 72.0% control). Isolated cardiac myocytes from these mice showed decreased calcium transients and fractional shortening after electrical stimulation. To determine the molecular basis for these alterations in excitation-contraction coupling, we first measured total sarcoplasmic reticulum calcium stores and calcium-dependent force generation in isolated muscle fibers, which were normal, suggesting a defect in calcium cycling. Analysis of gene expression demonstrated normal expression of most genes known to be involved in myocardial calcium cycling, with the exception of the ryanodine receptor binding protein FKBP12.6, which was expressed at increased levels in the conditional knockout hearts. Treatment of the isolated knockout myocytes with FK506, which inhibits the association of FKBP12.6 with the ryanodine receptor, restored contractile function. These findings demonstrate that conditional deletion of CHF1/Hey2 in the myocardium leads to abnormalities in calcium handling mediated by FKBP12.6 that predispose to pressure overload-induced heart failure.

AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility

Impaired systolic function, resulting from acute injury or congenital defects, leads to cardiac complications and heart failure. Current therapies slow disease progression but do not rescue cardiac function. We previously reported that elevating the cellular 2 deoxy-ATP (dATP) pool in transgenic mice via increased expression of ribonucleotide reductase (RNR), the enzyme that catalyzes deoxy-nucleotide production, increases myosin-actin interaction and enhances cardiac muscle contractility. For the current studies, we initially injected wild-type mice retro-orbitally with a mixture of adeno-associated virus serotype-6 (rAAV6) containing a miniaturized cardiac-specific regulatory cassette (cTnT(455)) composed of enhancer and promotor portions of the human cardiac troponin T gene (TNNT2) ligated to rat cDNAs encoding either the Rrm1 or Rrm2 subunit. Subsequent studies optimized the system by creating a tandem human RRM1-RRM2 cDNA with a P2A self-cleaving peptide site between the subunits. Both rat and human Rrm1/Rrm2 cDNAs resulted in RNR enzyme overexpression exclusively in the heart and led to a significant elevation of left ventricular (LV) function in normal mice and infarcted rats, measured by echocardiography or isolated heart perfusions, without adverse cardiac remodeling. Our study suggests that increasing RNR levels via rAAV-mediated cardiac-specific expression provide a novel gene therapy approach to potentially enhance cardiac systolic function in animal models and patients with heart failure.

An optimized and simplified system of mouse embryonic stem cell cardiac differentiation for the assessment of differentiation modifiers.

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation.

Fast and sensitive HPLC–MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells

Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC-MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10min. Calibration curves of all dNTPs ranged from 62.5 to 2500fmol injections and demonstrated excellent linearity (r2>0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was <25% for all points, including the lower limit of quantification (LLOQ). The inter-day accuracy was within 12% of expected concentration for the LLOQ and within 7% for all other points on the calibration curve. The intra-day accuracy was within 22% for the LLOQ and within 11% for all points on the curve. Compared to existing methods, this study presents a faster and more sensitive method for dNTP quantification.

Heart failure with preserved ejection fraction and skeletal muscle physiology

Heart failure with preserved ejection fraction (HFpEF) accounts for half of all heart failure in the USA, increases in prevalence with aging, and has no effective therapies. Intriguingly, the pathophysiology of HFpEF has many commonalities with the aged cardiovascular system including reductions in diastolic compliance, chronotropic defects, increased resistance in the peripheral vasculature, and poor energy substrate utilization. Decreased exercise capacity is a cardinal symptom of HFpEF. However, its severity is often out of proportion to changes in cardiac output. This observation has led to studies of muscle function in HFpEF revealing structural, biomechanical, and metabolic changes. These data, while incomplete, support a hypothesis that similar to aging, HFPEF is a systemic process. Understanding the mechanisms leading to exercise intolerance in this condition may lead to strategies to improve morbidity in both HFpEF and aging.

Regulation of MMP10 Expression by the Transcription Factor CHF1/Hey2 is Mediated by Multiple E Boxes

The cardiovascular restricted bHLH transcription factor CHF1/Hey2 has been reported to play an important role in regulation of vascular smooth muscle phenotype and gene expression, but the downstream target genes that mediate these effects have not been completely elucidated. We have previously found that loss of CHF1/Hey2 in vascular smooth muscle cells leads to dysregulated expression of the matrix metalloproteinase gene MMP10 after treatment with PDGF. Here we report that loss or knockdown of CHF1/Hey2 in vascular smooth muscle cells leads to increased expression and activity of MMP10 at baseline, suggesting a direct effect of CHF1/Hey2 on MMP10 promoter regulation. To test this hypothesis, we assessed the effects of CHF1/Hey2 on a 2.5 kb MMP10 promoter region upstream of the transcriptional start site. We found that this region contains multiple elements including 12 E-boxes that mediate constitutive activity and repression by CHF1/Hey2 in 293T cells and A7r5 smooth muscle cells. Surprisingly, mutation of these E-boxes not only abolished CHF1/Hey2 repression, but also diminished constitutive expression. In addition, we observed that some of these mutations unmasked an activator function for CHF1/Hey2, which has not been previously described. These findings support the hypothesis that CHF1/Hey2 is an important regulator of MMP10 expression.

Translation of Cardiac Myosin Activation With 2-Deoxy-ATP to Treat Heart Failure Via an Experimental Ribonucleotide Reductase-Based Gene Therapy

Summary Despite recent advances, chronic heart failure remains a significant and growing unmet medical need, reaching epidemic proportions carrying substantial morbidity, mortality, and costs. A safe and convenient therapeutic agent that produces sustained inotropic effects could ameliorate symptoms and improve functional capacity and quality of life. The authors discovered that small amounts of 2-deoxy-ATP (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin cross-bridge cycling with greater force generation during each contraction. They describe the rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy that up-regulates ribonucleotide reductase, the rate-limiting enzyme for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with no toxicity or safety concerns identified to date. Further animal studies are being conducted with the goal of testing this agent in patients with heart failure.

Gene Therapy for Nonischemic Cardiomyopathy: Moving Forward by Learning From Lessons of the Past

H eart failure (HF) is an epidemic affecting more than 6 million Americans, and many more worldwide (1). Pharmacological and device-based therapies have improved outcomes of patients with HF, but these patients still experience significant morbidity and have poor prognosis (1,2). With better understanding of the complex molecular pathways involved in HF, treatment strategies can become more targeted, precise, and successful. Gene therapy, the transfer of recombinant genetic material to increase levels of a specific protein, manipulates pathways that are difficult to target pharmacologically. It can also decrease off-target effects associated with other treatment options by using viral vectors with low immunogenicity, high transduction specificity for striated muscle, and cardiac-specific regulatory cassettes. To date, the targets for gene therapy have included the Ca2þ regulatory proteins (3–5), b-adrenergic system (6), angiogenesis (7), stress response pathways (8), stem cell homing (9), and myosin activation (10,11). Myosin targeting is the most downstream of these approaches, directly increasing the activity of the motor protein that determines systolic pressure development by a small adjustment in the adenosine nucleotide pool of cardiomyocytes (12–14).