Troponin I Tyrosine Phosphorylation Beneficially Accelerates Diastolic Function.

BACKGROUND: A healthy heart is able to modify its function and increase relaxation through post-translational modifications of myofilament proteins. While there are known examples of serine/threonine kinases directly phosphorylating myofilament proteins to modify heart function, the roles of tyrosine (Y) phosphorylation to directly modify heart function have not been demonstrated. The myofilament protein TnI (troponin I) is the inhibitory subunit of the troponin complex and is a key regulator of cardiac contraction and relaxation. We previously demonstrated that TnI-Y26 phosphorylation decreases calcium-sensitive force development and accelerates calcium dissociation, suggesting a novel role for tyrosine kinase-mediated TnI-Y26 phosphorylation to regulate cardiac relaxation. Therefore, we hypothesize that increasing TnI-Y26 phosphorylation will increase cardiac relaxation in vivo and be beneficial during pathological diastolic dysfunction. METHODS: The signaling pathway involved in TnI-Y26 phosphorylation was predicted in silico and validated by tyrosine kinase activation and inhibition in primary adult murine cardiomyocytes. To investigate how TnI-Y26 phosphorylation affects cardiac muscle, structure, and function in vivo, we developed a novel TnI-Y26 phosphorylation-mimetic mouse that was subjected to echocardiography, pressure-volume loop hemodynamics, and myofibril mechanical studies. TnI-Y26 phosphorylation-mimetic mice were further subjected to the nephrectomy/DOCA (deoxycorticosterone acetate) model of diastolic dysfunction to investigate the effects of increased TnI-Y26 phosphorylation in disease. RESULTS: Src tyrosine kinase is sufficient to phosphorylate TnI-Y26 in cardiomyocytes. TnI-Y26 phosphorylation accelerates in vivo relaxation without detrimental structural or systolic impairment. In a mouse model of diastolic dysfunction, TnI-Y26 phosphorylation is beneficial and protects against the development of disease. CONCLUSIONS: We have demonstrated that tyrosine kinase phosphorylation of TnI is a novel mechanism to directly and beneficially accelerate myocardial relaxation in vivo.

The lack of Troponin I Ser-23/24 phosphorylation is detrimental to in vivo cardiac function and exacerbates cardiac disease.

Troponin I (TnI) is a key regulator of cardiac contraction and relaxation with TnI Ser-23/24 phosphorylation serving as a myofilament mechanism to modulate cardiac function. Basal cardiac TnI Ser-23/24 phosphorylation is high such that both increased and decreased TnI phosphorylation may modulate cardiac function. While the effects of increasing TnI Ser-23/24 phosphorylation on heart function are well established, the effects of decreasing TnI Ser-23/24 phosphorylation are not clear. To understand the in vivo role of decreased TnI Ser-23/24 phosphorylation, mice expressing TnI with Ser-23/24 mutated to alanine (TnI S23/24A) that lack the ability to be phosphorylated at these residues were subjected to echocardiography and pressure-volume hemodynamic measurements in the absence or presence of physiological (pacing increasing heart rate or adrenergic stimulation) or pathological (transverse aortic constriction (TAC)) stress. In the absence of pathological stress, the lack of TnI Ser-23/24 phosphorylation impaired systolic and diastolic function. TnI S23/24A mice also had an impaired systolic and diastolic response upon stimulation increased heart rate and an impaired adrenergic response upon dobutamine infusion. Following pathological cardiac stress induced by TAC, TnI S23/24A mice had a greater increase in ventricular mass, worse diastolic function, and impaired systolic and diastolic function upon increasing heart rate. These findings demonstrate that mice lacking the ability to phosphorylate TnI at Ser-23/24 have impaired in vivo systolic and diastolic cardiac function, a blunted cardiac reserve and a worse response to pathological stress supporting decreased TnI Ser23/24 phosphorylation is a modulator of these processes in vivo.

Mouse Model of Spinal Cord Hypoperfusion with Immediate Paralysis Caused by Endovascular Repair of Thoracic Aortic Aneurysm.

BACKGROUND: A clinically relevant mouse model of thoracic endovascular aortic repair-induced ischemic spinal cord injury has been lacking since the procedure was first employed in 1991. The hypothesis was that ligation of mouse intercostal arteries would simulate thoracic endovascular aortic repair-induced ischemic spinal cord injury and behavioral deficit. The aim was to create a mouse model of thoracic endovascular aortic repair-induced spinal cord hypoperfusion by ligating five pairs of mouse intercostal vessels. METHODS: Mice were divided into sham (n = 53) and ligation (n = 60) groups. The procedures called for double ligation of three pairs and single ligation of two pairs of thoracic intercostal arteries in adult C57BL/6 mice. A laser Doppler probe was used in vivo on the spinal cords and intercostal arteries to document the extent of arterial ligation and spinal cord hypoperfusion. The Basso Mouse Scale for Locomotion, histological studies, and electron microscopy demonstrated postligation locomotive and histopathological changes. RESULTS: Ligation induced a significant and instantaneous drop in blood flow in the intercostal arteries (% change; mean = -63.81; 95% CI, -72.28 to -55.34) and the thoracic spinal cord (% change; mean = -68.55; 95% CI, -80.23 to -56.87). Paralysis onset was immediate and of varying degree, with behavioral deficit stratified into three groups: 9.4% exhibited severe paralysis, 37.5% moderate paralysis, and 53.1% mild paralysis at day 1 (n = 32; P < 0.001). Mild and moderate paralysis was transient, gradually improving over time. Severe paralysis showed no improvement and exhibited a higher mortality rate (83%; n = 15 of 18) compared to moderately (33%; n = 6 of 18) and mildly (24%; n = 6 of 25) paralyzed mice (P < 0.001). The overall ligation group survival rate (84%; n = 46 of 55) was significantly lower than the sham group (100%; n = 48 of 48) with P = 0.003. CONCLUSIONS: The mouse model generates reproducible spinal cord hypoperfusion and accompanying histopathological ischemic spinal cord damage. The resulting anatomical changes and variable behavioral deficits mimic the variability in radiological and clinical findings in human patients.

A Novel Endocrine Role for the BAT-Released Lipokine 12,13-diHOME to Mediate Cardiac Function.

BACKGROUND: Brown adipose tissue (BAT) is an important tissue for thermogenesis, making it a potential target to decrease the risks of obesity, type 2 diabetes, and cardiovascular disease, and recent studies have also identified BAT as an endocrine organ. Although BAT has been implicated to be protective in cardiovascular disease, to this point there are no studies that identify a direct role for BAT to mediate cardiac function. METHODS: To determine the role of BAT on cardiac function, we utilized a model of BAT transplantation. We then performed lipidomics and identified an increase in the lipokine 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME). We utilized a mouse model with sustained overexpression of 12,13-diHOME and investigated the role of 12,13-diHOME in a nitric oxide synthase type 1 deficient (NOS1-/-) mouse and in isolated cardiomyocytes to determine effects on function and respiration. We also investigated 12,13-diHOME in a cohort of human patients with heart disease. RESULTS: Here, we determined that transplantation of BAT (+BAT) improves cardiac function via the release of the lipokine 12,13-diHOME. Sustained overexpression of 12,13-diHOME using tissue nanotransfection negated the deleterious effects of a high-fat diet on cardiac function and remodeling, and acute injection of 12,13-diHOME increased cardiac hemodynamics via direct effects on the cardiomyocyte. Furthermore, incubation of cardiomyocytes with 12,13-diHOME increased mitochondrial respiration. The effects of 12,13-diHOME were absent in NOS1-/- mice and cardiomyocytes. We also provide the first evidence that 12,13-diHOME is decreased in human patients with heart disease. CONCLUSIONS: Our results identify an endocrine role for BAT to enhance cardiac function that is mediated by regulation of calcium cycling via 12,13-diHOME and NOS1.

Brown adipose tissue prevents glucose intolerance and cardiac remodeling in high-fat-fed mice after a mild myocardial infarction.

BACKGROUND: Obesity increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes (T2D) after myocardial infarction (MI). Brown adipose tissue (BAT) is important to combat obesity and T2D, and increasing BAT mass by transplantation improves glucose metabolism and cardiac function. The objective of this study was to determine if BAT had a protective effect on glucose tolerance and cardiac function in high-fat diet (HFD) fed mice subjected to a mild MI. METHODS: Male C57BL/6 mice were fed a HFD for eight weeks and then divided into Sham (Sham-operated) and +BAT (mice receiving 0.1 g BAT into their visceral cavity). Sixteen weeks post-transplantation, mice were further subdivided into ±MI (Sham; Sham-MI; +BAT; +BAT-MI) and maintained on a HFD. Cardiac (echocardiography) and metabolic function (glucose and insulin tolerance tests, body composition and exercise tolerance) were assessed throughout 22 weeks post-MI. Quantitative PCR (qPCR) was performed to determine the expression of genes related to metabolic function of perigonadal adipose tissue (pgWAT), subcutaneous white adipose tissue (scWAT), liver, heart, tibialis anterior skeletal muscle (TA); and BAT. RESULTS: +BAT prevented the increase in left ventricle mass (LVM) and exercise intolerance in response to MI. Similar to what is observed in humans, Sham-MI mice developed IGT post-MI, but this was negated in +BAT-MI mice. IGT was independent of changes in body composition. Genes involved in inflammation, insulin resistance, and metabolism were significantly altered in pgWAT, scWAT, and liver in Sham-MI mice compared to all other groups. CONCLUSIONS: BAT transplantation prevents IGT, the increase in LVM, and exercise intolerance following MI. MI alters the expression of several metabolic-related genes in WAT and liver in Sham-MI mice, suggesting that these tissues may contribute to the impaired metabolic response. Increasing BAT may be an important intervention to prevent the development of IGT or T2D and cardiac remodeling in obese patients post-MI.

Etiology-dependent impairment of relaxation kinetics in right ventricular end-stage failing human myocardium.

BACKGROUND: In patients with end-stage heart failure, the primary etiology often originates in the left ventricle, and eventually the contractile function of the right ventricle (RV) also becomes compromised. RV tissue-level deficits in contractile force and/or kinetics need quantification to understand involvement in ischemic and non-ischemic failing human myocardium. METHODS AND RESULTS: The human population suffering from heart failure is diverse, requiring many subjects to be studied in order to perform an adequately powered statistical analysis. From 2009-present we assessed live tissue-level contractile force and kinetics in isolated myocardial RV trabeculae from 44 non-failing and 41 failing human hearts. At 1 Hz stimulation rate (in vivo resting state) the developed active force was not different in non-failing compared to failing ischemic nor non-ischemic failing trabeculae. In sharp contrast, the kinetics of relaxation were significantly impacted by disease, with 50% relaxation time being significantly shorter in non-failing vs. non-ischemic failing, while the latter was still significantly shorter than ischemic failing. Gender did not significantly impact kinetics. Length-dependent activation was not impacted. Although baseline force was not impacted, contractile reserve was critically blunted. The force-frequency relation was positive in non-failing myocardium, but negative in both ischemic and non-ischemic myocardium, while the ß-adrenergic response to isoproterenol was depressed in both pathologies. CONCLUSIONS: Force development at resting heart rate is not impacted by cardiac pathology, but kinetics are impaired and the magnitude of the impairment depends on the underlying etiology. Focusing on restoration of myocardial kinetics will likely have greater therapeutic potential than targeting force of contraction.

TGF-ß1 affects cell-cell adhesion in the heart in an NCAM1-dependent mechanism.

The contractile property of the myocardium is maintained by cell-cell junctions enabling cardiomyocytes to work as a syncytium. Alterations in cell-cell junctions are observed in heart failure, a disease characterized by the activation of Transforming Growth Factor beta 1 (TGFß1). While TGFß1 has been implicated in diverse biologic responses, its molecular function in controlling cell-cell adhesion in the heart has never been investigated. Cardiac-specific transgenic mice expressing active TGFß1 were generated to model the observed increase in activity in the failing heart. Activation of TGFß1 in the heart was sufficient to drive ventricular dysfunction. To begin to understand the function of this important molecule we undertook an extensive structural analysis of the myocardium by electron microscopy and immunostaining. This approach revealed that TGFß1 alters intercalated disc structures and cell-cell adhesion in ventricular myocytes. Mechanistically, we found that TGFß1 induces the expression of neural adhesion molecule 1 (NCAM1) in cardiomyocytes in a p38-dependent pathway, and that selective targeting of NCAM1 was sufficient to rescue the cell adhesion defect observed when cardiomyocytes were treated with TGFß1. Importantly, NCAM1 was upregulated in human heart samples from ischemic and non-ischemic cardiomyopathy patients and NCAM1 protein levels correlated with the degree of TGFß1 activity in the human cardiac ventricle. Overall, we found that TGFß1 is deleterious to the heart by regulating the adhesion properties of cardiomyocytes in an NCAM1-dependent mechanism. Our results suggest that inhibiting NCAM1 would be cardioprotective, counteract the pathological action of TGFß1 and reduce heart failure severity.

Designing proteins to combat disease: Cardiac troponin C as an example.

Throughout history, muscle research has led to numerous scientific breakthroughs that have brought insight to a more general understanding of all biological processes. Potentially one of the most influential discoveries was the role of the second messenger calcium and its myriad of handling and sensing systems that mechanistically control muscle contraction. In this review we will briefly discuss the significance of calcium as a universal second messenger along with some of the most common calcium binding motifs in proteins, focusing on the EF-hand. We will also describe some of our approaches to rationally design calcium binding proteins to palliate, or potentially even cure cardiovascular disease. Considering not all failing hearts have the same etiology, genetic background and co-morbidities, personalized therapies will need to be developed. We predict designer proteins will open doors for unprecedented personalized, and potentially, even generalized medicines as gene therapy or protein delivery techniques come to fruition.

Obligatory role of neuronal nitric oxide synthase in the heart's antioxidant adaptation with exercise.

Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso-redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso-redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO2max. This study demonstrates that exercise results in a positive shift in the nitroso-redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

Combined troponin I Ser-150 and Ser-23/24 phosphorylation sustains thin filament Ca(2+) sensitivity and accelerates deactivation in an acidic environment.

The binding of Ca(2+) to troponin C (TnC) in the troponin complex is a critical step regulating the thin filament, the actin-myosin interaction and cardiac contraction. Phosphorylation of the troponin complex is a key regulatory mechanism to match cardiac contraction to demand. Here we demonstrate that phosphorylation of the troponin I (TnI) subunit is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia decreases intracellular pH resulting in depressed binding of Ca(2+) to TnC and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations we measured individual TnI Ser-150 (S150D), Ser-23/24 (S23/24D) and combined (S23/24/150D) pseudo-phosphorylation effects on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca(2+) binding to TnC regardless of TnI composition, TnI S150D attenuated this decrease rendering it similar to non-phosphorylated TnI at normal pH. The dissociation of Ca(2+) from TnC was unaltered by pH such that TnI S150D remained slow, S23/24D remained accelerated and the combined S23/24/150D remained accelerated. This effect of the combined TnI Ser-150 and Ser-23/24 pseudo-phosphorylations to maintain Ca(2+) binding while accelerating Ca(2+) dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate thin filament deactivation and maintain Ca(2+) sensitive activation. These data suggest that TnI Ser-150 phosphorylation induced attenuation of the pH-dependent decrease in Ca(2+) sensitivity and its combination with Ser-23/24 phosphorylation to maintain accelerated thin filament deactivation may impart an adaptive role to preserve contraction during acidic ischemia pH without slowing relaxation.

Effects of insulin resistance on skeletal muscle growth and exercise capacity in type 2 diabetic mouse models.

Type 2 diabetes mellitus is associated with an accelerated muscle loss during aging, decreased muscle function, and increased disability. To better understand the mechanisms causing this muscle deterioration in type 2 diabetes, we assessed muscle weight, exercise capacity, and biochemistry in db/db and TallyHo mice at prediabetic and overtly diabetic ages. Maximum running speeds and muscle weights were already reduced in prediabetic db/db mice when compared with lean controls and more severely reduced in the overtly diabetic db/db mice. In contrast to db/db mice, TallyHo muscle size dramatically increased and maximum running speed was maintained during the progression from prediabetes to overt diabetes. Analysis of mechanisms that may contribute to decreased muscle weight in db/db mice demonstrated that insulin-dependent phosphorylation of enzymes that promote protein synthesis was severely blunted in db/db muscle. In addition, prediabetic (6-wk-old) and diabetic (12-wk-old) db/db muscle exhibited an increase in a marker of proteasomal protein degradation, the level of polyubiquitinated proteins. Chronic treadmill training of db/db mice improved glucose tolerance and exercise capacity, reduced markers of protein degradation, but only mildly increased muscle weight. The differences in muscle phenotype between these models of type 2 diabetes suggest that insulin resistance and chronic hyperglycemia alone are insufficient to rapidly decrease muscle size and function and that the effects of diabetes on muscle growth and function are animal model-dependent.

Maternal exercise preserves offspring cardiovascular health via oxidative regulation of the ryanodine receptor.

OBJECTIVE: The intrauterine environment during pregnancy is a critical factor in the development of obesity, diabetes, and cardiovascular disease in offspring. Maternal exercise prevents the detrimental effects of a maternal high fat diet on the metabolic health in adult offspring, but the effects of maternal exercise on offspring cardiovascular health have not been thoroughly investigated. METHODS: To determine the effects of maternal exercise on offspring cardiovascular health, female mice were fed a chow (C; 21% kcal from fat) or high-fat (H; 60% kcal from fat) diet and further subdivided into sedentary (CS, HS) or wheel exercised (CW, HW) prior to pregnancy and throughout gestation. Offspring were maintained in a sedentary state and chow-fed throughout 52 weeks of age and subjected to serial echocardiography and cardiomyocyte isolation for functional and mechanistic studies. RESULTS: High-fat fed sedentary dams (HS) produced female offspring with reduced ejection fraction (EF) compared to offspring from chow-fed dams (CS), but EF was preserved in offspring from high-fat fed exercised dams (HW) throughout 52 weeks of age. Cardiomyocytes from HW female offspring had increased kinetics, calcium cycling, and respiration compared to CS and HS offspring. HS offspring had increased oxidation of the RyR2 in cardiomyocytes coupled with increased baseline sarcomere length, resulting in RyR2 overactivity, which was negated in female HW offspring. CONCLUSIONS: These data suggest a role for maternal exercise to protect against the detrimental effects of a maternal high-fat diet on female offspring cardiac health. Maternal exercise improved female offspring cardiomyocyte contraction, calcium cycling, respiration, RyR2 oxidation, and RyR2 activity. These data present an important, translatable role for maternal exercise to preserve cardiac health of female offspring and provide insight on mechanisms to prevent the transmission of cardiovascular diseases to subsequent generations.

Neuronal nitric oxide synthase is indispensable for the cardiac adaptive effects of exercise.

Exercise results in beneficial adaptations of the heart that can be directly observed at the ventricular myocyte level. However, the molecular mechanism(s) responsible for these adaptations are not well understood. Interestingly, signaling via neuronal nitric oxide synthase (NOS1) within myocytes results in similar effects as exercise. Thus, the objective was to define the role NOS1 plays in the exercise-induced beneficial contractile effects in myocytes. After an 8-week aerobic interval training program, exercise-trained (Ex) mice had higher VO(2max) and cardiac hypertrophy compared to sedentary (Sed) mice. Ventricular myocytes from Ex mice had increased NOS1 expression and nitric oxide production compared to myocytes from Sed mice. Remarkably, acute NOS1 inhibition normalized the enhanced contraction (shortening and Ca(2+) transients) in Ex myocytes to Sed levels. The NOS1 effect on contraction was mediated via greater Ca(2+) cycling that resulted from increased phospholamban phosphorylation. Intriguingly, a similar aerobic interval training program on NOS1 knockout mice failed to produce any beneficial cardiac adaptations (VO(2max), hypertrophy, and contraction). These data demonstrate that the beneficial cardiac adaptations observed after exercise training were mediated via enhanced NOS1 signaling. Therefore, it is likely that beneficial effects of exercise may be mimicked by the interventions that increase NOS1 signaling. This pathway may provide a potential novel therapeutic target in cardiac patients who are unable or unwilling to exercise.

Tropomyosin Ser-283 pseudo-phosphorylation slows myofibril relaxation.

Tropomyosin (Tm) is a central protein in the Ca(2+) regulation of striated muscle. The αTm isoform undergoes phosphorylation at serine residue 283. While the biochemical and steady-state muscle function of muscle purified Tm phosphorylation have been explored, the effects of Tm phosphorylation on the dynamic properties of muscle contraction and relaxation are unknown. To investigate the kinetic regulatory role of αTm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild-type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants in insect cells. Purified Tm's regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca(2+) binding to troponin C (TnC) in reconstituted thin filaments did not differ between the 3 Tm's, however disassociation of Ca(2+) from filaments containing pseudo-phosphorylated Tm was slowed compared to wild-type Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics. These data demonstrate that Tm pseudo-phosphorylation slows deactivation of the thin filament and muscle force relaxation dynamics in the absence of dynamic and steady-state effects on muscle activation. This supports a role for Tm as a key protein in the regulation of muscle relaxation dynamics.

Expression levels of sarcolemmal membrane repair proteins following prolonged exercise training in mice.

Membrane repair is a conserved cellular process, where intracellular vesicles translocate to sites of plasma membrane injury to actively reseal membrane disruptions. Such membrane disruptions commonly occur in the course of normal physiology, particularly in skeletal muscles due to repeated contraction producing small tears in the sarcolemmal membrane. Here, we investigated whether prolonged exercise could produce adaptive changes in expression levels of proteins associated with the membrane repair process, including mitsugumin 53/tripartite motif-containing protein 72 (MG53/TRIM72), dysferlin and caveolin-3 (cav3). Mice were exercised using a treadmill running protocol and protein levels were measured by immunoblotting. The specificity of the antibodies used was established by immunoblot testing of various tissue lysates from both mice and rats. We found that MG53/TRIM72 immunostaining on isolated mouse skeletal muscle fibers showed protein localization at sites of membrane disruption created by the isolation of these muscle fibers. However, no significant changes in the expression levels of the tested membrane repair proteins were observed following prolonged treadmill running for eight weeks (30 to 80 min/day). These findings suggest that any compensation occurring in the membrane repair process in skeletal muscle following prolonged exercise does not affect the expression levels of these three key membrane repair proteins.

Micro-dystrophin gene therapy demonstrates long-term cardiac efficacy in a severe Duchenne muscular dystrophy model.

Micro-dystrophin gene replacement therapies for Duchenne muscular dystrophy (DMD) are currently in clinical trials, but have not been thoroughly investigated for their efficacy on cardiomyopathy progression to heart failure. We previously validated Fiona/dystrophin-utrophin-deficient (dko) mice as a DMD cardiomyopathy model that progresses to reduced ejection fraction indicative of heart failure. Adeno-associated viral (AAV) vector delivery of an early generation micro-dystrophin prevented cardiac pathology and functional decline through 1 year of age in this new model. We now show that gene therapy using a micro-dystrophin optimized for skeletal muscle efficacy (AAV-µDys5), and which is currently in a clinical trial, is able to fully prevent cardiac pathology and cardiac strain abnormalities and maintain normal (>45%) ejection fraction through 18 months of age in Fiona/dko mice. Early treatment with AAV-µDys5 prevents inflammation and fibrosis in Fiona/dko hearts. Collagen in cardiac fibrotic scars becomes more tightly packed from 12 to 18 months in Fiona/dko mice, but the area of fibrosis containing tenascin C does not change. Increased tight collagen correlates with unexpected improvements in Fiona/dko whole-heart function that maintain impaired cardiac strain and strain rate. This study supports micro-dystrophin gene therapy as a promising intervention for preventing DMD cardiomyopathy progression.

Effects of increased systolic Ca(2+) and ß-adrenergic stimulation on Ca(2+) transient decline in NOS1 knockout cardiac myocytes.

We have previously shown that the main factor responsible for the faster [Ca(2+)](i) decline rate with ß-adrenergic (ß-AR) stimulation is the phosphorylation of phospholamban (PLB) rather than the increase in systolic Ca(2+) levels. The purpose of this study was to correlate the extent of augmentation of PLB Serine(16) phosphorylation to the rate of [Ca(2+)](i) decline. Thus, ventricular myocytes were isolated from neuronal nitric oxide synthase knockout (NOS1(-/-)) mice, which we observed had lower basal PLB Serine(16) phosphorylation levels, but equal levels during ß-AR stimulation. Ca(2+) transients (Fluo-4) were measured in myocytes superfused with 3mM extracellular Ca(2+) ([Ca(2+)](o)) and a non-specific ß-AR agonist isoproterenol (ISO, 1µM) with 1mM [Ca(2+)](o). This allowed us to get matched Ca(2+) transient amplitudes in the same myocyte. Similar to our previous work, Ca(2+) transient decline was significantly faster with ISO compared to 3mM [Ca(2+)](o), even with matched Ca(2+) transient amplitudes. Interestingly, when we compared the effects of ISO on Ca(2+) transient decline between NOS1(-/-) and WT myocytes, ISO had a larger effect in NOS1(-/-) myocytes, which resulted in a greater percent decrease in the Ca(2+) transient RT(50). We believe this is due to a greater augmentation of PLB Serine16 phosphorylation in these myocytes. Thus, our results suggest that not only the amount but the extent of augmentation of PLB Serine(16) phosphorylation are the major determinants for the Ca(2+) decline rate. Furthermore, our data suggest that the molecular mechanisms of Ca(2+) transient decline is normal in NOS1(-/-) myocytes and that the slow basal Ca(2+) transient decline is predominantly due to decreased PLB phosphorylation.

Antiquated ejection fraction: Basic research applications for speckle tracking echocardiography.

For years, ejection fraction has been an essentially ubiquitous measurement for assessing the cardiovascular function of animal models in research labs. Despite technological advances, it remains the top choice among research labs for reporting heart function to this day, and is often overstated in applications. This unfortunately may lead to misinterpretation of data. Clinical approaches have now surpassed research methods, allowing for deeper analysis of the tiers of cardiovascular performance (cardiovascular performance, heart performance, systolic and diastolic function, and contractility). Analysis of each tier is crucial for understanding heart performance, mechanism of action, and disease diagnosis, classification, and progression. This review will elucidate the differences between the tiers of cardiovascular function and discuss the benefits of measuring each tier via speckle tracking echocardiography for basic scientists.